Organic Chemicals : An Environmental Perspective - Chapter 7 doc

Neilson, Alasdair H. "Ecotoxicology" Organic Chemicals : An Environmental Perspective Boca Raton: CRC Press LLC,2000 7 Ecotoxicology SYNOPSIS The basic input required for assessing the toxicity of xenobiotics is summarized and includes data both on the exposure to the toxicant and an evaluation of its biological effect in terms of numerically determined end points. A brief discussion is directed to the choice of test species, to the range of acceptable end points, and to the choice of media for laboratory tests. Some comments are provided on the commonly used tests using single organisms that include representatives of algae, crustaceans, and fish with emphasis on assays for sublethal effects. Brief descriptions are given of less widely used assays using rotifers, mayflies, tadpoles, and sea urchins. Assays for evaluating toxicity in sediments and toward terrestrial organisms are discussed, and procedures for detecting genotoxic and estrogenic effects are noted. A discussion is presented on multicomponent test systems including different types of mesocosms. Attention is directed to the important question of metabolism by the test organisms with emphasis on fish. The use of biomarkers is briefly discussed and includes application of both biochemical and physiological parameters. Some cautionary comments are given on the application of these to feral fish. Attention is briefly drawn to the application of toxicity-equivalent factors and the role of epidemiology. It is suggested that a hierarchical approach to evaluating toxicity could be used, and that assays at the higher levels are justified if no effects are observed at the lower ones. Any system should be flexible and should be able to incorporate studies of partition and additional factors relevant to a particular environment. Introduction Previous chapters have been devoted to the distribution and persistence of xenobiotics after discharge into the aquatic environment. This chapter is devoted to the effect of xenobiotics on aquatic organisms. Its depth and ori- entation should be clearly recognized: like Chapter 2 on analytical procedures, this chapter is not directed to the professional ecotoxicologist. The aim has been to provide an overview of the kinds of bioassays that are being used ©2000 CRC Press LLC ©2000 CRC Press LLC in environmental research and to indicate a few of the areas to which further attention might profitably be directed. No attempt has been made to provide protocols for standardized procedures, nor to indicate which of the many possible assay procedures are acceptable to the administrative authorities that issue discharge permits. Attention is directed to detailed presentation of procedures aimed at determining the ecological effects of xenobiotics on the structure of populations and communities (Petersen and Petersen 1989) and procedures for community testing (Landner et al. 1989). Toxicology may be defined as the science of poisons, and has traditionally been devoted to the effect of poisons on higher organisms including humans and domestic animals. The cardinal concepts are those of dose, which was introduced by Paracelsus in the 16th century, and the correlation of effect with the chemical nature of the toxicant promulgated by Mattieu Orfila in the 19th century. Toxicology is concerned with four interacting elements—the cause, the organism, the effect, and the consequence. The term ecotoxicology has been coined to include the effect of toxicants on biota both in natural ecosystems and in laboratory test systems: these may evaluate the effect on a wide spectrum of organisms ranging from bacteria through algae and crustaceans to vertebrates. In addition, attention has increasingly been directed to the application of a number of parameters in routine clinical use, and to their adaptation for use both in laboratory experiments and in evaluations using feral fish; for example, the levels of specific enzymes, morphological changes in organs such as liver, and blood parameters have been successfully used. The two approaches are not, of course, mutually exclusive. Humans are a predator of organisms at higher trophic levels so that, for example, the consumption of fish provides a mechanism whereby humans may be indirectly exposed to xenobiotics. In this case, the critical question therefore is the degree of contamination of fish by toxicants; the mechanisms whereby xenobiotics may be accumulated in biota have been discussed in Section 3.1, and the metabolism of these by higher aquatic biota will be reviewed briefly later in Section 7.5. There is an enormous literature on human toxicology from which many useful ideas applicable to ecotoxicology may be gleaned, and these can profitably be adapted with only minor modi- fication. In human toxicology, a number of basic problems have been extensively explored and these include: 1. The fundamental issue of the relation between the dose of a toxicant and the response elicited; 2. The vexatious question of the existence or otherwise of threshold concentrations below which toxicity is not displayed; 3. The statistical design of experiments in toxicology. It is appropriate therefore to make brief reference to some essentially popular accounts in which these are illustrated by readily understood examples ©2000 CRC Press LLC (Ottoboni 1984; Rodricks 1992) in addition to the substantial discussions presented in the classic text by Casarett and Doull (Klaasen et al. 1986). Most of the present discussion will be illustrated by examples from experiments with pure organic compounds, but it should be appreciated that the discharge of single compounds into the aquatic environment is exceptional: almost invariably the effluents consist of a complex mixture of compounds and these are generally evaluated as nonfractionated effluents or occasion- ally on the basis of the effect of their major components. The question of syn- ergistic or antagonistic effects therefore remains essentially unresolved. Toxicity equivalent factors (TEFs) have been used to provide an overall estimate of toxicity in situations where mixtures of compounds are present, and this is discussed again in Section 7.7.1. Briefly, the toxicity of each component is evaluated using a given test system and this value is multiplied by the concentration of that component in the mixture; these values are then summed to provide an estimate of the toxicity of the mixture. The problem of assessing effects on natural ecosystems is so complex that it is generally simplified to a greater or lesser extent: experiments may be con- ducted in the laboratory or outdoors in model ecosystems, and a plethora of single species have been used for assaying biological effects. These assays attempt to encompass various trophic levels, and differencies in physiology and metabolism. For example, representatives of algae are generally used to assess effects on photosynthesis and on primary production, crustaceans and fish may be used to evaluate effects on secondary producers, while communities may be used to explore interactions among components of natural ecosystems. In the final analysis, however, there are three well-defined stages in all test systems: 1. Exposure of the test organism(s) to the toxicant; 2. Evaluation of the effect(s) in terms of numerically accessible end points; 3. Analysis of the data to provide a single value representing the biological effect (toxicity). In the past, many industrial effluents were significantly toxic so that tests relying on acute toxicity to fish were routinely used: these usually involved exposure to the toxicant for a maximum of 96 h. Rainbow trout were traditionally used and the results were reported as LD 50 values. With increased demand for less toxic effluents before discharge into aquatic systems and increased appreciation of the complexity of ecosystem effects (Rosenthal and Alderdice 1976), assays for acute toxicity have gradually been replaced by considerably more-sophisticated test systems. A review has been given that discusses not only the broad mechanisms whereby PAHs exert their toxicity on aquatic organisms, but the cardinal issue of bioavailability (van Brum- melen et al. 1998). ©2000 CRC Press LLC It is desirable to distinguish clearly and appreciate the differences between the various terms; in this account, the following usage has been adopted: Acute implies that the organism does not survive the exposure and often—although not necessarily—implies a short-term exposure. It should be appreciated that organisms at various stages of development may be used, and that earlier stages will generally display greater sensitivity to the xenobiotic. Subacute or sublethal implies that the test organism survives exposure, but is nonetheless impaired in some specific way: a test may, for example, examine the effect of a toxicant on growth or reproduction. An old though stimulating review with valuable references to the basic literature is available (Sprague 1971). Chronic tests aim at examining the effect of prolonged exposure and will therefore of necessity examine subacute effects. Considerable ambiguity surrounds the application of the term chronic , and this has been carefully analyzed by Suter et al. (1987); the length of the exposure is not rigorously defined, but should probably be related to both the growth rate and the life expectation of the test organism. Life-cycle tests using, for example, fish, clearly represent a truly chronic assay. The introduction of the term subchronic and its use in the context of relatively short term tests lasting 7 days (Norberg and Mount 1985; Norberg-King 1989) seems therefore regrettable. Reproduction tests may be directed to a single generation or, especially for fish, to a complete life cycle: growth of fish from spawn to maturity followed by growth of the next generation. The term fecundity has been used for tests that examine early stages in the development of the test organism. There has been increased interest in the pathology of organisms exposed to xenobiotics, and tumor induction in fish is noted in Sections 7.3.3 and 7.5.1. Particular concern has been expressed over the effect of exposure to xenobiotics on the early life stages of fish, and this may be illustrated by investiga- tions with 2,3,7,8-tetrachlorodibenzo[1,4]dioxin. Fertilized eggs of lake trout ( Salvelinus namaycush ) were exposed for 48 h to the toxicant and then maintained in flowing water; subcutaneous hemorrhages developed in embryos and fry, and survivors displayed severe edemas and necroses (Spitsbergen et al. 1991). Broadly similar observations were made (Walker et al. 1992) when the toxicant was injected into the fertilized eggs of rainbow trout ( Oncorhyn- chus mykiss ). Injection of 4-chloroaniline into the fertilized eggs of zebra fish ( Brachydanio rerio ) resulted in delayed hatching and a large number of dose- related cytological alterations in the proximal renal tubule of 4- and 6-day old fish (Oulmi and Braunbeck 1996). The exclusive use of single-species test systems has been the subject of justified criticism (Cairns 1984), and it should be pointed out that fundamental ©2000 CRC Press LLC objections have been raised to the application of conventional bioassays to predictive assessment. A valuable critique has been provided (Maltby and Calow 1989) in which the limitations of conventional approaches were carefully delineated: it was suggested that the intrinsic limitations of inductive procedures make these of low predictive value except in restricted circumstances. Attention is directed to some general issues. 1. The biological level of the system used for assay may range from the subcellular to the individual to the population, and the interpretation of the results should take this into account. For example, tests at the subcellular level cannot consider the important issue of transport into the cell, and assays with individual organisms cannot validly be extrapolated to effects on populations. 2. The effect of a potential toxicant is a function of a number of factors: (a) the organism that is exposed and its trophic level, (b) its physiology and biochemistry including the capacity for repair or excretion, and (c) the stage in its life cycle. 7.1 Choice of Test Species in Laboratory Tests Broadly, three different philosophies may be adopted although these should not be regarded as mutually exclusive. 1. Internationally recognized organisms using standardized protocols may be used, and for judicial purposes may be obligatory. These procedures have the advantage of enabling comparison with extensive published data, although their relevance to a specific ecosystem may be restricted. 2. Alternatively, use may be made of indigenous species, in which case there can be no doubt of their relevance to the ecosystem from which they were isolated: standardized protocols must exist for these organisms also, and cloned cultures of taxonomically defined individuals should be used. An interesting example is afforded by the widespread European use of zebra fish ( Brachidanio rerio ) as a test organism. This is, of course, a tropical fish and it may reason- ably be questioned whether this is appropriate for application to the cold-water environment of northern Europe. A study using 3,4- dichloroaniline has revealed, however, that in early life-stage studies there was no significant difference between the results from tests using zebra fish and those using perch ( Perca fluviatilis ) that is widely distributed in Europe (Schäfers and Nagel 1993). This investigation alone clearly does not provide a nihil obstat to the use of zebra fish but provides valuable support for its widespread application in ecotoxicology. ©2000 CRC Press LLC 3. Feral organisms have sometimes been used for each test series and stocks of these have not been maintained in the laboratory. This is, however, a questionable procedure for at least three reasons: • Variations in the natural population may remain unnoticed. • Experiments may be restricted to particular seasons of the year. • In view of the virtual global dissemination of xenobiotics including potentially toxic organochlorines and PAHs, the test organisms may already have been exposed to background levels of such toxicants. It should be emphasized that the choice of test organism is not primarily determined by the requirement for maximum sensitivity to toxicants: indeed, extreme sensitivity may be a disadvantage if it results in problems of repeat- ability or reproducibility. On the other hand, it should be clearly appreciated that some groups of organisms may be significantly more sensitive than oth- ers to a given class of toxicant—that may have a common mode of action. For example, the substituted diphenyl ether pyrethroids permethrin, fenvalerate, and cypermethrin were generally more toxic to marine invertebrates than to marine fish, and among the former the mysid Mysidopsis bahia was the most sensitive (Clark et al. 1989). There exist also technical issues of considerable importance. 1. Some organisms such as strains of algae or cultures of crustaceans may be maintained in the laboratory as stocks: this has the advantage that putatively unaltered test strains are always available, but it is important that additional stress or selection is not imposed during maintenance. 2. Many species of fish may be available from commercial breeders, and this avoids extensive labor in keeping such stocks. On the other hand, absolute uniformity cannot be guaranteed and genetic variations are clearly possible. One possible danger results from the use of antibiotics by fish breeders to maintain healthy stocks free from microbial infection. 3. Relatively little attention has been directed to genetic variation within specific taxa. Examples of the care which should be exer- cised are provided by studies with the midge Chironomus tentans, the water flea Daphnia magna , and the viviparous fish Poeciliopsis monacha-lucida . a. Analysis of gel electrophoresis patterns of a number of glyco- lytic enzymes in different strains of C. tentans was used to assess a number of relevant genetic parameters including heterozy- gosity, percent polymorphic loci, and genetic distance within the populations (Woods et al. 1989). The observed variations between strains of the same organism from different sources ©2000 CRC Press LLC were considerable, and the results strongly underscore the critical importance of taking this into consideration in assessing the effects of toxicants on different populations of the same organism. b. Clonal variations in various strains of D. magna to cadmium chloride and 3,4-dichloroaniline were examined (Baird et al. 1990) in both acute and in 21-day life-cycle (chronic) tests. There were wide differences in results from the acute tests, particularly for cadmium chloride, although these were relatively small for the life-cycle tests; it was therefore concluded that different mechanisms of toxicity operate in the two test systems and this illustrates, in addition, the advantage of including both kinds of assay. c. Cytochrome P-450 CYP2E1 mediates dealkylaton of nitrosodi- alkylamines, and its activity among P. lucida-monacha hybrids varied markedly: values in liver microsomes ranging from 0.4 to 3.6 µg 6-hydroxychlorzoxazone/min/mg protein (Crivello and Schultz 1995). It should be noted that fish in the genus Poeciliopsis are unisexual, and that as many as 20 different “hemi- clones” have been identified in river systems. All these results not only reinforce the conclusions on the possible significance of genetic variations in test species within the same taxon, but also indicate the subtleties in expression of toxicity that may be revealed under different exposure conditions. A commendable development is therefore the assessment of the genetic structure in the mayfly Cloeon triangulifer that has been proposed as a suitable assay organism (Sweeney et al. 1993). Since the analysis of alloenzyme composition for enzymes representing different genetic foci is well developed and generally straightforward, more widespread application of this technique could profitably be made to other populations of organisms already used for bioassay. 7.2 Experimental Determinants There are a number of important experimental considerations which affect both the design of the experiments and the interpretation of the data, and some of the most important will therefore be briefly summarized. It should be noted that in order to display its effect the potential toxicant must be trans- ported into the cells and that the compound may then be metabolized. In assays for toxicity, no distinction can therefore be made between transport into the cell, toxicity of the compound supplied, or that of potential metabo- lites: these effects are assayed collectively and indiscriminately. The situation ©2000 CRC Press LLC has therefore certain features in common with that of bioconcentration, which are discussed in Section 3.1. Indeed, because of their close relation, it may be expedient to examine the potential for bioconcentration and bioaccu- mulation, metabolism and excretion, and toxicity in the same experiment . 7.2.1 Exposure Exposure conditions should simulate as far as possible those to which the organisms will be exposed in the environment that is being evaluated. Aquatic Organisms The most widely used exposure of the test organisms is to aqueous solutions of the toxicant prepared in media that are suitable for their growth and reproduction; this represents the situation for many potential toxicants, although attention is briefly drawn later to alternative procedures that have been applied to compounds with poor water solubility. Defined synthetic mineral media in deionized organic-free water are generally used for the sake of reproducibility and since this minimizes the possible ameliorating effect of organic components in natural waters. An interesting departure from the conventional practice of reporting toxicant concentrations in the test medium is provided by a study with sea urchin genetes and embryos in which esti- mates of toxicity were based on concentrations of the toxicant in the embryos (Anderson et al. 1994). This has also been used for terrestrial organisms that are discussed in Section 7.3.6. Different exposure protocols have been used: (1) static systems without renewal of the toxicant during exposure, (2) semistatic systems in which the medium containing the toxicant is renewed periodically during the test, and (3) continuous flow-through systems in which the concentration of the toxicant is essentially constant. In the first procedure—and to a lesser extent in the second—toxicant concentrations will not remain constant during exposure, and the range of exposure concentrations during the test could advan- tageously be reported. In any case, analytically controlled concentrations of pure compounds should be provided since some of them may be unstable under the conditions used for testing. Whereas exposure for a predetermined time is acceptable for experiments using crustaceans and fish, this cannot be done for growth tests using algae since an unpredictable lag phase may exist before growth commences. The length of the lag phase may provide useful information on adaptation to the toxicant, but growth must be measured during the whole of the test and into the stationary phase. A particularly troublesome problem arises with compounds having only low solubility in water, and this is particularly acute if the compound is only slightly toxic since high concentrations are then necessary to elicit a response from the test organism. Solutions of such compounds have been prepared in water-miscible organic solvents such as acetone, ethanol, dimethyl sulfoxide, or dimethylformamide, which are added to the test medium. Although the ©2000 CRC Press LLC toxicity of these solvents can readily be evaluated, a much greater uncer- tainty surrounds the true state of the toxicant: Is a true solution attained or merely a suspension of finely divided particles? It has also been shown that the acute toxicity of three xenobiotics toward a number of crustacean and rotifers was influenced by the organic solvent independently of the possible effect of the solvent alone (Calleja and Persoone 1993). It is therefore prefera- ble to use saturated solutions of the toxicant in the test medium: these may conveniently be prepared by passing the test medium through a column containing glass beads or other sorbants coated with the toxicant (Veith and Comstock 1975; Billington et al. 1988). Although this is the method of choice, preparation of large volumes for testing may be impractical although a design incorporating continuous flow has been developed and is clearly attractive (Veith and Comstock 1975). There is an additional problem that may be encountered with compounds that have extremely low water solubility: in an investigation on the uptake of highly chlorinated dibenzo[1,4]diox- ins by fish, it was found that the concentrations in saturated solutions prepared by this procedure exceeded the established water solubility of the octachloro congener (Muir et al. 1986). It was hypothesized that the compound was associated with low concentrations of dissolved organic carbon in the water, and that the very low BCF values that were measured could be the result of the poor bioavailability of the compound; this is equally relevant to the toxicity and is an issue to which further attention should be devoted. The organic C content of the water, although low, may be sufficient to medi- ate associations with low concentrations of toxicants. There are, however, situations in which organisms in natural systems may not be exposed to the essentially constant concentrations of the toxicant used in flow-through laboratory systems. This may be the case in quite different field situations: 1. Accidental spills that result in sudden and temporarily high concentrations of the toxicant; 2. Anadromous and catadromous fish that may be exposed temporarily to a plume of toxicant on their way to the spawning ground; 3. Nonstationary fish that are therefore exposed to varying concentrations of a toxicant. The question then arises of the extent to which any nonlethal effect is revers- ible after removal of the toxicant. The answer seems to be that in the few cases which have been examined, this may indeed be the case: all of them have examined phenolic compounds, one (McCahon et al. 1990) using the crustacean Asellus aquaticus , one assessing respiratory/cardiovascular effects on rainbow trout (Bradbury et al. 1989), and the third (Neilson et al. 1990) using the embryo/larvae assay with zebra fish ( Brachydanio rerio ). The last of these has been developed into a protocol that is modeled on the standard bioconcentration procedure in which a period of depuration is included after exposure to the toxicant. [...]... human breast cancer cell line MCF -7 transiently cotransfected with the Gal4-human ER anf and a Gal4responsive luciferase reporter (17m5-G-Luc) was used to analyze activities relative to 17 -estradiol in fractionated samples of urban air particulates Although activity was shown by benzo[a]pyrene, benzo[a]anthracene, and chrysene, this was only 1/1000 to 1/10,000 that of 17 -estradiol (Clemons et al 1998)... treatment plant effluents (Section 2.2.3) has been examined (Desbrow et al 1998) The results showed that the activity of 4-tert-octyl phenol was only from 1/100 to 1/1000 that of 17 -estradiol c A range of flavanoids was examined and their estrogenic potency was between 4000 and 4 million times lower than that of 17 -estradiol (Breinholt and Larsen 1998) 2 An estrogen-responsive human breast cancer cell... to as wide a range of toxicants as is realistic The systematic collection and availability of these basic data for all test organisms would be extremely valuable 7. 3 7. 3.1 Test Systems: Single Organisms Aquatic Organisms A wide range of organisms is available, and many of them have been extensively evaluated with structurally diverse organic compounds The choice of organism and the design of the study... results for one cold-water fish, one warm-water fish, and an invertebrate It is critically important to assess the significance of the differences in the response of various fish to the same toxicant: for example, how significant is ©2000 CRC Press LLC the difference between the 96-h acute toxicity of chlorothalonil (2,4,5,6-tetrachloro-1,3-dicyanobenzene) to channel catfish (52 µg/l) and to rainbow trout... exposure on filter paper was higher than for the other routes of exposure 7. 2.2 End Points Any parameter that can be assessed numerically may be used, and these are generally adapted to the test organism and to experimental accessibility An ©2000 CRC Press LLC interesting survey (Maltby and Calow 1989) of papers during the periods pre-1 979 and 1 979 to 19 87 using single-species laboratory tests revealed... concept of pollution-induced community tolerance (PICT) (Blanck et al 1988) whereby exposure to a toxicant results in the elimination of sensitive species and the dominance of tolerant ones: quantification is achieved by using short-term assays for the inhibition of photosynthesis under laboratory conditions (Blanck and Wängberg 1988) One of the attractive features of the system is that it can be used in... Chironomus including C tentans, C decorus, and C riparius have been quite extensively used (Nebeker et al 1984), and these organisms have been incorporated into microcosms (Fisher and Lohner 19 87) Two end points have been effectively used: adult emergence and growth of larvae (Nebeker et al 1988) using C tentans Both acute toxicity and effects on reproduction (Kosalwat and Knight 1987a,b) have been examined... and the different fish that have been used in early lifestage evaluations, it may be mentioned that the Californian grunion (Leuresthes tenuis) has been employed for assessing the toxicity of chlorpyrifos (O,Odiethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate (Goodman et al 1985) and pike (Esox lucius) for 2,3 ,7, 8-tetrachlorodibenzo[1,4]dioxin (Helder 1980) A truly chronic test using zebra fish and... Embryos and tadpoles of three species of frogs (Rana sylvatica, Bufo americanus, and R clamitans) were used to examine the toxicity of endosulfan in a 96-h assay (Berrill et al 1998) Tadpoles showed abnormal behavior including paralysis during exposure Postexposure mortality was greater for 2-week-old than for newly hatched tadpoles, and occurred at the lowest concentrations evaluated—68, 41, and 53... need for, and the effectiveness of, remediation are discussed briefly here All these assays are subject to the limitations noted for aquatic organisms in Section 7. 3.1, and, as for assays for sediment and soil toxicity (Section 7. 3.2), the importance of association with the organic matrix and bioavailability plays a cardinal role in the interpretation and evaluation of the results The important issues . all test organisms would be extremely valuable. 7. 3 Test Systems: Single Organisms 7. 3.1 Aquatic Organisms A wide range of organisms is available, and many of them have been extensively. "Ecotoxicology" Organic Chemicals : An Environmental Perspective Boca Raton: CRC Press LLC,2000 7 Ecotoxicology SYNOPSIS The basic input required for assessing the toxicity of xenobiotics is. of the toxicant is essentially constant. In the first procedure—and to a lesser extent in the second—toxicant concentrations will not remain constant during exposure, and the range of exposure

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