319 17 Organic Pollutants Future Prospects 17.1 INTRODUCTION During the second half of the 20th century, it was discovered that a number of organic pollutants (OPs) were having harmful side effects in natural ecosystems, prominent among which were chemicals that combined high toxicity (lethal or sublethal) with marked biological persistence. Examples such as dieldrin, DDT, TBT, and methyl mercury are represented in Part 2 of the present book. Following these discover- ies, restrictions and bans on the release of these chemicals into the environment were introduced in many countries. Persistent organochlorine (OC) insecticides, for example, were withdrawn from many uses and replaced by less persistent OP and carbamate insecticides. More stringent legislation was brought in to control the pro- duction and marketing of new chemicals, with clearer guidelines for environmental risk assessment. Particularly strict rules were applied to new pesticides—something of a double-edged weapon. This tightening of regulations has reduced the risk of new pesticides creating new problems, but it may also have impeded the discovery and registration of newer, more environmentally friendly compounds by making research and development too expensive. In spite of the immediate advantages tighter regula- tions bring, they can, in the long run, be counterproductive. Since the introduction of these restrictions and bans on persistent pesticides, it has been discovered that other less persistent compounds can also cause environmental problems. Some highly toxic insecticides, including the carbamate aldicarb used as a granular formulation, and the organophosphorous compounds carbophenothion and chlorfenvinphos used as seed dressings, have been responsible for poisoning incidents on agricultural land (Hardy 1990). Also, tributyl tin, an antifouling agent used in marine paints, has been shown to have serious effects upon aquatic mollusks, including oysters and dog whelks (Chapter 8, Section 8.3 of this book). So, with the tightening of the rules, other compounds of lesser persistence have been subject to restrictions and bans. At the same time, some new pesticides have come on to the market that are regarded as being more environmentally friendly. Among the insec- ticides, newer pyrethroids and neonicotinoids fall into this category. With these developments, it would appear that many of the more obvious envi- ronmental problems relating to particular compounds or groups of compounds have now disappeared. At least, this seems to be so in the developed world where there are now strict controls of environmental pollution that are reasonably well enforced. However, this does not necessarily apply to third-world countries where there is not such strict control. One consequence of this trend in developed countries has been © 2009 by Taylor & Francis Group, LLC 320 Organic Pollutants: An Ecotoxicological Perspective, Second Edition for ecotoxicological research and the development of ecotoxicity testing procedures to move toward strategies for testing the toxicity of complex mixtures, the compo- nents of which are usually at low concentration. More attention, for example, has been paid to environmental contamination by low levels of pharmaceuticals. Where these have been found to be present in complex mixtures, questions have been asked about the possibility of potentiation of toxicity. Among pharmaceuticals, EE2 has been the subject of particular recent attention because of its ability to cause endocrine disruption in sh, as has been described in Chapter 15. Low levels of mixtures of beta blockers, such as propranolol, metoprol, and nadolol have been detected in surface waters, and there have been investigations of their possible effects on aquatic invertebrates (Huggett et al. 2002). Veterinary medicines, too, have come under scrutiny: for example, the dramatic effects of diclofenac on vultures, which will be discussed shortly. Many questions remain to be answered about the possible ecological effects of complex mixtures of pharma- ceuticals and veterinary medicines. This trend toward studying the effects of low levels of chemicals existing in com- plex mixtures has been evident in much of the third part of the present text. Some researchers have gone so far as to suggest that ecotoxicology might now be seen as an aspect of stress ecology: that the toxic action of environmental chemicals is just one of a number of stress factors to which free-living organisms are exposed, and that stress factors should be considered as a whole when studying populations (Van Straalen 2003). This approach has scientic merit, and the desirable situation may exist in some areas of the world where the effect of pollutants is no more important than that of other stress factors. However, there are other areas where this is not the case, and serious problems of pollution still exist—areas where, in other words, chemical stress substantially outweighs other stress factors and is the driving force behind changes in population numbers or the genetic composition of populations. Even in North America, there are areas, some of them Superfund sites, where serious pollution still exists, and certain organic pollutants are having effects on natural populations. In the present text, examples were given of ongoing problems in certain areas with high levels of organomercury (Chapter 8, Section 8.2) and of PCBs and dioxins (Chapters 6 and 7). More dramatically, there have been other instances, sometimes with chemicals not previously considered as important pollutants, in countries less well developed than the United States or Canada. For example, a few years ago, a pollution problem came to light in India and Pakistan that was as disturbing for the Zoroastrian community as it was for conservationists. Three species of vulture declined rapidly during the decade 1997–2006 (Green, Taggart, and Das 2006). The species affected were Gyps bengalensis, Gyps indicus, and Gyps tenuirostris. Further investigation has strongly implicated the nonsteroidal antiinammatory drug diclofenac in the death of these birds over a wide area (Schultz et al. 2004 and Green, Taggart, and Das 2006). Many of the birds found dead over this large area contained residues of diclofenac. Many also had visceral gout, which is a condition strongly associated with diclofenac tox- icity. They evidently obtained residues of this compound by feeding on dead cattle that had recently been dosed with it. A toxicological investigation into the uptake of diclofenac by vultures concluded that more than 10% of the birds could acquire a © 2009 by Taylor & Francis Group, LLC Organic Pollutants 321 lethal dose in a single meal if they were feeding on cattle that had died within two days of receiving of the drug. So, once again, a chlorinated compound that shows a fair degree of persistence in vertebrates has been implicated in lethal poisoning and population decline in the eld. Speaking more widely, there continue to be cases of gross misuse of pesticides and other organic pollutants in some parts of the world, including on humans who use them. In India, for example, there are still reports of organophosphorous poisoning of spray operators working in crops such as cotton. We know little of the ecological consequences of the misuse of such pesticides. The following sections will attempt to look ahead to likely future problems with organic pollution, to probable changes in the ways in which it is studied and monitored, and in the tests and strategies used for environmental risk assessment of organic chemicals. 17.2 THE ADOPTION OF MORE ECOLOGICALLY RELEVANT PRACTICES IN ECOTOXICITY TESTING Currently, the environmental risk assessment of chemicals for registration purposes depends on the comparison of two things: (1) An estimate (sometimes a measure) of the environmental concentration of a chemical, and (2) an estimate of the envi- ronmental toxicity of this chemical. Environmental concentration is difcult to esti- mate, especially for mobile species in terrestrial ecosystems. In the present example, the estimation of environmental toxicity is expressed as a concentration, which may be a No Observable Effect Concentration (NOEC) or an LC 50 for the most sensitive organism found in a series of ecotoxicity tests. Very seldom is the species used in the toxicity test one of those most at risk in the natural environment. Nearly always a surrogate is used, and there is the immediate question of species differences in sus- ceptibility, which are largely unknown. In the case of birds, for example, two or three species are used in ecotoxicity testing, whereas some 8700 species exist in nature. For further discussion of these issues, see Chapter 6 in Walker et al. (2000), Calow (1993), and Walker (1998b). Because of the high levels of uncertainty involved, the estimate of environmental toxicity is divided by a large safety factor, commonly 1000. Following these computations, there is perceived to be a risk if the estimate of environmental concentration (1) exceeds the estimate of environmental toxicity (2). The limitations of this approach are not difcult to appreciate (see, for example, Kapustka, Williams, and Fairbrother 1996). It is based on the approach to risk assess- ment used in human toxicology and has been regarded as the best that can be done with existing resources. It is concerned with estimating the likelihood that there will be a toxic effect upon a sensitive species following the release of a chemical into the environment. With the very large safety factors that are used, it may well seriously overestimate the risks presented by some chemicals. More fundamentally, it does not address the basic issue of effects upon populations, communities, or ecosystems. Small toxic effects may be of no signicance when it comes to possible harmful effects at these higher levels of biological organization, where population numbers are often controlled by density-dependent factors (Chapter 4). Also, it does not deal with the question of indirect effects. As mentioned earlier (Chapter 14), standard © 2009 by Taylor & Francis Group, LLC 322 Organic Pollutants: An Ecotoxicological Perspective, Second Edition environmental risk assessment of herbicides would have given no indication that they could be the indirect cause of a decline of the grey partridge on agricultural land. There has been growing pressure from biologists for the development of more ecologically relevant end points when carrying out toxicity testing for the purposes of environmental risk assessment (see Walker et al. 1998b, and Chapter 12 in Walker et al. 2000). In concept, populations will decline when pollutants, directly or indi- rectly, have a sufciently large effect on rates of mortality and/or rates of recruitment to reduce population growth rate (Chapter 4, Section 4.4). Thus, sublethal effects, such as on reproduction or behavior, can be more important than lethal ones. If pol- lutant effects can be quantied in this way, for example, through the use of biomarker assays for toxic effect (see Chapter 15, Section 15.4), then better risk assessment is made possible by including them in appropriate population models. In practice, this approach is still at an early stage of development; it is a research strategy that can only be used in a few cases, and cannot yet deal with the large numbers of com- pounds submitted for risk assessment. Nevertheless, looking at the problem from this more fundamental point of view does suggest certain improvements that could be made in the protocols for environmental risk assessment. A large proportion of the resources currently being spent on the determination of LD 50 levels for birds or LC 50 levels of sh could be diverted to more relevant test- ing procedures. At best these values give only a rough indication of lethal toxicity in a small number of species. A ranking of compounds with respect to toxicity, for example, low toxicity, moderate toxicity, etc., is good enough for such a crude and empirical approach; knowing particular values a little more precisely does practically nothing to improve the quality of this type of environmental risk assessment where the uncertainties are so big. In the rst place, greater consideration of ecological aspects before embarking on testing should lead to the selection of more appropri- ate species, life stages, and end points in the testing protocol. It might be useful, for example, to include tests on behavioral effects if testing a neurotoxic pesticide, or of reproductive effects if testing a compound that can disturb steroid metabolism. These are mechanisms the operation of which might be expected to have adverse ecological effects. In a number of instances, population declines have been the consequence of reproductive failure (e.g., effects of p,pb-DDE on shell thickness of raptors, effects of PCBs and other polychlorinated compounds on reproduction of sh-eating birds in the Great Lakes, and the effects of TBT on the dog whelk). Effects on behavior may affect breeding and feeding and lead to population decline. In some species there may be good reasons for looking at the toxicity of certain types of compounds in early developmental stages (e.g., avian embryos, larval stages of amphibians) rather than adults. In short, testing protocols should be more exible so that there can be a greater opportunity for expert judgment rather than follow- ing a rigid set of rules. Knowledge of the metabolism and the mechanism of action of a new chemical may suggest the most appropriate end points in toxicity testing. Indeed, mechanistic biomarkers can provide better and more informative end points than lethality; they can be used to monitor progression through sublethal (including subclinical) effects before lethal tissue concentrations are reached. An approach that has gained attention recently is the use of model ecosys- tems: microcosms, mesocosms, and macrocosms for testing chemicals (Chapter 4, © 2009 by Taylor & Francis Group, LLC Organic Pollutants 323 Section 4.5). Of these, mesocosms have stimulated the greatest interest. In these, replicated and controlled tests can be carried out to establish the effects of chemicals upon the structure and function of the (articial) communities they contain. The major problem is relating effects produced in mesocosms to events in the real world (see Crossland 1994). Nevertheless, it can be argued that mesocosms do incorporate certain relationships (e.g., predator/prey) and processes (e.g., carbon cycle) that are found in the outside world, and they test the effects of chemicals on these. Once again, the judicious use of biomarker assays during the course of mesocosm studies may help to relate effects of chemicals measured by them with similar effects in the natural environment. During the period leading up to the implementation of the REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) proposals by the European Commission in 2006, there was considerable public debate about the procedures that are laid down in it for ecotoxicity testing of industrial chemicals (Walker 2006). One strongly debated issue relevant to the present discussion was the operation of the tiered testing system recommended in it. The recommended testing protocols were determined by the level of production or importation of particular chemicals. Although this may be a convenient system to operate from a bureaucratic point of view, it inevitably has encountered a good deal of criticism from scientists, includ- ing the U.K. Royal Commission on Environmental Pollution. The biggest objection is that the scale of production or exportation of a chemical bears a very uncertain relationship to the actual exposure of free-living organisms to it, and it is the latter issue that is important in the context of environmental risk assessment. From an eco- logical point of view, testing protocols should be decided on the basis of estimated environmental exposure. Despite these objections, a tiered system linked to annual production has now been accepted by the European Commission. The improvement of testing procedures is a slow business. 17.3 THE DEVELOPMENT OF MORE SOPHISTICATED METHODS OF TOXICITY TESTING: MECHANISTIC BIOMARKERS The judicious use of mechanistic biomarkers can, in theory, overcome many of the basic problems associated with establishing causality. In the eld, they can be used to measure the extent to which pollutants act upon wild species through dened toxic mechanisms, thus giving more insight into the sublethal as well as lethal effects of chemicals. Most important, they can provide measures of the integrated effects of mixtures of compounds operating through the same mechanism, measures that take into account potentiation at the toxicokinetic level (Chapter 13, Section 13.4). With the advent of new biomarker technology such as that of the “omics” (See Chapter 4, Box 4.2), they can be used to study the effect of complex mixtures containing chemicals working through contrasting mechanisms of action (see Chapters 15 and 16). In theory, biomarkers can provide the vital link between known levels of expo- sure and changes in mortality rates or recruitment rates; estimates of mortality rates and recruitment rates so obtained can then be incorporated into population mod- els (Chapter 4, Section 4.3). More explicitly, graphs can be generated that link a © 2009 by Taylor & Francis Group, LLC 324 Organic Pollutants: An Ecotoxicological Perspective, Second Edition biomarker response to a population parameter, as has already been achieved with eggshell thinning in the sparrowhawk induced by p,pb-DDE and imposex in the dog whelk caused by TBT. Currently, because of the shortage of appropriate biomarker assays, this approach lies largely in the realm of research and cannot be applied to most problems with environmental chemicals. At the practical level, an ideal mechanistic biomarker should be simple to use, sensitive, relatively specic, stable, and usable on material that can be obtained by nondestructive sampling (e.g., blood or skin). A tall order, no doubt, and no bio- marker yet developed has all of these attributes. However, the judicious use of com- binations of biomarkers can overcome the shortcomings of individual assays. The main point to emphasize is that the resources so far invested in the development of biomarker technology for environmental risk assessment has been very small (cf. the investment in biomarkers for use in medicine). Knowledge of toxic mechanisms of organic pollutants is already substantial (especially of pesticides), and it grows apace. The scientic basis is already there for technological advance; it all comes down to a question of investment. As mentioned earlier, the development of bioassay techniques is one important aspect of biomarker technology. Cell lines have been developed for species of interest in ecotoxicology, for example, birds and sh (Pesonen et al. 2000), and have some- times been genetically manipulated (e.g., with incorporation of receptors and reporter genes) to facilitate their employment as biomarker assays (Walker 1998b). In principle, it should be possible to conserve the activities of enzymes concerned with detoxication and activation in these cellular systems so that the toxicokinetics of the in vitro assay are comparable to those of the living animal. Bioassays with such cellular systems could be developed for species of ecotoxicological interest that are not available for ordinary toxicity testing, which would go some ways in overcoming the fundamental problem of interspecies differences in toxicity. One difculty encountered with cell lines has been that of gene expression. Enzymes concerned with detoxication or activa- tion have sometimes not been expressed in cell systems. However, work with geneti- cally manipulated cell lines has begun to overcome this problem (Glatt et al. 1997). Interest has been expressed in the possibility of using biomarker assays as a part of risk assessment for regulatory purposes, and some workers have suggested tiered testing procedures that follow this approach (see, for example, Handy et al. 2003). It is to be hoped that regulatory schemes, such as that of REACH (see European Union 2003), will be sufciently exible to incorporate new assays and testing strategies as the science advances. 17.4 THE DESIGN OF NEW PESTICIDES It is not surprising that many of the organic pollutants that have caused environmental problems have been pesticides. Pesticides are designed with a view to causing dam- age to pests, and selectivity between pests and other organisms can only be achieved to a limited degree. In designing new pesticides, manufacturers seek to produce compounds of greater efcacy, cost effectiveness, and environmental safety than are offered by existing products (for an account of the issues involved in the develop- ment of new, safer insecticides, see Hodgson and Kuhr 1990). Sometimes the driving © 2009 by Taylor & Francis Group, LLC Organic Pollutants 325 force behind pesticide innovation is to overcome a developing resistance problem when existing products become ineffective against major pests. It may also be to pro- vide a product that is more environmentally safe than those currently on the market. Innovation, however, is to some extent hampered by escalating costs—not least, the costs associated with ecotoxicity testing and environmental risk assessment. With the rapid growth of knowledge in the eld of biochemical toxicology, it is becoming increasingly possible to design new pesticides based on structural models of the site of action—the QSAR (Quantitative Structure–Activity Relationship) approach (see Box 17.1). Sophisticated computer graphic systems make life easier for the molec- ular modeler. The discovery and development of EBI fungicides as inhibitors of certain forms of P450 provide an example of the successful application of this approach. There has also been rapid growth in understanding of the enzyme systems that metabolize pesticides and other xenobiotics (see Chapter 2 of this book, and Hutson and Roberts 1999). As more is discovered about the mechanisms of catalysis by P450-based monooxygenases, esterases, glutathione-S-transferases, etc., it becomes easier to predict the routes and rates of metabolism of pesticides. In principle, it has become easier to design readily biodegradable pesticides that have better selectivity than existing products (there are large species differences in metabolism that can be exploited). It should also be possible to design pesticides that not only overcome resistance but are also selectively toxic toward resistant strains. BOX 17.1 QUANTITATIVE STRUCTURE–ACTIVITY RELATIONSHIPS (QSARS) There has long been an interest in mathematical relationships between chemi- cal structure and toxicity, and the development of models from them that can be used to predict the toxicity of chemicals (see Donkin, Chapter 14 in Vol. 2 of Calow 1993). If considering groups of compounds that share the same mode of action, much of the variation in toxicity between different molecules is related to differences in cellular concentration when the same dose is given. In other words, toxicokinetic differences are of primary importance in determining selective toxicity (see Chapter 2, Section 2.2). The simplest situation is repre- sented by nonspecic narcotics, which include general anesthetics. Toxicity here is related to the relatively high concentrations that the compounds reach in biological membranes, and is not due to any specic interaction with cel- lular receptors (see Chapter 2, Section 2.3). Simple models can relate chemical properties to both cellular concentration and toxicity. Good QSARs have been found for narcotics when using descriptors for lipophilicity such as log K ow . For example, the following equation relates the hydrophobicity of members of a group of aliphatic, aromatic, and alicyclic narcotics to their toxicity to sh. log 1/LC 50 = 0.871 log K ow − 4.87 (Konemann 1981) Other much more toxic compounds operating through specic biochemical mechanisms (e.g., OP anticholinesterases) cannot be modeled in this way. If © 2009 by Taylor & Francis Group, LLC 326 Organic Pollutants: An Ecotoxicological Perspective, Second Edition Also of continuing interest is the identication of naturally occurring compounds that have biocidal activity (Hodgson and Kuhr 1990, Copping and Menn 2000, Copping and Duke 2007). These may be useful as pesticides in their own right, or they may serve as models for the design of new products. Examples of natural prod- ucts that have already served this function include pyrethrins, nicotine, rotenone, plant growth regulators, insect juvenile hormones, precocene, avermectin, ryano- dine, and extracts of the seed of the neem tree (Azadirachta indica) (see Copping and Duke 2007, Otto and Weber 1992, and Hodgson and Kuhr 1990). It is likely that natural products will continue to be a rich source of new pesticides or models for new commercial products in the years ahead. A vast array of natural chemical weap- ons have been produced during the evolutionary history of the planet, and many are still awaiting discovery (Chapter 1). 17.5 FIELD STUDIES Ecotoxicology is primarily concerned with effects of chemicals on populations, communities, and ecosystems, but the trouble is that eld studies are expensive and difcult to perform and can only be employed to a limited extent. In the main, environmental risk assessment of pesticides and certain other chemicals has to be toxicity were to be plotted against log K ow , such compounds would be repre- sented as “outliers” in relation to the straight line provided by the data for the narcotics (Lipnick 1991). Their toxicity would be much greater than predicted by the simple hydrophobicity model for the narcotics. For such compounds, more sophisticated QSAR equations are required that bring in descriptors for chemical properties relating, for example, to their ability to interact with a site of action. An example: of such an equation relates the properties of OPs to their toxicity to bees (Vighi et al. 1991): log1/LD 50 = 1.14 log K ow − 0.28 [log K ow ] 2 + 0.28 2 X − 0.76 2 Xox − 1.09Y3 + 0.096[Y3] 2 +12.29 where X and Y are chemical descriptors for reactivity with the active site of cholinesterase. The K ow is for the active “oxon” form of an OP. In general, it is easier to use models such as these to predict the distribu- tion of chemicals (i.e., relationship between exposure and tissue concentration) than it is to predict their toxic action. The relationship between tissue concen- tration and toxicity is not straightforward for a diverse group of compounds, and depends on their mode of action. Even with distribution models, however, the picture can be complicated by species differences in metabolism, as in the case of models for bioconcentration and bioaccumulation (see Chapter 4). Rapid metabolism can lead to lower tissue concentrations than would be pre- dicted from a simple model based on K ow values. Thus, such models need to be used with caution when dealing with different species. © 2009 by Taylor & Francis Group, LLC Organic Pollutants 327 accomplished by other means. With the registration of pesticides, large-scale eld studies are occasionally carried out to resolve questions that turn up in normal risk assessment (Somerville and Walker 1990) but are far too expensive and time con- suming to be used with any regularity. Lack of control of variables and the difculty of achieving adequate replication are fundamental problems. However, the develop- ment of new strategies, and the development of new biomarker assays could pave the way for more informative and cost-effective investigations of the effects of pollutants in the eld. Small-scale eld studies and semi-eld studies are used for risk assess- ment of pesticides to bees (Thompson and Maus 2007). That said, long-term case studies of pollution by chemicals can give important insights into problems with other similar chemicals that may arise later on. Cases in point include long-term studies that have been carried out on persistent lipophilic compounds such as OC insecticides, PCBs, organomercury compounds, and organo- tin compounds, which have been described in this second section of this book. With the advance of science, results from well-conducted eld studies can be looked at retrospectively to gain new insights—with the benet of hindsight. In the nal analy- sis, the natural environment is too complex to just make simple predictions with laboratory-based models, and there is no adequate substitute for hard data from the real world. It is important that long term in-depth studies of pollution of the natural environment continue. The use of biotic indices in environmental monitoring is one way of identifying existing/developing pollution problems in the eld (see Chapter 11 in Walker et al. 2000). Such ecological proling can ag up structural changes in communities that may be the consequence of pollution. For example, the RIVPACS system can iden- tify changes in the macroinvertebrate communities of freshwater systems (Wright 1995). It is important that adverse changes found during biomonitoring are followed up by the use of biomarker assays (indicator organisms or bioassays or both) and chemical analysis to identify the cause. As noted earlier, improvements in biomarker technology should make this task easier and cheaper to perform. Biomarker assays can be used to establish the relationship between the levels of chemicals present and consequent biological effects both in controlled eld studies (e.g., eld trials with pesticides) and in the investigation of the biological conse- quences of existing or developing pollution problems in the eld. In the latter case, clean organisms can be deployed to both clean and polluted sites in the eld, and biomarker responses measured in them. Organisms can be deployed along pollu- tion gradients so that dose-response curves can be obtained for the eld for com- parison with those obtained in the laboratory. An example of this approach was the deployment of Mytilus edulis along PAH gradients in the marine environment and the measurement of scope for growth (Chapter 9, Section 9.6). The challenge here is to take the further step and relate biomarker responses to population parameters so that predictions of population effects can be made using mathematical models. The predictions from the models can then be compared with the actual state of the populations in the eld. The validation of such an approach should lead to its wider employment in the general eld of environmental risk assessment. © 2009 by Taylor & Francis Group, LLC 328 Organic Pollutants: An Ecotoxicological Perspective, Second Edition 17.6 ETHICAL QUESTIONS There has been growing opposition to the use of vertebrate animals for toxicity testing. This has ranged from the extremism of some animal rights organizations to the reasoned approach of the Fund for the Replacement of Animals in Medical Experiments (FRAME), and the European Centre for the Validation of Alternative Methods (ECVAM) (see Balls, Bridges, and Southee 1991, issues of the jour- nal ATLA, and publications of ECVAM at the Joint Research Centre, Ispra, Italy). FRAME, ECVAM, and related organizations advocate the adoption of the principles of the three Rs, namely, the reduction, renement, and replacement of testing proce- dures that cause suffering to animals. Regarding ecotoxicity testing, these proposals gain some strength from the criti- cisms raised earlier to existing practices in environmental risk assessment. There is a case for making testing procedures more ecologically relevant, and this goes in hand with attaching less importance to crude measures of lethal toxicity in a few species of birds and sh (Walker 1998b). The savings made by a substantial reduction in the numbers of vertebrates used for “lethal” toxicity testing could be used for the development and subsequent use of testing procedures that do not cause suffering to animals and are more ecologically relevant. Examples include sublethal tests (e.g., on behavior or reproduction), tests involving the use of nondestructive biomarkers, the use of eggs for testing certain chemicals, and the renement of tests with meso- cosms. Rigid adherence to xed rules would prolong the use of unscientic and outdated practices and slow down much-needed improvements in techniques and strategies for ecotoxicity testing. Better science should, for the most part, further the aims of the three Rs. 17.7 SUMMARY With the restrictions and bans placed in developed countries on a considerable num- ber of environmental chemicals—especially on persistent and/or highly toxic pesti- cides—many serious pollution problems have been resolved and more attention has come to be focused on the effects of mixtures of organic pollutants, often at quite low concentrations. It has been argued by some that chemical pollution should be seen as part of stress ecology, that chemicals should be considered together with other stress factors to which free-living organisms are exposed. While this trend has been marked in developed countries, it has not necessarily been true of other countries where there is less control of environmental pollution by chemicals, and there are still some serious problems with certain organic pollutants. With improvements in scientic knowledge and related technology, there is an expectation that more environmentally friendly pesticides will continue to be intro- duced, and that ecotoxicity testing procedures will become more sophisticated. There is much interest in the introduction of better testing procedures that work to more ecologically relevant end points than the lethal toxicity tests that are still widely used. 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As mentioned earlier (Chapter 14), standard © 2009 by Taylor & Francis Group, LLC 322 Organic Pollutants: An Ecotoxicological Perspective, Second Edition environmental. biochemical mechanisms (e.g., OP anticholinesterases) cannot be modeled in this way. If © 2009 by Taylor & Francis Group, LLC 326 Organic Pollutants: An Ecotoxicological Perspective, Second Edition Also. Taylor & Francis Group, LLC 328 Organic Pollutants: An Ecotoxicological Perspective, Second Edition 17. 6 ETHICAL QUESTIONS There has been growing opposition to the use of vertebrate animals for