Relationship of plasma sex steroid concentrations in female fathead minnows to reproductive success and population status

Concentration and/or production of sex steroids such as 17β-estradiol (E2) and testosterone (T) in fish have commonly been measured in field studies concerned with endocrine-active chemicals. There is a reasonable mechanistic basis for using E2 or T as biomarkers, as chemicals can alter steroid production through both direct and indirect effects on the hypothalamic-pituitary-gonadal (HPG) axis. There is uncertainty, however, as to what changes in steroid status may mean relative to apical endpoints, such as reproduction, that directly affect population status. In this study, we analyzed data from fathead minnow (Pimephales promelas) reproduction studies in which decreases in fecundity were associated with depressed steroid production as a result of chemical exposure. Although the chemicals acted on the HPG axis through different mechanisms, reproductive effects appeared to be expressed through a common pathway, depression of vitellogenin production in females. Plasma concentrations of E2 or T in the females were significantly, positively correlated with fecundity. Linear regression models describing the relationship between E2 or T concentrations and relative fecundity were linked to a population model to predict population trajectories of fathead minnows exposed to chemicals that inhibit steroid production. For example, a population existing at carrying capacity and exposed to a chemical stressor(s) that causes a 50% decrease in E2 production was predicted to exhibit a 92% decrease in population size over a 5-year period. Results of our analysis illustrate a conceptual framework whereby a commonly measured biomarker, sex steroid status, could be linked to individual- and population-level effects in fish.     

Effects of copper on development of the fathead minnow,Pimephales promelas Rafinesque

Embryos of the fathead minnow, Pimephales promelas Rafinesque, were exposed to total copper concentrations (Cu_T) of 0.6, 61, 113, 204, 338 and 621 μg/l from 5 to 10 h post-fertilization through 2 days post-hatch. A decrease in hatching time was observed with increasing total copper concentration but there was no decrease in embryonic developmental rate. Therefore, embryos hatched at earlier stages of development. Significant (P ≤ 0.05) declines in percent survival and percent total hatch were observed at 621 μg/l Cu_T) but not at 338 μg/l Cu_T or lower concentrations. The percentage of embryos with abnormalities was greater at 338 and 621 μg/l Cu_T than at 204 μg/l Cu_T and lower concentrations.Individuals exposed to copper during early development were then exposed to the same test concentrations for 28 days post-hatch. Survivors at 113 through 338 μg/l Cu_T were at earlier stages of development than were control fish. The percentage of fish surviving decreased with increasing copper concentration over the range 61 through 204 μg/l Cu_T. The percentage of fish surviving at 204 μg/l Cu_T was not significantly different from that at 338 μg/l Cu_T, and there were no survivors at 621 μg/l Cu_T. Surviving larvae at all copper concentrations from 61 through 621 μg/l Cu_T showed decreased length, weight and coefficient of condition compared to controls. The percentage of larvae with abnormalities increased significantly with increasing copper concentration. The calculated 96-h LC₅₀ (larval stage) was 250 μg/l Cu_T and the 28-day LC₅₀ (larval stage) was approximately 123 μg/l Cu_T.     

An optimised experimental test procedure for measuring chemical effects on reproduction in the fathead minnow, Pimephales promelas

The production of viable offspring is fundamental to the survival of any population. Tests that quantify effects on reproduction can, therefore, inform on the potential for long-term health effects of exposure to endocrine active chemicals. Surprisingly little is known, however, about the reproductive capacity of laboratory fish species used for chemical testing. As an example, the fathead minnow, Pimephales promelas, is widely used in chronic assessments of reproductive toxicology, and is readily induced to reproduce in captivity, yet there is little agreement on the reproductive capacity (egg number) of this species. For this species, the notable variation in reported estimates of egg number might relate to differences in the methods of egg collection adopted by many laboratories. To investigate this hypothesis, reproduction was assessed in a total of 200 pair-breeding fathead minnow, using egg collection methods that included the addition of trays placed beneath an inverted U-shaped PVC tile that is conventionally used alone for egg collection. The results demonstrated that the placement of a mesh-screened egg collection tray, beneath the spawning tile, increased estimates of the egg number by 25-67%. In addition, adopting the mesh-screened tray reduced variation in egg number between pairs, within an experiment, from >50% to <30% and variation between experiments was reduced from 53% to 7%. Adoption of the revised system for egg collection shows that egg number in the fathead minnow is considerably more consistent than frequently reported and is a highly robust endpoint against which chemical effects can be challenged effectively.     

Reproductive impairment and induction of alkaline-labile phosphate, a biomarker of estrogen exposure, in fathead minnows (Pimephales promelas) exposed to waterborne 17β-estradiol

The objectives of this research were: (1) to assess the effects of waterborne 17β-estradiol [E2; (17β)-estra-1,3,5(10)-triene-3,17-diol; CAS RN 50-28-2] on the reproduction of fathead minnows (Pimephales promelas) as a benchmark to which xeno-estrogens can be compared, and (2) to correlate the effects on reproductive function with plasma vitellogenin expression, measured as alkaline-labile phosphorous. Histopathological changes were also noted but are reported elsewhere. Duplicate groups of six fish (3 male and 3 female) were exposed to waterborne E2 at nominal concentrations of 10, 1, and 0.1 nM (2724, 272.4, and 27.24 ng l⁻¹) administered via a flow-through proportional diluter apparatus for 19 days. An ethanol carrier solvent was used at a final tank concentration of 1 ppm v/v in the treated tanks and in the solvent control tanks; the latter did not receive E2. Duplicate control tanks received neither ethanol nor E2. Dissolved E2 concentrations, measured throughout the exposure period using an ELISA, averaged 79% of nominal concentrations in the treated tanks. ELISA-detectable concentrations of E2 were found in all tanks (ranging from 3.5 to 15 ng E2 l⁻¹), including the control and solvent control tanks, which indicated that fish in the untreated tanks may have been the source of some E2. The EC50 (concentration expected to cause 50% effect), based on measured E2 concentrations, for inhibition of egg production was 120 ng E2 l⁻¹ (log₁₀ EC50=2.08±1.22, ±S.E.). The EC50 for induction of vitellogenin (measured as plasma alkaline-labile phosphate) in males was 251 ng E2 l⁻¹ (log₁₀ EC50=2.40±0.33, ±S.E.). No vitellogenin induction plateau was observed in females, therefore no EC50 could be calculated. Egg production, expressed as eggs laid per female, was significantly correlated with plasma vitellogenin in both males (linear r²=0.46, P<0.03) and females (linear r²=0.81, P<0.0004), though the relationship was stronger with female plasma vitellogenin expression than with males. The primary effect of E2 exposure on female fathead minnows appeared to be alteration of the timing of recrudescence including vitellogenin production. Spawning was inhibited in a way that indicated that exposure to waterborne E2 may have ‘reset' the cycle of recrudescence toward the beginning of the oogenic cycle. Vitellogenin induction in male fathead minnows was strongly correlated with E2 exposure, but less so with egg production. The results of this experiment link a biochemical indicator of waterborne estrogen exposure, vitellogenin, with a reproductive performance indicator, egg production, an important parameter affecting fish populations in the environment.     

The effects of methimazole on development of the fathead minnow, Pimephales promelas, from embryo to adult.

The importance of thyroid hormones in regulating early developmental processes of many amphibian and fish species is well known, but the impacts of exposure to disrupters of thyroid homeostasis during the embryo-larval-juvenile transitions are unclear. To investigate these impacts, fathead minnows, Pimephales promelas, were exposed to a model thyroid axis disrupter, methimazole, an inhibitor of thyroid hormone synthesis, at control (0), 32, 100, and 320 mug/l, starting at <24-h postfertilization, for 28, 56, and 83/84 days postfertilization (dpf). Thyroid disruption was evident at 28 dpf, when survival was significantly reduced by 32 or 100 mug/l methimazole concomitant with a reduced thyroxine (T(4)) content. However, the T(3) content of these fish was similar to that of control fish, and body mass was unaffected (as in all groups), suggesting compensatory mechanisms overcame reduced T(4) synthesis. At the highest concentration of methimazole (320 mug/l), activation of feedback mechanisms on the hypothalamic-pituitary-thyroid axis was suggested by the normal T(4) content after 28 dpf exposure to methimazole, although triiodothyronine (T(3)) content of these fish was significantly reduced. The generally less pronounced disruption of thyroid hormone homeostasis after 56 days exposure to methimazole also suggests compensatory mechanisms in juvenile/adult fish that may regulate T(4) content, despite exposure to methimazole at 32 or 100 mug/l (in fish held in 320 mug/l methimazole, the T(4) content was significantly higher than in controls). Whole body T(3) content at 56 dpf was significantly depressed only in fish held in 100 mug/l methimazole. By 83/84 dpf, length, body mass, and thyroid hormone concentrations were similar in all experimental groups and controls, indicating that adult fish may achieve regulation of their thyroid axis despite prolonged exposures to thyroid disruptors throughout early development.     

Effects of environmental exposure to diazepam on the reproductive behavior of fathead minnow,Pimephales promelas

Pharmaceutical drugs are continuously discharged into the aquatic environment primarily through wastewater discharge; therefore, their possible effects on wildlife is a reason of concern. Diazepam is a widely prescribed benzodiazepine drug used to treat insomnia and anxiety disorders, and it has been found in wastewater effluents worldwide. The present study tested the effects of diazepam on fecundity and the reproductive behavior of the fathead minnow, Pimephales promelas, a fish that exhibits male parental care. Sexually mature fathead minnows were housed at a ratio of one male and two females per tank and exposed to nominal (measured) concentrations of 0, 0.1 (0.14 ± 0.06), 1.0 (1.04 ± 0.15), 10 (13.4 ± 1.5) µg L^?1 for 21 days. Fish receiving the low diazepam treatment had significantly larger clutches than fish receiving the highest concentration but neither were different from controls. Diazepam exposure was not associated with a significant change in fertilization rate, hatchability or time to hatch, but a trend toward a higher number of eggs/day was observed in fish exposed to the low diazepam concentration relative to those exposed to the medium concentration. There were no significant differences in any of the behaviors analyzed when responses were averaged over time. The results showed that exposure to diazepam at concentrations as high as 13 µg L^?1 did not significantly impact the reproductive behavior of fathead minnow. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 561–568, 2016.     

Effects of estrogens and antiestrogens on gene expression of fathead minnow (Pimephales promelas) early life stages

Endocrine disrupting chemicals (EDCs) are known to contaminate aquatic environments and alter the growth and reproduction of organisms. The objective of this study was to evaluate the sensitivity and utility of fathead minnow (Pimephales promelas) early life‐stages as a model to measure effects of estrogenic and antiestrogenic EDCs on physiological and gene expression endpoints relative to growth and reproduction. Embryos (<24‐h postfertilization, hpf) were exposed to a potent estrogen (17α‐ethinyl estradiol, EE₂, 2, 10, and 50 ng L^?1); a weak estrogen (mycotoxin zearalenone, ZEAR, same concentrations as above); an antiestrogen (ZM 189, 154; 40, 250, and 1000 ng L^?1); and to mixtures of EE₂ and ZM until swim‐up stage (～170 hpf). Exposure to all concentrations of ZEAR and to the lowest concentration of ZM resulted in increased body sizes, whereas high concentrations of EE₂ decreased body sizes. There was a significant increase in the frequency of abnormalities (mostly edema) in larvae exposed to all concentrations of EE₂, and high ZEAR, and EE₂ + ZM mixture groups. Expression of growth hormone was upregulated by most of the conditions tested. Exposure to 50 ng L^?1 ZEAR caused an induction of insulin‐like growth factor 1, whereas exposure to 40 ng L^?1 ZM caused a downregulation of this gene. Expression of steroidogenic acute regulatory protein gene was significantly upregulated after exposure to all concentrations of EE₂ and luteinizing hormone expression increased significantly in response to all treatments tested. As expected, EE₂ induced vitellogenin expression; however, ZEAR also induced expression of this gene to similar levels compared to EE₂. Overall, exposure to EE₂ + ZM mixture resulted in a different expression pattern compared to single exposures. The results of this study suggest that an early life stage 7‐day exposure is sufficient to recognize and evaluate effects of estrogenic compounds on gene expression in this fish model. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

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.     

The toxicity of acetylenic alcohols to the fathead minnow, Pimephales promelas: narcosis and proelectrophile activation

1. The 96-h LC50 values for 16 acetylenic alcohols in the fathead minnow (Pimephales promelas) were determined using continuous-flow diluters. The measured LC50 values for seven tertiary propargylic alcohols agreed closely with the QSAR predictions based upon data for other organic non-electrolytes acting by a narcosis mechanism. 2. Four primary and four secondary propargylic alcohols were 7 to 4600 times more toxic than the respective narcotic toxicity estimated by QSAR. Metabolic activation to electrophilic alpha,beta-unsaturated propargylic aldehydes or ketones is proposed to account for the increased toxicity. 3. 3-Butyn-1-ol and 4-pentyn-2-ol, primary and secondary homopropargylic alcohols, were 320 and 160, respectively, times more toxic than predicted. In this case an activation step involving biotransformation to an allenic electrophile intermediate was proposed.     

Exposure to exogenous 17beta-oestradiol disrupts p450aromB mRNA expression in the brain and gonad of adult fathead minnows (Pimephales promelas)

Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (P450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17β-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of P450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on P450aromB mRNA expression in the gonads; E2 up-regulated P450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, P450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on P450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish.     

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.     

Life-cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and demasculinizes males

Forty-eight hours after fertilization, fathead minnow (Pimephales promelas) eggs were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE2) at nominal concentrations of 0.32 and 0.96 ng/L and measured concentrations of 3.5, 9.6, and 23 ng/L. The fish were observed through the larval, juvenile, and adult stages. Growth, secondary sex characteristics, the liver somatic index, the gonadosomatic index, and fecundity were examined after several lengths of exposure. No significant changes were seen in fry or juvenile growth from 8 to 30 days posthatch (dph). An increase in the ovipositor index (a female secondary sex characteristic) was the most sensitive early response at 60 dph and was seen in fish exposed to EE2 concentrations > or = 3.5 ng/L. Continuation of the EE2 exposure until 150 dph, through maturation and reproduction, allowed measurement of two sensitive end points: decreased egg fertilization and sex ratio (skewed toward females), both of which were significantly affected at the lowest EE2 concentration tested, 0.32 ng/L. The next most sensitive end point was demasculinization (decreased male secondary sex characteristic index) of males exposed to an EE2 concentration of 0.96 ng/L. The effects of low concentrations of EE2 (0.32 and 0.96 ng/L) were manifested in male fish (decreased male sex characteristics and reduced egg fertilization success), whereas female fish showed no changes in the gonadosomatic index. Exposure to higher EE2 concentrations negatively affected females, as shown by a reduced gonadosomatic index at 150 dph in fish exposed to > or =3.5 ng/L EE2. Although there were some end points that showed changes at 60 dph, the reproductive end points and external sex characteristics measured in mature fish at 150 dph were more sensitive, with response thresholds of EE2 ranging from 0.32 to 0.96 ng/L. The concentrations of EE2 that negatively affected fathead minnows were similar to or lower than those detected in many municipal wastewater effluents. In conclusion, life-cycle exposure of fathead minnows proved to be a very sensitive bioassay, and responses were seen at concentrations of less than 1 ng/L, which are environmentally relevant concentrations of EE2.     

Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas).

Cytochrome P450 aromatase (CYP19) is a key enzyme in vertebrate steroidogenesis, catalyzing the conversion of C19 androgens to C18 estrogens such as beta-estradiol (E2). The objective of this study was to assess effects of the CYP19 inhibitor fadrozole on fathead minnow (Pimephales promelas) reproductive endocrinology and physiology in a short-term reproduction assay proposed for identifying specific classes of endocrine-disrupting chemicals. A concentration-dependent reduction in fecundity was observed in fish exposed for 21 days to water concentrations of fadrozole ranging from 2 to 50 microg/l. Consistent with the expected mechanism of action, there was a significant inhibition of brain aromatase activity in both male and female fathead minnows exposed to fadrozole. In females, this inhibition was accompanied by a concentration-dependent decrease in plasma E2 and vitellogenin concentrations; the latter observation is consistent with the fact that activation of the estrogen receptor by E2 initiates hepatic vitellogenin production in oviparous vertebrates. Histological assessment of ovaries from females exposed to fadrozole indicated a decrease in mature oocytes and an increase in preovulatory atretic follicles. Exposure of male fathead minnows to fadrozole significantly increased plasma concentrations of the androgens testosterone (T) and 11-ketotestosterone (KT) and resulted in a marked accumulation of sperm in the testes. Results of this study indicate that the proposed fathead minnow assay should effectively identify test chemicals as potential aromatase inhibitors, both in the context of their reproductive toxicity and the specific mechanism of action. These results also should be of utility in assessing the potential ecological risk of CYP19 inhibitors, in particular in the context of relating alterations in subcellular indicators of endocrine function (changes in steroids, proteins) to adverse consequences in the whole organism.     

Defining the chronic impacts of atenolol on embryo-larval development and reproduction in the fathead minnow (Pimephales promelas)

Atenolol is a beta-adrenergic receptor antagonist ('beta-blocker') widely used for the treatment of angina, glaucoma, high blood pressure and other related conditions. Since atenolol is not appreciably metabolized in humans, the parent compound is the predominant excretory product, and has been detected in sewage effluent discharges and surface waters. Consequently, atenolol has been chosen as a reference pharmaceutical for a European Union-funded research consortium, known as ERAPharm (http://www.erapharm.org), which focused on the fate and effects of pharmaceuticals in the environment. Here, we present data generated within this project from studies assessing population-relevant effects in a freshwater fish species. Using fathead minnows (Pimephales promelas) as a standard OECD test species, embryo-larval development (early life stage or ELS) and short-term (21 d) adult reproduction studies were undertaken. In the ELS study, the 4d embryo NOEC(hatching) and LOEC(hatching) values were 10 and >10mg/L, respectively, and after 28 d, NOEC(growth) and LOEC(growth) values were 3.2 and 10mg/L, respectively (arithmetic mean measured atenolol concentrations were >90% of these nominal values). In the short-term reproduction study, NOEC(reproduction) and LOEC(reproduction) values were 10 and >10mg/L, respectively (mean measured concentrations were 77-96% of nominal values), while the most sensitive endpoint was an increase in male fish condition index, giving NOEC(condition index) and LOEC(condition index) values of 1.0 and 3.2mg/L, respectively. The corresponding measured plasma concentration of atenolol in these fish was 0.0518 mg/L. These data collectively suggest that atenolol has low chronic toxicity to fish under the conditions described, particularly considering the low environmental concentrations reported. These data also allowed the assessment of two theoretical approaches proposed as predictors of the environmental impact of human pharmaceuticals: the Huggett 'mammalian-fish leverage model'; and the acute:chronic ratio (ACR). The Huggett model gave a measured human: fish effect ratio (ER) of 19.3 for atenolol, which compared well with the predicted ER of 40.98. Moreover, for an ER of 19.3, the model suggests that chronic testing may be warranted, and from our resultant effects data, atenolol does not cause significant chronic effects in fathead minnow at environmentally realistic concentrations. The calculated ACR for atenolol is >31.25, which is far lower than that of 17 alpha-ethinylestradiol and other potent steroidal oestrogens, thus further supporting the observed low toxicity. The data produced for atenolol here fit well with both approaches, but also highlight the importance of generating 'real' experimental data with which to calibrate and validate such models.     

Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas).

Many chemicals that adversely affect reproduction and/or development do so through multiple pathways within the reproductive tract and hypothalamic-pituitary-gonadal axis. Notable in this regard are fungicides, such as prochloraz or fenarimol, which in mammals have the potential to impact endocrine function through inhibition of CYP enzymes involved in steroid metabolism, as well as through antagonism of the androgen receptor(s). The objective of our studies was to assess the effects of prochloraz and fenarimol on reproductive endocrine function in a model small fish species, the fathead minnow (Pimephales promelas), using both in vitro and in vivo assays. The two fungicides inhibited in vitro CYP19 aromatase activity in brain and ovarian homogenates from the fish, with prochloraz exhibiting a greater potency than fenarimol. Prochloraz and fenarimol also bound competitively to the cloned fathead minnow androgen receptor expressed in COS-1 cells. The two fungicides significantly reduced fecundity of the fish in a 21-day reproduction assay at water concentrations of 0.1 (prochloraz) and 1.0 (fenarimol) mg/l. The in vivo effects of prochloraz on plasma steroid (17beta-estradiol, testosterone, 11-ketotestosterone) and vitellogenin (an estrogen-responsive protein) concentrations, as well as on gonadal histopathology, were consistent with inhibition of steroidogenesis. Fenarimol also affected several aspects of endocrine function in vivo; however, the suite of observed effects did not reflect either aromatase inhibition or androgen receptor antagonism. These studies contribute to a better mechanistic understanding of the extrapolation of effects of endocrine-disrupting chemicals across vertebrate classes.     

Assessing effects of a mining and municipal sewage effluent mixture on fathead minnow (Pimephales promelas) reproduction using a novel, field-based trophic-transfer artificial stream

The Junction Creek watershed, located in Sudbury, ON, Canada receives effluent from three metal mine wastewater treatment plants, as well as a municipal wastewater (MWW) discharge. Effects on fish have been documented within the creek (decreased egg size and increased metal body burdens). It has been difficult to identify the cause of the effects observed due to the confounded nature of the creek. The objectives of this investigation were to assess the: (1) effects of a mine effluent and municipal wastewater (CCMWW) mixture on fathead minnow (FHM; Pimephales promelas) reproduction in an on-site artificial stream and (2) importance of food (Chironomus tentans) as a source of exposure using a trophic-transfer system. Exposures to CCMWW through the water significantly decreased egg production and spawning events. Exposure through food and water using the trophic-transfer system significantly increased egg production and spawning events. Embryos produced in the trophic-transfer system showed similar hatching success but increased incidence and severity of deformities after CCMWW exposure. We concluded that effects of CCMWW on FHM were more apparent when exposed through the water. Exposure through food and water may have reduced effluent toxicity, possibly due to increased nutrients and organic matter, which may have reduced metal bioavailability. More detailed examination of metal concentrations in the sediment, water column, prey (C. tentans) and FHM tissues is recommended to better understand the toxicokinetics of potential causative compounds within the different aquatic compartments when conducting exposures through different pathways.     

Validation of the REA bioassay to detect estrogenic activity in the water cycle

Endocrine disrupting compounds (EDCs) with estrogenic potency contaminate water and might eventually cause adverse effects to the aquatic environment. Many estrogenic compounds are not completely removed by wastewater treatment systems and, together with the run-off from agricultural areas, they enter surface waters. Chemical analytical methods to determine these compounds are usually expensive and laborious. Therefore, screening bioassays which are able to detect compounds based on their effects offer a solution for prior selection of samples that need to be chemically analyzed. In this study, the REA (RIKILT yeast Estrogen bioAssay), which has been developed to detect estrogenic compounds in calf urine and animal feed at RIKILT, is validated at the Water Board Laboratory of Waterproef for water samples. According to EC Decision 2002/657, detection capability CCβ, specificity and stability have to be determined for the internal validation of a qualitative screening test. In addition, surface water and effluent samples were analyzed to further demonstrate the applicability of the validated test procedure. Results demonstrate that the REA assay is reproducible and specific for estrogenic compounds in water and meets the criteria as prescribed in EC Decision 2002/657. The assay was sensitive enough to detect estrogenic activity of pollutants in water with a limit of quantification (LOQ) below 1 ng EEQ/L. This means that samples can be compared with preliminary threshold levels for drinking water and surface waters (7 and 1 ng EEQ/L, respectively). The stability of estrogenic activity in water samples is at least 4 weeks, when stored at 4 °C.