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The uses and abuses of ecotoxicology
Marine
Plymouth, U.K.), working on Neanthes larvae under laboratory conditions, have shown that SCE frequencies may be more useful than chromosomal aberrations for measuring genetic damage induced by low levels of irradiation (X-rays, 6°Co gamma rays). More detailed investigations, however, both of the responses and the factors affecting them, are of paramount importance before any direct cause-effect relationship between SCEs and environmental radiation can be considered proven. Understandably, Mytilus edulis, the common mussel and major pollution indicator species, has received its share of attention in terms of SCE studies. Both adults (D. R. Dixon & K. R. Clarke, loc. cir.) and larvae (F. L. Harrison & I. M. Jones, 1982, Mutation Res., 105, 235242) have been shown to yield adequate numbers of dividing cells for SCE analysis; although in adults the rate of cell division (in gill tissues) was only sufficient when the animals were actively growing. In common with other aquatic organisms, the cells of Mytilus edulis were shown to be very sensitive to a wide variety of chemical mutagens and carcinogens at low concentrations. However, with both these life-history stages there are serious practical
MarinePollutionBulletin, Vol. 14. No. 8, pp. 284-288.1983 Printed in Great Britain
Pollution
Bulletin
difficulties associated with their application in the field, namely small size in the case of the larvae and low natural rates of cell division in the case of the adult organism. A much more general limitation on the use of the SCE method under field conditions is the requirement for cells to be exposed to BrdU for two replication cycles before a response can be detected. While there may be ways around this problem, e.g. the use of implanted BrdU pellets such as are routinely used in some mammalian laboratories or, as was tried on a marine subject (P. T. Stromberg et al., 1981, NOAA TechnicalMemorandum OMPA-10, 43 pp.), dispensing with the field treatment of BrdU altogether (thus relying upon a residual effect due to mutagens remaining in the tissues for some time after collection), these approaches do not represent a complete answer to the problem. It seems likely, therefore, that SCE will remain for the most part an important laboratory method for detecting chromosomal disturbances resulting from contact between cells and environmental mutagens and carcinogens.
D. R. DIXON
0 0 2 5 - 3 2 6 X / 8 3 $3,(X) ÷ 0.00 © 1983 PerL,amon Pre~,s IId.
Viewpoint is a column which allows authors to express their own opinions about current events.
The Uses and Abuses of Ecotoxicology D. C. MONK Dr David Monk is a biologist employed by the Environmental Control Centre of BP International. For the past six years he has been involved in various capacities in assessing the environmental impacts of the oil and chemical industries. During the past two decades a new name - ecotoxicology has been added to the list of disciplines which can be included under the banner of environmental science. Whilst ecotoxicology is concerned with the fate and effects of contaminants in ecosystems, the aspect which has received most attention, and with which ecotoxicology has become particularly identified, is the laboratory testing of chemicals in order to predict the hazards that they pose to the environment. The enormous growth of activity in this field has been prompted partly by an increased scientific interest in the way that pollutants are distributed in and affect biological systems, but largely by an international proliferation of actual and threatened legislative activity which entails 'ecotoxicological' evaluation of potential environmental contaminants. To well known regulatory applications of aquatic toxicity testing such as the approval of pesticides, oil 284
spill dispersants and industrial wastes to be dumped at sea can be added the determination of tax payable on industrial discharges (in parts of Europe), the monitoring of discharged effluent quality (e.g. in Canada and the USA), the establishment of water quality criteria and the assessment and notification of the environmental hazards posed by new chemicals prior to marketing or manufacture (in many parts of the world). The development of standard test methods and methods for the subsequent evaluation of the results has involved international organizations such as the Organization for Economic Co-operation and Development (OECD) and the International Standards Organization as well as national bodies and the EEC. The worrying aspect of all this is that legislative respectability tends to endow the results of laboratory testing with a significance and precision which is not always merited. Although there is little doubt that testing of this sort can be
Volume 14/Number 8/August 1983
useful within limited and clearly defined objectives, it is important that the scientists who devise and use the tests make it clear to the legislators and administrators, who have now become deeply involved in the subject, just what the fundamental limitations of their efforts are. Laboratory testing of the environmental effects of particular contaminants is bound to increase in future years and attention will increasingly spread from freshwater, which is the medium supporting most of the currently standard test species and procedures. The development of standard test methodologies for marine organisms is likely to be a particularly active area in the next few years and it is hence worthwhile considering how the fundamental difficulties inherent in interpreting the results of bioassays limit the degree to which they can be of use in achieving the objectives of legislators and administrators as well as scientists. A good starting point in this is an acknowledgement that there is really nothing new about ecotoxicology.
History and Politics The first aquatic invertebrate bioassays were probably those carried out in 1816 by Beudant, who studied the effects of seawater on freshwater invertebrates and freshwater on marine invertebrates. These were followed during the nineteenth century by other studies concerning the effects of salts on invertebrates and by the end of the century the first tests of the effects of chemicals had been carried out and the first dose-response curves drawn (Anderson, 1980). Increasing numbers of such studies were carried out during the twentieth century and following the Second World War the production of large numbers of pesticides and the resulting pesticide regulatory schemes led to a boom in toxicity testing using both invertebrate and fish species. During this period more attention was being paid to the degree to which toxicants (particularly chlorinated hydrocarbons and surfactants) degrade in the environment and the ways in which they become partitioned between the various components of ecosystems and the resulting environmental distribution. There was also an increasing realization of the shortcomings of acute toxicity studies and more and more effort was put into developing chronic tests which looked at the entire lifecycle of the organism, sub-lethal tests which in some cases made use of an increasing understanding of the precise mode of action of the toxicant, and multi-species testing which attempted to mimic complex ecosystem interactions in laboratory microcosms. During the 1960s and 1970s a heightened awareness of the hazards posed by chemicals in the environment was brought about by several well publicized incidents. These included methyl mercury poisoning of humans in Japan as a result of eating contaminated fish from Minimata Bay, reproductive failure of seals and mink in several parts of the world caused by polychlorinated biphenyls and various problems caused by organochlorine pesticides-kills of terns and eiders in the Wadden Sea, declines in certain predatory bird populations throughout much of the north temperate zone and mortalities of juvenile salmonids in various parts of the world. Such incidents continue to receive wide publicity to the present day, particularly where the ultimate recipient of the contaminant may be
man. Examples are current concern over the effects of seed-dressings on bird and human populations and a well publicized international debate concerning dioxincontaminated herbicides. Public concern over the hazards posed by chemicals in the environment has been translated in many countries of the world into legislative measures designed to prevent further occurrence of incidents of the type mentioned. The Toxic Substances Control Act (in the USA), the EEC 'Sixth Amendment' (of a 1967 Directive relating to the packaging, labelling and classification of dangerous substances) and many other laws have in common a requirement to submit information to authorities concerning the effects of new chemicals on human health and the environment. These pieces of legislation have entailed the development of standardized methods for predicting the fate and effects of substances in the environment and have provided a major impetus for the integration, development and co-ordination of the previous strands of research in these areas. They have also been largely responsible for the adoption of a new t e r m - ecotoxicology - for an old science. A problem in the development of suitable test methodologies is that many of the available methods are related to specific problems and not designed for common application. Furthermore, different techniques have been developed in different countries and, left to themselves, those countries would doubtless formulate very different test requirements. In order to prevent the resulting confusion the OECD has been attempting to develop a package of standard tests which will be acceptable in all OECD countries as adequate for assessing the hazards of new substances prior to marketing. This package contains requirements for an acute toxicity test against a fish species, a test for effects on the reproduction of Daphnia and an algal species, and simple tests for biodegradation and bioaccumulation (as well as more extensive tests for effects on human health). This package is seen as a minimum requirement and chemicals which appear to pose a potential hazard may require much more extensive testing. This could include chronic and sub-chronic toxicity tests using a wide range of species, tests for effects on reproduction, tests for bioaccumulation and complex biodegradation tests. OECD have already published some of the simpler test procedures (OECD, 1981) and more are in the pipeline. All this may seem to be straying somewhat from environmental science, but the point is that scientists have been, are, and will increasingly be, enmeshed with administrators and legislators in attempting to control the environmental hazards posed by chemicals. Whilst the objective of these activities is clearly desirable, it is also essential to keep in mind some of the fundamental difficulties involved so that large amounts of time, effort and money are not wasted in producing meaningless data.
Biological Organization and Toxic Effects The common theme of testing of the type discussed here is the exposure of a biological system to a contaminant, usually in the laboratory, with the objective of making a prediction concerning the effec~ of that contaminant on other biological systems. The problem is that contaminants 285
Marine Pollution Bulletin generally exert their effects at the biochemical level, often by inhibition of enzymes or interruption of the function of other macromolecules, but effects which are normally considered to matter in the environment occur at the opposite end of the spectrum of biological organization - changes in (some) populations, communities and ecosystems. The resultant necessity to extrapolate between different levels of biological organization is the essential difference between human toxicology and ecotoxicology and has led to greatly diversified efforts to find a suitable way of linking effects which can be readily measured and ascribed to particular contaminants with those which actually matter. Effects on communities and ecosystems can best be studied by directly looking at changes in those communities and ecosystems. Monitoring the environmental impact of particular sources of pollution in just this way has been common practice for many years, but such methods can obviously only be used after contamination has occurred and any changes found can rarely be ascribed to specific substances. During the past few years studies of this type have, however, contributed to an increasing appreciation of the considerable resilience of most ecosystems and of the degree to which ecosystems can recover from environmental 'damage' following cessation of exposure to many pollutants. These are fundamental points to consider in assessing the hazards posed by potential pollutants but they cannot generally be allowed for in the design of laboratory bioassays. The interpretation of laboratory results in the light of what we know about the natural environment hence becomes particularly difficult. Test methods which come closest to looking at changes which are of genuine environmental significance are the 'big-bag' experiments carried out in Scotland and North America, which have looked at the effects of various pesticides, metals and hydrocarbons in marine enclosures, and equivalent intertidal enclosures now being used by the Norwegian Institute for Water Research to study the effects of oil pollution on the littoral communities of soft and hard substrates. Such studies can obviously yield valuable results but are clearly impractical as a routine means of assessing the hazards posed by large numbers of chemicals. In an effort to find a more practical way of bridging the gap between biochemistry and ecosystems a gamut of methods from enzyme inhibition to laboratory microcosms has evolved. None of these are perfect, though they all have their uses, and it is worth considering just what can be achieved by some of the alternatives.
Acute Lethality Testing The most commonly used levels of biological organization at which toxicity is assessed are those of individuals and populations, and the most common form of test looks at mortality in small population sub-samples over short periods. The drawbacks of such techniques have long been recognized - indeed, some ecologists would dismiss them as entirely u s e l e s s - b u t they nevertheless form the backbone of the standard procedures that have been drawn up for the regulatory purposes previously discussed. This is hardly surprising as such tests are relatively simple, cheap and practical to carry out and an 286
immense background literature exists on the topic. A further irresistible attraction for regulatory purposes is that answers (LCs0s or percentage mortalities at given concentrations) can be produced in the form of a single figure. The disadvantages of such techniques, however, fall into two categories - those inherent in the particular procedures used and those associated with the wider significance of the results obtained. In the first category the dependence of test results on physical and chemical test conditions is well known. Water movement, water hardness, temperature, pH, photoperiod, species used, degree of acclimation, the age of the organisms, synergisms and antagonisms between toxicants, various minor differences in laboratory procedure and the genetic stock of the species used have all been found to affect test results, and it is to be expected that test results produced by different laboratories will vary somewhat. These problems are generally well recognized and are not insurmountable if they are always borne in mind when considering test results. Useful data other than LC~0s should be produced by tests of this type, particularly the shape of the toxicity curve and the occurrence of a threshold toxic concentration. These can add considerably to an understanding of the way the toxicant is affecting the individuals and population sample concerned. This does not help, however, with the second category of problem in acute toxicity testing - relating the results to real environmental problems. Even if an accurate and reproducible LCs0 is obtained, the great complexity of populations, communities and ecosystems and the biological and chemical mechanisms by which they interact means thai it is almost impossible to understand the significance of the results at these levels. A simple example of this is the effect of density dependent mortality. Even where a large proportion of a population is killed by a toxicant there is no guarantee that this will have a long term effect on the population as a decreased mortality rate among the survivors and their offspring as a result of reduced population density may result in rapid recovery of the population. It is for this reason, for instance, thai mortality of larval fish following oil spills is likely to have to be on a massive scale before recruitment to fishery stocks is affected (GESAMP, 1977). Further problems arise in comparing actual levels of exposure in the field and the laboratory. Chemicals tend to behave in very complex ways in the environment. They may volatilize, adsorb on to surfaces, be passively or actively taken up by living organic material (including the food of affected organisms), be passively absorbed by dead organic material, or they may form complexes with other substances. Their own chemical speciation may be complex and highly dependent on environmental conditions. The actual concentration to which animals have been exposed in the laboratory during testing is often not known and the concentrations to which organisms are likely to be exposed in the field may be extremely hard to estimate. Few attempts have been made to correlate acute toxic effects with ecological changes observed in the field, and this is an area of research in need of furtherence. The existing evidence indicates that some correlations of the 'order of magnitude' variety may be possible, but the complexity of ecosystems is likely to render any more sophisticated interpretation impossible (Buikema et al., 1981; Hansen &
Volume 14/Number8/August 1983 Garton, 1982). Acute toxicity testing hence represents the crudest possible means of obtaining a preliminary evaluation of the possible environmental hazard posed by chemicals. However, such tests are practical and most of their drawbacks are well known. Before dismissing such techniques as useless it is necessary to look at what can be achieved by the alternatives. The most obvious way of extending the usefulness of toxicity tests is to lengthen the period over which they are carried out. This overcomes the problems caused by exposure periods being too short for toxic effects to be fully realized, but overcomes none of the other objections to acute toxicity testing. Indeed, the toxicity of many substances increases little after 96 hours exposure, so little additional useful data may be generated. The other main options are to devise tests which look at other levels of biological organization or tests which integrate most of the toxic responses looked at in other tests.
Sub-lethal Testing It has frequently been pointed out that acute tests in which the observed response is death do not take into account sub-lethal effects which occur at lower concentrations and which may have considerable ecological significance. For this reason acute lethality tests are often considered insufficiently sensitive as a means of evaluating the potential effects of contaminants. Whilst it is clearly true that effects will occur which do not result in the death of an individual, the ecological significance of tests based on these effects is particularly difficult to understand. Some extremely sensitive procedures have been devised which make use of biological responses at biochemical or physiological levels, or look at changes in the behaviour of individuals (e.g. Miller et al., 1982; Christensen et al., 1982) and such techniques are frequently recommended for use as routine means for assessing pollutant effects. Extrapolation of the results of such tests to higher levels of biological organization is even more difficult than the extrapolation of acute lethality data, however, as the number of steps, and hence the number of assumptions and the complexity of the extrapolation, is increased. Some of the most useful sub-lethal techniques are those which are directly related to the mode of action of the toxicant. One of the best known examples is the inhibition of acetyl-cholinesterase by organo-phosphorous pesticides The inhibition of this enzyme is well known as the means by which these pesticides disrupt the nervous systems of target (and non-target) organisms and can be easily measured in a wide range of organisms including man. Monitoring cholinesterase levels in blood serum has obvious applications in monitoring human exposure to these chemicals and similar approaches have been used in monitoring exposure of fish (e.g. Weiss, 1959). Even in a case like this, where cause and effect are relatively well understood at lhe biochemical level, the value of the technique is largely limited to that of a kind of biological 'meter' for detecting contamination. Indeed, such techniques may be the only meter available where the precise nature of the contaminant is unknown or where exposure is being assessed retrospectively. Whilst sub-lethal changes
must ultimately have some links with ecological processes, these links are so poorly understood that interpretation of the results of most sub-lethal tests beyond their significance as indicators of exposure is largely limited to their implications for the individuals concerned. The most important use of sub-lethal techniques is in increasing our fundamental understanding of the way in which toxicants exert their effects. By looking directly at biochemical and physiological changes, toxic mechanisms can be studied without the complications introduced by higher levels of biological complexity. Cause and effect relationships can be clearly elucidated and, hopefully, useful generalizations developed. Understanding and hence prediction of dose-response relationships and synergisms and antagonisms is dependent on an understanding of the mode of action of toxicants at physiological and biochemical levels as well as detailed knowledge of the behaviour of contaminants in the environment and the way in which they are transported to the sites of toxic action. Hence, such studies, whilst not normally of direct value in assessing the environmental hazards posed by contaminants, can play a major role in research which will hopefully lead to an increased understanding of the way toxicants work and the development of generalizations and useful predictive techniques. An opportunity to understand the links between the mode of action of toxicants and changes in communities could arise where a particular species has been extensively studied at many levels of biological organization. The mussel Mytilus edulis falls into this category and a programme which looks at the effects of pollutants at biochemical, physiological and individual levels has been developed (see Bayne et al., 1980). A significant aspect of this technique is that it can be used to determine stress responses of mussels in the field and the opportunity hence arises for an integrated study in which the effects of known contaminants can be examined at all levels from biochemistry to communities and the results related to contaminant residue measurements, perhaps more of which have been carried out on mussels than any other organisms. Such an integrated programme could yield many interesting results concerning the mechanisms by which contaminants affect communities and could eventually pinpoint those effects which are most significant in terms of changes at the community level.
Reproduction An optimum type of screening test would clearly be one which was sensitive to sub-lethal effects, but in which these were integrated and expressed in terms of probable effects at the highest possible level of biological organization. Tests which last the lifecycle of the organism concerned and in which the results are expressed as a change in reproductive fitness (which will probably have implications at the population level) seem to fit the bill most closely. Much work has been carried out on 'lifecycle' tests but a problem can be that the lifecycle of many organisms is so long as to make these tests unwieldly for routine purposes. This can be overcome by choosing an organism with a very short lifecycle and such a test has been adopted by the OECD (OECD, 1981), using changes in the reproductive rate of Daphnia over fourteen 287
Marine Pollution Bulletin
days. This test, not surprisingly, is an extremely sensitive one but the problem remains that it is unlikely to be possible to relate a possible reduction in a Daphnia population to anything that is particularly important in the real world the assumption that because such a reduction may occur there is bound to be some sort of wider environmental implication is not necessarily justified. Research which relates changes in reproductive fitness to population changes of the same species in the field would be of great value in increasing the value of these potentially useful test procedures.
Laboratory Microcosms Tests at higher levels of biological organization have been probably less extensively used than the other methods discussed, but the problems involved in developing them as standard test systems would be considerably greater than those experienced with many other types of test because of the increased complexity of the systems. A major problem even in fairly simple systems is maintaining the stability which is necessary for standardized procedures but which never occurs in the real world. In order for laboratory microcosms to be practical propositions they must necessarily be kept reasonably simple and this in turn means that the results are still hard to relate to the natural environment. If the system is small enough to manage conveniently there may be insufficient organisms present to sample adequately or predators may have to be removed in order to maintain adequate numbers at lower trophic levels. If the system is larger and more complex it may lose its convenience as a means of routine assessment. In spite of these problems laboratory microcosms may be useful in following up particular problems which have been identified in other ways. They are not suitable for routine screening and should preferably be purpose designed for each problem as it arises. They may be particularly useful in studies of the fate of chemicals where the dose can be carefully controlled and a budget for the substance concerned accurately determined within the system. Studies of effects are likely to be more difficult to carry out, less reproducible and of only slightly more relevance to the real world than the more conventional tests discussed previously.
Conclusions Whilst it is desirable that the probable effects of contaminants in the environment are assessed before the contaminants are discharged, efforts to make the assessment will inevitably be severely hampered by the yawning gap between simple laboratory tests and enormously complex ecosystems. This gap imposes severe restraints on the interpretation and uses of laboratory testing which are only too familiar to most scientists. However, the deep involvement of legislators and administrators in the field and the
288
probable future growth in interest specifically in marine testing means that it is worthwhile reiterating a few of the basic principles involved: (11 There is no such thing as a universally applicable test or test organism and to look for such things is wasted effort. (2) Almost every test ever devised has some use, but this is usually very limited. Failure to recognize and state the limitations can lead to an enormous waste of time, money and resources and misunderstandings with those who make and administer the rules. (3) It is probably impossible to predict ecological effects from laboratory test data and laboratory tests do not in general set out to make such a prediction. If it is accepted that it is desirable to screen the hazards posed by substances in the environment, simple acute toxicity tests are as good a starting point as any for making a first crude estimate of effects, particularly if other data on the behaviour of the substance in the environment are available. More sensitive tests are best devised by looking at effects on reproduction, but even these will not allow a prediction of ecological effects. Many other types of test are available and are best 'purpose designed' to meet individual requirements. Looked at in this context, the approach recommended by OECD and used in the EEC '6th amendment' can be seen as a reasonable way to satisfy increasing demands for some preliminary screening of the hazards posed by chemicals in the environment. Sensible use of the data obtained and the correct design of further tests to evaluate potential environmental effects will depend upon a clear understanding of the fundamental limitations of the test procedures available.
Anderson, B. G. (19801. Aquatic invertebrates in tolerance investigations from Aristotle to Naumann. In Aquatic Invertebrate Bioassavs. ASTM STP 715 (A. L. Buikema, Jr. & John Cairns, Jr., eds.), pp. 3-35. American Society for Testing of Materials, Philadelphia. Bayne, B. L., Brown, D. A., Harrison, F. & Yevich, P. D. (19801. Mussel health. In The International Mussel Watch, pp. 163-235. National Academy of Sciences, Washington D.C. Buikema, A. L., Kennedy, J., Benfield, E. F., Cairns, J. 8,: Hendricks, A. C. ( 1981 ). Prediction ~[ Environrnental Perturbation hv A cute and Chronic Toxici(v Tests. American Petroleum Institute, Washington, D.C. Christensen, G. M., Olson, D. & Riedel, B. (19821. Chemical effects on the activity of eight enzymes: a review and a discussion relevant to environmental monitorinm Envir. Res. 29, 247-255. GESAMP (19771. Joint Group of Experts on the Scientific Aspects of Marine Pollution. Impact of oil on the marine environmenl. Rep. Slud. GESAMP 6. Hansen, S. R. & Garton, R. R, (19821. Ability of standard toxicity Iesls to predict the effects of the insecticide dit3ubenzuron on laboratory stream communities. Can. J. Fish aquat. Sci. 39, 1273-1288. Miller, D+ C., Lang, W. H., Greaves, J. O. B. & Wilson+ R. S. (19821. In;'esligations in aquatic behavioural toxicology using a computeriscd video quantification system. In Aquatic Toxicology and Ha=ard Assessment: Fifth Conference, ASTM STP 766, pp. 206-220. American Society for Testing of Materials, Philadelphia. Organization for Economic Co-operation and Developmenl (1981). OECD Guidelines f o r Testin~ o f ChemicaZs, OECD, Paris. Weiss, C. M. (1959). Response of fish to sub lethal exposures of organic phosphorus insecticides. Sewage indust. Was'tes 31,580-593.
Plymouth, U.K.), working on Neanthes larvae under laboratory conditions, have shown that SCE frequencies may be more useful than chromosomal aberrations for measuring genetic damage induced by low levels of irradiation (X-rays, 6°Co gamma rays). More detailed investigations, however, both of the responses and the factors affecting them, are of paramount importance before any direct cause-effect relationship between SCEs and environmental radiation can be considered proven. Understandably, Mytilus edulis, the common mussel and major pollution indicator species, has received its share of attention in terms of SCE studies. Both adults (D. R. Dixon & K. R. Clarke, loc. cir.) and larvae (F. L. Harrison & I. M. Jones, 1982, Mutation Res., 105, 235242) have been shown to yield adequate numbers of dividing cells for SCE analysis; although in adults the rate of cell division (in gill tissues) was only sufficient when the animals were actively growing. In common with other aquatic organisms, the cells of Mytilus edulis were shown to be very sensitive to a wide variety of chemical mutagens and carcinogens at low concentrations. However, with both these life-history stages there are serious practical
MarinePollutionBulletin, Vol. 14. No. 8, pp. 284-288.1983 Printed in Great Britain
Pollution
Bulletin
difficulties associated with their application in the field, namely small size in the case of the larvae and low natural rates of cell division in the case of the adult organism. A much more general limitation on the use of the SCE method under field conditions is the requirement for cells to be exposed to BrdU for two replication cycles before a response can be detected. While there may be ways around this problem, e.g. the use of implanted BrdU pellets such as are routinely used in some mammalian laboratories or, as was tried on a marine subject (P. T. Stromberg et al., 1981, NOAA TechnicalMemorandum OMPA-10, 43 pp.), dispensing with the field treatment of BrdU altogether (thus relying upon a residual effect due to mutagens remaining in the tissues for some time after collection), these approaches do not represent a complete answer to the problem. It seems likely, therefore, that SCE will remain for the most part an important laboratory method for detecting chromosomal disturbances resulting from contact between cells and environmental mutagens and carcinogens.
D. R. DIXON
0 0 2 5 - 3 2 6 X / 8 3 $3,(X) ÷ 0.00 © 1983 PerL,amon Pre~,s IId.
Viewpoint is a column which allows authors to express their own opinions about current events.
The Uses and Abuses of Ecotoxicology D. C. MONK Dr David Monk is a biologist employed by the Environmental Control Centre of BP International. For the past six years he has been involved in various capacities in assessing the environmental impacts of the oil and chemical industries. During the past two decades a new name - ecotoxicology has been added to the list of disciplines which can be included under the banner of environmental science. Whilst ecotoxicology is concerned with the fate and effects of contaminants in ecosystems, the aspect which has received most attention, and with which ecotoxicology has become particularly identified, is the laboratory testing of chemicals in order to predict the hazards that they pose to the environment. The enormous growth of activity in this field has been prompted partly by an increased scientific interest in the way that pollutants are distributed in and affect biological systems, but largely by an international proliferation of actual and threatened legislative activity which entails 'ecotoxicological' evaluation of potential environmental contaminants. To well known regulatory applications of aquatic toxicity testing such as the approval of pesticides, oil 284
spill dispersants and industrial wastes to be dumped at sea can be added the determination of tax payable on industrial discharges (in parts of Europe), the monitoring of discharged effluent quality (e.g. in Canada and the USA), the establishment of water quality criteria and the assessment and notification of the environmental hazards posed by new chemicals prior to marketing or manufacture (in many parts of the world). The development of standard test methods and methods for the subsequent evaluation of the results has involved international organizations such as the Organization for Economic Co-operation and Development (OECD) and the International Standards Organization as well as national bodies and the EEC. The worrying aspect of all this is that legislative respectability tends to endow the results of laboratory testing with a significance and precision which is not always merited. Although there is little doubt that testing of this sort can be
Volume 14/Number 8/August 1983
useful within limited and clearly defined objectives, it is important that the scientists who devise and use the tests make it clear to the legislators and administrators, who have now become deeply involved in the subject, just what the fundamental limitations of their efforts are. Laboratory testing of the environmental effects of particular contaminants is bound to increase in future years and attention will increasingly spread from freshwater, which is the medium supporting most of the currently standard test species and procedures. The development of standard test methodologies for marine organisms is likely to be a particularly active area in the next few years and it is hence worthwhile considering how the fundamental difficulties inherent in interpreting the results of bioassays limit the degree to which they can be of use in achieving the objectives of legislators and administrators as well as scientists. A good starting point in this is an acknowledgement that there is really nothing new about ecotoxicology.
History and Politics The first aquatic invertebrate bioassays were probably those carried out in 1816 by Beudant, who studied the effects of seawater on freshwater invertebrates and freshwater on marine invertebrates. These were followed during the nineteenth century by other studies concerning the effects of salts on invertebrates and by the end of the century the first tests of the effects of chemicals had been carried out and the first dose-response curves drawn (Anderson, 1980). Increasing numbers of such studies were carried out during the twentieth century and following the Second World War the production of large numbers of pesticides and the resulting pesticide regulatory schemes led to a boom in toxicity testing using both invertebrate and fish species. During this period more attention was being paid to the degree to which toxicants (particularly chlorinated hydrocarbons and surfactants) degrade in the environment and the ways in which they become partitioned between the various components of ecosystems and the resulting environmental distribution. There was also an increasing realization of the shortcomings of acute toxicity studies and more and more effort was put into developing chronic tests which looked at the entire lifecycle of the organism, sub-lethal tests which in some cases made use of an increasing understanding of the precise mode of action of the toxicant, and multi-species testing which attempted to mimic complex ecosystem interactions in laboratory microcosms. During the 1960s and 1970s a heightened awareness of the hazards posed by chemicals in the environment was brought about by several well publicized incidents. These included methyl mercury poisoning of humans in Japan as a result of eating contaminated fish from Minimata Bay, reproductive failure of seals and mink in several parts of the world caused by polychlorinated biphenyls and various problems caused by organochlorine pesticides-kills of terns and eiders in the Wadden Sea, declines in certain predatory bird populations throughout much of the north temperate zone and mortalities of juvenile salmonids in various parts of the world. Such incidents continue to receive wide publicity to the present day, particularly where the ultimate recipient of the contaminant may be
man. Examples are current concern over the effects of seed-dressings on bird and human populations and a well publicized international debate concerning dioxincontaminated herbicides. Public concern over the hazards posed by chemicals in the environment has been translated in many countries of the world into legislative measures designed to prevent further occurrence of incidents of the type mentioned. The Toxic Substances Control Act (in the USA), the EEC 'Sixth Amendment' (of a 1967 Directive relating to the packaging, labelling and classification of dangerous substances) and many other laws have in common a requirement to submit information to authorities concerning the effects of new chemicals on human health and the environment. These pieces of legislation have entailed the development of standardized methods for predicting the fate and effects of substances in the environment and have provided a major impetus for the integration, development and co-ordination of the previous strands of research in these areas. They have also been largely responsible for the adoption of a new t e r m - ecotoxicology - for an old science. A problem in the development of suitable test methodologies is that many of the available methods are related to specific problems and not designed for common application. Furthermore, different techniques have been developed in different countries and, left to themselves, those countries would doubtless formulate very different test requirements. In order to prevent the resulting confusion the OECD has been attempting to develop a package of standard tests which will be acceptable in all OECD countries as adequate for assessing the hazards of new substances prior to marketing. This package contains requirements for an acute toxicity test against a fish species, a test for effects on the reproduction of Daphnia and an algal species, and simple tests for biodegradation and bioaccumulation (as well as more extensive tests for effects on human health). This package is seen as a minimum requirement and chemicals which appear to pose a potential hazard may require much more extensive testing. This could include chronic and sub-chronic toxicity tests using a wide range of species, tests for effects on reproduction, tests for bioaccumulation and complex biodegradation tests. OECD have already published some of the simpler test procedures (OECD, 1981) and more are in the pipeline. All this may seem to be straying somewhat from environmental science, but the point is that scientists have been, are, and will increasingly be, enmeshed with administrators and legislators in attempting to control the environmental hazards posed by chemicals. Whilst the objective of these activities is clearly desirable, it is also essential to keep in mind some of the fundamental difficulties involved so that large amounts of time, effort and money are not wasted in producing meaningless data.
Biological Organization and Toxic Effects The common theme of testing of the type discussed here is the exposure of a biological system to a contaminant, usually in the laboratory, with the objective of making a prediction concerning the effec~ of that contaminant on other biological systems. The problem is that contaminants 285
Marine Pollution Bulletin generally exert their effects at the biochemical level, often by inhibition of enzymes or interruption of the function of other macromolecules, but effects which are normally considered to matter in the environment occur at the opposite end of the spectrum of biological organization - changes in (some) populations, communities and ecosystems. The resultant necessity to extrapolate between different levels of biological organization is the essential difference between human toxicology and ecotoxicology and has led to greatly diversified efforts to find a suitable way of linking effects which can be readily measured and ascribed to particular contaminants with those which actually matter. Effects on communities and ecosystems can best be studied by directly looking at changes in those communities and ecosystems. Monitoring the environmental impact of particular sources of pollution in just this way has been common practice for many years, but such methods can obviously only be used after contamination has occurred and any changes found can rarely be ascribed to specific substances. During the past few years studies of this type have, however, contributed to an increasing appreciation of the considerable resilience of most ecosystems and of the degree to which ecosystems can recover from environmental 'damage' following cessation of exposure to many pollutants. These are fundamental points to consider in assessing the hazards posed by potential pollutants but they cannot generally be allowed for in the design of laboratory bioassays. The interpretation of laboratory results in the light of what we know about the natural environment hence becomes particularly difficult. Test methods which come closest to looking at changes which are of genuine environmental significance are the 'big-bag' experiments carried out in Scotland and North America, which have looked at the effects of various pesticides, metals and hydrocarbons in marine enclosures, and equivalent intertidal enclosures now being used by the Norwegian Institute for Water Research to study the effects of oil pollution on the littoral communities of soft and hard substrates. Such studies can obviously yield valuable results but are clearly impractical as a routine means of assessing the hazards posed by large numbers of chemicals. In an effort to find a more practical way of bridging the gap between biochemistry and ecosystems a gamut of methods from enzyme inhibition to laboratory microcosms has evolved. None of these are perfect, though they all have their uses, and it is worth considering just what can be achieved by some of the alternatives.
Acute Lethality Testing The most commonly used levels of biological organization at which toxicity is assessed are those of individuals and populations, and the most common form of test looks at mortality in small population sub-samples over short periods. The drawbacks of such techniques have long been recognized - indeed, some ecologists would dismiss them as entirely u s e l e s s - b u t they nevertheless form the backbone of the standard procedures that have been drawn up for the regulatory purposes previously discussed. This is hardly surprising as such tests are relatively simple, cheap and practical to carry out and an 286
immense background literature exists on the topic. A further irresistible attraction for regulatory purposes is that answers (LCs0s or percentage mortalities at given concentrations) can be produced in the form of a single figure. The disadvantages of such techniques, however, fall into two categories - those inherent in the particular procedures used and those associated with the wider significance of the results obtained. In the first category the dependence of test results on physical and chemical test conditions is well known. Water movement, water hardness, temperature, pH, photoperiod, species used, degree of acclimation, the age of the organisms, synergisms and antagonisms between toxicants, various minor differences in laboratory procedure and the genetic stock of the species used have all been found to affect test results, and it is to be expected that test results produced by different laboratories will vary somewhat. These problems are generally well recognized and are not insurmountable if they are always borne in mind when considering test results. Useful data other than LC~0s should be produced by tests of this type, particularly the shape of the toxicity curve and the occurrence of a threshold toxic concentration. These can add considerably to an understanding of the way the toxicant is affecting the individuals and population sample concerned. This does not help, however, with the second category of problem in acute toxicity testing - relating the results to real environmental problems. Even if an accurate and reproducible LCs0 is obtained, the great complexity of populations, communities and ecosystems and the biological and chemical mechanisms by which they interact means thai it is almost impossible to understand the significance of the results at these levels. A simple example of this is the effect of density dependent mortality. Even where a large proportion of a population is killed by a toxicant there is no guarantee that this will have a long term effect on the population as a decreased mortality rate among the survivors and their offspring as a result of reduced population density may result in rapid recovery of the population. It is for this reason, for instance, thai mortality of larval fish following oil spills is likely to have to be on a massive scale before recruitment to fishery stocks is affected (GESAMP, 1977). Further problems arise in comparing actual levels of exposure in the field and the laboratory. Chemicals tend to behave in very complex ways in the environment. They may volatilize, adsorb on to surfaces, be passively or actively taken up by living organic material (including the food of affected organisms), be passively absorbed by dead organic material, or they may form complexes with other substances. Their own chemical speciation may be complex and highly dependent on environmental conditions. The actual concentration to which animals have been exposed in the laboratory during testing is often not known and the concentrations to which organisms are likely to be exposed in the field may be extremely hard to estimate. Few attempts have been made to correlate acute toxic effects with ecological changes observed in the field, and this is an area of research in need of furtherence. The existing evidence indicates that some correlations of the 'order of magnitude' variety may be possible, but the complexity of ecosystems is likely to render any more sophisticated interpretation impossible (Buikema et al., 1981; Hansen &
Volume 14/Number8/August 1983 Garton, 1982). Acute toxicity testing hence represents the crudest possible means of obtaining a preliminary evaluation of the possible environmental hazard posed by chemicals. However, such tests are practical and most of their drawbacks are well known. Before dismissing such techniques as useless it is necessary to look at what can be achieved by the alternatives. The most obvious way of extending the usefulness of toxicity tests is to lengthen the period over which they are carried out. This overcomes the problems caused by exposure periods being too short for toxic effects to be fully realized, but overcomes none of the other objections to acute toxicity testing. Indeed, the toxicity of many substances increases little after 96 hours exposure, so little additional useful data may be generated. The other main options are to devise tests which look at other levels of biological organization or tests which integrate most of the toxic responses looked at in other tests.
Sub-lethal Testing It has frequently been pointed out that acute tests in which the observed response is death do not take into account sub-lethal effects which occur at lower concentrations and which may have considerable ecological significance. For this reason acute lethality tests are often considered insufficiently sensitive as a means of evaluating the potential effects of contaminants. Whilst it is clearly true that effects will occur which do not result in the death of an individual, the ecological significance of tests based on these effects is particularly difficult to understand. Some extremely sensitive procedures have been devised which make use of biological responses at biochemical or physiological levels, or look at changes in the behaviour of individuals (e.g. Miller et al., 1982; Christensen et al., 1982) and such techniques are frequently recommended for use as routine means for assessing pollutant effects. Extrapolation of the results of such tests to higher levels of biological organization is even more difficult than the extrapolation of acute lethality data, however, as the number of steps, and hence the number of assumptions and the complexity of the extrapolation, is increased. Some of the most useful sub-lethal techniques are those which are directly related to the mode of action of the toxicant. One of the best known examples is the inhibition of acetyl-cholinesterase by organo-phosphorous pesticides The inhibition of this enzyme is well known as the means by which these pesticides disrupt the nervous systems of target (and non-target) organisms and can be easily measured in a wide range of organisms including man. Monitoring cholinesterase levels in blood serum has obvious applications in monitoring human exposure to these chemicals and similar approaches have been used in monitoring exposure of fish (e.g. Weiss, 1959). Even in a case like this, where cause and effect are relatively well understood at lhe biochemical level, the value of the technique is largely limited to that of a kind of biological 'meter' for detecting contamination. Indeed, such techniques may be the only meter available where the precise nature of the contaminant is unknown or where exposure is being assessed retrospectively. Whilst sub-lethal changes
must ultimately have some links with ecological processes, these links are so poorly understood that interpretation of the results of most sub-lethal tests beyond their significance as indicators of exposure is largely limited to their implications for the individuals concerned. The most important use of sub-lethal techniques is in increasing our fundamental understanding of the way in which toxicants exert their effects. By looking directly at biochemical and physiological changes, toxic mechanisms can be studied without the complications introduced by higher levels of biological complexity. Cause and effect relationships can be clearly elucidated and, hopefully, useful generalizations developed. Understanding and hence prediction of dose-response relationships and synergisms and antagonisms is dependent on an understanding of the mode of action of toxicants at physiological and biochemical levels as well as detailed knowledge of the behaviour of contaminants in the environment and the way in which they are transported to the sites of toxic action. Hence, such studies, whilst not normally of direct value in assessing the environmental hazards posed by contaminants, can play a major role in research which will hopefully lead to an increased understanding of the way toxicants work and the development of generalizations and useful predictive techniques. An opportunity to understand the links between the mode of action of toxicants and changes in communities could arise where a particular species has been extensively studied at many levels of biological organization. The mussel Mytilus edulis falls into this category and a programme which looks at the effects of pollutants at biochemical, physiological and individual levels has been developed (see Bayne et al., 1980). A significant aspect of this technique is that it can be used to determine stress responses of mussels in the field and the opportunity hence arises for an integrated study in which the effects of known contaminants can be examined at all levels from biochemistry to communities and the results related to contaminant residue measurements, perhaps more of which have been carried out on mussels than any other organisms. Such an integrated programme could yield many interesting results concerning the mechanisms by which contaminants affect communities and could eventually pinpoint those effects which are most significant in terms of changes at the community level.
Reproduction An optimum type of screening test would clearly be one which was sensitive to sub-lethal effects, but in which these were integrated and expressed in terms of probable effects at the highest possible level of biological organization. Tests which last the lifecycle of the organism concerned and in which the results are expressed as a change in reproductive fitness (which will probably have implications at the population level) seem to fit the bill most closely. Much work has been carried out on 'lifecycle' tests but a problem can be that the lifecycle of many organisms is so long as to make these tests unwieldly for routine purposes. This can be overcome by choosing an organism with a very short lifecycle and such a test has been adopted by the OECD (OECD, 1981), using changes in the reproductive rate of Daphnia over fourteen 287
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days. This test, not surprisingly, is an extremely sensitive one but the problem remains that it is unlikely to be possible to relate a possible reduction in a Daphnia population to anything that is particularly important in the real world the assumption that because such a reduction may occur there is bound to be some sort of wider environmental implication is not necessarily justified. Research which relates changes in reproductive fitness to population changes of the same species in the field would be of great value in increasing the value of these potentially useful test procedures.
Laboratory Microcosms Tests at higher levels of biological organization have been probably less extensively used than the other methods discussed, but the problems involved in developing them as standard test systems would be considerably greater than those experienced with many other types of test because of the increased complexity of the systems. A major problem even in fairly simple systems is maintaining the stability which is necessary for standardized procedures but which never occurs in the real world. In order for laboratory microcosms to be practical propositions they must necessarily be kept reasonably simple and this in turn means that the results are still hard to relate to the natural environment. If the system is small enough to manage conveniently there may be insufficient organisms present to sample adequately or predators may have to be removed in order to maintain adequate numbers at lower trophic levels. If the system is larger and more complex it may lose its convenience as a means of routine assessment. In spite of these problems laboratory microcosms may be useful in following up particular problems which have been identified in other ways. They are not suitable for routine screening and should preferably be purpose designed for each problem as it arises. They may be particularly useful in studies of the fate of chemicals where the dose can be carefully controlled and a budget for the substance concerned accurately determined within the system. Studies of effects are likely to be more difficult to carry out, less reproducible and of only slightly more relevance to the real world than the more conventional tests discussed previously.
Conclusions Whilst it is desirable that the probable effects of contaminants in the environment are assessed before the contaminants are discharged, efforts to make the assessment will inevitably be severely hampered by the yawning gap between simple laboratory tests and enormously complex ecosystems. This gap imposes severe restraints on the interpretation and uses of laboratory testing which are only too familiar to most scientists. However, the deep involvement of legislators and administrators in the field and the
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probable future growth in interest specifically in marine testing means that it is worthwhile reiterating a few of the basic principles involved: (11 There is no such thing as a universally applicable test or test organism and to look for such things is wasted effort. (2) Almost every test ever devised has some use, but this is usually very limited. Failure to recognize and state the limitations can lead to an enormous waste of time, money and resources and misunderstandings with those who make and administer the rules. (3) It is probably impossible to predict ecological effects from laboratory test data and laboratory tests do not in general set out to make such a prediction. If it is accepted that it is desirable to screen the hazards posed by substances in the environment, simple acute toxicity tests are as good a starting point as any for making a first crude estimate of effects, particularly if other data on the behaviour of the substance in the environment are available. More sensitive tests are best devised by looking at effects on reproduction, but even these will not allow a prediction of ecological effects. Many other types of test are available and are best 'purpose designed' to meet individual requirements. Looked at in this context, the approach recommended by OECD and used in the EEC '6th amendment' can be seen as a reasonable way to satisfy increasing demands for some preliminary screening of the hazards posed by chemicals in the environment. Sensible use of the data obtained and the correct design of further tests to evaluate potential environmental effects will depend upon a clear understanding of the fundamental limitations of the test procedures available.
Anderson, B. G. (19801. Aquatic invertebrates in tolerance investigations from Aristotle to Naumann. In Aquatic Invertebrate Bioassavs. ASTM STP 715 (A. L. Buikema, Jr. & John Cairns, Jr., eds.), pp. 3-35. American Society for Testing of Materials, Philadelphia. Bayne, B. L., Brown, D. A., Harrison, F. & Yevich, P. D. (19801. Mussel health. In The International Mussel Watch, pp. 163-235. National Academy of Sciences, Washington D.C. Buikema, A. L., Kennedy, J., Benfield, E. F., Cairns, J. 8,: Hendricks, A. C. ( 1981 ). Prediction ~[ Environrnental Perturbation hv A cute and Chronic Toxici(v Tests. American Petroleum Institute, Washington, D.C. Christensen, G. M., Olson, D. & Riedel, B. (19821. Chemical effects on the activity of eight enzymes: a review and a discussion relevant to environmental monitorinm Envir. Res. 29, 247-255. GESAMP (19771. Joint Group of Experts on the Scientific Aspects of Marine Pollution. Impact of oil on the marine environmenl. Rep. Slud. GESAMP 6. Hansen, S. R. & Garton, R. R, (19821. Ability of standard toxicity Iesls to predict the effects of the insecticide dit3ubenzuron on laboratory stream communities. Can. J. Fish aquat. Sci. 39, 1273-1288. Miller, D+ C., Lang, W. H., Greaves, J. O. B. & Wilson+ R. S. (19821. In;'esligations in aquatic behavioural toxicology using a computeriscd video quantification system. In Aquatic Toxicology and Ha=ard Assessment: Fifth Conference, ASTM STP 766, pp. 206-220. American Society for Testing of Materials, Philadelphia. Organization for Economic Co-operation and Developmenl (1981). OECD Guidelines f o r Testin~ o f ChemicaZs, OECD, Paris. Weiss, C. M. (1959). Response of fish to sub lethal exposures of organic phosphorus insecticides. Sewage indust. Was'tes 31,580-593.