Principales impactos antrópicos y sus efectos

sobre la comunidad de peces del lago Titicaca

Mario Monroy López

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 Departamento de Biología Animal

Programa de Doctorado de Biodiversidad H0G01

Principales impactos antrópicos y sus efectos sobre la comunidad de peces del

lago Titicaca.

Memoria presentada por Mario Monroy López para optar por el grado de doctor

por la Universidad de Barcelona

El doctorando El director de tesis

Mario Monroy López Dr. Adolfo de Sostoa Fernández

Profesor Titular

Departamento de Biología Animal

Universidad de Barcelona
Diseño:

©Rubén Monroy

www.rubenmonroy.com
 A mis padres,
a mi hermano,

a los pobladores del lago Titicaca

y a todas las personas que luchan por su conservación.

“Si tienes la paciencia de la tierra, la pureza del agua y la justicia del viento, entonces eres
libre”.
Paulo Coelho
AGRADECIMIENTOS
Esta tesis además de haber sido un proceso enriquecedor en el campo científico también
lo fue en el campo personal. Durante este largo camino recorrido lleno de vivencias y anécdo-
tas, son muchas las personas a las que tengo agradecer por haberme ayudado a conseguir este
gran objetivo y haber sido mi apoyo y mi aliento en todos los buenos y malos momentos de
esta aventura que finalmente ha llegado a buen puerto.
En primer lugar me gustaría agradecer a mi director Adolfo de Sostoa. Muchas gracias
Adolfo por la confianza y por dejarme trabajar en tu grupo. Desde los inicios, pasando datos
biológicos y posteriormente, cuando me ofreciste la oportunidad de poder combinar el trabajo
de máster con las faenas de pesca por los ríos catalanes, me fui sumergiendo en el mundo de
la ictiología dulceacuícola. Gracias a ello, desarrollamos parte del proyecto del lago Titicaca
y compartimos unas cuantas campañas y congresos por Perú y Bolivia en viajes muy enrique-
cedores. Por lo tanto, sin todas estas oportunidades esta tesis no hubiera sido posible. Segura-
mente que algunas veces habrás tenido que consultar mi continuidad con la almohada y con
María, así que le hago extenso mi agradecimiento jejeje. En Perú y Bolivia fueron muchas las
personas e instituciones que hicieron posible la toma de los datos y la logística de las campa-
ñas. Especialmente mis más sinceros agradimientos a los miembros de la Agencia Española
de Cooperación Internacional (AECID), el Ministerio de Producción de Perú, la Autoridad
Binacional del lago Titicaca (ALT), el Instituto del Mar de Perú (IMARPE) y el Proyecto
Especial Binacional del lago Titicaca (PELT), especialmente a Itziar, Kalen, Jordi, Orieta y
Ernesto. Por otro lado, agradezco principalmente a Silvia y Patricia que nos ayudaron de par-
te del Institut de Recerca i Tecnología Agroalimentaries (IRTA) en los muestreos.
Retomando el tema de los profesores, me gustaría dar las gracias a todos los “profes”
con los cuales tuve algún trabajo de campo, me enseñaron a utilizar alguna técnica, me dieron
algún consejo, o al menos me dirigieron un saludo en el pasillo del departamento! Principal-
mente gracias a Carles Nadal y Xavier Ferrer por su interés en el desarrollo de mi tesis y en
las historias de sus viajes, a Dolors Vinyoles, Domingo Rodríguez, Narcís Prat y María Rie-
radevall por los momentos compartidos en las salidas de las prácticas de gestión de fauna
acuática y del proyecto del río Ripoll, a Lluís Jové por sus opiniones sobre el artículo de isó-
topos, a Humbert Salvadó por su paciencia en enseñarme a manejar la cámara para tomar las
fotografías en el microscopio y para la identificación de las "Trichodinas" y a Isabel Navarro
por facilitar la transición entre la tesina de máster de acuicultura y el cambio al grupo de ic-
tiología.
Pasando a los compañeros del despacho, tengo que hacer una mención especial a mi gran
amigo Alberto Maceda. Primero que todo, a nivel científico he aprendido muchísimo de ti y
eres un ejemplo a seguir, gracias por todas las codirecciones, correcciones (más rojo no puede
haber en una hoja) y consejos que han sido la base fundamental para la presentación de esta
tesis. Por otro lado, durante todo el tiempo que compartimos el despacho siempre fuiste un
apoyo incondicional como amigo y psicólogo de cabecera jejejej (tienes que publicar unas
memorias del departamento algún día!). No tengo palabras para agradecer por los buenos mo-
mentos vividos: viajes, campañas, anécdotas, etc., y por mostrarme la filosofía gallega: "be
water my friend que tarde o temprano todo cae por su propio peso". Espero que algún día re-
greses a la facultad como profe porque tienes mucha madera para eso. Otras personas que
pasaron por el despacho y que compartieron mis inicios en el grupo fueron Thais, Alex
(fantasma), Cons y Edgar a quienes recuerdo con cariño porque con ellos di mis primeros
pasos como "peixatero". Posteriormente han ido y venido varios alumnos que han hecho cola-
boraciones (Laura, Marc, Yutzil, Martí, Mireia (Mare de Déu dels castellers jejeje), tesinas de
licenciatura (Sergi) y trabajos de Máster (Elisenda, Lorena, Oriol) y con los cuales ha sido un
placer compartir experiencias. Entre ellos, Yutzil, Sergi y Oriol, muchísimas gracias por la
gran ayuda con parte del trabajo de laboratorio de la tesis. Similarmente quiero hacer otra
mención especial a mis dos actuales compañeros de despacho y estudiantes de doctorado la
Nicole y el Oriol, los cuales me han ayudado en el día a día de la tesis. Con la Nico, además
de las campañas de su tesis, hemos compartido varios momentos buenos como los bailes de
salsa, varias cenas, fiestas y reuniones así que sólo me queda decir Chichichi Lelele viva Chi-
le lelé. Con el Ori casi igual que con Nicole (eso si nunca será mi pareja de baile, ni siquiera
de sardana jejeje) pero hasta terminamos jugando futbol en el mismo equipo! Ha sido todo
un placer compartir tan buenos momentos con ustedes y espero que saquen esa tesis muy
pronto.
 Como ya han pasado varios años en el departamento (no hay mal que por bien no ven-
ga), he conocido un montón de personas de todas las nacionalidades y de los cuales quiero
acordarme en estos agradecimientos. Sin un orden particular, quiero agradecer a mis amigos
Adolfo San Juan (gracias por la conexión con el grupo de ictiólogos y los amigos del máster
de biodiversitat), Pablo (espero que te den el DNI ecuatoriano algún día! y gracias por el pas-
tel de jaiba y las atenciones por Chile), Aina (sólo diosa), Mariancilla (mamacilla tu tierrinha
me encantó), Raquel (nunca olvidaré nuestros viajes), Juan Pablo, Karina, Juan Carlos y Vir-
ginia (por el viaje a Marruecos y las fiestas), Miguel (por las “croquetas” de Blanes), Chelina
(por las excursiones) y a Desy, Juli, Marquitos, Margarita, Chuck, Alí, Fernando, Clarice,
Alicia, Anna por los buenos momentos pasados. En cuanto a la "vieja guardia" de la cual ya
hago parte, un sincero agradecimiento al Albert Cama (por los tiempos de birra), Isa (porque
has dejado un legado en los diseños de tesis difícil de superar), Fran R., Raül y Rocío (por los
inicios en los isótopos), Owen (por los viajes y los buceos), la White (por sus idas de olla),
Olatz (por todos los buenos momentos que pasamos, aunque quedan todavía muchas playas
por visitar; ánimos con esa tesis Peque que ya falta muy poco!), Gemma (por los ánimos y tu
carácter de lucha), Luigi (porque nos enseñaste que si uno se lo propone se puede conseguir
cualquier cosa), Eloy (por la amistad y por tu disponibilidad para ayudar) y Manolo (por los
censos en la facultad jejeje, por tantas tardes de risas y por dar la nota diferente en el departa-
mento) y en general a Javi C., Laura C., Aida, Marc (Batman), Marc, Inés, María, Noel, Ser-
gi, Jenny, Laura (por el interés en el desarrollo de la tesis).
Junto a ellos, fueron entrando diferentes becarios y alumnos con los cuales compartí un
sinfín de historias y que me aportaron, la mayor parte del tiempo, muchas alegrías. Muchas
gracias a Edu (por tus ganas de lucha y tu disposición para resolver los problemas estadísticos
en R, eres un crack y me evitaste muchas úlceras), Débora (que rico, no digo más!), Fabi (por
tu espíritu de hacer las cosas mejores), Urtxi (por encontrar el verdadero sentido de las MA-
NOVAS), Teresinha (por tu constancia y por estar siempre para todo, eres una gran científica
y una gran persona, no lo olvides! y un placer verte desfilar por los pasillos del departamen-
to), Néstor (nos echamos unas buenas risas mi niiiiiiiññññio), Heather (por iluminar nuestras
vidas jeje y por los viajes), Jóse (por mantener bendecido al rebaño no sólo con agua bendi-
ta!, muchos ánimos que al final lo conseguirás), Laura S. (por tu risa contagiosa que nos
alegró más de una tarde gris), Zuzy (por tu modo zen y las ayudas con los scripts de R), Vero
(por tu ingenuidad y tu esfuerzo que es todo un ejemplo a seguir), Irene (por compartir parte
de tu mundo paralelo conmigo, por el “coñacao” y por las ayudas con los gráficos, explota
esa vocación!), Rosana (por tu forma diferente de ver la vida, que chimba Paisandú), Karla
(por tu alegría y solidaridad), Francesc (por tus enseñanzas mitológicas), Jaime (por tu senci-
llez y amabilidad), Fernanda (por tu naturalidad y porque tu llegada ha sido un bálsamo para
afrontar la última etapa de esta tesis “mesmo”, nos quedan muchos “deberes” por hacer), Ca-
rolina (por ser el respaldo colombiano en el departamento), Toni cubano (porque remando
llegarás jeje), Laurita (por tu positivismo que se contagia y tu forma de ser), Núria (por tu
amistad, alegría y las cavas), Helena y Moni (por todas las excursiones y los buenos ratos que
pasamos en la montaña) y en general a Alex, Sandra, Mar1, Mar2, Oriol, Carme, Víctor H.,
Daniela, Marcel, Morgana, Lyssette, Giulia, Eudald, Elisa, Elizabeth, Diego y Adriana por
interesarse en cómo iba el desarrollo de la tesis. Siguiendo con el personal de la universidad,
me gustaría agradecer a Joan por sus charlas apasionadas sobre fútbol y las nuevas incorpora-
ciones del departamento jeje, a las secretarias por sus labores que muchas veces pasan des-
apercibidas y porque siempre tuvieron para mí una sonrisa pese a darles más trabajo, a la
Helen por sus historias mientras trabajaba, a José Luis y Miguel del bar por su simpatía y sus
bromas en la cafetería y a los técnicos de la uni los cuales siempre han tenido la amabilidad
de perder unos minutos de su tiempo para explicarme muchas metodologías y procedimientos
de laboratorio, en especial a Raquel, Maite 1, Maite 2, Toni, Paco, Pilar, Pau, Iraima, Jordi,
Laia, Jessica.
Fuera de la universidad, me gustaría agradecer también a los amigos que he conocido
durante todo este tiempo en España, los cuales además, involucran una parte sentimental y
fueron mi salida de escape en los momentos difíciles. Por una parte, agradezco especialmente
a los amigos del Máster de Acuicultura de Gran Canarias principalmente a Tania, Chili ,
Pims y Eze por demostrarme su cariño y nunca perder el contacto a pesar de la distancia, así
como también a la familia Martín que es como mi familia adoptiva. Por otro lado, muchísi-
mas gracias a los amigos de Barcelona los cuales hicieron esta parte de mi vida una de las
mejores, principalmente al “parche” (Helene, Oscar, Joyce, Gabriel, Karen, Rodrigo, Lucía,
Alejo, Seve y Víctor) y a otras personas como Sofi y Lara, que vivieron muy de cerca el
transcurrir de esta tesis.
Para finalizar, me gustaría dar las gracias a mis padres y mi familia por su sacrificio y
apoyo infinito para que haya podido cumplir mi sueño de viajar y estudiar en el exterior. Han
sido años muy duros lejos de mi familia pero que al final han valido la pena. Todo esto es por
y para ustedes.
TABLA DE CONTENIDO

INTRODUCCIÓN GENERAL………………………………………………………………13

OBJETIVOS……………………………………………………………………………….....41

INFORME DEL DIRECTOR………………………………………………………………..45

CAPÍTULO 1. Trophic interactions between native and introduced fish species in a littoral

fish community ………………….……………………………………………………………...51

CAPÍTULO 2. Metal concentration in water, sediment and four fish species from Lake Ti-

ticaca reveals a large-scale environmental concern…...………………………………….....71

CAPÍTULO 3. Blood analyses in three fish species indicate the presence of cytotoxic and

genotoxic compounds in Lake Titicaca…………………………...………………………...105

CAPÍTULO 4. Gillnet selectivity in the offshore ichthyic community from Lake Titicaca…133

DISCUSIÓN GENERAL…………………………………………………………………...157

CONCLUSIONES GENERALES………………………………………………………….171

BIBLIOGRAFÍA…………………………………………………………………………....177

ANEXOS……………………………………………………………………………………203
Introducción general
 INTRODUCCIÓN GENERAL

El agua dulce es el recurso más explotado por la humanidad ocupando sólo el 0.8% de la
superficie y el 0.01% del volumen total de agua del planeta (Gleick, 1996; Helfman, 2007). A
pesar de estos bajos porcentajes, los ecosistemas dulceacuícolas albergan cerca del 6% de las
especies animales descritas, abarcando aproximadamente un 40% de los peces y un tercio de
la diversidad de vertebrados a nivel mundial (incluyendo anfibios, reptiles acuáticos y mamí-
feros) (Dudgeon et al., 2006; Helfman, 2007; Elosegui & Sabater, 2009). Dentro de éstos eco-
sistemas, los ríos y lagos son los más impactados a escala global (Dudgeon et al., 2006; Helf-
man, 2007; Leprieur et al., 2008; Hinton-Taylor et al., 2009; Vörösmarty et al., 2010) (Fig.1),
siendo mucho mayor el decrecimiento de su biodiversidad que en los ecosistemas terrestres
más afectados (Sala et al., 2000).

Fig. 1. Mapa del patrón de riesgo para el consumo humano de agua y la biodiversidad acuática.
Modificado de Vörösmarty et al. (2010).

Los principales factores de amenaza para la biodiversidad de los ecosistemas dulceacuí-

colas pueden ser agrupados en cinco categorías: la introducción de especies exóticas, la des-
trucción o degradación del hábitat, la contaminación del agua, la modificación del caudal y la
sobrepesca (Fig.2) (Allan & Flecker, 1993; Dudgeon et al., 2006). Estos impactos antropogé-
nicos pueden derivar en la extinción de especies y en profundos cambios en la estructura bio-

14
 Introducción general

lógica a diferentes niveles jerárquicos alterando los flujos de energía de los ecosistemas
(Covich et al., 2004; Duffy et al., 2007; Cucherousset et al., 2012). Esta situación ha generado
en los últimos años un proceso de homogenización de las especies a escala global, que resulta
más evidente en las regiones tropicales y que no solo tiene repercusiones a escala ecológica
(ej. alteraciones de la red alimentaria) sino que también puede influir a nivel sociológico (ej.
implicaciones en la salud humana) y económico (ej. pesquerías) (Hall & Mills, 2000; Balirwa
et al., 2003; Jones et al., 2010).

Fig. 2. Principales amenazas sobre la biodiversidad de los cuerpos de agua dulce. Los cambios
ambientales a escala global tales como deposición de nitrógeno, calentamiento y variaciones en
precipitación y escorrentía son asumidos sobre todas estas categorías. Modificado de Dudgeon et
al. (2006).

El mantenimiento de las comunidades acuáticas se sustenta en las relaciones ecológicas

entre las especies y el medio ambiente (Margalef, 1983; Preston, 2002; Sandin & Solimini,
2009). Por lo tanto, cualquier alteración tanto biótica como abiótica que afecte el equilibrio
homeostático de las especies o de su entorno puede ser una posible causa de estrés (Maceda-

15
 Veiga, 2011). Sin embargo, la sola presencia de un agente estresor en el ambiente acuático (ej.
metales pesados, pesticidas) no es indicadora de que se estén produciendo efectos negativos.
Esto se debe a que el desarrollo de los efectos del estrés dependerá de la intensidad y la dura-
ción del agente estresor (Fig. 3) y de las características específicas de cada especie e individuo
para adaptarse y recuperarse de la perturbación (Adams & Greeley, 2000; Archard et al.,
2012; de Castilhos, 2012). Si el estrés es intermitente o constante durante un largo período de
tiempo, es considerado como estrés crónico y si es generado por un estímulo intenso de corta
duración, es denominado agudo (Fig. 4).

(-)
(+)

Éxito poblacional
Estructura
Crecimiento

Relevancia ecológica
Tiempo de respuesta

Reproductivo
Bioenergético
Histopatológico

Fisiológico
Bioquímico
Biomolecular

(+)
(-)

Sub-organismo Individuo Población

Nivel biológico

Fig. 3. Tiempo transcurrido para la detección de algunos efectos del estrés y la relevancia ecológi-
ca de las alteraciones detectadas a escala de sub-organismo, individuo y población (adaptado de
Adams & Greeley, 2000).

No obstante, los organismos sometidos a estrés pueden presentar una serie de mecanis-
mos fisiológicos para mitigar los efectos generados por los cambios ambientales (proceso de
aclimatación) e incluso desarrollar un proceso de adaptación que implica cambios genéticos a

16
 Introducción general

lo largo del tiempo (Mason, 2002; Medina et al., 2007; Øverli et al., 2007; Moiseenko, 2010;
Archard et al., 2012). Sin embargo, para explorar las rutas de entrada de los agentes estresores
y conocer la relevancia ecológica que tienen los impactos antrópicos en una especie o grupo
funcional, se debe conocer de antemano su estructura trófica en el ecosistema (Dudgeon et
al., 2006). Por lo tanto, el establecimiento de las preferencias tróficas de una población es
esencial para proponer medidas de gestión, facilitando la predicción de cambios y consecuen-
cias en la dinámica poblacional de la fauna salvaje.

Estrés

Crónico Agudo

Sistema endocrino Sistema inmune Sistema nervioso

Ajustes fisiológicos Desajustes bioquímicos Cambios en el comportamiento

Aclimatación Patologías y disfunciones fisiológicas Cambios a escala de población

Mecanismos evolutivos Muerte

Adaptación

Fig. 4. Esquema general de los efectos del estrés en peces (adaptado de Maceda-Veiga, 2011).

Uso de isótopos estables para determinar interacciones tróficas

En los últimos años se ha incrementado el uso del análisis de isótopos estables (SIA),
principalmente de los isótopos de carbono (13C/12C, δ13C) y nitrógeno (15N/14N, δ15N), para
explorar las preferencias alimentarias y las relaciones tróficas de los sistemas acuáticos a

17
 través de la estructura de las redes alimentarias y evaluar los efectos de la introducción de es-
pecies en un ecosistema (Vander Zanden & Rasmussen, 1996; Vander Zanden et al., 1997,
1999; Post, 2002; Fry, 2006) (Cuadro 1). Los isótopos estables pueden dar una información
complementaria a los análisis tradicionales de dieta, dilucidando la estructura de la red ali-
menticia y las relaciones tróficas entre las especies (Vander Zanden & Rasmussen, 1996,
2001; Post, 2002; Bearhop et al., 2004). Los análisis de dieta a través del método tradicional
presentan varias limitaciones que pueden interferir en la interpretación de los resultados tales
como sesgos de distinta magnitud, dependiendo del tipo de comida analizada. Esto se debe a
que dichos estudios se basan en observaciones directas las cuales, generalmente, están sesga-
das hacia la presa más conspicua, subestimando las presas más digeridas o con mayor dificul-
tad para su identificación (Bearhop et al., 2004). Adicionalmente, éste tipo de análisis sólo
permite determinar la información de un momento específico de la dieta del individuo y no
provee información de los recursos utilizados en el pasado (Vander Zanden & Rasmussen,
2001; Vander Zanden & Vadeboncoeur, 2002).
Aunque SIA no provee el detalle taxonómico que puede dar el método tradicional de
análisis de contenidos estomacales, evita los sesgos dados por el grado de digestión de las pre-
sas ya que sólo tiene en cuenta la comida asimilada (Hobson, 1999; Bearhop et al., 2004;
Clarke et al., 2005). Además, SIA puede proveer una estima integrada de la dieta asimilada
por un cierto período de tiempo dependiendo del tipo de tejido evaluado (Hobson, 1999). Esto
es posible debido a que los isótopos estables son incorporados a través de la dieta aumentando
un 1‰ en el δ13C de un consumidor con respecto a su presa (France, 1995; Vander Zanden &
Rasmussen, 2001; Clarke et al., 2005). En particular, las medidas de δ13C son indicadoras de
la fuente de productividad primaria de carbono ya que proveen información espacial sobre la
localidad donde se alimenta una especie determinada (Vander Zanden et al., 1997, 1999; Van-
der Zanden & Rasmussen, 2001). Esto se debe a que el δ13C es generalmente mayor en los
consumidores litorales de los ecosistemas acuáticos (principalmente en lagos) que en los pelá-
gicos, por la mayor interacción con ecosistemas terrestres con altos contenidos de carbono
(France, 1995; Vander Zanden et al., 1997). De igual forma, los organismos que viven en zo-
nas profundas tienden a presentar un δ13C más negativo con respecto a los pelágicos, debido al

18
 Introducción general

Cuadro 1. Qué son los isótopos estables?

Los isótopos son las diferentes formas de un elemento químico que se diferencian en el
número de neutrones de su núcleo y que por lo tanto varían en masa atómica. Se dividen en
estables (cuando la relación entre protones y neutrones es la adecuada para tener una estabi-
lidad nuclear) o inestables (radioactivos) los cuales tienen tendencia a desintegrarse (Fig. 5).
Los isótopos más utilizados en estudios de ecología trófica son los correspondientes a los
elementos de carbono (C) y nitrógeno (N) debido a su abundancia y a que están presentes
en numerosos procesos naturales (Peterson & Fry, 1987; West, 2006).
La forma más abundante de carbono es el isótopo más liviano el 12C (98.9%) y la me-
nor es la del isótopo más pesado 13C (1.10%). Por su parte el nitrógeno presenta dos isóto-
pos el 14N (99.63%) y el 15N que se encuentra en muy poca proporción (0.37%) (Fry, 2006).
La diferencia en masa (peso) genera que los diferentes isótopos se comporten de manera
diferente en las reacciones bioquímicas en las que intervienen, tendiendo los isótopos más
livianos a reaccionar de forma más rápida, generando una separación. Debido a esto, la pro-
porción isotópica del consumidor varía respecto a su dieta quedando reflejada en sus teji-
dos.

Fig. 5. Formas isotópicas del carbono.

decrecimiento de la producción primaria al aumentar la profundidad (Hecky & Hesslein,

1995; Vander Zanden & Rasmussen, 1996).

19
 La ratio de los isótopos estables puede también cambiar entre la dieta y el consumidor
debido a diferencias en el fraccionamiento durante la asimilación en los procesos metabólicos
(e.j. respiración, excreción) o en la digestión, principalmente en organismos herbívoros, debi-
do a la presencia de complejos carbohidratos como la celulosa o la lignina (McCutchan et al.,
2003). Por lo tanto, los individuos que se alimentan de plantas vasculares (incluyendo organis-
mos omnívoros) tienden a presentar un mayor fraccionamiento que los individuos que presen-
tan otras dietas (e.j. organismos carnívoros). Este fraccionamiento metabólico puede hacer
variar los ratios isotópicos de diversos tejidos entre individuos debido a su composición, prin-
cipalmente por el porcentaje de lípidos (Hobson & Clark, 1992, Focken & Becker, 1998). Los
lípidos usualmente están empobrecidos en 13C en relación a la proteína y los carbohidratos por
el proceso de fraccionamiento durante la oxidación del piruvato a acetyl coenzima A (Focken
& Becker, 1998). Por lo tanto, los valores de δ13C son más bajos en muestras con alto con-
tenido de lípidos (Focken & Becker, 1998; McCutchan et al., 2003).
Por otro lado, las medidas de δ15N sirven para identificar el nivel trófico del consumidor,
ya que existe un incremento progresivo de 3-4‰ en δ15N a medida que se avanza de nivel
trófico (Peterson & Fry, 1987; Vander Zanden & Rasmussen, 2001; Vanderklift & Ponsard,
2003; Clarke et al., 2005). La proporción isotópica del 15N/14N aumenta a lo largo de la ca-
15
dena trófica debido a una menor intervención del N en las reacciones bioquímicas, por lo
que su análisis permite determinar el nivel trófico del consumidor (Kelly, 2000). Ya que el
nitrógeno excretado es menor en 15N en relación a la dieta, el fraccionamiento trófico debería
ser mayor entre animales que se alimentan de dietas con alto contenido en nitrógeno
(McCutchan et al., 2003). No obstante, el uso de los isótopos estables para describir las rela-
ciones tróficas y el estado ecológico de una especie, debe estar acompañado de otras he-
rramientas que integren los mecanismos de variabilidad en el funcionamiento del ecosistema
(Vander Zanden et al., 1997).

Evaluación del estado de los ecosistemas lacustres

Actualmente, la cuantificación del nivel de degradación de los lagos se puede expresar en
parámetros como la hidrología (indicadores hidromorfológicos), la calidad del agua

20
 Introducción general

(indicadores químicos) o la producción pesquera (indicadores biológicos o bioindicadores)

(Lyons et al., 2000). Un bioindicador es definido como un organismo cuya presencia, ausen-
cia, comportamiento o alguna otra característica da información de las condiciones ambienta-
les de su hábitat (de Castilhos, 2012). Tradicionalmente, esta cuantificación en ecosistemas
acuáticos se realiza en base al análisis de bioindicadores en comunidades de flora, macroin-
vertebrados y peces que reflejan el estado ecológico de la zona que habitan (ej. Barbour et al.,
1999; Prat & Munné, 2000; Ricart et al., 2010; Jorgensen et al., 2010). Respecto a este tipo de
indicadores, uno de los más importante es el índice de integridad biótica (IBI), que utiliza los
peces como bioindicadores del estado del ecosistema (Karr, 1981), aunque existen muy pocos
IBIs adecuados para lagos (ej. Harig & Bain, 1998; Jennings et al., 1998; Lyons et al., 2000).
Por lo tanto, el desarrollo de otro tipo de herramientas de diagnóstico, principalmente en gran-
des lagos, es necesario para evaluar el estado de sus poblaciones.
En comparación a otros grupos de animales (ej. macroinvertebrados, diatomeas), los pe-
ces presentan diversas características que los hacen ideales como organismos indicadores del
estado de salud de los ecosistemas acuáticos (Simon, 1999; Damásio et al., 2007; Benejam et
al., 2008; Jorgensen et al., 2010). Los peces han colonizado la mayoría de los ambientes de
agua dulce y son el grupo más estudiado de la fauna acuática, pudiendo integrar efectos de
larga duración en el ecosistema debido a que tienen ciclos de vida longevos (generalmente
entre 2-10 años), se encuentran distribuidos en diversos hábitats, presentan tamaños más gran-
des que otras especies y ocupan varios niveles de la red trófica (Benejam, 2008; Maceda-
Veiga, 2011). Estos atributos son ideales para el seguimiento de procesos de bioacumulación
y biomagnificación de sustancias nocivas a lo largo de la red trófica (Barbour et al., 1999;
Ribeiro et al., 2005; Damásio et al., 2007; Barata et al., 2010).
A pesar de que la exposición a estresores ambientales (ej. contaminación por metales pe-
sados) puede causar efectos adversos en los peces (ej. alteraciones metabólicas, reducción del
potencial reproductivo y de crecimiento), no todas las especies presentan la misma susceptibi-
lidad a los diferentes impactos y contaminantes (Kennard et al., 2005; Maceda-Veiga & de
Sostoa, 2011; Maceda-Veiga et al., 2013). Estos efectos son a menudo difíciles de detectar en
organismos salvajes porque tienden a manifestarse sólo después de largos períodos de tiempo

21
 cuando el proceso destructivo es generalmente irreversible (de Castilhos, 2012). Por lo tanto, es
preciso y necesario identificar de forma temprana, mediante el uso de biomarcadores, los parámetros
que adviertan de los posibles impactos de los estresores antes de que comiencen a ser críticos.
(Maceda-Veiga et al., 2010; de Castilhos, 2012) (Fig. 3). Los biomarcadores son útiles para
identificar las primeras etapas de efectos biológicos (Vasseur & Cossu-Leguille, 2003), para
determinar las fuentes de contaminación de un contaminante (Van der Oost et al., 2003; Ma-
ceda-Vaiga et al., 2010, 2013) y para evaluar la relación entre la exposición de un contami-
nante y el efecto que genera en una diana específica (Houston, 1997; Galloway et al., 2004)
(Cuadro 2).
Aparte de los típicos parámetros morfológicos (ej. índices de condición o histopatología),
los principales biomarcadores en peces pueden incluir enzimas, proteínas, hormonas, paráme-
tros inmunológicos, neurológicos, genotóxicos, histopatológicos y hematológicos (Tabla 1).
La selección apropiada de un biomarcador requiere un cuidadoso estudio de su especificidad y
sensibilidad para tener una mayor precisión en la evaluación del riesgo producido por un es-
tresor ambiental, ya que muchas variables pueden simplemente mostrar interacciones ines-
pecíficas con el organismo bioindicador (WHO, 1993; Hagger et al., 2006). Sin embargo, de-
be tenerse en cuenta que muchos parámetros bióticos (ej. especie, sexo, edad, condición física
y reproductiva) y abióticos (ej. temperatura, salinidad, pH, dieta) pueden influenciar el rango
fisiológico de un biomarcador y su respuesta bajo un estrés ambiental (Sánchez & Porcher,
2009). Esta situación conlleva a que la evaluación de la idoneidad de un biomarcador tenga
que ser combinada con la respuesta a factores ecológicos y ambientales así como otros bio-
marcadores tales como la evaluación histopatológica (Ribeiro et al., 2005; Koca et al., 2008),
el factor de condición (Bervoets & Blust, 2003) o índices organosomáticos (Van der Oost et
al., 2003). Por lo tanto, deben establecerse conexiones entre los niveles externos de exposi-
ción de un estresor, los niveles internos de contaminación en el tejido y los efectos adversos
que causan en los organismos (de Castilhos, 2012).

Biomarcadores hematológicos para determinar estresores ambientales

La monitorización de los efectos generados por estresores ambientales en especies vivas
debe realizarse procurando utilizar herramientas de fácil uso, económicas y que eviten el mal-

22
 Introducción general

Cuadro 2. Diferentes tipos de biomarcadores.

Un biomarcador es una variación bioquímica, celular, fisiológica o de comporta-

miento que puede ser medida en un tejido, fluido corporal u organismo, y que provea
una respuesta a la exposición y/o efecto de agentes tóxicos o cualquier otra fuente de
estrés (Van Gestel & Van Brummelen, 1996; Adams, 2002; Di Giulio & Hinton, 2008;
Sánchez & Porcher, 2009). De acuerdo a la Organización Mundial de la Salud (WHO,
1993) existen tres tipos de biomarcadores:
Biomarcador de efecto: cualquier medida bioquímica, fisiológica, de comporta-
miento u otra alteración de un organismo que dependiendo de su magnitud, pueda ser
reconocida o asociada a un posible riesgo para la salud.
Biomarcador de exposición: cualquier sustancia exógena, su metabolito o el pro-
ducto de una interacción entre un agente xenobiótico y una célula o molécula diana que
pueda ser medida en algún compartimento de un organismo.
Biomarcador de susceptibilidad: indica de la predisposición o capacidad para res-
ponder a la exposición de una sustancia xenobiótica específica.

trato de los organismos como es el caso de los biomarcadores no específicos (Smith & Dar-
wall, 2006; Moyle et al., 2011; Maceda-Veiga et al., 2013). Dentro de éstos, los biomarcado-
res que actúan con un menor tiempo de respuesta y que presentan mayor relevancia ecológica
(ej. nivel molecular), tales como los parámetros hematológicos, han sido ampliamente acepta-
dos como buenos indicadores de disfunciones fisiológicas en medicina humana (ver WHO,
1993) y están siendo cada vez más utilizados en el monitoreo de peces. Tal es el caso del por-
centaje de hematocrito y hemoglobina (ej. Roche & Bogé, 1996; Maceda-Veiga et al., 2010,
2013), presencia de micronúcleos (ej. Ayllon & Garcia-Vazquez, 2001; Grisolia & Starling,
2001; Çavas et al., 2005), deformidades nucleares de los eritrocitos (ENAs) (ej. Pacheco &
Santos, 2002; Bolognesi & Hayashi, 2011; Maceda-Veiga et al., 2010, 2013), nivel de glucosa
y cortisol (Roche & Bogé, 1996) o la respuesta de algunas transaminasas o fosfatasas en el
suero (ej. Agrahari et al., 2007; Öner et al., 2008).

23
 Tabla 1. Principales biomarcadores usados para la evaluación toxicológica del estado de salud de
los peces (Modificado de Sánchez & Porcher, 2009).

Biomarcador Función Sensibilidad

Enzima implicada en la transmisión

Acetilcolinesterasa Organosfosfatos
nerviosa
Actividad EROD Biotransformación de enzimas PCBs, PAHs y dioxinas
Actividad de las lisozi- Salud del organismo en
Factor de resistencia a enfermedades
mas general
Análisis de agregados Primera línea de defensa del sistema Múltiples contaminantes
de macrófagos inmune incluyendo PAHs
Hormona liberada como respuesta al Compuestos genotóxicos
Cortisol
estrés y metales pesados

Contiene las instrucciones genéticas Compuestos genotóxicos

Daño del ácido desoxi-
usadas en el desarrollo y funciona- incluyendo PAHs y meta-
rribonucleico
miento de los organismos les pesados

Deformidades nucleares Compuestos genotóxicos

Transporte de oxígeno en la sangre
de los eritrocitos y metales pesados
Estabilidad de los liso- Desempeñan importantes funciones Salud del organismo en
somas en el hígado general
Proteína de protección contra el Metales pesados e induc-
Metalotioneina
estrés oxidativo tores del estrés oxidativo

Precursor de la yema del huevo, nor- Compuestos disruptores

Vitelogenina
malmente sintetizada por hembras endocrinos estrogénicos

EROD: Ethoxyresorufin-O-dietilasa
PCBs: Bifenilos policlorados
PAHs: Hidrocarburos aromáticos policíclicos

24
 Introducción general

En este contexto, la sangre de los peces está formada por dos tipos principales de células:
los eritrocitos (glóbulos rojos) con una función primaria de transporte de sustancias y elemen-
tos (ej. O2, CO2, glucosa) y los leucocitos (glóbulos blancos) que desempeñan una función de
defensa contra sustancias extrañas o agentes infecciosos, los cuales están divididos en neutró-
filos, basófilos o eosinófilos si son polinucleares o en linfocitos y monocitos si son mononu-
cleares (Fänge, 1992). No obstante, la sangre también está constituida por fragmentos de célu-
las llamados trombocitos (plaquetas) que inician la formación de coágulos o trombos en caso
de hemorragia. Sin embargo, algunas células sanguíneas varían en estructura y función según
la especie (Fänge, 1992). Tal es el caso de los eritrocitos de los peces de agua dulce, los cuales
son nucleados y que en la mayoría de los casos presentan una forma elíptica a diferencia de
los eritrocitos de peces elasmobranquios o de mamíferos (Fänge, 1992). La presencia del
núcleo en los eritrocitos, los convierte en buenos candidatos para evaluar los efectos genotóxi-
cos de un contaminante, ya que la expresión de malformaciones nucleares pueden ser atribui-
das a daños en el ADN (de Castihos, 2012). Tales anomalías se producen por irregularidades
en el proceso de división celular (ej. ruptura de cromosomas, repartición desigual de la croma-
tina entre las células hijas) y su posterior expresión en genes mutantes por la exposición a
compuestos genotóxicos (Guilherme et al., 2008; Di Giulio & Hinton, 2008; Maceda-Veiga et
al., 2010). Adicionalmente, debido a que varios estresores ambientales generan la producción
y liberación de eritrocitos inmaduros, así como de leucocitos en el torrente sanguíneo, su pro-
porción puede ser utilizada como biomarcador (Rowan, 2007). Similarmente, la simple pro-
porción de eritrocitos degenerados permite detectar procesos citolíticos (muerte celular), así
como la proporción de eritrocitos inmaduros y degenerados permite inferir la eritrocinesis
(formación de nuevos eritrocitos) y su eliminación, principalmente por el bazo (Pacheco &
Santos, 2002; Maceda-Veiga et al., 2010). Junto con el perfil de eritrocitos, la fórmula leuco-
citaria es indicadora del estado de salud de los peces ya que define el porcentaje de los dife-
rentes tipos de glóbulos blancos, los cuales se presentan en mayor o menor grado en el sistema
inmune de acuerdo al tipo de agente estresor (Houston, 1997; Davis et al., 2008; Maceda-
Veiga et al., 2010). Tanto los efectos citotóxicos como los genotóxicos han sido demostrados
en eritrocitos y leucocitos en algunos contaminantes tales como hidrocarburos aromáticos po-

25
 licíclicos (PAHs) (Rowan, 2007), herbicidas (Guilherme, et al., 2010) o complejos de xeno-
bióticos y metales pesados (Pacheco & Santos, 2002; Maceda-Veiga et al., 2010, 2013).

Bioacumulación por metales pesados

Entre los contaminantes anteriormente nombrados, los metales pesados conciernen una
gran preocupación ambiental ya que pueden ser bioacumulados en varios compartimentos del
ecosistema a medida que se avanza en el nivel trófico de la red alimentaria (Amundsen et al.,
1997; Ivanciuc et al., 2006). Todos los metales pesados se encuentran en los medios acuáticos
en formas coloidales (que dan lugar a hidróxidos), partículas minerales (sólidos en suspen-
sión) o en fases disueltas (cationes o iones complejos) (Alloway, 2013). Estos pueden ser
esenciales, cuando son requeridos por el organismo (ej. Cu, Zn, Co, Fe), siendo tóxicos a altas
concentraciones, o no esenciales, si no tienen un efecto vital o beneficioso en el organismo
(ej. Cd, Hg, Pb) (Miller et al., 1992; Amundsen et al., 1997; Kojadinovic et al., 2007). Sin
embargo, la asimilación, sensibilidad, lugar de acumulación y la habilidad para regular los
metales pesados pueden variar entre especies según el tipo de elemento (Tabla 2) (Amundsen
et al., 1997; Canli & Atli, 2003; Kojadinovic et al., 2007; Adhikari et al., 2009). La acumula-
ción de los metales pesados en los peces puede variar debido a varios factores fisicoquímicos
(ej. temperatura, pH, materia orgánica) y biológicos (ej. preferencias de hábitat, comporta-
miento alimenticio, edad, tasas de crecimiento) (Mason et al., 2000; Kojadinovic et al., 2007),
incluso en grandes lagos con características parecidas (Chen et al., 2000; Ward et al., 2012).
Por lo tanto, para conocer las especies más susceptibles a la contaminación se deben identifi-
car los principales problemas y focos de transmisión que permitan proponer medidas de con-
servación.

La ictiofauna del lago Titicaca como modelo de estudio

Biodiversidad y principales características del lago Titicaca
El lago Titicaca se encuentra sobre la cordillera de los Andes entre los países de Perú y
Bolivia a una altitud de 3810 m sobre el nivel del mar. Es el lago de agua dulce más grande de
Sur América con una superficie aproximada de 8559 km2 y un volumen de 932 km3 (Vila et
al., 2007). Se encuentra dividido en dos grandes subcuencas: el lago Mayor que ocupa el 84 %

26
 Tabla 2. Principales estructuras anatómicas de acumulación, fuentes de emisión y efectos potenciales de algunos de los metales pe-
sados más estudiados en peces (de acuerdo a Seymore et al., 1996; Amundsen et al., 1997; Rashed, 2001; Farkas et al., 2002; Canli &
Atli, 2003; Çogun et al., 2006; Kojadinovic et al., 2007; Adhikari et al., 2009; Maceda-Veiga et al., 2012; Alloway, 2013).
Metal Acumulación Efecto potencial Fuentes de emisión
Preservantes para madera y papel, abonos, proceso de elabo-
Cobre Piel, esqueleto, Daños en ADN, reducción de talla,
ración de textiles
(Cu) hígado peso y producción de huevos
Interactúa con otros químicos produ-
Piel,
Zinc ciendo cambios metabólicos, bajo Corrosión de objetos galvanizados, pinturas, uso de carbón y
esqueleto,
desarrollo gonadal, decrecimiento del petróleo
hígado
(Zn) páncreas y talla del cuerpo

Reduce la producción de huevos, in- Baterías recargables de Ni/Cd, fertilizantes,

cremento de la susceptibilidad a enfer- estabilizadores en plástico y PVC, pigmentos en pinturas,
Cadmio
Hígado, riñón medades y daños a nivel oxidativo e galvanización, catalizadores y conservadores en la industria
(Cd)
histopatológico del plástico, uso de carbón y petróleo, aleaciones

Daños en el ADN, lípidos y proteínas, Fundición primaria y secundaria de metales, elaboración de

toxicidad hematológica y renal, decre- soldaduras, industria electrónica, aditivos de gasolina y lubri-
Mercurio
Hígado cimiento de los niveles de hematocri- cantes, pesticidas, fertilizantes, baterías, producción de loza
(Hg)
to, tasa de crecimiento y producción vidriada, cauchos, pinturas
de huevos, aumento de deformidades
Extracción de oro, plata y cobre, fundición primaria y secun-
daria de metales, producción de carbón y coque, combustión
Efectos reproductivos adversos, daños
de carburantes en la generación de electricidad, industria ma-
Plomo tisulares principalmente en riñón,
Hígado derera y de producción de plástico, incineración de residuos
(Pb) estrés oxidativo
peligrosos y biológico-infecciosos, uso de pigmentos de pin-
tura, fungicidas

Pigmentos y pinturas, catálisis de petróleo e industria quími-

Cobalto Esqueleto y
Necrosis celular ca, secante para pinturas, cables de acero de neumáticos
(Co) riñón
Hemosiderosis, daños en ADN y
Hierro Corrosión de objetos galvanizados
Músculo peroxidación de lípidos
(Fe)

27
Introducción general
 de la superficie total y el lago Menor que corresponde al 16% restante con unas profundidades
máximas de 280 y 40 m, respectivamente (Fontúrbel, 2008).
El clima de la zona circunlacustre está sujeto a grandes variaciones diarias de temperatu-
ra (generalmente, con valores que pueden oscilar a lo largo del año entre -2 y 25°C), fuertes
vientos y una alta radiación solar sin presentar una marcada estacionalidad (Helbling et al.,
2001; Vila et al., 2007). Sus aguas presentan bajas temperaturas (menores a 15°C) y altas sali-
nidades (inferiores a 1 mg l-1), afectadas por un período de lluvias que ocurre entre los meses
de diciembre a marzo, con una precipitación anual que varía entre los 200 y los 1400 mm
(Myers et al., 2000; ALT, 2003).
La cuenca del lago Titicaca es endorreica y sus principales ríos tributarios son el río Ra-
mis (76 m3 s-1), río Ilave (69 m3 s-1), río Coata (42 m3 s-1) y río Huancané (20 m3 s-1) ubicados
en territorio peruano y el río Suches (11 m3 s-1), ubicado en la parte boliviana (ALT, 2003;
Ministerio de la Producción & AECID, 2008). El único río efluente del lago es el río De-
saguadero (35 m3 s-1) que transporta el agua hasta el lago Uru-Uru y el salar de Coipasa
(Ministerio de la Producción & AECID, 2008). La cuenca del lago cumple un papel funda-
mental en la conservación de diversos grupos taxonómicos albergando una gran riqueza de
especies endémicas. Destaca la importancia que tienen sus biotopos o hábitats en los que se
desarrollan más de 60 especies de aves (40 de ellas permanentes en el lago) y aproximada-
mente unas 26 especies de peces sujetos a confirmación taxonómica (Fig.6 y Tabla 2). Debido
a su gran biodiversidad, el lago fue declarado sitio RAMSAR de importancia mundial para la
conservación de humedales (Fontúrbel, 2008).
En el lago se distinguen tres tipos de hábitats: la zona litoral compuesta por macrófitas,
principalmente Schoenoplectus californicus ssp. tatora y Juncus articus ssp. andicola
(llamados totorales) y donde habitan una gran cantidad de insectos y anfípodos, la zona bénti-
ca compuesta principalmente por moluscos y anfípodos y la zona pelágica, rica en cladóceros,
(Lauzanne, 1991; Vila et al., 2007). Se considera que cerca del 56% superficie total del lago
se encuentra a una profundidad menor a 5 m lo cual resalta la importancia de esta zona en la
biodiversidad de las especies acuáticas (Wirrman, 1991).

28
 Introducción general

Bryozoa *

Porifera

Cnidaria

Platyhelmintes *

Annelida

Mollusca *

Crustacea

Amphibia *

Pisces *

Aves

0 10 20 30 40 50 60 70
Número de especies

Fig. 6. Número de especies de algunos de los principales grupos zoológicos del lago Titicaca. Da-
tos fueron tomados de Dejoux & Iltis (1991) y Gobierno Regional de Puno (2008).
*Datos sujetos a confirmación genética por presentar subespecies o hibridización

Especies ícticas posibles bioindicadoras del estado ecológico del lago

Junto con los anfibios, los peces continentales son los vertebrados más amenazados del
mundo, siendo las especies tropicales las que presentan un mayor porcentaje de riesgo
(Groombridge & Jenkins, 2000; Dudgeon et al., 2006; Helfman, 2007; Vié, et al., 2009). Este
grado de amenaza es mayor en el caso de sistemas lénticos de agua, como lo son los lagos,
donde habitan especies especialistas con áreas reducidas o delimitadas por el tamaño y las
condiciones del cuerpo de agua (ej. la extinción de más de cien especies de cíclidos en el lago
Victoria y Malawi, ver Fry et al., 1999; Aloo, 2003; Balirwa et al., 2003). Descensos simila-
res en las pesquerías y pérdidas en la biodiversidad podrían ocurrir en otros grandes lagos con

29
 fuerte influencia antrópica, principalmente en aquellos con poblaciones endémicas y amenaza-
das, ubicados en regiones con condiciones ambientales extremas. Tal es el caso del lago Titi-
caca, en el que más del 80% de su fauna íctica es endémica y donde el mal uso de los recursos
naturales han derivado en una reducción de la biomasa íctica cercana al 45% en los últimos 30
años (Vila et al., 2007; Aliaga & Segura, 2013). Esta situación ha generado que la mayoría de
las especies de peces se encuentren catalogadas en cinco diferentes niveles de amenaza (casi
amenazada, vulnerable, en peligro, en peligro crítico y extinto) de acuerdo a los criterios pro-
puestos en el Libro Rojo de especies amenazadas generado por la Unión Internacional para la
Conservación de la Naturaleza (IUCN) (Van Damme et al., 2009; ver Tabla 3).

Híbridos
Lauzanne
Subespecies
(1991)
Especies nuevas
descritas
Total de especies
Parenti descritas
(1984)

Tchernavin
(1944)

0 10 20 30
Número de especies

Fig. 7. Número de especies ícticas descritas en algunos de los trabajos taxonómicos realizados en
el lago Titicaca.

La ictiofauna nativa del lago Titicaca está representada por sólo dos géneros: Orestias
(Cyprinodontidae) también llamados “carachis” y dos especies de bagres filtradores Tricho-
mycterus rivulatus “suche” y Trichomycterus dispar “mauri”, pertenecientes a la familia Tri-
chomycteridae, aunque la revisión preliminar genética llevada a cabo por de Sostoa et al.

30
 Tabla 3. Categorización de las especies nativas de peces del lago Titicaca de acuerdo a los niveles de amenaza (no evaluada=NE,
casi amenazada=NT, vulnerable=VU, en peligro=EN, en peligro crítico=CR y extinto=EX) propuestos en el Libro Rojo de vertebra-
dos de Bolivia (Van Damme et al., 2009). Las especies del género Orestias fueron agrupadas en complejos según sus afinidades filo-
genéticas de acuerdo a Parenti (1984). Las especies marcadas con asterisco son las especies reconocidas por Lauzanne (1991).

Especie Categoría Especie Categoría

Trichomycterus rivulatus Valenciennes 1846 VU Complejo gilsoni

Trichomycterus dispar Tschudi 1846 NE *Orestias gilsoni Tchernavin 1944 VU
Complejo agassii *Orestias imarpe Parenti 1984 VU
*Orestias agassii Valenciennes 1846 VU *Orestias minimus Tchernavin 1944 VU
*Orestias albus Valenciennes 1846 EN *Orestias mooni Tchernavin 1944 NE
Orestias farfani Parenti 1984 NE *Orestias robustus Parenti 1984 VU
Orestias frontosus Cope 1876 NE *Orestias taquiri Tchernavin 1944 NE
*Orestias jussiei Valenciennes 1846 NE *Orestias tchernavini Lauzanne 1981 VU
*Orestias luteus Valenciennes 1846 VU *Orestias tomcooni Parenti 1984 VU
*Orestias olivaceus Garman 1895 NE *Orestias uruni Tchernavin 1944 VU
Orestias puni Tchernavin 1944 NE Complejo mulleri
Orestias rotundipinnis Parenti 1984 NE *Orestias crawfordi Tchernavin 1944 VU
Orestias tschudii Parenti 1984 NE *Orestias gracilis Parenti 1984 VU
Complejo cuvieri *Orestias incae Garman 1895 VU
*Orestias cuvieri Valenciennes 1846 EX *Orestias mulleri Valenciennes 1846 VU
*Orestias forgeti Lauzane 1981 VU *Orestias sp. (O. olivaceus sensu Lauzanne 1982) NE
*Orestias ispi Lauzanne 1981 NT *Orestias tutini Tchernavin 1944 VU
*Orestias pentlandii Valenciennes 1846 CR

31
Introducción general
 (2010) sólo mantiene una especie de bagre (T. rivulatus). No obstante, el género más contro-
vertido ha sido Orestias ya que desde su establecimiento por parte de Valenciannes en 1846,
se han presentado discrepancias en el número de especies descritas por parte de diversos auto-
res, debido a que algunas de las especies han sido catalogadas a partir de estadios juveniles o
de ligeras variaciones morfológicas de las poblaciones (de Sostoa et al., 2010; Fig.7).
El género Orestias se caracteriza por una gran plasticidad fenotípica y una alta diversidad
genética (Lüssen et al., 2003). Los “carachis” ocupan diferentes nichos en la zona litoral,
bentónica y pelágica del lago, lo que hace posible que ocurra una división de los recursos y se
generen especies especialistas por determinados tipos de alimento (Lauzanne 1982; Vaux et
al., 1988; Lauzanne, 1991; Lüssen et al., 2003; Maldonado et al., 2009). Ahora bien, para uti-
lizar los peces como indicadores biológicos es necesario un soporte biológico y ecológico de
las especies para conocer los atributos funcionales, rangos de tolerancia y respuestas frente a
diversas clases de estrés (Benejam, 2008) e identificar los posibles agentes que puedan afectar
el desarrollo normal de las especies.

Principales problemas del lago Titicaca

El lago Titicaca tipifica la problemática de los cinco principales factores de amenaza para
la biodiversidad descritos anteriormente. El crecimiento descontrolado de las poblaciones cir-
cunlacustres, el incremento del turismo sin planificación, el mal uso de los recursos naturales
y la débil legislación de la autoridad competente han potenciado estos efectos adversos en el
ecosistema (Fontúrbel, 2008).

Introducción de especies
A partir de 1940 algunas especies pertenecientes a la familia de los salmónidos tales co-
mo la trucha común (Salmo trutta), trucha lacustre (Salvelinus namaycush), la trucha de arro-
yo (Salvelinus fontinalis) y la trucha arco iris (Oncorhynchus mykiss) fueron introducidas en
el lago Titicaca con el fin de mejorar la pesca comercial (Vila et al., 2007; Ministerio de la
Producción & AECID, 2008). Posteriormente, en el año 1946 una especie de la familia Aterí-
nidae, el pejerrey argentino Odontesthes bonariensis, fue introducido en el lago Poopó remon-

32
 Introducción general

tando hasta el lago Titicaca a través del río Desaguadero (Ministerio de la Producción & AE-
CID, 2008). A finales de los años 70’s se aprobó la incorporación de jaulas para cultivo de
salmónidos, principalmente de trucha arco iris (Ministerio de la Producción & AECID, 2008;
de Sostoa et al., 2010).
Actualmente, sólo la trucha arco iris y el pejerrey argentino son las únicas especies intro-
ducidas que se han adaptado con éxito a las condiciones del lago. Gran parte de su éxito pasa
por el hecho de que ambas especies son carnívoras, pudiendo depredar otros peces y quizás
también, por la competencia trófica con las especies nativas, lo cual puede generar alteracio-
nes en la estructura y composición de las comunidades (Vaux et al., 1988; Loubens, 1989;
Modenutti et al., 1998; Vila et al., 2007). Esta situación puede verse agravada debido al mal
manejo de los desechos originados por las piscifactorías artesanales y la utilización de espe-
cies nativas (principalmente O. ispi y otras especies de pequeño tamaño) como materia prima
para la alimentación de los cultivos de trucha (de Sostoa et al., 2010). Adicionalmente, la in-
troducción de especies exóticas podría ser la causa principal de la introducción de algunas
epizootias como la enfermedad del punto blanco, causada por el protozoo ciliado Ichthyopht-
hirius multifiliis, la cual causó la muerte de aproximadamente unos 18 millones de peces nati-
vos del lago en 1981 (Wurtsbaugh & Alfaro, 1988). Sin embargo, actualmente se desconocen
los efectos de ambas especies introducidas sobre la estructura trófica de las comunidades nati-
vas.

Destrucción del hábitat

Los tapetes de macrófitas o totorales constituyen un ecosistema de primordial importan-
cia para el desarrollo de muchas especies, no sólo de peces, sino de otros muchos grupos zo-
ológicos, ya que son utilizados como zona de reproducción, refugio y alimentación
(Northcote, 1991; Northcote, 2000; Constantini et al., 2004; Vila et al., 2007; Fontúrbel,
2008). Sin embargo, en los últimos años el porcentaje de su cobertura ha disminuido drástica-
mente (ej. de 1970 a 1990 se redujo en aproximadamente un 55% la cobertura total)
(Northcote, 1991). Esta reducción ha sido atribuida principalmente a la eutrofización del agua,
a la alimentación del ganado con dichas macrófitas y a la quema de totorales para obtener una

33
 mayor área para el cultivo (Northcote, 1991; Fontúrbel, 2008).

Contaminación del agua

Pese a ser considerado un lago oligotrófico, algunos procesos puntuales de contamina-
ción orgánica por vertidos directos o escorrentía de zonas agrícolas han generado eutrofiza-
ción en bahías con un bajo recambio de agua, cercanas a grandes ciudades (ej. bahía de Puno)
o a desembocaduras de ríos (ej. río Ramis) (Northcote et al., 1989; Northcote, 1991; Paredes
& Gonfiantini, 1999; Revollo et al., 2003; Constantini et al., 2004; Cruz et al., 2006; Fontúr-
bel, 2008; Gobierno Regional de Puno, 2008). Esto se debe principalmente a un deficiente
tratamiento de las aguas residuales y alcantarillado que no cubren a la totalidad de los habitan-
tes de la cuenca (Revollo et al., 2003), siendo la ciudad de El Alto (Bolivia) la única de las
principales ciudades de la cuenca del lago con un sistema de tratamiento para las aguas resi-
duales (ALT, 2003). Esta situación ha generado diversos procesos eutróficos caracterizados
por la presencia de macrófitas acuáticas flotantes (principalmente Lemna gibba, ver Fontúrbel,
2008) o blooms de algas (Vila et al., 2007) que han deteriorado la calidad del agua y que han
generado junto a otros factores, altos contenidos de parásitos en los peces, principalmente de
los platelmintos Diplostomum sp., y Ligula intestinalis (Lauzanne, 1991).
Sin embargo, el principal problema de contaminación de las aguas del lago es la activi-
dad minera debido a que la zona es rica en varios metales tales como oro (Au), plata (Ag),
estaño (Sn), cobre (Cu), plomo (Pb), zinc (Zn) y otros productos no metálicos como fosfatos,
carbón y material de construcción, los cuales son explotados principalmente mediante méto-
dos artesanales (Revollo et al., 2003). La actividad minera produce gran cantidad de desechos
minerales utilizados en el proceso de refinamiento que son almacenados en depósitos a cielo
abierto y los cuales, al oxidarse, producen un proceso de lixiviación que genera metales pesa-
dos (Gammons et al., 2006). Estos metales son generalmente arrastrados por lluvias o esco-
rrentías a los principales ríos tributarios del lago o a las zonas agrícolas y ganaderas generan-
do un grave problema de contaminación (Revollo et al., 2003). Este problema toma mayor
relevancia si se considera que el lago Titicaca provee agua potable para más de tres millones
de personas (Revollo et al., 2003). Por tales razones, es muy importante identificar los conta-

34
 Introducción general

minantes más comunes (p.e. fármacos, tensoactivos, metales pesados) y determinar los posi-
bles efectos sobre los diferentes compartimentos del ecosistema y el riesgo sobre las poblacio-
nes humanas que viven en áreas contaminadas (Barbour et al., 1999; Damásio et al., 2007,
2008; Benejam et al., 2008). Por lo tanto, es necesario conocer si algunas especies sirven co-
mo bioindicadoras de la contaminación y si algunos biomarcadores sirven como herramientas
de evaluación de la calidad ambiental del lago.

Sobrepesca
La pesquería que se ejerce en el lago Titicaca es principalmente de carácter artesanal y
frecuentemente con embarcaciones propulsadas a vela o remo lo que limita la navegación a
sitios cercanos a la costa que normalmente no superan los 5 km de distancia con respecto a la
orilla (Paca et al., 2003; Ministerio de la Producción & AECID, 2008). Las zonas con mayor
frecuencia de pesca son la bahía interior de Puno y la parte norte del lago entre las localidades

A
C

B E

Fig. 8. Métodos artesanales de pesca del lago Titicaca (Modificado de Paca et al., 2003). (A) Maja-
ña, (B) Sakaña, (C) Sapuro, (D) Kapikawana, (E) nasa o Kullancha (nótese que los nombres de
los artes provienen de vocablos aymaras o quechuas).

35
36
Fig. 9. Desembarque anual de la flota pesquera (T) de las especies más abundantes del lago Titicaca. Datos de 1982 al 2006 fueron
obtenidos de Ministerio de la Producción y AECID (2008) y a partir del 2007, de IMARPE (2008, 2010). Nótese que la escala es lo-
garítmica.

10000

1000

Orestias sp.
O. ispi
100 T. rivulatus
O. bonariensis
O. mykiss

10

Volumen de capturas (Toneladas métricas)

1
1982 1990 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Años
 Introducción general

de Pusi y Huancané (Ministerio de la Producción & AECID, 2008). Sin embargo, a pesar de
que el número de pescadores ha ido disminuyendo en estas zonas en los últimos años, el au-
mento de la demanda de pescado ha propiciado un remplazo de los artes nativos de pesca (Fig.
8) y la sofisticación tanto de embarcaciones de mayor calado con motores fuera de borda co-
mo de los artes y aparejos de pesca (Vila et al., 2007; Fontúrbel, 2008; Ministerio de la Pro-
ducción & AECID, 2008).

Fig. 10. Diferentes hábitats del lago Titicaca y distribución de sus especies ícticas más caracterís-
ticas (modificado de Lauzanne, 1991 y Ministerio de la Producción & AECID, 2008).

Debido a que actualmente no existe ninguna normativa de ordenamiento pesquero global

para el lago Titicaca y que el control sobre las tallas mínimas y artes ilegales es prácticamente
inexistente, el uso de nuevas tecnologías puede haber generado sobrepesca, la cual puede ser
una de las posibles causas de la drástica disminución de muchas de las especies ícticas del
lago (Vila et al., 2007). De hecho, los volúmenes de capturas totales entre 1982 y 2010 han

37
 disminuido cerca de un 90% (Fig. 9). Esta situación puede verse agravada debido a que el ma-
yor esfuerzo de pesca se realiza en la zona litoral (muchas veces con redes de malla que no
respetan las tallas mínimas de captura), donde habitan los juveniles de la mayoría de las espe-
cies y los adultos de especies de menor tamaño, que pueden ser la base de las redes alimenta-
rias (Ministerio de la Producción & AECID, 2008) (Fig. 10).
Los principales artes de pesca utilizados en la zona litoral son las redes de arrastre, gene-
ralmente el chinchorro o las redes tipo caída como la atarraya (Paca et al., 2003). Las redes de
enmalle de tipo cortina o algunas modificaciones de ésta, también llamadas redes de seno o de
bolsa, son las de uso más generalizado en el lago y generalmente, se emplean pasando la zona
de totoral (Fig. 9). En aguas más profundas, los métodos más utilizados son las redes agalle-
ras, así como también los palangres y espineles que utilizan generalmente especies nativas
como carnada (Paca et al., 2003). Esta dramática situación requiere la estandarización de
métodos de pesca en el lago que permitan la sostenibilidad de las pesquerías del lago y que
generen bases para crear normativas que conlleven a la conservación de las especies.

Cambios en el caudal o nivel del agua

La cuenca del lago Titicaca recibe una fuerte radiación solar con una tasa anual de 533
calorías/cm2 por día lo que genera altas tasas de evaporación (Dejoux & Iltis, 1991; ALT,
2003). Esta situación puede verse agravada por el calentamiento global y el aumento en la
demanda de agua del lago debido al crecimiento de las poblaciones circunlacustres (Fontúrbel,
2008). El calentamiento global ha afectado el período de lluvias variando entre años, siendo
más secos en el período influenciado por el fenómeno de “El Niño” y con temporadas de pre-
cipitaciones más largas, con el fenómeno de “La Niña” (Garreaud et al., 2003). Estas altera-
ciones han generado en el último siglo un descenso en el nivel de agua de aproximadamente
unos 6.3 m lo cual ha podido generar impactos en los ciclos del agua, perdidas de biodiversi-
dad, salinización y desertificación (ALT, 2003).

Desconocimiento del estado actual de la comunidad íctica

A pesar del alto valor endémico, la estructura trófica de la comunidad de peces nativos
del lago Titicaca está pobremente estudiada y pocos estudios proveen evidencias sobre su eco-

38
 Introducción general

logía (ej. Lauzanne, 1982; Vaux et al., 1988; Maldonado et al., 2009) dejándose de lado las
especies de menor valor económico (de Sostoa et al., 2010). Sin embargo, los grandes impac-
tos antrópicos acontecidos en las últimas décadas en el lago podrían haber generado cambios
en la estructura trófica de las poblaciones de peces y por ende en el ecosistema.
Esta situación genera la necesidad de conocer el estado de las poblaciones, su ecología,
relaciones tróficas y estrategias alimentarias para implementar medidas de conservación que
eviten la extinción de las especies nativas y ayuden a paliar la problemática ambiental y social
relacionada con los impactos ambientales y la sobreexplotación de los recursos pesqueros,
mediante medidas de mejora ambiental y el uso sostenible de los recursos. Adicionalmente, el
conocimiento de dichos impactos sobre otros compartimentos del ecosistema (ej. agua, sedi-
mentos) es fundamental para tomar medidas que eviten el riesgo por consumo y sean la base
para el mejoramiento de los recursos acuáticos. En este sentido, el establecimiento de biomar-
cadores que sirvan como herramientas de diagnóstico que permitan detectar los efectos antes
de que tengan consecuencias nocivas para los peces, principalmente mediante técnicas poco
invasivas de evaluación, son indispensables para la conservación de sus comunidades.

39
Objetivos
 OBJETIVOS
Objetivo General
Conocer el estado de conservación de las poblaciones ícticas del lago Titicaca ampliando
el conocimiento de los efectos que ejercen la introducción de especies y la contaminación por
metales pesados en la regresión de sus densidades, proponiendo nuevas técnicas para el diag-
nóstico de los posibles impactos antrópicos y para la sostenibilidad de sus pesquerías.

Objetivos específicos
1. Explorar las interacciones tróficas entre las especies de peces nativas e introducidas
del lago Titicaca.
Para tal fin, se hicieron análisis de contenidos estomacales de especies de las que no se
conocía su tipo de dieta y se complementó con información sobre el tipo de hábitat, formán-
dose grupos funcionales de especies de acuerdo a características ecológicas similares. Estos
grupos se relacionaron con el análisis de isótopos estables de δ13C y δ15N para conocer su
posición trófica y observar si las especies que los componen, presentaban segregación
espacial. Además, se examinaron los efectos de las especies exóticas sobre las nativas rela-
cionando el solapamiento de nicho con la posible depredación o la competencia por recursos
(ver Capítulo I).

2. Analizar la concentración de metales pesados en agua, sedimento y cuatro de las prin-

cipales especies de importancia pesquera en el lago Titicaca.
Se definieron las especies y el tejido con la más alta bioacumulación de Cu, Zn, Cd, Hg,
Pb, Co y Fe en varias localidades identificadas como contaminadas por diferentes actividades
antrópicas en comparación con localidades referencia. Así mismo, se determinó si dichas con-
centraciones estaban correlacionadas con parámetros fisicoquímicos o biológicos de las espe-
cies y si sobrepasaban los estándares para consumo humano con el fin de detectar posibles
amenazas para la salud y establecer prioridades de conservación (ver Capítulo II).

3. Conocer el efecto que ejerce la contaminación por metales pesados en varios paráme-

42
 Objetivos

tros hematológicos y validar su uso como indicadores del estado de salud de tres especies
de peces nativos.
Se evaluó la idoneidad de biomarcadores en relación a los efectos genotóxicos y citotóxi-
cos de los metales y la respuesta a factores biológicos y ambientales, así como a otros biomar-
cadores tales como la evaluación histopatológica. Así mismo, se probó una técnica no invasi-
va para diagnosticar los efectos crónicos subletales de los metales antes de que puedan llegar a
ser perjudiciales para la conservación de las especies ícticas (ver Capítulo III).

4. Determinar los mejores modelos de selectividad de redes para las especies más abun-
dantes en la zona de aguas abiertas del lago estableciendo las luces de malla más adecua-
das para la pesca sostenible mediante redes agalleras.
Para este fin, se determinaron las variaciones en las capturas en cinco de las principales
zonas de pesca del lago de acuerdo a diferentes luces de malla. Adicionalmente se evaluaron
las capturas entre redes agalleras pelágicas y bentónicas distribuidas a diferentes profundida-
des para conocer la composición de cada tipo de red (ver Capítulo IV).

43
Informe del director
 INFORME DEL DIRECTOR
El doctorando Mario Monroy López presenta su tesis doctoral titulada “Principales im-
pactos antrópicos y sus efectos sobre la comunidad de peces del lago Titicaca”.

El director de tesis, el Dr. Adolfo de Sostoa Fernández, informa que la presente tesis doc-
toral consta de cuatro trabajos científicos a modo de capítulos. Los capítulos están publicados,
enviados o en evaluación para envío a revistas científicas internacionales reconocidas en el
Science Citation Index (SCI). A continuación se detalla la contribución del doctorando en ca-
da uno de ellos, así como el factor de impacto según el Thomson Institute for Scientific Infor-
mation:

CAPÍTULO 1
Monroy, M., Maceda-Veiga, A., Caiola, N., de Sostoa, A. 2014. Trophic interactions between
native and introduced fish species in a littoral fish community. Journal of Fish Biology, 185,
1693-1706.
Factor de impacto (2014): 1.83
El doctorando ha participado en el procesamiento de las muestras en el laboratorio, el análisis
de los datos y la redacción científica.

CAPÍTULO 2
Monroy, M., Maceda-Veiga, A., de Sostoa, A. 2014. Metal concentration in water, sediment
and four fish species from Lake Titicaca reveals a large-scale environmental concern. Science
of the Total Environment, 487, 233-244.
Factor de impacto (2014): 3.25
El doctorando ha participado en el diseño del muestreo, la recogida y procesamiento de las
muestras en el laboratorio, el análisis de los datos y en la redacción científica.

CAPÍTULO 3
Monroy, M., Maceda-Veiga, A., Cano, O., de Sostoa, A. Blood analyses in three fish species

46
 Informe del director

indicate the presence of cytotoxic and genotoxic compounds in Lake Titicaca.

Pendiente de envío para ser sometido a Aquatic Toxicology.
Factor de impacto (2014): 3.7
El doctorando ha participado en el diseño del muestreo, la recogida y procesamiento de las
muestras en el laboratorio, el análisis de los datos y en la redacción científica.

CAPÍTULO 4
Monroy, M., Maceda-Veiga, A., Gálvez-López, E., Rodríguez-Climent, S., Caiola, N., de
Sostoa, A. Gillnet selectivity in the offshore ichthyic community from Lake Titicaca.
Pendiente de envío para ser sometido a Canadian Journal of Fisheries and Aquatic Science.
Factor de impacto (2014): 2.2
El doctorando ha participado en el diseño del muestreo, la recogida y análisis de los datos y en
la redacción científica.

Los coautores participantes en los artículos que componen esta tesis no han utilizado ni
implícita ni explícitamente ninguno de estos trabajos para la elaboración de otras tesis docto-
rales.

Adolfo de Sostoa Fernández

Barcelona, 25 de Septiembre de 2014.

47
Capítulos
 CHAPTER 1
Trophic interactions between native and introduced fish
species in a littoral fish community

Mario Monroy1, Alberto Maceda-Veiga2, Nuno Caiola3, Adolfo de Sostoa1

1
Department of Animal Biology and Biodiversity Research Institute (IRBIO), University of
Barcelona, E-08028 Barcelona, Spain.
2
Cardiff School of Biosciences, Cardiff University, CF10 3AX, Cardiff, United Kingdom.
3
Aquatic Ecosystems, IRTA, Sant Carles de la Ràpita, Spain.
Chapter 1

RESUMEN
En este estudio se exploraron las interacciones tróficas entre 15 especies nativas de pe-
ces y dos introducidas, el pejerrey (Odontesthes bonariensis) y la trucha arco iris
(Oncorrhynchus mykiss), capturadas en una de las áreas de mayor importancia pesquera del
lago Titicaca, integrando los conocimientos de ecología tradicional con el análisis de isóto-
pos estables (SIA). SIA sugirió la existencia de seis grupos tróficos en la comunidad de pe-
ces, basados en las firmas isotópicas de δ13C y de δ15N. Esto fue respaldado por evidencias
ecológicas que ilustraron una marcada segregación espacial entre grupos, aunque con niveles
tróficos similares para la mayoría de grupos nativos. Basados en los análisis de elipses bayes-
ianas, el solapamiento de nicho parece ocurrir entre los más pequeños pejerreyes (<90 mm) y
las especies bentopelágicas (31.6%) y entre la especie pelágica nativa Orestias ispi y grandes
pejerreyes (39%), así como también con la trucha arco iris (19.7%). Adicionalmente, los
modelos de mezcla bayesianos sugirieron que O. ispi y las especies epipelágicas parecen ser
las presas principales de las dos especies introducidas. Este estudio revela la conexión trófica
existente entre las especies nativas e introducidas de peces y demuestra la utilidad de combi-
nar SIA con el conocimiento aportado por la ecología tradicional para poder entender las
relaciones entre especies con hábitos alimenticios similares.

Palabras clave
Peces endémicos, red alimenticia, especies introducidas, isótopos estables.

52
 Trophic relationships between ichthyic community

ABSTRACT
The trophic interactions between 15 native and two introduced fish species, silverside
Odonthestes bonariensis and rainbow trout Oncorhynchus mykiss, collected in a major fish-
ery area at Lake Titicaca were explored by integrating traditional ecological knowledge and
stable isotope analyses (SIA). SIA suggested the existence of six trophic groups in this fish
community based on δ13C and δ15N signatures. This was supported by ecological evidence
illustrating marked spatial segregation between groups but a similar trophic level for most
native groups. Based on Bayesian ellipse analyses, niche overlap appeared to occur between
small silversides (<90 mm) and benthopelagic native species (31.6%), and between the native
pelagic killifish Orestias ispi and large silversides (39%) or rainbow trout (19.7%). In addi-
tion Bayesian mixing models suggested that O. ispi and epipelagic species are likely to be the
main prey items for the two introduced fish species. This study reveals a trophic link between
native and introduced fish species, and demonstrates the utility of combining both SIA and
traditional ecological knowledge to understand trophic relationships between fish species
with similar feeding habits.

Key words
Endemic fish; food web; introduced species; stable isotopes.

53
Chapter 1
INTRODUCTION
reshwater ecosystems are ex-
periencing a worldwide biodi-
versity crisis, which is particu-
larly evident in the conserva-
tion status of many fish species (Dudgeon
et al., 2006; IUCN, 2008). Common
threats to freshwater ichthyofauna are
habitat degradation, the introduction of
exotic species and overfishing (Dudgeon
et al., 2006; IUCN, 2008). These anthro-
pogenic impacts can lead to species ex-
tinction but may also cause profound
changes in community structure, thus af-
fecting ecosystem functioning (Duffy et
al., 2007; Cucherousset et al., 2012). Tra-
ditional ecological approaches such as
observational studies of species behaviour
and gut content analyses can detect
changes in species performance that could
be associated with ecosystem functioning
(see Petchey & Gaston, 2006). However,
such approaches may fail to detect subtle
modifications in the structure of commu-
nities especially when the species exam-
ined belong to a highly diverse fish com-
munity and most species seem to have
relatively similar feeding behaviour
(Vander Zanden & Vadeboncoeur, 2002;
54
Quevedo et al., 2009). Consequently, in-
ferring the food web structure of fish spe-
cies exhibiting generalist and similar feed-
ing behaviour in large water bodies such
as lakes requires the integration of infor-
mation provided by traditional ecology
and food web tracers (Vander Zanden et
al., 1999; Post, 2002; Clarke et al., 2005).
Stable isotope analyses of C and N
(hereafter δ13C and δ15N, respectively) are
food web tracers that are widely applied in
studies of fish ecology in lakes (Vander
Zanden et al., 1999; Post, 2002). The pre-
dictive enrichment in δ15N throughout tro-
phic levels (i.e. 3–4 ‰) enables the deter-
mination of fish trophic position, and the
similarity in δ13C between diet and con-
sumer allows the determination of fish
foraging habitat (Vander Zanden et al.,
1997). Stable isotope analyses may then
guide management strategies by showing
evidence of the differential effect of intro-
duced species on the native fish commu-
nity by trophic competition or predation.
Indeed the introduction of the piscivorous
silverside Odontesthes bonariensis
(Valenciennes 1835) and rainbow trout
Oncorhynchus mykiss (Walbaum 1792)
into Lake Titicaca in the 1940s to enhance

fisheries resources for local human com-
munities typifies one of these situations.
Over a period of 32 years (1979–2011)
the biomass of native fish stocks has de-
clined by 45% (see Vila et al., 2007;
Aliaga & Segura, 2013), and observa-
tional studies suggest that predation by
introduced fish species is likely to be one
of the main causative factors (Vaux et al.,
1988; Loubens, 1989; Vila et al., 2007).
However, trophic competition is also
likely to occur because juveniles of both
native and introduced species feed on
macroinvertebrates (Vaux et al., 1988).
The present study aimed to explore
the trophic interactions between native
and introduced fish species in Lake Titi-
Figure 1. Map of the study area with the sampling locations (L1: Kapara, L2: Pusi, L3: Olojpata,
L4: Kauta) examined in “Lago Mayor” in February 2010. Original map provided by National
Aeronautics and Space Administration (NASA®).
Trophic relationships between ichthyic community
caca by integrating the information pro-
vided by traditional ecology and stable
isotope analysis. Previous studies address-
ing the native fish species’ diet by gut
content analyses have suggested that these
species show a relatively similar trophic
spectrum. Therefore, all fish species
should have a similar trophic level, limit-
ing trophic competition by spatial segre-
gation. Finally, the differential effect on
native fish species of the two introduced
fish species in terms of predation and
niche overlap was examined.
MATERIAL AND METHODS
Study area
55
Chapter 1
Lake Titicaca is the largest freshwa-
ter lake in South America, covering a sur-
face area of approximately 8559 km2. It is
one of the highest lakes worldwide, at an
elevation of 3810 m (Vila et al., 2007)
(Fig. 1). It is divided into two large ba-
sins: “Lago Mayor”, which reaches a
maximum of 285 m in depth, and “Lago
Menor”, with a maximum depth of 40 m.
Lake Titicaca is mainly an oligotrophic
lake characterised by permanent hyper-
haline cool water due to geographic fea-
tures and the lack of strong seasonality
(Myers et al., 2000; Vila et al., 2007).
Littoral areas have the characteristic
macrophyte carpet dominated by reeds of
totora Schoenoplectus californicus ssp.
tatora Meyer 1829 and juncus Juncus ar-
ticus ssp. andicola Willdenow 1799, as
well other macrophyte species of the ge-
nus Chara, Potamogeton, Myriophyllum,
Nitella and Ruppia (Lauzanne, 1991; Vila
et al., 2007). Although the macrophyte
carpet only covers 20% of the total lake
surface, it acts as an important feeding
and breeding area for the majority of na-
tive species in the lake (Lauzanne, 1991).
The native fish community in Lake Titi-
caca has a high degree of endemicity and
56
is dominated by members of the killifish
genus Orestias (Teleostei, Cyprinodonti-
dae) and the benthic catfish Tricho-
mycterus rivulatus Valenciennes 1846
(Doadrio I., com. pers.). A variety of an-
thropogenic impacts such as the introduc-
tion of alien species, overfishing and sew-
age discharges from urban areas and min-
ing activities may have led many native
species to be listed as vulnerable, criti-
cally endangered or extinct based on the
International Union of Conservation of
Nature (Vila et al., 2007; Van Damme et
al., 2009; Monroy et al., 2014a).
Sampling procedure
Fish were collected in February 2010
by local fishermen using benthic nylon
gillnets with a mesh size of 10–30 mm in
four open bays (Kapara Pusi, Olojpata and
Kauta) across 20 km of Lago Mayor (Fig.
1). The selection of this study area en-
abled to explore the trophic interactions in
an area of high fish diversity (17 out of
the 26 fish species reported), maximising
the chances of species interaction in an
environment with little influence from
other anthropogenic impacts other than
the presence of introduced fish species
 Trophic relationships between ichthyic community
(see Ministerio de la Producción &
AECID, 2008). As pollution may mod-
ify the structure of fish communities, the
good water quality of the area surveyed
was corroborated by determining stan-
dard water physico-chemistry variables
as detailed in Table I. All fish species
were commercial species captured in the
littoral area of the four bays surveyed so
there is observational evidence of the
possibility of species interactions. Fish
were identified to species level, meas-
ured (total length, LT, mm) and approxi-
2
mately 3 cm of white muscle tissue was
dissected from the mid-dorsal region of
each fish. As the taxonomic composition
of some fish species’ diet was known,
only the digestive tracts of killifish
Orestias gracilis Parenti 1984, Orestias
tomcooni Parenti 1984, Orestias fronto-
sus Cope 1876, Orestias incae Garman
1895, Orestias uruni Tchernavin 1944,
and Orestias farfani Parenti 1984 were
fixed in 10% formaldehyde for gut con-
tent analyses (see Table II for details).
Three samples of zooplankton and
benthic invertebrates were collected at
each location to determine the isotopic
composition of potential fish prey. Zoo-
plankton samples were obtained by pull-
ing a 250-µm mesh net through a 1-m
water column deep net and benthic
macroinvertebrates were collected using
dip nets. Prey samples were then classi-
fied in three categories: zooplankton,
chironomids and amphipods. All sam-
ples were frozen at -80°C and trans-
ported frozen in dry ice to the facilities
at the University of Barcelona (Spain).
Sample preparation and stable isotope
analyses
Fish and prey samples were freeze-
dried, ground to a powder and lipid-
extracted with three-baths chloroform-
methanol (2:1) (Logan et al., 2008).
Samples were then homogenised and
weighed to the nearest μg in ultra-clean
tin capsules for δ13C and δ15N determi-
nation at the Scientific-Technical Ser-
vices of the University of Barcelona.
Samples were analysed in a Thermo-
Finnigan Flash 1112 (CE Elantech,
Lakewood, NJ, USA) elemental analyser
coupled to a Delta-C isotope ratio mass
spectrometer via a CONFLO III inter-
face (Thermo Finnigan MAT, Bremen,
Germany). IAEA reference materials
57
Chapter 1

TABLE 1. Geographical location (Lat: latitude, Lon: longitude) of four sampling sites (L1: Kapara, L2: Pusi, L3: Olojpata, L4:

chemical variables such as temperature (T, °C), dissolved oxygen (DO2, mg l-1 ), conductivity (C, µS cm-1), general hardness (GH),
Kauta) at Lake Titicaca with information on the total number of species captured (S) and the water quality analyses of physico-
were applied and inserted every 12

17

17
16

17
S
samples to calibrate the system. Sta-

<0.20

<0.20
<0.20

<0.20
PO4 b
13
ble isotope ratios of C/12C and
15
N/14N were expressed as the rela-

NO3 b
carbonate hardness (CH), and ammonia (NH3), nitrite (NO2), nitrate (NO3) and phosphate (PO4) measured in mg l-1.

3
2

3
tive difference per mil (‰), using the
equation: δX= [RSample * (RStandard)-1 -

<0.02

<0.02
<0.02

<0.02
NO2b

1] * 1000, where δX is δ15N or δ13C,

13
R the corresponding ratio C/12C or
NH3b

0.1

0.1
0.1

0.1
15
N/14N and RStandard the ratio of the
international reference Vienna Pee
CHb

Dee Belemnite for carbon and AIR

6

7
6

for nitrogen. Replicate assays of stan-

GHb

17

17
16

17

dards indicated analytical measure-

ment errors of ±0.1 ‰ and ±0.3 ‰
pHa

8.2

8.3
8.3

8.3

for δ13C and δ15N, respectively.

1304

1299
1316

1295
Ca

Data analyses
DO2a

measured using a digital multiparametric probe YSI® 553

Defining fish groups by integrating

6.3
6.2
6.3
6.4

measured using a colorimetric test kit VISOCOLOR®

the information provided by tradi-

16.1

tional ecology and stable isotopic

16

16
16
Ta

signatures
69°51’28’’
69°53’43’’
69°51’47’’
69°51’43’’

Homogeneous fish groups based

(West)
Lon.

on the information provided by sta-

ble isotope analyses and traditional
15°33’07’’
15°28’62’’
15°25’07’’
15°23’02’’

ecology were created in order to fa-

(South)
Lat.

cilitate the interpretation of the

graphical output and to guarantee the
accuracy of the analytical procedures
Location

L1
L2
L3
L4

used in determining niche overlap

a
b

58

and prey-consumer relationships (see be-
13 15
low). First, a δ C-δ N bi-plot space was
performed to group fish based on the
similarities in their isotopic signatures, as
fish showing similar isotopic signatures
are more likely to have similar ecological
attributes. Secondly, the groups created by
the isotopic space were refined using the
ecological information available for these
species gathered via an extensive litera-
ture review (Lauzanne, 1982; Parenti,
1984; Vaux et al., 1988; Loubens, 1989;
Lauzanne, 1991; Northcote, 2000; Paca et
al., 2002; Vila et al., 2007; Maldonado et
al., 2009) (see Table II for details). At this
stage, results from stable isotopic analyses
were visually assessed with the previous
ecological knowledge about these species.
This comparison also enabled to assign
poorly studied species to the most similar
fish group in terms of isotopic signatures
matching their ecological attributes (see
Table II for further details). For those spe-
cies whose gut contents were analysed,
the dominant prey items in the gut were
determined. After integrating this infor-
mation fish were classified into groups as
follows: (I) epipelagic and strictly zoo-
planktophagous species, (II) pelagic and
Trophic relationships between ichthyic community
mainly zooplanktophagous species, (III)
benthopelagic and mainly omnivorous
species, and (IV) littoral species that
mainly feed on insects and amphipods.
Introduced fish species were independ-
ently sorted into two groups correspond-
ing to O. bonariensis (V) and O. mykiss
(VI). The least abundant species (n<5)
were removed from trophic analyses to
increase the confidence of the results, al-
though the relevant data are shown in the
δ13C-δ15N bi-plot space (Fig. 2).
Exploring trophic interactions using
stable isotope analysis
Using non-parametric statistics as the
data lacked normality and homogeneity of
variances according to the Shapiro and
Barlett tests, the mean δ13C and δ15N sig-
natures of the six fish groups were com-
pared using the Kruskal-Wallis test fol-
lowed by Wilcoxon’s test for pair-wise
comparisons. Ontogenetic changes in the
use of habitat and the trophic level were
explored using the Spearman rank correla-
tion between LT and δ13C and δ15N, re-
spectively. Isotopic niche overlap between
fish groups was assessed using standard
ellipse areas (SEA) (‘siber’ function in R)
59
Chapter 1
(Jackson et al., 2011). Briefly, ellipses
13 15
reflect the covariance of the δ C and δ N
signatures, and are defined by their shape
and size, with the mean of the isotopic
signatures determining their location in
the bi-plot isotopic space (Jackson et al.,
2011). Unlike the Convex Hull method
(Layman et al., 2007), SEA is recom-
mended when there is no balanced design
(i.e. sample sizes vary between fish
groups) and sample sizes are small (<50
samples) (Jackson et al., 2011; Syväranta
et al., 2013).
The relative contribution of the four
native fish groups and invertebrate
sources in the diet of the two introduced
fish species was explored using Bayesian
mixing models (‘siar’ function in R)
(Parnell et al., 2008). The Bayesian ap-
proach incorporates more sources of vari-
ability and estimates various prey items
ingested by a consumer, thus offering a
more reliable picture of its diet (Jackson
et al., 2011). This modelling approach,
however, is very sensitive to differences
in the trophic discrimination factors ex-
hibited by consumers (McCutchan et al.,
2003; Bond & Diamond, 2011). As there
was no previous study addressing the dis-
60
crimination factor of silverside, the accu-
racy of results was increased assuming the
discrimination factors of 2.33 for Δδ15N
and 2.22 for Δδ13C observed in rainbow
trout for the mixing model (Pinnegar &
Polunin, 1999). The different trophic be-
haviour of large and small specimens of
O. bonariensis was studied with individu-
als above 90 mm due to likely be mainly
piscivorous (Fig. 2). The dietary prefer-
ences for O. mykiss were only explored in
large specimens due to collected individu-
als were all adults bigger than 149 mm.
All statistical analyses were performed in
R v.2.15 (R Development Core Team
2012) and the libraries MASS (Venables
& Ripley, 1999) and SIAR (Parnell et al.,
2008) with default settings. The level of
significance was α= 0.05.
RESULTS
Fish groups defined by traditional eco-
logical knowledge and stable isotopic
signatures
A total of 150 individuals belonging
to 17 fish species were captured in the
littoral area of Lake Titicaca with the aim
of addressing the food web structure of
the littoral fish community. Based on the
 TABLE 2. Characterization of the fish groups (FG) based on the information provided by traditional ecological analyses through an
extensive literature review (Lauzanne, 19821, Parenti, 19842, Vaux et al., 19883, Loubens, 19894, Lauzanne, 19915, Northcote, 20006,
Paca et al., 20027; Vila et al., 20078, Maldonado et al., 20099). For the species whose gut content was examined (PO) we present the per-
centage of individuals per species for each food category.

FG Species Habitat Diet Source

I Orestias gilsoni Epipelagic Zooplankton 1, 2
I Orestias gracilis * Zooplankton (100%) PO
I Orestias tomcooni * Zooplankton (100%) PO
II Orestias ispi Pelagic Zooplankton, eggs 1, 2, 5, 6, 7, 8
III Orestias mulleri Benthopelagic Zooplankton, amphipods, molluscs, insects, plant detritus 1, 2, 3, 4, 5, 8
III Orestias frontosus * Zooplankton (85.71%), amphipods (71.42%) PO
III Orestias forgeti * Zooplankton 1, 2, 8
III Orestias agassii Benthic, pe- Amphipods, zooplankton, insects, plant detritus 1, 2, 4, 5, 7, 8, 9
lagic, littoral
III Orestias albus Benthopelagic Molluscs, fish, zooplankton 1, 2, 8
III Orestias luteus Benthic Amphipods, molluscs, insects, algae, eggs, fish 1, 2, 5, 6, 8, 9
III Orestias crawfordi Benthopelagic Amphipods, molluscs, zooplankton 1, 2, 5, 8
III Trichomycterus rivulatus Benthic Amphipods, molluscs, insects, eggs, algae, plant detritus 7, 8
III Orestias incae Benthopelagic Molluscs (100%), insects (33.33%) 2, PO
IV Orestias uruni Littoral Insects (100%), molluscs (83.33%), amphipods (66.66%), 2, PO
zooplankton (50%)
IV Orestias farfani * Insects (100%), amphipods (88.88%) PO
V Odontesthes bonariensis Pelagic, littoral Amphipods, zooplankton, insects, fish 3, 8
VI Oncorhynchus mykiss Pelagic, littoral Amphipods, zooplankton, insects, fish, frogs 4, 8

* No information available

61
Trophic relationships between ichthyic community
Chapter 1
information provided by traditional eco-
logical studies and the species location
within the δ13C-δ15N bi-plot space, the 17
fish species captured were sorted into six
groups: the epipelagic planktophagous
group (I) composed of Orestias tomcooni,
Orestias gilsoni Tchernavin 1944 and
Orestias gracilis; the pelagic and mainly
zooplanktophagous group (II) containing
Orestias ispi Lauzanne 1981; the ben-
thopelagic group composed of mainly om-
nivorous species (III) formed by Orestias
incae, Trichomycterus rivulatus, Orestias
crawfordi Tchernavin 1944, Orestias lu-
teus Valenciennes 1846, Orestias albus
Valenciennes 1846, Orestias agassii
(Valenciennes 1846), Orestias forgeti
Lauzanne 1981, Orestias mulleri Valen-
ciennes 1846 and Orestias frontosus; the
littoral shallow water group that mainly
feed on insects and amphipods (IV) com-
posed of Orestias farfani and Orestias
uruni; and the introduced fish species,
Odontesthes bonariensis and Oncorhyn-
chus mykiss, in groups V and VI, respec-
tively (Fig. 2).
The designation of six fish groups
was supported by the significant differ-
13
ences observed in the mean δ C (KW,
62
χ2= 101.81; df=5; P<0.001) and δ15N sig-
natures (KW, χ2= 62.28, df=5, P<0.001).
Specifically, the δ15N values did not differ
significantly between most native groups,
possibly because all such fish species
have a similar trophic spectrum (e.g. am-
phipods and zooplankton) as demon-
strated by gut content analyses. Intro-
duced fish species showed the highest
δ15N values with evidence of ontogenetic
dietary changes in silversides (Spearman’s
rho = 0.81, n= 26; P<0.001). In particular,
individuals below 90 mm showed lower
δ15N values than larger individuals
(W=160, P<0.001) (Fig. 2). For δ13C,
there were significant differences between
all fish groups with the exception of
groups containing the pelagic species (II)
and the introduced O. mykiss (VI) (P=
0.79), possibly because variation in the
foraging areas exists for most fish groups.
Nonetheless, variation in δ13C also had an
ontogenetic component because there was
a negative relationship between LT and
δ13C for O. ispi (Spearman’s rho= -0.84,
n= 8; P<0.01), O. gracilis (Spearman’s
rho= -0.76, n= 10; P= 0.01), O. gilsoni
(Spearman’s rho= -0.66, n= 10; P= 0.04)
and O. bonariensis (Spearman’s rho= -
 Trophic relationships between ichthyic community

0.39, n= 26; P= 0.04). For silverside the Exploring niche overlap and prey-
increase in size was also related to an in- consumer relationships using stable iso-
crease in δ15N (Table III). tope analyses
Bayesian ellipse analyses revealed that

Figure 2. Mean δ15N and δ13C values of 15 native fish species, the two introduced species and
potential invertebrates used as food by these fish species. Native fish species were classified into
groups as follows to facilitate the interpretation of the analyses (see methods): (I) composed of
Orestias gracilis (gra), Orestias tomcooni (tom), Orestias gilsoni (gil); (II) containing Orestias ispi
(isp); (III) Orestias frontosus (fro), Orestias mulleri (mul), Orestias forgeti (for), Orestias luteus
(lut), Orestias crawfordi (cra), Orestias incae (inc) and Trichomycterus rivulatus (riv); and (IV)
composed of Orestias uruni (uru) and Orestias farfani (far). Red circles represent food sources
such as chironomids (chi), amphipods (amp), and zooplankton (zoo). Note that large and small
(<90 mm) individuals of Odontesthes bonariensis (group V) are shown in blue and green trian-
gles, respectively, and that Oncorhynchus mykiss (group VI) is shown in grey triangles. Error
bars represent standard deviation.

63
64
TABLE 3. List of species and total number of fish captured for each species in the lake, mean fish length (LT, mm) and size range
Chapter 1

(minimum-maximum, mm). The relationship between LT and δ13C or δ15N is also indicated using the Spearman rank correlation
coefficient (rho).

Species N LT LT range δ13C (‰) rho P-value δ15N (‰) rho P-value
Orestias gilsoni 10 95.05 56-112 -20.07±0.38 -0.66 <0.05 8.88±1.31 -0.45 >0.05
Orestias gracilis 10 52.07 47-57 -20.10±0.52 -0.76 <0.05 9.01±0.66 0.40 >0.05
Orestias tomcooni 9 48.67 39-55 -19.72±0.45 0.38 >0.05 8.51±0.76 0.19 >0.05
Orestias ispi 8 83.80 80-90 -18.82±0.71 -0.84 <0.01 10.24±0.67 -0.55 >0.05
Orestias mulleri 6 81.80 70-91 -18.35±0.57 0.57 >0.05 8.12±0.61 -0.28 >0.05
Orestias frontosus 7 144.57 128-168 -18.42±0.66 0.50 >0.05 8.43±0.64 -0.32 >0.05
Orestias forgeti 4 62.60 60-65 -18.29±0.27 0.20 >0.05 7.77±0.23 0.00 >0.05
Orestias agassii 7 134.27 112-163 -18.09±1.08 -0.60 >0.05 8.8±1.53 0.67 >0.05
Orestias albus 10 125.40 84-167 -17.41±0.84 -0.29 >0.05 8.48±1.93 0.59 >0.05
Orestias luteus 10 134.92 115-148 -17.30±0.83 -0.21 >0.05 7.30±0.81 -0.23 >0.05
Orestias crawfordi 9 76.30 59-85 -16.81±0.48 0.19 >0.05 7.40±0.49 -0.15 >0.05
Trichomycterus rivulatus 8 134.12 65-159 -16.90±0.88 -0.49 >0.05 7.94±1.14 -0.01 >0.05
Orestias incae 3 52.33 48-60 -16.37±0.17 -0.50 >0.05 6.64±0.37 1.00 >0.05
Orestias uruni 6 42.16 39-46 -15.88±0.42 -0.60 >0.05 5.87±0.42 0.75 >0.05
Orestias farfani 9 135.66 95-162 -15.10±0.48 -0.68 0.05 6.41±0.52 -0.43 >0.05
Odontesthes bonariensis 26 158.00 40-422 -17.33±1.51 -0.39 <0.05 10.76±0.86 0.81 <0.001
Oncorhynchus mykiss 8 200.00 149-268 -18.98±0.48 -0.16 >0.05 10.40±0.61 0.47 >0.05
 Trophic relationships between ichthyic community

Figure 3. Density plot showing the confidence intervals (95, 75, and 50%) of the relative propor-
tion of fish groups consumed by (a) Oncorhynchus mykiss, (b) Odontesthes bonariensis and (c)
Odontesthes bonariensis below 90 mm in length. Values were corrected by a discrimination factor
of 2.33‰ for δ15N and 2.22 for δ13C according to Pinnegar and Polunin (1999).

65
Chapter 1
large silversides overlapped with pelagic
fish (group II) by 39% and that small
silversides overlapped with benthopelagic
fish (group III) by 31.57%. These analy-
ses also showed an overlap of 19.66%
between rainbow trout and pelagic fish
(group II). The location of silversides and
rainbow trout along the δ13C axis demon-
strated the existence of spatial segregation
(Fig. 2). In this regard, large silversides
δ13C values were similar to those observed
in pelagic (group II) and benthopelagic
13
fish (group III), while rainbow trout δ C
values were more similar to those ob-
served in pelagic fish (group II) (P>0.05
for all pair-wise comparisons). In order to
determine the relative contribution of na-
tive fish groups to the diet of the two in-
troduced fish species Bayesian mixing
models were applied including four native
fish groups and invertebrate items as food
sources. The results revealed that pelagic
fish groups are likely to dominate the diet
of large specimens of both introduced fish
species, while chironomids appear to be
the main prey item for small silversides
(Fig. 3).
DISCUSSION
This study examined the trophic in-
66
teractions between 15 native fish species
and two introduced fish species in a litto-
ral area of Lake Titicaca illustrating that
stable isotope analyses and traditional
ecological studies can give complemen-
tary information in studies of food web
structure (Vander Zanden & Vadebon-
coeur, 2002; Clarke et al., 2005; Quevedo
et al., 2009). Additionally, this is the first
time than ecological attributes of six na-
tive species are explored to know the dif-
ferential effect of the introduction of
silversides and rainbow trout on the native
fish community.
Niche partitioning of the native fish com-
munity
As predicted, most native fish groups
showed a similar trophic level with
marked spatial segregation, possibly asso-
ciated with a decrease in trophic competi-
tion. The pelagic native O. ispi was the
only native fish group showing higher
δ15N values than the remaining native fish
groups. This could be attributed to the
occasional consumption of eggs by this
fish species (Paca et al., 2002). An addi-
tional reason for the high δ15N of O. ispi
might be that the pelagic condition en-
ables O. ispi to feed on prey items from

food webs differing in baseline levels of
15 15
δ N, which may lead to a higher δ N
(Hobson, 1999; Vander Zanden & Ras-
mussen, 2001; Bearhop et al., 2004).
Likewise, the large deviations observed in
13
the δ C values of T. rivulatus, O. luteus
and O. agassii could be associated with
the consumption of prey from different
feeding grounds. Current findings may be
related to the fact that different morpho-
types have been described for these three
species, these being particularly evident
for O. agassii but somewhat difficult to
differentiate in all cases by gross morpho-
logical examination due to hybridisation
(Parenti, 1984; Lauzanne, 1991;
Maldonado et al., 2009). Nonetheless,
certain trophic specialisation appears to be
common in native ichthyofauna because
an exhaustive morphological study of the
native species’ feeding structures (e.g.
pharyngeal dentition, gill raker number)
seems to support the notion that Orestias
species could prey on different zooplank-
ton groups (Northcote, 2000). In turn, al-
though details regarding the trophic spe-
cialization of the native fish species were
not identified in the current study, the
variations in the isotopic signatures may
be related to foraging specializations al-
Trophic relationships between ichthyic community
ready reported in fish fauna from other
great lakes (e.g. cichlids from Lake Ma-
lawi or Lake Victoria, Fry et al., 1999;
Balirwa et al., 2003, respectively).
Whilst all native species considered
in this study were described as littoral
species with the exception of O. ispi, the
low δ13C values observed in the named
epipelagic group (I) could be compatible
with its pelagic condition. Although the
reasons behind these low δ13C values re-
main largely unknown due to poor knowl-
edge about the ecology of these species,
their pelagic condition seems to be sup-
ported by the similarities observed be-
tween the δ13C values of these species and
those of the pelagic O. ispi (Parenti, 1984;
Vaux et al., 1988; Villwock, 1998; Vila et
al., 2007). An alternative explanation for
the low δ13C values of this fish group is
that all these fish species are strictly zoo-
planktivorous, and zooplanktonic organ-
isms often display high variation in δ13C
values (Cabana & Rasmussen, 1996).
Therefore, the δ13C values of species be-
longing to the epipelagic group may be
driven by the isotopic signature of zoo-
plankton even when all three species in-
habit the littoral area. This is consistent
with the pelagic condition of O. ispi be-
67
Chapter 1
cause this species has been observed for-
aging in the littoral area, so the relative
contribution of littoral and pelagic items
to its diet may explain the similar isotopic
composition of this group and littoral spe-
cies (Villwock, 1998).
The isotopic composition of the na-
tive species was also related to ontoge-
netic changes in the species’ use of forag-
13
ing grounds. As the δ C gradient is asso-
ciated with enrichment in δ13C in littoral
primary producers relative to offshore
producers (France, 1995), fish species that
move from littoral areas to offshore areas
when they age show a negative relation-
13
ship between LT and δ C. The negative
relationship observed between LT and
δ13C for O. ispi, O. gracilis, O. gilsoni and
O. bonariensis supports the field observa-
tion suggesting that the littoral macro-
phyte carpet is crucial for the juvenile
stages of fish species at Lake Titicaca,
even for the introduced silversides.
Trophic interactions between native and
introduced fish species
As previously reported (e.g. Vaux et
al., 1988; Loubens, 1989), silversides and
rainbow trout are the top fish predators at
68
Lake Titicaca. As large specimens of both
introduced fish species seem to prey
mainly on native pelagic fish groups,
silversides and rainbow trout appear to
depict spatial segregation according to
δ13C, thus minimising trophic interactions.
In addition, Bayesian ellipse analyses sug-
gested that large silverside specimens
show niche overlap with the pelagic na-
tive group II. This interaction seems to be
explained by a predator–prey relationship
as suggested by the mixing model analy-
ses. Interestingly, the current findings
suggested that silversides become mainly
piscivorous when smaller than the 200
mm total length suggested by Vaux et al.
(1988).
Unlike in silversides, the changes in
the foraging behaviour of rainbow trout
were not addressed because all individuals
captured were within the size range of
piscivorous individuals (149–268 mm)
reported in previous studies and consistent
with the δ15N values observed in the cur-
rent study. Lauzanne (1991) reported that
rainbow trout mainly feeds on O. ispi and
O. agassii at Lake Titicaca. However, a
prey–consumer interaction would be rep-
resented by an increase in the δ15N value
 Trophic relationships between ichthyic community

of rainbow trout compared to that of these
two species. The current findings instead
only demonstrated an overlap in the iso-
topic signatures of rainbow trout and O.
ispi. As stated above, this might be ex-
plained by the nature of the O. ispi diet
coupled with the fact that the isotopic sig-
nature of both fish species may be more
influenced by the pelagic than the littoral
food chain (e.g. Quevedo et al., 2009).
In conclusion, this study reveals a trophic
link between native and introduced spe-
cies and demonstrates the utility of com-
bining both SIA and traditional ecological
knowledge to understand trophic relation-
ships in fish communities with a pool of
species exhibiting similar feeding habits.
ment of Production of Peru, mainly for I.
González, K. Su and J. Vera. The authors
are also grateful to F. Ramírez, L. Jover,
E. García-Galea, T. Militão, R. Ramos
and M. García for useful suggestions on
the isotopic analysis and data interpreta-
tion as well T. Evans from the Language
Service at the University of Barcelona for
revising the English. AMV was funded by
a Marie Curie Fellow (EC-Funded ‘Para-
Tox’ project nº 327941).

ACKNOWLEDGMENTS
The authors would like to thank the
staff of the Binational Special Project of
Lake Titicaca (PELT), the Sea of Peru
Institute (IMARPE) and the Lake Titicaca
Agency (ALT), for their invaluable field
and laboratory assistance, especially O.
Flores and E. Yujra. Funding and logistic
support was provided by the Spanish
Agency for International Cooperation and
Development (AECID) and the Depart-

69
 CHAPTER 2
Metal concentration in water, sediment and four fish species from Lake
Titicaca reveals a large-scale environmental concern

Mario Monroy1, Alberto Maceda-Veiga2, Adolfo de Sostoa1

1
Department of Animal Biology and Biodiversity Research Institute (IRBIO), University of
Barcelona, E-08028 Barcelona, Spain.
2
Cardiff School of Biosciences, Cardiff University, CF10 3AX, Cardiff, United Kingdom.
 Chapter 2

RESUMEN
Aunque la intensa actividad minera y la descarga de aguas residuales son las mayores
fuentes de entrada de metales en el lago Titicaca, el riesgo que genera la contaminación por
metales pesados ha sido pobremente estudiado. En este estudio comparamos las concentra-
ciones de Cu, Zn, Cd, Hg, Pb, Co y Fe en agua, sedimento y dos tejidos (hígado y músculo)
de cuatro especies de peces (Odontesthes bonariensis, Orestias luteus, Orestias agassii y
Trichomycterus rivulatus) en las áreas de pesca más importantes del lago Titicaca. La con-
centración de Pb en el agua en los sitios de descarga de los principales ríos y la mayoría de
los elementos analizados en los peces capturados en este estudio, con la excepción de Co y
Fe, excedieron los umbrales de seguridad establecidos por la legislación internacional. Las
más altas concentraciones de metales fueron observadas en las especies bentopelágicas y el
hígado fue identificado como el principal tejido donde se depositan todos los metales con la
excepción del mercurio. El patrón de bioacumulación en los peces fue débilmente relacio-
nado con las concentraciones de metales en el ambiente, con la excepción del Hg en la loca-
lidad más contaminada, en parte explicado por las diferencias en el rol metabólico de los
elementos esenciales y no esenciales y la influencia de otros factores tales como la ecología
de las especies y caracteres individuales en la bioacumulación de la mayoría de los metales.
Como la contaminación por metales abarca toda el área de estudio y fueron detectadas altas
concentraciones de metales en las cuatro especies de peces estudiadas, instamos a las auto-
ridades a aplicar medidas de control para el consumo de agua y pescado, así como evaluar
los efectos de la contaminación por metales en la salud de los peces.

Palabras clave
Peces endémicos, contaminación minera, pesquerías, metales pesados, bioacumulación.

72
 Metal concentrations in Lake Titicaca

ABSTRACT
Although intensive mining activity and urban sewage discharge are major sources of
metal inputs to Lake Titicaca, the risk posed by metal pollution to wildlife and human popu-
lations has been poorly studied. In this study we compared the concentrations of Cu, Zn, Cd,
Hg, Pb, Co, and Fe in water, sediment, and two tissues (liver and muscle) of four fish spe-
cies (Odontesthes bonariensis, Orestias luteus , Orestias agassii, and Trichomycterus rivu-
latus) across important fishery areas in Lake Titicaca. The concentration of Pb in water at
the discharge sites of the main rivers and of most elements, with the exception of Co and Fe,
in all fish collected in this study exceeded the safety thresholds established by international
legislation. The highest metal concentrations were observed in benthopelagic species, and
liver tissue was identified as the main depository for all metals with the exception of mer-
cury. The metal bioaccumulation pattern in fish was weakly related to the metal concentra-
tions in the environment with the exception of Hg at the most polluted location, partly ex-
plained by the different metabolic role of essential and non-essential elements and the influ-
ence of other factors such as species’ ecology and individual traits in the bioaccumulation of
most metals. As metal pollution extended across the study area and high metal concentra-
tions were detected in all four fish species, we urge the authorities to enforce legislation for
water and fish consumption and to evaluate the effects of metal pollution on fish health.

Keywords
Endemic fish, mining contamination, fisheries, heavy metals, bioaccumulation.

73
 Chapter 2
INTRODUCTION
he conservation of inland
fisheries is an ecological,
economic and social con-
cern given the conservation
status of many fish species and the
strong dependence of human populations
living around lakes on fish resources as a
major source of protein (Welcomme,
2001). Common threats to inland fisher-
ies are overharvesting, the introduction
of exotic species and a wide range of
anthropogenic habitat disturbances such
as water pollution (Allan & Flecker,
1993; Dudgeon et al., 2006). The input
of sewage discharge to rivers is often the
main input of pollution into large lakes
(Carpenter & Cottingham, 2002; Dudg-
eon et al., 2006). To assess the conse-
quences of sewage pollution for wildlife
and humans while increasing our under-
standing about pollutant dynamics in
lakes, it is crucial to determine the varia-
tion in the concentration of contaminants
over space and across ecosystem com-
partments.
The effect of sewage discharge on
aquatic ecosystems primarily depends on
the mixture of organic (e.g. household
74
products, drugs) and inorganic (e.g. ac-
ids, heavy metals) contaminants that
they contain (Pinto, 2009). Heavy metals
are of particularly high environmental
risk because of their long-term persis-
tence in nature and possible bioaccumu-
lation and biomagnification (Demirak et
al., 2006; Ivanciuc et al., 2006; Agah et
al., 2009; Uysal et al., 2009). Metal
sources other than sewage are atmos-
pheric deposition, geologic weathering
and the run-off from adjacent agricul-
tural areas (e.g. pesticides, fertilizers)
(Demirak et al., 2006; Uysal et al., 2009;
Bereswill et al., 2013). However, ele-
ments such as copper (Cu), zinc (Zn),
cobalt (Co) and iron (Fe) cannot be con-
sidered a water quality hazard unless
they reach high concentrations since
they are necessary for animal life (Miller
et al., 1992; Canli & Atli, 2003). In con-
trast, other elements such as cadmium
(Cd), mercury (Hg) and lead (Pb) always
behave as toxic elements in organisms
(Miller et al., 1992; Amundsen et al.,
1997; Kojadinovic et al., 2007).
Sediments are the main sink of met-
als in lakes and sediment transport along
the upstream–downstream river gradient,

especially in high flow periods, is one of
the main pathways of metal input into
these ecosystems (Alloway, 2013). Be-
cause sediments concentrate metals and
the concentration of these elements in
sediment is less variable than in water,
sediments are suitable for monitoring the
long-term metal deposition in ecosys-
tems (MacDonald et al., 2000; Alloway,
2013). However, measuring metal con-
centrations either in water or sediment
does not provide information on the risk
posed by metal bioaccumulation or bio-
magnification (Ricart et al., 2010; Ma-
ceda-Veiga et al., 2013). These proc-
esses are firstly driven by metal avail-
ability for the biota (i.e. bioavailability),
which in turn is related to water vari-
ables, such as pH, oxygen concentration,
water hardness and temperature, and
sediment characteristics, such as organic
carbon content (Kotze et al., 1999; Canli
& Atli, 2003; Adhikari et al., 2009).
However, species traits such as trophic
position, age, body size or home range
also modify metal bioaccumulation pat-
terns (Mason et al., 2000; Gammons et
al., 2006; Kojadinovic et al., 2007), il-
lustrating that a combination of sentinel
Metal concentrations in Lake Titicaca
species with different ecological attrib-
utes will provide the best picture of the
risk posed by metal pollution for the bi-
ota (Jorgensen, 2011).
Fish are suitable bioindicators for
metal pollution because they occupy a
range of trophic levels and they have a
known ability to concentrate pollutants
(e.g. pesticides, biphenyls, heavy metals)
(Manirakiza et al., 2002; Bervoets &
Blust, 2003; Agarwal et al., 2007). The
life span of fish also enables detection of
the consequences of pollution over long
time periods compared to other bioindi-
cators, such as macroinvertebrates and
diatoms (Jorgensen, 2011). In addition,
because many species, including hu-
mans, consume fish as part of their diet,
fish reflect best the consequences of
metal pollution in lakes for wildlife and
humans. Although metal bioaccumula-
tion patterns have been described for
many freshwater fish species (e.g. Oreo-
chromis mossambicus, Kotze et al.,
1999; Tilapia nilotica, Rashed, 2001),
even lakes with similar inputs of metals
can produce fish with widely different
metal concentrations, due to variation in
biological, geochemical, and environ-
75
 Chapter 2
mental factors that affect metal uptake
and accumulation (Chen et al., 2000;
Ward et al., 2012).
The situation in Lake Titicaca typi-
fies the global decline in inland fisheries
due to anthropogenic perturbations, in-
cluding the discharge of mining waste,
with a 45% reduction in native fish bio-
mass over the last 30 years, and many
species now listed as threatened accord-
ing to the International Union for the
Conservation of Nature (Loubens, 1989;
Revollo et al., 2003; Vila et al., 2007;
Gobierno Regional de Puno, 2008;
Aliaga & Segura, 2013). This lake is an
important breeding and wintering area
for up to 60 bird species (e.g., sandpi-
pers, egrets, waterfowl, grebes) includ-
ing some endemisms such as the titicaca
flightless grebe (Rollandia microptera).
In addition 18 amphibian species inhabit
this lake, including the endemic Titicaca
water frog Telmatobius culeus, and a
growing number of fish species, cur-
rently standing at 26 species, pending
genetic confirmation (Lauzanne, 1991;
Sarmiento & Barrera, 2003; Costa, 2003;
Vila et al., 2007; Gobierno Regional de
Puno, 2008). The native fish community
76
is dominated by members of the killifish
genus Orestias (Teleostei, Cyprinodonti-
dae) and the catfish Trichomycterus
rivulatus, with no marked breeding sea-
son (Vila et al., 2007). Fisheries re-
sources are economically important as a
source of income and food for local
communities around Lake Titicaca. In
addition Lake Titicaca provides three
million people with drinking water,
which is also used for agricultural pur-
poses (Revollo et al., 2003).
Previous studies addressing the en-
vironmental aspects of metal pollution in
Lake Titicaca have focused on mercury
in its main tributary, the Ramis River
(Gammons et al., 2006), but a more
comprehensive study assessing the mag-
nitude of metal pollution in this area has
been lacking. The present study com-
pared the concentrations of Cu, Cd, Zn,
Hg, Pb, Co and Fe in water, sediment
and fish across the main fishery areas in
Lake Titicaca. Since the main rivers
have been portrayed as the main sources
of metal pollution, we expected that
sediments and fish at the discharge sites
would have the highest metal concentra-
tions, and that the metal concentrations
 Metal concentrations in Lake Titicaca

In fish would be in turn driven by environ-
mental (organic matter content) and spe-
cies traits (size, age, species ecology). The
sentinel species were the native and ben-
thopelagic killifishes Orestias agassii and
Orestias luteus, the native and benthonic
catfish T. rivulatus, and the introduced
and pelagic silverside Odontesthes bon-
ariensis. All species are omnivorous with
the exception of silverside, which mainly
feeds on macroinvertebrates or fish de-
pending on the silversides’ size (Vaux et
al., 1988; Vila et al., 2007). Previous stud-
ies have suggested that metal concentra-
tion is highest in species living close to
the sediment or in species feeding on high
trophic levels (Roméo et al., 1999; Mason
et al., 2000; Agarwal et al., 2007; Kojadi-
novic et al., 2007; Yilmaz et al., 2007;
Agah et al., 2009). Therefore, we pre-
dicted that benthonic and fish predators
would have the highest metal load. Fi-
nally, we discuss the implications of our
findings for the development of manage-
ment strategies at Lake Titicaca.

Fig. 1. Location of the two reference sites (R1 and R2) and the seven known polluted sites (L1-
L7) in Lake Titicaca where metal analyses were conducted in water, sediment and fish for the
current study. Original map provided by National Aeronautics and Space Administration
(NASA®).

77
 Chapter 2
MATERIAL AND METHODS
Study area
Lake Titicaca, located between Peru
and Bolivia at 3810 m above sea level, is
the largest freshwater lake in South
2
America with 8559 km of surface area
3
and a water volume of 932 km . The lake
is divided into two large water bodies,
the “Lago Mayor” and the “Lago Me-
nor”, with a maximum depth of approxi-
mately 280 and 40 m, respectively. Lake
Titicaca is a cold oligotrophic lake with a
high salt concentration due to the geol-
ogy of the basin (e.g. carbonate/
bicarbonates, chlorides) (Arze
Quintanilla, 1991). The water chemistry
is relatively constant across seasons and
sites, with the exception of the rainy pe-
riod between December and March, and
those sites close to the mouth of the main
tributaries, the Ramis, Coata and Ilave
Rivers (Vila et al., 2007). These rivers
represent the main source of metal pollu-
tion in the Lake due to the location of
mining areas upstream (Gammons et al.,
2006). However, agricultural areas close
to the rivers or surrounding Lake Titicaca
could be potential source of contamina-
tion (e.g. pesticides, fertilizers).
78
In order to gain an overall picture of
the metal pollution in Lake Titicaca, we
collected water, sediment and fish from
two supposed non-polluted areas (R1:
Huencalla Bay and R2: Tamán Bay) and
seven known polluted locations (L1:
Mouth of Ilave River, L2: Barco-
Chucuito, L3: Mouth of Ramis River, L4:
Pusi, L5: Mouth of Coata River, L6:
Mohokachi and L7: Inka Chaka Bay)
across the main fishery areas of Lake
Titicaca in November 2010 (Fig. 1). All
sampling sites were located in Lago
Mayor with the exception of L6 which
& was located in Lago Menor. These sam-
pling sites were selected based on an ex-
tensive literature review, interviews with
local communities and our personal ob-
servations (Supplementary material: Ta-
ble S1).
Water quality and sediment characteris-
tics
General water quality variables were
determined at each sampling site using a
digital YSI® 556 MPS multiprobe for
temperature (ºC), conductivity (µS cm-1),
dissolved oxygen (mg L-1) and pH, and
the colorimetric test kit VISOCOLOR®

for ammonia (NH3, mg L-1), nitrite (NO2,
mg L-1), nitrate (NO3, mg L-1) and phos-
-1
phate (PO4, mg L ) concentrations, and
general (ºGH) and carbonate hardness
(ºKH). For metal analysis, triplicate wa-
ter samples were collected at each loca-
tion in 250-mL polyethylene bottles pre-
cleaned with nitric acid (10%) for 24 h
and rinsed three times with ultrapure wa-
ter. Water samples were immediately
filtered through a 0.45-µm Millipore
membrane filter and kept acidified with
nitric acid (pH ~2) until metal concentra-
tions were determined (see below).
At each sampling three random sedi-
ment samples were collected at a 10-m
deep in the mid region of the nine bays
surveyed using a grab sampler of 325
2
cm . These samples were air-dried and
sieved through a 100-µm mesh. pH was
determined using a Thermo pH Orion
Star A221 in a suspension of 10 g of soil
and 25 mL of distilled water that was
shaken for 15 min and left to settle for 30
min (Pansu & Gautheyrou, 2007). In ad-
dition, three subsamples (~1.5 g) per
sampling site were combusted at 540 °C
for 4 h to determine the percentage of
organic matter content by measuring the
Metal concentrations in Lake Titicaca
weight difference before and after com-
bustion.
Fish sampling and collection of biologi-
cal data
Fish were collected by local fisher-
men using gillnets with a mesh-size rang-
ing from 10 to 30 mm length, set over-
night in each bay. All individuals were
identified to species level and a random
sub-sample of 10 individuals per species
at each location was used for metal
analysis. As the juveniles of these species
(<90 mm total length, LT) could not be
reliably identified, they were excluded
from the analyses. The ten fish used for
metal analyses were euthanized with an
overdose of MS-222 (3-aminobenzoic
acid ethyl ether, Sigma-Aldrich®, see
Noga, 2010 for details), measured to the
nearest mm (LT) and weighed to the near-
est 0.01 g (total body wet weight, WT).
Fish were then dissected, sexed by a vis-
ual gross examination of gonads and a
sample of muscle (ca. 500 mg) below the
dorsal fin and the whole liver were stored
for metal analyses in polypropylene vials
previously pre-cleaned with nitric acid
(10%) and rinsed three times in water.
79
 Chapter 2
Muscle was selected to determine the risk
posed by metal pollution to humans and
liver because it is a key organ in detoxifi-
cation processes and is targeted in metal
accumulation (Miller et al., 1992). For
age determination six to eight scales per
fish were also collected from above the
lateral line and posterior to the dorsal fin
area (see Carmona-Catot et al., 2011 for
details on ageing criteria using scales).
Scales were cleared in KOH for 12 h,
rinsed in distilled water and examined
using a Microbox® reader for the deter-
mination of year annuli. For T. rivulatus,
which lacks scales, we used the age
groups established by Paca et al. (2003)
based on fish length histograms as a
proxy measure of fish age since alterna-
tive tissues for fish ageing, such as oto-
liths, could not be examined due to logis-
tic constraints.
Determination of metal concentration in
water, sediment and fish
Water, sediments and fish samples
were frozen at -20ºC, transported in poly-
styrene boxes embedded in dry ice to the
University of Barcelona (Spain) and kept
for 1 month at the same temperature until
80
analysis. Metal concentrations in water
were directly determined in acidified so-
lutions prepared in the field. For sedi-
ments a total attack with aqua regia was
carried out by adding 6 mL HCl and 2
mL HNO3 to 500 mg of well-
homogenised sediment sample. This mix-
ture was digested in closed 60-mL Teflon
vessels using a Milestone Ethos Plus mi-
crowave digestion system at 200°C fol-
lowing ISO 11466. Fish tissues were first
freeze-dried and ground to a powder to
facilitate homogenisation. Fish samples
were then acid-digested in an oven at 90°
C for 12 h according to their weight as
follows: 3 mL HNO3 and 1 mL H2O2
added to 100 mg tissue, 2 mL HNO3 and
1 mL H2O2 added to samples weighing
over 50 mg, and 1 mL HNO3 and 0.5 mL
H2O2 added to samples weighing under
50 mg, using reagents of Instra quality (J.
T. Baker®).
Metal concentrations were deter-
mined at the Technical Services of the
University of Barcelona using a Perkin-
Elmer OPTIMA-3200 RL Inductively
Coupled Plasma Optical Spectrometer
(ICP-OES) for iron (Fe, µg L-1) and a
Perkin-Elmer ELAN-6000 Inductively

Coupled Plasma Mass Spectrometer (ICP
-MS) for zinc (Zn, µg L-1), cadmium (Cd,
-1 -1
µg L ), total mercury (Hg, µg L ), lead
(Pb, µg L-1), and cobalt (Co, µg L-1), us-
ing standard procedures (Maceda-Veiga
et al., 2013 for details). Blanks and a cer-
tified reference material for soils
(WQB1, National Research Institute,
Canada) and fish (DORM-3, National
Research Council Canada) were also
processed in each batch of digestions to
provide quality control data. The metal
concentrations in blanks were always
below the detection limits, and the metal
recovery rate for soil and fish samples
was always above 90% (see Supplemen-
tary material: Table S2 for details). Metal
concentrations under the detection limits
were replaced by half the detection limit
before the statistical analysis (Maceda-
Veiga et al., 2012; 2013). Heavy metal
concentrations in sediments and fish are
presented as the mean ± standard devi-
ance (SD) in mg g-1 on a dry weight (dw)
basis. We also estimated the water con-
tent of both tissues to facilitate the con-
version of metal concentration from dry
to wet weight (24.58%, 23.48%, 23.65%
and 25.76% for muscle and 25.82%,
Metal concentrations in Lake Titicaca
24.45%, 24.78% and 27.12% for liver of
O. bonariensis, O. luteus, O. agassii and
T. rivulatus, respectively), based on 10
samples of each tissue and species.
Data analysis
Water, sediment and fish variables
were tested for normality and homogene-
ity of variances using Shapiro-Wilk and
Levene’s tests, respectively, and para-
metric and non-parametric statistics were
then applied accordingly. The mean
metal concentrations either in water or
sediment and sediment features (organic
matter and pH) were compared between
locations using a one-way ANOVA fol-
lowed by Tukey HSD post-hoc test for
pair–site comparisons. The relationships
between metal concentrations, other wa-
ter chemistry variables (temperature, con-
ductivity, dissolved oxygen, pH, general
and carbonate hardness, and ammonia,
nitrite, nitrate and phosphate concentra-
tions) and sediment characteristics were
explored using Spearman’s rank correla-
tion analysis (rho). Differences in mean
metal concentrations between species,
tissues and locations were compared us-
ing a three-way ANOVA. Where signifi-
81
 Chapter 2
cance was observed, one-way ANOVA
was applied followed by Tukey HSD
post-hoc test for paired comparisons be-
tween locations or species. As LT and WT
were strongly correlated (Pearson’s cor-
relation coefficient, r >0.9), LT was con-
sidered in this study as a more direct
measure of fish size than weight. To ex-
plore the relative influence of fish attrib-
utes on the observed metal bioaccumula-
tion pattern in fish, sampling site, tissue,
species, age, sex and LT was combined in
a general lineal model (GLM) fitted to
each metal concentration (log-
transformed) with a Gaussian error distri-
bution. The best models were selected
using a manual stepwise backward dele-
tion of non-significant terms from the full
global models containing all fixed factors
and interactions (i.e. including covariate
effects). The significance of each factor
was checked using an F-test (‘Anova’
function in R). Finally, to describe the
main sources of variation in metal con-
centrations in fish and sediments, two
principal component analyses (PCAs)
were independently applied to each metal
data set. The “varimax” rotation method
was used to increase the interpretation of
82
axes and the number of PCA axes was
determined using Kaiser's rule
(eigenvalue≥1) (Legendre & Legendre,
1998). The synthetic gradients built
(henceforth, PCbio and PCenv scores) also
made the degree of divergence between
metal concentrations in fish and sedi-
ments visible, and facilitated the inter-
linking between the metal concentrations
in the environment and fish. Pearson’s
correlation coefficient (r) was calculated
to determine pair-wise relationships be-
tween the synthetic gradients built with
metal concentrations.
All statistical analyses were per-
formed using the R package (R Develop-
ment Core Team, 2012) and the libraries
car, vegan and MASS. GLM assumptions
were checked by examining standardised
residuals using qq plots and plotting fit-
ted versus predicted values.
RESULTS
Metal concentrations in water and sedi-
ments and their relationship with envi-
ronmental variables
The mean metal concentrations in
water differed significantly between loca-
tions for all elements determined

(ANOVA, p<0.001) with the exception
of Hg, which remained below the detec-
-1
tion limits at all locations (<0.2 µg L ;
F8,18=1, p=0.46) (Table 1). The peak con-
centrations of Cu, Cd, Pb and Co were
significantly associated with the dis-
charge site of the River Ramis (Tukey
post-hoc, p<0.05), while the highest val-
ues of Zn and Fe were respectively ob-
served at locations L2 and L6 (Tukey
post-hoc, p<0.05), without any river ef-
fect (Table 1). As the highest conductiv-
ity values were observed at the discharge
site of the River Ramis, conductivity was
also positively associated with metals
such as Cu (r=0.85, p=0.003) and Pb
(r=0.75, p=0.01). Nitrate concentration
was also positively related to Cu (r=0.66,
p=0.04) and Cd (r=0.74, p=0.02) levels in
water, possibly because of the spatial co-
occurrence of the slight increase in ni-
trate concentration and that of these two
elements in Barco-Chucuito (Table 1).
The mean metal concentrations in
sediments also varied significantly be-
tween locations (ANOVA, p<0.001), as
did organic matter content (ANOVA,
F8,18= 437500, p=0.001) and pH
(ANOVA, F8,18= 16706, p=0.001) (Table
Metal concentrations in Lake Titicaca
2). Specifically, the highest organic mat-
ter concentrations and lowest pH were
observed at Mohokachi (Lago Menor)
and those sites close to the discharge sites
of the Ramis and Coata Rivers in Lago
Mayor (Table 2; Tukey post-hoc,
p<0.05). This explains why organic mat-
ter content was also positively related to
the concentration of most elements: Cu
(r= 0.64, p<0.001), Zn (r= 0.51,
p=0.006), Cd (r= 0.57, p=0.001) and Co
(r= 0.51, p=0.006). In addition, pH was
negatively related to the concentration of
organic matter (r=-0.58, p=0.001), and all
metal concentrations in sediments (all, r>
-0.66, p<0.001) with the exception of Co
(r= -0.16, p=0.4).
Determinants of metal bioaccumulation
in fish and their relationship with envi-
ronmental concentrations
The concentrations of all metals ana-
lysed differed significantly between spe-
cies (ANOVA, F3,696>9.72, p<0.01), tis-
sues (ANOVA, F1,696>50.45, p<0.01) and
locations (ANOVA, F8,696>3.03, p<0.01).
Liver was the main metal depository for
all elements with the exception of Hg
(Fig. 2). The tissue analysed was indeed
83
84
Chapter 2

Table 1. Water quality of the nine bays surveyed in Lake Titicaca. Descriptive results are shown for general indicators of water
quality but mean metal concentrations (µg l-1± standard deviation) and detection limits are reported for each element. The specific
legislation of Health Canada (2012 x), US EPA (2009 y), and BOE (2011z) to establish the safety threshold for each water quality
variable is also included. The letters (a, b, c, d, e, f, g, h, i) group sites by heavy metal, considered homogenous at p<0.05.

Detection Legislation
Location R1 R2 L1 L2 L3 L4 L5 L6 L7
limits thresholds
Temperature 
15 15 15 16 15 15 15 17 15 0 -
(ºC)
Dissolved
oxygen  5.7 6.1 6.6 5.5 5.3 6.2 11 5.7 6.9 0 -
-1
(mg l )
Conductivity 
1207 1239 1239 1318 1754 1258 1570 1540 1280 0 -
(µS cm-1)

pH 8.2 8.2 8.2 8.2 8 8.3 8 8.9 8.8 1 5-9 y

General hard-
17 20 17 18 20 16 5 16 18 1 -
ness (ºdH)

Carbonate
hardness 6 7 8 5 7 6 3 4 7 1 -
(ºKH)

Ammonia
-1
0.1 0.1 <0.02 0.1 0.1 0.1 0.1 0.1 <0.02 0.02 0.1x
(mg l )
 Continue Table 1
Detection Legislation
Location R1 R2 L1 L2 L3 L4 L5 L6 L7
limits thresholds
Nitrites 
<0.02 <0.02 <0.02 <0.02 <0.02 <0.02 0.1 <0.02 <0.02 0.02 1x
(mg l-1)
Nitrates
1 1 1 4 2 3 2 1 1 1 10 y
(mg l-1)
Phosphates
<0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 0.2 -
(mg l-1)

Cu 0.63a±  0.27b±  2.45c±  8.93d±  16.84e±  7.28f±  11.09g±  7.43f±  5.47h± 

0.2 1300 y
(mg l-1) 0.005 0.008 0.01 0.01 0.004 0.007 0.02 0.02 0.02
Zn 7.11a±  58.46b±  23.67c±  117.32d±  47.48e±  31.42f±  51.09g±  45.77h±  20.1i± 
0.2 5000 y
(mg l-1) 0.01 0.03 0.01 0.02 0.01 0.02 0.02 0.02 0.02
Cd 0.06b±  0.07b,c±  0.11d±  0.06b,c,e±  0.07b,c±  0.05b,e,f±  0.04f± 
˂0.01a ˂0.01 a 0.02 5y
(mg l-1) 0.006 0.005 0.004 0.005 0.001 0.005 0.001
Hg
˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a 0.2 2y
(mg l-1)
Pb 2.57a  0.18b±  3.07c±  14.44d±  32.39e±  19.34f±  24.43g±  15.62h±  14.35d± 
0.02 0y
(mg l-1) ±0.01 0.005 0.006 0.02 0.01 0.01 0.02 0.01 0.02
Co 0.05a±  0.05a±  0.25b±  0.12c±  0.28b±  0.12c±  0.19d±  0.15e± 0.05a± 
-1
0.1 5z
(mg l ) 0.008 0.008 0.005 0.006 0.01 0.01 0.01 0.005 0.008
a b c d e f g h
Fe 74.08 ±  13.43 ±  111.6 ±  25.33 ±  40.78 ±  52.12 ±  19.37 ±  211.08 ± 21.61i± 
10 300 y
(mg l-1) 0.01 0.01 0.02 0.01 0.01 0.009 0.01 0.005 0.01
Metal concentrations in Lake Titicaca

85
86
Chapter 2

Table 2. Sediment characteristics and mean metal concentration (µg g-1 dw ± standard deviation) at each sampling site surveyed
in Lake Titicaca. The safety thresholds reported for metal concentrations in sediments are based on MacDonald et al. (2000). The
letters (a, b, c, d, e, f, g, h, i) group sites by environmental variable, considered homogenous at p<0.05.

Reference
Legislation
Location R1 R2 L1 L2 L3 L4 L5 L6 L7 material
thresholds
WQB1

Organic
1.85a± 1.19b± 5.96c± 3.66d± 35.8e± 2.67f± 23.66g± 21.3h± 4.45i±
matter - -
0.04 0.01 0.01 0.05 0.02 0.03 0.05 0.02 0.01
(%)

8.43a± 7.95b± 7.31c± 8.12d± 5.13e± 6.9f± 7.05g± 7.51h± 8.12i±

pH - -
0.01 0.01 0.005 0.005 0.01 0.01 0.02 0.01 0.01

Cu 3.3a± 3.07b± 10.39c± 20.99d± 41.09e± 27.21f± 25.77g± 14.17h± 9.95i± 79.6±
149
(µg g-1) 0.002 0.002 0.004 0.002 0.002 0.01 0.002 0.003 0.004 19.1

Zn 9.1a± 4.32b± 19.04c± 71.09d± 323.24e± 73.89f± 65.61g± 11.97h± 46.57i± 275±
459
(µg g-1) 0.003 0.001 0.001 0.002 0.006 0.002 0.002 0.002 0.002 58

Cd 0.11b,e± 1.98c± 0.21d± 0.13b± 0.18d± 0.08e± 2±

˂0.01a ˂0.01a ˂0.01a 4.98
(µg g-1) 0.01 0.01 0.005 0.01 0.005 0.01 0.91

Hg 0.78b± <0.1a± 1.09±

˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a ˂0.1a 1.06
(µg g-1) 0.01 0.001 0.15

Pb 15.82a± 2.16b± 8.33c± 17.65d± 74.97e± 25.82f± 16.61g± 5.47h± 10.45i± 83.7±
128
(µg g-1) 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.004 0.002 22.3

Co 3.77a± 1.62b± 5.69c± 10.3d± 8.68e± 4.76f± 10.1g± 1.75h± 6.28i± 20.1±
-
(µg g-1) 0.001 0.003 0.004 0.002 0.001 0.004 0.004 0.002 0.002 9.3

Fe 162.44a± 87.79b± 246.81c± 319.19d± 433.38e± 374.98f± 290.79g± 58.05h± 199.72i± 47358±
-
(µg g-1) 0.002 0.002 0.002 0.002 0.002 0.001 0.001 0.002 0.003 10536
 Metal concentrations in Lake Titicaca

Fig. 2. Mean metal concentrations in muscle (white boxes) and liver (grey boxes) in the native
benthopelagic (O. agassii and O. luteus), the native benthonic (T. rivulatus) and the pelagic in-
troduced species (O. bonariensis) collected from nine study sites in Lake Titicaca. Discontinuous
lines indicate the safety thresholds for metal concentration established by international legisla-
tion for human consumption (Cu, Cd, Hg and Pb according to the EC, 2006; and Zn based on
FAO/WHO, 1998). The letters (a, b, c, d) group sites by variable, considered homogenous at
p<0.05. Note that metal concentrations in males and females are grouped by location.

87
 Chapter 2
the main factor explaining the variation in
all metal concentrations with the excep-
tion of Hg and Pb when individual traits
were incorporated into the analysis (Table
4). Together with tissue, the sentinel spe-
cies used for the metal analysis was also a
significant contributor for most elements
(Table 4), illustrating that native ben-
thopelagic species (O. luteus and O. agas-
sii) generally had the highest metal con-
centrations, followed by the catfish (T.
rivulatus) (Fig. 2). Specifically, O. luteus
had the highest mean values of Cu
(108.84±85.75 µg g-1 dw), Hg (0.8±0.93
-1 -1
µg g dw) and Co (0.64±0.4 µg g dw),
O. agassii contained the highest concen-
trations of Zn (102.22±21.36 µg g-1 dw),
and T. rivulatus had the highest values of
-1
Cd (0.15±0.18 µg g dw), Pb (0.06±0.08
-1 -1
µg g dw) and Fe (266.1± 146.56 µg g
dw) (Fig. 2).
As the fish used for metal analyses
spanned a wide range of sizes and ages
(Table 3), GLMs were employed to ex-
plore the relative influence of individual
traits (LT, gender and age) compared to the
location of sampling sites in explaining the
variation in metal bioaccumulation (Table
88
4). Interestingly, location was the most
significant contributor to Hg bioaccumula-
tion but the interaction between species
and location was also significant for most
elements, showing that the metal bioac-
cumulation pattern observed in all four
species differed across the study area (Fig.
3). Individual traits appeared to have a low
influence on the concentration of all ele-
ments in fish with the exception of Zn, Co
and Fe, for which gender was a significant
predictor but with a marginal effect (Table
4) and no clear trend observed in metals
between males and females across species
(Table 5). None of the GLM models re-
tained length or age as a significant ex-
planatory variable (Table 4).
To further examine the metal bioac-
cumulation pattern in fish and in the sedi-
ments of the locations surveyed we gener-
ated two PCA analyses for each data set,
which produced two significant axes for
fish (PC1bio, PC2bio) and environment
(PC1env, PC2env) that together explained,
respectively, 61.32% and 91.80% of the
variation in metal concentrations (Fig. 4
and Supplementary material: Table S3).
PC1bio accounted for 37.31% of the varia-
 Metal concentrations in Lake Titicaca

Fig. 3. Variation in mean metal concentrations in the nine bays surveyed and in the four fish
species examined (O. bonariensis, black; O. luteus, dark grey; O. agassii, grey and T. rivulatus,
white boxes) using the liver as the sentinel tissue for the assessment of all metals with the excep-
tion of Hg. Discontinuous lines indicate the safety thresholds for each metal established by in-
ternational legislation for human consumption (Cu, Cd, Hg and Pb according to the EC, 2006;
and Zn based on FAO/WHO, 1998). Metal concentrations are plotted without log-
transformation to facilitate the interpretation of axes.
89
 Chapter 2

Fig. 4. Principal component analysis of the metal concentrations in the environment and fish.
Scores are according to location either in sediments (a) or fish (b), and only by species (c).

90
 Table 3. Sample size (N), mean and standard deviation of body length (LT mm) and weight (W g), minimum and maximum range,
and details of gender and ages of the four fish species in Lake Titicaca used for metal analysis.

Min- Age (years)

Species Gender N LT W Min-Max
Max
0+ 1+ 2+ 3+ 4+ 5+ 6+ 7+

♂ 52 210.65±24.83 166-280 58.32±21.56 25.25-121.18 0 47 5 0 0 0 0 0

O. bonariensis
♀ 37 228.02±37.27 165-375 85.83±47.44 25.54-278.08 0 28 8 1 0 0 0 0

♂ 46 120.95±18.2 94-155 44.51±18.77 17.06-79.71 0 9 3 3 3 23 5 0

O. luteus
♀ 39 131.94±20.41 90-164 62.51±24.85 16.84-105.18 0 5 1 1 1 19 10 2

♂ 27 121.44±22.93 90-160 31.11±16.74 11.12-61.57 0 6 9 4 6 2 0 0

O. agassii
♀ 63 133.71±22.26 90-170 42.86±18.95 9.31-90 0 4 14 19 13 13 0 0

♂ 39 158.15±15.64 121-181 40.39±11.19 18.73-60.37 0 4 35 0 0 0 0 0

T. rivulatus
♀ 44 164.97±18.54 122-204 44.82±12.56 19.21-69.16 0 2 39 0 3 0 0 0
Metal concentrations in Lake Titicaca

91
 Chapter 2

Table 4. Results of the final GLM models for all metals (Cu, Zn, Cd, Hg, Pb, Co and Fe) deter-
mined in the four fish species (O. bonariensis, O. luteus, O. agassii, T. rivulatus) collected in
Lake Titicaca. Note that only significant variables highlighted in previous full models with all
explanatory variables and interactions (see methods) are shown.

Metal Fish attributes SS Df F p-value

Species 182.02 3 101.34 <0.001
Tissue 993.11 1 1658.82 <0.001
Cu
Species x Location 23.94 24 1.66 0.02
Residuals 393.34 657
Species 23.7 3 59.09 <0.001
Location 3.36 8 3.14 0.001
Zn Tissue 105.73 1 790.63 <0.001
Gender 0.639 1 4.77 0.02
Residuals 90.94 680
Species 0.31 3 26.9 <0.001
Location 0.2 8 6.79 <0.001
Cd
Tissue 0.85 1 221.17 <0.001
Residuals 2.53 657
Species 1.98 3 21.13 <0.001
Location 34.02 8 135.82 <0.001
Hg Tissue 8.75 1 279.67 <0.001
Species x Location 6.48 24 8.62 <0.001
Residuals 20.57 657
Species 0.08 3 15.89 <0.001
Location 0.07 8 5.4 <0.001
Pb Tissue 0.02 1 15.72 <0.001
Species x Location 0.14 24 3.3 <0.001
Residuals 1.16 657
Species 1.96 3 33.75 <0.001
Tissue 17.69 1 910.87 <0.001
Co Gender 0.43 1 22.5 <0.001
Species x Gender 0.44 3 7.62 <0.001
Residuals 13.3 685
Species 45.58 3 38.26 <0.001
Tissue 1068.64 1 2691.94 <0.001
Fe Gender 3.05 1 7.67 <0.001
Species x Location 82.03 32 6.45 <0.001
Residuals 260.42 656

92
 Table 5. Sample size (N) and mean metal concentration (µg g-1 dw ± standard deviation) in relation to species (Sp; O. bonariensis: 1,
O. luteus: 2, O. agassii: 3, T. rivulatus: 4) gender (G) and tissue (muscle, M and liver, L) in Lake Titicaca. Note that only significant
differences were observed for concentrations of Zn, Co and Fe from Table 4.

Cu Zn Cd Hg Pb Co Fe
Sp G N
M L M L M L M L M L M L M L

1.3±  7.65±  38.45±  50.43±  0.007±  0.02±  0.74±  0.3±  0.02±  0.01±  0.03±  0.2±  23.68±  164.94± 
♂ 52
1.8 7.07 15.55 17.27 0.007 0.01 0.49 0.55 0.02 0.01 0.07 0.11 43.84 73.2
1
0.93±  5.46±  28.63±  44.22±  0.007±  0.02±  0.84±  0.34±  0.02±  0.01±  0.04±  0.17±  21.1±  138.69± 
♀ 37
1.31 3.47 9.16 15.66 0.008 0.01 0.48 0.57 0.05 0.01 0.07 0.07 36.46 56.67

1.26±  118.42±  30.6±  77.4±  0.006±  0.14±  0.76±  0.16±  0.02±  0.04±  0.01±  0.82±  12.31±  170.9± 
♂ 46
1.03 85.35 9.86 20.68 0.003 0.003 0.89 0.15 0.02 0.09 0.008 0.44 5.74 119.23
2
1.26±  97.54±  29.32±  83.37±  0.007±  0.08±  0.86±  0.2±  0.01±  0.04±  0.01±  0.44±  13.99±  112.24± 
♀ 39
1 85.93 6.67 24.78 0.003 0.003 0.99 0.2 0.02 0.05 0.009 0.24 7.02 57.72

1.06±  83.18±  32.94±  103.93±  0.003±  0.07±  0.67±  0.47±  0.009±  0.01±  0.02±  0.75±  13.37±  261.02± 
♂ 27
0.54 77.87 10.29 17.2 0.004 0.004 1.12 0.75 0.01 0.01 0.01 0.48 5.01 197.61
3
0.96±  65.55±  31.67±  101.49±  0.004±  0.04±  0.44±  0.36±  0.02±  0.02±  0.01±  0.42±  15.83±  194.43± 
♀ 63
0.44 44.85 9.16 23 0.004 0.004 0.66 0.66 0.02 0.02 0.01 0.28 14.64 125.46

1.9±  10.89±  49.17±  87.97±  0.006±  0.14±  0.44±  0.19±  0.02±  0.06±  0.02±  0.4±  22.66±  304.82± 
♂ 39
0.75 4.82 15.26 11.93 0.007 0.007 0.38 0.24 0.03 0.07 0.01 0.19 12.46 170.36
4
2.05±  12.03±  46.2±  90.36±  0.004±  0.15±  0.43±  0.2±  0.04±  0.07±  0.02±  0.42±  21±  231.77± 
♀ 44
1.4 10.06 15.85 19.57 0.004 0.004 0.45 0.4 0.09 0.09 0.01 0.23 7.47 112.9
Metal concentrations in Lake Titicaca

93
 Chapter 2
tion and loaded on Cu, Zn, Cd, Hg, Co
and Fe concentrations, PC1bio accounted
for 37.31% of the variation and loaded on
Cu, Zn, Cd, Hg, Co and Fe concentra-
tions, while PC2bio explained 24.01% of
variation and was mainly related to Pb
concentration. In contrast, PC1env ac-
counted for 79.22% of the variation and
loaded on Cu, Zn, Cd, Hg, Pb and Fe
concentrations, while PC2env accounted
for 12.59% of variation and was mainly
related to Co concentration. These envi-
ronmental metal synthetic gradients were
poorly and non-significantly correlated
with the metal bioaccumulation pattern
observed in fish summarised in PC1bio
(PC1env: r=0.54, p=0.13; PC2env: r=-0.54,
p=0.13) and PC2bio (PC1env: r=-0.11,
p=0.77; PC2env: r=-0.04, p=0.91) (Fig. 4).
DISCUSSION
Metal concentrations in water and sedi-
ments and relationship with other envi-
ronmental variables
Water chemical analyses showed
that the Ramis River was the main source
of metal pollution in Lake Titicaca with
peak concentrations of Cu, Cd, Pb and
Co observed at this location. Specifically,
94
the Pb concentration exceeded the safety
thresholds established by legislation by
30-fold (US EPA, 2009). This peak in Pb
might be related to the use of fuel addi-
tives and lubricants enriched in Pb in the
mining and industrial areas located up-
stream, since no hunting practices using
Pb are present in this area (Revollo et al.,
2003; Gammons et al., 2006). Baseline
locations generally had fairly low con-
centrations of all elements determined
but our results also showed that all pol-
luted locations showed a degree of metal
pollution (e.g. Cu, Zn, Pb, Co) regardless
of their proximity to the main rivers. The
release of domestic wastewaters might
increase the metal concentrations in these
areas because a wide variety of house-
hold products, such as cleaning materials,
toothpaste and cosmetics, can contain
trace concentrations of Cu, Zn, Pb and Fe
(Sörme & Lagerkvist, 2002; Alloway,
2013), and only large urban areas around
Lake Titicaca have proper sewage treat-
ment plants (Revollo et al., 2003). None-
theless, metals are refractive to biological
degradation and are also present in natu-
ral systems receiving the effluents from
sewage treatment plants (Maceda-Veiga

et al., 2012). In addition, the run-off from
adjacent agricultural areas might also be
responsible for Cu, Zn, Cd, Hg and Pb
release since these are often present in
fertilizers or pesticides (Alloway, 2013).
However, our study was unable to detect
any other evidence of agricultural prac-
tices (e.g. eutrophication) despite the fact
that excessive use of nitrogenous com-
pounds as fertilizers is a growing concern
at Lake Titicaca (Fontúrbel, 2008), possi-
bly because of the instability of these
compounds in the environment compared
to metals and/or the dilution associated
with the rain that fell during some of our
surveys.
The contribution of the main tribu-
taries to metal pollution in Lake Titicaca
was better reflected by the spatial varia-
tion in metal concentration in sediments.
Sediments enriched in organic matter act
as good sinks for metals compared to
water because humic substances present
in sediments create metal complexes
(Rognerud & Fjeld, 2001; Alloway,
2013). An increase in organic matter con-
tent was particularly evident in the cur-
rent study at the discharge site of the
River Ramis, which in turn would ex-
Metal concentrations in Lake Titicaca
plain the high metal concentrations ob-
served at this site. The high river flow
conditions two weeks before our survey
was performed might also have increased
the release of sediments from upstream
areas down to the mouth. In addition the
low pH observed at these sampling sites
might be due to the decomposition of the
organic matter in sediments or the sew-
age discharge from the mining activities
(e.g. Galán et al., 2003 in Spain or Alpers
et al., 2005 in California). Because the
concentration of some of the metals ana-
lysed was moderately high, with some
exceeding the safety thresholds estab-
lished by international legislation (i.e. Pb
in water), and as the long-term release of
metals from contaminated sediments is
well known, negative effects are ex-
pected in aquatic and terrestrial organ-
isms as long as metals are able to incor-
porate into the biota (MacDonald et al.,
2000).
Metal bioaccumulation in fish
The metal concentrations in all fish
species collected exceeded the threshold
established by the European (EC, 2001,
2006) and American (FAO/WHO, 1998)
95
 Chapter 2
legislation with the exception of Pb, Co
and Fe. This finding is consistent with
Gammons et al. (2006) and IMARPE and
FONCHIP (2010), who also detected Hg
levels in O. bonariensis and T. rivulatus
above the safety threshold at the mouth
of the Ramis River and at other locations
around the lake. Although the cross-study
comparison of metal bioaccumulation in
fish species from other water ecosystems
is challenging, the mean concentrations
of Cu Zn, Hg, Co and Fe observed in the
current study were higher than those re-
ported in the liver of Abramis brama for
-1
Cu (49.07 µg g dw) and in muscle for
Hg (0.09 µg g-1 dw) in Lake Balanton
(Farkas et al., 2002), in the liver of Labeo
-1
rohita for Cu (1.03 µg g dw), Zn (1.03
-1
µg g dw) and in muscle for Hg (0.07 µg
g-1 dw) in Lake of Bhopal (Malik et al.,
2010) or in the liver of Tilapia nilotica
-1
for Cu (7.5 µg g dw), Zn (2.28 µg g
-1
dw), Co (0.31 µg g dw) and Fe (4.58 µg
g-1 dw) in Lake Nasser (Rashed, 2001).
However, Cd and Pb concentrations were
lower than those reported in the liver of
-1
Abramis brama for Cd (1.72 µg g dw)
and Pb (2.22 µg g-1 dw) from Lake
Balanton (Farkas et al., 2002), the liver
96
of Labeo rohita for Cd (0.52 µg g-1 dw)
and Pb (1.26 µg g-1 dw) in Lake of Bho-
pal (Malik et al., 2010) or even in muscle
of Lates marie for Cd (0.25 µg g-1 dw)
and Pb (4.96 µg g-1 dw) in Lake Tanga-
nyika (Chale, 2002).
The bioaccumulation pattern of all
elements in fish at Lake Titicaca was
mainly associated with tissue and sentinel
species effects. Of the tissues, the liver
was confirmed as the best metal deposi-
tory for all elements determined with the
exception of Hg (Farkas et al., 2002;
Gammons et al., 2006; Çogun et al.,
2006). This could be because Hg has a
high affinity for the sulphydryl groups of
proteins (Miller et al., 1992; Mason et
al., 2000), as well as because the muscle
contains low levels of binding proteins
(i.e. metallothioneins) compared to the
liver (Roméo et al., 1999; Karadede et
-1
al., 2004; Kojadinovic et al., 2007). In
terms of species, our results corroborated
our predictions and the highest metal
concentrations were observed in O. lu-
teus, O. agassii and T. rivulatus, possibly
because they live close to the sediments
(Roméo et al., 1999; Agarwal et al.,
2007; Yilmaz et al., 2007). However,

pelagic species such as O. bonariensis
often showed a similar metal bioaccumu-
lation pattern (e.g. Hg) to native ben-
thopelagic species, possibly reflecting the
different metabolic dynamics of essential
and non-essential elements in fish species
coupled with the influence of other indi-
vidual traits.
The spatial variation in metal con-
centrations in fish showed that the levels
of non-essential elements (Cd, Hg, Pb) in
fish were generally more consistent with
the peaks in metal concentrations in the
environment than essential elements. In
this regard, the most polluted locations
were Barco-Chucuito (located inside
Puno Bay) and, as predicted, the mouth
of Ramis River. The considerable varia-
tion in the concentration of essential ele-
ments could be due to the greater regula-
tion of the uptake and excretion of essen-
tial elements compared to that of non-
essential elements (Kojadinovic et al.,
2007). However, our results also showed
that essential elements (e.g. Cu) can bio-
accumulate when environmental concen-
trations are high, thereby becoming a
pollution source, just as non-essential
elements do (Chale, 2002; Farkas et al.,
Metal concentrations in Lake Titicaca
2002; Demirak et al., 2006; Maceda-
Veiga et al., 2013). Likewise, an increase
in essential elements in a given individ-
ual from a polluted location might also be
attributed to the participation of Cu, Zn,
Co and Fe in metabolic functions (e.g.
detoxification processes) to cope with
pollution effects (e.g. oxidative stress)
(Bervoets & Blust, 2003; Çogun et al.,
2006). Because non-essential elements
are better tracers of pollution than essen-
tials and because all species showed in-
creased Hg concentration at the most pol-
luted location regardless of their swim-
ming behaviour (i.e. pelagic/benthonic),
our results suggest that all species, in-
cluding the apparent highly mobile O.
bonariensis, are resident in this area, al-
though the home range of all four species
is currently unknown. Nonetheless, the
metal bioaccumulation pattern observed
in all four fish species should be consid-
ered mostly poorly related to the environ-
mental metal concentrations as suggested
by the pair-wise correlation analysis be-
tween PCA scores.
An additional explanation for the
sometimes similar bioaccumulation pat-
tern observed in pelagic and benthope-
97
 Chapter 2
lagic species is that all four species can
feed on bottom drift invertebrates and
may ingest sediment or suspended or-
ganic matter when they feed (Lauzanne,
1982; Parenti, 1984; Loubens, 1989; Vila
et al., 2007). Differences in the relative
proportion of items in fish diets with a
variable metal content could have deter-
mined the observed metal concentration
in fish species (i.e. killifish/catfish)
(Mason et al., 2000; Kojadinovic et al.,
2007; Agah et al., 2009; Maceda-Veiga
et al., 2013). In the case of O. bonarien-
sis the high concentration of Hg observed
in some locations relative to other species
could also be attributed to its high trophic
position (i.e. biomagnification) (Mason et
al., 2000; Kojadinovic et al., 2007; Agah
et al., 2009). In this regard, the mean
length of O. bonariensis individuals ex-
amined in the current study (217.8 mm)
suggests that they are mainly piscivorous
(above 200 mm, see Vaux et al., 1988; or
above 90 mm, Monroy et al., 2014b).
Likewise, a high metal concentration in
O. luteus could be attributed to the fact
that this fish species occasionally feeds
on fish juveniles (Monroy et al., 2014b).
Alternatively, the more active metabo-
98
lism of pelagic species could also lead to
an increase in metal uptake compared to
benthopelagic fish (Kojadinovic et al.,
2007). However, this hypothesis seems
unlikely owing to the fact that the metal
bioaccumulation in pelagic species was
not consistently higher than that in the
benthopelagic species across the study
area. Species occurrence in deep and
poorly oxygenated waters, as is the case
for O. bonariensis, is also likely to in-
crease Hg bioaccumulation since these
environmental conditions favour methy-
lation and hence Hg accumulation
(Gammons et al., 2006; Kojadinovic et
al., 2007).
Individual traits made a minor con-
tribution towards explaining the variation
in the concentration of most elements in
all four fish species. Previous studies
have suggested that age and fish size
could increase metal accumulation due to
the long-term exposure of individuals to
pollution (Gammons et al., 2006), the
metabolic activity of individuals (Canli &
Atli, 2003), ontogenetic changes in fish
diet (Mason et al., 2000) or a reduction in
the detoxification ability of old fish
(Kojadinovic et al., 2007). The concen-

trations of these essential elements could
be associated with differing metabolic
activities or the different gonad develop-
mental stage of males and females
(Kojadinovic et al., 2007). As gonads
were not weighed in this study, the effect
of gonad development cannot be ruled
out, although an apparently similar devel-
opmental stage was observed in the field.
Metal bioaccumulation in fish in Lake
Titicaca may also be affected by factors
other than those mentioned above, in-
cluding metal speciation (Ivanciuc et al.,
2006; Adkikari et al., 2009), the rate of
export of metals through other food web
compartments (Amundsen et al., 1997;
Bervoets & Blust, 2003; Kojadinovic et
al., 2007) and species physiology, and
therefore this study cannot explain the
likely mechanisms associated with the
bioaccumulation pattern we observed in
these fish species. Finally, we note that
although the present study focused on
metals, exposure to other toxic com-
pounds present in sewage waters (e.g.
drugs, pesticides) could also have been
responsible for the observed bioaccumu-
lation patterns.
Metal concentrations in Lake Titicaca
Concluding remarks and management
actions
This study demonstrates contrasting
metal bioaccumulation patterns in water,
sediments and fish, suggesting caution
when inferring the risk posed by metal
pollution based on analysis of a single
ecosystem compartment. Most elements
in fish and Pb in water were above the
safety threshold, illustrating the large-
scale metal pollution at Lake Titicaca.
Given that both humans and wildlife (e.g.
piscivorous birds and amphibians) de-
pend on the fisheries resources in the lake
and high Hg levels in blood of humans
living close to mining areas have been
already reported (Hurtado et al., 2006),
we would encourage the authorities to
enforce legislation to reduce metal pollu-
tion in Lake Titicaca. Limiting species
consumption would be a better manage-
ment strategy than the prohibition of fish-
ing in certain areas since pollution by
metals seems to extend across the study
area. Further studies should focus on the
ecology of fish species, metal speciation
and metal export from contaminated ar-
eas through other components of the food
web to increase our knowledge about the
99
 Chapter 2

likely mechanisms behind the observed ship (Para-Tox project no 327941).

metal bioaccumulation. As the species
affected have a high conservation value,
the evaluation of the risk posed by pollu-
tion in Lake Titicaca to fish health is also
mandatory.
SUPPLEMENTARY MATERIAL
Supplementary material is shown in
the Tables S1, S2 and S3.

ACKNOWLEDGMENTS
This work and protocols were
funded and approved by the Spanish
Agency for International Cooperation
and Development (AECID) and the De-
partment of Production of Peru trough
rhe Hispanic-Peruvian Cooperation Pro-
gram (PCHP) (projects no FBG305168
and FBG 306311). We thank O. Flores,
E. Yujra and the rest of the crew at the
Binational Special Project of Lake Titi-
caca (PELT), the Sea of Peru Institute
(IMARPE) and the Lake Titicaca Agency
(ALT), for field and laboratory assis-
tance. We also thank to Dr. Carsten
Müller for useful insights on metal pollu-
tion in lakes, Toffa Evans from the Lan-
guage Service at the University of Barce-
lona for the English revision and three
anonymous referees for valuable com-
ments on the manuscript. AMV is cur-
rently funded by a Marie Curie Fellow-

100
 Metal concentrations in Lake Titicaca

Table S1. Sampling sites with details of the geographical location and classified according to the
source of pollution (0= non-contaminated, 1= urban and industrial sewage waters, 2= mining,
3= residual waters from ancient oil extraction, 4= tourism) based on own analyses1, personal
communications (PELT2) or previous studies (see Northcote et al., 19893; UNEP, 19964; Gam-
mons et al., 20065; Fontúrbel, 20086; Ministerio de la Producción & AECID, 20087; IMARPE
& FONCHIP, 20108).

Location UTM x UTM y Source of pollution Literature evidence

R1. Huencalla Bay 421389 8240932 0 1, 2

R2. Tamán Bay 413150 8272466 0 1, 2

L1. Mouth of Ilave River 453200 8228489 1 8

L2. Barco –Chucuito 404380 8243906 1, 4 3, 8

L3. Mouth of Ramis River 418134 8304874 1, 2 4, 5, 7, 8

L4. Pusi 404900 8288919 3 4, 7

L5. Mouth of Coata River 467731 8203022 1, 2 3, 4

L6. Mohokachi 499441 8202696 1 6

L7. Inka Chaka Bay 490817 8218142 1, 4 6

Table S2. Metal recovery rates (%) for sediment and fish samples from Lake Titicaca.

Compartment Cu Zn Cd Hg Pb Co Fe

Sediment 95 101 90 90 102 90 106

Fish 92 90 93 90 90 - 90

101
 Chapter 2

Table S3. Loadings for axes PC1env, PC2env, PC1bio and PC2bio according to the two PCAs built
with the metal concentrations in the environment (PCenv) and fish (PCbio).

Metal PC1env PC2env PC1bio PC2bio

Cu 0.55 0.31 1.85 1.21
Zn 0.88 0.42 0.46 0.13
Cd 1.17 0.35 1.27 -0.13
Hg 1.82 -0.66 -0.39 0.14
Pb 0.63 0.17 1.23 -1.90
Co 0.25 0.34 1.00 0.33
Fe 0.34 0.14 0.44 -0.14

102
Metal concentrations in Lake Titicaca

103
 CHAPTER 3
Blood analyses in three fish species indicate the presence of
cytotoxic and genotoxic compounds in Lake Titicaca

Mario Monroy1, Alberto Maceda-Veiga2, Oriol Cano1, Adolfo de Sostoa1

1
Department of Animal Biology and Biodiversity Research Institute (IRBIO), Uni-
versity of Barcelona, E-08028 Barcelona, Spain.
2
Cardiff School of Biosciences, Cardiff University, CF10 3AX, Cardiff, United
Kingdom.
 Chapter 3

RESUMEN
Los ecosistemas de agua dulce han experimentado una larga historia de impactos antrópi-
cos incluyendo las descargas de aguas residuales las cuales amenazan la vida salvaje y la sa-
lud humana. El entendimiento de la respuesta fisiológica de las especies frente a la polución
es crucial para desarrollar estrategias efectivas de manejo y frenar la pérdida de biodiversidad.
En el presente estudio, fue explorada la idoneidad de marcadores letales y no letales (variables
histológicas y hematológicas, respectivamente), para evaluar el impacto de la descarga de
aguas residuales sobre tres especies nativas de peces (Orestias luteus, Orestias agassii y
Trichmycterus rivulatus) en el lago Titicaca. Estas especies mostraron los más altos valores
del índice hepatosomático (HSI), incrementando el porcentaje de eritrocitos con anormali-
dades nucleares (ENAs), inmaduros (IE) y cariorreicos (KE) así como también monocitosis,
principalmente en el sitio de descarga del contaminado río Ramis. Todos los patrones de los
biomarcadores sanguíneos fueron débilmente asociados a las características de cada individuo,
siendo la especie y la localidad los factores más significativos para su variabilidad. Adicional-
mente, varias relaciones positivas fueron registradas entre las variables hematológicas y las de
calidad del agua, descritas principalmente por la conductividad, oxígeno disuelto y los nitritos,
así como también, por algunos metales pesados tales como Zn, Cd, Hg y Co. Evaluaciones
histopatológicas evidenciaron la degradación de las células debido a la alta presencia de
núcleos periféricos y vacuolización de los hepatocitos, confirmando los efectos adversos. Por
lo tanto, este estudio sugiere la conveniencia de usar la hematología como un procedimiento
de muestreo no letal para monitorear la salud del ecosistema, siendo ambas especies de los
bentopelágicos Orestias, más sensibles a la contaminación por metales que el bentónico T.
rivulatus.

Palabras clave
Muestreo no letal, conteo diferencial de leucocitos, genotoxicidad, citotoxicidad, histología
del hígado.

106
 Blood biomarkers of pollution of fish species from Lake Titicaca

ABSTRACT
Freshwater ecosystems have experienced a long history of anthropogenic impacts including
sewage discharges that threaten wildlife and human health. Understanding the physiological
response of fish species to pollution is crucial to develop effective management strategies to
stem biodiversity loss. In the current study, it was explored the suitability of lethal and non-
lethal markers, histological and haematological variables, respectively, to assess the impact of
sewage discharges on three native fish species (Orestias luteus, Orestias agassii, and Trich-
mycterus rivulatus) in Lake Titicaca. These species showed higher values of hepatosomatic
index (HSI), increasing the percentage of eythrocytic nuclear abnormalities (ENAs), immature
(IE) and karyorheic erythrocytes (KE) and monocytosis mainly at the discharge site of the
polluted Ramis River. All blood biomarkers pattern were weakly associated to individual
traits being specie and location the most significant contributors to their variability. In addi-
tion, several positive relationships were registered between both haematological and water
quality variables described mainly by conductivity, dissolved oxygen and nitrites as well as
some heavy metals such as Zn, Cd, Hg and Co. Histopathological evaluations evidenced cell
degradation due to higher presence of peripheral nuclei and vacuolization in hepatocytes con-
firming adverse effects. Therefore, this study suggests the suitability of haematology as non-
lethal sampling procedure to monitor ecosystem health, being both benthopelagic Orestias
species more sensitive to metal pollution than the benthic T. rivulatus.

Keywords
Non-lethal sampling, differential leucocyte count, genotoxicity, citotoxicity, liver histology.

107
 Chapter 3
INTRODUCTION
he input of sewage discharges
is still a leading cause of
habitat degradation in fresh-
water ecosystems (Frenzilli et
al., 2004; Dudgeon et al., 2006). Sewage
discharges contain a mixture of xenobiotics
(e.g. organochlorines, drugs, pesticides,
and heavy metals) that sewage treatments
plants are unable to eliminate completely
which could generate adverse physiological
effects and changes in aquatic organism
life-history traits (Faller et al., 2003;
Damásio et al., 2008). Such effects could
be magnified by the direct exposition to
sewage that has not been treated (e.g. run-
off from agricultural areas, atmospheric
deposition, illegal spill-offs), mainly in less
-developed countries (Dudgeon et al.,
2006). However, these changes are difficult
to predict due to connections must be de-
fined between external levels of exposure
to contaminant, internal levels of contami-
nation, and the effects generated in the or-
ganism at different biological levels (i.e.
molecular, subcellular, cellular/tissue)
(Pacheco & Santos, 2002; de Castilhos,
2012). In this context, recognizing early-
warning signals or biomarkers, before that
108
potential destructive process generated by
xenobiotics becomes irreversible is crucial
to have a greater understanding of species
tolerance to environmental pollution
(Maceda-Veiga et al., 2010, de Castilhos,
2012).
Traditionally, the methods to identify
the effects of contamination on fauna imply
the sacrifice of animals but the current con-
servation status of many species and ethics
argue the need for validate non-lethal diag-
nostic tools (Smith & Darwall, 2006; Ma-
ceda-Veiga et al., 2013). In this sense,
blood markers are portrayed as good indi-
cators of physiological dysfunctions (e.g.
metabolic changes, interactions of toxic
metals with oxygen) in aquatic species
(Houston, 1997; Pacheco & Santos, 2002;
Elahee & Bhagwant, 2007). The cheapest
but highly informative option to obtain in-
formation on individuals’ health from
blood is through the examination of blood
films (Canfield, 1998). These blood films
consist in the evaluation of cell biometry,
the leukocyte differential count, and the
nuclear abnormalities (ENAs) (Canfield,
1998). Such ENAs are mainly due to xeno-
biotics exposition which can generate by-
products (i.e. oxyradicals) linking to mac-
 Blood biomarkers of pollution of fish species from Lake Titicaca
romolecules (e.g. proteins, DNA) that
could result in cell injury and diseases eas-
ily recognised in blood cells (Pacheco &
Santos, 2002; Bolognesi et al., 2006). Ac-
cording to the form, the most common
ENAs have been classified as notched
(NE), lobed (LE) and micronuclei (MN)
(Ayllon & Garcia-Vazquez, 2000; Pacheco
& Santos, 2002; Guilherme et al., 2008).
However, the use of blood biomarkers
should be complemented with other bio-
markers (e.g. histopathology or organoso-
matic indices) to corroborate the adverse
effects at different levels (i.e. cellular, tis-
sue, or organ) (Van der Oost et al., 2003;
Neff & Cargnelli, 2004; Maceda-Veiga et
al., 2013).
Fish are reliable indicators of xenobi-
otic pollution because they can concentrate
pollutants in their tissues, they occupy vari-
ous trophic levels, and they are key species
in trophic chains (Agarwal et al., 2007;
Jorgensen et al., 2010). Moreover, fish re-
spond quickly to environmental pollutants
reflecting long-term impacts (in compari-
son with macroinvertebrates and diatoms)
which make them in good sentinels for
evaluating the risk for human populations
living in polluted areas (Bolognesi et al.,
2006; Benejam et al., 2008; Jorgensen et
al., 2010).
The situation of fish species at Lake
Titicaca exemplifies the strong pressure
that occur over freshwater fauna worldwide
generated by several anthropogenic pertur-
bations such as water pollution, habitat
degradation, overexploitation of the fishing
resources, and introduction of exotic spe-
cies (Modenutti et al., 1998; Vila et al.,
2007; Monroy et al., 2014a, 2014b). This
scenario could be the cause of the diminu-
tion of 45% in fish biomass in the last 32
years (see Vila et al., 2007; Aliaga &
Segura, 2013) and that several species from
the lake are catalogued as threatened (Van
Damme et al., 2009). Thus, we selected
three native species from Lake Titicaca as
sentinels, the benthopelagic killifishes
Orestias agassii and Orestias luteus, and
the benthonic catfish Trichomycterus rivu-
latus. Specifically, the current study ex-
plores the suitability of blood films as non-
lethal sampling procedure to obtain reliable
information on the health status of fish spe-
cies in Lake Titicaca. As far as we are
aware, this is the first time that a blood cell
profile, the hepatosomatic index and histol-
ogy have been studied in any fish specie at
109
 Chapter 3
Lake Titicaca and related with pollution.
Since the main rivers have been portrayed
as the main sources of metal pollution at
Lake Titicaca (see Monroy et al., 2014a),
and both killifish are more sensible to their
bioaccumulation, we expected that indi-
viduals of these species captured at the dis-
charge sites would have the highest
genotoxic and cytotoxic effects. Such ef-
fects could generate histopathological
changes that they will drive to different
responses by the immune system (Houston,
1997; Davis et al., 2008; Cazenave et al.,
2009) reflecting the impact of xenobiotic
exposition in the native fauna.
MATERIAL AND METHODS
Study Area
Lake Titicaca is one of the biggest
lakes in the world and the largest in South
America covering a surface area of ap-
proximately 8559 km2. It is located at an
altitude of 3810 m in the south central An-
des (Vila et al., 2007; Miller et al., 2010)
(Fig. 1).
The lake is divided in two sub-basins,
the “Lago Mayor” and the “Lago Menor”
with a maximum depth of 280 m and 40 m,
respectively. This lake is characterised by
110
cool water and high salinity due to the ge-
ology of the basin. The water chemistry is
relative constant across all year except the
rainy period between December and March
(Vila et al., 2007).
Whilst Lake Titicaca has in general
oligotrophic waters, several pollution prob-
lems are localized in some affluent rivers
and specific bays, mainly in closed areas
with low water circulation (Paredes &
Gonfiantini, 1999). According to a previ-
ous metal pollution study (see Monroy et
al., 2014a), we sampled in two non-
polluted areas (R1, R2) and seven known
polluted locations (L1, L2, L3, L4, L5, L6
and L7) across the main fishery areas of
Lake Titicaca in November 2010 (Fig. 1).
These sampling sites were selected based
on an extensive literature review, inter-
views with local communities and our per-
sonal observations (see Monroy et al.,
2014a). Note that although Monroy et al.
(2014a) determined bioaccumulation in
four fish species in the present study we
only use 3 species (O. luteus, O. agassii,
and T. rivulatus) due to the difficulty to
capture living Odontesthes bonariensis
individuals.
 Blood biomarkers of pollution of fish species from Lake Titicaca

Fig. 1. Map of the sampling sites which include two named baseline locations (R1: Huencalla
Bay and R2: Tamán Bay) and the 7 known polluted sites (L1: Mouth of Ilave River, L2: Barco-
Chucuito, L3: Mouth of Ramis River, L4: Pusi, L5: Mouth of Coata River, L6: Mohokachi and
L7: Inka Chaka Bay) in Lake Titicaca. Original map provided by National Aeronautics and
Space Administration (NASA®).

Environmental parameters hardness (°CH).

Temperature (°T, °C), pH, conductiv-
ity (mS cm-1) and dissolved oxygen (DO2,
Fish sampling
mg l-1) were measured in situ in each local-
Fish samples were collected in No-
ity using digital YSI® 553 multi-probe
vember 2010 trough gillnets ranging from
system, whereas a colorimetric test kit VI-
10 to 30 mm length, set overnight in each
SOCOLOR® was used for ammonia (NH3,
bay (Table 1 and Fig.1). From each sam-
mg l-1), nitrites (NO2, mg l-1), nitrates
pling site and species, 20 individuals were
(NO3, mg l-1), phosphates (PO4, mg l-1),
anaesthetized using MS-222 (3-
general hardness (°GH) and carbonate

111
 Chapter 3
aminobenzoic acid ethyl ether, Sigma-
Aldrich®). Peripheral blood was collected
by caudal vein puncture using heparinised
syringes 25G. Blood samples were kept at
4°C in polystyrene boxes with ice until
analyses at PELT laboratory in Chucuito,
Peru.
Fish were measured to the nearest mm
(total length, LT) and total weight (WT) and
liver weight (WL) were recorded to mg. As
LT and WT were strongly correlated (r>0.9)
LT was considered in this study as a more
direct measure of fish size than weight.
Additionally, hepato-somatic index (HSI)
was obtained by using the formula HSI=
-1
(WL* WT )*100.
Haematological measurement
A drop of homogenized blood of each
individual was smeared on microscopic
slide pre-cleaned in 70% alcohol. Each
blood film was air-dried, fixed and stained
with Diff-Quick® stain. Differential leuko-
cytes counts (DLCs) were performed
counting trough the microscope 100 leuko-
cytes per slide at 1000x magnification. One
thousand erythrocytes with the same mag-
nification were observed. Immature (IE)
and senescent erythrocytes (KE) percent-
112
ages were differentiated morphologically
according to (Takashima & Hibiya, 1995),
as well the erythrocytes with nuclear ab-
normalities (ENAs) according to Houston
(1997), Pacheco and Santos (2002); Guil-
herme et al. (2008) (Fig. 2). Additionally,
ten fields per slide were photographed and
examined at 40x magnification in order to
measure nuclear and cellular length of 100
erythrocytes (per slide) using Sigma Scan
Pro® software. The nuclear cytoplasmatic
ratio (NCR) was calculated to infer the ma-
turity state of erythrocytes (Takashima &
Hibiya, 1995), dividing the diameter of the
nucleus and the total cell lenght using the
minor cell axis.
Histopathological evaluation
Ten individuals per specie at each lo-
cation were euthanized with exposure to
overdose of MS-222. Specimens were dis-
sected and a small liver section was fixed
in 10% neutral buffered formalin. Samples
of liver were dehydrated with ethanol,
cleared in xylene and embedded in paraffin
wax. Three sections of 5 µm thickness
were cut at different levels of the tissue and
stained with haematoxylin and eosin on
slides. One hundred hepatocytes per slide
 Blood biomarkers of pollution of fish species from Lake Titicaca

A B C D

E F G H
Fig. 2. Morphological features of haematological variables (40x magnification) studied in Lake
Titicaca. The blood profile is composed by erythrocytes (A) immature, (B) lobbed, (C) notched,
(D) micronuclei, (E) senescent, as well leukocytes as (F) neutrophils, (G) lymphocytes and (H)
monocytes.
spread in 3 fields were counted and exam- no nuclei as previously described in Cabal-
ined in the microscopy to 1000x magnifica- lero et al. (2004).
tion. The structure of liver tissue was
evaluated semi-quantitatively by ranking to Ethical note
each hepatocyte a five-graded category: 0= This study was authorized by Depart-
centred cellular nuclei and pink cytoplasm; ment of Production of Peru as well as by
1= peripheral nuclei and pink cytoplasm; the responsible entities to conservation and
2= centred nuclei and white cytoplasm; 3= management of the natural resources in
peripheral nuclei and white cytoplasm; 4= Lake Titicaca: ALT, IMARPE and PELT.
Blood extraction protocols were evaluated

113
 Chapter 3
and validated in other freshwater fish spe-
cies of similar size (i.e. Barbus haasi and
Squalius laietanus) by the Autonomous
Government of Catalonia (Departament de
Medi Ambient i Habitatge - Generalitat de
Catalunya).
Statistical analyses
Haematological (ENAs, LE, NE, MN,
IE, KE, NCR, monocytes, neutrophyls,
lymphocytes, HSI) and biological (LT,
weight, HSI) variables were tested for nor-
mality and homogeneity of variances using
Shapiro-Wilk and Levenne’s test, respec-
tively. Parametric and non-parametric sta-
tistics were applied accordingly to the ful-
filment of above mentioned assumptions.
Differences between biological variables
by gender and sampling location were ob-
tained trough two-way ANOVA followed
by Tukey HSD post-hoc test for pair–site
comparisons. Similarly, differences by lo-
cations were determined in both HSI and
haematological variables trough one-way
ANOVA. Otherwise, differences by sam-
pling locations between erythrocyte meas-
urements and between histopathological
categorization were obtained applying
Kruskal-Wallis followed by Wilcoxon post
114
-hoc test for pair-site comparisons. To ex-
plore the relative influence of fish attrib-
utes on blood markers, sampling site, spe-
cies, age, gender and LT (assuming quasi-
binomial error distribution) as well HSI
and NCR (assuming Gaussian family) were
incorporated in a general lineal model
(GLM) using the “cbind” function. The
best models were selected using a manual
stepwise backward deletion of non-
significant terms from the full global mod-
els containing all fixed factors and interac-
tions (i.e. including covariate effects). The
significance of each factor was checked
using an F-test (“Anova” function in R).
Detailed descriptive statistic of metal
bioaccumulation in water, sediments and
fish species are included in Monroy et al.,
2014a). Indications appear trough the
manuscript wherever it is necessary. In or-
der to describe the main sources of varia-
tion in heavy metals found in water (Cu,
Zn, Cd, Pb, Co, Fe) a principal component
analyses (PCA) was applied to environ-
mental variables (°T, pH, conductivity,
DO2, NH3, NO2, NO3, PO4, °GH, °CH).
The number of PCA axes was determined
by Kaiser’s rule (eigenvalue ≥ 1) and the
“varimax” rotation was incorporated to
 Blood biomarkers of pollution of fish species from Lake Titicaca
increase the interpretation of axes
(Legendre & Legendre, 1998). The rela-
tionship of blood variables and HSI with
respect to environmental variables
(described by the synthetic gradients PC1
and PC2) and heavy metals found in fish
was explored by using Pearson correlation
coefficient (r). All statistical analyses were
performed using the R package (R Devel-
opment Core Team, 2012) and the library
car with a level of significance of p= 0.05.
Model assumptions were checked by ex-
amining standardised residuals using qq
plots and plotting fitted versus predicted
values.
RESULTS
Haematological biomarkers and relation
with biological and environmental vari-
ables
Principal component analysis grouped
in two axes all environmental variables
measured (Table 1). PC1 accounted for
35% of the variation and was mainly re-
lated with Cu, Zn, Cd, Pb and Co while
PC2 accounted for 23% of variation ex-
plaining mainly the dissolved oxygen and
nitrites (Table 1).
The fish used for haematological
analyses spanned a wide range of sizes,
weights and ages (Table 2). In the current
study, females were bigger than males in
LT and WT in all three species (ANOVA,
p<0.05) as well in HSI with the exception
of T. rivulatus (ANOVA, F8, 53=1.88, p=
0.08 for males and F8, 95= 1.93, p= 0.06 for
females) (Table 2 and 3). In particular,
lower LT and WT were obtained in males of
O. luteus from L3 (106±17.84 mm,
31.64±21.56 g, respectively), males of O.
agassii from L4 (101.87± 16.94 mm,
17.35±11.72 g, respectively) and males of
T. rivulatus from baseline location R1
(133±16.97 mm, 22.07±4.72 g, respec-
tively) (Table 3). In the case of HSI, higher
values were obtained in individuals of all
studied species captured in L3 (Tukey test,
p<0.05).
All three fish species increased the
percentage of eythrocytic nuclear abnor-
malities, immature and karyorheic erythro-
cytes mainly at the discharge site of Ramis
River , as well MN frequency in the mouth
of Coata River (Tukey, p<0.05) (Fig. 2). T.
rivulatus evidenced erythrocytes with big-
ger cellular length (13.17±1.31 µm) with
respect to O. luteus and O. agassii
(10.48±0.96 and 10.22±1.11, respectively),
115
 Chapter 3

Table 1. Loadings for axes PC1 and PC2 according to the principal component analysis (PCA)
built with physicochemical variables measured in water. Note that Hg was not included in analy-
ses due to all its values were under detection limits.

Physicochemical variable PC1 PC2

Temperature 0.36 0.54

Dissolved oxygen 0.06 -0.94

Conductivity 0.81 -0.1
pH -0.21 0.5
General hardness -0.28 0.82
Carbonate hardness -0.62 0.32
Ammonia 0.68 0.09
Nitrite 0.31 -0.89
Nitrate 0.52 -0.07
Cu 0.91 -0.11
Zn 0.42 0
Cd 0.91 -0.02
Pb 0.84 -0.19
Co 0.76 -0.01
Fe 0.18 0.6

suggesting physiological differences be- sii.

tween species. Accordingly, NCR showed
statistical differences between locations in
O. luteus (K-W X2= 81.37, df= 8, p<0.001),
2
O. agassii (K-W X = 311.8, df= 8,
2
p<0.001) and T. rivulatus (K-W X = 40.46,
df= 8, p<0.001). Highest NCR values were
found in the mouth of Ilave River in O.
luteus and T. rivulatus individuals as well
as in Barco-Chucuito and Pusi in O. agas-
Differential leukocyte count indicated
an increase in monocytes and a decrease in
lymphocytes in mouth of Ramis River
mainly in O. luteus and O. agassii (Fig. 3).
Additionally, an increase of neutrophils
was observed in Pusi, being considerably
higher in T. rivulatus with respect to Ores-
tias species (ANOVA, p<0.01).
GLMs were used to explore the rela-

116
 Blood biomarkers of pollution of fish species from Lake Titicaca

Table 2. Sample size (N), mean and standard deviation of body length (LT mm) and weight (W g), minimum and maximum range,
and details of gender and ages of the three fish species Orestias luteus (OL), Orestias agassii (OA) and Trichomycterus rivulatus (TR)
tive influence of individual traits (specie,

7+

0
1
0
gender, LT, and age) and sampling location

6+

11

18

0
1
0
in order to explain the variation in blood

5+

52

44

20

0
2
Age (years) biomarkers (Table 4). Specie, location and
4+

12
39
10
the interaction between both variables
7

0
were the most significant contributors to
3+

42
15
4

2
blood biomarkers (Fig. 2 and 3). Individual
2+

53

86
23
17
6

traits seem to have a weak influence on the

1+

12

biomarkers due to GLMs retained gender

5

4
4
6

and age for HSI and micronucleus, and

0+

0
0
0

only gender for neutrophils as significant

16.84-105.18

9.31-103.88

18.26-60.37

19.21-83.83
11.12-61.57

explanatory variables (Table 3).

Min-Max

15.49-87

To analyse further the relationships

between haematological parameters and
both environmental variables and fish
47.77±17.52

61.91±21.07

40.04±11.26

47.85±14.94
31.3±15.26

44.6±18.61

heavy metals Spearman’s correlation were

W
used for haematological parameter analyses in Lake Titicaca.

applied (Table 5). Specifically, HSI was

correlated with water heavy metals for O.
Min-Max

121-192

122-213

luteus (rho= 0.96, p= <0.001) and T. rivu-

90-160

90-197
91-155

90-164

latus (rho= 0.78, p= 0.01) and Hg for O.

agassii (Table 5). Furthermore, red blood
122.62±19.55

136.73±20.93
133.34±16.61

158.58±16.23

168.45±19.25
123.9±16.41

biomarkers (i.e. IE, KE, MN, NE, LE,

LT

ENAs) were also correlated with water

physicochemical parameters and some
heavy metals found in fish as such as Zn
130

104
92

78

50

62
N

and Cd in O. luteus, and Cu and Co in T.

Gender

rivulatus (Table 5). Likewise, the percent-

♂

age of neutrophils was positively corre-

lated with the environmental gradient PC1
Species

OA
OL

TR

117
118
Table 3. Mean values ± standard deviation of total length (LT, mm), body weight (W, g) and hepatosomatic index (HSI) of 3 fish spe-
Chapter 3

cies Orestias luteus (OL), Orestias agassii (OA) and Trichomycterus rivulatus (TR) obtained in polluted areas (L1-L7) and reference
areas (R1 and R2) from Lake Titicaca, according to Monroy et al. (2014a). The letters (a, b, c, d, ab, bc, cd, abc, bcd, abcd) grouped
sites by environmental variable considered homogenous at p<0.05.

Specie Variable Gender R1 R2 L1 L2 L3 L4 L5 L6 L7

♂ 123.83abc 139.08c 110.66ab 129.6bc 106a 123.86bc 125.91bc 134.14c 135.44c
±5.67 ±7.72 ±18.36 ±5.94 ±17.84 ±14.31 ±5.68 ±10.58 ±6.28
LT
♀ 139ab 137 ab 134.87ab 137.06b 111.33a 122.2ab 133.25ab 130.69ab 142.72b
±16 ±13.97 ±15.62 ±9.17 ±26.25 ±8.78 ±10.13 ±21.86 ±9.81
♂ 39.42abc 63.49d 33.82ab 58.93cd 31.64a 46.68abcd 49.49bcd 59.22cd 60.5cd
±6.99 ±9.24 ±17.06 ±9.44 ±21.56 ±15.9 ±6.69 ±5.19 ±8.25
OL W
♀ 57.54ab 63.93ab 57.4ab 73.78b 39.97a 46.62ab 63.31ab 60.16ab 69.06ab
±29.37 ±20.44 ±20 ±17.02 ±32.34 ±9.75 ±17.33 ±21.48 ±12.33
♂ 1.03abc 0.99ab 0.89a 1.53abcd 2.25d 1.6c 1.46bc 1.25abc 1.33abc
±0.23 ±0.13 ±0.18 ±0.47 ±0.9 ±0.21 ±0.41 ±0.23 ±0.28
HSI
♀ 1.24ab 0.96a 1.03a 2.42cd 3.27d 1.95abc 2.37cd 1.95bc 1.74abc
±0.36 ±0.37 ±0.23 ±0.64 ±1.17 ±0.31 ±0.5 ±0.57 ±0.28
♂ 136.75bc 126.5abc 128.66abc 128abc 120.77ab 101.87a 115ab 128abc 156.66c
±16.56 ±2.12 ±16.86 ±11.31 ±19.7 ±16.94 ±10.79 ±15.82 ±4.16
LT
♀ 134.93abc 141.33c 131.14abc 129.16abc 143cd 115.58a 126.27abc 139bc 162.11d
±11.15 ±6.57 ±22 ±19.03 ±19.08 ±18.05 ±27.98 ±21.01 ±8.6
♂ 34.74abc 33.11abc 43.56bc 36.01abc 29.86ab 17.35a 24.1b 31.69b 60.06c
±10.24 ±0.34 ±17.41 ±10.46 ±15.03 ±11.72 ±8.21 ±10.3 ±2.08
OA W
♀ 38.79abc 48.92c 49.16c 37.08abc 49.23c 25.66a 28.87ab 45.16bc 69.83d
±8.89 ±7.82 ±19.18 ±15.76 ±19.5 ±11.59 ±15.54 ±19.14 ±8.34
♂ 0.97a 1.42abc 1.14ab 1.83abc 2.23c 1.63ab 1.38ab 1.69bc 1.33ab
±0.2 ±0.14 ±0.08 ±0.19 ±0.34 ±0.22 ±0.23 ±0.62 ±0.36
HSI
♀ 1.43a 1.56a 1.63ab 2.17c 2.26c 1.63ab 2.14c 2bc 2.23c
±0.27 ±0.18 ±0.52 ±0.34 ±0.23 ±0.29 ±0.46 ±0.46 ±0.32
 Blood biomarkers of pollution of fish species from Lake Titicaca

(rho= 0.86, p= 0.002), whereas PC2 was

141.16ab

161.8abc

40.42abc
27.87ab
±11.21

±10.01
±14.4

±5.47

±0.37

±0.64
0.81a

1.2a
L7 positively related to the percentage of
monocytes (rho=-0.73, p= 0.03) and lym-
176.08bcd
159.75cd

51.3bcde
39.78cd
±11.09

±21.03

±13.16
phocytes (rho= 0.73, p= 0.03). On the

±8.25

±0.15
0.82a

1.15a
±0.4
L6

other hand, neutrophils of O. agassii and

monocytes of T. rivulatus were related
169.57bcd

46.84bcd
172.25d
±17.74

±11.79
50.85d
±16.8

±9.44

±0.43

±0.41
1.03a

1.07a
with Hg (rho= 0.77, p= 0.01 and rho= 0.71,
L5

p= 0.03, respectively), as well neutrophils

of T. rivulatus were related with Co (rho=
140.81ab

154.22ab

27.42ab

35.17ab
±12.78

±13.79

±7.66

±9.66

±0.23

±0.51
1.07a

1.34a
L4

0.7, p= 0.04) (Table 5).

171.75d

183.83d
±16.53

±13.33
49.95d

64.09e
±7.24

±6.36

±0.29

±0.33
1.23a

1.19a

Histopathological evaluation
L3

The liver from the individuals of all

fish species collected at baseline locations
166.12d

183.08d
±16.06

±12.73
45.75d

57.6de
±7.49

±6.16

±0.53

±0.32
0.94a

1.36a
L2

(R1 and R2) showed normal mural archi-

tecture, with hepatocytes presenting in big-
163.55abcd
170.33d

45.5abcd
±14.95
46.81d
±6.94

±6.05

0.83a

1.01a

ger proportion a centred and spherical nu-

±8.7

±0.2

±0.4
L1

clei and a pink homogenous cytoplasm

(Fig 4a and see also Supplementary mate-
169.85bcd
161.16cd

52.83cde
±11.56

±13.71
43.55d
±4.07

±4.57

±0.12

±0.17
0.73a

0.97a
R2

rial: Annex 1). In contrast, hepatocytes

structure from fish collected in polluted
locations presented in bigger proportion
147.75a
±16.97

±14.66
22.07a

31.27a
±4.72

±0.05

±0.08
0.97a
133a

0.9a
R1

±8

peripheral nuclei and pink cytoplasm (see

Supplementary material: Table S1). Addi-
Gender

tionally, hepatic damage symptoms as such

♂

as vacuolization were also detected in pol-

luted locations (Fig. 4). The presence of
Variable
Continue Table 3.

HSI
LT

parasites (Fig. 4f) in polluted sites was also

detected but significance was not reached
Specie

TR

in any of studied species (p>0.05).

119
 Chapter 3

Fig. 2. Frequency of erythrocytes (A) with nuclear abnormalities, ENAs (B) micronuclei, MN,
(C) notched, NE, (D) lobed, LE (E) immature, IE, (F) kariorrheyc (KE) and (G) nuclear cytoplas-
matic ratio, NCR, in the 3 species studied (Orestias luteus, dark grey; Orestias agassii, grey; and
Trichomycterus rivulatus, white) at each sampling site at the Lake Titicaca. Sites were independ-
ently grouped by sites.

120
 Blood biomarkers of pollution of fish species from Lake Titicaca

A B

C D

Fig. 3. Differential leukocyte count (A) monocytes, (B) lymphocytes, (C) neutrophils obtained in
males, (D) neutrophils obtained in females of the 3 species studied (Orestias luteus, dark grey;
Orestias agassii, grey; and Trichomycterus rivulatus, white) at each sampling site at the Lake Titi-
caca. Note that frequency of neutrophils was displayed by gender according to Table 4.

lation in its ichthyic community (Monroy

DISCUSSION
The current study provides the first
empirical evidence of the presence of
genotoxic and cytotoxic compounds in
Lake Titicaca, and illustrates the suitability
of blood smears as a non-lethal diagnostic
procedure in three fish species. The dis-
charging site of tributaries around the Lake
were identified as the main sources of wa-
ter pollution containing heavy metals and
other compounds such as pesticides, sup-
porting previous data on metal bioaccumu-
et al., 2014a).
Haematological parameters as biomarkers
of sewage pollution
The presence of xenobiotics in water
can generate stress in fish species and
stimulate the immunological function of
their leukocytes (Thrall, 2004; Davis et al.,
2008). As the general response observed in
all three fish species exposed to sewage
effluents was monocytosis and only neutro-
philia was detected in the case of the cat-
fish, the present study supports the diag-

121
 Chapter 3

Table 4. Results of the final GLM models showing the influence of some biological variables and
location with haematological parameters such as erythrocytes (with nuclear abnormalities,
ENAs; lobed, LE; notched, NE; micronuclei, MN; immature, IE; karyorheics, KE; nuclear cyto-
plasmatic ratio, NCR) and from leukocytes (monocytes, M; neutrophils, N; and lymphocytes, L)
determined in the three fish species collected in Lake Titicaca. All models were built with quasi-
bionomial family with the exception of HSI and NCR which were obtained with Gaussian family.
Note that only significant variables highlighted in previous full models with all explanatory vari-
ables and interactions (see methods) are shown.

Biomarker Variable SS Df F p-value

Species 26.48 2 91.24 <0.001
Location 42.67 8 36.75 <0.001
Gender 12.95 1 89.29 <0.001
Age 2.97 6 3.41 0.002
HSI Species x location 7.93 16 3.41 <0.001
Species x gender 1.21 2 4.18 0.01
Location x age 16.29 38 2.95 <0.001
Location x gender 49.56 8 2.46 0.01
Residuals 49.88 434
Species 6.01 2 11.17 <0.001
Location 12.27 8 5.7 <0.001
Gender 2.37 1 8.84 0.003
MN
Age 3.52 6 2.18 0.04
Species x location 10.71 16 2.48 0.001
Residuals 129.6 482
Species 574.51 2 215.69 <0.001
Location 173.87 8 16.32 <0.001
NE
Species x location 61.28 16 2.87 <0.001
Residuals 651.23 489
Species 500.42 2 175.12 <0.001
Location 204.22 8 17.86 <0.001
LE
Species x location 68.09 16 2.97 <0.001
Residuals 698.65 489
Species 1005.1 2 238.5 <0.001
Location 369.98 8 21.94 <0.001
ENAs
Species x location 104.7 16 3.1 <0.001
Residuals 1030.37 489

122
 Blood biomarkers of pollution of fish species from Lake Titicaca

Continue Table 4.
Biomarker Variable SS Df F p-value
Location 80.29 8 7.21 <0.001
IE Species x location 76.91 18 3.07 <0.001
Residuals 680.35 489
Species 321.72 2 106.34 <0.001
Location 170.88 8 14.12 <0.001
KE
Species x location 94.09 16 3.88 <0.001
Residuals 739.68 489
Species 2677.78 2 229.39 <0.001
Location 858.91 8 18.39 <0.001
M
Species x location 230.57 16 2.46 0.001
Residuals 2854.08 489
Specie 6809.5 2 1084.38 <0.001
Location 261.3 8 10.4 <0.001
N Gender 47.8 1 15.21 <0.001
Species x location x gender 267.9 42 2.03 <0.001
Residuals 1450.6 462
Location 897.3 8 16.16 <0.001
L Species x location 478.2 18 3.82 <0.001
Residuals 3393.5 489
Species 0.6 2 902.9 <0.001
Location 0.01 8 5.83 <0.001
NCR
Species x location 0.03 16 6.59 <0.001
Residuals 0.16 489

nostic value of monocytes (instead of neu-
trophils alone) in the differential leucocyte
counts as also pointed out by Davis et al
(2008). Particularly, the highest frequen-
cies of monocytes were detected in L3 due
to the high charge of heavy metal by min-
ing activity and in L5 by the xenobiotics
brought by sewage discharges from Juliaca
city industry (Northcote et al., 1989; Mi-
nisterio de la Producción & AECID, 2008).
Moreover, highest frequencies of neutro-
phils were observed in L4 and L7. Such
locations are influenced by residual waters
from ancient oil extraction and contamina-
tion by the bad use of diesel and other sub-
stances derived from oil (e.g. fuel, addi-
tives) used in boats with outboard engine
(Fontúrbel, 2008; Ministerio de la Produc-

123
 Chapter 3

Table 5. Spearman’s correlation coefficients (rho) and p-values (p) between haematological
biomarkers, the synthetic environmental gradients built by principal component analyses (PC1
and PC2) and metal bioaccumulation in Orestias luteus, Orestias agassii and Trichomycterus
rivulatus.
Biomarker Specie Metal/PCA rho p
HSI 1 Hg 0.71 0.03
HSI 1 1 0.96 <0.001
MN 1 1 0.7 0.04
NE 1 1 0.83 0.004
LE 1 1 0.91 0.001
ENAs 1 1 0.83 0.008
KE 1 Zn 0.73 0.03
KE 1 Cd 0.73 0.03
MN 2 2 -0.72 0.02
NE 2 1 0.8 0.01
LE 2 1 0.78 0.01
ENAs 2 1 0.76 0.02
M 2 2 -0.73 0.03
N 2 Hg 0.77 0.01
N 2 1 0.86 0.002
L 2 2 0.73 0.03
HSI 3 1 0.78 0.01
NE 3 Co 0.73 0.03
LE 3 Cu -0.81 0.01
M 3 Hg 0.71 0.03
N 3 Co 0.7 0.04
L 3 2 0.81 0.01

ción & AECID, 2008). quency of karyorrheic erythrocytes (KE)

Besides of chronic stress reported in
fish species, prolonged exposition to oil
and other xenobiotics (e.g. hydrocarbons,
nitrogen-oxygen compounds, pesticides,
heavy metals) produce carcinogenic and
mutagenic effects in erythrocytes (Pacheco
& Santos, 2002; Baršiene et al., 2004,
2006; Vanzella et al., 2007). Such effects
were supported by an increase in the fre-
and ENAs (including NE, LE, MN) mainly
at the discharge sites of the main tributary
rivers (i.e. L3 and L5). Both NE and LE
evidenced a pattern of response similar to
ENAs in all three species studied, but they
were not related with MN frequency as it
was reported by Bolognesi et al. (2006). To
cope cellular anomalies and cytotoxic ef-
fects fish species may respond releasing

124
 Blood biomarkers of pollution of fish species from Lake Titicaca

Fig. 4. Sections of hepatic tissue of 3 species sampled showing: (A) liver with normal hepatic ar-
chitecture and bile duct (O. agassii), (B) swollen parenchyma with vacuolization and capillary
distension (T. rivulatus), (C) acute vacuolization demonstrating (macrovesicular) steatosis (O. lu-
teus), (D) hepatocytes with pyknotic or peripheral nuclei or without nuclei (O. luteus), (E) hepato-
cytes lost their normal polygonal structure with indistinct cell membrane and interstitial si-
yncytial cells (O. agassii), (F) presence of parasites (O. agassii).

125
 Chapter 3
erythrocytes in circulation and increasing
immature erythrocytes (IE), (Mazon et al.,
2002) as such it happens in polluted L3.
Additionally, it is possible that morphology
of erythrocytes may change depending of
the intensity and duration of exposition
(Takashima & Hibiya, 1995). However,
NCR did not show a clear pattern between
polluted and baseline locations in any of
three fish species since these latter had not
higher percentages. On the contrary, the
highest NCR percentages for all three spe-
cies were found in locations L1 and L2,
respectively, indicating erythrocytes with
large nuclei and the supposed presence of
IE and KE (Maceda-Veiga et al., 2010).
Given that this was not concordant with
our results (i.e. higher IE and KE values
were obtained in L3), NCR percentage is
not a good biomarker of sewage pollution
in the species studied.
In the current study, location and spe-
cie were the individual traits that contrib-
uted the most in order to explain the varia-
tion in haematological frequencies. These
differences may are related to variability in
physiological status, bioaccumulation, me-
tabolism, DNA repair mechanisms and
anomalies expressions between species
126
(Frenzilli et al., 2004; Bolognesi et al.,
2006). However, the weak expression of
nuclear anomalies in T. rivulatus could be
due to damaged erythrocytes are easier rec-
ognised for elimination instead of forming
non-lethal lessions (Guilherme et al., 2008;
Singh & Srivastava, 2010). On the other
hand, gender and age have been secondary
factors affecting the MN and neutrophils
frequency as well the HSI values. This
could be related with the metabolic activity
which is usually higher in young individu-
als than in older fish (Canli & Atli, 2003),
differences in bioaccumulation of some
heavy metals with age, and physiological
changes between males and females
(mainly in reproduction) (Kojadinovic et
al., 2007). As gonads of fish species were
not analyzed in the field, the effect of re-
production cannot be ruled out, although
all three species are partial spawners with
no marked breeding season (Vila et al.,
2007).
The effects of sewage waters in eryth-
rocytes and differential leukocyte count
were supported with histopathological
evaluations of liver revealing metabolic
dysfunctions in all three species from pol-
luted locations. Such dysfunctions, in the
 Blood biomarkers of pollution of fish species from Lake Titicaca
current study, consisted in the presence of
pyknotic nucleus and several degenerative
alterations in the architecture of hepato-
cytes (i.e. swollen parenchyma and intersti-
tial syncytial cells) (Almeida et al., 2005;
Lukin et al., 2011; Agamy, 2012). Addi-
tionally, capillary distension was observed,
may indicating an increase in blood flow
due to the high concentrations of sewage
pollutants as heavy metals reported in the
same species (Monroy et al., 2014a). Liver
intracellular degenerative process gener-
ated by the exposition to xenobiotics could
derive in metabolic disorders resulting in
acute vacuolization as it was evidenced in
polluted sites (Pacheco & Santos, 2002;
Almeida et al., 2005; Lukin et al., 2011).
Hepatomegaly, as indicated by the ob-
served increase in HSI in the most polluted
locations (i.e. L3), is related with the above
mentioned histological anomalies (Datta et
al., 2007; Di Giulio & Hinton, 2008). This
variability may be related with mobiliza-
tion or accumulation of fat in liver and the
use of energy reserves to cope pollution
(Di Giulio & Hinton, 2008). However, it is
possible that such accumulation of fat may
also be generated by several factors that
affect Lake Titicaca fish community such
as alterations in the food web or compe-
tence by food items with exotic species
(Vaux et al., 1988; Monroy et al., 2014b).
The physiologic effects detected in
this study were positively related with
some heavy metals such as Cu, Zn, Cd, Hg
and Co according to the species, as well
with specific and non-specific indicators of
pollution (i.e. ammonia and conductivity,
respectively). Such results are in agreement
with the reported by Monroy et al.,
(2014a), generating a big concern since all
above mentioned metals, with the excep-
tion of Co, exceeded the threshold estab-
lished for fish consumption (EC, 2006).
Specifically, Zn and Cd in O. luteus and Co
in T. rivulatus were positively related to the
frequency of KE, meanwhile there was a
negative relationship between Cu and LE
in T. rivulatus, supporting that exposition
of xenobiotics could generate cytotoxic and
genotoxic alterations (Houston et al., 1993;
Kaviraj & Das, 1994; Kori-Siakpere &
Ubogu, 2008). In the case of leukocytes,
Co was positively related to neutrophils
and Hg to monocytes in T. rivulatus, as
well nitrites and dissolved oxygen concen-
trations influenced mostly the increment of
monocytes and lymphocytes in O. agassii
127
 Chapter 3
and T. rivulatus, respectively. These results
suggest that water conditions are producing
stress and triggering the leukocyte action
(Houston. 1997; Das et al., 2004; Strunjak-
Perovic et al., 2009). Additionally, it is
possible that water pollution propitiates the
developing of other organisms (e.g. para-
sites) which could affect differential leuko-
cyte count (Davis et al., 2008; Maceda-
Veiga et al., 2013). However, although
parasites were observed in some liver’ indi-
viduals, external inspection of fish bodies
did not reveal any macroscopic parasite nor
the presence of eosinophils which are asso-
ciated in the defence against pathogen or-
ganisms (Kiesecker, 2002). Despite of this,
high contents of some fish parasites as the
platyhelminthes Diplostomum sp., and
Ligula intestinalis has been reported due to
residual waters mainly from big cities and
animal-raising areas surrounding the lake
(Lauzanne, 1991).
Concluding remarks
This study reflected an integrative vi-
sion of the effects of environmental vari-
ables and metal pollution over the response
of several biomarkers of three fish species
from Lake Titicaca at different biological
levels such as subcellular (i.e. cytogenic
128
effects) and tissular (histopathological ef-
fects). We validated the use of the fre-
quency of ENAs (NE, LE, MN), IE, KE,
and three kinds of leukocytes as non-lethal
biomarkers of sewage pollution being both
Orestias species more sensitive to xenobi-
otics as heavy metals, than T. rivulatus.
However, due to the majority of heavy
metals studied are above of the safety
thresholds (Monroy et al., 2014a) and their
concentrations generate cytotoxic and
genotoxic effects in fish species, we urge to
authorities to strengthen controls and im-
prove legislations to reduce pollution prob-
lems in Lake Titicaca. Further studies
should include a better understanding of
the biochemical responses of species to
xenobiotics considering sensitivity to DNA
damage induction and the temporal vari-
ability of the haematological parameters.
Therefore, the exploration of other haema-
tological or biochemical parameters (e.g.
hematocrit, haemoglobin; serum responses)
could be very important to the evaluation
of fish species health and the conservation
of their communities at Lake Titicaca.
ACKNOWLEDGEMENTS
This study was funded by the Spanish
Agency for International Cooperation and
 Blood biomarkers of pollution of fish species from Lake Titicaca

Development (AECID) and the Department

of Production of Peru trough the Hispanic-
Peruvian Cooperation Program (PCHP).
We thank O. Flores, E. Yujra and the rest
of the crew at the Binational Special Pro-
ject of Lake Titicaca (PELT), the Sea of
Peru Institute (IMARPE) and the Lake Titi-
caca Binational Agency (ALT), for field
assistance in Peru and Bolivia, as well to
the undergrad students S. Vargas and Y.
Castán for laboratory assistance in Barce-
lona. We also thank to E. García-Galea for
useful suggestions on the manuscript.
AMV is currently funded by a Marie Curie
Fellowship (Para-Tox project no 327941).

129
130
SUPPLEMENTARY MATERIAL
Chapter 3

Table S1. Histopathological categorization of livers of Orestias luteus (OL), Orestias agassii (OA) and Trichomycterus rivulatus (TR)
by sampling location considered polluted areas (L1-L7) and reference areas (R1 and R2) at Lake Titicaca according to Monroy et
al. (2014a). Average score (maximum-minimum scores) are reported in 100 hepatocytes per individual (n). Scores were assigned as
follow: (0) centred cellular nuclei and pink cytoplasm, (1) peripheral nuclei and pink cytoplasm, (2) centred nuclei and white cyto-
plasm, (3) peripheral nuclei and white cytoplasm, (4) no nuclei (modified from Caballero et al., 2004). Sites were independently
grouped per species using the letters (a, b, c, d, ab, bc, cd, de, abc, bcd) according to significance (p<0.05).

Categorization Species N R1 R2 L1 L2 L3 L4 L5 L6 L7
29.6ab 42.3cd
58.1e 56.5e 49.2de 19.9a 32.2bc 40.9cd 37bc
OL 10 (37- (49-
(72-39) (69-35) (70-40) (28-10) (39-23) (48-33) (45-28)
20) 33)
56.7bc 63.3c
53.1b 63.8c 60.1bc 39.7a 53.8b 39.3a 56.9bc
0 OA* 10 (67- (68-
(59-46) (69-52) (71-49) (49-30) (63-40) (44-31) (69-50)
46) 59)
51.2cd 55d
65.7e 68.6e 63.4e 32.2a 43.2bc 39.4ab 54.2d
TR 10 (58- (67-
(75-58) (75-61) (69-60) (45-17) (51-29) (46-35) (65-45)
40) 41)
14.9a 19.5abc 15.6ab 27.2cd 29.6d 33.7d 25bcd 10.5a 16.2ab
OL 10
(18-12) (38-13) (19-11) (37-3) (39-2) (39-20) (34-12) (26-3) (24-5)
30.1a 26a
31.6ab 25.8a 25.5a 30.8ab 31.2ab 38b 27.5a
1 OA 10 (35- (36-
(42-23) (34-20) (31-18) (45-23) (36-28) (50-30) (40-21)
25) 20)
25.5ab
18.2a 17.7a 18.5a 26.2ab 27.9b 25ab 24.7ab 20.8ab
TR 10 (34-
(23-9) (25-6) (31-14) (38-6) (34-24) (29-20) (38-19) (39-3)
20)
 Continue Table S1

Categorization Species N R1 R2 L1 L2 L3 L4 L5 L6 L7
11ab 9.6a 16.2abc 21.1bc 9.3a 8a 8.2a 29c 14.4ab
OL* 10
(20-7) (14-2) (22-1) (26-14) (16-5) (14-0) (15-2) (39-12) (35-2)
4.7a 3.2a 4.4a 4.2a 6.1a 5.1a 4.9a 4.6a 3.9a
2 OA 10
(11-0) (7-0) (9-0) (9-0) (9-3) (9-2) (7-1) (8-1) (8-0)
8.9ab 9.8abc 12.2abc 11abc 13.4bc 12abc 15.1c 7.6a 7.5a
TR 10
(13-4) (15-3) (19-5) (16-2) (18-8) (17-8) (19-12) 14-0) (14-1)
10.2a 9.1a 9.6a 10.7a 20.7b 12.1ab 11.5ab 13.3ab 14.2ab
OL 10
(24-2) (28-2) (17-2) (18-2) (42-8) (19-3) (21-2) (21-6) (24-3)
5.1a 3.9a 5.5a 5a 5.3a 3.5a 6.8a 4.3a 3.2a
3 OA 10
(9-1) (8-1) (10-0) (10-0) (9-1) (8-0) (11-3) (7-0) (6-0)
0.3a 1.2a 1.9ab 5.3abc 13.9d 6.5abc 7.9bcd 9.2cd 8bcd
TR* 10
(2-0) (11-0) (7-0) (12-0) (22-4) (15-0) (18-0) (19-0) (16-2)
5.8a 5.3a 9.4ab 11.4ab 20.5b 14ab 14.4ab 10.2ab 12.9ab
OL 10
(17-0) (13-1) (25-0) (32-0) (46-4) (42-2) (39-0) (28-0) (38-0)
5.5a 3.3a 4.5a 4a 18.1b 6.4a 11ab 6.7a 3.6a
4 OA 10
(11-0) (18-0) (12-0) (13-0) (28-2) (24-2) (26-3) (16-0) (7-0)
6.9ab 2.7a 4a 7ab 14.3b 10.4ab 12.6ab 4.3ab 8.7ab
TR 10
(22-2) (7-0) (8-0) (16-1) (45-4) (23-0) (25-1) (10-0) (30-0)

*log+1 transformed
Blood biomarkers of pollution of fish species from Lake Titicaca

131
 CHAPTER 4
Gillnet selectivity in the offshore ichthyic community from Lake Titicaca

Mario Monroy1,2, Alberto Maceda-Veiga3, Eloy Gálvez-López1, Silvia Rodríguez-Climent4,

Nuno Caiola4, Adolfo de Sostoa1,2
1
Department of Animal Biology, University of Barcelona, E-08028 Barcelona, Spain.
2
Biodiversity Research Institute (IRBIO), University of Barcelona, E-08028 Barcelona, Spain.
3
Cardiff School of Biosciences, Cardiff University, CF10 3AX, Cardiff, United Kingdom.
4
Aquatic Ecosystems, IRTA, Sant Carles de la Ràpita, Spain.
 Chapter 4

RESUMEN
Redes agalleras multimalla, bentónicas y pelágicas, fueron colocadas en cinco de las más
importantes localidades de pesca del lago Titicaca para determinar la selectividad del tamaño de
malla en tres profundidades diferentes. Cada red agallera estuvo compuesta de paneles distribui-
dos aleatoriamente, oscilando desde los 5 a los 29 mm de longitud (10 y 40 paneles para las redes
bentónicas y mesopelágicas, respectivamente). Las capturas por unidad de esfuerzo (CPUE) fue-
ron comparadas entre localidades, tipos de red agallera y profundidades, mostrando diferencias
significativas, excepto entre profundidades. La red agallera bentónica estuvo asociada a una más
alta diversidad de especies y la red agallera mesopelágica produjo las más altas capturas por su-
perficie de red (lo cual también incluyó las especies con el más alto valor económico). Adicional-
mente, la selectividad de redes fue estudiada ajustando cinco modelos (“normal fixed,” “normal
scale”, “gamma”, “lognormal” e “inverse Gaussian”) en las ocho especies más abundantes,
usando el método SELECT (“Share Each Length’s Catch Total”). El modelo “normal fixed” obtu-
vo los mejores ajustes en tres especies (Orestias albus Orestias imarpe, y Oncorhynchus mykiss),
sugiriendo que las proporciones de éstas no cambian geométricamente con la talla corporal. Los
mejores modelos para las otras cinco especies fueron “inverse Gaussian” para Odontesthes bona-
riensis y Orestias mulleri, “gamma” para Orestias luteus y “normal scale” para Orestias agassii y
Orestias ispi, principalmente debido a sus características morfológicas. Comparando la longitud
modal prevista para los mejores modelos de ajuste, de acuerdo a la longitud de la madurez sexual
de cada especie, se demostró que la talla ideal de malla para capturar individuos maduros de O.
mykiss, O. bonariensis y O. ispi fue 29, 24 y 8 mm, respectivamente. En las otras especies la se-
lectividad de tallas no pudo ser relacionada con la madurez sexual debido a la falta de informa-
ción o a que las capturas estuvieron principalmente compuestas por individuos inmaduros
(probablemente como consecuencia de la sobrepesca). Para evitar un colapso en las pesquerías
continentales es recomendable implementar restricciones temporales en la pesca de las especies
nativas, limitando los métodos de pesca no selectivos con el tamaño. Adicionalmente, la pesquería
del lago Titicaca debería enfocarse a zonas de aguas abiertas, a partir de los 10 m de profundidad
de la zona litoral en áreas desprovistas de macrófitas, las cuales son usadas como área de cría y
alimentación por muchas especies nativas en peligro.

Palabras clave
Método SELECT, redes multimalla, pesca continental, Orestias sp.

134
 Gillnet selectivity in Lake Titicaca

ABSTRACT
Benthic and mesopelagic multimesh gillnets were set in five of the most important fishing
localities in Lake Titicaca to determine mesh size selectivity at three different depths. Each gillnet
was composed of randomly-distributed panels ranging from 5 to 29 mm bar length (10 and 40
panels for benthic and mesopelagic gillnets, respectively). Catch per unit effort (CPUE) were
compared between localities, gillnet types and depths, showing significant differences, except
among depths. The benthic gillnet was associated with more species-diverse catches, while the
mesopelagic gillnet produced higher catches per net surface (which also included species with
higher economic value). Additionally, gillnet selectivity was studied fitting five models (normal
fixed, normal scale, gamma, lognormal, and inverse Gaussian) in the eight more abundant species
using the SELECT (Share Each Length’s Catch Total) method. The normal fixed model produced
the best fit in three species (Orestias albus, Orestias imarpe, and Oncorhynchus mykiss) suggest-
ing that the proportions of these species do not change geometrically to body size. Best model fit
for the other five species were inverse Gaussian for Odontesthes bonariensis and Orestias mulleri,
gamma for Orestias luteus, and normal scale for Orestias ispi and Orestias agassii, probably due
to their morphological features. Comparing predicted modal lengths of the best-fitting models
with length at sexual maturity of each species, showed that the ideal mesh sizes to catch O.
mykiss, O. bonariensis and O. ispi mature individuals were 29, 24, and 8 mm, respectively. In the
other species size selectivity could not be related to sexual maturity, either due to lack of informa-
tion or to high proportion of immature individuals catches (probably an overfishing consequence).
In order to avoid the collapse of inland fisheries, it is recommended that temporary restrictions on
native species fishing should be implemented, limiting size-unselective fishing methods. Addi-
tionally, fisheries in Lake Titicaca should focus on the offshore zone, away from the 10 m deep
littoral zone of macrophytes’ carpets, which is used as breeding and feeding area by several
threatened species.

Keywords
SELECT method, multimesh nets, inland fisheries, Orestias sp.

135
 Chapter 4
INTRODUCTION
reshwater ecosystems not
only present the highest spe-
cies richness, but also in-
clude the most endangered
ecosystems in the world (Dudgeon et al.,
2006). Although surface freshwater habi-
tats only represent around 0.01% of the
world’s water, they house approximately
40% of the global fish diversity (Gleick,
1996; Helfman, 2007). Habitat degrada-
tion, introduction of alien species, water
contamination, flow modification, and
overfishing are considered the main
threats to the conservation of global fish
fauna (Dudgeon et al., 2006). The interac-
tion of those threat categories has resulted
in fish population declines around the
globe, with a tendency towards higher
biodiversity losses in tropical latitudes
(Groombridge & Jenkins, 2000). Gener-
ally, fisheries are rarely ‘sustainable’, in-
ducing serial depletion and disequilibrium
in ecosystems, which makes their proper
management a key factor to maintain spe-
cies biodiversity (Pauly et al., 2002).
However, due to a homogenization proc-
ess, freshwater fisheries are experiencing
a drastic decline in both their abundance
136
and diversity of their species worldwide.
Rehabilitation of native fish communities
and their fisheries is a daunting task, espe-
cially in ecosystems with a high number
of endemic species. Such is the case of
Lake Titicaca’s ichthyofauna, which, ac-
cording to recent evaluations (Lauzanne,
1991; Sarmiento & Barrera, 2003; Costa,
2003; Vila et al., 2007), is composed of
26 species, including the Andean endemic
genera Orestias and Trichomycterus, and
two introduced species, the rainbow trout
(Oncorhynchus mykiss) and the silverside
(Odontesthes bonariensis). Most of Lake
Titicaca’s endemic fish species are cur-
rently classified as endangered (Van
Damme et al., 2009). A high population
growth around the lake has resulted in
both the pollution of its waters and over-
fishing, which have drastically reduced
fish landings (Vila et al., 2007). In fact,
over the last 30 years annual landings
have decreased 88.6% in introduced spe-
cies and 92.1% in native species
(IMARPE, 2008, 2010; Ministerio de la
Producción & AECID, 2008). This trend
has been accompanied by a reduction of
both mean catching size and spawning
stock biomass (Chura, 2013). In addition,

acoustic evaluations of the ichthyic com-
munity have shown a 45 % reduction in
native fish abundance (Vila et al., 2007;
Aliaga & Segura, 2013).
The incorporation of new technolo-
gies to the traditional fisheries of Lake
Titicaca has contributed to the reduction
of the fishing catch effort, but has also
altered and lowered native fish popula-
tions (Paca et al., 2003), probably due to
their weak regulations and scanty control
by local authorities. Traditional fishing
techniques involve a broad variety of fish-
ing gears with a poorly known size-
selectivity, such as small bag nets (usually
carried out by women and children along
the coast), long-lines, trawls, trammel nets
and gillnets (Paca et al., 2003; Ministerio
de la Producción & AECID, 2008). Spe-
cifically, gillnets are one of the most fre-
quently used fishing methods, since they
are cheap, easy to use, and can be em-
ployed at any depth regardless of bottom
characteristics (Hovgård & Lassen, 2000).
Additionally, the selective nature of gill-
nets allows the assessment of fishing ef-
fects on exploited stocks (McAuley et al.,
2007). The size of fish caught with this
technique can be controlled by modifying
Gillnet selectivity in Lake Titicaca
the gillnet mesh size, which allows the
estimation of retention curves (defined as
the relative probability of a fish of a given
length to be caught using a particular
mesh size). These retention curves can be
used to calculate an optimal mesh size to
produce higher catch frequencies (Millar,
2000; Dos Santos et al., 2003). Therefore,
understanding commercial fishing gears
size-selectivity is essential to refine esti-
mation in modelling inland fisheries and
avoid their collapse, mainly by preventing
the catch of juvenile fish (Wileman et al.,
1996; Welcomme, 2011).
Fisheries in Lake Titicaca are pre-
dominantly located in the littoral area due
to its high productivity and accessibility
(Treviño et al., 1989; Ministerio de la
Producción & AECID, 2008). Macro-
phytes carpets prevail in this zone, where
juveniles and small-sized native species
share habitats (Monroy et al., 2014b).
This way, fishing in littoral areas may
have a negative impact on the functioning
of their ecosystems, such as disruption of
nursery functions or trophic cascading,
which can lead to extinctions (Blaber et
al., 2000). Based on local fish landings,
the littoral fish stocks of Lake Titicaca are
137
 Chapter 4
strongly depleted (Ministerio de la Pro-
ducción & AECID, 2008; IMARPE,
2009; 2010). Thus, further fishing efforts
should focus on open-water zones, where
the more abundant species inhabit (mainly
Orestias ispi and the two mentioned intro-
duced species). In order to improve the
sustainability of fisheries and reduce the
overfishing impact, a size-selectivity
analysis should be urgently conducted to
regulate the fishing of the most vulnerable
species.
The main objective of the present
work is to provide such an analysis.
Therefore, the current composition and
distribution of the Lake Titicaca ichthyo-
fauna was studied at three different depths
in five of the most important fishing
zones. Since most fish species in Lake
Titicaca present small body sizes and ben-
thopelagic feeding habits (Monroy et al.,
2014b), we expected higher catch rates
with benthic nets and also that these rates
would diminish with increasing depth.
Additionally, gillnet selectivity was mod-
elled in the most abundant species in or-
der to propose management measures to
preserve native fish species.
138
MATERIAL AND METHODS
Study area
Lake Titicaca is one of the largest
freshwater lakes in the world, with a
maximum length of 150 km and a maxi-
mum width of 80 km, and covering a sur-
face area of approximately 8559 km2 and
a volume of 930 km3 (Revollo et al.,
2003). This endorheic lake is located in
the Andes, between Peru and Bolivia, at
an altitude of 3810 m (Vila et al., 2007)
(Fig. 1). Lake Titicaca is divided in two
sub-basins: the “Lago Mayor”, which
represents 84% of total surface and
reaches a maximum of 285 m deep, and
the “Lago Menor” with a maximum depth
of 40 m (Fontúrbel, 2008).
Currently, fishing activities in Lake
Titicaca take place at the littoral area,
with a maximum distance of 5 km away
from the coast line (Ministerio de la Pro-
ducción & AECID, 2008). Approximately
20% of the total lake surface is covered
by macrophytes’ carpets, which are pri-
marily composed of totora reeds
(Schoenoplectus californicus ssp. tatora),
totorilla reeds (Juncus articus ssp. andi-
cola), and several species of characean
algae (Chara sp., Nitella sp.), pondweeds

(Potamogeton sp.), watermilfoils
(Myriophyllum sp.), and ditch-grasses
(Ruppia sp.) (Lauzanne, 1991; Vila et al.,
2007). These carpets extend up to 10 m
deep around the lake and provide a feed-
ing and nursery zone for most native spe-
cies (Lauzanne, 1991; Northcote, 2000;
Vila et al., 2007; Monroy et al., 2014b).
The rest of the lake, where the most con-
sumed and abundant pelagic fish species
inhabit (i.e. those species with higher eco-
nomic value), is mostly devoid of macro-
phytes (Lauzanne, 1991). Between the
macrophytes’ proliferation limit and the
rest of the lake exists a transition zone
populated by species that alternate be-
tween littoral and open-water habitats
(Lauzanne, 1991; Monroy et al., 2014b).
Data collection
To assess open-water fishing in Lake
Titicaca while avoiding littoral fishing
interference, two different kinds of mul-
timesh nylon gillnets (benthic and
mesopelagic nets, Pokorný®) were set in
five of the most important fishing locali-
ties (Tilali, L1; Moho, L2; Pusi, L3; Po-
mata, L4; and Inka-Chaka, L5; Fig. 1) at
depths of 10, 20, and 30 meters by local
Gillnet selectivity in Lake Titicaca
fishermen in February 2011. These gill-
nets were installed at each depth thanks to
the adjustable length of the semi-floating
lines between the load line and the
weights. Thus, the benthic gillnet touched
the lake bottom, while the mesopelagic
gillnet was suspended several meters
above it. Depths were estimated using a
Hondex PS-7 Portable Depth Sounder
(Speedtech Instruments, Great Falls, US-
VA). All nets were set three times in each
sampling location in the late afternoon,
and hauled 12 hours later in the next
morning. Each time, the estimates of rela-
tive fish density (catch per unit of effort,
CPUE) were registered.
Benthic gillnets had a surface line of
50 m and a bottom line of 60 m, with a
height of 1.5 m, and were composed of
ten different randomly-distributed panels
with a variety of mesh sizes (bar lengths,
in mm: 5, 6.25, 8, 10, 12.5, 15.5, 19.5, 21,
24, 29). Mesopelagic gillnets had the
same surface- and bottom-line dimensions
than pelagic gillnets, but were 6 m high
and consisted of forty panels randomly
distributed (same bar lengths). Captured
fishes were identified to the species level
following Parenti (1984), and the total
139
 Chapter 4

Fig. 1. Map of Lake Titicaca showing the fishing localities studied: Tilali (L1), Moho (L2), Pusi
(L3), Pomata (L4), and Inka-Chaka (L5). Original map provided by the National Aeronautics
and Space Administration (NASA®).

length (TL, mm) and weight (W, g) of
each specimen was recorded.
Statistical analyses and gillnet selectivity
Catch per unit effort (CPUE) was
calculated for each gillnet type at the dif-
ferent localities and depths. For each case,
a CPUE matrix was constructed indicat-
ing how many fish of each species (rows)
were caught in each mesh size (columns).
Since catch effort was the same in all
cases (12 hours), catch data was not stan-
dardized to unit time.
Normality and homoscedasticity of
data were tested using the Shapiro-Wilk
test and Levene’s test, respectively. Since
CPUE data did not conform to a normal
distribution, permutational multivariate
analyses of variance (PERMANOVA)
were used to test whether significant dif-
ferences between localities and gillnet
types existed. Depth was nested within

140

gillnet type. Differences in CPUE were
assessed using the Bray-Curtis measure of
dissimilarity, with 1000 permutations in
each test. Differences between CPUEs
according to locality and gillnet type were
graphically confirmed with a principal
coordinates analysis (PCoA) using Bray-
Curtis as the ordination method (“vegdist”
function in R).
Gillnet selectivity can be estimated
using both direct and indirect methods,
depending on whether previous knowl-
edge of population structure exists (Millar
& Holst, 1997; Millar & Fryer, 1999).
The most widespread of the indirect ap-
proaches is the SELECT method
(acronym for Share Each Length’s Catch
Total), which is mainly used to estimate
gillnet retention curves based on com-
parative catch data in size-selective analy-
sis (Millar & Holst, 1997; Millar & Fryer,
1999; Millar, 2000). This method is a
generalized linear model (GLM) that de-
scribes the probability of fish to be caught
according to its body size and the mesh
size of the net (Millar & Holst, 1997). As
in other GLM methods, the relationship
among its parameters can be modelled
using several functions (e.g. normal,
Gillnet selectivity in Lake Titicaca
gamma) (Millar & Holst, 1997; Millar,
2000; Carol & García-Berthou, 2007;
Rodríguez-Climent et al., 2012). The dif-
ferent properties of these functions can be
related to several fish retention causes and
to changes in fish growth patterns (Dos
Santos et al., 2003; Erzini et al., 2003;
Carol & García-Berthou, 2007; Rodríguez
-Climent et al., 2012).
Five models were fitted using the
SELECT method in the eight species
more frequently caught: normal fixed,
normal scale, lognormal, gamma and in-
verse Gaussian (see Millar, 2000, and
Rodríguez-Climent et al., 2012, for a de-
tailed description of these models). Both
normal fixed and normal scale models are
based on the normal distribution, whereas
the other three are skewed curves with
positive asymmetry (Carol & García-
Berthou, 2007; Rodríguez-Climent et al.,
2012). In all models but normal fixed,
geometric similarity between meshes and
between individuals of the same fish spe-
cies is assumed. Thus, since gillnet cap-
ture depends only in the relative geometry
of mesh and fish, the selectivity curves for
each mesh size should also be similar
(i.e., both its modal length and spread
141
 Chapter 4
should increase proportionally to mesh
size). This is known as Baranov’s princi-
ple of geometric similarity (Baranov,
1948). In the normal fixed model, the
spread of selectivity curves is fixed, and
thus geometric similarity does not apply
(Millar & Fryer, 1999).
The SELECT method fits these mod-
els using a maximum-likelihood algo-
rithm, assuming that the number of fishes
caught for each length class and mesh size
are independent Poisson random variables
(although this method is robust to viola-
tions of the Poisson assumption; Millar,
2000). Goodness of fit was evaluated by
referring model deviances to a chi-
squared distribution and, for each species,
the model with the lowest deviance was
considered as the best fit (Dos Santos et
al., 2003; Erzini et al., 2003).
All statistical analyses were per-
formed using the libraries lawstat, vegan
and the SELECT-R of the R software
package (R Development Core Team,
2012). In all analyses, the alternative hy-
pothesis was accepted at α<0.05.
RESULTS
A total of 2069 individuals belonging
142
to 12 species were caught in the 5 locali-
ties studied at Lake Titicaca (Table 1).
Among these species, Orestias crawfordi,
Orestias incae, Orestias uruni, and
Trichomycterus rivulatus, were extremely
rare (n<10; Table 1), while O. ispi was the
most abundant and widespread species.
The benthic gillnet was better in re-
flecting the species composition than the
mesopelagic gillnet (12 and 3 species,
respectively). However, CPUEs were
higher in the mesopelagic gillnet, in
which 603 individuals of the pelagic spe-
cies O. bonariensis, O. mykiss and O. ispi
were caught (Table 1). Since the benthic
gillnet is 4 times larger than the mesope-
lagic gillnet, the 1466 individuals caught
with the former would correspond to
366.5 individuals if gillnet surfaces were
equalled. The PERMANOVA revealed
significant differences in CPUEs between
localities (p<0.001) and gillnet types
(p<0.001) (Table 2, Fig. 2). Furthermore,
the interaction between these factors was
also significant (p<0.001), indicating that
the species caught with each gillnet varied
between localities. However, the effect of
depth on CPUE was not significant in ei-
ther gillnet (p= 0.07).
 Table 1. Total catches in Lake Titicaca (N) by species, gillnet type and mesh size. Data combines the catches of all localities and all
depths. For each species, mean ± standard deviation (SD) and range (Min-Max) is given for both total length and weight. Addition-
ally, the length at sexual maturity (LSM) is presented, based on previous studies (Loubens, 1989a; Doadrio, 2002b; IMARPE &
FONCHIP, 2010c).

Mesh size Total length Weight SM

(mm) (mm) (g) (mm)
Specie
Max- Max-
N 5 6.25 8 10 12.5 15.5 19.5 21 24 29 Mean Mean
Min Min
Benthic net
O. bonariensis 4 0 0 0 0 0 3 1 0 0 0 238±12.35 222-250 85.88±17.92 59.81-100.58 >222 c
O. mykiss 1 0 0 0 0 0 1 0 0 0 0 205 205 51.71 51.71 >100 b
O. agassii 29 0 0 9 13 7 0 0 0 0 0 75.86±10.204 57-93 5.94±2.36 2.63-11.28 >104 c
O. albus 176 0 0 41 59 76 0 0 0 0 0 71.73±9.109 53-86 6.04±2.28 2.14-10.23 >100 a
O. crawfordi 4 0 0 1 1 2 0 0 0 0 0 69.5±7.93 60-77 5.99±2.16 3.14-7.71 -
O. imarpe 258 46 59 145 7 1 0 0 0 0 0 51.93±8.17 32-78 2.19±1.12 0.52-7.16 -
O. incae 2 0 0 0 2 0 0 0 0 0 0 67.5±4.94 64-71 5.31±1.61 4.17-6.45 -
O. ispi 451 300 109 37 5 0 0 0 0 0 0 55.99±8.46 34-82 1.54±0.84 0.44-6.37 >63c
O. luteus 297 48 13 32 113 75 16 0 0 0 0 66.09±14.71 32-95 5.42±3.47 0.47-16.15 >107 a
O. mulleri 241 2 5 73 44 38 72 7 0 0 0 78.42±16.17 40-120 8.86±5.85 1.02-28.93 -
O. uruni 2 0 0 2 0 0 0 0 0 0 0 62±9.89 55-69 3.94±1.67 2.76-5.13 -
T. rivulatus 1 0 0 0 1 0 0 0 0 0 0 118 118 15.76 15.76 > 126c
Mesopelagic net
O. bonariensis 98 0 0 0 1 2 25 19 29 15 7 211.64±30.57 150-315 63.33±31.56 11.08-178.08 >225 c
O. mykiss 13 0 0 0 0 2 3 0 1 3 4 217.23±62.13 148-336 149.02±157.71 27.35-484 >100 b
O. ispi 492 31 32 384 45 0 0 0 0 0 0 64.1±5.12 45-87 2.06±0.45 0.82-4.37 >63c
Gillnet selectivity in Lake Titicaca

143
 Chapter 4

(Table 3; Fig. 2). For Orestias albus,

Table 2. Results of the permutational multi-
variate analysis of variance
(PERMANOVA) on CPUE. The effect of
locality, gillnet type (net), and depth
(nested within gillnet type; Net x Depth)
was assessed, as well as the interaction be-
tween locality and gillnet type (Locality x
Net). All effects were highly significant ex-
cept for the interaction. Abbreviations: df,
degrees of freedom; F, Fischer statistic.
Variables df F R2 p indicated no significant deviation from
Orestias imarpe and O. mykiss, the nor-
mal fixed model provided the best fit.
However, inverse Gaussian was the model
with lowest deviance for O. bonariensis
and Orestias mulleri. Similarly, the nor-
mal scale provided the best fit for Ores-
tias agassii and O. ispi, while the same
was true for the Gamma model for O. lu-
teus. In all species, goodness-of-fit tests

Locality 4 2.44 0.23 <0.001 the observed catches for the predictions of
the best models (p>0.35), demonstrating
Net 1 2.28 0.21 <0.001
the accuracy of the models (Table 3). As a
Locality x Net 4 1.80 0.16 <0.001
final remark, the lognormal model did not
Net x Depth 4 1.11 0.10 0.07
present the lowest deviations for any of

Residuals 20 0.27
the species (i.e., it was never the best fit
model).

Total length increased with mesh In several of the studied species (e.g.

size in all captured fish species (Fig. S1), O. agassii, O. albus, O. luteus), the mesh

in agreement with Baranov’s principle of size in which most of the individuals were

geometric similarity (Baranov, 1948; caught did not coincide with the mesh

Rodríguez-Climent et al., 2012). Addi- size associated to the length of sexual ma-

tionally, larger mesh sizes were also asso- turity of those species (Table 1). That is,

ciated with higher variability of catches, in most species the majority of individuals

particularly in species with a high number caught were immature specimens. There-

of catches (e.g. Orestias luteus, O. ispi). fore, mesh sizes lower than 15.5 mm

Gillnet selectivity varied between the should not be used to fish O. agassii and

eight species more frequently caught O. albus, neither bar lengths under 19.5

144
 Gillnet selectivity in Lake Titicaca

Table 3. Gillnet selectivity in the eight more abundant species of Lake Titicaca tested using the
SELECT method. For each species and each fitted model, the estimates of parameters 1 and 2
are given (these parameters are different for each model; e.g. Rodríguez-Climent et al., 2012).
Note that model deviance (Dev) measures goodness of fit, with the lowest value (in bold) indicat-
ing the best fit. Other abbreviations: df, degrees of freedom of the associated chi-squared statis-
tic; p, p-value of the chi-squared test (p< 0.05, marked by an asterisk (*), indicates lack of fit).

Specie Model Parameter 1 Parameter 2 Dev df p

Normal fixed 11.1 27.56 198.16 352 1

Normal scale 11.77 2.35 199.82 352 1
O. bonariensis Gamma 59.61 0.19 195.36 352 1
Lognormal 4.75 0.12 193.69 352 1
Inverse Gaussian 11.76 693.29 193.63 352 1
Normal fixed 9.47 25.82 20.98 50 1
Normal scale 10.24 2.23 23.63 50 1
O. mykiss Gamma 47.92 0.21 23.1 50 1
Lognormal 4.83 0.14 22.92 50 1
Inverse Gaussian 10.2 484.84 22.91 50 1
Normal fixed 7.58 8.87 30.02 34 0.66
Normal scale 7.76 0.62 24.46 34 0.77
O. agassii Gamma 79.45 0.09 27.8 34 0.76
Lognormal 4.13 0.11 29.66 34 0.68
Inverse Gaussian 7.83 549.06 29.89 34 0.66
Normal fixed 6.77 4.34 30.33 60 1
Normal scale 6.88 0.18 34.49 60 1
O. albus Gamma 263.03 0.02 33.54 60 1
Lognormal 4 0.06 33.16 60 1
Inverse Gaussian 6.86 1803.2 33.16 60 1
Normal fixed 7.22 5.23 103.68 134 0.97
Normal scale 7.46 0.54 107.27 134 0.95
O. imarpe Gamma 98.25 0.07 107.31 134 0.95
Lognormal 3.61 0.1 108.82 134 0.94
Inverse Gaussian 7.45 707.83 109.18 134 0.94

145
 Chapter 4

Continue Table 3.

Specie Model Parameter 1 Parameter 2 Dev df P

Normal fixed 10.36 6.8 172.24 136 0.01*

Normal scale 10.63 1 91.97 136 1
O. ispi Gamma 101.19 0.1 101.82 136 0.98
Lognormal 3.97 0.1 107.87 136 0.96
Inverse Gaussian 10.67 1009.54 108.41 136 0.96
Normal fixed 6.57 7.86 286.23 278 0.35
Normal scale 7.05 0.68 288.16 278 0.32
O. luteus Gamma 70.05 0.1 285.95 278 0.35
Lognormal 3.55 0.12 286.65 278 0.34
Inverse Gaussian 7.05 477.21 286.69 278 0.34
Normal fixed 6.56 10.71 260.22 376 1
Normal scale 7.11 0.91 240.61 376 1
O. mulleri Gamma 53.74 0.13 237.56 376 1
Lognormal 3.56 0.13 237.67 376 1
Inverse Gaussian 7.11 371 237.43 376 1
PCoA 2

PCoA 1

Fig. 2. Principal coordinates analyses (PCoA) of CPUE data in Lake Titicaca. Data are coloured
by locality (L1: red, L2: blue, L3: green, L4: grey, L5: black), and grouped by gillnet type
(benthonic: black ellipse, pelagic: blue ellipse).

146
 Gillnet selectivity in Lake Titicaca

Odontesthes bonariensis Oncorhynchus mykiss

Orestias agassii Orestias albus

Relative Retention

Orestias imarpe Orestias ispi

Orestias luteus Orestias mulleri

Total lenght (mm)

Fig. 3. Best-fitted gillnet selectivity curve for each of the eight most abundant fish species of Lake
Titicaca. The dashed grey line indicates length of sexual maturity (in mm). Coloured curves indi-
cate catch probability, with each colour representing one of the mesh sizes with which specimens
of that species were caught, in ascending order (see Table 2).

147
 Chapter 4

Odontesthes bonariensis Oncorhynchus mykiss

4 3
19.5 24
3 12.5
2
15.5 21
2
10 29 15.5 24 21 29
1
1
12.5
0 0
150 197 219 239 148 165 226 336

Orestias albus Orestias agassii

25 3
12.5
20
8 10 12.5
2
15 10
8
10
1
5
0
Number of fish

0
53 65 76 86 57 68 78 85
Orestias imarpe Orestias ispi
20 75
8 8
60
15 5
45
10 5 6.25
30
5 6.25
10 15
12.5 10
0 0
32 42 52 62 34 50 65 80

Orestias luteus Orestias mulleri

25 10 8
10
20 8 15.5
12.5 10
15 5 6
10 4 19.5
8 5 6.25 12.5
5 6.25 2
15.5
0 0
32 52 68 83 40 70 90 113

Total lenght (mm)

Fig. 4. Number of individuals caught in each gillnet size (mm) per length class for the eight
most abundant fish species in Lake Titicaca.

148

mm for O. luteus. Similarly, O. ispi and
O. bonariensis were mostly captured as
immature individuals, since they were
caught with mesh sizes under 8 mm and
29 mm, respectively (Fig. 3). On the other
hand, mature individuals of O. mykiss
could be captured using the 21 mm mesh
(Table 1, Fig. 4), but most of the captured
specimens were caught in the 24 mm
mesh (Figs. 3, 4). Finally, the catches of
O. imarpe and O. mulleri, which were
higher for the 8 mm mesh (Table 1, Fig.
4), were not related to sexual maturity due
to the scarce knowledge on the biology of
those species.
DISCUSSION
Gillnet types, localities, and their inter-
action
From the 26 species that currently
inhabit Lake Titicaca, gillnet catches evi-
denced that only eight species are rela-
tively abundant between 10 and 30 m
depth. Among these eight species, five
were mostly caught with benthic gillnets
(O. agassii, O. albus, O. imarpe, O. luteus
and O. mulleri), while the other three spe-
cies were mainly fished with mesopelagic
gillnets (O. bonariensis, O. mykiss and O.
Gillnet selectivity in Lake Titicaca
ispi). Accordingly, significant differences
in the CPUEs were obtained between gill-
net types in the present study. The fact
that no significant differences were found
between depths further supports the dif-
ferences in faunistic composition between
the pelagic and benthic fish communities.
This finding contrasts with those of previ-
ous studies in deep lakes (CEN, 2005;
Prchalová et al., 2008; Vasěk et al.,
2009), in which depth did influence the
CPUEs. The fact that the depth effect be-
comes more marked after 30 m depth
could explain these differences, since pre-
vious studies with echo-sounder traces
have reported relatively larger fish
schools at depths of 25-50 m and higher
numbers of smaller schools in the upper
25 m of the water column (Vaux et al.,
1988).
Differences between localities are
probably related to the higher biodiversity
and abundance attributed to the northern
shore of Lake Titicaca, relative to the
southern area (Ministerio de la Produc-
ción & AECID, 2008). Accordingly, lo-
calities L1 to L3, all sited at the northern
shore, have significantly higher CPUEs
than the southern L4 and L5 (Table S1).
149
 Chapter 4
Additionally, the proximity of tributary
rivers and streams could also be related to
high CPUEs, since river discharge cause
changes in several physicochemical pa-
rameters of the ecosystem (e.g. turbidity,
salinity, ion concentrations; Lampert &
Sommer, 1997; Wetzel, 2001). These
changes, together with the additional in-
put of nutrients from the river, are associ-
ated with an increase in species richness
(Bunn & Arthington, 2002). Conse-
quently, this could influence prey distri-
bution and their biomass composition
(Willis & Magnuson., 2000). Both the
Suches and Ramis rivers discharge on the
northeastern area of Lake Titicaca, where
L1 to L3 are located.
Regarding the significant interaction
between gillnet type and locality, in most
localities either the benthic or the mesope-
lagic gillnet produced markedly higher
CPUEs (Table S1). These differences
could be caused by a myriad of factors,
such as bottom characteristics, prey distri-
bution, or distance from the shoreline. For
instance, habitats closest to the shoreline
usually have higher species richness
(Beisner et al., 2006), which could ex-
plain higher CPUEs from benthic gillnets
150
in some localities.
Gillnet selectivity models
In three of the studied fish species
(O. albus, O. imarpe and O. mykiss) the
normal fixed model provided the best fit.
This model does not assume geometric
similarity in fish proportions as they in-
crease in body size. For instance, as they
grow older, their girths may increase
faster than their length (Carol & García-
Berthou, 2007), or their dimensions could
increase with a factor significantly differ-
ent from 0.33 (geometric scaling; Alexan-
der, 2003). Additionally, if a species at-
tains a maximum body size, it would no
longer scale to bar length as suggested by
Baranov’s principle (Carol & García-
Berthou, 2007). In the case of O. mykiss
and O. albus, an alternative explanation
for the better fit of the normal fixed model
could be the observed dietary changes
with increasing size (from insects to other
fish species) (Loubens, 1989; Vila et al.,
2007). Since eating fish provides more
nutrients and energy than an insectivorous
diet, these changes could lead to different
growth rates, which would explain the
lack of conformity to geometric similar-

ity.
Although in several previous gillnet
selectivity studies the normal scale model
provided the best fit in most of the species
analysed (Poulsen et al., 2000; Carol &
García-Berthou, 2007; Rodríguez-Climent
et al., 2012), in the present study that was
true only for O. agassii and O. ispi. Mod-
els based on the normal distribution tend
to provide the best fit when most fish are
gilled or wedged (i.e., when they get stuck
on the mesh due to the girth of their gill-
cover or body, respectively) (Hamley,
1975). Thus, the present results suggest
that this is the case for most of the studied
species: O. albus, O. imarpe, O. mykiss,
O. agassii and O. ispi. The case of O.
agassii deserves special consideration,
since different morphotypes and hybridi-
sation have been described for this species
(Parenti, 1984; Lauzanne, 1991;
Maldonado et al., 2009), which may in-
fluence its catchability. The three mor-
photypes described are related to body
shape, preferred habitat, and diet speciali-
sation (Lauzanne, 1982). The littoral mor-
photype presents the smallest individuals,
which are predominantly phytophagous;
the largest individuals are pelagic, present
Gillnet selectivity in Lake Titicaca
a slender body and feed on zooplankton;
and those from the deep zone are more
robust and specialized in algae, chi-
ronomid larvae and ostracods (Lauzanne,
1982). Although these morphotypes were
not discriminated in the present study, if
most of the O. agassii specimens corre-
sponded to the pelagic morphotype, its
slender body shape would explain the best
-fit of a normal model.
Inverse Gaussian and gamma models
provided the best fit for O. bonariensis
and O. mulleri and for O. luteus, respec-
tively. These models are based on skewed
curves and thus tend to provide a better fit
in species for which entangled fish repre-
sent a significant amount of the catch,
since these results in right-skewed catch
data (Hamley, 1975; Dos Santos et al.,
2003; Erzini et al., 2003). Fish entangle-
ment occurs when they are wrapped into
the netting, held by pockets of netting, or
attached to the netting by teeth, fins,
spines or other body protrusions and is
thus less size-dependent than gilling,
wedging and snagging (Hickford &
Schiel, 1996). However, previous studies
suggest that the morphological character-
istics influencing retention by entangle-
151
 Chapter 4
ment (e.g. robust body, presence of pro-
truding spines or teeth) do change with
fish size (Reis & Pawson, 1999; Campos
& Fonseca, 2003; Fonseca et al., 2005;
Rodríguez-Climent et al., 2012).
As a final remark on the relationship
between morphological characteristic of
fish and their catchability, the absence of
scales should also be considered. The
only species in the lake with this trait is T.
rivulatus, which previous studies have
described as a species with low mobility
living in the transition zone (Vila et al.,
2007). The low CPUE of this species sug-
gests that gillnet catches underestimate its
abundance. Thus, others fishing methods
(e.g. basket traps) could be more effective
for its catch. In agreement with this, spe-
cies with more swimming activity had
higher CPUEs (e.g. O. ispi, O. bonarien-
sis). However, catching efficiency may
also vary with other factors, such as sam-
pling conditions (e.g. time of day, soak
time), environmental properties (e.g.
transparency of water, conductivity, tem-
perature), and stock density of species
(Specziár et al., 2013), which were not
evaluated in the present study.
The present study has shown that
152
gillnet fishing gears can be used for moni-
toring the length distribution of the stock
of a particular species. Specifically, by
using the size-selectivity of the gears to
adjust the length distribution of catches
(Millar & Holst, 1997). With the excep-
tion of O. imarpe and O. mulleri, from
which the LSM is unknown, mature indi-
viduals were caught almost exclusively
for the pelagic species studied (i.e., O.
bonariensis, O. mykiss and O. ispi). The
relative absence of mature individuals of
the benthopelagic native species was un-
expected, since fish that live in deeper
waters show bigger sizes and several na-
tive species have been reported as abun-
dant until 30 m depth (Parenti, 1984;
Vaux et al., 1988; Northcote, 2000). This
finding could be related to the fact that
many fisheries harvest fish of a particular
size range due to the size-selective char-
acteristics of the fishing gears employed
(Santos et al., 2006). This type of exploi-
tation has been previously reported to
modify the pattern of size distribution of
vulnerable species and also to reduce their
average LSM and age structure (Botsford
et al., 1997; Goñi, 1998).
Besides overfishing, the low catches

of O. crawfordi, O. incae, O. uruni and T.
rivualtus could also be related to a reduc-
tion of fish stock caused by anthropogenic
threats such as contamination (Gammons
et al., 2006; Monroy et al., 2014a), spe-
cies introduction (Monroy et al., 2014b),
and habitat destruction (Northcote, 1991).
This panorama may be driving towards an
accelerated homogenization process of the
native fish fauna, as it has occurred in
other big lakes (e.g. Lake Victoria, Gold-
schmidt et al., 1993).
Concluding remarks
The alarming depletion of fish land-
ings in Lake Titicaca requires urgent leg-
islations to regulate fish extractions. Gill-
net selectivity studies, such as the present
one, are potentially of great relevance in
future fisheries management, since they
provide a basis from which design mesh
size restrictions according to minimum
sizes at which fish reach sexual maturity
(Bromaghin, 2005). For this, further stud-
ies should be conducted in order to know
the biology of all species (mainly in those
in which MLS is unknown), and also
whether there is seasonality in the fishing
parameters of each species (e.g. spatial
Gillnet selectivity in Lake Titicaca
distribution, temporal variations in fish
abundance).
In order to avoid the collapse of
inland fisheries and the extinction of na-
tive species (to conserve fish diversity),
temporary restrictions on fishing should
be implemented, and the use of size-
unspecific fishing methods should be lim-
ited. Moreover, it is also necessary to con-
trol fleet size and fishing landings in order
to reduce overfishing. An increase in
mean minimum catch size could also be
beneficial to the recovery of endangered
species. Additionally, fisheries should
focus on the offshore zone, away from the
macrophytes’ carpet and thus from the
breeding and feeding area of several
threatened species. As shown in the pre-
sent study, mesopelagic gillnets used far
from this littoral area have a higher yield
than benthic gillnets, both in catches per
net surface and in the economic value of
the species caught.
ACKNOWLEDGMENTS
We are sincerely grateful to the staff
of the Binational Special Project of Lake
Titicaca (PELT), the Sea of Peru Institute
(IMARPE), and the Lake Titicaca Bina-
153
 Chapter 4

tional Agency (ALT), for their invaluable
field and laboratory assistance, especially
to Orieta Flores, Ernesto Yujra and
Patricia Ornelas. Funding and logistic
support was provided by the Spanish
Agency for International Cooperation and
Development (AECID) and the Depart-
ment of Production of Peru. Thus, thanks
to Itziar González, Kalen Su and Jordi
Vera. We also thank Nicole Colin and
Eduardo García-Galea for valuable sug-
gestions over data interpretation.

SUPPLEMENTARY MATERIAL
Table S1. Catch per unit of effort (CPUE) by species and gillnet type (B: benthic, MP:
mesopelagic). Note that data from all depths and mesh sizes was combined for each gillnet
type (see Table 2).

Tilali Moho Pusi Pomata Inka Chaka

Locality
(L1) (L2) (L3) (L4) (L5)
Species / Gillnet type B MP B MP B MP B MP B MP
Oncorhynchus mykiss 0 0 2 20 0 22 1 26 1 30

Odontesthes bonariensis 0 0 0 0 0 4 1 9 0 0

Orestias agassii 1 0 0 0 25 0 1 0 2 0

Orestias albus 3 0 0 0 172 0 0 0 1 0

Orestias crawfordi 0 0 0 0 0 0 0 0 4 0

Orestias imarpe 158 0 5 0 44 0 9 0 42 0

Orestias incae 2 0 0 0 0 0 0 0 0 0

Orestias ispi 310 56 99 426 31 5 3 1 8 4

Orestias luteus 40 0 1 0 173 0 83 0 0 0

Orestias mulleri 190 0 8 0 26 0 9 0 8 0

Orestias uruni 2 0 0 0 0 0 0 0 0 0
Trichomycterus rivulatus 0 0 1 0 0 0 0 0 0 0

154
 Gillnet selectivity in Lake Titicaca

350
300 a. y = 5.7643x + 85.983 b. y = 7.9764x + 47.713
2 300 2
Total Lenght (mm) 250 R = 0.5683 R = 0.7506
250
200
200
150 150
100 100
50 50
0 0
5 10 15 20 25 30 5 10 15 20 25 30
100 100
c. y = 4.6015x + 29.926 d. y = 4.6573x + 22.302
2
80
Total Lenght (mm)

80 2
R = 0.5791 R = 0.8555

60 60

40 40

20 20

0 0
5 7 9 11 13 5 7 9 11 13

100 100
e. y = 5.3631x + 13.656 f. y = 3.7116x + 35.022
2
2 R = 0.507
Total Lenght (mm)

80 R = 0.7554 80

60 60

40 40

20 20

0 0
4 6 8 10 12 4 6 8 10
140
y = 4.8669x + 18.692 140 y = 4.2383x + 29.309
100 120 h. y = 4.2383x + 29.309
g. 2
R = 0.903 120
2
R2 = 0.8154
Total Lenght (mm)

100 R = 0.8154
80 100
80
60 80
60
60
40
40
40
20 20
20
0 00
4 6 8 10 12 14 16 44 88 12
12 16
16 20
20
Bar Lenght (mm) Bar Lenght
Bar Lenght (mm)
(mm)
Fig. S1. Linear regression of total length of fish on gillnet bar length in the eight most abundant
species of Lake Titicaca. (a) Odonthestes bonariensis, (b) Oncorhynchus mykiss, (c) Orestias agas-
sii, (d) O. albus, (e) O. imarpe, (f) O. ispi, (g) O. luteus, (h) O. mulleri. Note that both benthic and
mesopelagic data from Table 1 were pooled.

155
Discusión general
 DISCUSIÓN
Los ecosistemas andinos están considerados como unos de los más amenazados a nivel
mundial (Anderson et al., 2011) y su ictiofauna nativa es una de las más vulnerables debido al
alto grado de endemicidad y a la gran variedad de impactos antrópicos que les afectan (Vila et
al., 2007). Por lo tanto, conocer el estado ecológico de sus comunidades y los efectos produci-
dos por tales impactos es determinante para el manejo y conservación de sus especies (Groom
et al., 2006; Dudgeon et al., 2006; Helfman, 2007).
Los trabajos realizados en la presente memoria ponen de manifiesto la dramática situa-
ción de las poblaciones ícticas del lago Titicaca, pero al mismo tiempo suponen un avance
importante en la conservación de sus especies, ya que exploran nuevas técnicas de diagnóstico
de los principales problemas y proponen medidas de manejo para sus pesquerías. En primer
lugar, en esta tesis se ha explorado el estado actual de la ictiofauna del lago Titicaca y las rela-
ciones tróficas entre las especies introducidas y las nativas mediante el uso del análisis de isó-
topos estables (SIA) (Capítulo 1). Una vez determinada la estructura íctica actual del lago, se
estudiaron los efectos causados por los crecientes problemas de contaminación
(principalmente por metales pesados) en algunas de las especies más consumidas y en algunos
compartimentos del ecosistema (agua y sedimentos) (Capítulo 2). Estos resultados desvelaron
que las concentraciones de metales pesados sobrepasan los límites permitidos por entidades
reguladoras internacionales (ej. FAO/WHO, 1998; EC, 2001, 2006) para el consumo, lo que
generó que se probara la aplicación de biomarcadores hematológicos como herramientas no
invasivas para la detección de los desajustes causados por la contaminación de xenobióticos,
antes de que tengan consecuencias letales en las poblaciones de peces (Capítulo 3). Todos
estos factores antrópicos han generado en las últimas décadas un descenso en las pesquerías
del lago, de las que dependen un importante número de familias de ambos países limítrofes
de su cuenca (Vila et al., 2007; Ministerio de la Producción & AECID, 2008). Así pues, se
utilizaron dos tipos de redes de muestreo científico para dilucidar los mejores tamaños de ma-
lla para capturar individuos maduros de las especies más abundantes, con la finalidad de me-
jorar la gestión de los recursos de forma sostenible (Capítulo 4). Los resultados aportan infor-
mación novedosa que puede ser aplicada en la monitorización del estado de salud y en el ma-

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 Discusión general

nejo de las comunidades ícticas en beneficio de su conservación.

A continuación se discuten de manera breve los aspectos más relevantes de cada uno de
los trabajos realizados como paso previo a las conclusiones generales.

Relaciones tróficas entre las especies introducidas y nativas de la comunidad íctica litoral
Caracterizar las relaciones tróficas de las especies ícticas de grandes lagos requiere la
integración de la biología y ecología proveída por estudios tradicionales que integren los cam-
bios en las cadenas alimenticias y contemplen la influencia de factores externos que puedan
alterar el equilibrio del ecosistema (Vander Zanden et al., 1999; Post, 2002; Clarke et al.,
2005). En este sentido, los isótopos estables han demostrado ser una herramienta útil para di-
lucidar las relaciones tróficas entre especies (Vander Zanden & Vadeboncoeur, 2002; Clarke
et al., 2005; Quevedo et al., 2009). Sin embargo, la interpretación de sus resultados debe
hacerse acompañada del análisis de presas, obtenido a través del contenido estomacal, espe-
cialmente en grandes lagos con amplia diversidad de hábitats y comunidades de presas, así
como complejas relaciones entre especies, como es el caso del lago Titicaca.
Para simplificar la complejidad de las interacciones entre especies y entender las conse-
cuencias de los impactos antrópicos en las redes alimentarias del lago, es preciso analizar las
especies utilizando grupos funcionales compuestos por especies con características similares
en el ecosistema (Trites, 2003; Hoeinghaus et al., 2007). El mantenimiento de estos grupos
funcionales garantiza el funcionamiento de las comunidades y por lo tanto, cualquier altera-
ción se verá reflejada en la pérdida del equilibrio del ecosistema (Elosegui & Sabater, 2009;
Jorgensen et al., 2010). Estas alteraciones en la estructura de la comunidad pueden ser mitiga-
das, en cierto modo, por otras especies del mismo grupo con similares características. Sin em-
bargo, los individuos que constituyen grupos funcionales con poca riqueza de especies, con un
especial interés en la conservación, pueden ser muy importantes para el funcionamiento del
ecosistema (Mason, 2002; Aroviita et al., 2010, Maceda-Veiga, 2011) y por lo tanto se debe
hacer énfasis en su estudio (ej. los organismos correspondientes al grupo I y IV, Capítulo 1).
En los últimos años han sido detectados grandes cambios en la comunidad íctica del lago,
pero actualmente aún se desconocen sus relaciones tróficas, especialmente en lo referente a la

159
 influencia que tienen las especies introducidas sobre las especies nativas. El problema de la
introducción de peces en el lago Titicaca data de varias décadas atrás, pero lamentablemente,
aparte de las descripciones de especies, no se tienen datos de las condiciones de las comunida-
des ícticas de aquel entonces. Este hecho dificulta la interpretación del efecto de las especies
introducidas, a pesar de la evidencia de regresión en las abundancias de las especies nativas,
incluyendo la extinción de Orestias cuvieri y las bajas abundancias de especies como O. tutu-
ni (muy esporádica desde la descripción de Tchernavin, 1944). Así mismo, O. pentlandii, O.
uruni, O. incae y O. minimus sólo han sido capturadas en un porcentaje menor al 0.001% del
total de individuos pescados durante el último estudio ictiológico realizado por de Sostoa et
al. (2010). Sin embargo, el uso de técnicas como los isótopos estables a través de diferentes
aplicaciones (ej. elipses Bayesianas o modelos de mezcla) ha permitido deducir que las espe-
cies introducidas están solapando el nicho trófico y compitiendo por los mismos recursos que
algunas de las especies nativas (Capítulo 1). No obstante, los presentes resultados deben ser
interpretados con cierta cautela, ya que al no disponer de estudios previos de cambio trófico
en las especies del lago, se asumieron valores generales que podrían generar subestimas o so-
breestimas producidas por la variabilidad individual. Estos valores pueden variar para cada
consumidor debido a factores tales como el estrés nutricional, calidad de la dieta, tamaño del
cuerpo, mecanismos excretores y tasas de alimentación (McCutchan et al., 2003; Vanderklift
& Ponsard, 2003).
Sin embargo, existen muchos ejemplos de los efectos adversos y los impactos a gran es-
cala que la introducción de especies alóctonas puede generar (ej. la perca del Nilo Lates niloti-
cus en el lago Victoria o los salmónidos en lagos y ríos del hemisferio sur, ver Rahel, 2002).
Dichas introducciones pueden hacer variar la abundancia y la distribución de las especies nati-
vas, alterar las redes alimentarias y hábitats, y cambiar los procesos del ecosistema debido a la
competencia por recursos y nichos, restringiendo la disponibilidad de alimento, el cual es un
factor limitante en la dinámica poblacional de muchas especies (Zambrano et al., 2001; Figue-
redo & Giani, 2005). Por lo tanto, es más que probable que en los últimos años la abundancia
de algunas de las presas más comunes del lago hayan cambiado, ya que algunas especies han
desaparecido o presentan muy bajas densidades y otras están consumiendo presas que usual-

160
 Discusión general

mente no estaban reportadas (ej. huevos en O. ispi o peces en O. luteus) (Capítulo 1). Sin em-
bargo, el nivel trófico similar que presentaron la mayoría de las especies (de acuerdo al estre-
cho margen de variación del δ15N) puede ser debido a que la gran mayoría de especies se ali-
mentan de ítems similares, lo que sugiere una menor presencia de especies especialistas. Aun-
que el presente trabajo no determinó con certeza la especialización trófica, la presencia de al-
gunas estructuras utilizadas para alimentación y las pequeñas variaciones en la firma isotópica
son indicadoras de una posible especialización (ej. los individuos epipelágicos planctónicos
del grupo I).
Por otro lado, la marcada segregación espacial determinada por las grandes variaciones
13
de δ C entre especies está relacionada con el diferente uso del hábitat y por lo tanto, con las
diferentes presas de las cuales se alimentan (Vander Zanden & Vadeboncoeur, 2002; Clarke et
al., 2005; Quevedo et al., 2009). Cambios en la distribución y abundancia de presas, gener-
ados por la introducción de especies alóctonas u otros factores antrópicos, quizás hayan gener-
ado que algunas especies tengan que explotar diferentes tipos de hábitats los cuales podrían
conducir a adaptaciones morfológicas para evitar competencia por recursos entre los indi-
viduos de una misma población (Bootsma et al., 1996; Svanbäck et al., 2008). Por con-
siguiente, los diferentes morfotipos reportados en O. agassii (Lauzanne, 1982) y la hibridi-
zación de algunas especies del género Orestias (Lauzanne, 1991; de Sostoa et al., 2010) po-
drían sustentar esta hipótesis (Capítulo 1).
Adicionalmente, existen indicios de que algunas especies, dependiendo de su tamaño,
puedan ocupar temporalmente ecotonos donde la competencia por alimento es menor o que
constituyan un mejor refugio contra los depredadores (ej. zona cubierta de macrófitas y totora-
les). Como consecuencia, a pesar de que el presente estudio se basó en la comunidad litoral,
diferentes conexiones con otras cadenas tróficas, con diferentes valores isotópicos, pueden
estar influyendo en la media isotópica de algunas especies (ej. O. ispi, O. bonariensis). Por lo
tanto, futuros estudios de isótopos estables en el lago deberían determinar las diferencias entre
las cadenas tróficas del lago (ej. litoral vs aguas abiertas), mediante el uso de “baselines” tra-
tando de abarcar la estación seca y de lluvias, con el fin de inferir mejor las relaciones tróficas
entre los individuos y poder comparar los datos con otros ecosistemas similares. De igual for-

161
 ma, futuros estudios experimentales en laboratorio deberían realizarse con la finalidad de co-
nocer el cambio trófico de la fauna íctica del lago.

Bioacumulación de metales pesados en peces y otros compartimentos del ecosistema

El lago Titicaca ejemplifica muy bien la problemática ambiental mundial generada por la
contaminación, principalmente debida a la descarga de aguas residuales provenientes de la
industria, agricultura y minería de los pueblos ribereños, lo cual ha contribuido a la degrada-
ción de sus ecosistemas acuáticos (Revollo et al., 2003; Gammons et al., 2006; Gobierno Re-
gional de Puno, 2008). Estas aguas residuales están compuestas por un gran porcentaje de ele-
mentos perjudiciales para el desarrollo de los seres vivos tales como los metales pesados, los
cuales pueden bioacumularse o biomagnificarse a través de las redes alimentarias (Demirak et
al., 2006; Agah et al., 2009; Uysal et al., 2009).
Los problemas de contaminación del lago se han incrementado en las últimas décadas
debido a la mala gestión de los recursos, el acelerado crecimiento de la población y el inapro-
piado servicio de alcantarillado y gestión de residuos que presentan la mayoría de los núcleos
urbanos circunlacustres (Revollo et al., 2003). Los resultados del presente trabajo mostraron
que las desembocaduras de los ríos (principalmente la del río Ramis) fueron los lugares donde
se encontraron las mayores concentraciones de metales pesados (Capítulo 2). Este hecho se
debe principalmente a que la cuenca de este río constituye una zona aurífera y polimetálica
que en su transcurso, pasa por varias minas y zonas de explotación (ej. Chaquiminas, Pampa
Blanca, Morrena Vizcacha o la Rinconada) las cuales no presentan un adecuado manejo de los
relaves mineros (Revollo et al., 2003). Adicionalmente, el uso de fertilizantes agrícolas en
zonas aledañas a los ríos ha podido ser uno de los factores principales de la gran acumulación
de metales pesados detectados tanto en agua como en sedimento y fauna acuática (Capítulo 2).
Las altas concentraciones de metales en sedimento estuvieron relacionadas con el contenido
en materia orgánica, posiblemente arrastrada por procesos de escorrentía y magnificada con
vertidos de las poblaciones ribereñas y actividades agropecuarias (Revollo et al., 2003; Go-
bierno Regional de Puno, 2008).
Por su parte, la acumulación de metales pesados en peces también presentó el mismo

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 Discusión general

patrón que en agua en las localidades cercanas a las desembocaduras de los ríos (Capítulo 2).
La mayor bioacumulación de metales se dio en las especies nativas que habitan cerca del fon-
do (especies bentónicas y bentopelágicas) en comparación con la encontrada en la especie
pelágica introducida, a pesar de tener ésta una relativa mayor posición trófica (Capítulo 1).
Sin embargo, todas las especies presentaron un patrón similar de bioacumulación en tejidos
(principalmente con los metales no esenciales) debido quizás a las diferencias metabólicas y a
las características de cada especie (ej. sexo).
El tejido hepático fue el de mayor bioacumulación de metales pesados en todas las espe-
cies estudiadas, con la excepción del Hg, cuyos valores más altos se presentaron en la muscu-
latura. La bioacumulación de metales en el hígado tiene una gran importancia debido a que
muchos pobladores consumen algunas especies de peces sin eviscerar (principalmente las del
género Orestias de pequeño tamaño). Dentro de los metales estudiados el Cu, Zn, Cd y Hg
medido en peces y el Pb determinado en agua excedieron los límites establecidos para su con-
sumo (FAO/WHO, 1998; EC, 2001, 2006, US EPA, 2009). La alta contaminación por metales
pesados tanto del agua del lago (que es consumida en la mayoría de los hogares sin ningún
tipo de tratamiento) como de las especies de peces que forman parte de la dieta alimenticia de
la población ribereña, constituyen un importante riesgo para la salud humana y pueden causar
infecciones, enfermedades respiratorias y gastrointestinales, mortalidad infantil y, en general,
disminución de la esperanza de vida (más baja que la tasa nacional) (ALT, 2003).
Sin embargo, ya que el presente estudio describe sólo una época puntual del año, los fu-
turos trabajos deberían abarcar la temporada seca y de lluvias con objeto de monitorear la va-
riabilidad de los factores que pueden afectar la disponibilidad de los metales pesados y su bio-
acumulación en el ecosistema. Hasta no conocer esta variabilidad en la concentración de me-
tales a lo largo del año, es difícil proponer estrategias de manejo, aunque los altos niveles de
algunos de los elementos estudiados y la posibilidad de su biomagnificación sugieren que se
debe limitar el consumo de por lo menos, las cuatro especies estudiadas, principalmente las
pescadas en las localidades cercanas a las desembocaduras de los ríos y grandes ciudades.
Consecuentemente, profundizar en el conocimiento de la dinámica de dispersión y bioacumu-
lación de estos metales es de suma importancia para dimensionar el alcance de sus efectos.

163
 Uso de biomarcadores para detectar la contaminación por xenobióticos en los peces del
lago Titicaca
Conocer el efecto que tiene la contaminación sobre los organismos es crucial para deter-
minar la tolerancia de las especies y las consecuencias de las alteraciones que pueden producir
en el ecosistema (Pacheco & Santos, 2002; de Castilhos, 2012). El reconocimiento de estos
efectos mediante el uso de biomarcadores hematológicos ha demostrado que puede ser de gran
utilidad para futuros monitoreos y estimaciones del estado de salud de la comunidad de peces
del lago. A pesar de que su utilidad ha sido comprobada en varios grupos de vertebrados (ej.
anfibios, reptiles, mamíferos) son pocos los valores de referencia que se tienen en peces para
cada uno de los parámetros hematológicos (Maceda-Veiga, 2011). Sin embargo, dada la gran
variabilidad de estos parámetros detectada por especie, la interpretación de sus posibles efec-
tos debe ser analizada con cautela (Houston, 1997; Rowan, 2007; Maceda-Veiga et al., 2013).
El presente estudio demostró que la contaminación por xenobióticos (ej. organoclorados,
drogas, metales pesados) acumulados en algunos de los compartimentos del ecosistema tales
como agua, sedimento y peces del lago Titicaca (Capítulo 2) pueden ser diagnosticados a
través parámetros hematológicos de peces (ej. anormalidades celulares o nucleares en eritroci-
tos, respuesta del sistema inmune), lo cual fue confirmado a través de otros biomarcadores
como el índice hepatosomático o la histología del hígado (Capítulo 3). Los mayores efectos
citotóxicos y genotóxicos (ej. frecuencias más altas de eritrocitos cariorreicos o con alteracio-
nes nucleares) y las mayores respuestas por parte del sistema inmune para combatir el des-
equilibrio homeostático (ej. frecuencias más altas de monocitos) fueron principalmente obser-
vados en los ejemplares capturados en la desembocadura del río Ramis (Capítulo 3), donde se
encontraron las mayores concentraciones de metales (Capítulo 2). En general, la frecuencia de
estos parámetros se incrementó al aumentar las concentraciones de algunos metales tales co-
mo Cu, Zn, Cd, Hg y Co y de variables ambientales como el amonio y la conductividad en el
caso de los eritrocitos (ej. ENAs), y de los nitritos y el oxígeno disuelto en el caso de los leu-
cocitos. Dentro de los ENAs, los eritrocitos lobulados (LE) y los mellados (NE) presentaron
un patrón de respuesta similar en todas las especies estudiadas, por lo que el simple conteo de
la anomalía basta como carácter diagnóstico en futuras investigaciones.

164
 Discusión general

Otro de los parámetros más utilizados en el diagnóstico hematológico es el recuento de

micronúcleos, los cuales se forman por aberraciones cromosómicas debidas a rupturas de los
cromosomas generadas en la replicación o repartición del ADN de las dos células hijas duran-
te la anafase mitótica (Ayllon & Garcia-Vazquez, 2001; Pacheco & Santos, 2002; Guilherme
et al., 2008). A pesar de que se detectó un incremento en la frecuencia de micronúcleos en las
localidades contaminadas, este trabajo evidencia que su recuento, por si sólo, no es tan sensi-
ble como otros biomarcadores (ej. ENAs, eritrocitos inmaduros o cariorreicos), ya que sus
frecuencias fueron muy bajas. Por otro lado, no se observaron diferencias significativas entre
el NCR de las estaciones control y las estaciones contaminadas, lo que sugiere que la morfo-
logía externa de los eritrocitos no se ve muy afectada por los efectos de la contaminación de-
bida a la acción genotóxica de algunos compuestos (Capítulo 3). Por lo tanto, NCR no es un
buen biomarcador para evaluar los problemas de contaminación de las especies estudiadas.
Varios factores tales como la edad, el sexo, la localidad y la especie influenciaron la ex-
presión de los biomarcadores (Capítulo 3). Esto se debe, principalmente, a la relación existen-
te entre la intensidad de la exposición a la contaminación a la cual estaban sometidos los indi-
viduos estudiados con respecto a las características individuales de las especies (ej. diferentes
tasas metabólicas, estatus fisiológico) (Frenzilli et al., 2004; Bolognesi et al., 2006). Tanto O.
luteus como O. agassii presentaron mayores efectos citotóxicos y genotóxicos en compara-
ción a T. rivulatus, por lo que ambas especies de carachis pueden ser mejores bioindicadoras
en futuros monitoreos de contaminación en la comunidad íctica del lago.
Las alteraciones hematológicas fueron corroboradas con el diagnóstico histopatológico
ya que el hígado es el principal órgano donde ocurren los procesos de detoxificación (Hinton
& Laurén, 1990; Facey et al., 2005; Koca et al., 2008; Costa et al., 2009). Los individuos de
todas las especies estudiadas las zonas más contaminadas presentaron varias anomalías (ej.
vacuolización de los hepatocitos, alta presencia de núcleos picnóticos y periféricos), relacio-
nadas con disfunciones metabólicas (Capítulo 3). Adicionalmente, estas alteraciones fueron
respaldadas por el aumento del índice gonadosomático (principalmente en ambas especies de
carachis) en las estaciones más contaminadas, sugiriendo que la contaminación por xenobióti-
cos puede estar generando un proceso de hipertrofia (Hinton & Laurén, 1990; Facey et al.,

165
 2005).
La alteración metabólica del hígado y el estrés generado por la presencia de xenobióticos
son consistentes con la fórmula leucocitaria, en las que se detectó monocitosis. La monocito-
sis de ambas especies de carachis evidencia una posible respuesta crónica al ser los macrófa-
gos los que predominan y van remplazando a los neutrófilos a medida que transcurre el proce-
so degenerativo (Olivares-Gordillo, 2001; Majno & Joris, 2004; Maceda-Veiga, 2011). Sin
embargo, es posible que la alteración de la frecuencia relativa de los neutrófilos pueda tam-
bién estar relacionada con la presencia de patógenos (Thrall, 2004) aunque no pudieron ser
detectados en el presente estudio.
Debido a los efectos anteriormente descritos, es posible suponer que si las concentracio-
nes de los diferentes compuestos se mantienen o se incrementan a lo largo del tiempo, generen
graves consecuencias en las poblaciones de peces y en el funcionamiento del ecosistema en
general. Por tal razón, futuros estudios dosis-respuesta experimentales en laboratorio deberían
realizarse para conocer los valores de referencia y su variabilidad en función de los cambios
ambientales y otros agentes estresores (ej. presencia de depredadores). A pesar de que los
parámetros hematológicos son biomarcadores inespecíficos, su implementación como herra-
mienta de diagnóstico puede considerarse de gran utilidad para la conservación de las comuni-
dades de peces.

Selectividad de las redes agalleras en la comunidad íctica de aguas abiertas del lago Titica-
ca
En los capítulos anteriores se ha puesto de manifiesto el efecto de diversos factores
antrópicos, los cuales han derivado en una drástica disminución de la ictiofauna del lago Titi-
caca, como ha sido sugerida por otros autores como Vila et al. (2007). Entre estos factores, la
sobrepesca es uno de los más preocupantes actualmente debido a que no existe ninguna nor-
mativa de ordenamiento pesquero específica extensible a todas las especies del lago, lo cual
ha generado la insostenibilidad de su pesca artesanal, principalmente realizada en la zona lito-
ral (Ministerio de la Producción & AECID, 2008). No obstante, en esta zona habitan la ma-
yoría de especies de menor tamaño y los estadios juveniles de un gran número de especies

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 Discusión general

(Lauzanne, 1991; Vila et al., 2007). Algunos peces predominantemente pelágicos como los
introducidos O. bonariensis y O. mykiss (que son los de mayor importancia comercial) y el
nativo O. ispi (que es la especie más abundante del lago), ocupan regularmente los hábitats de
aguas abiertas y, por lo tanto, son especies candidatas a una mayor explotación pesquera. Fi-
nalmente, aunque se sabe que algunas especies pueden vivir entre ambos ambientes, se desco-
nocen algunos aspectos de su biología pesquera y ecología (ej. distribución espacial, tamaños
de malla para su captura).
El presente estudio evidenció que actualmente doce especies habitan la franja comprendi-
da entre los 10 y los 30 m de profundidad del lago, de las cuales ocho fueron frecuentemente
capturadas por las redes agalleras multimalla (Capítulo 4). Este tipo de redes constituyen un
método de pesca pasivo ya que su éxito de captura depende de la actividad de los peces. Por
tal razón, hay que tener en cuenta que las especies que presentan escasa movilidad pueden
presentar menor capturabilidad y, por consiguiente, puede existir cierta subestima a la hora de
detectar cambios en sus poblaciones (Fonseca et al., 2005). Sin embargo, este tipo de muestre-
os con redes multimalla, son los utilizados en la captura científica, ya que aportan una valiosa
información sobre la dinámica poblacional en medios lacustres. Los resultados obtenidos
muestran que la estructura de tallas de algunas de estas especies estuvo sesgada hacia indivi-
duos juveniles (ej. O. agasii, O. albus, O. luteus), lo que evidencia que actualmente existen
fuertes impactos que están afectando la dinámica de sus poblaciones (ej. Vaux et al., 1988;
Wurtsbaugh & Alfaro, 1988; Northcote, 1991; Gammons et al., 2006). Esto puede ser, en par-
te, debido a que la fuerte presión de pesca ejercida genera una mayor captura, retirándose ini-
cialmente los individuos de tallas más grandes y remplazándose por individuos cada vez más
pequeños con crecimientos más rápidos, lo cual puede producir una ilusión de productividad
sostenida que no refleja los profundos cambios en la estructura de las poblaciones y de la red
trófica (Balirwa et al., 2003).
Las capturas por especie se ajustaron a diferentes modelos matemáticos que describieron
las causas de su retención en las mallas y sugirieron qué tipo de crecimiento presentan los in-
dividuos (Dos Santos et al., 2003; Erzini et al., 2003; Carol & García-Berthou, 2007; Rodrí-
guez-Climent et al., 2012). El tipo de retención está relacionado a la alta diversidad fenotípica

167
 de la ictiofauna del lago, la cual puede ser debida a las diferencias interespecíficas en las es-
trategias de alimentación de acuerdo a los hábitats que ocupan las especies (Maldonado et al.,
2009). Los mejores modelos para cada especie determinaron que los individuos de O. bona-
riensis, O. mulleri y O. luteus quedaron mayoritariamente enredados en la malla debido a la
presencia de espinas, dientes y/o escamas protuberantes, o bien a su tipo de natación. Por el
contrario, los de O. albus, O. agassii, O. imarpe, O. ispi y O. mykiss fueron interceptados
principalmente a nivel de las agallas o el resto del cuerpo (ver Dos Santos et al., 2003; Erzini
et al., 2003). Adicionalmente, es posible que los diferentes cambios de alimentación que pre-
sentan algunas especies a lo largo de su vida (ej. O. albus y O. mykiss) (Lauzanne, 1982; Lou-
bens, 1989; Lauzanne, 1991), influyan en su tipo de crecimiento y por ende, en la retención
por determinados tamaños de malla.
El análisis de selectividad de mallas demostró que las mallas menores a 15.5 mm para O.
agassii y O. albus, así como las menores a 19.5 mm para O. luteus, no deberían utilizarse para
pescar dichas especies, ya que incluyen la presencia de juveniles en sus capturas. Por el con-
trario, las mallas de 29, 24 y 8 mm para O. mykiss, O. bonariensis y O. ispi son las ideales
para pescar un mayor número de individuos maduros de especies pelágicas (Capítulo 4). Por
consiguiente, y debido a la disminución de la abundancia de la ictiofauna del lago en los últi-
mos años, el uso de los artes de pesca debería ser estandarizado a métodos que tengan en
cuenta una selectividad de redes estableciendo una talla mínima de captura de acuerdo a la
talla de madurez sexual. Tales métodos, en la medida de lo posible, no deberían utilizarse en
áreas con cobertura de macrófitas, ya que son lugares de freza y de alevinaje para muchas es-
pecies nativas (Lauzanne, 1991; Vila et al., 2007). Para tal fin, una reestructuración en la le-
gislación pesquera y una mayor supervisión en el control de los efectos antrópicos (p. ej. des-
cargas de xenobióticos, protección de hábitats, licencias para acuicultura) podrían ser claves
para la conservación de las especies del lago.

168
Discusión general

169
Conclusiones
 CONCLUSIONES

1. La ictiofauna del lago Titicaca presenta un nivel trófico similar pero al mismo tiempo una

marcada segregación espacial relacionada con el uso de diferentes hábitats y el consumo de

diferentes presas, habiendo en términos generales, especies más generalistas que especializa-

das por un determinado tipo de alimento.

2. La relación entre el δ13C y la longitud de talla de algunas especies (principalmente nativas)

demostró que la zona litoral cubierta por macrófitas es de vital importancia para el refugio y

alimentación de muchos de los juveniles e individuos de menor tamaño.

3. Además de los efectos generados por la depredación, las especies exóticas comparten los

mismos nichos tróficos con las especies nativas por lo que pueden competir por recursos ali-

menticios similares (principalmente con las especies pelágicas). Entre éstas, los individuos de

O. bonariensis con tallas mayores a 90 mm presentan un cambio de alimentación relacionado

con el consumo de peces.

4. La concentración de Pb en agua y la de Cu, Zn, Cd y Hg en los tejidos de O. bonariensis,

O. luteus. O. agassii y T. rivulatus sobrepasaron los límites establecidos para consumo huma-

no por lo que se aconseja un mayor control en las políticas de gestión del lago. Las mayores

concentraciones de dichos metales estuvieron asociadas a mayores contenidos de materia

orgánica y fueron encontradas en las localidades cercanas a los ríos tributarios influenciadas

por actividades mineras (principalmente en la desembocadura del río Ramis).

172
 Conclusiones generales

5. Las especies bentopelágicas son más susceptibles a la acumulación por metales pesados que

la especie pelágica (O. bonariensis) siendo el hígado en comparación con la musculatura el

mejor tejido diana para la bioacumulación de todos los metales estudiados a excepción del Hg.

Sin embargo, el patrón de bioacumulación estuvo poco relacionado con las concentraciones de

metales en el ambiente y fue mayormente influenciado por la ecología y las características

individuales de cada especie, principalmente en los metales no esenciales.

6. Los biomarcadores sanguíneos pueden ser utilizados como herramientas no letales en el

diagnóstico de los efectos generados por la contaminación causada por xenobióticos. Sin em-

bargo, para validar su uso en las especies del lago, se necesitan conocer los valores de referen-

cia de los parámetros hematológicos y su variabilidad temporal en experimentos de dosis-

respuesta.

7. La frecuencia relativa de eritrocitos con anormalidades nucleares ENAs (mellados, lobula-

dos, con micronúcleos), eritrocitos inmaduros, y degenerados, se incrementó al aumentar las

concentraciones de Cu, Zn, Cd, Hg y Co y algunas variables ambientales como los nitritos, el

oxígeno disuelto y la conductividad, lo que generó una respuesta por parte del sistema inmune

desencadenando un proceso de monocitosis.

8. A pesar de las altas concentraciones de xenobióticos detectadas y su posible acción ge-

notóxica, la morfología externa de los eritrocitos no se ve muy afectada por los efectos de la

contaminación, por lo que la ratio nucleo-citoplasmática no es un buen biomarcador del esta-

do de salud de las especies de peces estudiadas.

173
 9. El análisis histológico y el mayor índice hepatosomático obtenido en los individuos de las

tres especies evaluadas (O. luteus, O. agassii y T. rivulatus) confirmó que las anomalías ob-

servadas en los biomarcadores sanguíneos de las localidades más contaminadas son debidas a

disfunciones metabólicas.

10. El crecimiento de los individuos de O. albus, O. imarpe y O. mykiss no es proporcional-

mente geométrico a lo largo de su vida. Sin embargo, esto no interfiere en que estas especies

junto a O. ispi y O. agassii queden retenidas a través de las agallas a la hora de su captura. Por

el contrario, otras especies como O. bonariensis, O. mulleri y O. luteus quedan mayoritaria-

mente enredadas o enganchadas en las redes debido a su forma y actividad nadadora.

11. El alto porcentaje de especies endémicas, la baja riqueza de especies y el decrecimiento en

la abundancia íctica en los últimos años requiere la utilización de métodos selectivos como las

redes agalleras, principalmente fuera de la zona de macrófitas donde se encuentran las mayo-

res abundancias de especies con más alto valor comercial (ej. O. bonariensis, O. ispi y O. my-

kiss).

12. Las mejores tallas para la captura de individuos maduros de O. mykiss, O. bonariensis y

O. ispi son 29, 24 y 8 mm respectivamente. Sin embargo, para la gran mayoría de las especies

nativas es necesario hacer un estudio de su biología reproductiva para poder tomar medidas de

ordenamiento pesquero.

174
Conclusiones generales

175
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Anexos
Journal of Fish Biology (2014) 85, 1693–1706
doi:10.1111/jfb.12529, available online at wileyonlinelibrary.com

Trophic interactions between native and introduced fish

species in a littoral fish community

M. Monroy*†, A. Maceda-Veiga‡, N. Caiola§ and A. De Sostoa*

*Department of Animal Biology and Biodiversity Research Institute (IRBio), University of
Barcelona, E-08028, Barcelona, Spain, ‡Cardiff School of Biosciences, Cardiff University,
Cardiff CF10 3AX, U.K. and §Aquatic Ecosystems, IRTA, Sant Carles de la Ràpita, Spain

(Received 4 March 2014, Accepted 6 August 2014)

The trophic interactions between 15 native and two introduced fish species, silverside Odontesthes
bonariensis and rainbow trout Oncorhynchus mykiss, collected in a major fishery area at Lake Titicaca
were explored by integrating traditional ecological knowledge and stable-isotope analyses (SIA). SIA
suggested the existence of six trophic groups in this fish community based on 𝛿 13 C and 𝛿 15 N signatures.
This was supported by ecological evidence illustrating marked spatial segregation between groups, but
a similar trophic level for most of the native groups. Based on Bayesian ellipse analyses, niche overlap
appeared to occur between small O. bonariensis (<90 mm) and benthopelagic native species (31⋅6%),
and between the native pelagic killifish Orestias ispi and large O. bonariensis (39%) or O. mykiss
(19⋅7%). In addition, Bayesian mixing models suggested that O. ispi and epipelagic species are likely
to be the main prey items for the two introduced fish species. This study reveals a trophic link between
native and introduced fish species, and demonstrates the utility of combining both SIA and traditional
ecological knowledge to understand trophic relationships between fish species with similar feeding
habits.
© 2014 The Fisheries Society of the British Isles

Key words: endemic fish; food web; introduced species; Lake Titicaca stable isotopes.

INTRODUCTION
Freshwater ecosystems are experiencing a worldwide biodiversity crisis, which is
particularly evident in the conservation status of many fish species (Dudgeon et al.,
2006; IUCN, 2008). Common threats to freshwater ichthyofauna are habitat degrada-
tion, the introduction of exotic species and overfishing (Dudgeon et al., 2006; IUCN,
2008). These anthropogenic interactions can lead to species extinction, and may also
cause profound changes in community structure, thus affecting ecosystem functioning
(Duffy et al., 2007; Cucherousset et al., 2012). Traditional ecological approaches
such as observational studies of species behaviour and gut content analyses can detect
changes in species performance that could be associated with ecosystem functioning
(Petchey & Gaston, 2006). Such approaches may fail to detect subtle modifications in
the structure of communities, however, especially when the species examined belong
to a highly diverse fish community and most species appear to have relatively similar

†Author to whom correspondence should be addressed. Tel.: +34 934021041; email: monroylopezmario
@gmail.com

1693
© 2014 The Fisheries Society of the British Isles
1694 M . M O N R O Y E T A L.

feeding behaviour (Vander Zanden & Vadeboncoeur, 2002; Quevedo et al., 2009).
Consequently, inferring the food web structure of fish species exhibiting generalist and
similar feeding behaviour in large water bodies such as lakes requires the integration
of information provided by traditional ecology and food web tracers (Vander Zanden
et al., 1999; Post, 2002; Clarke et al., 2005).
Stable-isotope analyses (SIA) of C and N (𝛿 13 C and 𝛿 15 N) are food-web tracers that
are widely applied in studies of fish ecology in lakes (Vander Zanden et al., 1999;
Post, 2002). The predictive enrichment in 𝛿 15 N throughout trophic levels (i.e. 3–4‰)
enables the determination of fish trophic position, and the similarity in 𝛿 13 C between
diet and consumer allows the determination of fish foraging habitat (Vander Zanden
et al., 1997). SIAs may then guide management strategies by showing evidence of the
differential effect of introduced species on the native fish community by trophic com-
petition or predation. Indeed, the introduction of the piscivorous silverside Odontesthes
bonariensis (Valenciennes 1835) and rainbow trout Oncorhynchus mykiss (Walbaum
1792) into Lake Titicaca in the 1940s to enhance fisheries resources for local human
communities typifies one of these situations. Over a period of 32 years (1979–2011),
the biomass of native fish stocks has declined by 45% (Vila et al., 2007; Aliaga &
Segura, 2013), and observational studies suggest that predation by introduced fish
species is likely to be one of the main causative factors (Vaux et al., 1988; Loubens,
1989; Vila et al., 2007). Trophic competition is, however, also likely to occur because
juveniles of both native and introduced species feed on macroinvertebrates (Vaux
et al., 1988).
This study aimed to explore the trophic interactions between native and introduced
fish species in Lake Titicaca by integrating the information provided by traditional ecol-
ogy and SIA. Previous studies addressing the native fish species’ diet by gut content
analyses have suggested that these species show a relatively similar trophic spectrum.
Therefore, all fish species should have a similar trophic level, limiting trophic compe-
tition by spatial segregation. Finally, the differential effect on native fish species of the
two introduced fish species in terms of predation and niche overlap was examined.

MATERIALS AND METHODS

S T U DY A R E A
Lake Titicaca is the largest freshwater lake in South America, covering a surface area of c.
8559 km2 . It is one of the highest lakes worldwide, at an elevation of 3810 m (Vila et al., 2007)
(Fig. 1). It is divided into two large basins: Lago Mayor, which reaches a maximum of 285 m in
depth, and Lago Menor, with a maximum depth of 40 m. Lake Titicaca is mainly an oligotrophic
lake characterized by permanent hyperhaline cool water due to geographic features and lack of
strong seasonality (Myers et al., 2000; Vila et al., 2007). Littoral areas have the characteris-
tic macrophyte carpet dominated by reeds of totora Schoenoplectus californicus ssp. tatora and
juncus Juncus articus ssp. andicola, as well other macrophyte species of the genus Chara, Pota-
mogeton, Myriophyllum, Nitella and Ruppia (Lauzanne, 1991; Vila et al., 2007). Although the
macrophyte carpet only covers 20% of the total lake surface, it acts as an important feeding
and breeding area for the majority of native species in the lake (Lauzanne, 1991). The native
fish community in Lake Titicaca has a high degree of endemicity and is dominated by members
of the killifish genus Orestias (Teleostei, Cyprinodontidae) and the benthic catfish Trichomyc-
terus rivulatus Valenciennes 1846 (I. Doadrio, pers. comm.). A variety of anthropogenic events
such as the introduction of alien species, overfishing and sewage discharges from urban areas
and mining activities may have led many native species to be listed as vulnerable, critically

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
 T RO P H I C I N T E R AC T I O N S I N L A K E T I T I C AC A F I S H E S 1695
70°00´ 68°45´ 80° 60° 40°

Huancané
L4
L3 0°
L2
16°30´ L1
Juliaca
Lago Mayor Peru
Bolivia

Lake Titicaca
Puno Bolivia 20°

PERU Copacabana
16°18´
Lago Menor
La Paz 40°

0 10 20
Km

Fig. 1. Map of the study area with the sampling locations (L1, Kapara; L2, Pusi, L3, Olojpata; L4, Kauta)
examined in Lago Mayor in February 2010. Original map provided by National Aeronautics and Space
Administration (NASA).

endangered or extinct based on the International Union of Conservation of Nature (Vila et al.,
2007; Van Damme et al., 2009; Monroy et al., 2014).

S A M P L I N G P RO C E D U R E
Fishes were collected in February 2010 by local fishermen using benthic nylon gillnets with a
mesh size of 10–30 mm in four open bays (Kapara, Pusi, Olojpata and Kauta) across 20 km of
Lago Mayor (Fig. 1). The selection of this study area enabled exploration of the trophic inter-
actions in an area of high fish diversity (17 of the 26 fish species reported), maximizing the
chances of species interaction in an environment with little influence from other anthropogenic
impacts other than the presence of introduced fish species [Ministerio de la Producción de Perú
& Agencia Española de Cooperación Internacional (AECID, 2008)]. As pollution may modify
the structure of fish communities, the good water quality of the area surveyed was corrobo-
rated by determining standard water physico-chemistry variables as detailed in Table I. All fish
species were commercial species captured in the littoral area of the four bays surveyed so there
is observational evidence of the possibility of species interactions. Fishes were identified to
species level, measured (total length, LT , mm) and c. 3 cm2 of white muscle tissue was dissected
from the mid-dorsal region of each fish. As the taxonomic composition of some fish species’
diet was known, only the digestive tracts of killifish Orestias gracilis Parenti 1984, Orestias
tomcooni Parenti 1984, Orestias frontosus Cope 1876, Orestias incae Garman 1895, Orestias
uruni Tchernavin 1944 and Orestias farfani Parenti 1984 were fixed in 10% formaldehyde for
gut content analyses (Table II).
Three samples of zooplankton and benthic invertebrates were collected at each location to
determine the isotopic composition of potential fish prey. Zooplankton samples were obtained
by pulling a 250 μm mesh net through a 1 m water column and benthic macroinvertebrates were
collected using dip nets. Prey samples were then classified in three categories: zooplankton,
chironomids and amphipods. All samples were frozen at −80∘ C and transported frozen in dry
ice to the facilities at the University of Barcelona (Spain).

S A M P L E P R E PA R AT I O N A N D S TA B L E - I S O T O P E A N A LY S I S
Fish and prey samples were freeze-dried, ground to a powder and lipid-extracted with
three-baths of chloroform–methanol (2:1) (Logan et al., 2008). Samples were then homoge-
nized and weighed to the nearest μg in ultra-clean tin capsules for 𝛿 13 C and 𝛿 15 N determination
at the Scientific-Technical Services of the University of Barcelona. Samples were analysed in a

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
 1696

Table I. Geographical location of sampling sites at Lake Titicaca with information on the total number of species captured (S) and the water
quality analyses
Dissolved General Carbonate
Temperature oxygen Conductivity hardness hardness NH3 NO2 NO3 PO4
Location Latitude Longitude (∘ C)* (mg l−1 )* (𝜇S cm−1 )* pH* (∘ GH)† (∘ KH)† (mg l−1 )† (mg l−1 )† (mg l−1 )† (mg l−1 )† S

L1. Kapara 15∘ 33′ 07′′ S 69∘ 51′ 28′′ W 16⋅0 6⋅3 1304 8⋅2 17 6 0⋅1 <0⋅02 2 <0⋅20 16
L2. Pusi 15∘ 28′ 62′′ S 69∘ 53′ 43′′ W 16⋅1 6⋅2 1316 8⋅3 16 6 0⋅1 <0⋅02 3 <0⋅20 17
L3. Olojpata 15∘ 25′ 07′′ S 69∘ 51′ 47′′ W 16⋅0 6⋅3 1299 8⋅3 17 7 0⋅1 <0⋅02 3 <0⋅20 17
L4. Kauta 15∘ 23′ 02′′ S 69∘ 51′ 43′′ W 16⋅0 6⋅4 1295 8⋅3 17 7 0⋅1 <0⋅02 3 <0⋅20 17
M . M O N R O Y E T A L.

*Measured using a digital multiparametric probe YSI 553 (www.ysi.com).

†Measured using a colorimetric test kit VISOCOLOR (www.mn-net.com).

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 T RO P H I C I N T E R AC T I O N S I N L A K E T I T I C AC A F I S H E S 1697

Table II. Characterization of the fish groups (FG) based on the information provided by tra-
ditional ecological analyses through an extensive literature review. For the species whose gut
content was examined, the percentage of individuals per species for each food category is shown
FG Species Habitat Diet Source

I Orestias gilsoni Epipelagic Zooplankton Lauzanne (1982); Parenti

(1984)
I Orestias gracilis – Zooplankton (100%) Pers. obs.
I Orestias – Zooplankton (100%) Pers. obs.
tomcooni
II Orestias ispi Pelagic Zooplankton, eggs Lauzanne (1982, 1991);
Parenti (1984); Northcote
(2000); Paca et al.
(2002); Vila et al. (2007)
III Orestias mulleri Benthopelagic Zooplankton, amphipods, Lauzanne (1982, 1991);
molluscs, insects, plant Parenti (1984); Vaux
detritus et al. (1988); Loubens
(1989); Vila et al. (2007)
III Orestias – Zooplankton (85⋅71%), Pers. obs.
frontosus amphipods (71⋅42%)
III Orestias forgeti – Zooplankton Lauzanne (1982); Parenti
(1984); Vila et al. (2007)
III Orestias agassii Benthic, pelagic, Amphipods, zooplankton, Lauzanne (1982, 1991);
littoral insects, plant detritus Parenti (1984); Loubens
(1989); Paca et al.
(2002); Vila et al. (2007);
Maldonado et al. (2009)
III Orestias albus Benthopelagic Molluscs, fish, zooplankton Lauzanne (1982); Parenti
(1984); Vila et al. (2007)
III Orestias luteus Benthic Amphipods, molluscs, Lauzanne (1982, 1991);
insects, algae, eggs, fish Parenti (1984); Northcote
(2000); Vila et al. (2007);
Maldonado et al. (2009)
III Orestias Benthopelagic Amphipods, molluscs, Lauzanne (1982, 1991);
crawfordi zooplankton Parenti (1984); Vila et al.
(2007)
III Trichomycterus Benthic Amphipods, molluscs, Paca et al. (2002); Vila
rivulatus insects, eggs, algae, plant et al., 2007
detritus
III Orestias incae Benthopelagic Molluscs (100%), insects Parenti (1984); pers. obs.
(33⋅33%)
IV Orestias uruni Littoral Insects (100%), molluscs Parenti (1984); pers. obs.
(83⋅33%), amphipods
(66⋅66%), zooplankton
(50%)
IV Orestias farfani – Insects (100%), amphipods Pers. obs.
(88⋅88%)
V Odontesthes Pelagic, littoral Amphipods, zooplankton, Vaux et al. (1988); Vila
bonariensis insects, fish et al. (2007)
VI Oncorhynchus Pelagic, littoral Amphipods, zooplankton, Loubens (1989); Vila et al.
mykiss insects, fish, frogs (2007)

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
1698 M . M O N R O Y E T A L.

Thermo-Finnigan Flash 1112 (CE Elantech; www.ceelantech.com) elemental analyser coupled

to a Delta-C isotope ratio mass spectrometer via a CONFLO III interface (Thermo Finnigan
MAT; www.chemeurope.com). International Atomic Energy authority (IAEA) reference mate-
rials were applied and inserted every 12 samples to calibrate the system. Stable-isotope ratios of
13 C:12 C and 15 N:14 N were expressed as the relative difference per mil (‰), using the equation:

𝛿X = [RSample × (RStandard )−1 − 1] 1000, where 𝛿X is 𝛿 15 N or 𝛿 13 C, R the corresponding ratio

13 C:12 C or 15 N:14 N and R
Standard is the ratio of the international reference Vienna Pee Dee
Belemnite for carbon and air for nitrogen. Replicate assays of standards indicated analytical
measurement errors of ±0⋅1 and ±0⋅3‰ for 𝛿 13 C and 𝛿 15 N, respectively.

D ATA A N A LY S E S

Defining fish groups by integrating the information provided by traditional ecology

and stable-isotopic signatures
Homogeneous fish groups based on the information provided by SIAs and traditional ecology
were created in order to facilitate the interpretation of the graphical output and to guarantee the
accuracy of the analytical procedures used in determining niche overlap and prey–consumer
relationships. First, a 𝛿 13 C–𝛿 15 N bi-plot space was performed to group fish based on the simi-
larities in their isotopic signatures, as fishes showing similar isotopic signatures are more likely
to have similar ecological attributes. Secondly, the groups created by the isotopic space were
refined using the ecological information available for these species gathered via an extensive
literature review (Lauzanne, 1982, 1991; Parenti, 1984; Vaux et al., 1988; Loubens, 1989; North-
cote, 2000; Paca et al., 2002; Vila et al., 2007; Maldonado et al., 2009) (Table II). At this stage,
results from SIAs were visually assessed with the previous ecological knowledge about these
species. This comparison also enabled assignment of poorly studied species to the most similar
fish group in terms of isotopic signatures matching their ecological attributes (Table II). For those
species whose gut contents were analysed, the dominant prey items in the gut were determined.
After integrating this information, fishes were classified into groups as follows: (I) epipelagic
and strictly zooplanktophagous species, (II) pelagic and mainly zooplanktophagous species,
(III) benthopelagic and mainly omnivorous species and (IV) littoral species that mainly feed on
insects and amphipods. Introduced fish species were independently sorted into two groups cor-
responding to O. bonariensis (V) and O. mykiss (VI). The least abundant species (n < 5) were
removed from trophic analyses to increase the confidence of the results, although the relevant
data are shown in the 𝛿 13 C–𝛿 15 N bi-plot space (Fig. 2).

Exploring trophic interactions using SIA

Using non-parametric statistics as the data lacked normality and homogeneity of variances
according to the Shapiro and Barlett tests, the mean 𝛿 13 C and 𝛿 15 N signatures of the six fish
groups were compared using the Kruskal–Wallis (KW) test followed by Wilcoxon’s test for
pair-wise comparisons. Ontogenetic changes in the use of habitat and the trophic level were
explored using the Spearman rank correlation between LT and 𝛿 13 C and 𝛿 15 N, respectively. Iso-
topic niche overlap between fish groups was assessed using standard ellipse areas (SEA) (siber
function in R; www.r-project.org) (Jackson et al., 2011). Briefly, ellipses reflect the covariance
of the 𝛿 13 C and 𝛿 15 N signatures, and are defined by their shape and size, with the mean of the
isotopic signatures determining their location in the bi-plot isotopic space (Jackson et al., 2011).
Unlike the Convex Hull method (Layman et al., 2007), SEA is recommended when there is no
balanced design (i.e. sample sizes vary between fish groups) and sample sizes are small (<50
samples) (Jackson et al., 2011; Syväranta et al., 2013).
The relative contribution of the four native fish groups and invertebrate sources in the diet of
the two introduced fish species was explored using Bayesian mixing models (siar function in
R) (Parnell et al., 2008). The Bayesian approach incorporates more sources of variability and
estimates various prey items ingested by a consumer, thus offering a more reliable picture of its
diet (Jackson et al., 2011). This modelling approach, however, is very sensitive to differences
in the trophic discrimination factors exhibited by consumers (McCutchan et al., 2003; Bond
& Diamonds, 2011). As there was no previous study addressing the discrimination factor of

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 T RO P H I C I N T E R AC T I O N S I N L A K E T I T I C AC A F I S H E S 1699

14

12 II

I isp III
10
gra aga
fro
8 tom mul riv
for
IV
15N

gil
cra linc
6 alb lut far

uru
4

amp
2 chi
zoo
0
–21 –20 –19 –18 –17 –16 –15 –14
13C

Fig. 2. Mean 𝛿 15 N and 𝛿 13 C values of 15 native fish species from Lake Titicaca, the two introduced species and
potential invertebrates used as food by these fish species. Native fish species were classified into groups
to facilitate the interpretation of the analyses: (I) composed of Orestias gracilis (gra), Orestias tomcooni
(tom) and Orestias gilsoni (gil); (II) containing Orestias ispi (isp); (III) Orestias frontosus (fro), Orestias
mulleri (mul), Orestias forgeti (for), Orestias luteus (lut), Orestias crawfordi (cra), Orestias incae (inc),
Trichomycterus rivulatus (riv); (IV) composed of Orestias uruni (uru) and Orestias farfani (far). repre-
sent food sources such as chironomids (chi), amphipods (amp) and zooplankton (zoo). Note that large and
small (<90 mm total length) individuals of Odontesthes bonariensis (group V) are shown ( , ), and that
Oncorhynchus mykiss (group VI) is shown ( ). Error bars are s.d.

O. bonariensis, the accuracy of results was increased assuming the discrimination factors of
2⋅33 for 𝛥𝛿 15 N and 2⋅22 for 𝛥𝛿 13 C observed in O. mykiss for the mixing model (Pinnegar &
Polunin, 1999). The different trophic behaviour of large and small specimens of O. bonarien-
sis was studied with individuals >90 mm likely to be mainly piscivorous (Fig. 2). The dietary
preferences for O. mykiss were only explored in large specimens because collected individuals
were all adults of >149 mm. All statistical analyses were performed in R 2.15 (R Development
Core Team; www.r-project.org) and the libraries MASS (Venables & Ripley, 1999) and SIAR
(Parnell et al., 2008) with default settings. The level of significance was 𝛼 = 0⋅05.

RESULTS

F I S H G RO U P S D E F I N E D B Y T R A D I T I O NA L E C O L O G I C A L
K N O W L E D G E A N D S TA B L E - I S O T O P I C S I G N AT U R E S
A total of 150 individuals belonging to 17 fish species were captured in the littoral
area of Lake Titicaca with the aim of addressing the food web structure of the littoral
fish community. Based on the information provided by traditional ecological stud-
ies and the species location within the 𝛿 13 C–𝛿 15 N bi-plot space, the 17 fish species
captured were sorted into six groups: the epipelagic planktophagous group (I) com-
posed of O. tomcooni, Orestias gilsoni Tchernavin 1944 and O. gracilis; the pelagic
and mainly zooplanktophagous group (II) containing Orestias ispi Lauzanne 1981; the

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
1700 M . M O N R O Y E T A L.

benthopelagic group composed of mainly omnivorous species (III) formed by O. incae,

T. rivulatus, Orestias crawfordi Tchernavin 1944, Orestias luteus Valenciennes 1846,
Orestias albus Valenciennes 1846, Orestias agassii (Valenciennes 1846), Orestias for-
geti Lauzanne 1981, Orestias mulleri Valenciennes 1846 and O. frontosus; the littoral
shallow-water group that mainly feed on insects and amphipods (IV) composed of O.
farfani and O. uruni and the introduced fish species, O. bonariensis and O. mykiss, in
groups V and VI, respectively (Fig. 2).
The designation of six fish groups was supported by the significant differences
observed in the mean 𝛿 13 C (KW, 𝜒 2 = 101⋅81, d.f. = 5, P < 0⋅001) and 𝛿 15 N signatures
(KW, 𝜒 2 = 62⋅28, d.f. = 5, P < 0⋅001). Specifically, the 𝛿 15 N values did not differ
significantly between most native groups, possibly because all such fish species have
a similar trophic spectrum (e.g. amphipods and zooplankton) as demonstrated by
gut content analyses. Introduced fish species showed the highest 𝛿 15 N values with
evidence of ontogenetic dietary changes in O. bonariensis (Spearman’s 𝜌 = 0⋅81,
n = 26, P < 0⋅001). In particular, individuals <90 mm showed lower 𝛿 15 N values than
larger individuals (W = 160, P < 0⋅001) (Fig. 2). For 𝛿 13 C, there were significant
differences between all fish groups with the exception of groups containing the pelagic
species (II) and the introduced O. mykiss (VI) (P > 0⋅05), possibly because variation
in the foraging areas exists for most fish groups. Nonetheless, variation in 𝛿 13 C also
had an ontogenetic component because there was a negative relationship between LT
and 𝛿 13 C for O. ispi (Spearman’s 𝜌 = −0⋅84, n = 8, P < 0⋅01), O. gracilis (Spearman’s
𝜌 = −0⋅76, n = 10, P < 0⋅01), O. gilsoni (Spearman’s 𝜌 = −0⋅66, n = 10, P < 0⋅05)
and O. bonariensis (Spearman’s 𝜌 = −0⋅39, n = 26, P < 0⋅05). For O. bonariensis, the
increase in size was also related to an increase in 𝛿 15 N (Table III).

E X P L O R I N G N I C H E O V E R L A P A N D P R E Y–C O N S U M E R
R E L AT I O N S H I P S U S I N G S I A S
Bayesian ellipse analyses revealed that large O. bonariensis overlapped with pelagic
fishes (group II) by 39% and that small O. bonariensis overlapped with benthopelagic
fishes (group III) by 31⋅57%. These analyses also showed an overlap of 19⋅66%
between O. mykiss and pelagic fishes (group II). The location of O. bonariensis and O.
mykiss along the 𝛿 13 C axis demonstrated the existence of spatial segregation (Fig. 2).
In this regard, large O. bonariensis 𝛿 13 C values were similar to those observed in
pelagic (group II) and benthopelagic fishes (group III), while O. mykiss 𝛿 13 C values
were more similar to those observed in pelagic fishes (group II) (P > 0⋅05 for all
pair-wise comparisons). In order to determine the relative contribution of native fish
groups to the diet of the two introduced fish species, Bayesian mixing models were
applied including four native fish groups and invertebrate items as food sources.
The results revealed that pelagic fish groups are likely to dominate the diet of large
specimens of both introduced fish species, while chironomids appear to be the main
prey item for small O. bonariensis (Fig. 3).

DISCUSSION
This study examined the trophic interactions between 15 native fish species and
two introduced fish species in a littoral area of Lake Titicaca illustrating that SIAs

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 T RO P H I C I N T E R AC T I O N S I N L A K E T I T I C AC A F I S H E S 1701

Table III. List of species and total number of fish captured (n) for each species in Lake Tit-
caca, mean fish length (LT ) and size range (minimum–maximum). The relationship between
LT and 𝛿 13 C or 𝛿 15 N (mean ± s.d.) is also indicated using the Spearman rank correlation
coefficient 𝜌*
LT LT range
Species n (mm) (mm) 𝛿 13 C (‰) 𝜌 P-value 𝛿 15 N (‰) 𝜌 P-value

Orestias gilsoni 10 95⋅05 56–112 −20⋅07 ± 0⋅38 −0⋅66 <0⋅05 8⋅88 ± 1⋅31 −0⋅45 >0⋅05
Orestias gracilis 10 52⋅07 47–57 −20⋅10 ± 0⋅52 −0⋅76 <0⋅05 9⋅01 ± 0⋅66 0⋅40 >0⋅05
Orestias 9 48⋅67 39–55 −19⋅72 ± 0⋅45 0⋅38 >0⋅05 8⋅51 ± 0⋅76 0⋅19 >0⋅05
tomcooni
Orestias ispi 8 83⋅80 80–90 −18⋅82 ± 0⋅71 −0⋅84 <0⋅01 10⋅24 ± 0⋅67 −0⋅55 >0⋅05
Orestias mulleri 6 81⋅80 70–91 −18⋅35 ± 0⋅57 0⋅57 >0⋅05 8⋅12 ± 0⋅61 −0⋅28 >0⋅05
Orestias 7 144⋅57 128–168 −18⋅42 ± 0⋅66 0⋅50 >0⋅05 8⋅43 ± 0⋅64 −0⋅32 >0⋅05
frontosus
Orestias forgeti 4 62⋅60 60–65 −18⋅29 ± 0⋅27 0⋅20 >0⋅05 7⋅77 ± 0⋅23 0⋅00 >0⋅05
Orestias agassii 7 134⋅27 112–163 −18⋅09 ± 1⋅08 −0⋅60 >0⋅05 8⋅8 ± 1⋅53 0⋅67 >0⋅05
Orestias albus 10 125⋅40 84–167 −17⋅41 ± 0⋅84 −0⋅29 >0⋅05 8⋅48 ± 1⋅93 0⋅59 >0⋅05
Orestias luteus 10 134⋅92 115–148 −17⋅30 ± 0⋅83 −0⋅21 >0⋅05 7⋅30 ± 0⋅81 −0⋅23 >0⋅05
Orestias 9 76⋅30 59–85 −16⋅81 ± 0⋅48 0⋅19 >0⋅05 7⋅40 ± 0⋅49 −0⋅15 >0⋅05
crawfordi
Trichomycterus 8 134⋅12 65–159 −16⋅90 ± 0⋅88 −0⋅49 >0⋅05 7⋅94 ± 1⋅14 −0⋅01 >0⋅05
rivulatus
Orestias incae 3 52⋅33 48–60 −16⋅37 ± 0⋅17 −0⋅50 >0⋅05 6⋅64 ± 0⋅37 1⋅00 >0⋅05
Orestias uruni 6 42⋅16 39–46 −15⋅88 ± 0⋅42 −0⋅60 >0⋅05 5⋅87 ± 0⋅42 0⋅75 >0⋅05
Orestias farfani 9 135⋅66 95–162 −15⋅10 ± 0⋅48 −0⋅68 0⋅05 6⋅41 ± 0⋅52 −0⋅43 >0⋅05
Odontesthes 26 158⋅00 40–422 −17⋅33 ± 1⋅51 −0⋅39 <0⋅05 10⋅76 ± 0⋅86 0⋅81 <0⋅001
bonariensis
Oncorhynchus 8 200⋅00 149–268 −18⋅98 ± 0⋅48 −0⋅16 >0⋅05 10⋅40 ± 0⋅61 0⋅47 >0⋅05
mykiss

*Note that this list includes two species removed from ellipse analyses due to the reduced sample size (n < 5).

and traditional ecological studies can give complementary information about studies
of food web structure (Vander Zanden & Vadeboncoeur, 2002; Clarke et al., 2005;
Quevedo et al., 2009). Additionally, this is the first time that ecological attributes of
six native species are explored to investigate the differential effect of the introduction
of O. bonariensis and O. mykiss on the native fish community.

N I C H E PA RT I T I O N I N G O F T H E N AT I V E F I S H C O M M U N I T Y
As predicted, most of the native fish groups showed a similar trophic level with
marked spatial segregation, possibly associated with a decrease in trophic competition.
The pelagic native O. ispi was the only native fish group showing higher 𝛿 15 N values
than the remaining native fish groups. This could be attributed to the occasional con-
sumption of eggs by this fish species (Paca et al., 2002). An additional reason for the
high 𝛿 15 N of O. ispi might be that the pelagic condition enables O. ispi to feed on prey
items from food webs differing in baseline levels of 𝛿 15 N, which may lead to a higher
𝛿 15 N (Hobson, 1999; Vander Zanden & Rasmussen, 2001; Bearhop et al., 2004).
Likewise, the large deviations observed in the 𝛿 13 C values of T. rivulatus, O. luteus
and O. agassii could be associated with the consumption of prey from different feeding
grounds. The current findings may be related to the fact that different morphotypes have

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
1702 M . M O N R O Y E T A L.

(a) 1

0·8

0·6

0·4

0·2

(b) 1

0·8
Proportion

0·6

0·4

0·2

0
I I III
n ds od
s
oup upI p p IV
nkto o mi ip
Gr ro ou ou pla on ph
G Gr Gr o ir m
Zo Ch A
(c) 1

0·8

0·6

0·4

0·2

0
Zooplankton Chironomids Amphipods
Source

Fig. 3. Density plot showing the confidence intervals (95, 75 and 50%) of the relative proportion of Lake Titicaca
fish groups consumed by (a) Oncorhynchus mykiss, (b) Odontesthes bonariensis and (c) O. bonariensis
<90 mm in total length. Values were corrected by a discrimination factor of 2⋅33‰ for 𝛿 15 N and 2⋅22‰ for
𝛿 13 C according to Pinnegar & Polunin (1999).

been described for these three species, being particularly evident for O. agassii, but
somewhat difficult to differentiate in all cases by gross morphological examination due
to hybridization (Parenti, 1984; Lauzanne, 1991; Maldonado et al., 2009). Nonetheless,
certain trophic specialization appears to be common in native ichthyofauna because an
exhaustive morphological study of the native species’ feeding structures (e.g. pharyn-
geal dentition and gill raker number) appears to support the notion that Orestias species
could prey on different zooplankton groups (Northcote, 2000). In turn, although details
regarding the trophic specialization of the native fish species were not identified in

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
 T RO P H I C I N T E R AC T I O N S I N L A K E T I T I C AC A F I S H E S 1703

this study, the variations in the isotopic signatures may be related to foraging special-
izations already reported in fish fauna from other great lakes [e.g. cichlids from Lake
Malawi or Lake Victoria, Fry et al. (1999); Balirwa et al. (2003), respectively].
Whilst all native species considered in this study were described as littoral species
with the exception of O. ispi, the low 𝛿 13 C values observed in the epipelagic group (I)
could be compatible with its pelagic condition. Although the reasons behind these low
𝛿 13 C values remain largely unknown due to poor knowledge about the ecology of these
species, their pelagic condition appears to be supported by the similarities observed
between the 𝛿 13 C values of these species and those of the pelagic O. ispi (Parenti,
1984; Vaux et al., 1988; Villwock, 1998; Vila et al., 2007). An alternative explanation
for the low 𝛿 13 C values of this fish group is that all these fish species are strictly zoo-
planktivorous, and zooplanktonic organisms often display high variation in 𝛿 13 C values
(Cabana & Rasmussen, 1996). Therefore, the 𝛿 13 C values of species belonging to the
epipelagic group may be driven by the isotopic signature of zooplankton even when all
three species inhabit the littoral area. This is consistent with the pelagic condition of O.
ispi because this species has been observed foraging in the littoral area, so the relative
contribution of littoral and pelagic items to its diet may explain the similar isotopic
composition of this group and littoral species (Villwock, 1998).
The isotopic composition of the native species was also related to ontogenetic
changes in the species’ use of foraging grounds. As the 𝛿 13 C gradient is associated
with enrichment in 𝛿 13 C in littoral primary producers relative to offshore producers
(France, 1995), fish species that move from littoral areas to offshore areas when they
age show a negative relationship between LT and 𝛿 13 C. The negative relationship
observed between LT and 𝛿 13 C for O. ispi, O. gracilis, O. gilsoni and O. bonariensis
supports the field observation suggesting that the littoral macrophyte carpet is cru-
cial for the juvenile stages of fish species at Lake Titicaca, even for the introduced
O. bonariensis.

T R O P H I C I N T E R A C T I O N S B E T W E E N N AT I V E A N D
I N T RO D U C E D F I S H S P E C I E S
As previously reported (Vaux et al., 1988; Loubens, 1989), O. bonariensis and O.
mykiss are the top fish predators at Lake Titicaca. As large specimens of both intro-
duced fish species appear to prey mainly on native pelagic fish groups, O. bonariensis
and O. mykiss appear to depict spatial segregation according to 𝛿 13 C, thus minimiz-
ing trophic interactions. In addition, Bayesian ellipse analyses suggested that large O.
bonariensis specimens show niche overlap with the pelagic native group II. This inter-
action appears to be explained by a predator–prey relationship as suggested by the
mixing model analyses. Interestingly, the current findings suggested that O. bonarien-
sis become mainly piscivorous when smaller than the 200 mm LT suggested by Vaux
et al. (1988).
Unlike in O. bonariensis, the changes in the foraging behaviour of O. mykiss were not
addressed because all individuals captured were within the size range of piscivorous
individuals (149–268 mm) reported in previous studies and consistent with the 𝛿 15 N
values observed in this study. Lauzanne (1991) reported that O. mykiss mainly feeds on
O. ispi and O. agassii at Lake Titicaca. A prey–consumer interaction, however, would
be represented by an increase in the 𝛿 15 N value of O. mykiss compared to that of these
two species. The current findings instead only demonstrated an overlap in the isotopic

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 85, 1693–1706
1704 M . M O N R O Y E T A L.

signatures of O. mykiss and O. ispi. As stated above, this might be explained by the
nature of the O. ispi diet coupled with the fact that the isotopic signature of both fish
species may be more influenced by the pelagic than the littoral food chain (Quevedo
et al., 2009).
In conclusion, this study reveals a trophic link between native and introduced species
and demonstrates the utility of combining both SIA and traditional ecological knowl-
edge to understand trophic relationships in fish communities with a pool of species
exhibiting similar feeding habits.

The authors would like to thank the staff of the Binational Special Project of Lake Titicaca
(PELT), the Sea of Peru Institute (IMARPE) and the Lake Titicaca Agency (ALT) for their
invaluable field and laboratory assistance, especially O. Flores and E. Yujra. Funding support
was provided by the Spanish Agency for International Cooperation and Development (AECID)
and the Department of Production of Peru and by I. González, K. Su and J. Vera provided valu-
able logistic support. The authors are also grateful to F. Ramírez, L. Jover, E. García-Galea,
T. Militão, R. Ramos and M. García for useful suggestions on the isotopic analysis and data
interpretation, as well as to T. Evans from the Language Service at the University of Barcelona
for revising the English. A.M.-V. was funded by a Marie Curie Fellow (EC-Funded ‘Para-Tox’
project no. 327941).

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 Science of the Total Environment 487 (2014) 233–244

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Metal concentration in water, sediment and four fish species

from Lake Titicaca reveals a large-scale environmental concern
Mario Monroy a,⁎, Alberto Maceda-Veiga b, Adolfo de Sostoa a
a
Department of Animal Biology and Biodiversity Research Institute (IRBIO), University of Barcelona, E-08028 Barcelona, Spain
b
Cardiff School of Biosciences, Cardiff University, CF10 3AX Cardiff, United Kingdom

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Pb content in water exceeded interna-

tional safety thresholds.
• Sediments enriched in organic matter
are a good sink for metals.
• High levels of Cu, Zn, Cd and Hg were
found in 4 fish species from Lake Titicaca.
• Benthopelagic species showed highest
metal concentrations.
• The metal bioaccumulation pattern was
mainly associated with tissue and species.

a r t i c l e i n f o a b s t r a c t

Article history: Although intensive mining activity and urban sewage discharge are major sources of metal inputs to Lake
Received 28 February 2014 Titicaca, the risk posed by metal pollution to wildlife and human populations has been poorly studied. In this
Received in revised form 28 March 2014 study we compared the concentrations of Cu, Zn, Cd, Hg, Pb, Co, and Fe in water, sediment, and two tissues
Accepted 29 March 2014
(liver and muscle) of four fish species (Odontesthes bonariensis, Orestias luteus, Orestias agassii, and Trichomycterus
Available online xxxx
rivulatus) across important fishery areas in Lake Titicaca. The concentration of Pb in water at the discharge sites of
Editor: D. Barcelo the main rivers and of most elements, with the exception of Co and Fe, in all fish collected in this study exceeded
the safety thresholds established by international legislation. The highest metal concentrations were observed in
Keywords: benthopelagic species, and liver tissue was identified as the main depository for all metals with the exception of
Endemic fish mercury. The metal bioaccumulation pattern in fish was weakly related to the metal concentrations in the
Mining contamination environment with the exception of Hg at the most polluted location, partly explained by the different metabolic
Fisheries role of essential and non-essential elements and the influence of other factors such as species' ecology and
Heavy metals individual traits in the bioaccumulation of most metals. As metal pollution extended across the study area and
Bioaccumulation
high metal concentrations were detected in all four fish species, we urge the authorities to enforce legislation
for water and fish consumption and to evaluate the effects of metal pollution on fish health.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. Tel.: +34 93 402 1041; fax: +34 93 403 4426. The conservation of inland fisheries is an ecological, economic and
E-mail address: monroylopezmario@gmail.com (M. Monroy). social concern given the conservation status of many fish species and

http://dx.doi.org/10.1016/j.scitotenv.2014.03.134
0048-9697/© 2014 Elsevier B.V. All rights reserved.
234 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244
the strong dependence of human populations living around lakes on fish
resources as a major source of protein (Welcomme, 2001). Common
threats to inland fisheries are overharvesting, the introduction of exotic
species and a wide range of anthropogenic habitat disturbances such as
water pollution (Allan and Flecker, 1993; Dudgeon et al., 2006). The
input of sewage discharge to rivers is often the main input of pollution
into large lakes (Carpenter and Cottingham, 2002; Dudgeon et al.,
2006). To assess the consequences of sewage pollution for wildlife and
humans while increasing our understanding about pollutant dynamics
in lakes, it is crucial to determine the variation in the concentration of
contaminants over space and across ecosystem compartments.
The effect of sewage discharge on aquatic ecosystems primarily
depends on the mixture of organic (e.g. household products, drugs)
and inorganic (e.g. acids, heavy metals) contaminants that they contain
(Pinto, 2009). Heavy metals are of particularly high environmental risk
because of their long-term persistence in nature and possible bioaccu-
mulation and biomagnification (Demirak et al., 2006; Ivanciuc et al.,
2006; Agah et al., 2009; Uysal et al., 2009). Metal sources other than sew-
age are atmospheric deposition, geologic weathering and the run-off
from adjacent agricultural areas (e.g. pesticides, fertilizers) (Demirak
et al., 2006; Uysal et al., 2009; Bereswill et al., 2013). However, elements
such as copper (Cu), zinc (Zn), cobalt (Co) and iron (Fe) cannot be con-
sidered a water quality hazard unless they reach high concentrations
since they are necessary for animal life (Miller et al., 1992; Canli and
Atli, 2003). In contrast, other elements such as cadmium (Cd), mercury
(Hg) and lead (Pb) always behave as toxic elements in organisms
(Miller et al., 1992; Amundsen et al., 1997; Kojadinovic et al., 2007).
Sediments are the main sink of metals in lakes and sediment trans-
port along the upstream–downstream river gradient, especially in high
flow periods, is one of the main pathways of metal input into these
ecosystems (Alloway, 2013). Because sediments concentrate metals
and the concentration of these elements in sediment is less variable
than in water, sediments are suitable for monitoring the long-term
metal deposition in ecosystems (MacDonald et al., 2000; Alloway,
2013). However, measuring metal concentrations either in water or
sediment does not provide information on the risk posed by metal bioac-
cumulation or biomagnification (Ricart et al., 2010; Maceda-Veiga et al.,
2013). These processes are firstly driven by metal availability for the
biota (i.e. bioavailability), which in turn is related to water variables,
such as pH, oxygen concentration, water hardness and temperature,
and sediment characteristics, such as organic carbon content (Kotze
et al., 1999; Canli and Atli, 2003; Adhikari et al., 2009). However, species
traits such as trophic position, age, body size or home range also modify
metal bioaccumulation patterns (Mason et al., 2000; Gammons et al.,
2006; Kojadinovic et al., 2007), illustrating that a combination of sentinel
species with different ecological attributes will provide the best picture
of the risk posed by metal pollution for the biota (Jorgensen, 2011).
Fish are suitable bioindicators for metal pollution because they occupy
a range of trophic levels and they have a known ability to concentrate
pollutants (e.g. pesticides, biphenyls, heavy metals) (Manirakiza et al.,
2002; Bervoets and Blust, 2003; Agarwal et al., 2007). The life span of
fish also enables detection of the consequences of pollution over long
time periods compared to other bioindicators, such as macroinverte-
brates and diatoms (Jorgensen, 2011). In addition, because many species,
including humans, consume fish as part of their diet, fish reflect best the
consequences of metal pollution in lakes for wildlife and humans.
Although metal bioaccumulation patterns have been described for
many freshwater fish species (e.g. Oreochromis mossambicus, Kotze
et al., 1999; Tilapia nilotica, Rashed, 2001), even lakes with similar inputs
of metals can produce fish with widely different metal concentrations,
due to variation in biological, geochemical, and environmental factors
that affect metal uptake and accumulation (Chen et al., 2000; Ward
et al., 2012).
The situation in Lake Titicaca typifies the global decline in inland
fisheries due to anthropogenic perturbations, including the discharge
of mining waste, with a 45% reduction in native fish biomass over the
last 30 years, and many species now listed as threatened according to
the International Union for the Conservation of Nature (Loubens, 1989;
Revollo et al., 2003; Vila et al., 2007; Gobierno Regional de Puno, 2008;
Aliaga and Segura, 2013). This lake is an important breeding and winter-
ing area for up to 60 bird species (e.g., sandpipers, egrets, waterfowl,
grebes) including some endemisms such as the titicaca flightless grebe
(Rollandia microptera). In addition 18 amphibian species inhabit this
lake, including the endemic Titicaca water frog Telmatobius culeus, and
a growing number of fish species, currently standing at 26 species, pend-
ing genetic confirmation (Lauzanne, 1991; Sarmiento and Barrera, 2003;
Costa, 2003; Vila et al., 2007; Gobierno Regional de Puno, 2008). The
native fish community is dominated by members of the killifish genus
Orestias (Teleostei, Cyprinodontidae) and the catfish Trichomycterus
rivulatus, with no marked breeding season (Vila et al., 2007). Fisheries
resources are economically important as a source of income and food
for local communities around Lake Titicaca. In addition Lake Titicaca
provides three million people with drinking water, which is also used
for agricultural purposes (Revollo et al., 2003).
Previous studies addressing the environmental aspects of metal pollu-
tion in Lake Titicaca have focused on mercury in its main tributary, the
Ramis River (Gammons et al., 2006), but a more comprehensive study
assessing the magnitude of metal pollution in this area has been lacking.
The present study compared the concentrations of Cu, Cd, Zn, Hg, Pb, Co
and Fe in water, sediment and fish across the main fishery areas in Lake
Titicaca. Since the main rivers have been portrayed as the main sources
of metal pollution, we expected that sediments and fish at the discharge
sites would have the highest metal concentrations, and that the metal
concentrations in fish would be in turn driven by environmental (organic
matter content) and species traits (size, age, species ecology). The senti-
nel species were the native and benthopelagic killifishes Orestias agassii
and Orestias luteus, the native and benthonic catfish T. rivulatus, and the
introduced and pelagic silverside Odontesthes bonariensis. All species are
omnivorous with the exception of silverside, which mainly feeds on
macroinvertebrates or fish depending on the silversides' size (Vaux
et al., 1988; Vila et al., 2007). Previous studies have suggested that
metal concentration is highest in species living close to the sediment or
in species feeding on high trophic levels (Roméo et al., 1999; Mason
et al., 2000; Agarwal et al., 2007; Kojadinovic et al., 2007; Yilmaz et al.,
2007; Agah et al., 2009). Therefore, we predicted that benthonic and
fish predators would have the highest metal load. Finally, we discuss
the implications of our findings for the development of management
strategies at Lake Titicaca.
2. Material and methods
2.1. Study area
Lake Titicaca, located between Peru and Bolivia at 3810 m above sea
level, is the largest freshwater lake in South America with 8559 km2 of
surface area and a water volume of 932 km3. The lake is divided into
two large water bodies, the “Lago Mayor” and the “Lago Menor”, with
a maximum depth of approximately 280 and 40 m, respectively. Lake
Titicaca is a cold oligotrophic lake with a high salt concentration due
to the geology of the basin (e.g. carbonate/bicarbonates, chlorides)
(Arze and Quintanilla, 1991). The water chemistry is relatively con-
stant across seasons and sites, with the exception of the rainy period
between December and March, and those sites close to the mouth of
the main tributaries, the Ramis, Coata and Ilave Rivers (Vila et al.,
2007). These rivers represent the main source of metal pollution in
the Lake due to the location of mining areas upstream (Gammons
et al., 2006). However, agricultural areas close to the rivers or sur-
rounding Lake Titicaca could be potential source of contamination
(e.g. pesticides, fertilizers).
In order to gain an overall picture of the metal pollution in Lake
Titicaca, we collected water, sediment and fish from two supposed
non-polluted areas (R1: Huencalla Bay and R2: Tamán Bay) and seven
 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244
known polluted locations (L1: Mouth of Ilave River, L2: Barco-Chucuito,
L3: Mouth of Ramis River, L4: Pusi, L5: Mouth of Coata River, L6:
Mohokachi and L7: Inka Chaka Bay) across the main fishery areas of
Lake Titicaca in November 2010 (Fig. 1). All sampling sites were located
in Lago Mayor with the exception of L6 which was located in Lago Menor.
These sampling sites were selected based on an extensive literature
review, interviews with local communities and our personal observations
(Supplementary material: Table S1).
2.1.1. Water quality and sediment characteristics
General water quality variables were determined at each sampling
site using a digital YSI® 556 MPS multiprobe for temperature (°C),
conductivity (μS cm−1), dissolved oxygen (mg L−1) and pH, and the
colorimetric test kit VISOCOLOR® for ammonia (NH3, mg L−1), nitrite
(NO2, mg L−1), nitrate (NO3, mg L−1) and phosphate (PO4, mg L−1)
concentrations, and general (°GH) and carbonate hardness (°KH). For
metal analysis, triplicate water samples were collected at each location
in 250-mL polyethylene bottles pre-cleaned with nitric acid (10%) for
24 h and rinsed three times with ultrapure water. Water samples were
immediately filtered through a 0.45-μm Millipore membrane filter and
kept acidified with nitric acid (pH ~2) until metal concentrations were
determined (see below).
At each sampling three random sediment samples were collected at
a 10-m deep in the midregion of the nine bays surveyed using a grab
sampler of 325 cm2. These samples were air-dried and sieved through
a 100-μm mesh. pH was determined using a Thermo pH Orion Star
A221 in a suspension of 10 g of soil and 25 mL of distilled water that
was shaken for 15 min and left to settle for 30 min (Pansu and
Gautheyrou, 2007). In addition, three subsamples (~1.5 g) per sampling
site were combusted at 540 °C for 4 h to determine the percentage of
organic matter content by measuring the weight difference before and
after combustion.
Fig. 1. Location of the two reference sites (R1 and R2) and the seven known polluted sites (L1–L7) in Lake Titicaca where metal analyses were conducted in water, sediment and fish for the
current study.
Original map provided by National Aeronautics and Space Administration (NASA®).
235
2.2. Fish sampling and collection of biological data
Fish were collected by local fishermen using gillnets with a mesh-
size ranging from 10 to 30 mm length, set overnight in each bay. All
individuals were identified to species level and a random sub-sample
of 10 individuals per species at each location was used for metal analy-
sis. As the juveniles of these species (b90 mm total length, LT) could not
be reliably identified, they were excluded from the analyses. The ten fish
used for metal analyses were euthanized with an overdose of MS-222
(3-aminobenzoic acid ethyl ether, Sigma-Aldrich®, see Noga, 2010 for
details), measured to the nearest mm (LT) and weighed to the nearest
0.01 g (total body wet weight, WT). Fish were then dissected, sexed
by a visual gross examination of gonads and a sample of muscle
(ca. 500 mg) below the dorsal fin and the whole liver were stored for
metal analyses in polypropylene vials previously pre-cleaned with nitric
acid (10%) and rinsed three times in water. Muscle was selected to
determine the risk posed by metal pollution to humans and liver
because it is a key organ in detoxification processes and is targeted in
metal accumulation (Miller et al., 1992). For age determination six to
eight scales per fish were also collected from above the lateral line and
posterior to the dorsal fin area (see Carmona-Canot et al., 2011 for
details on ageing criteria using scales). Scales were cleared in KOH for
12 h, rinsed in distilled water and examined using a Microbox® reader
for the determination of year annuli. For T. rivulatus, which lacks scales,
we used the age groups established by Paca et al. (2003) based on fish
length histograms as a proxy measure of fish age since alternative
tissues for fish ageing, such as otoliths, could not be examined due to
logistic constraints.
2.3. Determination of metal concentration in water, sediment and fish
Water, sediments and fish samples were frozen at − 20 °C,
transported in polystyrene boxes embedded in dry ice to the
236 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244
University of Barcelona (Spain) and kept for 1 month at the same tem-
perature until analysis. Metal concentrations in water were directly
determined in acidified solutions prepared in the field. For sediments a
total attack with aqua regia was carried out by adding 6 mL HCl and
2 mL HNO3 to 500 mg of well-homogenised sediment sample. This mix-
ture was digested in closed 60-mL Teflon vessels using a Milestone Ethos
Plus microwave digestion system at 200 °C following ISO 11466. Fish
tissues were first freeze-dried and ground to a powder to facilitate
homogenisation. Fish samples were then acid-digested in an oven at
90 °C for 12 h according to their weight as follows: 3 mL HNO3 and
1 mL H2O2 added to 100 mg tissue, 2 mL HNO3 and 1 mL H2O2 added
to samples weighing over 50 mg, and 1 mL HNO3 and 0.5 mL H2O2
added to samples weighing under 50 mg, using reagents of Instra quality
(J. T. Baker®).
Metal concentrations were determined at the Technical Services of the
University of Barcelona using a PerkinElmer OPTIMA-3200 RL Inductively
Coupled Plasma Optical Spectrometer (ICP-OES) for iron (Fe, μg L−1) and
a PerkinElmer ELAN-6000 Inductively Coupled Plasma Mass Spectrome-
ter (ICP-MS) for zinc (Zn, μg L−1), cadmium (Cd, μg L−1), total mercury
(Hg, μg L−1), lead (Pb, μg L−1), and cobalt (Co, μg L−1), using standard
procedures (Maceda-Veiga et al., 2013 for details). Blanks and a certified
reference material for soils (WQB1, National Research Institute, Canada)
and fish (DORM-3, National Research Council Canada) were also proc-
essed in each batch of digestions to provide quality control data. The
metal concentrations in blanks were always below the detection limits,
and the metal recovery rate for soil and fish samples was always above
90% (see Supplementary material: Table S2 for details). Metal concentra-
tions under the detection limits were replaced by half the detection limit
before the statistical analysis (Maceda-Veiga et al., 2012, 2013). Heavy
metal concentrations in sediments and fish are presented as the mean
± standard deviance (SD) in mg g−1 on a dry weight (dw) basis. We
also estimated the water content of both tissues to facilitate the conver-
sion of metal concentration from dry to wet weight (24.58%, 23.48%,
23.65% and 25.76% for muscle and 25.82%, 24.45%, 24.78% and 27.12%
for liver of O. bonariensis, O. luteus, O. agassii and T. rivulatus, respectively),
based on 10 samples of each tissue and species.
2.4. Data analysis
Water, sediment and fish variables were tested for normality and
homogeneity of variances using Shapiro–Wilk and Levene's tests,
respectively, and parametric and non-parametric statistics were then
applied accordingly. The mean metal concentrations either in water or
sediment and sediment features (organic matter and pH) were com-
pared between locations using a one-way ANOVA followed by Tukey
HSD post-hoc test for pair–site comparisons. The relationships between
metal concentrations, other water chemistry variables (temperature,
conductivity, dissolved oxygen, pH, general and carbonate hardness,
and ammonia, nitrite, nitrate and phosphate concentrations) and sedi-
ment characteristics were explored using Spearman's rank correlation
analysis (rho). Differences in mean metal concentrations between spe-
cies, tissues and locations were compared using a three-way ANOVA.
Where significance was observed, one-way ANOVA was applied follow-
ed by Tukey HSD post-hoc test for paired comparisons between loca-
tions or species. As LT and WT were strongly correlated (Pearson's
correlation coefficient, r N 0.9), LT was considered in this study as a
more direct measure of fish size than weight. To explore the relative
influence of fish attributes on the observed metal bioaccumulation
pattern in fish, sampling site, tissue, species, age, sex and LT was com-
bined in a general lineal model (GLM) fitted to each metal concentration
(log-transformed) with a Gaussian error distribution. The best models
were selected using a manual stepwise backward deletion of non-
significant terms from the full global models containing all fixed factors
and interactions (i.e. including covariate effects). The significance of
each factor was checked using an F-test (‘Anova’ function in R). Finally,
to describe the main sources of variation in metal concentrations in fish
and sediments, two principal component analyses (PCAs) were indepen-
dently applied to each metal data set. The “varimax” rotation method was
used to increase the interpretation of axes and the number of PCA axes
was determined using Kaiser's rule (eigenvalue ≥ 1) (Legendre and
Legendre, 1998). The synthetic gradients built (henceforth, PCbio and
PCenv scores) also made the degree of divergence between metal concen-
trations in fish and sediments visible, and facilitated the interlinking
between the metal concentrations in the environment and fish. Pearson's
correlation coefficient (r) was calculated to determine pair-wise relation-
ships between the synthetic gradients built with metal concentrations.
All statistical analyses were performed using the R package (R Core
Team, 2012) and the libraries car, vegan and MASS. GLM assumptions
were checked by examining standardised residuals using qq plots and
plotting fitted versus predicted values.
3. Results
3.1. Metal concentrations in water and sediments and their relationship
with environmental variables
The mean metal concentrations in water differed significantly
between locations for all elements determined (ANOVA, p b 0.001)
with the exception of Hg, which remained below the detection limits
at all locations (b0.2 μg L−1; F8,18 = 1, p = 0.46) (Table 1). The peak
concentrations of Cu, Cd, Pb and Co were significantly associated with
the discharge site of the River Ramis (Tukey post-hoc, p b 0.05), while
the highest values of Zn and Fe were respectively observed at locations
L2 and L6 (Tukey post-hoc, p b 0.05), without any river effect (Table 1).
As the highest conductivity values were observed at the discharge site
of the River Ramis, conductivity was also positively associated with
metals such as Cu (r = 0.85, p = 0.003) and Pb (r = 0.75, p = 0.01).
Nitrate concentration was also positively related to Cu (r = 0.66, p =
0.04) and Cd (r = 0.74, p = 0.02) levels in water, possibly because of
the spatial co-occurrence of the slight increase in nitrate concentration
and that of these two elements in Barco–Chucuito (Table 1).
The mean metal concentrations in sediments also varied significant-
ly between locations (ANOVA, p b 0.001), as did organic matter content
(ANOVA, F8,18 = 437,500, p = 0.001) and pH (ANOVA, F8,18 = 16,706,
p = 0.001) (Table 2). Specifically, the highest organic matter concentra-
tions and lowest pH were observed at Mohokachi (Lago Menor) and
those sites close to the discharge sites of the Ramis and Coata Rivers in
Lago Mayor (Table 2; Tukey post-hoc, p b 0.05). This explains why
organic matter content was also positively related to the concentra-
tion of most elements: Cu (r = 0.64, p b 0.001), Zn (r = 0.51, p =
0.006), Cd (r = 0.57, p = 0.001) and Co (r = 0.51, p = 0.006). In
addition, pH was negatively related to the concentration of organic
matter (r = -0.58, p = 0.001), and all metal concentrations in sediments
(all, r N −0.66, p b 0.001) with the exception of Co (r = −0.16, p = 0.4).
3.2. Determinants of metal bioaccumulation in fish and their relationship
with environmental concentrations
The concentrations of all metals analysed differed significantly
between species (ANOVA, F3,696 N 9.72, p b 0.01), tissues (ANOVA,
F1,696 N 50.45, p b 0.01) and locations (ANOVA, F8,696 N 3.03, p b 0.01).
Liver was the main metal depository for all elements with the exception
of Hg (Fig. 2). The tissue analysed was indeed the main factor explaining
the variation in all metal concentrations with the exception of Hg and Pb
when individual traits were incorporated into the analysis (Table 4).
Together with tissue, the sentinel species used for the metal analysis
was also a significant contributor for most elements (Table 4), illustrating
that native benthopelagic species (O. luteus and O. agassii) generally had
the highest metal concentrations, followed by the catfish (T. rivulatus)
(Fig. 2). Specifically, O. luteus had the highest mean values of Cu
(108.84 ± 85.75 μg g − 1 dw), Hg (0.8 ± 0.93 μg g − 1 dw) and Co
(0.64 ± 0.4 μg g−1 dw), O. agassii contained the highest concentrations
 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244 237

Water quality of the nine bays surveyed in Lake Titicaca. Descriptive results are shown for general indicators of water quality but mean metal concentrations (μg L−1 ± standard deviation) and detection limits are reported for each element. The
specific legislation of Health Canada (2012x), US EPA (2009y), and BOE (2011z) to establish the safety threshold for each water quality variable is also included. The letters (a, b, c, d, e, f, g, h, i) group sites by heavy metal, considered homogenous
of Zn (102.22 ± 21.36 μg g−1 dw), and T. rivulatus had the highest

Legislation thresholds
values of Cd (0.15 ± 0.18 μg g−1 dw), Pb (0.06 ± 0.08 μg g−1 dw) and
Fe (266.1 ± 146.56 μg g−1 dw) (Fig. 2).
As the fish used for metal analyses spanned a wide range of sizes and
ages (Table 3), GLMs were employed to explore the relative influence of

1300 y
5000 y
5–9y

300y
10 y
0.1x
individual traits (LT, gender and age) compared to the location of sam-

1x

5y
2y
0y
5z
–
–
–

–
–

–
Detection limits pling sites in explaining the variation in metal bioaccumulation
(Table 4). Interestingly, location was the most significant contributor
to Hg bioaccumulation but the interaction between species and location
was also significant for most elements, showing that the metal bioaccu-

0.02

0.02
0.02

0.02
mulation pattern observed in all four species differed across the study

0.2
0.2

0.2

0.1
0.2

10
1
0

1
0

area (Fig. 3). Individual traits appeared to have a low influence on the
concentration of all elements in fish with the exception of Zn, Co and

14.35d ± 0.02
0.05a ± 0.008
0.04f ± 0.001

21.61i ± 0.01
5.47h ± 0.02
20.1i ± 0.02
Fe, for which gender was a significant predictor but with a marginal
effect (Table 4) and no clear trend observed in metals between males
b0.02
b0.02
1280

b0.2

0.1a
and females across species (Table 5). None of the GLM models retained
6.9

8.8
18
15
L7

length or age as a significant explanatory variable (Table 4).

211.08h ± 0.005
0.05b,e,f ± 0.005 To further examine the metal bioaccumulation pattern in fish and in
45.77h ± 0.02

15.62h ± 0.01
0.15e ± 0.005
7.43f ± 0.02

the sediments of the locations surveyed we generated two PCA analyses

for each data set, which produced two significant axes for fish (PC1bio,
b0.02
1540

PC2bio) and environment (PC1env, PC2env) that together explained,

b0.2

0.1a
5.7

8.9

0.1
16
17
L6

respectively, 61.32% and 91.80% of the variation in metal concentrations

(Fig. 4 and Supplementary material: Table S3). PC1bio accounted for
0.07b,c ± 0.001
11.09g ± 0.02
51.09g ± 0.02

24.43g ± 0.02

19.37g ± 0.01
0.19d ± 0.01

37.31% of the variation and loaded on Cu, Zn, Cd, Hg, Co and Fe concen-
trations, while PC2bio explained 24.01% of variation and was mainly
related to Pb concentration. In contrast, PC1env accounted for 79.22% of
1570

b0.2

0.1a
0.1
0.1
15
11
L5

the variation and loaded on Cu, Zn, Cd, Hg, Pb and Fe concentrations,
5
3
8

while PC2env accounted for 12.59% of variation and was mainly related
0.06b,c,e ± 0.005

52.12f ± 0.009

to Co concentration. These environmental metal synthetic gradients

7.28f ± 0.007
31.42f ± 0.02

19.34f ± 0.01
0.12c ± 0.01

were poorly and non-significantly correlated with the metal bioaccu-

mulation pattern observed in fish summarised in PC1bio (PC1env: r =
b0.02

b0.1a
1258

b0.2

0.54, p = 0.13; PC2 env : r = − 0.54, p = 0.13) and PC2 bio (PC1 env :
6.2

8.3

0.1
16
15
L4

r = − 0.11, p = 0.77; PC2env: r = − 0.04, p = 0.91) (Fig. 4).

16.84e ± 0.004

0.11d ± 0.004
47.48e ± 0.01

32.39e ± 0.01

40.78e ± 0.01
0.28b ± 0.01

4. Discussion
b0.02

b0.1a
1754

b0.2
5.3

0.1
20
15
L3

7
8

4.1. Metal concentrations in water and sediments and relationship with

117.32d ± 0.02
0.07b,c ± 0.005

other environmental variables

14.44d ± 0.02

25.33d ± 0.01
0.12c ± 0.006
8.93d ± 0.01

Water chemical analyses showed that the Ramis River was the main
b0.02

b0.1a
1318

b0.2

source of metal pollution in Lake Titicaca with peak concentrations of

5.5

8.2

0.1
18
16
L2

Cu, Cd, Pb and Co observed at this location. Specifically, the Pb concen-

tration exceeded the safety thresholds established by legislation by
0.06b ± 0.006

0.25b ± 0.005
23.67c ± 0.01

3.07c ± 0.006

111.6c ± 0.02
2.45c ± 0.01

30-fold (US EPA, 2009). This peak in Pb might be related to the use of
fuel additives and lubricants enriched in Pb in the mining and industrial
b0.02
b0.02

b0.1a
1239

b0.2

areas located upstream, since no hunting practices using Pb are present

6.6

8.2
17
15
L1

in this area (Revollo et al., 2003; Gammons et al., 2006). Baseline loca-
0.27b ± 0.008
58.46b ± 0.03

0.18b ± 0.005

13.43b ± 0.01
0.05a ± 0.008

tions generally had fairly low concentrations of all elements determined

but our results also showed that all polluted locations showed a degree
b0.01 a

of metal pollution (e.g. Cu, Zn, Pb, Co) regardless of their proximity to
b0.02

b0.1a
1239

b0.2
6.1

8.2

0.1

the main rivers. The release of domestic wastewaters might increase

R2

20
15

the metal concentrations in these areas because a wide variety of house-

0.63a ± 0.005

0.05a ± 0.008
74.08a ± 0.01

hold products, such as cleaning materials, toothpaste and cosmetics, can

7.11a ± 0.01

2.57a ± 0.01

contain trace concentrations of Cu, Zn, Pb and Fe (Sörme and Lagerkvist,

b0.01a
b0.02

2002; Alloway, 2013), and only large urban areas around Lake Titicaca
b0.1a
1207

b0.2
5.7

8.2

0.1
R1

17
15

have proper sewage treatment plants (Revollo et al., 2003). Nonethe-

6

less, metals are refractive to biological degradation and are also present
Dissolved oxygen (mg L−1)

Carbonate hardness (°KH)

in natural systems receiving the effluents from sewage treatment plants

Conductivity (μS cm−1)

General hardness (°dH)

(Maceda-Veiga et al., 2012). In addition, the run-off from adjacent agri-

Phosphates (mg L−1)
Ammonia (mg L−1)

cultural areas might also be responsible for Cu, Zn, Cd, Hg and Pb release
Nitrates (mg L−1)
Temperature (°C)

Nitrites (mg L−1)

since these are often present in fertilizers or pesticides (Alloway, 2013).

Hg (μg L−1)
Cu (μg L−1)

Cd (μg L−1)
Zn (μg L−1)

Co (μg L−1)
Pb (μg L−1)

Fe (μg L−1)

However, our study was unable to detect any other evidence of agricul-
at p b 0.05.

Location

tural practices (e.g. eutrophication) despite the fact that excessive use of
Table 1

nitrogenous compounds as fertilizers is a growing concern at Lake

pH

Titicaca (Fontúrbel, 2008), possibly because of the instability of these

238 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244

Sediment characteristics and mean metal concentration (μg g−1 dw ± standard deviation) at each sampling site surveyed in Lake Titicaca. The safety thresholds reported for metal concentrations in sediments are based on MacDonald et al. (2000).
compounds in the environment compared to metals and/or the dilution

Legislation thresholds
associated with the rain that fell during some of our surveys.
The contribution of the main tributaries to metal pollution in Lake
Titicaca was better reflected by the spatial variation in metal concentra-
tion in sediments. Sediments enriched in organic matter act as good

4.98
1.06
sinks for metals compared to water because humic substances present

149
459

128
–
–

–
–
in sediments create metal complexes (Rognerud and Fjeld, 2001;
Reference material WQB1 Alloway, 2013). An increase in organic matter content was particularly
evident in the current study at the discharge site of the River Ramis,
which in turn would explain the high metal concentrations observed

47358 ± 10536
79.6 ± 19.1 at this site. The high river flow conditions two weeks before our survey

1.09 ± 0.15
83.7 ± 22.3
20.1 ± 9.3
275 ± 58 was performed might also have increased the release of sediments from
2 ± 0.91 upstream areas down to the mouth. In addition the low pH observed at
these sampling sites might be due to the decomposition of the organic
–
–

matter in sediments or the sewage discharge from the mining activities

199.72i ± 0.003
46.57i ± 0.002

10.45i ± 0.002

(e.g. Galán et al., 2003 in Spain or Alpers et al., 2005 in California).

9.95i ± 0.004

6.28i ± 0.002
0.08e ± 0.01
4.45i ± 0.01
8.12i ± 0.01

Because the concentration of some of the metals analysed was moder-

ately high, with some exceeding the safety thresholds established by
0.1a

international legislation (i.e. Pb in water), and as the long-term release

L7

of metals from contaminated sediments is well known, negative effects

14.17h ± 0.003
11.97h ± 0.002

58.05h ± 0.002
5.47h ± 0.004
1.75h ± 0.002
0.18d ± 0.005

are expected in aquatic and terrestrial organisms as long as metals are

21.3h ± 0.02
7.51h ± 0.01

able to incorporate into the biota (MacDonald et al., 2000).

0.1a
L6

4.2. Metal bioaccumulation in fish

290.79g ± 0.001
25.77g ± 0.002
65.61g ± 0.002

16.61g ± 0.001
23.66g ± 0.05

10.1g ± 0.004
0.13b ± 0.01
7.05g ± 0.02

The metal concentrations in all fish species collected exceeded the

threshold established by the European (European Commission, 2001,
0.1a

2006) and American (FAO/WHO, 1998) legislation with the exception

L5

of Pb, Co and Fe. This finding is consistent with Gammons et al. (2006)
374.98f ± 0.001
73.89f ± 0.002

25.82f ± 0.001
b0.1a ± 0.001
0.21d ± 0.005

and IMARPE and FONCHIP (2010), who also detected Hg levels in

27.21f ± 0.01

4.76f ± 0.004
2.67f ± 0.03
6.9f ± 0.01

O. bonariensis and T. rivulatus above the safety threshold at the mouth

of the River Ramis and at other locations around the lake. Although the
cross-study comparison of metal bioaccumulation in fish species from
L4

other water ecosystems is challenging, the mean concentrations of Cu

323.24e ± 0.006

433.38e ± 0.002
41.09e ± 0.002

74.97e ± 0.001

Zn, Hg, Co and Fe observed in the current study were higher than those
8.68e ± 0.001
0.78b ± 0.01
35.8e ± 0.02
5.13e ± 0.01

1.98c ± 0.01

reported in the liver of Abramis brama for Cu (49.07 μg g−1 dw) and in
The letters (a, b, c, d, e, f, g, h, i) group sites by environmental variable, considered homogenous at p b 0.05.

muscle for Hg (0.09 μg g−1 dw) in Lake Balanton (Farkas et al., 2002),
in the liver of Labeo rohita for Cu (1.03 μg g−1 dw), Zn (1.03 μg g−1
L3

dw) and in muscle for Hg (0.07 μg g−1 dw) in Lake of Bhopal (Malik
319.19d ± 0.002

et al., 2010) or in the liver of Tilapia nilotica for Cu (7.5 μg g−1 dw), Zn
20.99d ± 0.002
71.09d ± 0.002

17.65d ± 0.001
8.12d ± 0.005

10.3d ± 0.002
0.11b,e ± 0.01
3.66d ± 0.05

(2.28 μg g−1 dw), Co (0.31 μg g−1 dw) and Fe (4.58 μg g−1 dw) in
Lake Nasser (Rashed, 2001). However, Cd and Pb concentrations were
b0.1a

lower than those reported in the liver of Abramis brama for Cd (1.72 μg
L2

g−1 dw) and Pb (2.22 μg g−1 dw) from Lake Balanton (Farkas et al.,
2002), the liver of Labeo rohita for Cd (0.52 μg g−1 dw) and Pb (1.26 μg
246.81c ± 0.002
10.39c ± 0.004
19.04c ± 0.001
7.31c ± 0.005

8.33c ± 0.001
5.69c ± 0.004

g−1 dw) in Lake of Bhopal (Malik et al., 2010) or even in muscle of

5.96c ± 0.01

Lates marie for Cd (0.25 μg g−1 dw) and Pb (4.96 μg g−1 dw) in Lake Tan-
b0.01a
b0.1a

ganyika (Chale, 2002).

L1

The bioaccumulation pattern of all elements in fish at Lake Titicaca

87.79b ± 0.002

was mainly associated with tissue and sentinel species effects. Of the
3.07b ± 0.002
4.32b ± 0.001

2.16b ± 0.001
1.62b ± 0.003
1.19b ± 0.01
7.95b ± 0.01

tissues, the liver was confirmed as the best metal depository for all
elements determined with the exception of Hg (Farkas et al., 2002;
b0.01a
b0.1a

Gammons et al., 2006; Çogun et al., 2006). This could be because Hg

R2

has a high affinity for the sulphydryl groups of proteins (Miller et al.,
162.44a ± 0.002

1992; Mason et al., 2000), as well as because the muscle contains low
15.82a ± 0.001
3.77a ± 0.001
1.85a ± 0.04
8.43a ± 0.01
3.3a ± 0.002
9.1a ± 0.003

levels of binding proteins (i.e. metallothioneins) compared to the liver

(Roméo et al., 1999; Karadede et al., 2004; Kojadinovic et al., 2007).
b0.01a
b0.1a

In terms of species, our results corroborated our predictions and the

R1

highest metal concentrations were observed in O. luteus, O. agassii and

Organic matter (%)

T. rivulatus, possibly because they live close to the sediments (Roméo

et al., 1999; Agarwal et al., 2007; Yilmaz et al., 2007). However, pelagic
Hg (μg g−1)
Cu (μg g−1)

Cd (μg g−1)
Zn (μg g−1)

Co (μg g−1)
Pb (μg g−1)

Fe (μg g−1)

species such as O. bonariensis often showed a similar metal bioaccumula-

Location

tion pattern (e.g. Hg) to native benthopelagic species, possibly reflecting

Table 2

the different metabolic dynamics of essential and non-essential elements

pH

in fish species coupled with the influence of other individual traits.

 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244 239

Fig. 2. Mean metal concentrations in muscle (white boxes) and liver (grey boxes) in the native benthopelagic (O. agassii and O. luteus), the native benthonic (T. rivulatus) and the pelagic
introduced species (O. bonariensis) collected from nine study sites in Lake Titicaca. Discontinuous lines indicate the safety thresholds for metal concentration established by international
legislation for human consumption (Cu, Cd, Hg and Pb according to the EC, 2006; and Zn based on FAO/WHO, 1998). The letters (a, b, c, d) group sites by variable, considered homogenous
at p b 0.05. Note that metal concentrations in males and females are grouped by location.

The spatial variation in metal concentrations in fish showed that the
levels of non-essential elements (Cd, Hg, Pb) in fish were generally
more consistent with the peaks in metal concentrations in the environ-
ment than essential elements. In this regard, the most polluted locations
were Barco–Chucuito (located inside Puno Bay) and, as predicted, the
mouth of Ramis River. The considerable variation in the concentration
of essential elements could be due to the greater regulation of the
uptake and excretion of essential elements compared to that of non-
essential elements (Kojadinovic et al., 2007). However, our results also
showed that essential elements (e.g. Cu) can bioaccumulate when envi-
ronmental concentrations are high, thereby becoming a pollution source,
just as non-essential elements do (Chale, 2002; Farkas et al., 2002;
Demirak et al., 2006; Maceda-Veiga et al., 2013). Likewise, an increase
in essential elements in a given individual from a polluted location
might also be attributed to the participation of Cu, Zn, Co and Fe in met-
abolic functions (e.g. detoxification processes) to cope with pollution
effects (e.g. oxidative stress) (Bervoets and Blust, 2003; Çogun et al.,
2006). Because non-essential elements are better tracers of pollution
240 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244

Table 3
Sample size (N), mean and standard deviation of body length (LT mm) and weight (W g), minimum and maximum range, and details of gender and ages of the four fish species in Lake
Titicaca used for metal analysis.

Species Gender N LT Min–max W Min–max Age (years)

0+ 1+ 2+ 3+ 4+ 5+ 6+ 7+

O. bonariensis ♂ 52 210.65 ± 24.83 166–280 58.32 ± 21.56 25.25–121.18 0 47 5 0 0 0 0 0

♀ 37 228.02 ± 37.27 165–375 85.83 ± 47.44 25.54–278.08 0 28 8 1 0 0 0 0
O. luteus ♂ 46 120.95 ± 18.2 94–155 44.51 ± 18.77 17.06–79.71 0 9 3 3 3 23 5 0
♀ 39 131.94 ± 20.41 90–164 62.51 ± 24.85 16.84–105.18 0 5 1 1 1 19 10 2
O. agassii ♂ 27 121.44 ± 22.93 90–160 31.11 ± 16.74 11.12–61.57 0 6 9 4 6 2 0 0
♀ 63 133.71 ± 22.26 90–170 42.86 ± 18.95 9.31–90 0 4 14 19 13 13 0 0
T. rivulatus ♂ 39 158.15 ± 15.64 121–181 40.39 ± 11.19 18.73–60.37 0 4 35 0 0 0 0 0
♀ 44 164.97 ± 18.54 122–204 44.82 ± 12.56 19.21–69.16 0 2 39 0 3 0 0 0

than essentials and because all species showed increased Hg concentra- (i.e. killifish/catfish) (Mason et al., 2000; Kojadinovic et al., 2007; Agah
tion at the most polluted location regardless of their swimming behaviour et al., 2009; Maceda-Veiga et al., 2013). In the case of O. bonariensis
(i.e. pelagic/benthonic), our results suggest that all species, including the the high concentration of Hg observed in some locations relative
apparent highly mobile O. bonariensis, are resident in this area, although to other species could also be attributed to its high trophic position
the home range of all four species is currently unknown. Nonetheless, (i.e. biomagnification) (Mason et al., 2000; Kojadinovic et al., 2007;
the metal bioaccumulation pattern observed in all four fish species should Agah et al., 2009). In this regard, the mean length of O. bonariensis indi-
be considered mostly poorly related to the environmental metal concen- viduals examined in the current study (217.8 mm) suggests that they are
trations as suggested by the pair-wise correlation analysis between mainly piscivorous (above 200 mm, see Vaux et al., 1988; or above
PCA scores. 90 mm, Monroy et al., in preparation). Likewise, a high metal concentra-
An additional explanation for the sometimes similar bioaccumula- tion in O. luteus could be attributed to the fact that this fish species
tion pattern observed in pelagic and benthopelagic species is that occasionally feeds on fish juveniles (Monroy et al., in preparation). Alter-
all four species can feed on bottom drift invertebrates and may ingest natively, the more active metabolism of pelagic species could also lead to
sediment or suspended organic matter when they feed (Lauzanne, an increase in metal uptake compared to benthopelagic fish (Kojadinovic
1982; Parenti, 1984; Loubens, 1989; Vila et al., 2007). Differences in the et al., 2007). However, this hypothesis seems unlikely owing to the fact
relative proportion of items in fish diets with a variable metal content that the metal bioaccumulation in pelagic species was not consistently
could have determined the observed metal concentration in fish species higher than that in the benthopelagic species across the study area.
Species occurrence in deep and poorly oxygenated waters, as is the
case for O. bonariensis, is also likely to increase Hg bioaccumulation
since these environmental conditions favour methylation and hence Hg
Table 4
Results of the final GLM models for all metals (Cu, Zn, Cd, Hg, Pb, Co and Fe) determined in accumulation (Gammons et al., 2006; Kojadinovic et al., 2007).
the four fish species (O. bonariensis, O. luteus, O. agassii, T. rivulatus) collected in Lake Individual traits made a minor contribution towards explaining the
Titicaca. Note that only significant variables highlighted in previous full models with all variation in the concentration of most elements in all four fish species.
explanatory variables and interactions (see methods) are shown.
Previous studies have suggested that age and fish size could increase
Metal Fish attributes SS Df F p-value metal accumulation due to the long-term exposure of individuals to
Cu Species 182.02 3 101.34 b0.001
pollution (Gammons et al., 2006), the metabolic activity of individuals
Tissue 993.11 1 1658.82 b0.001 (Canli and Atli, 2003), ontogenetic changes in fish diet (Mason et al.,
Species x Location 23.94 24 1.66 0.02 2000) or a reduction in the detoxification ability of old fish (Kojadinovic
Residuals 393.34 657 et al., 2007). In the current study fish size and age might have been
Zn Species 23.7 3 59.09 b0.001
secondary factors affecting metal bioaccumulation, possibly because
Location 3.36 8 3.14 0.001
Tissue 105.73 1 790.63 b0.001 they were not evenly distributed across the polluted locations given the
Gender 0.639 1 4.77 0.02 likely effect of fisheries pressure. In terms of gender, there was no clear
Residuals 90.94 680 difference between males and females in the bioaccumulation pattern
Cd Species 0.31 3 26.9 b0.001
of the essential elements Zn, Co and Fe in most species However, studies
Location 0.2 8 6.79 b0.001
Tissue 0.85 1 221.17 b0.001
on other fish species, such as skipjack tuna Katsuwonus pelamis and com-
Residuals 2.53 657 mon dolphinfish Coryphaena hippurus have reported the effect of gender
Hg Species 1.98 3 21.13 b0.001 on Zn and Fe bioaccumulation (Kojadinovic et al., 2007). The concentra-
Location 34.02 8 135.82 b0.001 tions of these essential elements could be associated with differing meta-
Tissue 8.75 1 279.67 b0.001
bolic activities or the different gonad developmental stage of males and
Species x Location 6.48 24 8.62 b0.001
Residuals 20.57 657 females (Kojadinovic et al., 2007). As gonads were not weighed in this
Pb Species 0.08 3 15.89 b0.001 study, the effect of gonad development cannot be ruled out, although an
Location 0.07 8 5.4 b0.001 apparently similar developmental stage was observed in the field. Metal
Tissue 0.02 1 15.72 b0.001 bioaccumulation in fish in Lake Titicaca may also be affected by factors
Species x Location 0.14 24 3.3 b0.001
Residuals 1.16 657
other than those mentioned above, including metal speciation (Ivanciuc
Co Species 1.96 3 33.75 b0.001 et al., 2006; Adhikari et al., 2009), the rate of export of metals through
Tissue 17.69 1 910.87 b0.001 other food web compartments (Amundsen et al., 1997; Bervoets and
Gender 0.43 1 22.5 b0.001 Blust, 2003; Kojadinovic et al., 2007) and species physiology, and there-
Species x Gender 0.44 3 7.62 b0.001
fore this study cannot explain the likely mechanisms associated with
Residuals 13.3 685
Fe Species 45.58 3 38.26 b0.001 the bioaccumulation pattern we observed in these fish species. Finally,
Tissue 1068.64 1 2691.94 b0.001 we note that although the present study focused on metals, exposure
Gender 3.05 1 7.67 b0.001 to other toxic compounds present in sewage waters (e.g. drugs, pesti-
Species x Location 82.03 32 6.45 b0.001 cides) could also have been responsible for the observed bioaccumulation
Residuals 260.42 656
patterns.
 M. Monroy et al. / Science of the Total Environment 487 (2014) 233–244 241

Fig. 3. Variation in mean metal concentrations in the nine bays surveyed and in the four fish species examined (O. bonariensis, black; O. luteus, dark grey; O. agassii, grey and T. rivulatus,
white boxes) using the liver as the sentinel tissue for the assessment of all metals with the exception of Hg. Discontinuous lines indicate the safety thresholds for each metal established
by international legislation for human consumption (Cu, Cd, Hg and Pb according to the EC, 2006; and Zn based on FAO/WHO, 1998). Metal concentrations are plotted without
log-transformation to facilitate the interpretation of axes.

5. Concluding remarks and management actions
This study demonstrates contrasting metal bioaccumulation pat-
terns in water, sediments and fish, suggesting caution when inferring
the risk posed by metal pollution based on analysis of a single ecosys-
tem compartment. Most elements in fish and Pb in water were above
the safety threshold, illustrating the large-scale metal pollution at
Lake Titicaca. Given that both humans and wildlife (e.g. piscivorous
birds and amphibians) depend on the fisheries resources in the lake
and high Hg levels in blood of humans living close to mining areas
have been already reported (Hurtado et al., 2006), we would encour-
age the authorities to enforce legislation to reduce metal pollution in
Lake Titicaca. Limiting species consumption would be a better man-
agement strategy than the prohibition of fishing in certain areas
since pollution by metals seems to extend across the study area.
Further studies should focus on the ecology of fish species, metal
speciation and metal export from contaminated areas through other
components of the food web to increase our knowledge about the
likely mechanisms behind the observed metal bioaccumulation. As
the species affected have a high conservation value, the evaluation
of the risk posed by pollution in Lake Titicaca to fish health is also
mandatory.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2014.03.134.
Conflict of interest
Authors declare that they have no financial and personal relationships
with other people or organizations that can inappropriately influence or
be perceived to influence their work.
Acknowledgements
This work and protocols were funded and approved by the Spanish
Agency for International Cooperation and Development (AECID) and
the Department of Production of Peru trough the Hispanic-Peruvian
Cooperation Program (PCHP) (projects no FBG305168 and FBG306311).
We thank O. Flores, E. Yujra and the rest of the crew at the Binational
 242

Table 5
Sample size (N) and mean metal concentration (μg g−1 dw ± standard deviation) in relation to species (O. bonariensis: 1, O. luteus: 2, O. agassii: 3, T. rivulatus: 4) gender and tissue in Lake Titicaca. Note that only significant differences were observed
for concentrations of Zn, Co and Fe from Table 4.

Species Gender N Cu Zn Cd Hg Pb Co Fe

Muscle Liver Muscle Liver Muscle Liver Muscle Liver Muscle Liver Muscle Liver Muscle Liver

1 ♂ 52 1.3 ± 1.8 7.65 ± 7.07 38.45 ± 15.55 50.43 ± 17.27 0.007 ± 0.007 0.02 ± 0.01 0.74 ± 0.49 0.3 ± 0.55 0.02 ± 0.02 0.01 ± 0.01 0.03 ± 0.07 0.2 ± 0.11 23.68 ± 43.84 164.94 ± 73.2
♀ 37 0.93 ± 1.31 5.46 ± 3.47 28.63 ± 9.16 44.22 ± 15.66 0.007 ± 0.008 0.02 ± 0.01 0.84 ± 0.48 0.34 ± 0.57 0.02 ± 0.05 0.01 ± 0.01 0.04 ± 0.07 0.17 ± 0.07 21.1 ± 36.46 138.69 ± 56.67
2 ♂ 46 1.26 ± 1.03 118.42 ± 85.35 30.6 ± 9.86 77.4 ± 20.68 0.006 ± 0.003 0.14 ± 0.003 0.76 ± 0.89 0.16 ± 0.15 0.02 ± 0.02 0.04 ± 0.09 0.01 ± 0.008 0.82 ± 0.44 12.31 ± 5.74 170.9 ± 119.23
♀ 39 1.26 ± 1 97.54 ± 85.93 29.32 ± 6.67 83.37 ± 24.78 0.007 ± 0.003 0.08 ± 0.003 0.86 ± 0.99 0.2 ± 0.2 0.01 ± 0.02 0.04 ± 0.05 0.01 ± 0.009 0.44 ± 0.24 13.99 ± 7.02 112.24 ± 57.72
3 ♂ 27 1.06 ± 0.54 83.18 ± 77.87 32.94 ± 10.29 103.93 ± 17.2 0.003 ± 0.004 0.07 ± 0.004 0.67 ± 1.12 0.47 ± 0.75 0.009 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 0.75 ± 0.48 13.37 ± 5.01 261.02 ± 197.61
♀ 63 0.96 ± 0.44 65.55 ± 44.85 31.67 ± 9.16 101.49 ± 23 0.004 ± 0.004 0.04 ± 0.004 0.44 ± 0.66 0.36 ± 0.66 0.02 ± 0.02 0.02 ± 0.02 0.01 ± 0.01 0.42 ± 0.28 15.83 ± 14.64 194.43 ± 125.46
4 ♂ 39 1.9 ± 0.75 10.89 ± 4.82 49.17 ± 15.26 87.97 ± 11.93 0.006 ± 0.007 0.14 ± 0.007 0.44 ± 0.38 0.19 ± 0.24 0.02 ± 0.03 0.06 ± 0.07 0.02 ± 0.01 0.4 ± 0.19 22.66 ± 12.46 304.82 ± 170.36
♀ 44 2.05 ± 1.4 12.03 ± 10.06 46.2 ± 15.85 90.36 ± 19.57 0.004 ± 0.004 0.15 ± 0.004 0.43 ± 0.45 0.2 ± 0.4 0.04 ± 0.09 0.07 ± 0.09 0.02 ± 0.01 0.42 ± 0.23 21 ± 7.47 231.77 ± 112.9

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