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Environmental Toxicology and Chemistry, Vol. 24, No. 10, pp.

25002508, 2005
q 2005 SETAC
Printed in the USA
0730-7268/05 $12.00 1 .00




Research and Technology, Melbourne Water Corporation, GPO Box 4342, Melbourne, Victoria 3001, Australia
Centre for Environmental Stress and Adaptation Research, La Trobe University, Bundoora, Victoria 3086, Australia
Centre for Environmental Stress and Adaptation Research, Department of Zoology, University of Melbourne, Parkville,
Victoria 3010, Australia

( Received 10 January 2005; Accepted 24 March 2005)

AbstractHighmolecular weight (.C16) hydrocarbons (HMWHs) are common pollutants in sediments of freshwater systems,
particularly urban water bodies. No sediment quality guidelines exist for total hydrocarbons; more emphasis is placed on polyaromatic
hydrocarbons, the most toxic component of hydrocarbons. A field-based microcosm experiment was conducted to determine whether
unpolluted sediments spiked with synthetic motor oil impair freshwater macroinvertebrate assemblages. Total petroleum hydrocarbon
(TPH) concentrations of 860 mg/kg dry weight significantly increased the abundance of Polypedilum vespertinus and Cricotopus
albitarsis and decreased the abundance of Paratanytarsus grimmii adults (all Chironomidae), whereas TPH concentrations ranging
from 1,858 to 14,266 mg/kg produced a significant reduction in the total numbers of taxa and abundance, with significant declines
in the abundance of nine chironomid taxa. About 28% of water bodies surveyed in urban Melbourne, Australia, had TPH concen-
trations in sediments likely to cause ecological impairment, and about 14% of the water bodies surveyed are likely to have reduced
species richness and abundance. Therefore, HMWHs can be a significant pollutant in urban water bodies. Freshwater sediment
quality guidelines should be developed for this ubiquitous urban pollutant.

KeywordsBioindicators Sediment contamination Chironomidae Microcosms Urban pollution

INTRODUCTION organisms, they can alter the physical properties of sediments,

Hydrocarbons are common anthropogenic pollutants in the coat and smother organisms [14], and contribute to organic
waters and sediments of urban water bodies that are mainly enrichment of sediments [15].
derived from crude oil (gasoline, kerosene, fuel oil, mineral Sediment quality guidelines have been proposed for PAHs
oil, and asphalt) [1]. It is estimated that 1.7 to 8.8 million [1618], but not for other hydrocarbon compounds that com-
metric tons of petroleum oils are released into the environment monly occur in urban sediments. This raises the question of
annually [2]. Major sources of petroleum hydrocarbon pol- whether sediment quality guidelines for just PAHs provide an
lution include atmospheric deposition from vehicle and in- adequate indication of whether hydrocarbon-polluted sedi-
dustrial emissions, asphalt degradation and leakage of oils ments are affecting the health of aquatic ecosystems. There-
from vehicles [1,3] and gray water [4]. fore, the need to investigate the ecological effects of sediments
Petroleum or crude oil is a complex mixture of many hy- polluted with HMWHs, but containing low concentrations of
drocarbons that mainly consist of saturated predominantly PAHs, is warranted.
straight-chain alkanes, small amounts of slightly branched al- A field-based microcosm method (following [19]) was used
kanes, cycloalkanes, and aromatics [5]. The aromatic com- to determine whether sediments contaminated by HMWHs,
ponents of oils, particularly polyaromatic hydrocarbons but not PAHs, affect the macroinvertebrates that can inhabit
(PAHs), are generally assumed to be the most toxic component these sediments. Macroinvertebrates would appear to be good
of petroleum hydrocarbons and are generally the focus of field indicators of sediment pollution because many taxa are as-
surveys (e.g., [68]). Heterocyclics may be a major determi- sociated with sediments. Clean sediments and sediments
nant of toxicity in water polluted with weathered distillate oils spiked with four different concentrations of weathered syn-
[9]. Synthetic motor oils have also become more widely used thetic motor oil were used to describe how the composition
in vehicles and industry in past decades (e.g., http:// and abundance of macroinvertebrate assemblages can change
www.mobil1.com). with increased levels of hydrocarbon pollution in sediments.
Lowmolecular weight hydrocarbons are more soluble, and The sediments spiked with synthetic motor oil contained low
therefore more toxic, to aquatic organisms than those of higher levels of PAHs but elevated concentrations of total petroleum
molecular weight [10]. Highmolecular weight hydrocarbons hydrocarbons (TPHs) within the C15 to C36 range (hence
(HMWHs) have very limited solubility in water and are more referred to as HMWHs), which is similar to what is found
likely to sorb onto soil [11]. Recalcitrant aliphatics and other when HMWHs are exposed to weathering [1922].
HMWHs are the primary hydrocarbon fractions found in sed-
The purpose of this investigation was to determine whether
iment [12] and are considered to have a low toxicity potential
sediments polluted with HMWHs, but with low levels of PAHs,
[13]. Although HMWHs might not be directly toxic to aquatic
can severely impair aquatic ecosystems and whether the de-
* To whom correspondence may be addressed velopment of sediment quality guidelines for HMWHs is war-
(vin.pettigrove@melbournewater.com.au). ranted. Finally, to determine the ecological significance of
Effects of hydrocarbons on macroinvertebrates Environ. Toxicol. Chem. 24, 2005 2501

HMWH pollution on urban aquatic ecosystems, the TPH con- ranged into five blocks, with each block containing six mi-
centrations present in sediments in this experiment are com- crocosms (two Glynns Wetland sediments and one each of the
pared to those present in urban streams, drains, and wetlands four motor oilspiked treatments). Briefly, insects that emerge
in Melbourne, Australia. Melbourne is a good location to as- from the wetland mate and then randomly lay eggs within the
sess the contribution of urban runoff to petroleum hydrocarbon microcosms. This method provides a unique means of gath-
pollution in freshwater sediments because it has separate storm ering information on the effects of sediment quality on mac-
water and sewerage systems, and spills of sewage into the roinvertebrates in lentic habitats, particularly for indigenous
storm water system rarely occur. species that cannot be easily reared in the laboratory. Further
details regarding the method used for the field-based experi-
MATERIALS and METHODS ment are described by Pettigrove and Hoffmann [19], except
Design of microcosm experiments the following modifications were made to collect adults emerg-
Nonpolluted sediments were collected from Glynns Wet- ing from the microcosms. Fine 100% nylon elastic mesh (Cool
land, located approximately 30 km east of Melbourne, Aus- Catst stockings, Coolaroo, Victoria, Australia) was placed
tralia. These sediments are used as reference sediments in this over each microcosm once emergence from the microcosms
experiment because previous experiments have demonstrated was evident (after 50 d). This mesh was sufficiently fine (,100
that Glynns Wetland sediments can support a variety of mac- mm) to prevent new insects from colonizing the microcosms
roinvertebrate taxa [19]. Glynns Wetland sediments were and to contain emerged insects within the microcosms, but
spiked with four different concentrations of synthetic motor sufficiently coarse to allow ventilation for reducing conden-
oil (Mobil 1t 10W-40, Melbourne, Australia). The spiked sed- sation within the microcosms. Adult insects that emerged from
iments were thoroughly mixed in a blender for 30 min and the microcosms were collected with aspirators approximately
stored for two months (to allow weathering) before being once a week. These were stored dry in 1.5-ml Eppendorf tubes
placed in field-based microcosms. Ten replicate microcosms at 48C until they were processed in the laboratory.
were set up for the nonpolluted Glynns Wetland sediment, and The experiment was terminated 88 d after the mesh had
five replicates were set up for the remaining four motor oil been placed over the microcosms, when most species had com-
treatments. A larger number of replicates were set up for pleted their life cycle and few individuals were emerging from
Glynns Wetland sediment to increase the statistical power of the microcosms. The only species that did continue to emerge
comparisons between a nonpolluted sediment and several pol- in large numbers at this time was the parthenogenetic [24]
luted sediments. chironomid Paratanytarsus grimmii. Adults of this species
Synthetic motor oils are manufactured from a variety of were observed laying fertile eggs within the tanks soon after
synthetic hydrocarbons and nonhydrocarbon organic com- emergence. No other species was able to successfully breed
pounds. The main ingredient in the synthetic motor oil tested within the confines of the microcosms once these had been
was polyalphaolefins (http://www.mobil1.com), which bind covered with mesh. After the experiment was terminated, re-
tightly to sediments, have low bioavailability, and are rela- maining larvae in the microcosms were collected with a 64-
tively nontoxic compared with mineral oils, diesel fuel, and mm mesh net and stored in 100% ethanol.
internal olefins [23]. The polyalphaolefins were selected as a Common macroinvertebrate taxa were identified to the low-
surrogate of HMWH pollution because their low bioavail- est possible taxonomic unit. Dytiscidae larvae and chironomid
ability and toxicity provide a conservative estimate of the po- adults, pupae, and larvae were identified with the use of keys
tential effects of hydrocarbon pollution in freshwater sedi- by Pettigrove and Hoffmann [19]. Hemiptera [25], Hydrop-
ments. tilidae larvae [26], and Ephemeroptera nymphs [27] were iden-
The PAH concentrations in sediments from each treatment tified with the use of keys by Anderson and Weir [25], Wells
were analyzed according to the U.S. Environmental Protection [26], and Dean and Suter [27]. Molecular markers (following
Agency (U.S. EPA) Method 8270C (http://www.epa.gov/ [28]) were also examined in some species of Chironomidae to
epaoswer/hazwaste/test/pdfs/8270c.pdf). Volatile petroleum confirm species identification.
hydrocarbons (C6C9) were extracted by purge and trap (U.S.
Data analyses
EPA Method 5030B; http://www.epa.gov/epaoswer/hazwaste/
test/pdfs/5030b.pdf) and analyzed according to U.S. EPA All analyses were conducted with SPSS V 11.0 for Win-
Method 8260B (http://www.epa.gov/epaoswer/haswaste/test/ dows (Version 11.5, SPSS, Chicago, IL, USA). Because the
pdfs/8260b.pdf). Semivolatiles were analyzed according to biological data exhibited skewed distributions, nonparametric
U.S. EPA Method 8015B (http://www.epa.gov/epaoswer/ tests were conducted to compare species abundance between
hazwaste/test/pdfs/8015b.pdf). All analyses were conducted by treatments. Two-tailed MannWhitney U tests were undertak-
the Australian Laboratory Services, which is accredited by the en to compare the total number of taxa and individuals between
National Association of Testing Authorities, Australia (http:// treatments. A second test was conducted on the total number
www.nata.asn), to conduct these tests. Blanks, checks, and of individuals present, excluding P. grimmii, because this spe-
spikes were conducted as part of quality control procedures cies dominated the number of individuals present and obscured
for all analyses. Sediments that were analyzed for TPHs were other trends in faunal abundance.
spiked with TPH/benzene, toluene, ethylbenzene, and xylene Nonparametric KruskalWallis and JonckheereTerpstra
compound surrogates. The recovery of all surrogates was with- tests were undertaken to determine whether significant differ-
in 70 to 130% of the initial concentration, thus indicating a ences occurred in the abundance of each common taxon be-
satisfactory recovery of these compounds. tween each of the five treatments. It is hypothesized that the
The microcosms (20-L polypropylene tanks with 750 g of abundance of common taxa gradually declines with increased
sediment and 15 L of filtered water from the wetland) were HMWH pollution. To test this hypothesis, a JonckheereTerps-
placed along the edge of Glynns Wetland. A random block tra test of each common taxon was conducted to determine
design was used in the experiment. The microcosms were ar- whether abundance declined significantly with a gradient of
2502 Environ. Toxicol. Chem. 24, 2005 V. Pettigrove and A. Hoffmann

increased pollution. An alternative hypothesis is that moderate Table 1. Total petroleum hydrocarbon (TPH) concentrations (dry wt)
separated into carbon number range in reference sediment (treatment
levels of pollution increase the abundance of some common
0) and sediment spiked with motor oil (treatments 14)
taxa and higher levels of pollution decrease abundance. In
these instances, the abundance of a taxon would not vary in TPH treatment (mg/kg)
an a priori manner. Therefore, KruskalWallis tests were used Carbon no.
to test whether significant differences in abundance occurred Range 0 1 2 3 4
in each common taxon between treatments. C6C9 ,4 ,4 ,4 ,4 ,4
Two-tailed MannWhitney U tests were conducted in a C10C14 ,25 26 ,25 ,25 38
sequential manner to explore what concentrations of TPH in C15C28 ,50 316 727 1,811 5,580
sediments produced significant changes in the total numbers C29C36 ,50 518 1,131 2,821 8,648
Total TPH ,125 860 1,858 4,632 14,266
of individuals, total number of taxa, and abundance of common
taxa along a gradient of HMWH pollution in sediments. In
these tests, treatment 0 was first compared with treatment 1. RESULTS
If there was no significant difference between these treatments,
they were compared with treatment 2. If there was a significant Chemical analysis of sediments
difference between treatments 0 and 1, then only treatment 1 The TPH concentrations in sediments ranged from below
was compared with treatment 2. This process continued until detection limits (,125 mg/kg) in nonpolluted Glynns Wetland
comparisons had been undertaken between all treatments. sediment (treatment 0) to 860, 1,858, 4,632, and 14,266 mg/
An evaluation of sublethal responses to environmental kg in treatments 1, 2, 3, and 4, respectively (Table 1). The C6
stress might provide a better early warning system of ecolog- to C9 TPH fraction was below the detection limit of 4 mg/kg
ical impairment than metrics based on species presence and in all samples. The TPHs present were typically from the large
abundance [29]. Growth is often chosen as a response variable carbon fractions: 38 to 39% of the TPHs were in the C15 to
to measure sublethal effects because it is easy to quantify and C28 fraction and 60 to 61% in the C29 to C36 fraction.
integrates a suite of biochemical and physiological effects into All 16 PAHs analyzed in these sediments were below the
a metric that is often linked to individual fitness [30]. The detection limit of 0.25 mg/kg in all treatments. Therefore, all
emergence rates of adult Cricotopus albitarsis were compared treatment sediments were below the available freshwater sed-
between treatments 0 to 3 to determine whether HMWH pol- iment TEC [18] and ERL [17] sediment quality guidelines,
luted sediments produce sublethal stress, as indicated by re- indicating that harmful effects from PAH pollution are unlikely
tarded rates of emergence. This assessment was confined to to be observed.
C. albitarsis because it was the only species, except for P.
grimmii (which could breed within closed microcosms), in Common macroinvertebrate taxa
which .10 adults emerged from at least four treatments. Two- A total of 36 taxa and 19,499 individuals were collected
tailed MannWhitney U tests were conducted in a sequential from the 30 microcosms used in this experiment. The fauna
manner to explore whether sediments polluted with TPHs de- was dominated by the chironomid P. grimmii, which repre-
layed adult emergence, as indicated by the time taken for adults sented 74.9% of all individuals collected. A list of the common
to first emerge and for 50% of adults to emerge from each taxa present in this experiment and the number of microcosms
treatment. The sequential process is the same used to compare they inhabited are presented in Table 2. The fauna was dom-
changes in total numbers of individuals, total numbers of taxa, inated by Chironomidae (Diptera), and species from the He-
and abundance of common taxa along a gradient of HMWH miptera, Coleoptera, Ephemeroptera, and Trichoptera were
pollution in sediments. also common. Fifteen taxa (P. grimmii, Procladius villosi-
manus, Mesovelia sp., Tasmanocoenis sp., Micronecta annae,
Comparison of experimental TPH concentrations with Kiefferulus martini, Necterosoma pencillatum, Hellyethira
field data sp., Larsia albiceps, Tanytarsus fuscithorax, Polypedilum
vespertinus, C. albitarsis, Cladopelma curtivalva, Polype-
Total petroleum hydrocarbon concentrations present in the dilum watsoni, and Cladotanytarsus bispinosus) were present
experimental treatments were compared with field data col- in six or more microcosms.
lected by the watershed management authority for Melbourne
(Melbourne Water) from urban streams, drains, and wetlands Comparison of number of taxa between treatments
(including lakes) in the Melbourne region. These data were The median number of taxa in microcosms ranged from 11
used to determine the proportion of urban aquatic ecosystems per microcosm in treatment 2, to 0 per microcosm in treatment
likely to show levels of pollution comparable to that found in 4 (Fig. 1A). The numbers of taxa decline significantly between
the field-based experiments. Total petroleum hydrocarbon sed- treatments 0, 1 and 2, and treatment 3 (MannWhitney U test,
iment concentrations were measured at wadeable (,2 m) p , 0.01) and between treatments 3 and 4 (MannWhitney U
depths from 104 sites in streams, 112 sites on subterranean test, p , 0.01). Therefore, TPH concentrations between 1,858
drains, and 269 wetlands and lakes. The method for collection and 4,632 mg/kg in sediments reduced species richness, and
of sediments is detailed in Pettigrove and Hoffmann [19], ex- concentrations exceeding 4,632 mg/kg led to a severely im-
cept that unfiltered sediments were used for the analysis of poverished fauna.
TPHs. Sediments were also analyzed for PAHs; however, these
data are not presented because .99% of sediments had PAH Comparison of number of individuals between treatments
concentrations below the threshold effect concentrations The median number of individuals per treatment ranged
(TEC) [18] and effects range low (ERL) [17] sediment quality from 1,568 per microcosm in treatment 2 to 0 per microcosm
guidelines, indicating that harmful PAH effects are potentially in treatment 4, as reported in Figure 1B (error bars represent
rare. the 25th and 75th percentiles for each treatment). The number
Effects of hydrocarbons on macroinvertebrates Environ. Toxicol. Chem. 24, 2005 2503

Table 2. List of the most abundant invertebrate taxa, percentage of total abundance, and the number of microcosms the taxon inhabited

Taxon Family/Order No. of individuals % Total abundance No. of microcosms

1 Paratanytarsus grimmii Chironomidae 13,930 71.40 24

2 Procladius paludicola Chironomidae 1,774 9.09 22
3 Mesovelia sp. Hemiptera 1,486 7.62 6
4 Tasmanocoenis sp. Ephemeroptera 528 2.71 13
5 Polypedilum nubifer Chironomidae 299 1.53 3
6 Micronecta annae Hemiptera 154 0.79 6
7 Kiefferulus martini Chironomidae 153 0.78 8
8 Necterosoma pencillatum Coleoptera 144 0.73 10
9 Hellyethira simplex Trichoptera 127 0.65 6
10 Larsia albiceps Chironomidae 112 0.57 19
11 Tanytarsus fuscithorax Chironomidae 112 0.57 11
12 Coelopynia pruinosa Chironomidae 102 0.52 3
13 Polypedilum vespertinus Choronomidae 98 0.50 12
14 Cricotopus albitarsis Chironomidae 88 0.45 16
15 Cladopelma curtivalva Chironomidae 78 0.40 11
16 Polypedilum watsoni Chironomidae 74 0.38 9
17 Oligochaeta 64 0.33 1
18 Cladotanytarsus bispinosus Chironomidae 35 0.18 11
19 Cryptochironomus delfinicus Chironomidae 33 0.17 2

of individuals between treatments 3 and 4 declined signifi-

cantly (MannWhitney U test, p , 0.01), but with no signif-
icant differences between remaining treatments.
Because the number of individuals present is dominated by
P. grimmii, which was observed laying fertile eggs within the
confines of the microcosms, another analysis was conducted
comparing the number of individuals between treatments with
all data, except P. grimmii. With the exclusion of P. grimmi,
the numbers of individuals with increasing TPH sediment con-
centrations from treatment 0 (median 5 178 individuals/mi-
crocosm) to treatment 4 (median 5 0 individuals/microcosm)
reduces progressively (Fig. 1C). Significant declines in the
number of individuals occur between treatments 0 and 1 and
treatment 2 (MannWhitney U test, p , 0.001), and treatments
2 and 3 and treatment 4 (MannWhitney U test, p 5 0.028).
Therefore, TPH sediment concentrations between 860 and
1,858 mg/kg can reduce macroinvertebrate abundance, and
TPH sediment concentrations exceeding 1,858 mg/kg will lead
to further reductions in macroinvertebrate abundance.

Comparison of abundance of common taxa between

TPH treatments
Paratanytarsus grimmii was the most abundant taxon col-
lected during this experiment and was present in 24 of the 30
microcosms (Table 2). Large numbers of larvae were present
in many microcosms when the experiment was concluded. This
species is parthenogenetic [25], and adults that emerged from
the microcosms were observed laying fertile eggs within the
confines of the microcosms. The abundance of P. grimmii
adults and larvae was significantly different between treat-
ments (KruskalWallis test; Table 3). Although P. grimmii
adults and larvae were abundant in treatments 0 to 3 (Fig. 2A),
their abundance was significantly lower in treatment 4 than in
other treatments (MannWhitney U test, p 5 0.001). Para-
tanytarsus grimmi occurred in 92% of microcosms in treat-
ments 0 to 3 but was only present in one of the five microcosms
used for treatment 4.
Fig. 1. The average number of taxa (A), average number of individuals A higher sensitivity to HMWH pollution is apparent in P.
(B), and average number of individuals excluding Paratanytarsus grimmii when only adults are analyzed (JonckheereTerpstra
grimmii (C) per microcosm in total petroleum hydrocarbon (TPH)
treatments. The error bars indicate the 25th and 75th percentiles, and test; Table 3). The median number of individuals per treatment
the asterisks indicate data sets are significantly ( p , 0.05) different ranged from 66.4 individuals/microcosm in treatment 0, to 0
from one another. individuals/microcosm in treatment 4 (Fig. 2B). The number
2504 Environ. Toxicol. Chem. 24, 2005 V. Pettigrove and A. Hoffmann

Table 3. Results of KruskalWallis and JonckheereTerpsta tests tini, and P. watsoni declines significantly with increased TPH
determining whether significant differences exist between the pollution (JonckheereTerpstra; Table 3). Abundance and fre-
abundance of common taxa between all total petroleum hydrocarbon
treatmentsa quency of occurrence of C. curtivalva are similar between
treatments 0, 1, and 2, whereas it is absent in treatments 3 and
Jonckheere 4 (Fig. 3). A further significant decline in abundance of C.
Taxon Terpstra KruskalWallis curtivalva occurred between treatments 0, 1, and 2, and treat-
Cricotopus albitarsis NS 0.004 ments 3 and 4 (MannWhitney U test, p 5 0.03). Significantly
Cladotanytarsus bispinosus NS NS fewer P. villosimanus occurred in treatment 2 than in treat-
Cladopelma curtivalva 0.016 NS ments 0 and 1 (MannWhitney U test, p 5 0.004), and sig-
Procladius villosimanus ,0.001 ,0.001 nificantly fewer individuals were present in treatment 3 than
Kiefferulus martini 0.012 NS in treatment 2 (MannWhitney U test, p 5 0.042). Kiefferulus
Larsia albiceps 0.002 0.009
Mesovelia sp. NS NS martini was present in only eight microcosms, but seven of
Micronecta annae NS NS these are from treatments 0 or 1, and one from treatment 2
Necterosoma pencillatum NS NS (Fig. 3). Abundance declined significantly between treatments
Paratanytarsus grimmii (all) NS 0.017 0 and 1 and the remaining treatments (MannWhitney U test,
Paratanytarsus grimmii (adults) ,0.001 ,0.001
Polypedilum vespertinus NS 0.021 p 5 0.011). Polypedilum watsoni was present in 75% of mi-
Polypedilum watsoni 0.009 0.074 crocosms used in treatments 0 and 1 but was present in only
Tasmanocoenis sp. NS NS one microcosm used in treatment 2 and absent in treatments
Tanytarsus fuscithorax NS 0.048 3 and 4 (Fig. 3). Polypedilum watsoni abundance was signif-
a NS 5 not significant.
icantly lower in treatments 2, 3, and 4 compared with treat-
ments 0 and 1 (MannWhitney U test, p 5 0.012).
The abundance of C. albitarsis, P. vespertinus, Larsia al-
of adults present in treatments 1 and 2 was significantly lower biceps, and Tanytarsus fuscithorax differ between treatments
than those present in treatment 0 (MannWhitney U test, p 5 (KruskalWallis tests; Table 3), but these species tended to be
0.046), and adult abundance was significantly lower in treat- most abundant in sediments with low to moderate levels of
ment 3 than in treatments 1 and 2 (MannWhitney U test, p TPH pollution (Fig. 3). This suggests that moderate levels of
5 0.006). Therefore, although the larvae are able to exist in HMWH pollution in sediments might provide an advantage
the more polluted sediments (treatments 13), even low levels for these species. Cricotopus albitarsis abundance was sig-
of TPH pollution (e.g., treatment 1) are sufficient to reduce nificantly higher in treatments 1 and 2 than in treatment 0
adult emergence. (MannWhitney U test, p 5 0.002); significantly higher in
The abundance of C. curtivalva, P. villosimanus, K. mar- treatments 1, 2, and 3 than in treatment 0 (MannWhitney U
test, p 5 0.012); and significantly lower between treatments
1, 2, and 3 and treatment 4 (MannWhitney U test, p 5 0.008).
Polypedilum vespertinus was more abundant in treatments 1
and 2 than in treatment 0 (MannWhitney U test, p 5 0.027)
and more abundant in treatments 1 and 2 than in treatments
3 and 4 (MannWhitney U test, p 5 0.009). Larsia albiceps
and T. fuscithorax were most abundant in treatments 2 and 1,
respectively, but not higher than in treatment 0. Larsia albi-
ceps was less abundant in treatments 3 and 4 than in treatments
0, 1, and 2 (MannWhitney U test, p 5 0.002). Tanytarsus
fuscithorax was less abundant in treatments 2 and 3 than in
treatments 0 and 1 (MannWhitney U test, p 5 0.015).
The abundance of C. bispinosus, Mesovelia sp., M. annae,
N. pencillatum, and Tasmanocoenis sp. between treatments
was not significantly different by either the JonckheereTerps-
tra or KruskalWallis test. Statistical power was limited for
analyzing Mesovelia sp. and M. annae because they were
present in six of the 30 microcosms. Cladotanytarsus bispi-
nosus, N. pencillatum, and Tasmanocoenis sp. were able to
tolerate HMWH-polluted sediments at levels present in treat-
ment 3, but not treatment 4 (Fig. 3), suggesting they are af-
fected by TPH sediment concentrations between 4,632 and
14,266 mg/kg.

Adult emergence rate

Adult C. albitarsis emerged from several treatments and
were tested for delayed emergence with increased HMWH
pollution in sediments. The median value for emergence was
between 6 and 9 d after the microcosms were closed to aerial
Fig. 2. The effects of total petroleum hydrocarbon (TPH) pollution
on abundance and frequency of occurrence of adults and larvae (A) colonization for treatments 0, 1, and 2, whereas the median
and adults (B) of Paratanytarsus grimmii. The asterisks indicate data emergence for treatment 3 was 14 d (Fig. 4). Emergence com-
sets are significantly ( p , 0.05) different from one another. menced in treatments 0, 1, and 2 between 1 and 5 d after the
Effects of hydrocarbons on macroinvertebrates Environ. Toxicol. Chem. 24, 2005 2505

Fig. 3. The effect of total petroleum hydrocarbon (TPH) pollution on the abundance and occurrence of 12 common taxa present in the microcosms.
The bars illustrate the average number of individuals per microcosm and lines represent the proportion of microcosms per treatment in which
that taxon was present. The asterisks indicate data sets are significantly ( p , 0.01) different from one another.
2506 Environ. Toxicol. Chem. 24, 2005 V. Pettigrove and A. Hoffmann

Table 4. The percentage of urban wetlands, drains, and streams in

Melbourne, Australia, with total petroleum hydrocarbon (TPH)
concentrations present in the sediments that exceed the TPH
concentrations in the treatments used in the field-based experiment

Water bodies affected (%)

No. TPH (mg/kg) Streams Drains Wetlands Total

1 .860 23.1 34.8 26.4 27.6

2 .1,858 6.7 19.6 14.9 14.2
3 .4,632 2.9 1.8 6 4.3
4 .14,266 0 0 1.1 0.6
n 104 112 269 485
Fig. 4. The cumulative numbers of Cricotopus albitarsis adults that
emerged from treatments 1, 2, and 3 and unpolluted sediment (treat-
ment 0). Day 0 5 day when microcosms were covered with a mesh
to prevent further aerial colonization by insects. Treatment 0 (l);
Treatment 1 (- v -); Treatment 2 (m); Treatment 3 (m). Ecological implications
Urban water bodies are affected by a variety of pollutants,
including heavy metals [31,32], hydrocarbons [1,3], pharma-
microcosms were closed, whereas no emergence occurred in ceuticals, hormones, and other organic wastewater chemicals
treatment 3 until 14 d afterwards. The difference in the time [33]. Polluted urban aquatic sediments could have a direct
required for adults to first emerge from treatments 0, 1, and effect on the occurrence and abundance of macroinvertebrates
2 and treatment 3 was significant (MannWhitney U test, p in water bodies in urban areas [19]. To improve the ecological
5 0.025), but the differences in median emergence times be- health of these systems, it is important to identify the primary
tween treatments were not significant. This delayed first emer- pollutants affecting the biota so that prudent pollution man-
gence of adults in treatment 3 indicates C. albitarsis in treat- agement strategies can be developed. This is the first study to
ment 3 were stressed from HMWH polluted sediments, al- use the field-based microcosm method developed by Pettigrove
though the numbers of adults that emerged from treatment 3 and Hoffmann [19] that isolates the effect of one type of sed-
was similar to those in treatments 0 to 2. iment pollution (HMWHs) on macroinvertebrate occurrence
and abundance.
Critical TPH sediment concentrations The synthetic motor oil added to clean sediments produced
a form of hydrocarbon pollution similar to that in urban streams
Some taxa (C. albitarsis, P. vespertinus, L. albiceps, and
containing low levels of heavy metals and PAHs, with sedi-
T. fuscithorax) appeared to have an advantage with low-level
ment quality guidelines indicating that harmful effects from
TPH sediment pollution (860 mg/kg in treatment 1). The only
PAHs are unlikely. Furthermore, these sediments contained
indication that any of the common taxa were stressed by a
low concentrations of small-chain carbon (C6C9) substances
TPH sediment concentration of 860 mg/kg was that signifi-
and a large proportion of HMWHs (C15C36).
cantly fewer P. grimmi adults emerged from treatments 1 and
Caution must be taken when comparing the results of the
2 than from treatment 0. At a TPH concentration of 1,858 mg/
field-based microcosm experiment, in which only synthetic
kg (treatment 2), the abundance of some species (P. villosi-
motor oil was tested, with TPH concentrations measured in
manus and K. martini) declined significantly compared with
urban sediments. Total petroleum hydrocarbon concentration
nonpolluted and less polluted sediments. Total petroleum hy-
data cannot be used to quantitatively estimate environmental
drocarbon sediment concentrations of 4,632 mg/kg (treatment
conditions because sediments with the same TPH concentra-
3) produced significantly lower total numbers of taxa and in-
tion can represent very different compositions and very dif-
dividuals, and significant declines in the abundance of C. cur-
ferent risks to the environment. Furthermore, some TPH meth-
tivalva, P. villosimanus, P. watsoni, P. vespertinus, L. albi-
ods are subject to interference from naturally occurring ma-
ceps, and T. fuscithorax. Only small numbers of a few taxa
terials, such as animal and vegetable oils, which could result
were able to inhabit microcosms with TPH sediment concen-
in artificially high reported TPH concentrations [34]. Synthetic
trations of 14,266 mg/kg (treatment 4), thus indicating that
motor oil containing polyalphaolefins was considered a good
this level of pollution severely affects aquatic ecosystems.
indicator of TPHs in sediments because their low bioavail-
ability and toxicity [23] would provide a conservative estimate
TPH concentrations in urban sediments
of the potential effects of petroleum hydrocarbon pollution in
The TPH concentrations present in the treatments used in freshwater sediments. Further field-based experiments are be-
this experiment are similar to those found in many urban water ing conducted to assess the effects of other HMWHs (mineral
bodies in Melbourne, Australia (Table 4). oil, diesel, and biodiesel) on benthic macroinvertebrates. It is
More than 25% of sediments from water bodies in the Mel- anticipated that these studies will help to confirm which TPH
bourne region exceed the concentrations present in treatment concentrations in the field might suggest ecological impair-
1, whereas ,5% exceed levels present in treatment 3. A high ment in freshwater ecosystems.
percentage (34.8%) of urban drain sediments exceed TPH con- Given the results of this study with synthetic motor oil, it
centrations present in treatment 1, but few drains had concen- is predicted that TPH sediment concentrations of 860 mg/kg
trations exceeding those present in treatment 3. A larger pro- in urban bodies of freshwater could impair resident ecosys-
portion of wetlands (including lakes) than drains or streams tems. Low-level (860 mg/kg) TPH-polluted sediments might
had TPH sediment concentrations exceeding those found in increase abundance of opportunistic species, whereas TPH
treatments 3 and 4. concentrations between 860 and 1,870 mg/kg are likely to
Effects of hydrocarbons on macroinvertebrates Environ. Toxicol. Chem. 24, 2005 2507

reduce the abundance of TPH pollutionsensitive taxa. Total concentrations in the field-based experiment have a similar
petroleum hydrocarbon concentrations .1,870 mg/kg are pre- effect to those in the field, at least 28% of water bodies in
dicted to lead to more substantial losses in species presence Melbourne, Australia, are likely to be affected by HMWH
and abundance. pollution in sediments, and this percentage could be even high-
The loss of macroinvertebrates, as occurred in treatments er for two reasons. First, it is possible that TPH concentrations
2, 3, and 4, would reduce the amount of food available for lower than 860 mg/kg (the lowest concentration tested in this
fish and other animals that feed on macroinvertebrates. For experiment) could affect other aquatic macroinvertebrates.
example, a nonbreeding adult platypus consumes on the order Second, changes in the occurrence and abundance of species
of 15 to 28% of its body mass per day to accommodate a daily appear to be less sensitive measures of stress than the use of
energy expenditure of approximately 850 to 1,100 kg/d [35], metrics, such as numbers of adults that emerge from micro-
with the food intake of a female in late lactation rising to about cosms and growth rates (e.g., adult emergence rates/devel-
three times her daily consumption when not lactating [36]. The opment rates). More severe ecological impacts (reduced abun-
availability of macroinvertebrates could be a major factor lim- dance of HMWH pollutionsensitive species and reduced spe-
iting their distribution in urban streams [37]. Therefore, it is cies richness) are predicted to occur in 14.2% of urban water
hypothesized that HMWH pollution (where TPH sediment bodies in Melbourne.
concentrations exceed between 860 and 1,860 mg/kg) would Even although HMWH-polluted sediments are widespread
severely affect predatory organisms that have a direct or in- in urban water bodies, no sediment quality guidelines for the
direct reliance on macroinvertebrates as a source of food. protection of aquatic ecosystems exist for these substances
because they are nontoxic [10,13]. However, because we
How do HMWHs affect macroinvertebrates?
have demonstrated that sediments polluted with nontoxic
The toxic effects of hydrocarbons is usually evaluated in HMWHs affect freshwater macroinvertebrate assemblages, the
terms of the presence of PAHs because these substances are development of freshwater sediment quality guidelines for
considered to be the most toxic substances present (e.g., [6 these substances is warranted. The concentrations of TPHs
8]). However, the presence of relatively nontoxic HMWHs provide a good indication of the potential effects these con-
could influence organisms in several ways. Direct coating with taminants have on aquatic ecosystems. A better indicator of
oil does not biochemically interfere with cellular activities. HMWH pollution might become available once the ways that
The primary effects are smothering and mechanical interfer- substances affect macroinvertebrate assemblages have been
ence with activities such as movement and feeding [14]. Phys- identified and once further work is conducted to determine the
ical alterations to sediments from petroleum hydrocarbon pol- effects of mineral oil and other common petroleum hydrocar-
lution might also include increased sediment oxygen demand bon pollutants on benthic macroinvertebrates.
from microbial activity, a variety of chemical oxidation pro-
cesses, and a subsequent reduction in the aerobic layer in CONCLUSION
sediments [38]. Increased organic enrichment can also occur
Highmolecular weight hydrocarbons are widespread pol-
as a result of sediments being polluted with hydrocarbons [15],
lutants in urban bodies of freshwater. The aquatic macroin-
and this might have led to the increased abundance of oppor-
vertebrates in the field-based experiments were affected by
tunistic species in treatment 1.
HMWHs as low as 840 mg/kg. If the TPH concentrations
The findings of this study of HMWH pollution in freshwater
measured in the field have properties similar to the synthetic
sediments must be applied with caution to estuarine and marine
oil used in the experiments, then about 28% of aquatic eco-
systems. A number of factors can contribute to the greater
systems in urban water bodies in Melbourne, Australia, could
susceptibility of freshwater benthic ecosystems to the effects
be impaired by HMWH pollution in sediments. A TPH con-
of HMWH pollution than benthic marine ecosystems. Biotur-
centration of 840 mg/kg in sediments might be a reasonable
bation in a nonpolluted marine environment effectively mixes
interim guideline value to indicate possible ecological im-
the surface sediment layer about 10 cm deep [39], whereas
pairment. Sediment quality guidelines for just PAHs provide
the freshwater macroinvertebrate infauna is comparatively
an inadequate indication of whether petroleum hydrocarbon
small and generates little bioturbation of sediments. High
polluted sediments are affecting the health of freshwater eco-
molecular weight hydrocarbons adsorbed to sediments are
systems. Watershed management authorities should recognize
more likely to accumulate in anoxic sediments, in which they
that HMWH-polluted sediments can affect aquatic ecosystem
have lower biodegradation rates [40]. Therefore, HMWHs are
health and that appropriate actions need to be taken to mitigate
more likely to accumulate in freshwater than in marine sedi-
the presence of these pollutants in urban freshwater systems.
ments. Highmolecular weight hydrocarbons might also be
more widely dispersed in an open marine environment than
in small urban water bodies, resulting in a lower propensity AcknowledgementWe are grateful to Jacqueline Ward, Renae Ayres,
for TPH concentrations in marine sediments to reach concen- and David Sharley, who assisted with field work; Andrew Weeks for
comments on the draft manuscript; and Parks Victoria (Kew, Victoria),
trations present in urban sediments. For example, between Jan- which provided access to Glynns Wetland. Supported by the Austra-
uary 19 and 30, 1991, an estimated 10.8 million barrels of oil lian Research Council via the Special Research Centre scheme.
were spilled by Iraqi troops into the Arabian Gulf near the
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