what is meant by validation of predictions based on laboratory toxicity tests?

Hydrobiologia 137: 271 - 278, 1986 O Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands

What is meant by validation of predictions based on laboratory toxicity tests?

John Cairns, Jr. University Center for Environmental Studies and Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U S A .

Keywords: toxicity testing, bioassays, predictions, validation, hazard assessment

Abstract

For years, estimates of hazard to aquatic ecosystems resulting from exposure to toxic chemicals have been made from laboratory toxicity tests without substantive validation of their accuracy in natural systems. The mere absence of gross damage (e.g., fish kills) does not constitute scientifically justifiable validation. The validation process should be systematic and orderly. It should also make provision for confirmation at more than one level of biological organization. A series of validation proposals are included in this article.

Introduction

The term validation (or confirmation of predictions of responses in natural systems generally to potentially toxic chemicals) based on laboratory generated data is regularly used in environmental hazard evaluation literature, but the word appears to mean many things to many people. It is particularly important that we each communicate exactly how we are using the term since it is unlikely that a consensus on what constitutes validation will be reached in the near future. This is particularly important because of two general usages of the term that are vastly different.

There are those who believe that by carrying out laboratory tests on the most sensitive species their results will be validated if no deaths of indigenous species in natural systems are recorded after exposure to concentrations at the no-observable- effects level or less. There may have been no- observed effects on the indigenous species because: (1) field studies were inadequate because they co- vered only a limited number of species or a limited number of responses, (2) an application factor was used that reduced the concentration so strikingly that adverse biological effects became highly im- probable, or (3) the chemical fate (and often bio-

availability) and effects in the natural system were quite different from those in the laboratory test system, thereby reducing either the chemical concentration or the effects well below those studied in the laboratory (the reverse could also be true).

Others feel that validation requires a high correspondence between the results in the laboratory and in the field. That is, the response threshold in both systems should be quite similar, and the probability of harm resulting from exposure to various concentrations should be predicted with precision. The important issue in validation is the ability to predict the relationship between the response of the artificial laboratory system and the natural system.

Natural system validation of predictions based on laboratory evidence

Although the field of aquatic toxicity testing might be traced as far back as Aristotle (who trans- ferred fish from freshwater to sea water and observed them), its real development began shortly after World War 11. Even so, it might appear rather odd that scientifically justifiable validation of predictions based on single species laboratory tests has not been a major activity in the history of the

field and is not carried out to any significant extent today, although much more attention is now being given to this problem. One reason may be that the assumptions (e.g., that single species can be used to predict responses at higher levels of biological organization) are not explicitly stated, and another reason is that the people in the field have been so busy fighting emergency situations and disasters that the philosophical underpinnings did not re- ceive the attention they deserved. In addition, checks could be made on the accuracy of laboratory toxicity tests in the sense of the ability to repli- cate them within a single laboratory or among a group of laboratories. Presumably, testers thought that if the numbers generated were accurate in the sense of being repeatable and the error quantifia- ble, the predictions based on them, even for much more complex systems, were sound. Furthermore, people who were comfortable making the laboratory tests were generally not comfortable in the field and vice versa. The National Research Council report (1981) mentions that single species tests would be of greatest value if used in combination with tests that can pi-ovide data on population interac- tions and ecosystem processes. More important, the Executive Summary states 'research and development should be directed toward designing and validating test systems and procedures that will de- tect changes in ecosystem and population attributes.' The National Research Council intended only to examine the adequacy of the present strategy for testing for effects of chemicals on ecosystems, it did not provide a detailed discussion of the validation process. Since I consider toxicity tests to be one of the key elements in predicting the hazard to ecosystems of various chemicals appearing in them (or likely to be introduced into them), the question of the form validation should take deserves considerable attention. It is worth em- phasizing, that despite the drawbacks of toxicity tests, there is no instrument devised by man that will directly measure toxicity! Toxicity tests are the only method available. Living material must be used for this purpose because analyzing a chemical concentration will not give an accurate prediction of toxicity since species respond differently to the same concentration of a chemical, and even the same species will respond differently to the same concentration if environmental conditions are not similar. Therefore, knowing chemical concentrations is essential, but these measurements, however

precise, are not of themselves adequate predictors of toxicity. There is no substitute for living material in a toxicity test, and, therefore, it behooves us to learn how to use the living material well.

The most sensitive species

It seems to me that the only justification for at- tempting to find 'the most sensitive species' for toxicity tests is the perceived inability to predict the response of organisms in natural systems from laboratory evidence. If this response could be accurately predicted, it would not really matter whether the organisms in the laboratory tests were more or less sensitive than the indigenous organisms because the response of the latter could be accurately predicted and regulatory strategy based on this response. Therefore, the only justification for using 'the most sensitive species' is the conviction that extrapolations from laboratory to field results cannot be made with precision and that one must incorporate the safety factor into the toxicity test itself rather than carrying this out as a separate operation. Ideally, the determination of the probability of harm should be a scientific exercise requiring probablistic evidence and the determination of how much risk to take (i.e., degree of safety) is a social judgment based on an entirely different set of values and practices (e.g., Cairns, 1980). The deci- sion process would be vastly improved if these two activities could be separated. If they were, it would make no difference whether the test species was more or less sensitive than the indigenous species in the receiving system as long as the response of the latter groups could be accurately predicted from the response of the test species.

One might argue that the incorporation of a very large safety factor (by using the most sensitive species) is a good thing because risks are reduced to practically nothing, and this is justifiable whatever the costs. However, industrial repr~sentatives and, increasingly of late, society as a whole resent spend- ing money for additional waste treatment when no evident biological benefits can be realized. More important, the determination of acceptable risk is not the sole prerogative of scientists but should in-- volve citizens or their representatives making deci- sions based on scientific evidence that is communi- cated effectively.

Single species vs multispecies toxicity testing

In a recent issue of BioScience, Weis (1985) argued that community tests were not worthwhile because the use of the most sensitive species automatically protects the entire community because they 'may be the most sensitive if one chooses the species and parameters carefully.' Unfortunately, abundant evidence is contrary to this idea (Cairns, in press). For example, Niederlehner et al. (1985) demonstrated that protozoan colonization rates from a species pool epicenter were affected by 1 pg Cd I-' although the species on the epicenter itself (i.e., the source pool) exposed to 9.5 pg Cd 1k' were not significantly different from the control in several important characteristics. In short, an ecologically important process (e.g., colonization) was dramatically altered at a concentration 'safe' to the survival of the species in the original community. The important question is could the effect of cadmium on colonization rate have been predicted from other evidence, particularly the response of the most sensitive species? In the case of the protozoan community just mentioned, it could not have despite the fact that a very large array of species in quite different taxonomic groups within the Protozoa were represented. In this particular case, a critic might say they were only examined to see if they were alive (i.e., presence, absence data). This is precisely the point. How many more tests would one have to carry out on the 'most sensitive species' to find that end point that responded at the same threshold concentration as the colonization rate? It is precisely because we cannot make assumptions of the type made by Weis (1985) that the validation process must be explicitly stated. Those areas where one may have confidence will then be identified, as will the areas where the outcome is less certain.

Practically all toxicity tests carried out since they came into use as management tools in the late 1940s and early 1950s have been based on single species. Although this is now changing, only rarely does one encounter a multispecies toxicity test being used by an industry or a regulatory agency. The professional literature, however, which only a few years ago rarely had an article on multispecies testing, now has many such articles (e.g., Bishop et al., 1983). The theoretical importance of multispecies aggregations has been advanced by O h m (1953) for many years. At each level of biological organi-

zation, proceeding from subcellular through cell, tissue, whole organism, population, community, and ecosystem, new properties are added that cannot be effectively studied at lower levels of organization.

Even when multispecies tests are used, they are often at the end of a protocol (Dickson et al., 1979). There are compelling economic, as well as scientific, reasons for carrying out multispecies toxicity testing (Cairns, 1983a). It is worth noting that as of the time this is being prepared, no formally endorsed standard methods (e.g., those of the Ameri- can Society for Testing and Materials or in Stan- dard Methods for the Evaluation of Water and Wastewater for multispecies toxicity testing exist. However, a number of provisional methods appear in the symposium jointly sponsored by the Ecologi- cal Society of America and the Society for En- vironmental Toxicology and Chemistry on Mul- tispecies Toxicity Testing (Cairns, 1985). Additional provisional methods can be found in Cairns et al. (1985) and Cairns & Pratt (1985).

The validation process

With these problems in mind, I have designed an illustrative three-level validation process for a variety of circumstances, including the use of both 'sensitive species' and indigenous species. In all cases, the main focus is on the reliability of predictions of responses in natural systems from the .use of surrogate species or indigenous species in test systems that frequently lack environmental realization. The intent of this discussion is to focus attention on the validation problem and not to provide detailed methodologies for carrying out validation or confirmation. However, for those wishing some illustrative material on methodology, Cairns & Cherry (1983) provide an example of validation at the single species level of biological testing. A comparison between species richness and colonization rates in terms of their sensitivity to cadmium can be found in Niederlehner et al. (1985).

A series of commonly, and not so commonly, used predictors of toxicity follow with some consideration to how they might be validated.

I. Toxicity tests where the test species is not resi- dent in the ecosytem into which the waste is being discharged or introduced

Level 1. At the very least, validation should demonstrate that an important indigenous species in the receiving system has a response threshold reasonably close to or more tolerant than the test species. The importance could be determined on an ecological, recreational, or commercial basis. If the waste discharge is from an industry, validation might take the form of field studies of an important indigenous species. If the test is for a chemical of more widespread use, such as agricultural chemicals likely to enter as non-point source discharges, one might carry out the validation in microcosms, mesocosms, or field enclosures with a reasonable array of species likely to be exposed. Presumably, the results would be more persuasive if end points other than lethality, such as reproductive behavior and success, growth, or respiration, were used. Level 2. Demonstration that an array of species from a variety of taxonomic groups and trophic levels indigenous to the receiving system do not suffer deleterious effects (e.g., lethality, reproductive, or growth) at no-effect concentrations predicted by the laboratory test. One might use a primary pro- ducer, a detritus processor, a grazer, and a carni- vore for this purpose. If a point source discharge is available, the validation in a natural system should not be difficult. If not, a surrogate system should be used. Level 3. One might use an attribute, such as energy flow, nutrient cycling, or some other process involving more than one species, as confirmation that the protection included attributes at the higher levels of biological organization than single species. There will be substantial increased costs as one goes to the higher levels of validation.

11. Use of an indigenous toxicity testing species thought to be 'representative' or 'sensitive'

Level 1. Determination in the natural system or a surrogate of the natural system that the species responds the same way in a more complex natural system as it does under laboratory conditions.. If there is an existing point source discharge, the validation should be relatively simple (e.g., Cairns & Cherry, 1983). If no natural study site is available,

a surrogate system should be used with adequate ecological complexity.

Levels 2 and 3. Demonstration similar to that required for the non-indigenous test species as described above.

III. Validation of results from a microcosm or mesocosm designed to simulate ecologically important characteristics of a particular ecosystem

Level 1. Presumably, each microcosm or mesocosm would simulate one or more important attributes of the ecosystems. Therefore, direct validation of a laboratory microcosm could easily be carried out in the area of a point source discharge by using the same end points used in the laboratory microcosm tests. This is a determination of correspondence very similar to that used for single species; the only difference being that this is a higher level of biological organization or a more complex test system. If there is no discharge and one has to be simulated, a field enclosure could be used in the particular lake or other ecosystem for which the prediction has been made. As was the case in the earlier illustrative validations, if the correspondence between the laboratory results and the field results is good, the results of the laboratory tests can be considered validated. If there is an ecologically, as well as a statistically, significant difference, the factors causing this difference should be identified. These factors should be included in the microcosm tests if at all possible and new predictive tests should be run. At the very least, there should be some evidence that the explanation for the differences in results between the laboratory and natural system is credible.

If a field enclosure in a natural system is used and there is a point source discharge or an area where the non-point source runoff is higher than the rest of the ecosystem, validation can be carried out as already described using the same end points in both the natural and the artificial system. If there is no entry of the chemical into the ecosystem, provisional validation can probably be achieved by using a series of field enclosures in another part of the ecosystem. As was the case in earlier illustrations, if the correspondence is high, one can consider that an acceptable provisional validation has been produced. If the correspondence is not partic-

ularly good, some explanation for the cause of the differences should be developed and some means of experimentally confirming that the factors identified were in fact causing the differences in results would be most helpful.

Level 2. As was the case in earlier illustrations of Level 2, end points characteristic of whatever level of biological organization is being studied, in addition to the ones actually used as end points in the laboratory system or field enclosure, should be used. In short, the prediction of 'safety' for other attributes than the ones directly measured should be determined. Level 1 validation merely deter- mines that the attributes measured are responding as predicted in a natural system. For Level 2 validation, one should determine that attributes at the same level of biological organization other than those measured directly in the test system do not suffer deleterious effects in the natural system. Level 3. As was the case with the preceding illustrations, Level 3 validation should consist of show- ing that levels of biological organization other than those for which end points were developed in the test system are protected. In a sense, this validation is intended to confirm or falsify the assumption or hypothesis that the end point chosen (or end points chosen) was such a key attribute of the natural system that all other important attributes, or a high percentage of them, were also protected.

Acceptance of a toxicity test as a standard method

If a toxicity test involving aquatic organisms cannot always be accurately used to predict some important events in natural systems, running such a test may conform to legal requirements but the exercise has nothing to do with protecting natural systems. Evidence of the quality and extent of the predictions should be a part of the requirements for acceptance as a standard method. The term standard method is used here to include those legally recognized as such and, more important, with a recognized system of professional endorsement involving a significant number of scientists from a variety of organizations. For example, in North America, formally endorsed toxicity tests appear in Standard Methods for the Examination of Water and Wastewater and in various publications of the

American Society for Testing and Materials. Stan- dard Methods is produced by a series of highly qualified scientists selected by three prestigous, professional organizations with no particular regulatory or industrial bias. The American Society for Testing and Materials produces standard methods by consensus in which a single dissenting vote out of literally thousands of eligible voters can halt the progress of the standard method. In this latter instance, the person who votes negatively must provide an indication of the basis for the negative vote, and this is then judged persuasive or non-persuasive by the group. In Europe, such organizations as the European Inland Fisheries Advi- sories Commission serve a similar purpose. I have not discussed details of the OECD because much additional space would be required, and the validation problem appears to be quite similar although the genesis of the problem is not. Although toxicity tests espoused by regulatory agencies such as the US. Environmental Protection Agency are some- times referred to as standard methods or required methods, they are not subjected to anything ap- proaching the objective impartial, unbiased analysis of the groups cited. It is my opinion that putting bureaucratic convenience ahead of impartial, objective, scientific analysis should not be permitted and that regulatory agencies should not be allowed to use toxicity test methods not given at least provisional approval as a standard method. Giving the methods the aura of scientific sanctity that is justifiably bestowed following review in the larger academic community should not be permitted.

The validation evidence accompanying the standard method through the formal approval process should be explicit and detailed. The evidence on validation will delimit the conditions under which the tests can be used and document the precision of the predictions made from it. This is something badly needed in the toxicity testing standard methods publications because, even though the test may be carried out with great precision and high replicability, the extrapolations made from it to natural systems may be totally erroneous. The pity is that with the present system these errors in estimating environmental response to toxic materials go largely undetected unless the response is strik- ing. Subtle responses are probably missed mainly due to lack of widespread monitoring of structural and functional attributes of complex systems.

It will almost certainly be argued that the validation process is both more costly and more difficult than the toxicity test itself, particularly when the latter involves acute single species tests. Without question, the cost of the total package will be at least double the cost of the single species laboratory toxicity test. On the other hand, the value of the information generated will be enormously increased because one will have firm evidence on the amount of confidence one might place in the predictions based on the laboratory toxicity test. Kimerle (1979) has shown that extrapolations made from clean water laboratory evidence to the real world situations may be greatly in error in either direction. In other words, the toxicological effect may be either over- or underestimated for a complex series of reasons. Since Kimerle is employed as a research scientist by Montsano Corporation, it is clear that industry considers predictive accuracy important. The reason for this is quite clear since, if the toxicity effects are grossly overstated, industry will be forced to spend substantial sums of money for treatment costs that result in no demon- strable biological benefits. Conversely, industries might be responsible for a fish kill even while con- forming to the law if the toxic effects are understat- ed by the laboratory tests. Therefore, if one places the increased dost of toxicity testing due to validation in the perspective of the economic conse- quences of acting on inaccurate information, the additional cost of validation does not seem nearly as important as it does when one compares the validation cost with the cost of carrying out the toxicity test itself.

Most organizations that generate standard methods have provision for periodic updating and review of these methods. Experience with validation could be updated at the same time as the methodology itself. In this process, some attention should be given to the quality of the new information, especially in cases where conflicting results are obtained. For example, evidence appearing in a quality, peer reviewed professional journal should obviously be given more weight than evidence from the unreviewed, limited circulation 'gray' literature.

Interpreting the results of validation studies

Except for the simple cases of validation (e.g.,

Cairns & Cherry, 1983) where the same species is studied in both laboratory and natural systems and all studies are carried out at a single level of biological organization, considerable scientific judgment will be required in the intepretation of results. Even the next simplest case, that is the extrapolation from one species to another (the same level of biological organization), the characteristics used may appear similar on first glance but, on closer inspec- tion, may not be. For example, is a 50% reduction in cell division rate for diatoms equivalent to a 50% death rate in an acute fish toxicity test? If one can predict one event from the other with considerable accuracy, it does not matter, in terms of the deci- sion being made, whether they are similar or dis- similar. If there is considerable variability, one might reasonably wonder whether the extrapolation is justified. When one ventures into extrapolation from one level of biological organization to another (e.g., from single species to community) even more judgment is required. There is good reason to suspect that some of the previously widely held assumptions about predictions of complex system response from that of single species may not be valid. Another major problem in the validation process for widely used toxicity tests (i.e., those intended for use in the contigous United States) is the problem of biological species. As a matter of convenience, most species are identified by predominantly taxonomic characteristics. There is evidence that two sets of test organisms may have similar taxonomic characteristics (i.e., are structure- ly similar) but may be significantly different phys- iologically. This may cause problems when ex- trapolating from laboratory to field with the same species if the laboratory stock is from a different source than the field stock - a not uncommon sit- uation.

Discussion

Undoubtedly, the process of validation will pro- duce some very unpleasant surprises when some of the latest methods fail to justify their original promise and some of the older methods are found to be lacking in performance. On the other hand, some very pleasant surprises may surface as methods perform better than expected. The study cited earlier (Cairns & Cherry, 1983) was one such

instance: the correspondence between laboratory and field results was extraordinarily good, despite the fact that the environmental realism of the laboratory tests was not particularly high in terms of habitat replication, although it was in water quality replication. It is nearly platitudinous to state that the predictive capability of toxicity tests will never be what it can be unless extensive and thoughtful validation is carried out. Nevertheless, there are those who resist this process and claim that environmental protection will be adequate if the results of the present toxicity test are used carefully. A number of arguments are against this stand, which is based almost entirely on anecdotal or circumstantial evidence rather than rigorous scientific evidence. The most highly probable reason for this 'success,' if it is indeed a fact, is the over-protection resulting from use of an application factor that reduces the acceptable concentration an order of magnitude or more. This protects the environment but at the cost of expenditure of industrial waste treatment funds for little or no biological benefits. Almost certainly, this has occurred fairly frequently, and the capital costs alone for the improved waste treatment systems are usually more than an order of magnitude greater than the cost of more extensive biological testing, including validation. The second possibility, also probably occur- ring more frequently than we would care to admit, is that the catastrophes are unobtrusive. For example, the phenomenon of egg shell thinning caused for certain species of birds by DDT was neither predicted by laboratory tests nor immediately identified directly from field observations. Populations of these birds were in decline, and the egg shell thinning was eventually found as a consequence of the intensive effort to determine the reason for the decline. However, this particular environmental catastrophe was only noticed because most of the species of birds involved are 'highly visible' to hu- mans, and a number of bird watchers, both ama- teur and professional, keep careful track of their abundance, population recruitment, and the like through annual censuses, etc. If the same population decline occurred in aquatic systems involving unloved and generally unobserved organisms (but nevertheless extremely important in processing detritus, energy flow, and the like), it is highly im- probable that such catastrophes would be documented or even noticed. One can state that ab-

sence of a function or even absence of a group of relatively obscure or ecologically important organisms would not be quickly noticed because invad- ing species have not usually been noticed until they cause trouble or appear at a research site. We found the Asiatic clam Corbicula flurninea in some experimental continuous flow throughs adjacent to the New River at Glen Lyn, Virginia (Cherrry et al., 1980). This organism would probably not have been noticed in the initial stages of the invasion had these experimental systems not been in operation. Eventually, industrial cooling systems in nearby industries were clogged to the point of becoming a major obstacle to their operation. Such an important presence could have gone unremarked for a number of years had it not been discovered purely by accident in experimental systems. Surely the loss of a few species or an important functional characteristic of an ecosystem would be even more likely to go undetected in most situations since research sites in continuous operation are not abundant. Therefore, the argument that no catastrophes have resulted is based on extremely shaky evidence rather than scientific proof.

Even if the validation process shows the toxicity testing methodology to have some significant flaws, it is virtually certain that the alternative methods of controlling environmental impact are going to be vastly more seriously flawed. The two most prominent alternative methods (in fact, for some years, primary methods) in the United States are: (1) technology-based standards and (2) chemical standards. These alternatives are discussed at length in Cairns (1983b). However, the assumption that installing the best applicable or the best prac- ticable technology will automatically protect the ecosystem never deserved serious consideration because it totally ignored the biological response in the receiving system, which is the ultimate criterion for success. In short, this approach was never validated with biological evidence (in fact, it was designed to avoid gathering such evidence) but, had it been there, would undoubtedly have had serious flaws, particularly in overtreatment for industries located on large streams and undertreatment for industries located on small or very fragile aquatic ecosystem. The second alternative, namely chemical standards, ignores the well established fact that only biological material can be used to measure toxicity and knowing the chemical concentration in

a particular ecosystem does not enable one to predict with precision the biological response. This is so well established in the literature that it hardly needs documentation here, but readers in search of such evidence can find much of the older information in Water Quality Criteria of 1972 and in a variety of recent publications. Therefore, to paraphrase George Bernard Shaw, toxicity testing may not be all we hoped for but the alternatives are unthinka- ble. Although the field of toxicity testing dates to Aristotle, it has only in the last 40 years, command- ed major attention. As a consequence, it is quite likely that the growth process will impair some cherished beliefs. Protecting the environment is a major responsibility that must not depend on predictions that are not validated!

Acknowledgments

I appreciate the comments of John Sprague on a portion of an early draft of this manuscript. I am indebted to Darla Donald, Editorial Assistant, for getting this manuscript ready for publication and to Angela Miller for typing it. The manuscript was written at Rocky Mountain Biological Laboratory during the summer of 1985.

References

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Received 18 October 1985; in revised form 11 March 1986; ac- cepted 23 March 1986.

what is meant by validation of predictions based on laboratory toxicity tests?

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