Aquatic Toxicology 65 (2003) 187 /204 www.elsevier.

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Assessment of the use of biomarkers in aquatic plants for the evaluation of environmental quality: application to seagrasses
L. Ferrat a,*, C. Pergent-Martini a, M. Romeo b
b

EqEL, University of Corsica, BP 52, 20250 Corte, France R.O.S.E., University of Nice Sophia Antipolis, UMR INRA UNSA 1112, Parc Valrose, BP 71, 06108 Nice, France Received 17 April 2003; received in revised form 21 May 2003; accepted 7 June 2003

Abstract The use of aquatic plants as bio-indicators constitutes an irreplaceable tool for investigation in ecological research, applied to the conservation of littoral ecosystems. Today, studies in both the laboratory and the field have provided encouraging insights into the capacity of aquatic plants to act as biomonitors of environmental quality, through the use of biomarkers, and these are reviewed here. Photosynthetic activity, secondary metabolites, heat shock proteins, enzymes of detoxication, and oxidative stress biomarkers were measured in the case of various stressors, (e.g. light, thermal, hydric/haline stress, or herbicides, metals, organic contaminants). Most of them seem to be valuable and early markers of the environmental conditions, as demonstrated by experimentations carried out on Posidonia oceanica . Nevertheless, none can be in itself a valuable solution, and only a multiparametric approach, including both physiological biomarkers, biomarkers of general stress and more specific biomarkers seems to be appreciable in an ecotoxicological diagnostic. # 2003 Elsevier B.V. All rights reserved.
Keywords: Aquatic plants; Biomarkers; Oxidative stress; Detoxication; Glutathione S -transferase; Phenolic compounds

1. Introduction In the early XXth century, researchers proposed the use of living organisms in parallel with physico-chemical analyses (that do not provide either the threshold of sensitivity or the level of response of organisms to contamination, Amiard et al., 1998), to evaluate the state of a medium

* Corresponding author. Tel.: '/33-495-45-0146; fax: '/33495-46-2441. E-mail address: ferrat@univ-corse.fr (L. Ferrat).

(Blandin, 1986; Kolwitz and Marson, 1908; Kolwitz and Marson, 1909 in Amiard et al., 1998). These organisms are referred to as bio-indicator species, that is to say species or group of species, whom, by their presence and/or their abundance, are representative of one or more properties of the ecosystem in which they occur (Guelorget and Perthuisot, 1984). These species make it possible to determine, with precision, the impact and the progression of anthropic action on biocenosis vitality (Rainbow and Phillips, 1993). The bio-indicator species must be sedentary, of ecological importance, widespread

0166-445X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-445X(03)00133-4

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and widely studied and sensitive to environmental variations (Molfetas and Blandin, 1981). The use of bio-indicators constitutes an irreplaceable tool for investigation in ecological research, applied to the conservation of littoral ecosystems (Rainbow and Phillips, 1993). Despite of the crucial role of plants (aquatic or terrestrial) in ecosystems, these organisms have been underemployed for the diagnosis or prediction of the negative consequences of human activities, although physiological processes, biochemical response and mechanisms of adaptation or mortality can be used to evaluate the quality of a medium (Vangronsveld et al., 1998). These organisms are sedentary, sensitive to environmental variations and react, as first stages of the food chain, more rapidly to the presence of pollutants than organisms living at higher stages (Lovett Doust et al., 1994).

2. Aquatic plants as bio-indicators Belsher (1979) defined the indicator function of some groups of species, by their behavior under the impact of pollution (e.g. eutrophication). He recorded cases of regression (e.g. Phaeophyceae, Cryptonemiales, Ceramiales and Gigartinales), or even disappearance within the same group of species (e.g. Bonnemaisoniales, Gelidiales, Rhodymeniales), or on the contrary an increase (e.g. Protoflorideae or Bangiophyceae). In a general manner, Fucophyceae and Rhodophyceae have tended to disappear under the impact of pollution (Bellan et al., 1995). Similarly, the massive development of chlorophyceae (e.g. Ulva sp. and Enteromorpha sp.), observed in littoral lagoons or sheltered bays (e.g. Venice lagoon, Thessaloniki gulf, California salt marsh, etc.) has led to these organisms being considered as eutrophication bioindicators (Lazaridou et al., 1997; Sfriso and Marcomini, 1997; Fong et al., 1998). Cystoseira amentacea (Bory) and Cystoseira mediterranea (Sauvag.) have also been used as negative sentinel species for pollution (Bellan-Santini, 1966; Bellan, 1972). Bianchi and Peirano (1995) demonstrated that in Liguria, the ratio of magnoliophyta Cymodocea

nodosa (U.) Aschers./Posidonia oceanica (L.) Delile was correlated with water quality. This ratio varies from 0.3 in non-polluted waters to 0.8 in industrialized or urbanized waters. Many authors have noted a regression of P. oceanica meadows according to the degree of anthropization. Thus, given its wide range of distribution throughout the Mediterranean, P. oceanica would appear to be a potentially valuable bio-indicator of water quality. (Pergent, 1991) and has been used in bio-monitoring programs for the marine environment (Boudouresque et al., 2000). The use of macrophytes is notable in the biomonitoring of trace metal contamination (e.g. Fucus vesiculosus (L.), Ascophyllum nodosum (L.) Le Jol., Sargassum sp., Ulva lactuca, see synthesis in Rainbow and Phillips, 1993; Phillips, 1994). The mechanisms of accumulation of these metals have been studied under laboratory conditions (e.g. Padina gymnospora (Kutzing) Vickers and Ulva lactuca , Amado Filho et al., 1997), and in natural conditions: e.g. Caulerpa taxifolia (Vahl) C. Agardh (Gnassia-Barelli et al., 1995); Cystoseira sp. (Catsiki and Bei, 1992); Fucus vesiculosus (Ostapczuk et al., 1997); Padina pavonica (Campanella et al., 2001); Ulva lactuca (Catsiki and Papathanassiou, 1993); Ulva rigida C. Agardh (Favero et al., 1996). Even if magnoliophyta are less widely used as bio-indicators, particular attention must be paid to them with regard to their capacity to accumulate a wide range of pollutants such as organo-chlorine compounds (Chabert et al., 1984), artificial radionuclids (Florou et al., 1985; Calmet et al., 1991) and above all heavy metals (Malea and Haritonidis, 1989a). Several studies, generally in situ, have been carried out concerning the use of a wide diversity of magnoliophyta as bio-indicators of metallic contamination (Table 1). However, if these species integrate the quality of their environment, and constitute a good instrument of investigation, they generally respond belatedly, thus limiting their utilization for biomonitoring short term (daily to monthly) environmental variations (Rainbow and Phillips, 1993). This has led researchers to focus on the use of early symptoms, or biomarkers, defined as cellular,

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Table 1 Marine plant (particularly magnoliophyta) species employed as bio-indicators of metallic contamination Species Amphibolis Antarctica (Labill.) Sonder and Aschers. ex Aschers. Cymodocea nodosa Metals References

Cd, Cu, Fe, Mn, Pb, Zn Harris et al. (1979) Cr, Cu, Ni; Al; Ca, Cd, Catsiki and Panayotidis (1993), Malea (1993) Cu, Fe, K, Mg, Na, Pb, and Malea and Haritonidis (1995) Zn Cd, Cu, Pb, Zn Nienhuis (1986)

Cymodocea rotundata Ehrenb. and Hempr. ex Aschers C. serrulata (R. Brown) Aschers. and Magnus Enhalus acoroides (L. f) Royle Halodule uninervis (Fors.) Aschers. H. pinifolia (Miki) den Hartog Halophila ovalis (R. Br.) Hook. f Halophila stipulacea (Fors.) Aschers.

Al; Cd, Cu, Fe, K, Na, Pb, Zn; Cd; Al Heterozostera tasmanica (Martins ex Aschers.) den Hartog Cd, Cu, Fe, Mn, Pb, Zn; Cd Posidonia australis Hook. f Cd, Cu, Mn, Ni, Pb, Zn P. oceanica Cd, Cr, Cu, Pb, Zn; Hg; all Syringodium isoetifolium (Aschers.) Dandy Thalassia hem- Cd, Cu, prichii (Ehrenb.) Aschers. Thalassodendron ciliatum (Forrsk.) den Hartog Zostera marina (L.) Cd; Cd, Cd, Cu, Zostera muelleri Irmisch ex Aschers. Cd, Cu, Zn; Cu Pb, Zn

Malea and Haritonidis (1989b), Malea (1994a,b) and Malea and Haritonidis (1996) Harris et al. (1979) and Fabris et al. (1982) Ward (1987) Campanella et al. (2001) and Capiomont et al. (2000); see synthesis in Pergent-Martini and Pergent (2000) Nienhuis (1986)

Cu, Pb, Zn; Cr, Pb, Zn Fe, Mn, Pb,

Faraday and Churchill (1979), Brix et al. (1983) and Lyngby (1991) Harris et al. (1979) and Carter and Eriksen (1992)

molecular and biochemical changes induced by chemical pollutants, measurable in biological systems such tissues, cells and biological fluids (Lagadic et al., 1997; Mc Carthy and Shugart, 1990in Lagadic et al., 1998). Biomarkers are widely different in their significance and terminology (i.e. biomarkers of exposure, of effects, of stress, of alteration. (de Lafontaine et al., 2000), but in the context of this paper, we will categorize biomarkers according to two large types of responses: biomarkers of exposure and biomarkers of stress. Biomarker utilization in monitoring programs for environmental quality is increasingly common (see synthesis in Amiard et al. (1998)). In contrast to the simple measurement of contaminants accumulating in tissues, biomarkers can offer a more complete an biologically more relevant information on the potential impact of toxic pollutants on the health of organisms (van der Oost et al., 1996). They can provide information on the health of

organisms, and can be used as early warning signals for general or particular stress (Vangronsveld et al., 1998). From the temporal point of view, they can provide evidence of the dynamics of the patterns of variation of bio-available pollutant quantities, for the original molecules and degradation products (Amiard et al., 1998). Moreover, Lagadic et al. (1997) underlined the interest in measuring several biomarkers at the same time in the same organism, which allows a pertinent approach to evaluate the effects of pollutants on individuals.

3. Biomarkers in aquatic plants Today, studies focus on the identification of stress biomarkers. Aquatic plants, totally or partially submerged, have been studied principally from lagoon or estuarine ecosystems, under stress

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of various origin (e.g. light, thermal, hydric/haline stress, herbicides, metals, organic contaminants). Research on biomarkers is frequently carried out on the entire plant, or only on the leaves (for magnoliophyta) and there is little information on the stress response in the different parts of a plant. However, these responses can be very different, according to the degree of exposure and the physiological role of the different parts of the plant (Pflugmacher et al., 1999a). The principal biomarkers tested are measurable responses that occur in photosynthetic activity, enzymatic processes of nutrition, secondary metabolite synthesis, oxidative stress and/or detoxication mechanisms. 3.1. Modication of photosynthetic activity (Table 2) The abundance and distribution of aquatic plants is directly correlated with the quantity of light available. Light is essential to the survival of endogenous tissues, as they depend on the oxygen supply from photosynthesis performed by epigenous tissues. Chlorophyll fluorescence measurement is considered as a way to evaluate the biochemical and physiological state of plants. It is a reliable technique, easy to carry out, non-destructive and rapid (Kramer et al., 1987, Walker, 1988 in Vangronsveld et al., 1998). Chlorophyll content and chlorophyll fluorescence are used to highlight stress due to a single environmental factor or to a combination of different environmental factors (Table 2), but they also constitute potential biomarkers of anthropogenic stress (Table 2). 3.1.1. Chlorophyll uorescence The response of photosynthetic systems from various plant species has been established for a number of growth conditions and chemical products, and constitutes a reference base for the use of chlorophyll fluorescence to detect stress and its effects (Vangronsveld et al., 1998). Chlorophyll a fluorescence made it possible to detect, after 1 h exposure (i) in Zostera marina stress due to an infection by a pathogen (Ralph and Short, 2002), (ii) in Posidonia australis and Amphibolis antartica

stress caused by a combination of heat and desiccation (Seddon and Cheshire, 2001), (iii) in Halophila ovalis a stress induced by herbicides (Ralph, 2000) or by trace metals (Ralph and Burchett, 1998). Herbicides (e.g. atrazine, DCMU) act principally on their binding with the quinone protein Qb (the second electron acceptor in the PS II complex), inhibiting electron transport (Ralph, 2000). Trace metals (e.g. Cu) may act on water-splitting side of photosystem II and the electron transport chain (Ralph and Burchett, 1998). Copper and zinc showed greater effects on photosynthesis than lead and cadmium. The response to stress was proportional to the metal concentration and also a function of the time of exposure. However, Halophila ovalis was highly tolerant to metals, for the tested concentrations, thus limiting the interest of this species as a bioindicator (Ralph and Burchett, 1998). 3.1.2. Photosynthetic pigment concentration The analysis of photosynthetic pigment concentration generally confirms the results obtained by chlorophyll fluorescence measurements. Trace metals can substitute for the magnesium ion in the chlorophyll molecule, leading to the inability to catch photons and thus to a decrease in the photosynthetic activity. Generally, stressed plants increase their carotenoid concentration to provide protection against the formation of free radicals. A decrease in total chlorophyll and in the ratio chlorophyll/carotenoids are often observed. These variations in photosynthetic pigments under exposure to trace metals and herbicides have been observed for various species (e.g. Halophila ovalis, Ralph and Burchett, 1998; Ralph, 2000; Salvinia minima , planktonic diatoms, Table 2). For Salvinia minima contamination by Cr reduces all photosynthetic pigments, even carotenoids (Brent Nichols et al., 2000). For planktonic diatoms Fargasova (1999) showed a decrease of chlorophyll a , caused by oxidative stress due to Cu, and a decrease of chlorophyll c due to Zn (Rijstenbil et al., 1994a). In conclusion, trace metals may inhibit chlorophyll pigment biosynthesis and enzymes involved in this process. The same phenomena is observed with exposure to high irradiance, where photo-

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Table 2 Changes in photosynthetic activity as biomarkers of stress in aquatic plants Biomarkers Decrease of chlorophyll fluorescence Stress Infection by L. zosterae T8'/desiccation Light ( P/ o/) T8, Cu, triazine Species Zostera marina Posidonia australis , Amphibolis antartica Cladophora sp. Different species of Chlorophyta, Phaeophyta, Rhodophyta Photosynthetic pigments: decrease in ratio chlorophyll/carotenoids Cd, Cu, Zn, Pb, Fe Scenedesmus quadricauda (Turp.) Breb Ditylum brightwellii (T. West) Grunow in van Heurck Phoyosynthetic pigments'/ Chlorophyll fluorescence Cd, Cu, Pb, Zn Halophila ovalis Salvinia minima (Baker) Diuron [3-(3?,4?-dichlorophe- Halophila ovalis nyl)-1,1-dimethylurea; triazine Light Halophila ovalis Light Light, T8, salinity Chondrus crispus Stackh Halophila ovalis In vitro In vitro In vitro In vitro Kind of study In vitro In vitro In situ: Gulf of Saronikos (Gr) In situ: Cabo de Gata (Sp.) References Ralph and Short (2002) Seddon and Cheshire (2001) Hader et al. (1997) Hader et al. (1998)

In vitro

Fargasova (1999)

Rijstenbil et al. (1994b)

Ralph and Burchett (1998) Brent Nichols et al. (2000) Ralph (2000) Ralph and Burchett (1995) Yakovleva and Titlyanov (2001) Ralph (1999)

synthetic pigments (chlorophyll a'/b) decrease (Yakovleva and Titlyanov, 2001), and this only in oligotrophic conditions for P. oceanica (Alcoverro et al., 2001).

3.2. Enzymatic activities related to nutrients Plant nutrient metabolism can be influenced by various stressors, so the activity of enzymes involved in the assimilation of nitrogen (e.g. glutamine synthetase (GS), nitrate reductase (NR)) and phosphate (alkaline phosphatase, an enzyme hydrolyzing organic phosphate monoesters to inorganic phosphate) can be an interesting biomarker for their capacity to highlight a nutrient deficiency for plants under stressing conditions.

In aquatic ecosystems, decreases in irradiance are frequently observed, from minutes to months, in estuaries and lagoons. These conditions can cause severe stress. In the magnoliophyta Thalassia testudinum , Kraemer and Denis Hanisak (2000) reported (i) a decrease of soluble carbohydrate content in the leaves with a decrease of light, (in the same conditions, Peralta et al. (2002) reported a mobilization of these carbohydrates in the aboveground parts of Zostera noltii but Ruiz and Romero (2001) showed a depression in the rhizomes of P. oceanica ), (ii) an accumulation of nitrogenous and phosphorus nutrients during light exposure and a slow decrease during dark phase, and (iii) a decrease of GS activity in dark phase, and a rapid increase when returned to light. Physiological variations can be measured 24 h after a variation in light intensity.

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In the case of high irradiance stress, photoinhibition was demonstrated by a decrease of GS and NR activity in the freshwater Spirodela polyrhiza (Schwalbe et al., 1999). Ralph (1999), demonstrated in Halophila ovalis , that the combination of various forms of stress amplified the sensitivity of the plant to each of these individual factors. Alkaline phosphatase activity (APA) was studied in P. oceanica and Cymodocea nodosa , under experimental and natural conditions, for nutritive stress due to phosphorus deficiency (Perez and Romero, 1993; Invers et al., 1995). Perez and Romero (1993) showed that APA is correlated to phosphorus concentrations in the tissues, and/or the bio-availability of this element in the medium. An increase in APA was noted when the plant is growing in phosphorus limited medium. Values were higher in the older parts of the plant, and low but detectable in the roots (Perez and Romero, 1993). So APA could be a good indicator of phosphorous deficiency in aquatic plants and an indicator of the nutritional status in magnoliophyta (Hernandez et al., 1999). 3.3. Heat shock proteins All organisms respond to stress at the cellular level with the rapid synthesis of a small number of so-called stress proteins and a simultaneous inhibition of normal protein synthesis (Lewis et al., 2001). The protein synthesized were designated heat shock proteins (HSPs). The cellular stress response is induced by a range of stressors including pollutants (Mc Carthy and Shugart, 1990), and as primary protective response of cells, is potentially useful in environmental monitoring. Although some stress proteins are only found in cells responding to stressors, most are also present at lower concentrations under normal conditions (i.e. they are constitutively expressed) and play essential roles in cellular protein homeostasis by acting as molecular chaperones. This protects cells allowing them to recover and survive the stress (Lewis et al., 2001). Among stress proteins, HSP70 is a popular choice for biomarker research as this is the most highly conserved and widely studied of the HSPs

(Ryan and Hightower, 1996). These HSP70 have been evaluated as biomarkers of pollution, with research almost exclusively confined to animals (see synthesis in Lewis et al., 1998). Studies have been carried out in Enteromorpha intestinalis (Lewis et al., 1998, 2001) under exposure to a variety of stressors. The HSP70 response was affected by nutrient limiting conditions (reducing in fact protein metabolism), and copper induced an increase in HSP70 only under nutrient replete conditions. Herbicides failed to induce an increase of HSP70, suggesting that triazines may be weakly proteotoxic. Lewis et al. (2001) showed that HSP70 is only induced by stressors that are strongly proteotoxic. The authors concluded that HSP70 in E. intestinalis was not useful as a biomarker for copper and triazine. 3.4. Phenolic compounds Among secondary metabolites, phenolic compounds have been widely studied (Table 3). These compounds are of major importance in terms of protection in plant species despite the fact that they do not represent a primary function in physiological processes (Levin, 1971). They correspond to a wide range of chemical structures with at least an aromatic cycle (C6) carrying one or more hydroxyl groups (Waterman and Mole, 1994). A large number of these compounds have been identified and most of them are characteristic of only one or few species (Ernst and Peterson, 1994; Waterman and Mole, 1994), and are widely distributed in terrestrial higher plants, (BateSmith, 1968; Karolewski and Giertych, 1995; Giertych and Karolewski, 2000), and in aquatic species (Mc Clure, 1970; Zapata and Mc Millan, 1979; Quakenbush et al., 1986; Pip, 1992). Their qualitative and quantitative distribution varies among tissues and is influenced by growth conditions and the physiological state of the plants (Levin, 1971; Macheix, 1996). Phenolics play an important ecological role and their synthesis and storage are considered to be good indicators of stress such as grazing (Anderson and Velimirov, 1982; Steinberg, 1984, 1985; Ragan and Glombitza, 1986), infection by micro-

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Table 3 Studies realized on phenolic compounds as biomarkers of stress in aquatic plants Stress Species Kind of study References

Infection by L. zosterae , Zostera marina Porter and Muehlstein Interspecific competition P. oceanica

Hg Cu Grazing/phenolic compounds concentration

P. oceanica Ascophyllum nodosum

In situ: Roscoff, (Fce) Vergeer et al. (1995) and Vergeer and Develi (1997) In situ: Western Cuny et al. (1995), Agostini Mediterranean (Fr) et al. (1998) and Ferrat (2001) In situ: Livorno Agostini et al. (1998) and Mediterranean (It) Ferrat (2001) In vitro Toth and Pavia (2000) Anderson and Velimirov (1982) Steinberg (1984) Steinberg (1985) Steinberg and Paul (1990)

Different species of Chlorophyta, Phaeophyta, Rho- In vitro dophyta Alaria marginata Postels and Ruprecht Fucus vesiculosus (L.), Halidrys siliquosa (L.), Lyngbye Eisenia arborea Areschoug. Dictyota spiralis (Montagne), Dictyopteris australis (Sonder) Askensay, Lobophora variegata , (Lamouroux) Womersley Cystoseira trinodis , (P. Forsskal) C. Agardh Sargassum sp. Different species of Chlorophyta, Phaeophyta, Rhodophyta Sargassum sp.

Steinberg (1985) and Steinberg and Van Altena (1992) Steinberg et al. (1991) Pavia and Brock (2000)

Grazing, desiccation, UVB

Ascophyllum nodosum

In vitro

organisms (Ragan and Glombitza, 1986; Vergeer et al., 1995; Vergeer and Develi, 1997) and interand intra-specific competition (Mc Lachlan and Craigie, 1966). Most of these compounds make a significant contribution to the antioxidant activity of plants and have the capacity to bind heavy metals (Emmons et al., 1999) and are thus of major importance in the mechanisms of protection of plants against stress (Swain, 1977). In Zostera marina, infected by Labyrinthula zosterae , the concentration of phenolic compounds has been studied (Vergeer and Develi, 1997). The infection caused an increase of a phenolic compound, the caffeic acid, blocking the growth of L. zosterae . Under normal growth conditions, Zostera marina can protect itself, increasing its production of caffeic acid, but this energetic investment on behalf of defense against microorganisms did not occur when the plant is under limited growth conditions. The hypothesis that phenolic compounds can act as repellents against herbivores was suggested at the beginning of the last century (Hunger, 1902

in Schoenwaelder and Wiencke, 2000). It was first confirmed by various studies (see synthesis in Schoenwaelder and Wiencke, 2000). Among them, Steinberg (1984) showed a higher rate of production of phenolic compounds in the reproductive organs of marine species directly exposed to grazers. There was a negative correlation between the total phenolic concentration of plant tissues and their palatability for grazers (Anderson and Velimirov, 1982; Steinberg, 1984), but some studies have since demonstrated that this correlation is not systematic (Steinberg and Paul, 1990; Steinberg et al., 1991; Steinberg and Van Altena, 1992). In addition to these phenolic compounds, Smolders et al. (2000) measured lower tannin (complex phenolic compounds) concentrations in submerged than in emerged and floating plants. They explained these difference by greater exposure to pathogens or to grazers that led these plants to develop a strong chemical resistance and so an important energetic investment. Pavia and Brock (2000) supported in Ascophyllum nodosum

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an induced defense model for the production of phlorotannins, enhanced by exposure to grazing, UVB and desiccation. P. oceanica apparently does not increase phenolics in response to competition from invasive C. taxifolia. Neither Cuny et al. (1995) nor Agostini et al. (1998) showed any significant difference for water-soluble phenolic compounds in a site characterized by strong competition, compared with a site without C. taxifolia . Ferrat (2001) confirmed that potential stress, generated by competition with C. taxifolia , had a limited impact on the production of water-soluble phenolic compounds. However, an increase in tannin cells in blades from colonized sites justified reconsideration of the role of polymerized tannins in the mechanisms of response to competition. With respect to heavy metals, no modification in the levels of simple phenolic compounds was reported in P. oceanica contaminated by mercury (Agostini et al., 1998), nor in levels of polyphenolics in Ascophyllum nodosum contaminated by copper (Toth and Pavia, 2000). Two tendencies were apparent in P. oceanica (Ferrat, 2001). A decrease of total (simple and complex) or simple phenolic compounds was noted in plants coming from a contaminated site, in particular in the nonchlorophyllous basal parts of the leaves (sheaths). This suggests that complex phenolic compounds such as flavonoids, act as chelators and antioxidants, notably in the sheaths of the plant. Hydroxycinnamic acid derivates (caffeic acid, ferulic and p -coumaric acids) seemed to be involved to a lesser extent, because of their reduced antioxidant power compared to the flavonoids. Beyond their low antioxidant power, these compounds often appeared in the glycosilated form, further limiting their antioxidant activity (RiceEvans et al., 1999; Kahko nen et al., 1999; Ferrat, 2001). 3.5. Oxidative stress biomarkers (Table 4) Oxidative stress is first characterized by an oxidative burst, or a rapid and transient production of high quantities of reactive oxygen species (ROS: e.g. singlet oxygen, superoxide anion, hydrogen peroxide, hydroxyl and hydroperoxy-

radicals). The production of ROS is a natural phenomenon (Cossu et al., 1997), triggered by various external factors (Rijstenbil et al., 1994a), and generally reduced in plants under normal conditions of growth (Vangronsveld et al., 1997). The burst can be stimulated directly by various pollutants (Stegeman et al., 1992; Table 4) or indirectly by their metabolization (Cossu et al., 1997). Organic compounds and transition metals (e.g. Cu) were shown to be pro-oxidants and to accelerate the formation of oxy-radicals in plants (Salin, 1988), and their excess increased lipid peroxidation (loss of membrane integrity; Rijstenbil et al., 1994a). The oxidative burst constituted an early strategy of defense in plants (Wojtaszek, 1997), in response to various forms of stress (Ku pper et al., 2001). The presence of ROS triggered secondary reactions of defense that prevent the destructive oxidation of important metabolites (Subhadra et al., 1991). This antioxidant system was based on enzymatic (e.g. superoxide dismutase (SOD), peroxidase, catalase) and non-enzymatic mechanisms (e.g. thiol groups, flavones, phytoalexines, hydroquinones, vitamins; Cossu et al., 1997; Vangronsveld et al., 1997). The overproduction of ROS caused lipid peroxidation only when antioxidant defenses overlapped (Cossu et al., 1997). 3.5.1. Oxidative burst The phenomenon of oxidative burst has been widely studied in the case of the invasion of terrestrial plants by pathogens, but few studies have been carried out on the mechanisms of defense against pathogens in the marine environment (Potin et al., 1999). The first oxidative burst in marine species was revealed only in 1994 (Colen and Pedersen, 1994) in response to mechanical stress on Eucheuma platycladum . Studies have been carried out since in the case of an invasion by a pathogen in Chondrus crispus (Bouarab et al., 1999) and in Gracilaria conferta (Weinberger et al., 1999). 3.5.2. Lipid peroxidation Lipid peroxidation leads to the formation of degradation products such as alkanes and aldehydes (e.g. malone dialdehyde). Thiobarbituric

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Table 4 Changes in oxidative stress molecules as biomarkers of stress in aquatic plants (in vitro experiments) Biomarkers Kind of stress Species Eucheuma platycladum (Schmitz) Chondrus crispus Laminaria digitata (Hudson) Lamouroux Generation of lipid peroxidation (TBARS) Infection by a microorganism Cu, Zn, Cd Gracilaria conferta (Schousboe ex Montagne) P. oceanica Chlorella pyrenoidosa Kessler and Huss Lemna minor (L.) References Colen and Pedersen (1994) Bouarab et al. (1999) Kupper et al. (2001) Weinberger et al. (1999) and Ferrat (2001) Hamoutene et al. (1996) ` Vavilin et al. (1998) Roy et al. (1995) Subhadra et al. (1991) Ceratophyllum demersum (L.) P. oceanica Rama Devi and Prasad (1998) Ferrat (2001)

Generation of oxidative burst Mechanical stress (ROS) Infection by a microorganism

Induction of catalase, peroxidase, SOD; GR activities

HCB Cu, HgCl2, CH3HgCl HgCl2 Salinity

Hydrilla verticillata (L.f.) Royle, Najas sp. Rout and Shaw (2001) Chlorella pyrenoidosa Enteromorpha sp. Ceratophyllum demersum Ditylum brightwellii (T. West) Grunow in van Heurck, Thalassiosira pseudonana P. oceanica Vavilin et al. (1998) Rijstenbil et al. (1998) Rama Devi and Prasad (1998) Rijstenbil et al. (1994a,b) Hamoutene et al. (1996), Ferrat ` (2001) and Ferrat et al. (2003)

Decrease in thiol pool (GSH) Cu, Zn, Cd

acid reactive substances (TBARS) concentration is an interesting biomarker of metal-induced oxidative stress. Hamoutene et al. (1995, 1996) and ` Vavilin et al. (1998) noted a significant decrease of TBARS after exposure to trace metals in marine and freshwater species, respectively. TBARS increased in the foliar sheaths of P. oceanica after 48 h exposure to mercury chloride (HgCl2) at 0.01 and 0.1 mgHg l (1 as compared to the controls (Ferrat, 2001; Ferrat et al., 2002a). Paradoxically, at higher concentrations of mercury in the medium (1 mgHg l (1), a significant decrease in TBARS was noted in contaminated sheaths, compared to the controls. Non-enzymatic antioxidant molecules were suggested in counteraction to the oxidative stress generated by mercury.

3.5.3. Antioxidant enzymes Catalase accelerates the spontaneous dismutation reaction of hydrogen peroxide. An increase in its activity has been demonstrated in the presence of various pollutants (Table 4): e.g. peroxidase, glutathione reductase and SOD after 24 h exposure to organic pollutants significantly increased in Lemna minor (Roy et al., 1995). In Ceratophyllum demersum metal-induced oxidative stress increased antioxydant enzymes such as peroxidases, catalase and SOD (Rama Devi and Prasad, 1998). However, in Lemna minor, beyond a threshold of toxicity, these enzymatic activities decreased with increasing metal concentrations (Subhadra et al., 1991). Catalase activity increased significantly in P. oceanica following 48 h expo-

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sure to low concentrations of HgCl2 (0.01 mg l (1 and 0.1 mg l (1) as compared to controls (Ferrat, 2001; Ferrat et al., 2002b). In contrast, there was no significant difference between 1 mg l (1 and controls, and lipid peroxidation level was low. This result indicates that other systems of protection may be involved against oxidative stress (nonenzymatic induction of phytochelatins). When freshwater species are grown at high salt concentrations (Table 4), oxidative stress apparently induces catalase, SOD and peroxidase (Rout and Shaw, 2001). 3.5.4. Antioxidant molecules Concerning non-enzymatic mechanisms, research on biomarkers has focused on various compounds usually involved as intracellular binders for metals, or involved in their exclusion and their detoxication (e.g. vitamin E, thiol groups, particularly reduced glutathione (GSH) and phytochelatins). GSH and other non-protein-thiol groups (according to the determination method, authors consider that the analyzed molecules are glutathione or non-protein thiol compounds). Protect against oxy-radicals and pro-oxidant metals (Cu, Fe). When these molecules are not in sufficient quantities, antioxidant enzymes (e.g. SOD) can participate in the elimination of oxyradicals (Rijstenbil et al., 1994b). Glutathione (g L-glutamyl-L-cysteinyl-glycine) is involved as a co-substrate in conjugation reactions (with glutathione S -transferase: GST, see below), and is an important antioxidant in plants (Rijstenbil et al., 1994a,b; Hamoutene et al., 1995). In ` the same manner, homoglutathione (g L-glutamylL-cysteinyl-b-alanine), an alanine homologue of glutathione, can entirely or partially replace glutathione as antioxidant. Xenobiotics can modify quantities of reduced (GSH) or oxidized glutathione, indeed, in Ceratophyllum demersum , the introduction of metal into the medium caused a decrease in GSH levels proportional to its concentration (Rama Devi and Prasad, 1998). The comparison of non-protein thiol compound levels in sites contaminated by Hg, with those coming from pristine sites in the Mediterranean showed a significant decrease in reduced glu-

tathione in the foliar tissues (sheaths'/blades) of P. oceanica from the contaminated sites (Ferrat, 2001; Ferrat et al., 2003). These results are in agreement with the fact that GSH is involved in the binding of metals, single or polymerized into phytochelatins (Gekeler et al., 1989). Moreover, non-protein thiol levels were higher in blades than in sheaths (Ferrat, 2001; Ferrat et al., 2003). These processes appeared to be influenced by environmental factors, therefore GSH concentrations cannot be considered as specific indicators of oxidative stress due to a given pollutant (Rijstenbil et al., 1998). 3.6. Biomarkers of detoxication (Table 5) These biomarkers are different according to the toxic compounds that they can detoxify. Phytochelatins are reported to detoxify heavy metals whereas phase I and phase II enzymes of biotransformation metabolize also organic xenobiotics. 3.6.1. Phytochelatins The glutathione molecule above-mentioned is the precursor of phytochelatins or homophytochelatins (metalloisopeptides, (g-Glu-Cys)n-Gly with n0/2/8), peptides sequestrating trace metals (Leopold et al., 1999; Schmoger et al., 2000). The formation of these molecules is achieved by an enzymatic (g-glutamyl-cysteine synthetase) polymerization of glutathione or homoglutathione (Gekeler et al., 1989). These mechanisms have been demonstrated in terrestrial plants (Gekeler et al., 1989). Moreover, the increase of phytochelatin levels in macro- and micro-algae is a specific biomarker of heavy metal bioavailability and stress caused by these metals. Indeed, Skowronski et al. (1998) demonstrated phytochelatin synthesis in response to cadmium uptake in Vaucheria (xanthophyceae). Concerning micro-algae, experimental exposure to trace metals induced acute stress and a synthesis of phytochelatins in planktonic diatoms (Rijstenbil et al., 1994b), and Pawlik-Skowronska (2002) demonstrated, in 24 h, the production of Pb-induced phytochelatins in cells of Stichococcus bacillaris .

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3.6.2. Biotransformation enzymes The metabolism of xenobiotics proceeds in plants in three phases (Pflugmacher et al., 1999b): (i) a phase of functionalization consists of oxidation of the xenobiotic mostly by the system of cytochrome P450 monooxygenases, (ii) a second phase of conjugation facilitates the binding of the chemical pollutant to an endogenous substrate (glucuronic acid, glutathione, sulfates). The enzymes involved are generally transferases (e.g. uridinediphospho-glucuronosyl transferases, glutathione S -transferases or GSTs, sulfotransferases). An increase in the enzymatic activity during the first and second phase indicates exposure to xenobiotics (Roy et al., 1995), and (iii) the third phase consists of partitioning and storage processes in cellular walls or vacuoles. These detoxication mechanisms have been widely studied in agronomically important terrestrial plants and in particular in terms of resistance to herbicides, but there is little information on the activity of these enzymes in aquatic macrophytes (Pflugmacher et al., 1999b). Some studies have been carried out on herbicide (Tang et al., 1998) and hydrocarbon (Schrenk et al., 1998; Table 5) detoxification in freshwater species, and on the action of trace metals on marine species (Hamoutene et al., 1995; Ranvier et ` al., 2000; Ferrat, 2001; Ferrat et al., 2002a,c). 3.6.2.1. Phase I enzymes. The system of P450 cytochrome takes an important part in phase I enzymes. It is responsible for the oxidative metabolism of a wide variety of compounds, including xenobiotics as well as endogenous compounds (e.g. fatty acids). These hemoproteins are in fact only one of the various elements of the electron transport multienzymatic complex. The ethoxyresorufin O -deethylase (EROD) activity seems to be the most sensitive catalytic activity that can be used to measure an induction of cytochrome P450 (Hamoutene et al., 1996). In ` P. oceanica , these authors observed an inhibition of cytochrome P450 dependent enzymes in the case of a metallic contamination, but noted a high variability of the EROD measurement. Pflugmacher and Sandermann (1998), Pflugmacher et al. (1999a,b) have detected the existence of

cytochrome P450 oxygenases in various marine species. Activities of the cytochrome P450 monooxygenases system have been demonstrated for substrates such as fatty acids (e.g. lauric, palmitic, stearic acids) but also for xenobiotics like phenobarbital (Pflugmacher and Sandermann, 1998) and polychlorobiphenyls (PCBs) (Pflugmacher et al., 1999b). 3.6.2.2. Phase II enzymes. GSTs form a superfamily of multifunctional enzymes (mostly cytosolic), in animal like in vegetal organisms (Pascal and Scalla, 1999; Plaisance and Gronwald, 1999; Reade and Cobb, 1999). GSTs exist under homo or heterodimer forms of around 25 kDa molecular weight (Marrs, 1996). GSTs catalyze the conjugation reaction of reduced glutathione (GSH) with electrophilic substrates (Pascal and Scalla, 1999; Plaisance and Gronwald, 1999; Reade and Cobb, 1999). GSHsubstrate conjugates are more polar and less toxic (Marrs, 1996). Plant GSTs have been discovered as part of herbicide detoxication in terrestrial plants (Shimabukuro et al., 1970) and studied for their role in the selectivity towards these contaminants (Lamoureux et al., 1991, Plaisance and Gronwald, 1999). They appear to be responsible for tolerance to herbicides (Marrs, 1996; Reade and Cobb, 1999), but many authors have demonstrated their involvement in the detoxication of polycyclic aromatic hydrocarbons (PAHs), PCBs, heavy metals (see synthesis in Marrs, 1996). Various studies have revealed the existence of GST isozymes in various plants (Pascal and Scalla, 1999), with different degrees of specificity to herbicides (Timmermann, 1989). A significant influence of xenobiotic exposure has been demonstrated on the enzymatic activity of aquatic species. GST activity was significantly increased after exposure to PAHs (Schrenk et al., 1998), HCBs (Roy et al., 1995), and Pflugmacher et al. (1999b) also identified GST and O - and N glucosyltransferase activities in various marine species, in response to contamination by PCBs. Tang et al. (1998) showed, in freshwater species, the involvement of GST activity in the metabolism of atrazine. It seems that the species most tolerant

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Table 5 Induction of biotransformation molecules and enzymes by heavy metals or xenobiotics in aquatic plants Biomarkers Induction of phytochelatins Stress Heavy metals Species Ditylum brightwellii , Vaucheria Stichococcus bacillaris P. oceanica Kind of study In vitro References Rijstenbil et al. (1994a,b), Skowronski et al. (1998) and Pawlik-Skowronska (2002) Hamoutene et al. (1996) `

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187 /204

Induction of phase I enzymes: cyto- Cu chrome P450 monooxygenases EROD Phenobarbital

In vitro

Different species of Chlorophyta, Phaeophyta, Rhodophyta 3-Chlorobiphenyl Different species of Chlorophyta, Phaeophyta, Rhodophyta Chlamydomonas sp., Chlorella sp., Scenedesmus quadricauda Cyclotella sp., Synedra sp. P. oceanica

In vitro In vitro In vitro

Pflugmacher and Sandermann (1998) Pflugmacher et al. (1999a,b) Tang et al. (1998)

Induction of phase II enzymes: GSTs

Atrazine

Cu, HgCl2, CH3HgCl PAHs

Nuphar lutea , Lemna minor , Hippuris vulgaris , Potamogeton sp.

In vitro and in situ: WesHamoutene et al. (1996), Ranvier et al. ` tern Mediterranean (Fr, It) (2000), Ferrat et al. (2002a,c) and Ferrat (2001) Eastern European lakes Schrenk et al. (1998)

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of atrazine are those showing the highest levels of GST activities (Tang et al., 1998). Concerning the detoxification of heavy metals, studies have been carried out with Cd (Hamoutene ` et al., 1996), or Hg (Ranvier et al., 2000; Ferrat et al., 2002a,c), also showing an increase in GST activity in presence of heavy metal. Experimental contamination of P. oceanica by HgCl2 and CH3HgCl as a function of time (144 h), showed that the inorganic form of mercury preferentially induced GSTs (Ferrat et al., 2002a). In parallel, there was also an increase of GST activity in controls as a function of time. This last observation implied that GSTs are not specific to contamination by mercury, and responded as much to stress generated by conservation in the aquarium as to stress generated by mercury. Moreover, P. oceanica collected in a contaminated and a pristine site of the Western Mediterranean, showed increased GST activity from the contaminated site. An immunochemical study performed on these samples made it possible to determine the presence of A1/A1 isozyme (with chlorodinitrobenzene affinity, the exogenous substrate used for the experiment) only in the sheaths, compared to the blades. The induction was higher in the sheaths coming from the contaminated site as compared to the pristine one (Ferrat, 2001). Both in vitro and in vivo experiments (Ferrat et al., 2002a,c) have demonstrated that GST activities were always higher in the sheaths than in the blades. In the light of the results of the immunochemical study (Ferrat et al., 2002a), other GST isozymes may exist in the blades.

4. Conclusion In conclusion, the survey presented here shows the variety of the mechanisms of response that can be carried out by aquatic plants, in the case of the alteration of environmental conditions, whether of natural or anthropogenic origin. Among the biomarkers mentioned, markers of photosynthetic activity (chlorophyll fluorescence, photosynthetic pigments concentration), markers

of oxidative stress (lipid peroxidation, catalase, GSH) and of detoxication (GST, GSH, phytochelatins) seem to offer encouraging possibilities. They do indeed seem to be representative of the level of perturbation of the medium and of the health of the organisms. They could therefore be used for the early detection of alterations in water quality. Unfortunately, studies carried out on HSPs did not still show encouraging results for their utilization as stress markers. In addition to the species on which these mechanisms have been tested, seagrasses and particularly P. oceanica , have been revealed to be very sensitive with regard to variations in environmental quality, which is consistent with their use as classical bio-indicator species. Indeed, studies show that mercury contamination could provoke measurable damages like membrane degradation (lipid peroxidation), enzymatic inhibition, but that the plant could answer by mechanisms of protection (i) induced directly by mercury like biotransformation enzymes (GST), (ii) induced by oxygen reactive species (oxidative stress) like antioxidant enzymes (catalase), antioxidant and metal chelators molecules (GSH, phenolic compounds) very rapidly. So the study of various biomarkers in this species offers a good illustration of the multiplicity of the mechanisms involved and confirms the importance and the interest of a multiparametric approach, including both physiological biomarkers, biomarkers of general stress and more specific biomarkers. This multiparametric approach is highly recommended in ecotoxicology. Moreover, the identification of specific responses to a particular kind of stress in these plants, given their extensive range of distribution and their localization in the littoral zone, could make them effective tools for the early evaluation of the quality of this environment that is so exposed to human activities.

Acknowledgements The authors would like to thank Dr M. Paul for proof-reading the English.

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