The relative sensitivity of microtox®, daphnid,rainbow trout,and fathead minnow acute lethality tests

Relative sensitivity and correlations between the Microtox® test and three commonly used acute lethality bioassays (i.e., rainbow trout, fathead minnow, Daphnia) are reviewed and discussed. All relevant data available for comparison were separated and evaluated based on chemical groupings. Generally, Microtox was more sensitive than or as sensitive as the acute lethality tests for pure individual organics, but was less sensitive to most inorganics. Microtox was not as sensitive as acute lethality tests to effluents or leachates with a high component of insecticides, herbicides, inorganics, pharmaceuticals or textiles, or highly lipophilic contaminants. As the complexity and toxicity of industrial effluents increased, the correlations and sensitivity of Microtox increased, with a corresponding decrease in data variability. Additionally, limitations of the available data are that (1) comparisons of relative sensitivity varied with the compounds and organisms tested; (2) there was a lack of standardization in approach and a failure to provide adequate details on the origin of the cited toxicity data, which confused and obscured comparisons; and (3) published reviews commonly failed to identify fish species and test details for cited data.     

Species-related differences in the inhibition of brain acetylcholinesterase by paraoxon and malaoxon

Interspecies comparisons indicate that fish are relatively more resistant to acute intoxication with parathion and paraoxon than are rodents. In contrast, fish are more sensitive to malathion and malaoxon. The following investigation was designed to determine if species-related differences in the sensitivity of brain acetylcholinesterase (AChE) to inhibition by paraoxon and malaoxon could contribute to the interspecies differences in toxicity. Brain AChE activity was significantly greater in fathead minnows and rainbow trout than in rats and mice. The fathead minnow and rainbow trout IC50 values for paraoxon were 228- to 1879-fold greater than the corresponding values for rat and mouse. Similarly, the Ki (bimolecular inhibition constant) was 159- to 1663-fold greater in rodents than in fish, which reflected both a higher KA (association constant) and kp (phosphorylation constant) in rodents. The rodent IC50 values for malaoxon were 30-80% that of the fish IC50, and the Ki was 30-50% greater in rodents than in fish. These data suggest that the greater sensitivity of rodent brain AChE to inhibition by paraoxon may contribute to the greater toxicity of parathion and paraoxon in rodents than in fish. In contrast, the lack of correlation between the inhibition of brain AChE by malaoxon and species-related differences in acute I D50 suggests that other factors, such as the limited carboxylesterase activity in fish, may be responsible for this species selectivity.     

Gene expression profiles in fathead minnow exposed to 2,4-DNT: correlation with toxicity in mammals.

Toxicogenomics, the genome-wide analysis of gene expression to study the effect of toxicants, has great potential for use in environmental toxicology. Applied to standard test organisms, it has possible applications in aquatic toxicology as a sensitive monitoring tool to detect the presence of contaminants while providing information on the mechanisms of action of these pollutants. We describe the use of a complementary DNA (cDNA) microarray of the fathead minnow (Pimephales promelas) a standard sentinel organism in aquatic toxicology, to better understand the mechanisms of toxicity of 2,4-dinitrotoluene (2,4-DNT) which is released in the environment through military and industrial use. We have constructed a fathead minnow microarray containing 5000 randomly picked anonymous cDNAs from a whole fish cDNA library. Expression profiles were analyzed in fish exposed to 2,4-DNT for 10 days at three concentrations (11, 22, and 44 microM, respectively) below the measured median lethal concentration (58 microM). Sequence analysis of cDNAs corresponding to differentially expressed genes affected by exposure revealed that lipid metabolism and oxygen transport genes were prominently affected in a dose-specific manner. We measured liver lipids and demonstrate that lipid metabolism is indeed perturbed following exposure. These observations correlate well with available toxicological data on 2,4-DNT. We present possible modes of action of 2,4-DNT toxicity and suggest that fathead minnow cDNA microarrays can be useful to identify mechanisms of toxicity in fish and as a predictive tool for toxicity in mammals.     

Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas)

The steroid hormone progesterone (P4) is found at relatively high concentrations (～300 ng/L) in association with concentrated animal feeding operations (CAFOs). In an effort to better understand the potential endocrine disrupting effects of P4 in male fish, computer assisted sperm analysis (CASA) was used to evaluate the effects of this steroid on sperm motility in the fathead minnow (Pimephales promelas). The rationale for focusing on sperm motility is that certain progestins have been shown to bind to surface membrane receptors on fish spermatozoa and increase sperm swimming velocity. It was hypothesized, therefore, that sperm swimming velocity might be a useful indicator of progestin exposure in fish. Adult male fathead minnows (ages 6-12 months) were exposed to environmentally relevant doses of P4, both longer-term (1 week, in vivo exposure) and short-term (minutes, in vitro exposure). Sperm were then video recorded and analyzed by CASA. When fathead minnows were continuously exposed for 1 week to low levels of progesterone in vivo there was a significant dose-dependent reduction in sperm motility. There was no effect of short-term P4 exposure on fathead minnow sperm swimming characteristics. Additional research is required to elucidate the mechanism by which progesterone alters sperm swimming in the fathead minnow. With further validation, the fathead minnow sperm motility assay may be a useful tool to rapidly screen for endocrine disrupting chemicals in the aquatic environment.     

Sensitivity of early life stages of white sturgeon, rainbow trout, and fathead minnow to copper

Populations of white sturgeon (WS; Acipenser transmontanus) are in decline in several parts of the United States and Canada, attributed primarily to poor recruitment caused by degradation of habitats, including pollution with contaminants such as metals. Little is known about sensitivity of WS to contaminants or metals such as copper (Cu). Here, acute (96 h) mortalities of WS early life stages due to exposure to Cu under laboratory conditions are reported. Two standard test species, rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas), were exposed in parallel to determine relative sensitivity among species. Swim-up larvae [15 days post-hatch (dph)] and early juveniles (40?C45 dph) of WS were more sensitive to Cu (LC₅₀ = 10 and 9?C17 ??g/L, respectively) than were yolksac larvae (8 dph; LC₅₀ = 22 ??g/L) and the later juvenile life stage (100 dph; LC₅₀ = 54 ??g/L). WS were more sensitive to Cu than rainbow trout and fathead minnow at all comparable life stages tested. Yolksac larvae of rainbow trout and fathead minnow were 1.8 and 4.6 times, respectively, more tolerant than WS, while swim-up and juvenile life stages of rainbow trout were between 1.4- and 2.4-times more tolerant than WS. When plotted in a species sensitivity distribution with other fishes, the mean acute toxicity value for early life stage WS was ranked between the 1st and 2nd centile. The WS life stage of greatest Cu sensitivity coincides with the beginning of active feeding and close association with sediment, possibly increasing risk. WS early life stages are sensitive to aqueous copper exposure and site-specific water quality guidelines and criteria should be evaluated closely to ensure adequate protection.     

Characterization of responses to the antiandrogen flutamide in a short-term reproduction assay with the fathead minnow

A short-term reproduction assay with the fathead minnow (Pimephales promelas) has been developed to detect chemicals with the potential to disrupt reproductive endocrine function controlled by estrogen- and androgen-mediated pathways. The objective of this study was to use the assay to characterize responses of fathead minnow reproductive endocrinology and physiology to the mammalian antiandrogen, flutamide. Male and female fish were exposed to nominal (target) concentrations of 50 and 500 microg flutamide/l for 21-days, following which plasma steroid and vitellogenin concentrations were determined and gonadal morphology assessed. Fecundity of the fish was significantly reduced by exposure to a measured test concentration of 651 microg flutamide/l. In addition, embryo hatch was significantly reduced at this concentration. Qualitative histological assessment of ovaries from females exposed to flutamide indicated a decrease in mature oocytes and an increase in atretic follicles. Testes of males exposed to flutamide exhibited spermatocyte degeneration and necrosis. Concentration-dependent increases in plasma testosterone and vitellogenin concentrations were observed in the females. Flutamide also altered reproductive endocrinology of male fathead minnows. Males exposed to 651 microg flutamide/l exhibited elevated concentrations of beta-estradiol and vitellogenin. In summary, the results of this study with the fathead minnow demonstrate that flutamide affects reproductive endocrine function in fish and that the type of hormonal pattern and histopathology effects observed are consistent with an antiandrogenic mode-of-action. Consequently, our findings suggest that the 21-day reproduction assay utilizing fathead minnows is a sensitive short-term screening method for the detection of endocrine-disrupting chemicals, including antiandrogens.     

Relative sensitivity of algae,bacteria, invertebrates,and fish to phenol: Analysis of 234 tests conducted for more than 149 species

An analysis was conducted to estimate the relative sensitivities of algae, bacteria, fish, and invertebrates to one chemical—phenol. Results from 234 studies to estimate the toxicity (EC₅₀ or LC₅₀ value) of phenol to 1 rotifer, 3 algal species, 4 leech species, 9 worm species, 12 fish species, 13 water mite species, 20 bivalve species, 22 crustacean species, greater than 26 bacterial species, and 39 insect species were analyzed to estimate relative, not absolute, sensitivities within and between species and between groups of species. EC50 values from conducting ten 5-min Microtox® tests were very similar as were EC50 values from conducting two activated sludge respiration inhibition tests. Three-fold and thirty-fold differences were observed for two 120-min Motility and two Oxygen-depletion tests, respectively. Most of the fish within-species variability could be accounted for by differences in test temperatures. Most of the invertebrate within-species variability could be accounted for by lab-to-lab or test-to-test variability. Differences between bacterial tests to measure five physiological endpoints (bioluminescence, respiration, growth, dehydrogenase, motility) were estimated. Differences between the EC50 values for the two tests to measure bioluminescence were significant. However, none of the mean EC50 values from tests to measure five physiological end-points appeared to be significantly different. Mean LC50 values from conducting fish tests were compared; only the rainbow trout and fathead minnow were significantly different. A comparison of relative species sensitivity suggested that bluegills, daphnids, fathead minnows, featherbacks, giant gourami grass shrimp, guppies, P. phosphoreum in the 5-min Microtox® test or rainbow trout were among the more sensitive species to the acute effects of phenol. A comparison of groups of species suggested that caddisfly or dragonfly larvae, crustaceans, daphnids, fish, shrimp and water fleas were among the more sensitive species groups to the acute effects of phenol.     

Responses of fish exposed to a C9--11 linear alcohol ethoxylate nonionic surfactant in stream mesocosms

Bluegill sunfish (Lepomis macrochirus) and fathead minnow (Pimephales promelas) responses to a nonionic linear alcohol ethoxylate (LAE) surfactant were examined in 10-day laboratory and 30-day outdoor stream mesocosm experiments. Larval fathead minnow survival was determined in a 10-day laboratory study with nominal concentrations of 0, 1.0, 3.0, 7.0, 13.0 and 25.0 mg LAE l–1. Bluegill sunfish survival and growth and fathead minnow survival, reproduction and behaviour were evaluated in replicated stream mesocosms with mean measured concentrations of 0, 0.73, 2.04, 4.35, 5.7 and 11.24 mg LAE l–1. The 10-day laboratory No Observed Effect Concentration (NOEC) for larval fathead minnow survival was 1.0 mg l–1. The 30-day stream NOEC for bluegill sunfish survival and growth was 5.7 mg l–1. In the stream mesocosms, NOECs for adult fathead minnow survival, reproduction and behaviour were 2.04 mg l–1, 0.73 mg l–1 and 0.73 mg l–1, respectively. Surviving fathead minnows that had ceased reproduction following exposure to LAE in the streams re-established normal reproduction during a post- treatment observation period in the laboratory. Laboratory studies in conjunction with the stream mesocosm study yielded complementary results which provided a detailed assessment of threshold responses of fish to LAE     

Acute and chronic toxicity of nano-scale TiO2 particles to freshwater fish,cladocerans, and green algae,and effects of organic and inorganic substrate on TiO2 toxicity

This research evaluated the toxicity of TiO₂ nanoparticles to freshwater aquatic organisms and the effects of organic and inorganic material on TiO₂ toxicity. The fathead minnow was much less acutely sensitive to TiO₂ (LC50 500 mg/l and higher) than Ceriodaphnia dubia and Daphnia pulex (mean LC50 values 7.6 and 9.2 mg/l, respectively). Total organic carbon levels of 1.5 mg/l decreased TiO₂ acute toxicity to C. dubia (LC50 > 100 mg/l), but kaolinite clay decreased TiO₂ toxicity to a lesser extent. In chronic toxicity tests, the green algae Pseudokirchneriella subcapitata was more sensitive to TiO₂ (IC25 1–2 mg/l) than C. dubia (IC25 9.4–26.4 mg/l) and the fathead minnow (IC25 values over 340 mg/l). Study results indicate that the specific organisms exposed and the effects of water quality parameters on TiO₂ toxicity should be considered in hazard evaluations of this nanoparticle.     

Ecological effects testing under the Toxic Substances Control Act: Acrylamide

Analysis of acrylamide aquatic toxicity data submitted under section 4 of the Toxic Substances Control Act revealed that for acute toxicity studies with bluegill, fathead minnow, and rainbow trout, concentrations of acrylamide necessary to produce 50% mortality decreased by 50-70% as a function of increasing exposure from 1 to 4 days. Analysis of acute toxicity data for Mysidopsis bahia suggested that increases in mortality with increased exposure to acrylamide might also be observed in saltwater organisms. Ratios of acute (4-day) LC₅₀ values to chronic (28-day) maximum acceptable toxic concentrations for this saltwater invertebrate were 26 for parent and offspring survival, 115 for female dry weights, and 975 for male dry weights. These ratios illustrate that long-term acrylamide exposure to sensitive life stages of M. bahia produced adverse effects on reproduction and growth at acrylamide concentrations significantly lower than those suggested by acute LC₅₀ or EC₅₀ values.     

Aquatic toxicity of four alkylphenols (3-tert-butylphenol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol) and their binary mixtures to microbes, invertebrates, and fish

The acute and chronic toxicity of four simple alkylphenols with butyl and propyl substitutions was evaluated with aquatic microbes, invertebrates, and fish. These alkylphenols-3-tert-butylphenol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol-have been detected in various environmental media, but their impact on aquatic fauna has seldom been evaluated. Relative susceptibility to each phenolic varied by test species. The marine bacterium Vibrio fischeri was the most susceptible to the alkylphenols, up to 3 orders of magnitude more sensitive than species of higher trophic levels. For 4-isopropylphenol, the 5-min Microtox EC(50) value was 0.01 mg/L, whereas the EC(50) for Ceriodaphnia after a 48-h exposure was 10.1 mg/L. Notable differences in sensitivity to the alkylphenols was also observed with the Microtox assay: 4-isopropylphenol was > 200 times more toxic to V. fischeri than was 2-isopropylphenol (EC(50) = 2.72 mg/L). For V. fischeri, the mixture toxicity of the alkylphenols was additive in nature and was predicted by a concentration addition model. The energy of the lowest unoccupied molecular orbital (ELUMO) explained the observed toxicity of the individual alkylphenols to V. fischeri (r(2) = 0.92, p < 0.05). These results suggest that the mode of action of polar narcotic alkylphenols to V. fischeri is different than that of other test organisms, possibly because of the differences in the cell structure of the prokaryotic V. fischeri.     

Examining waterborne and dietborne routes of exposure and their contribution to biological response patterns in fathead minnow (Pimephales promelas)

The objectives of the current study were: (i) to gain a better understanding of the relative importance of water and diet as routes of exposure causing toxicity in fathead minnow (FHM) exposed to metal mining effluents (MME) using a full factorial water/food experimental design (Experiment 1), and (ii) to assess differences in the effects of food quality on toxicity by comparing FHM fed both a live and frozen diet of Chironomus dilutus (Experiment 2). The results showed significant increases in general water quality parameters (e.g., hardness, conductivity) and various metals in the effluent treatment waters compared to control waters, with maximum increase seen in the multi-trophic streams. Metals accumulation (Rb, Al, Se, Sr, Tl, Ce, Co, Cu, Pb) effects of both waterborne and multi-trophic exposures were significant in one or more fathead minnow tissue type (muscle, gonads, liver, larvae) relative to those in the control systems. Condition factor and liver somatic index (LSI) of FHM were also significantly affected in both exposures by one or both routes of exposure (water and/or diet). In addition, cumulative total egg production and cumulative spawning events were significantly affected by both waterborne and dietborne exposures, with maximum effect found in the multi-trophic streams. These results suggest that under environmentally relevant exposure conditions, trophic transfer of metals may lead to greater reproductive effects and increased metal toxicity in fish. It also indicates that metals are assimilated in tissues differently depending on the quality of the food (live vs. frozen). Overall, it appears that the multi-trophic bioassay provides an important link between the laboratory and field, which may allow for a more realistic assessment of the true impact of MME's in the environment.     

Mechanistic basis for estrogenic effects in fathead minnow (Pimephales promelas) following exposure to the androgen 17alpha-methyltestosterone: conversion of 17alpha-methyltestosterone to 17alpha-methylestradiol

Exposure of adult fathead minnows (Pimephales promelas) to the androgen 17alpha-methyltestosterone (MT) produces both androgenic and estrogenic effects, manifested as nuptial tubercle formation in females, and vitellogenin production in males and females, respectively. The present study was conducted to determine if the unanticipated estrogenic effects are produced by conversion of MT via aromatase activity to 17alpha-methylestradiol (ME2). Aromatase activity at the end of a 7-day waterborne MT exposure (20, 200microg/l) was significantly decreased in ovarian microsomes and brain homogenates from exposed fish, to about 30-50% of control activity. Although aromatase activity was decreased by 7 days, it is possible that the conversion of MT to ME2 occurred soon after initial exposure. In support of this, ME2 was detected in plasma samples of the fish following the 7-day exposure, confirming their ability convert the androgen MT to the estrogen ME2. The concentration of ME2 in plasma was within the range of plasma 17ss-estradiol (E2) found in control female fathead minnows (4-5ng/ml). These results, in conjunction with competitive binding assays that indicate ME2 binds to the fathead minnow estrogen receptor with a relative binding affinity of 68.3% of E2, support the hypothesis that aromatization of MT to ME2 contributes to the estrogenic effects in fathead minnows following exposure to this androgen.     

Measurement of the aquatic toxicity of volatile nitrosamines.

The acute toxicity of N-nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) was determined for three groups of aquatic organisms: algae, invertebrates, and fish. Toxicity of DMN and DEN to algae was assessed as a repression in the growth rate of either Selenastrum capricornutum or Anabaena flos-aquae in static bioassay tests. DMN and DEN concentrations of 1-10 ppm depressed algal growth in all cases. Invertebrate toxicity was determined in 96-h static bioassay tests with Dugesia dorotocephala and Gammarus limnaeus. The data indicated that these organisms are not highly susceptible to nitrosamine toxicity. The 96-h LC50s for D. dorotocephala were 1365 and 1490 ppm for DMN and DEN, respectively. Similar studies with G. limnaeus indicated LC50s of 330 and 500 ppm for DMN and DEN, respectively. Fish toxicity was also determined in 96-h statis bioassays with the fathead minnow (Pimephales promelas). Acute toxicities were calculated as LC50s of 940 and 775 ppm for DMN and DEN, respectively. Algae were calculated as LC50s of 940 and 775 ppm for DMN and DEN, respectively. Algae were quite sensitive to relative low levels of volatile nitrosamines, but higher organisms (invertebrates and fish) were relatively insensitive.     

Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro.

To establish the potential use of cell cultures as a simple and sensitive biological tool to detect environmental pollutants, six cell lines established from several fish species including GCF (grass carp fins), CIK (Ctenopharyngodon idellus kidney), EPC (epithelioma papulosum cyprini), CCO (channel catfish ovary), BB (brown bullhead caudal trunk) and FHM (fathead minnow muscle) were tested and compared for their cytotoxic sensitivity to four heavy metals: cadmium (Cd), chromium (Cr), zinc (Zn), and copper (Cu). Following a 24-h exposure to these metal salts at selected concentrations, test cells were characterized by morphology, viability and proliferation. Our results indicate that all these metal salts are cytotoxic to these fish cell lines, but at varied levels. Calculated inhibitory concentration (IC(50)) values revealed that the cytotoxicity of Cr and Cd was significantly more pronounced than that of the other two metal salts. Comparative analysis of these fish cell lines showed that C. idellus kidney (CIK) cells are the most sensitive cell line to copper, epithelioma papulosum cyprini (EPC) cells are more sensitive than other cells to Cr and Zn, while channel catfish ovary (CCO) cell line is the most sensitive one to Cd. In conclusion, CIK, EPC and CCO could potentially be sensitive bio-indicators for the initial monitoring and assessment of acute cytotoxicity of heavy metals in the aquatic environment.     

Toxicology evaluation of Atlantic Canadian seafood processing plant effluent

The purpose of this study was to carry out an acute aquatic toxicity assessment on select effluent samples from Atlantic Canadian seafood processing plants. Raw effluent acute aquatic toxicity for the flatfish and salmon effluents was assessed using the acute lethality test and Microtox? test. The effectiveness of dissolved air flotation treatment (DAF) in removing acute toxicity from these effluents was evaluated using the Microtox? test. The salmon effluent failed the acute lethality test using rainbow trout while the flatfish effluent showed acute toxicity in the Microtox? test with a 50% inhibiting concentration (IC₅₀) of 38.84%. Subsequent treatment by DAF of the flatfish and salmon effluents increased IC₅₀ values by 20% and 26% respectively. The findings of this study indicate that all of the processing effluents sampled showed characteristics that could potentially degrade effluent receiving waters and acute toxicity was demonstrated in the two raw finfish effluents. Application of DAF treatment was successful in significantly increasing Microtox? IC₅₀ values, thereby reducing acute toxicity, but failed to entirely remove acute toxicity. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

QSAR modelling of the ERL-D fathead minnow acute toxicity database

1. Regression analysis has been applied to examine the structure-activity relationships regarding the acute fish toxicity (96 h LC50 fathead minnow) of organic chemicals. The log P dependent baseline toxicity model has been confirmed for a data set composed of 618 compounds from 24 chemical classes associated with a putative common mode of action. 2. Covariance analysis of the discrete by class regression functions resulted in the combination of chemicals to subsets associated with their mode of action. Separate models were derived for nonpolar (Class I) and polar (Class II and III) compounds. Chemicals which are more toxic than estimated from the baseline model are identified.     

Estimation of toxicity to marine species with structure-activity models developed to estimate toxicity to freshwater fish

Structure-activity models which were developed to estimate toxicity of chemicals to freshwater fish were tested for use with an estuarine fish (Cyprinodon variegatus) and mysids (Mysidopsis bahia). Significant linear and polynomial relationships that correlated well existed between reported 96-h LC₅₀ values for each marine species and log P (log octanol/water partition coefficient). Good linear relationships were obtained when the 96-h LC₅₀ values for C. variegatus and M. bahia were regressed on water solubility (μmol/l). These models were compared to models developed for freshwater fish using log P and log S. Models using log P to estimate acute toxicity for two freshwater fish produced 96-h LC₅₀ values similar to those measured for C. variegatus and M. bahia, whereas, those models developed with water solubility produced 96-h LC₅₀ values similar to those for C. variegatus, but not for M. bahia. The data indicated that models developed with log P for freshwater fish can be used to estimate toxicity to C. variegatus for a minimum of 58% of the chemicals, whereas models using water solubility estimated toxicity to C. variegatus for a minimum of 77% of the chemicals within an order of magnitude for screening purposes. The calculated 96-h LC₅₀ values were compared to the measured values for each marine species and those measured for Pimephales promelas (fathead minnow) and Poecilia reticulata (guppy). Tests indicated generally that calculated 96-h LC₅₀ values were overestimates of the measured 96-h LC₅₀ values when models for freshwater fish were used to estimate toxicity to each marine species. More data are required for marine species to determine if highly significant relationships between marine and freshwater fish exist with comparisons using larger sample sizes.     

Androgenic and estrogenic effects of the synthetic androgen 17alpha-methyltestosterone on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) determined using the gonadal recrudescence assay

The effects of the androgen, 17alpha-methyltestosterone were assessed on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) using a gonadal recrudescence assay. In this assay, mature male and female fathead minnow, previously kept under simulated winter conditions (15 degrees C; 8:16 h light:dark regime) were transferred to simulated summer conditions (25 degrees C water temperature; 16:8 h light:dark regime) to induce gonadal recrudescence. To assess sexual development fish were exposed to nominal concentrations of 0, 0.1, 1, 5 and 50 microg/L 17alpha-methyltestosterone. After 3 weeks of chemical exposure, effects on condition (condition factor, CF), plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI) and gonad histology were investigated. Reproductive performance, including reproductive output (egg production), spawning behaviour, and fertilisation rate were measured over a subsequent 3-week-period in breeding adults maintained in clean water. 17alpha-Methyltestosterone had no effects on the condition of fish at any of the doses tested. 17alpha-Methyltestosterone induced both androgenic and estrogenic effects with females generally more affected by 17alpha-methyltestosterone than males: atretic follicles and male-specific sex characteristics (androgenic effect) were induced in females at > or = 0.1 and > or = 1 microg/L 17alpha-methyltestosterone, respectively. An inhibitory effect on ovary growth occurred at an exposure concentration of 50 microg/L 17alpha-methyltestosterone. In males 1 microg/L 17alpha-methyltestosterone induced a concentration-response induction of plasma vitellogenin (estrogenic effect) likely due to its conversion into 17alpha-methylestradiol, rather to the competition with endogenous steroids and their cross reactivity with the estrogen receptor. In the fish breeding studies, concentration-dependent reductions in egg number, fertilisation rate and increases in abnormal sexual behaviour in females were observed. All of these effects occurred at exposure concentrations of > or = 5 microg/L 17alpha-methyltestosterone. Thus, it could be assumed that the observed estrogenic effects in male fathead minnow were likely to the conversion of 17alpha-methyltestosterone into the estrogen 17alpha-methylestradiol, rather to the acting of 17alpha-methyltestosterone itself. In conclusion to this, showing hormonally activity of 17alpha-methyltestosterone in fish down to 100 ng/L, indicates that its potency was close to the range of several naturally occurring estrogens.     

Developing a list of reference chemicals for testing alternatives to whole fish toxicity tests

This paper details the derivation of a list of 60 reference chemicals for the development of alternatives to animal testing in ecotoxicology with a particular focus on fish. The chemicals were selected as a prerequisite to gather mechanistic information on the performance of alternative testing systems, namely vertebrate cell lines and fish embryos, in comparison to the fish acute lethality test. To avoid the need for additional experiments with fish, the U.S. EPA fathead minnow database was consulted as reference for whole organism responses. This database was compared to the Halle Registry of Cytotoxicity and a collation of data by the German EPA (UBA) on acute toxicity data derived from zebrafish embryos. Chemicals that were present in the fathead minnow database and in at least one of the other two databases were subject to selection. Criteria included the coverage of a wide range of toxicity and physico-chemical parameters as well as the determination of outliers of the in vivo/in vitro correlations. While the reference list of chemicals now guides our research for improving cell line and fish embryo assays to make them widely applicable, the list could be of benefit to search for alternatives in ecotoxicology in general. One example would be the use of this list to validate structure-activity prediction models, which in turn would benefit from a continuous extension of this list with regard to physico-chemical and toxicological data.