Camp. Biochem. Physid.
Vol. IOOC, No. l/2, pp. 69-75, 1991
0306.4492/91 $3.00 + 0.00 0 1991 Pergamon Press pk
Printed in Great Britain
MINI-REVIEW
ROLE OF PHYSIOLOGICAL ENERGETICS IN ECOTOXICOLOGY JOHNWIDDOWSand PETERDONKIN Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PLl 3DH, U.K. (Telephone: 0752 222772) (Received 1 October 1990) Abstract-l. The role of physiological energetic measurements combined with chemical analyses of contaminants in body tissues of mussels in fundamental toxicological studies and pollution monitoring programmes is outlined. 2. Important features of this toxicological approach are briefly reviewed, including aspects of bioaccumulation, sensitivity, quantitative concentration-response relationships, QSARs, mechanistic interpretation, ecological relevance, integration of the consequences of multiple mechanisms of toxicity and effects of contaminant mixtures and application to laboratory and field studies. 3. This review focuses particularly on recent advances in understanding and predicting the effects of complex mixtures of contaminants.-
INTRODUCTION
in ecotoxicology, namely to understand and predict the toxic effects of complex mixtures of environmental contaminants.
The ultimate objective of ecotoxicological studies is both to predict and diagnose the causes of biological/ ecological effects resulting from exposure to chemicals and other stressors in the environment. To meet this objective it is necessary to establish (a) relationships between the concentrations of chemical contaminants in the environment and in the tissues of biota, and (b) cause-effect relationships between [tissue] contaminant concentration and the resultant biological effects. Furthermore, interpretation of such relationships requires knowledge of the bioconcentration factors for chemicals and an understanding of their mode of toxic action. While there is no single biological effects measurement that can satisfy all environmental situations, the combination of chemical analysis of contaminant levels in the body tissues of mussels and the measurement of biological effects in terms of physiological energetics is ideally suited to such a “cause-effect” framework. Physiological energetics measurements not only provide information on the key processes of energy acquisition, energy expenditure and thus energy available for growth and reproduction, but also reflect some of the major mechanisms of toxicity. Important features of this toxicological approach include: bioaccumulation with minimal metabolic transformation of most organic contaminants; sensitivity to environmental levels of pollutants; quantitative [tissue] concentration-response relationships; mechanistic interpretation and ecological relevance; integration of effects of contaminant mixtures; biological integration of the consequences of multiple mechanisms of toxicity; and application to both laboratory and field studies. In this paper we shall briefly review these aspects, focusing particularly on the role of physiological energetics in addressing one of the major objectives
BIOACCUMULATION Mussels and other bivalves readily accumulate hydrophobic organic contaminants in their tissues with minimal metabolic transformation (Moore et al., 1989). Therefore complex tissue residues largely reflect changes in the quality and quantity of ambient environmental levels of contaminants (Burns and Smith, 1981) and thus mussels are widely used in pollution monitoring programmes (e.g. “Mussel Watch”, Farrington et al., 1983). This is in contrast to higher invertebrates and vertebrates which rapidly biotransform and excrete many xenobiotics, and under these circumstances body burdens do not simply reflect ambient levels of contaminants (Varanasi, 1989). SENSITIVITY Table 1 compares the relative sensitivities of lethal and sublethal physiological responses of mussels (Mytifus eddis), at different life stages, to environmentally important toxic contaminants such as a metal (copper), an organometal (tributyltin) and organic compounds (petroleum hydrocarbons). It shows that (a) the physiological components of the energy budget, which determine the energy available for growth, are responsive to environmental levels of pollutants, (b) the physiological energetic and growth responses are considerably more sensitive than lethal responses, and (c) the larval stages are apparently less sensitive to some contaminants than adult mussels (Beaumont et al., 1987). In addition, mussels and their physiological responses appear to be of greater, 69
J. WIDDOWS and P. D~NKIN
70
Table 1. Comparison of lethal and sublethal physiological responses of mussels (Mytilus eduh)
Toxicant
Biological response (50% of control)
CU CU CU CU CU CU
Lethal (15 days) Lethal (15 days) Lethal (39 days) Shell growth Shell growth Valve movement
CU
Clearance rate, SFG Lethal and shell growth (15 days) Lethal (5 days) Valve movement Shell growth SFG Lethal (6 hr)
TBT
TBT TBT TBT TBT Hvdrocarbons icrude oil1 H;drocarbons Lethal (4 days) (crude oil) Hydrocarbons Lethal (4 m) (diesel oil) Hydrocarbons Valve movement (crude oil) Hydrocarbons Shell growth (crude oil) Hydrocarbons Clearance rate, SFG (diesel oil) Clearance rate Hydrocarbons (aliphatics and aromatics log K,, < 5)
Water @g/l) concentration
Tissue (ads dry wt) concentration
Life stage
to selected toxicants
Reference
lo-20 /I g/l
Larvae Adults Adults Larvae Adults Adults
12pgll 0.1 fig/l
Adults Larvae
Beaumont ef al., 1987 Martin, 1979 Martin, 1979 Beaumont et al., 1987 Redpath, 1985 Davenport and Manley, 1978; Kramer et al., 1989 Widdows (unpublished data) Beaumont and Budd, 1984
>lOX IO)pg/l
Adults Adults Juveniles Adults Larvae
Page and Widdows, 1990 Kramer ef al., 1989 Strijmgren and Bongard, 1987 Page and Widdows, 1990 Craddock, 1977
I-10 x lO’pg/l
Adults
Craddock,
125 rgil
Adults
Widdows et al., 1987
Adults
Kramer et al., 1989
Adults
StrBmgren, 1986
150 rglg
Adults
Widdows er al., 1987
95 pglg
Adults
Donkin et al., 1989
4OOl&l 50 ;;/I-.
20&3/l
59 aglg
15Opgil
5Pl3ll
20 pg/g 10 rgll 6uall -.
0.1M/l
4 Icglg
6 x 10'pg/l 1.5 x 103/lg/l’ 30 rgil
1971
*Total concentration in microcapsules and water. Environmental ranges: Cu: In water: I to 30 rg/l (Davenport and Redpath, 1984); in tissue: 2.4 to 95 rg/g dry wt (Fowler and Oregioni, 1976). TBT: In water: 0.005 to 0.5 pg/l (Salazar and Salazar, 1991); in tissue: 0.1 to > 10 rg/g dry wt (Page and Widdows, 1990). Petroleum hydrocarbons: In water: 1-74 rg/l (Law, 1981); 19-56Opg/l (12-351 days after oil spill; Blackman and Law, 1980); in tissue: Aliphatic UCM l-270 rg/g dry wt; Aromatic UCM c3 to Ill rg/g dry wt; Selected PAH co.02 to 17Spg/g dry wt (Farrington et al., 1982, 1983).
or equal, sensitivity to environmental toxicants in comparison with other aquatic animals (see Donkin et al., 1989; Davenport and Redpath, 1984). One notable exception is the very sensitive and toxicantspecific “imposex” response of the gastropod, Nucella lupillus, to TBT. Imposex occurs at 0.005 pg TBT 1-l or 0.5 pg TBT gg’ dry wt (Gibbs et al., 1987), and is therefore an order of magnitude more sensitive than effects on the growth of mussels. QUANTITATIVECONCENTRATION-RESPONSE RELATIONSHIPS Tissue concentration-physiological response relationships derived from controlled laboratory and mesocosm studies, not only facilitate the identification of chemical contaminants causing effects recorded in the environment, but also enable biological effects to be predicted from environmental levels of contaminants in water and body tissues. Such toxicological research therefore provides the information necessary to establish an appropriate database for the toxicological interpretation of tissue residue data derived from chemical monitoring programmes. While it may be feasible to determine tissue concentration-response relationships for individual contaminants with specific mechanisms of toxicity, for example Cu (Redpath, 1985), TBT (Page and Widdows, 1990) and selected aromatic hydrocarbons (Widdows et al., 1987), it is clearly unrealistic to
examine the sublethal effects of the > lo4 individual organic contaminants which enter the environment. The application of Quantitative Structure-Activity Relationship (QSAR) approach (Hermens, 1986), which facilitates the prediction of toxicological properties of organic compounds from their chemical/ structural properties, is an important means of overcoming this problem (Donkin et al., 1989; Donkin and Widdows, 1990). Such QSAR studies have demonstrated that aromatic and aliphatic hydrocarbons with log K,, values of < 5 and < 6 respectively, inhibit ciliary feeding activity (clearance rate) by means of “nonspecific narcosis”, and have equal toxicity when expressed on the basis of toxicant concentration in the tissues. As a consequence, it is then possible to group structurally related compounds and predict bioconcentration factors (BCF) and toxic effects from their physicochemical properties. Current and future research is concerned with establishing QSARs for other major classes of organic compounds with different physicochemical properties and for other biological responses reflecting different mechanisms of toxicity. MECHANISMSOF TOXICITYREFLECTED IN PHYSIOLOGICAL
ENERGETICS
Physiological energetics and the disturbance of the mussel’s energy balance provide insight into, and integration of, some of the primary mechanisms of
Physiological energetics in ecotoxicology
that are both biologically and environmentally important. Major mechanisms of toxicity reflected in physiological energetics include: nonspecific narcosis affecting the ciliary feeding activity of bivalves (e.g. hydrocarbons, Donkin et al., 1989); neurotoxic effects on the neural control of gill cilia (e.g. dinoflagellate toxins, Widdows et al., 1979; Cu, Howell er al., 1984; TBT, Snoeij et al., 1987); uncoupling of oxidative phosphorylation causing an increase in respiration rate (e.g. TBT, Snoeij et al., 1987; Phenols, Buikema et al., 1979); inhibition of oxidative metabolism thus reducing respiration rate (e.g. DBT, Snoeij et al., 1987; hypoxia, Widdows et al., 1989); and toxic effects on membrane structure and function affecting processes of food digestion and absorption (e.g. hydrocarbons, Widdows et al., 1987). Physiological energetic responses are sensitive to important classes of environmental contaminants because they reflect these primary mechanisms of toxicity. toxicity
MECHANISTIC INTERPRETATION AND ECOLOGICAL RELEVANCE
Energetics offers a common currency [energy] enabling the consequences of primary toxic mechanisms at the cellular level to be translated into effects on growth, reproduction and survival at the individual and population levels. The ultimate effects at the higher levels of biological organisation are thus more readily interpreted and understood. Field and mesocosm studies have provided confirmation that the long-term consequences to growth and survival of individuals and the population can be predicted from measured effects on energy balance observed at the individual level (Gilfillan and Vandermeulen, 1978; Widdows et al., 1987). BIOLOGICAL INTEGRATION: TOXIC MECHANISMS AND CONTAMINANT MIXTURES
(a) Integration of the effects of different mechanisms of toxicity induced by a single compound Many toxicants induce effects via more than one mechanism of toxicity. Pentachlorophenol (PCP), for example, is both an uncoupler of oxidative phosphorylation, which results in enhanced oxygen consumption, and also induces narcosis which reduces ciliary feeding activity of the mussels. Table 2 illustrates the combined effect of PCP on these two components of the energy budget and the resultant effect on scope for growth (SFG) (Widdows and Donkin, unpublished data). Physiological energetics thus provides an integration of these separate toxic
71
effects as well as insight into the underlying mechanisms of toxicity. (b) Integration of eficts
of contaminant mixtures (structurally related toxicants with a common mode of action)
The toxic effects of structurally related organic compounds that form a single QSAR line, and therefore a common mechanism of toxicity, are predicted to be additive when present in a complex mixture (Kiinemann, 1981; Hermens, 1986). Oil is one of the best examples of a complex mixture of chemicals. QSAR studies have shown that aromatic hydrocarbons with log K,, values c 5 and aliphatic hydrocarbons with log K,, ~6 have equal toxicity (inducing narcosis) and form a common QSAR line (Donkin et al., 1989). Consequently, hydrocarbons above this “molecular weight cut-off” can be regarded as contaminants of significantly lower toxicity that are accumulated without exerting a direct toxic effect on the clearance rate of mussels. Experiments have confirmed the additive nature of toxicity of related compounds forming a single QSAR line. Mussels were exposed to mixtures of aromatic and aliphatic hydrocarbons where each compound was presented at a water (and tissue) concentration below the threshold required to significantly inhibit clearance rate; yet as a (total) mixture was predicted to reduce clearance rate by 25, 50 and 75%. The results (Table 3) show that there is close agreement between the observed and predicted relationships among clearance rate, water and tissue concentrations of hydrocarbons (Widdows and Donkin, unpublished data). Comparison between results of QSAR and mesocosm/field studies involving complex mixtures have highlighted a discrepancy between the [tissue] hydrocarbon concentration required to reduce clearance rate (ca. 100 pg hydrocarbons gg’ dry wt) and the measured [tissue] concentration of “selected hydrocarbons” causing such an effect in the field. It is apparent from the literature that the total petroleumderived body burden (hydrocarbons, substituted hydrocarbons and their polar oxygenated derivatives) is rarely measured in field studies and only a fraction of the “total toxic load” is routinely quantified in the chemical analysis of biota. Extraction and analytical procedures can lose a significant proportion of the more volatile toxic compounds (e.g. log K,, -C3; Farrington et al., 1988) and the unresolved complex mixture (UCM) component of chromatograms, which contains toxic compounds, is frequently not quantified. Furthermore, the precision and accuracy of some analytical procedures is inadequate for
Table 2. Relationships between concentration of pentachlorophenol in the water and tissues of mussels (MyNus edulis) and the effect on respiration, clearance rate and scope for growth Water concentration (fig 1-v 0.5 38 65
167 335 660 786
Tissue concentration mg kg-’ wet wt)
Respiration rate (% of control)
Clearance rate (% of control)
Scope for growth (J hr-‘)
0.5 2.9 4.6 9.8 19.3 36.4 42.1
101 118 131 149 191 134 45
100 74 78 67 35 3.4 2.9
15.17 9.22 9.42 6.43 - 1.80 -5.44 -
12
J. WDDOWSand P.
DONKIN
toxicological comparison (Farrington et al., 1988). Consequently, it is often necessary to use selected groups of compounds which can be analysed reliably, as “indicators” of the total petroleum-derived toxic load, and establish appropriate conversion factors (Widdows et al., 1987). (c) Integration of effects of contaminant mixtures (unrelated toxicants) The interactions between structurally unrelated toxicants, such as petroleum hydrocarbons and copper, and their combined effects on the growth of mussels have been studied. Both Striimgren (1986) and Widdows and Johnson (1988) have recorded a simple “proportional additive” effect on shell growth and clearance rate. However, Stromgren stated that the effects of oil and copper were less than additive, and were therefore antagonistic, and did not recognise the combined effects were simply additive on a proportional basis. Figure 1 shows the close agreement between observed and expected growth rate when expressed in terms of proportional additivity. Recent studies (Widdows, Page and Donkin, unpublished data) have examined the combined effects of two important and structurally different contaminants (e.g. hydrocarbons and TBT) which co-exist in many aquatic environments. Five interaction experiments (4 concentrations of naphthalene + TBT and nonane + TBT) were carried out to test the hypothesis of additivity. However, the results consistently demonstrated that the combined effect on clearance rate was markedly less than predicted on the basis of “proportional additivity”. In fact there was evidence of significant (P < 0.01) antagonism between TBT and hydrocarbons (Fig. 2). Field studies provide some circumstantial evidence in support of these experimental findings. For example, in some locations bivalves have been found surviving with very high and potentially lethal TBT [tissue] levels (5-10 pg TBT/g dry wt), and these harbours/marinas are also those sites with high inputs of hydrocarbons 8
hell growth as % Of oot$f
Observed Observed Obrerved Observed
Observed Predicted Observed Predicted Observed Predicted Observed Predicted
1.6mg oil /
3mg oil /
I * 3ug
I +3ug Cu /
Cu I
I
I
1.6mg oil / I + Bug Cu I
I
3mg oil I I* 6ug Cu / I Redrawn from Stromgren 1966
Fig. 1. Effect of combinations of two concentrations of oil and copper on the shell growth of mussels (Myfilus edulis); observed response (as % of control) and predicted response (based on proportional additivity). Redrawn from Striimgren (1986).
Physiological energetics in ecotoxicology
73
i5 ; ; L
Expt.
I
Fig. 2. Effect of naphthalene (Naph), tributyltin (TBT) and naphthalene i- TBT (interaction) on the clearance rate of Myth eduh. Pred: the predicted decline in clearance rate based on additivity. Mean & SE. From Widdows, Page and Donkin (unpublished data). (e.g. San Diego and Honolulu Harbours, Grovhoug 1986). At present there is no mechanistic explanation for this antagonism between hydrocarbons and TBT, but it is probably acting at the level of neural control of gill cilia. et al.,
FIELD APPLICATION IN RELATION POLLUTION ASSESSMENT
TO
The assessment of environmental pollution using physiological energetic measurements of bivalves in conjunction with analysis of contaminants in their tissues began with field studies by Gilfillan et al. (1977), Widdows et al. (1981) and Martin et al. (1984). Since the early field application of this approach, methodological development has continued and laboratory/mesocosm studies have enhanced toxicological understan~ng of [tissue] cont~inant concentration-response relationships. The more recent application of this approach is illustrated with reference to two pollution gradients investigated as part of Practical Biological Effects Workshops in Bermuda and Oslotjord. At the Bermuda Workshop, the effects on the physiological energetics of transplanted mussels (Arcu zebra) were quantified and related to a contiminant gradient in Hamilton Harbour, a “non-indust~alised” region receiving relativley low levels of contaminant inputs. Although tissue concentrations of hydrocarbons, their polar oxygenated derivatives, PCBs, TBT and Pb were all significantly correlated with reductions in SFG, toxicological in~~retation of the tissue residue data
extended the analysis beyond statistical correlations and indicated that hydrocarbons (+polar oxygenated derivatives) and TBT could explain the observed decline in SFG ~iddows et al., 1990). Furthermore, the overall reductian in SFG could be proportioned, such that at the most contaminated site TBT accounted for 21% and hydrocarbons for 79% of the observed effect. In contrast, the second study examined a pollution gradient associated with an “industrialised” environment (Langesundfjord). Here the effects were not only more marked, but they were greater than could be explained by the widespread ~nt~inants, hydrocarbons and TBT, thus indicating the presence of other important and unidentified toxicants (Fig. 3). The results also highlighted the importance of comparing effects at polluted sites with appropriate reference sites. The “cleanest” site in ~ngesund~ord was significantly contaminated by hydrocarbons and TBT and therefore a well established “uncontaminated reference” site in the Shetland Islands was used. This enabled the contaminated levels and effects at all sites in Langesun~ord to be placed in a broader context (Widdows and Johnson, 1988; Widdows and Donkin, 1989). Direct comparisons between populations/sites can generally be made during the summer growth period and over such distances, because mussel transplant experiments over > 1000 km have demonstrated that physiological responses and growth generally reflect environmental rather than genetic differences (Widdows and Salkeld, unpublished data; Kautsky et al., 1990).
14
J. WIDDOWSand P. DONKIN Mytitus edutis
Craddock D. R. (1977) Acute toxic effects of petroleum on Arctic and sub-Arctic marine organisms. In Eficts of Petroleum on Arctic and sub-Arctic Marine Environments and Organisms. Vol. II. Biological EfSects (Edited by
24-
Malins D. C.), pp. l-93. Academic Press, New York. Davenport J. and Manley A. R. (1978) The detection of heightened seawater copper concentrations by the mussel, My&s
edulis. J. mar.-biol. Ass. U.K. 58, 843-850.
Davennort J. and Rednath K. J. (1984) Conner and the . mussel Mytilus edulis L. In Toxins, Drugs and Pollutants in Marine Animals (Edited by Bolis et al.), pp. 176189.
16 -
I
__
Springer, Berlin. Donkin P. and Widdows J. (1990) Quantitative structureactivity relationships in aquatic invertebrate toxicology. Rev. Aquat. Sci. 2, 375-398.
Donkin P., Widdows J., Evans S. V., Worrall C. M. and Carr M. (1989) Quantitative structure-activity relationships for the effect of hydrophobic organic chemicals on rate of feeding by mussels (Mytilus edulis). Aquat. Toxicol. 14, 277-294.
“ilean
re erence’ (Shetland)
'ResfE9~d
(Langesundfjord
‘Ps$kttd’
: Oslo GEEP WI.9
Fig. 3. Effect of pollution on Scope for Growth (SGF) of Mytilus edulis from Langesundtjord in comparison to a “clean reference site” in the Shetland Islands. Partitioning and relative contribution of hydrocarbons and TBT towards observed decline in SFG at Langesundfjord (SFG measured under “standard&d conditions” and calculated for a ration level of 0.4mg particulate organic matter I-‘; data from Widdows and Johnson, 1988).
Farrington J. W., Davis A. C., Frew N. M. and Knap A. (1988) ICESjIOC intercomparison exercise on the determination of petroleum hydrocarbons in biological tissues (mussel homogenate). Mar. Pollut. Bull. 19, 372-380. Farrington J. W., Davis A. C., Frew N. M. and Rabin K. S. (1982) No. 2. Fuel oil compounds in Mytilus edulis. Retention and release after an oil spill. Mar. Biol. 66, 15-26.
Farrington J. W., Goldberg E. D., Riseborough R. W., Martin J. H. and Bowen V. T. (1983) U.S. “Mussel Watch” 19761978: an overview of the trace metal, DDE, PCB, hydrocarbon and artificial radionuclide data. Environ. Sci. Technol. 11, 490-496.
Fowler S. W. and Oreaioni B. (1976) Trace metals in mussels from the N.W. Mediterranean. bar. Poll. Bull. 7, 26-29. Gibbs P. E., Bryan G. W., Pascoe P. L. and Burt G. R. (1987) The use of the dog-whelk, Nucella lapillus, as an indicator or tributyltin (TBT) contamination. J. mar. biol. Ass. U.K. 67, 561-569.
has been made towards the long-term ecotoxicological objective of “predicting effects and interpreting the causes of observed effects”, further research is required to provide a more comprehensive toxicological interpretation of contaminant residues in body tissues. Future research within this toxicological framework needs to establish (a) QSARs for other classes of contaminants, (b) relationships for other important biological responses (e.g. reproductive processes), and (c) further understanding of the combined effects of complex contaminant mixtures. While significant
progress
REFERENCES Beaumont A. R. and Budd M. D. (1984) High mortality of the larvae of the common mussel at low concentrations of tributyltin. Mar. Poll. Bull. 15, 402405. Beaumont A. R., Tserpes G. and Budd M. D. (1987) Some effects of copper on the veliger larvae of the mussel, Mytilus edulis, and the scallop Pecten maximus (Mollusca, Bivalvia). Mar. environ. Res. 21, 299-309. Blackman R. A. A. and Law R. J. (1980) The Eleni V Oil Spill: Fate and effects of the oil over the first twelve months. Mar. Pollut. Bull. 11, 199-204. Buikema A. L., McGinniss M. J. and Cairns J. (1979) Phenolics in aquatic ecosystem: a selected review of recent literature. Mar. environ. Res. 2, 87-181. Burns K. A. and Smith J. L. (1981) Biological monitoring of ambient water quality: the case for using bivalves as sentinel organisms for monitoring petroleum pollution in coastal waters. Est. coast. Shelf Sci. 13, 433-443.
Gilfillan E. S.. Mavo D. W.. Pace D. S.. Donovan D. and Hanson S. ‘(1975) Effects’ of Larying’concentrations of petroleum hydrocarbons in sediments on carbon flux in Mya arenaria. In Physiological Responses of Marine Biota to Pollutants. (Edited by Vernberg F. J. et al.), pp. 299-314. Academic Press, New York. Gilfillan E. S. and Vandermeulen J. H. (1978) Alterations in growth and physiology in chronically oiled soft-shell clams, Mya &enaria,chronically oiled with Bunker C from Chedabucto Bav. Nova Scotia. 1970-1976. J. Fish. Res. Bd Can. 35, 630636.
Grovhoug J. G., Seligman P. F., Vafa G. and Fransham R. L. (1986) Baseline measurements of butvltin in U.S. harbors and estuaries. In Proceedings of the Organotin symposium of Marine Technology Society. Oceans 1986 Conference. pp. 1283-1288. Washington DC.
Hermens J. L. M. (1986) Quantitative structure-activity relationships in aquatic toxicology. Pestic. Sci. 17, 287-296.
Howell R., Grant A. M. and Maccoy N. E. J. (1984) Effect of treatment with reserpine on the change in filtration rate of Mytilus edulis subiected to dissolved copper. Mar. -_ Pollut. Bull. 15, 4364-39.
Kautskv N.. Johannesson K. and Tedenaren M. (1990) Gendtypic and phenotypic differences bet&een Baltic and North Sea populations of Mytilus edulis evaluated through reciprocal transplantations. I. Growth and morphology. Mar. Ecol. Prog. Ser. 59, 203-210. Ktinemann H. (1981) Quantitative structure-activity relationships in fish toxicity studies. Part I: Relationships for 50 industrial pollutants. Toxicology 19, 209-221. Kramer K. J. M., Jenner H. A. and de Zwart D. (1989) The valve movement response of mussels: a tool in biological monitoring. Hydrobiologia 188/189, 4334l3.
Physiological energetics in ecotoxicology Law R. J. (1981) Hydrocarbon concentrations in water and sediments from U.K. marine waters, determined by fluorescence spectroscopy. Mar. Pollut. Bull. 12, 153-157. Martin J. L. M. (1979) Schema of lethal action of copper on mussels. Bull. environ. Contam. Toxicol. 21, 808-814. Martin M., Ichikawa G., Goetzl J., Reyes M. de 10s and Stephenson M. D. (1984) Relationships between physiological stress and trace toxic substances in the Bay mussel, Mytilus edulis, from San Francisco Bay, California. Mar. environ. Res. 11, 91-110. Moore M. N., Livingstone D. R. and Widdows J. (1989) Hydrocarbons in marine mollusks: biological effects and ecological consequences. In Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment (Edited by Varanasi U.), pp. 291-328. CRC Press, Boca Raton, FL. Page D. S. and Widdows J. (1990) Temporal and spatial variation in levels of alkyltins in mussel tissues: a toxicological interpretation of field data. In 3rd International Organotin Symposium, April 1990, Monaco. @I press). Redpath K. J. (1985) Growth inhibition and recovery in mussels (Mytilus edulis) exposed to low copper concentrations. J. mar. biol. Ass. U.K. 65, 421431. Salazar M. H. and Salazar S. M. (1991) Mussel field studies: mortality, growth and bioaccumulation. In TributyltinEnvironmental Fate and Effects (Edited by Champ M. A. and Seligman P. F.). (In press). Snoeij N. J., Penninks A. H. and Seinen W. (1987) Biological activity of organotin compounds-an overview. Environ. Res. 44, 335-353. Strijmgren T. (1986) The combined effect of copper and hydrocarbons on the length growth of Mytilus edulis. Mar. environ. Res. 18, 251-258.
CBP IOCC/I-2-F
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Striimgren T. and Bongard T. (1987) The effect of tributyltin oxide on growth of Mytilus edulis. Mar. Polk. Bull. 18, 3&31. Varanasi U. (1989) Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. 328 pp. CRC Press, Boca Raton, FL. Widdows J., Burns K. A., Menon N. R., Page D. S. and Soria S. (1990) Measurement of physiological energetics (scope for growth) and chemical contaminants in mussels (Arca zebra) transplanted along a contaminant gradient in Bermuda. J. exp. mar. Biol. Ecol. 138, 99-117. Widdows J. and Donkin P. (1989) The application of combined tissue residue chemistry and physiological measurements of mussels (Mytitus edulis) for the assessment of environmental pollution. Hydrobiologia U&3/189, 455461. Widdows J., Donkin P. and Evans S. V. (1987) Physiological responses of Mytihts edulis during chronic oil exposure and recovery. mar. environ. Res. 23, 15-32. Widdows J. and Johnson D. (1988) Physiological energetics of Mytilus edulis: scope for growth. Mar. Ecol. Prog. Ser. 46, 113-121. Widdows J., Moore M. N., Lowe D. M. and Salkeld P. N. (1979) Some effects of a dinoflaeellate bloom (Gvrodinium aureoium) on the mussel, My&s edulis, J: mar. biol. Assoc. U.K. 59, 522-524. Widdows J.. Newell R. I. E. and Mann R. (1989) Effects of hypoxia and anoxia on survival, energy metabolism and feeding pf oyster larvae (Crassostrea virginica Gmelin). Biol. Bull. 177, 154-166. Widdows J., Phelps D. K. and Galloway W. (1981) Measurement of physiological condition of mussels transplanted along a pollution gradient in Narragansett Bay. Mar. environ. Res. 4, 181-194.
Vol. IOOC, No. l/2, pp. 69-75, 1991
0306.4492/91 $3.00 + 0.00 0 1991 Pergamon Press pk
Printed in Great Britain
MINI-REVIEW
ROLE OF PHYSIOLOGICAL ENERGETICS IN ECOTOXICOLOGY JOHNWIDDOWSand PETERDONKIN Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PLl 3DH, U.K. (Telephone: 0752 222772) (Received 1 October 1990) Abstract-l. The role of physiological energetic measurements combined with chemical analyses of contaminants in body tissues of mussels in fundamental toxicological studies and pollution monitoring programmes is outlined. 2. Important features of this toxicological approach are briefly reviewed, including aspects of bioaccumulation, sensitivity, quantitative concentration-response relationships, QSARs, mechanistic interpretation, ecological relevance, integration of the consequences of multiple mechanisms of toxicity and effects of contaminant mixtures and application to laboratory and field studies. 3. This review focuses particularly on recent advances in understanding and predicting the effects of complex mixtures of contaminants.-
INTRODUCTION
in ecotoxicology, namely to understand and predict the toxic effects of complex mixtures of environmental contaminants.
The ultimate objective of ecotoxicological studies is both to predict and diagnose the causes of biological/ ecological effects resulting from exposure to chemicals and other stressors in the environment. To meet this objective it is necessary to establish (a) relationships between the concentrations of chemical contaminants in the environment and in the tissues of biota, and (b) cause-effect relationships between [tissue] contaminant concentration and the resultant biological effects. Furthermore, interpretation of such relationships requires knowledge of the bioconcentration factors for chemicals and an understanding of their mode of toxic action. While there is no single biological effects measurement that can satisfy all environmental situations, the combination of chemical analysis of contaminant levels in the body tissues of mussels and the measurement of biological effects in terms of physiological energetics is ideally suited to such a “cause-effect” framework. Physiological energetics measurements not only provide information on the key processes of energy acquisition, energy expenditure and thus energy available for growth and reproduction, but also reflect some of the major mechanisms of toxicity. Important features of this toxicological approach include: bioaccumulation with minimal metabolic transformation of most organic contaminants; sensitivity to environmental levels of pollutants; quantitative [tissue] concentration-response relationships; mechanistic interpretation and ecological relevance; integration of effects of contaminant mixtures; biological integration of the consequences of multiple mechanisms of toxicity; and application to both laboratory and field studies. In this paper we shall briefly review these aspects, focusing particularly on the role of physiological energetics in addressing one of the major objectives
BIOACCUMULATION Mussels and other bivalves readily accumulate hydrophobic organic contaminants in their tissues with minimal metabolic transformation (Moore et al., 1989). Therefore complex tissue residues largely reflect changes in the quality and quantity of ambient environmental levels of contaminants (Burns and Smith, 1981) and thus mussels are widely used in pollution monitoring programmes (e.g. “Mussel Watch”, Farrington et al., 1983). This is in contrast to higher invertebrates and vertebrates which rapidly biotransform and excrete many xenobiotics, and under these circumstances body burdens do not simply reflect ambient levels of contaminants (Varanasi, 1989). SENSITIVITY Table 1 compares the relative sensitivities of lethal and sublethal physiological responses of mussels (Mytifus eddis), at different life stages, to environmentally important toxic contaminants such as a metal (copper), an organometal (tributyltin) and organic compounds (petroleum hydrocarbons). It shows that (a) the physiological components of the energy budget, which determine the energy available for growth, are responsive to environmental levels of pollutants, (b) the physiological energetic and growth responses are considerably more sensitive than lethal responses, and (c) the larval stages are apparently less sensitive to some contaminants than adult mussels (Beaumont et al., 1987). In addition, mussels and their physiological responses appear to be of greater, 69
J. WIDDOWS and P. D~NKIN
70
Table 1. Comparison of lethal and sublethal physiological responses of mussels (Mytilus eduh)
Toxicant
Biological response (50% of control)
CU CU CU CU CU CU
Lethal (15 days) Lethal (15 days) Lethal (39 days) Shell growth Shell growth Valve movement
CU
Clearance rate, SFG Lethal and shell growth (15 days) Lethal (5 days) Valve movement Shell growth SFG Lethal (6 hr)
TBT
TBT TBT TBT TBT Hvdrocarbons icrude oil1 H;drocarbons Lethal (4 days) (crude oil) Hydrocarbons Lethal (4 m) (diesel oil) Hydrocarbons Valve movement (crude oil) Hydrocarbons Shell growth (crude oil) Hydrocarbons Clearance rate, SFG (diesel oil) Clearance rate Hydrocarbons (aliphatics and aromatics log K,, < 5)
Water @g/l) concentration
Tissue (ads dry wt) concentration
Life stage
to selected toxicants
Reference
lo-20 /I g/l
Larvae Adults Adults Larvae Adults Adults
12pgll 0.1 fig/l
Adults Larvae
Beaumont ef al., 1987 Martin, 1979 Martin, 1979 Beaumont et al., 1987 Redpath, 1985 Davenport and Manley, 1978; Kramer et al., 1989 Widdows (unpublished data) Beaumont and Budd, 1984
>lOX IO)pg/l
Adults Adults Juveniles Adults Larvae
Page and Widdows, 1990 Kramer ef al., 1989 Strijmgren and Bongard, 1987 Page and Widdows, 1990 Craddock, 1977
I-10 x lO’pg/l
Adults
Craddock,
125 rgil
Adults
Widdows et al., 1987
Adults
Kramer et al., 1989
Adults
StrBmgren, 1986
150 rglg
Adults
Widdows er al., 1987
95 pglg
Adults
Donkin et al., 1989
4OOl&l 50 ;;/I-.
20&3/l
59 aglg
15Opgil
5Pl3ll
20 pg/g 10 rgll 6uall -.
0.1M/l
4 Icglg
6 x 10'pg/l 1.5 x 103/lg/l’ 30 rgil
1971
*Total concentration in microcapsules and water. Environmental ranges: Cu: In water: I to 30 rg/l (Davenport and Redpath, 1984); in tissue: 2.4 to 95 rg/g dry wt (Fowler and Oregioni, 1976). TBT: In water: 0.005 to 0.5 pg/l (Salazar and Salazar, 1991); in tissue: 0.1 to > 10 rg/g dry wt (Page and Widdows, 1990). Petroleum hydrocarbons: In water: 1-74 rg/l (Law, 1981); 19-56Opg/l (12-351 days after oil spill; Blackman and Law, 1980); in tissue: Aliphatic UCM l-270 rg/g dry wt; Aromatic UCM c3 to Ill rg/g dry wt; Selected PAH co.02 to 17Spg/g dry wt (Farrington et al., 1982, 1983).
or equal, sensitivity to environmental toxicants in comparison with other aquatic animals (see Donkin et al., 1989; Davenport and Redpath, 1984). One notable exception is the very sensitive and toxicantspecific “imposex” response of the gastropod, Nucella lupillus, to TBT. Imposex occurs at 0.005 pg TBT 1-l or 0.5 pg TBT gg’ dry wt (Gibbs et al., 1987), and is therefore an order of magnitude more sensitive than effects on the growth of mussels. QUANTITATIVECONCENTRATION-RESPONSE RELATIONSHIPS Tissue concentration-physiological response relationships derived from controlled laboratory and mesocosm studies, not only facilitate the identification of chemical contaminants causing effects recorded in the environment, but also enable biological effects to be predicted from environmental levels of contaminants in water and body tissues. Such toxicological research therefore provides the information necessary to establish an appropriate database for the toxicological interpretation of tissue residue data derived from chemical monitoring programmes. While it may be feasible to determine tissue concentration-response relationships for individual contaminants with specific mechanisms of toxicity, for example Cu (Redpath, 1985), TBT (Page and Widdows, 1990) and selected aromatic hydrocarbons (Widdows et al., 1987), it is clearly unrealistic to
examine the sublethal effects of the > lo4 individual organic contaminants which enter the environment. The application of Quantitative Structure-Activity Relationship (QSAR) approach (Hermens, 1986), which facilitates the prediction of toxicological properties of organic compounds from their chemical/ structural properties, is an important means of overcoming this problem (Donkin et al., 1989; Donkin and Widdows, 1990). Such QSAR studies have demonstrated that aromatic and aliphatic hydrocarbons with log K,, values of < 5 and < 6 respectively, inhibit ciliary feeding activity (clearance rate) by means of “nonspecific narcosis”, and have equal toxicity when expressed on the basis of toxicant concentration in the tissues. As a consequence, it is then possible to group structurally related compounds and predict bioconcentration factors (BCF) and toxic effects from their physicochemical properties. Current and future research is concerned with establishing QSARs for other major classes of organic compounds with different physicochemical properties and for other biological responses reflecting different mechanisms of toxicity. MECHANISMSOF TOXICITYREFLECTED IN PHYSIOLOGICAL
ENERGETICS
Physiological energetics and the disturbance of the mussel’s energy balance provide insight into, and integration of, some of the primary mechanisms of
Physiological energetics in ecotoxicology
that are both biologically and environmentally important. Major mechanisms of toxicity reflected in physiological energetics include: nonspecific narcosis affecting the ciliary feeding activity of bivalves (e.g. hydrocarbons, Donkin et al., 1989); neurotoxic effects on the neural control of gill cilia (e.g. dinoflagellate toxins, Widdows et al., 1979; Cu, Howell er al., 1984; TBT, Snoeij et al., 1987); uncoupling of oxidative phosphorylation causing an increase in respiration rate (e.g. TBT, Snoeij et al., 1987; Phenols, Buikema et al., 1979); inhibition of oxidative metabolism thus reducing respiration rate (e.g. DBT, Snoeij et al., 1987; hypoxia, Widdows et al., 1989); and toxic effects on membrane structure and function affecting processes of food digestion and absorption (e.g. hydrocarbons, Widdows et al., 1987). Physiological energetic responses are sensitive to important classes of environmental contaminants because they reflect these primary mechanisms of toxicity. toxicity
MECHANISTIC INTERPRETATION AND ECOLOGICAL RELEVANCE
Energetics offers a common currency [energy] enabling the consequences of primary toxic mechanisms at the cellular level to be translated into effects on growth, reproduction and survival at the individual and population levels. The ultimate effects at the higher levels of biological organisation are thus more readily interpreted and understood. Field and mesocosm studies have provided confirmation that the long-term consequences to growth and survival of individuals and the population can be predicted from measured effects on energy balance observed at the individual level (Gilfillan and Vandermeulen, 1978; Widdows et al., 1987). BIOLOGICAL INTEGRATION: TOXIC MECHANISMS AND CONTAMINANT MIXTURES
(a) Integration of the effects of different mechanisms of toxicity induced by a single compound Many toxicants induce effects via more than one mechanism of toxicity. Pentachlorophenol (PCP), for example, is both an uncoupler of oxidative phosphorylation, which results in enhanced oxygen consumption, and also induces narcosis which reduces ciliary feeding activity of the mussels. Table 2 illustrates the combined effect of PCP on these two components of the energy budget and the resultant effect on scope for growth (SFG) (Widdows and Donkin, unpublished data). Physiological energetics thus provides an integration of these separate toxic
71
effects as well as insight into the underlying mechanisms of toxicity. (b) Integration of eficts
of contaminant mixtures (structurally related toxicants with a common mode of action)
The toxic effects of structurally related organic compounds that form a single QSAR line, and therefore a common mechanism of toxicity, are predicted to be additive when present in a complex mixture (Kiinemann, 1981; Hermens, 1986). Oil is one of the best examples of a complex mixture of chemicals. QSAR studies have shown that aromatic hydrocarbons with log K,, values c 5 and aliphatic hydrocarbons with log K,, ~6 have equal toxicity (inducing narcosis) and form a common QSAR line (Donkin et al., 1989). Consequently, hydrocarbons above this “molecular weight cut-off” can be regarded as contaminants of significantly lower toxicity that are accumulated without exerting a direct toxic effect on the clearance rate of mussels. Experiments have confirmed the additive nature of toxicity of related compounds forming a single QSAR line. Mussels were exposed to mixtures of aromatic and aliphatic hydrocarbons where each compound was presented at a water (and tissue) concentration below the threshold required to significantly inhibit clearance rate; yet as a (total) mixture was predicted to reduce clearance rate by 25, 50 and 75%. The results (Table 3) show that there is close agreement between the observed and predicted relationships among clearance rate, water and tissue concentrations of hydrocarbons (Widdows and Donkin, unpublished data). Comparison between results of QSAR and mesocosm/field studies involving complex mixtures have highlighted a discrepancy between the [tissue] hydrocarbon concentration required to reduce clearance rate (ca. 100 pg hydrocarbons gg’ dry wt) and the measured [tissue] concentration of “selected hydrocarbons” causing such an effect in the field. It is apparent from the literature that the total petroleumderived body burden (hydrocarbons, substituted hydrocarbons and their polar oxygenated derivatives) is rarely measured in field studies and only a fraction of the “total toxic load” is routinely quantified in the chemical analysis of biota. Extraction and analytical procedures can lose a significant proportion of the more volatile toxic compounds (e.g. log K,, -C3; Farrington et al., 1988) and the unresolved complex mixture (UCM) component of chromatograms, which contains toxic compounds, is frequently not quantified. Furthermore, the precision and accuracy of some analytical procedures is inadequate for
Table 2. Relationships between concentration of pentachlorophenol in the water and tissues of mussels (MyNus edulis) and the effect on respiration, clearance rate and scope for growth Water concentration (fig 1-v 0.5 38 65
167 335 660 786
Tissue concentration mg kg-’ wet wt)
Respiration rate (% of control)
Clearance rate (% of control)
Scope for growth (J hr-‘)
0.5 2.9 4.6 9.8 19.3 36.4 42.1
101 118 131 149 191 134 45
100 74 78 67 35 3.4 2.9
15.17 9.22 9.42 6.43 - 1.80 -5.44 -
12
J. WDDOWSand P.
DONKIN
toxicological comparison (Farrington et al., 1988). Consequently, it is often necessary to use selected groups of compounds which can be analysed reliably, as “indicators” of the total petroleum-derived toxic load, and establish appropriate conversion factors (Widdows et al., 1987). (c) Integration of effects of contaminant mixtures (unrelated toxicants) The interactions between structurally unrelated toxicants, such as petroleum hydrocarbons and copper, and their combined effects on the growth of mussels have been studied. Both Striimgren (1986) and Widdows and Johnson (1988) have recorded a simple “proportional additive” effect on shell growth and clearance rate. However, Stromgren stated that the effects of oil and copper were less than additive, and were therefore antagonistic, and did not recognise the combined effects were simply additive on a proportional basis. Figure 1 shows the close agreement between observed and expected growth rate when expressed in terms of proportional additivity. Recent studies (Widdows, Page and Donkin, unpublished data) have examined the combined effects of two important and structurally different contaminants (e.g. hydrocarbons and TBT) which co-exist in many aquatic environments. Five interaction experiments (4 concentrations of naphthalene + TBT and nonane + TBT) were carried out to test the hypothesis of additivity. However, the results consistently demonstrated that the combined effect on clearance rate was markedly less than predicted on the basis of “proportional additivity”. In fact there was evidence of significant (P < 0.01) antagonism between TBT and hydrocarbons (Fig. 2). Field studies provide some circumstantial evidence in support of these experimental findings. For example, in some locations bivalves have been found surviving with very high and potentially lethal TBT [tissue] levels (5-10 pg TBT/g dry wt), and these harbours/marinas are also those sites with high inputs of hydrocarbons 8
hell growth as % Of oot$f
Observed Observed Obrerved Observed
Observed Predicted Observed Predicted Observed Predicted Observed Predicted
1.6mg oil /
3mg oil /
I * 3ug
I +3ug Cu /
Cu I
I
I
1.6mg oil / I + Bug Cu I
I
3mg oil I I* 6ug Cu / I Redrawn from Stromgren 1966
Fig. 1. Effect of combinations of two concentrations of oil and copper on the shell growth of mussels (Myfilus edulis); observed response (as % of control) and predicted response (based on proportional additivity). Redrawn from Striimgren (1986).
Physiological energetics in ecotoxicology
73
i5 ; ; L
Expt.
I
Fig. 2. Effect of naphthalene (Naph), tributyltin (TBT) and naphthalene i- TBT (interaction) on the clearance rate of Myth eduh. Pred: the predicted decline in clearance rate based on additivity. Mean & SE. From Widdows, Page and Donkin (unpublished data). (e.g. San Diego and Honolulu Harbours, Grovhoug 1986). At present there is no mechanistic explanation for this antagonism between hydrocarbons and TBT, but it is probably acting at the level of neural control of gill cilia. et al.,
FIELD APPLICATION IN RELATION POLLUTION ASSESSMENT
TO
The assessment of environmental pollution using physiological energetic measurements of bivalves in conjunction with analysis of contaminants in their tissues began with field studies by Gilfillan et al. (1977), Widdows et al. (1981) and Martin et al. (1984). Since the early field application of this approach, methodological development has continued and laboratory/mesocosm studies have enhanced toxicological understan~ng of [tissue] cont~inant concentration-response relationships. The more recent application of this approach is illustrated with reference to two pollution gradients investigated as part of Practical Biological Effects Workshops in Bermuda and Oslotjord. At the Bermuda Workshop, the effects on the physiological energetics of transplanted mussels (Arcu zebra) were quantified and related to a contiminant gradient in Hamilton Harbour, a “non-indust~alised” region receiving relativley low levels of contaminant inputs. Although tissue concentrations of hydrocarbons, their polar oxygenated derivatives, PCBs, TBT and Pb were all significantly correlated with reductions in SFG, toxicological in~~retation of the tissue residue data
extended the analysis beyond statistical correlations and indicated that hydrocarbons (+polar oxygenated derivatives) and TBT could explain the observed decline in SFG ~iddows et al., 1990). Furthermore, the overall reductian in SFG could be proportioned, such that at the most contaminated site TBT accounted for 21% and hydrocarbons for 79% of the observed effect. In contrast, the second study examined a pollution gradient associated with an “industrialised” environment (Langesundfjord). Here the effects were not only more marked, but they were greater than could be explained by the widespread ~nt~inants, hydrocarbons and TBT, thus indicating the presence of other important and unidentified toxicants (Fig. 3). The results also highlighted the importance of comparing effects at polluted sites with appropriate reference sites. The “cleanest” site in ~ngesund~ord was significantly contaminated by hydrocarbons and TBT and therefore a well established “uncontaminated reference” site in the Shetland Islands was used. This enabled the contaminated levels and effects at all sites in Langesun~ord to be placed in a broader context (Widdows and Johnson, 1988; Widdows and Donkin, 1989). Direct comparisons between populations/sites can generally be made during the summer growth period and over such distances, because mussel transplant experiments over > 1000 km have demonstrated that physiological responses and growth generally reflect environmental rather than genetic differences (Widdows and Salkeld, unpublished data; Kautsky et al., 1990).
14
J. WIDDOWSand P. DONKIN Mytitus edutis
Craddock D. R. (1977) Acute toxic effects of petroleum on Arctic and sub-Arctic marine organisms. In Eficts of Petroleum on Arctic and sub-Arctic Marine Environments and Organisms. Vol. II. Biological EfSects (Edited by
24-
Malins D. C.), pp. l-93. Academic Press, New York. Davenport J. and Manley A. R. (1978) The detection of heightened seawater copper concentrations by the mussel, My&s
edulis. J. mar.-biol. Ass. U.K. 58, 843-850.
Davennort J. and Rednath K. J. (1984) Conner and the . mussel Mytilus edulis L. In Toxins, Drugs and Pollutants in Marine Animals (Edited by Bolis et al.), pp. 176189.
16 -
I
__
Springer, Berlin. Donkin P. and Widdows J. (1990) Quantitative structureactivity relationships in aquatic invertebrate toxicology. Rev. Aquat. Sci. 2, 375-398.
Donkin P., Widdows J., Evans S. V., Worrall C. M. and Carr M. (1989) Quantitative structure-activity relationships for the effect of hydrophobic organic chemicals on rate of feeding by mussels (Mytilus edulis). Aquat. Toxicol. 14, 277-294.
“ilean
re erence’ (Shetland)
'ResfE9~d
(Langesundfjord
‘Ps$kttd’
: Oslo GEEP WI.9
Fig. 3. Effect of pollution on Scope for Growth (SGF) of Mytilus edulis from Langesundtjord in comparison to a “clean reference site” in the Shetland Islands. Partitioning and relative contribution of hydrocarbons and TBT towards observed decline in SFG at Langesundfjord (SFG measured under “standard&d conditions” and calculated for a ration level of 0.4mg particulate organic matter I-‘; data from Widdows and Johnson, 1988).
Farrington J. W., Davis A. C., Frew N. M. and Knap A. (1988) ICESjIOC intercomparison exercise on the determination of petroleum hydrocarbons in biological tissues (mussel homogenate). Mar. Pollut. Bull. 19, 372-380. Farrington J. W., Davis A. C., Frew N. M. and Rabin K. S. (1982) No. 2. Fuel oil compounds in Mytilus edulis. Retention and release after an oil spill. Mar. Biol. 66, 15-26.
Farrington J. W., Goldberg E. D., Riseborough R. W., Martin J. H. and Bowen V. T. (1983) U.S. “Mussel Watch” 19761978: an overview of the trace metal, DDE, PCB, hydrocarbon and artificial radionuclide data. Environ. Sci. Technol. 11, 490-496.
Fowler S. W. and Oreaioni B. (1976) Trace metals in mussels from the N.W. Mediterranean. bar. Poll. Bull. 7, 26-29. Gibbs P. E., Bryan G. W., Pascoe P. L. and Burt G. R. (1987) The use of the dog-whelk, Nucella lapillus, as an indicator or tributyltin (TBT) contamination. J. mar. biol. Ass. U.K. 67, 561-569.
has been made towards the long-term ecotoxicological objective of “predicting effects and interpreting the causes of observed effects”, further research is required to provide a more comprehensive toxicological interpretation of contaminant residues in body tissues. Future research within this toxicological framework needs to establish (a) QSARs for other classes of contaminants, (b) relationships for other important biological responses (e.g. reproductive processes), and (c) further understanding of the combined effects of complex contaminant mixtures. While significant
progress
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