Other Approaches to Reproductive Toxicity Assessment

The food ingestion/food chain model that generates HQs is one approach of several used to assess reproductive toxicity in nonhuman species. At present the HQ approach, essentially a dose comparison, is not even workable for two terrestrial animal groups, amphibians and reptiles, nor is it used for fish and other aquatic species. In the case of the amphibians and reptiles, there is a dearth of toxicity information of the dose-response type, but a greater difficulty to surmount is that of identifying the dominant mode of chemical uptake for these receptors. It is recognized that terrestrial ecological receptors, like man, have three operating routes of uptake - ingestion, inhalation, and dermal contact. With reasonable supports, it is assumed that the predominant route for birds and mammals is ingestion, and consequently ERAs do not even attempt to quantify for birds and mammals, the potential for reproductive effects (or any other systemic effects for that matter) that may stem from the other two routes. For amphibians and reptiles, where the integument is often quite moist, and where the animal lies closely appressed to the substrate, dermal contact and respiration through the skin may constitute the predominant route of chemical exposure and subsequent uptake. Should transdermal exposures constitute the primary concern, the requisite empirical toxicity studies to support reproductive assessments would first have to be conducted for there to be a useful and reliable assessment tool. Presently, amphibians and reptiles are not evaluated in ERAs altogether (i.e., for any toxicological endpoint), except in an occasional crude qualitative manner. This is somewhat surprising and unfortunate, in light of these receptors being understood to be sensitive bioindicators, and where many have argued that in response to man-induced environmental changes (pollution, primary among them), population declines are widespread, and species are vanishing. In the absence of an appropriate ingestion model, ecotoxicologists may turn to evaluating tissue concentrations of bioaccumulated chemicals (in specific organs, whole-body measures, bird eggs, or perhaps in the case of amphibians, in the gelatinous egg masses that undergo external development). The difficulty with such an approach is that it assumes that a higher tissue concentration is necessarily unhealthful, and there is little evidence to support such an assumption. With virtually the entirety of ecotoxicological databases being of the administered dose genre, little or no attention has been given to assigning effects, reproductive ones included, to tissue burden. The prospects are somewhat better for fish and other aquatic species, where efforts are underway to compile data sets that identify the principal organs that load chemicals, and to establish linkages of tissue concentrations and reproductive impairments. Despite efforts to do so, there appear to be many glaring examples of highly contaminated fish that although unquestionably unhealth-ful for the would-be human consumer, demonstrate no apparent reduction in fish health (as in fecundity). Examples would be alewife, shad, perch, and bass in the waters of the 40-mi-long Hudson River polychlorinated biphenyl (PCB) Superfund site, in upstate New York.

One common approach to assessing reproductive toxicity involves the use of 'toxicity tests' (also termed bioassays). Here, a standardized test species (e.g., fathead minnow, manure worm, the amphipod crustacean, Hyalella azteca) is exposed to a contaminated medium (such as site topsoil, whole effluent from a water treatment facility, or a specified dilution of the effluent) while under highly controlled laboratory conditions. Often the test endpoint, or one of several, is a reproductive one. Extreme care must be taken to ensure that the site-specific media samples satisfy the requirements to rear and maintain the test species. Certain invertebrates, for example, only fare well when species-specific sediment grain size specifications are met. Other species may only be able to tolerate a very narrow salinity range. Should essential life-supporting features of the contaminated site's media not well match those of the commercially available test species to be used, the situation may be ripe for drawing errant conclusions. A noteworthy and statistically significant negative change in a reproductive measure, such as reduced cocoon production in earthworms placed in jars of contaminated site soil (relative to the cocoon production rate of worms placed in jars of reference location soils), could have nothing to do with soil contaminant levels. Such toxicity testing is not without its share of drawbacks, and many of these reflect the dissimilarity of the imposed chemical exposures of the test organisms and the natural environmental exposures of receptors in the real world. Consider the case of a waterbody with several known contaminants in the shallow sediment's bioactive zone (the top several inches). Stakeholders might collectively agree to conduct a chronic freshwater toxicity test using the freshwater waterflea, Ceriodaphnia dubia. Since the established test for this species is one that monitors survival and the number of offspring produced in a water-column species, the sediment is first agitated in the laboratory to liberate to the column water that lies above the sediment, the contaminants that are bound to the sediment matrix. The 7-day static-renewal test to be run, where newly prepared water (the elutriate) can circulate through the test chambers each day, will require that ample site sediments have been brought to the laboratory beforehand. Although the test can detect statistically significant reductions in reproductive success, the following cannot be overlooked:

• the test species (Ceriodaphnia dubia in this hypothetical example) may not occur in the contaminated site's sediment;

• a 'column water' test species was used to speak to sediment-dwelling/exposed species;

• the commercially bred Ceriodaphnia sp. used in the test had no prior history of living amid contaminated water or sediment, unlike the macroinvertebrates that reside today in the waterbody's contaminated sediments (in all likelihood, the site sediments have been contaminated for several decades, and have consequently allowed vast opportunities for the site biota to adapt);

• a statistically significant difference, as in a measured reduction in the number of Ceriodaphnia sp. offspring produced, is not necessarily a 'biologically' significant difference for the test species;

• identification of valid, reproducible, and statistically significant reproductive impairment in the test organisms, does not necessarily mean that there is impairment in the actual site-exposed sediment-dwelling invertebrate species;

• there is enormous potential for error when extrapolating from 'failed' test responses; the actual waterbody-associated aquatic fauna, for whose protection a site cleanup could reasonably proceed (such as larger fish; cleanups do not occur for water fleas), may bear no ill-health effects, reproductive or otherwise (standard toxicity tests, like the Ceriodaphnia one discussed here, have no ability to speak to other, and especially higher-food-chain, aquatic species, e.g., fish).

To counterbalance the many uncertainties associated with toxicity testing, and in particular when evaluating water, it is recommended that the testing involve at least two species, as one fish, and one invertebrate. Still there are often problems associated with interpreting and applying toxicity test outcome information, and much of this reflects the desire to extrapolate from toxicity test species to higher trophic level species, that is, those for whose protection a site cleanup could realistically proceed. A stakeholder could argue that with only the pollution-tolerant invertebrates in a streambed being capable of reproducing normally, the fish that feed on these invertebrates are receiving a nutritionally compromised diet, and are consequently at risk of not receiving enough dietary energy such that they themselves can reproduce adequately. The only way to verify such an argument would be to conduct the empirical research to support the contention. In this case, not only would the nutritional value of the pollution-tolerant invertebrates need to be measured and possibly shown to be inferior, but it should be ascertained if in fact there is a depauperate resident fish population. Focused study might reveal that although the food base is predominantly or only comprised of pollution-tolerant forms, the biomass in the contaminated stream is actually greater than what was present prior to the contaminant releases. One of the more common misapplications of toxicity test outcome information concerns work with earthworms. Where the earthworms that have been exposed to a site's contaminated soil show reduced reproduction, there are those that would like to use the test results to estimate the corresponding reduction in the local songbird population. Such expressed wishful intentions overlook at least three realities: that the test was never designed to be used to make a population assessment for birds; that there is no way to relate an earthworm toxicity test outcome, such as reduced reproduction, to a corresponding impact in birds; and that with so much time having already elapsed at a site since it became contaminated, the local birds have rather assuredly adapted as necessary.

A review of toxicity testing for reproductive endpoints would be less than complete if it did not acknowledge the phenomenon of test subjects having imposed unnatural contaminant exposures. FETAX (frog embryo teratogenesis assay - Xenopus) is a 96 h whole embryo assay for detecting teratogenic (developmental) effects, and thereby an indirect reproduction assessment method. Frog embryos of the species Xenopus laevis are placed into aquaria with contaminated water, but just prior to this, the jelly coat of the embryos is carefully removed. This procedure, although well intended, vastly increases the likelihood that one or more contaminants in water will cause various malformations in the developing frogs. On the one hand, the philosophy behind the test is admirable - there is a keen interest in uncovering early on, the slightest potential for developmental malformations to occur. The counter-argument though is equally appreciated - what utility is there in evaluating the malformations, when in the real-world exposure, the jelly coat is not removed, but is rather intact? Should there be striking differences in response, as in the case where malformations are only observed in the embryos exposed to the contaminated water (and not in the control-aquarium embryos), such information comes only at the expense of having artificially tampered with nature. Given the prior discussion, it should be clear that toxicity testing for the purpose of enhancing our understanding of reproductive toxicity, whether in a contaminated site context or not, should only be applied in a weight-of-evidence, or lines-of-evidence, context.

For certain animal groups, there is the prospect of collecting somatic measurements that bear on reproductive capability, and then endeavoring to interpret the degree of reproductive well-being from the gathered information. Birds and smaller mammals at contaminated locations lend themselves to this work, where brood patches and placental scars are evaluated, respectively. Simplistically, fewer brood patches and fewer placental scars can indicate a reduction in the number of offspring produced. Caution must be exercised when reviewing the data though, because linking somatic differences such as these to particular site chemicals is not a straightforward process, and especially when most contaminated sites have multiple toxicants present. There is also the added possible complication of wrongly ascribing altered measures to specific contaminated sites when they in fact stem from chemical exposures that occurred tens and hundreds of miles away from the animal's point of capture, as in the case of migratory birds. A related but yet different approach in ecotoxicology's quest to identify clear somatic markers of chemically caused reproductive impairments, involves assessing the configuration and deployment of reproductive organs. Irregularities in the shape of a female rodent's uterine horns for example, can signify reduced reproductive capability.

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