Full Life-cycle Tests Research Articles

Ecological risk assessment and testing for endocrine disruption in the aquatic environment

Field observations of endocrine disruption (ED) in fish and other aquatic species have contributed to a global effort to establish test methods for detecting ED effects in wildlife species. For ecological risk assessment, validated tests are needed for Amphibia, fish and aquatic invertebrates, supported by a tiered approach incorporating mechanistic data and exposure characterisation. The potential for extrapolation of ED data from mammalian to aquatic species may be limited, however, due to significant physiological differences in function and regulation of hormone systems in (aquatic) lower vertebrates and invertebrates. Presently, the OECD is considering a tiered approach for ED risk assessment, incorporating a fish 14-day screening assay (Tier 1); fish development and reproduction tests (both Tier 2); and a fish full life-cycle test (Tier 3). For detection of (anti-)oestrogens, the yolk-precursor protein vitellogenin is an ideal biomarker of exposure and functionally equivalent biomarkers are being sought for (anti-)androgens in fish. At the two higher tiers, impacts are assessed in terms of apical endpoints (e.g. development, breeding behaviour and fecundity) and also gonadal histopathology. Validation of these higher tier tests should include comparison of sensitivity of biochemical and apical endpoints to optimise the value of biomarkers for predicting adverse health effects (e.g. impaired reproduction). The specificity of future OECD fish and amphibian test guidelines for endocrine disrupters needs further consideration through inclusion of mechanistic endpoints based on state-of-the-art molecular endocrinology.

Developmental stage-specific life-cycle bioassay for assessment of sediment-associated toxicant effects on benthic copepod production.

In chronic bioassays of sediment organic compounds, toxicant exposures often decline through time, such that the beginning of a test yields disproportionately higher exposures than the end. Thus, those life stages initiating a test often are exposed to the highest concentrations, and for rapidly maturing test fauna, this may lead to varying conclusions regarding compound toxicities depending on the initial life stage chosen. This problem can be addressed by comparative full life-cycle tests initiated with different test-organism life stages. Thus, a full life stage-to-life stage toxicity test was developed for the rapidly maturing meiobenthic copepod Amphiascus tenuiremis to assess the importance of developmental stage at the onset of sediment toxicant exposure relative to reproduction, net population growth, and sex and age structure. Tests were conducted with a model spiked-sediment insecticide, chlorpyrifos, for each of the major life stages (P1) of A. tenuiremis (nauplius, copepodite, and adult). Each P1 stage was allowed to mature and reproduce in low chlorpyrifos concentrations (6-33% of stage-specific 96-h LC50s; 4-22 ng chlorpyrifos/g dry sediment) for 26 d. Test endpoints were numbers of surviving adult females, males, eggs per female (clutch), first generation (F1) nauplii, F1 copepodites, F1 total production, and realized F1 production per surviving female. Only the copepodite P1 test showed a significant decline in survival of an adult age class: females declined by 28% at 22 ng/g. Reductions in total production ranged from 33-96% of controls from nauplius to adult. The P1 naupliar stage was most sensitive, with F1 production being 33-47% of that in controls. However, on a realized production per female basis, both the copepodite and naupliar P1 yielded significantly reduced F1s of 23 and 40% of controls at 11 and 22 ng/g.

Ecological Risk Assessment of Endocrine Disruptors

The European Centre for Ecotoxicology and Toxicology of Chemicals proposes a tiered approach for the ecological risk assessment of endocrine disruptors, integrating exposure and hazard (effects) characterization. Exposure assessment for endocrine disruptors should direct specific tests for wildlife species, placing hazard data into a risk assessment context. Supplementing the suite of mammalian screens now under Organization for Economic Cooperation and Development (OECD) validation, high priority should be given to developing a fish screening assay for detecting endocrine activity in oviparous species. Taking into account both exposure characterization and alerts from endocrine screening, higher tier tests are also a priority for defining adverse effects. We propose that in vivo mammalian and fish assays provide a comprehensive screening battery for diverse hormonal functions (including androgen, estrogen, and thyroid hormone), whereas Amphibia should be considered at higher tiers if there are exposure concerns. Higher tier endocrine-disruptor testing should include fish development and fish reproduction tests, whereas a full life-cycle test could be subsequently used to refine aquatic risk assessments when necessary. For avian risk assessment, the new OECD Japanese quail reproduction test guideline provides a valuable basis for developing a test to detecting endocrine-mediated reproductive effects; this species could be used, where necessary, for an avian life-cycle test. For aquatic and terrestrial invertebrates, data from existing developmental and reproductive tests remain of high value for ecological risk assessment. High priority should be given to research into comparative endocrine physiology of invertebrates to support data extrapolation to this diverse fauna.

Ecological risk assessment of endocrine disruptors.

The European Centre for Ecotoxicology and Toxicology of Chemicals proposes a tiered approach for the ecological risk assessment of endocrine disruptors, integrating exposure and hazard (effects) characterization. Exposure assessment for endocrine disruptors should direct specific tests for wildlife species, placing hazard data into a risk assessment context. Supplementing the suite of mammalian screens now under Organization for Economic Cooperation and Development (OECD) validation, high priority should be given to developing a fish screening assay for detecting endocrine activity in oviparous species. Taking into account both exposure characterization and alerts from endocrine screening, higher tier tests are also a priority for defining adverse effects. We propose that in vivo mammalian and fish assays provide a comprehensive screening battery for diverse hormonal functions (including androgen, estrogen, and thyroid hormone), whereas Amphibia should be considered at higher tiers if there are exposure concerns. Higher tier endocrine-disruptor testing should include fish development and fish reproduction tests, whereas a full life-cycle test could be subsequently used to refine aquatic risk assessments when necessary. For avian risk assessment, the new OECD Japanese quail reproduction test guideline provides a valuable basis for developing a test to detecting endocrine-mediated reproductive effects; this species could be used, where necessary, for an avian life-cycle test. For aquatic and terrestrial invertebrates, data from existing developmental and reproductive tests remain of high value for ecological risk assessment. High priority should be given to research into comparative endocrine physiology of invertebrates to support data extrapolation to this diverse fauna.

Development of a Reproductive Performance Test for Endocrine Disrupting Chemicals Using Pair-Breeding Fathead Minnows (Pimephales promelas)

Existing in vivo tests (with the exception of the full life-cycle test) are not adequate for assessing the reproductive effects of endocrine disrupting chemicals (EDCs) on fish, and hence the need for partial life-cycle tests has been recognized internationally. In this paper we describe the development of a short-term (6 week) reproductive performance test for EDCs using pair-breeding fathead minnows (Pimephales promelas). In the test, reproductive performance in paired fish is assessed over two 3 week periods, one with exposure to the test chemical and one without. The test is highly integrative and measures effects of exposure to chemicals on fecundity, gonadosomatic index (GSI), vitellogenin (VTG) induction, and secondary sexual characteristics (fat pad and tubercles in males). In this test, exposure to butyl benzyl phthalate (BBP) at a nominal concentration of 100 μg/L (measured concentration between 69 μg/L and 82 μg/L) had no discernible effects on reproductive performance. In contrast, all reproductive parameters measured were affected by exposure to 4-NP, albeit some (e.g. VTG induction and reduction in the prominence of secondary sexual characteristics − lowest effective dose between 0.65 μg/L and 8.1 ± 1 μg/L [measured]) were more sensitive than others (e.g. number of eggs and spawnings, where the lowest effective dose was between 8.1± 1 μg/L and 57.7 ± 3 μg/L [measured]). Concentrations of 4-NP at or above 48 μg/L [measured] inhibited reproduction completely.

Utility of a Full Life-Cycle Copepod Bioassay Approach for Assessment of Sediment-Associated Contaminant Mixtures

We compared a 21 day full life-cycle bioassay with an existing 14 day partial life-cycle bioassay for two species of meiobenthic copepods, Microarthridion littorale and Amphiascus tenuiremis. We hypothesized that full life-cycle tests would better simulate chronic exposures to copepods and provide more sensitive and realistic information about contaminant effects on growth of copepod populations. Copepods were exposed to sediments from two sites, non-contaminated North Inlet Estuary (NIE), SC (USA), and highly contaminated Shipyard Creek (SYC), SC, and measured for lethal and sublethal test points. Fourteen and 21 day bioassays showed similar negative effects in SYC sediments for both species. Survival and reproductive success were higher in the 14 day than in the 21 day bioassay for both species, in both sediment types. The lower 21 day overall response was not indicative of enhanced bioassay sensitivity. The 14 day bioassay provides sufficient f 1 generation information to detect negative population level responses to contaminant mixtures.

Longevity and reproduction of Ceriodaphnia dubia in receiving waters

AbstractSeven‐day tests with Ceriodaphnia dubia are commonly used to estimate toxicity of effluents or receiving waters but can sometimes yield no toxicity outcomes even if pollutants are present. We conducted two sets of full life‐cycle tests with C. dubia to (1) determine whether tests with longer exposure periods to low concentrations of contaminants in ambient water might reveal evidence of toxicity that could not be discerned from 7‐d tests and (2) determine the relative importance of water quality versus food as factors influencing C. dubia longevity and reproduction. In the first set of tests, C. dubia was reared in diluted mineral water (a negative control), water from a stream impacted by coal fly‐ash, or water from a retention basin containing sediments contaminated with mercury, other metals, and polychlorinated biphenyls. The second set of tests used water from the retention basin only. Before testing, though, water in the second set of tests was either filtered or not filtered, and food was either added or not added. Ceriodaphnia dubia longevity and reproduction did not differ much among the three water types in the first set of tests, but both longevity and reproduction were strongly affected by the filtering and food‐addition treatments in the second set of tests. Thus, C. dubia appeared to be relatively insensitive to general water quality factors but sensitive to food‐related factors. In five of the six full life‐cycle tests, lifetime reproduction by C. dubia could not be reliably predicted from reproduction data from the first 7 d of testing (R2 < 0.35, by regression analysis). The increase in predictability of lifetime reproduction of C. dubia as a function of test duration also differed among water types in the first set of tests and among treatments in the second set of tests. Thus, it may not be possible to reliably extrapolate the results of 7‐d tests with C. dubia to longer time scales.

Toxicity and bioaccumulation of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin in long‐term tests with the freshwater benthic invertebrates Chironomus tentans and Lumbriculus variegatus

AbstractTwo species of freshwater benthic invertebrates, Chironomus tentans and Lumbriculus variegatus, were exposed to three dietary concentrations of 2,3,7,8‐ tetrachlorodibenzo‐p‐dioxin (TCDD), and toxicity and bioaccumulation were determined. No toxic effects were observed in full life cycle tests with either species at tissue residue concentrations up to 9,533 ng TCDD/g lipid. The observed lack of sensitivity of the two species to TCDD was consistent with a presumed absence of the aryl hydrocarbon receptor in aquatic invertebrates. Predictions of lipid‐normalized tissue concentrations were made based on lipid‐normalized TCDD concentrations in the food and were within 15% of targeted concentrations in both species. Depuration studies indicated that TCDD elimination followed first‐order kinetics, with elimination rate constants of 0.0014 to 0.0022 h−1 for L. variegatus and 0.0070 to 0.0099 h−1 for C. tentans. Half‐lives ranged from 315 to 495 h in L. variegatus and from 70 to 99 h in C. tentans. The ability of invertebrates to accumulate relatively high concentrations of TCDD in the absence of toxic effects may be relevant to the transfer of contaminants through aquatic food webs to potentially sensitive vertebrate species.

Hepatic steatosis in zebra fish ( Brachydanio rerio) induced by long-term exposure to γ-hexachlorocyclohexane

In a full life cycle test, newly hatched eggs of zebra fish, Brachydanio rerio, were reared to sexual maturity under continuous exposure to 40, 80, 110, 130, and 150 μg/liter lindane, γ-hexachlorocyclohexane. The effects of lindane were investigated by recording behavior and survival of the F 0- and F 1-generation as well as morphological alterations in liver ultrastructure of F 0. Changes in peroxisomes were visualized by cytochemical staining for catalase activity with diaminobenzidine. Whereas behavioral changes can already be observed at 40 μg/liter, survival and number of eggs in F 0 as well as survival and growth of F 1 are unaffected by up to 80 μg/liter lindane. At concentrations ⩾ 110 μg/liter, survival of larvae is reduced already after 35 days, and mortality is 100% after 3 months. From 40 μg/liter, liver ultrastructure displays a microvesicular fatty vacuolation (steatosis) characterized by lipid deposition within the cisternae of the RER. At 40 μg/liter, this highly specific pathological change is accompanied by accumulation of hepatic macrovesicular triglyceride droplets, glycogen depletion, and the occurrence of club-shaped mitochondria. Additional alterations at 80 μg/liter comprise proliferation of SER in males and progressive fractionation of RER in females, stacking of club-shaped mitochondria, a conspicuous decrease in peroxisomal catalase activity, infiltration of macrophages into the liver parenchyma, and a significant stimulation of hepatocytic mitosis. Among several substances tested so far in zebra fish (4-nitrophenol, 4-chloroaniline 3,4-dichloroaniline, atrazine, lindane), lindane is the only compound inducing behavioral changes and hepatic steatosis in conjunction with a reduction in fertility. With regard to the relative sensitivity of the methods applied, behavioral and cytological studies appear more responsive to lindane exposure than survival studies.

Investigation of the long-term action of xenobiotics on fish with special regard to reproduction

A test is described which considers the action of xenobiotics on the reproduction of the zebrafish ( Brachydanio rerio). Successive reproduction cycles are investigated and the numbers of eggs released are the main criteria of the test. To evaluate the concentrations applied in the test, two short-term pretests are recommended. It is proposed that the reproduction test, in combination with the embryo-larva test as described here, may replace a full life-cycle test and might deliver more information than an extended embryo-larva test alone. Both tests could be performed within a considerably shorter time. The action of cadmium is used as an example to illustrate the tests.