Complexation and time-dependent accumulation of copper by larval fathead minnows (Pimephales promelas): implications for modeling toxicity

Mechanistic models predicting copper (Cu) toxicity to aquatic biota in natural waters require organic and inorganic water chemistry, and quantified values for Cu binding by sensitive biological receptors. In bioaccumulation experiments using larval fathead minnows (FHM; Pimephales promelas), we investigated time to asymptotic accumulation of Cu and quantified the conditional stability constants (binding affinity; log K(Cu-FHM)) and binding-site densities of Cu-FHM complexation. Cu bioaccumulation increased rapidly, approaching an asymptote in exposures longer than 12 h, indicating that Cu loading at 24 h is an appropriate exposure duration for modeling Cu complexation by larval FHM. Results of Langmuir and Scatchard analyses of other bioaccumulation experiments produced log K(Cu-FHM) values of 6.52, and binding-site densities of 0.39 micromol g(-1)dry weight. These whole-body log K(Cu-FHM) values are approximately an order of magnitude lower than those reported for adult FHM gills. However, binding-site densities for larval and adult FHM are similar. Under similar test conditions, comparable concentrations of aqueous Cu cause 50% mortality in adult and larval FHM suggesting that binding site densities determine comparable metal-tissue loadings and have greater influence on Cu bioavailability than binding affinity.     

Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas)

A lack of knowledge persists concerning the combination of kinetics on protein and mRNA levels of the most commonly used biomarker for estrogenic influences-vitellogenin (VTG). Consequently, male fathead minnows were exposed to 17alpha-ethinylestradiol (EE(2)) for 35 days, followed by an equally long depuration period in a flow-through system. VTG mRNA levels reached a plateau after 3 days of exposure, which remained stable until 3 days after EE(2) removal. Control levels were re-attained within 7 days of the depuration phase. VTG protein accumulated in the plasma following a two-phased model. The first phase depicting an exponential increase lasted 15 days and was followed by a saturation phase approaching a plateau of approximately 47 mg VTG/ml plasma. Clearance kinetics could be described by a two-compartment open model, with half-lives of 2.17 and 21.32 days for the alpha- and beta-phases, respectively. In addition, a high VTG protein synthesis rate seemed to adversely affect fitness and mortality of the fish.     

Influence of temperature and dissolved oxygen on the acute toxicity of profenofos to fathead minnows (Pimephales promelas)

The purpose of this study was to investigate the influence of temperature and dissolved oxygen levels on the acute toxicity of profenofos to fathead minnows (Pimephales promelas). Exposure conditions were as follows: normal temperature and normal dissolved oxygen (NTNO; 20 +/- 2 degrees C and 6.0-9.0 mg/L, respectively); normal temperature and low dissolved oxygen (NTLO; 20 +/- 2 degrees C and 1.7-2.6 mg/L, respectively); high temperature and normal dissolved oxygen (HTNO; 30 +/- 2 degrees C and 6.6-6.9 mg/L, respectively); high temperature and low dissolved oxygen (HTLO; 30 +/- 2 degrees C and 1.5-3.0 mg/L, respectively). Initial 96-h acute toxicity studies with profenofos were conducted at NTNO and HTLO exposure conditions. The 96-h LC50 at NTNO was 333 micrograms/L with 95% confidence limits ranging from 244 to 558 micrograms/L. However, the 96-h LC50 at HTLO was significantly lower at 21.5 micrograms/L with 95% confidence limits ranging from 17.4 to 28.8 micrograms/L. Acetylcholinesterase (AChE) activity was measured in the head and torso of surviving fish at 96-h. A weak dose-related decrease in AChE was observed at NTNO but no dose-response relationship was observed at HTLO exposure condition. Additional experiments were conducted using 50 micrograms/L profenofos at NTNO, NTLO, HTNO, and HTLO exposure conditions. Mortality, sublethal effects (erratic and hyperactive swimming), and AChE activity in the head and torso were measured at 2, 4, and 12-h following exposure to profenofos. No mortality or significant sublethal effects were observed in controls or profenofos-treated groups in NTNO and NTLO exposure conditions. However, significant mortality and sublethal effects were observed in profenofos-treated fish in HTNO at 12 h and at all time points in HTLO. Both high temperature and low dissolved oxygen, as well as combinations of high temperature and low dissolved oxygen significantly decreased AChE activity in control fish. Exposure to 50 micrograms/L profenofos in all exposure conditions further decreased AChE activity, but no apparent correlations between mortality and AChE activity were observed. These results suggest that the acute toxicity of profenofos to fathead minnows may be exacerbated during summer conditions in southern U.S. aquatic ecosystems.     

Lead-induced metabolic imbalances and feeding alterations in juvenile fathead minnows (Pimephales promelas)

Contaminant-induced feeding pattern changes may be a consequence or cause of changes in internal energy stores. Juvenile fathead minnows (Pimephales promelas; 25 ± 3 mm standard length) were exposed to 0.0., 0.5 or 1.0 ppm lead (Pb) during a 2-week preexposure and 2-week testing period (4 weeks total exposure). Changes in prey size selectivity, satiation levels, defecation rates, and weight were recorded. When simultaneously offered two prey sizes (10 2-day-old and 10 7-day-old Daphnia magna) control fish began switching from larger, more difficult-to-capture 7-day-old daphnids to smaller, easier-to-catch 2-day-old prey [analysis of variance (ANOVA) p < 0.06] by day 3. Fish exposed to 0.5 ppm Pb displayed a significant switching (ANOVA), p < 0.06) by day 10. Fish exposed to 1.0 ppm Pb did not significantly change their preference for 7-day-old daphnids. After 2 weeks of Pb exposure, control fish ate more flake food and did not satiate as quickly as exposed groups (ANOVA, p < 0.05). Fish exposed to 1.0 ppm Pb displayed reductions in amount of food remaining by day 4; control and 0.5 ppm Pb-exposed fish displayed a steady decline in the amount of food remaining. Daily defecation rates of Pb-exposed fish were significantly higher than controls (ANOVA, p < 0.05). Although all groups gained weight, there was a significantly smaller increase in Pb-exposed groups (ANOVA, p < 0.05). By altering prey size choices and rate of switching to energetically less costly prey, increasing perseveration (i.e., repeated efforts to capture single prey item) and visuo/psychomotor difficulty, shortening feeding bouts, and increasing defecation rates, Pb may cause metabolic imbalances in juvenile fish. © 1996 by John Wiley & Sons, Inc.     

Increased ovarian follicular apoptosis in fathead minnows (Pimephales promelas) exposed to dietary methylmercury

Exposure to environmentally relevant concentrations of dietary methylmercury impairs the reproduction of fish. Although specific mechanisms are unknown, recent research has linked altered reproduction in fish to the suppression of circulating levels of sex steroid hormones by methylmercury. We hypothesize that methylmercury induces apoptosis in steroidogenic gonadal cells in fish, thereby interfering with the synthesis of sex steroid hormones critical for the regulation of reproduction. To test this hypothesis, we chronically exposed fathead minnows (Pimephales promelas) to one of three diets contaminated with methylmercury: 0.06 microg Hg g(-1) (control), 0.87 microg Hg g(-1), and 3.93 microg Hg g(-1) dry weight. Apoptosis was evaluated histologically in ovaries of female fathead minnows by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Methylmercury significantly increased the number of apoptotic follicular cells in primary growth and cortical alveolus stage ovarian follicles. Ovarian follicular cells (i.e., granulosa, theca) are responsible for the production of 17beta-estradiol and other sex steroid hormones. Increased ovarian follicular apoptosis was related to suppressed 17beta-estradiol concentrations and smaller ovary size of female fathead minnows. Our results suggest increased apoptosis of steroidogenic gonadal cells as a possible mechanism for the suppression of sex steroid hormones and ultimately the impairment of reproduction in fish exposed to methylmercury.     

Histopathology of fathead minnow (Pimephales promelas) exposed to hydroxylated fullerenes

Hydroxylated fullerenes are reported to be very strong antioxidants, acting to quench reactive oxygen species, thus having strong potential for important and widespread applications in innovative therapies for a variety of disease processes. However, their potential for toxicological side effects is still largely controversial and unknown. Effects of hydroxylated fullerenes C₆₀(OH)₂₄ on the fathead minnow (Pimephales promelas) were investigated microscopically after a 72-hour (acute) exposure by intraperitoneal injection of 20 ppm of hydroxylated fullerenes per gram of body mass. Cumulative, semi-quantitative histopathologic evaluation of brain, liver, anterior kidney, posterior kidney, skin, coelom, gills and the vestibuloauditory system revealed significant differences between control and hydroxylated fullerene-treated fish. Fullerene-treated fish had much higher cumulative histopathology scores. Histopathologic changes included loss of cellularity in the interstitium of the kidney, a primary site of haematopoiesis in fish, and loss of intracytoplasmic glycogen in liver. In the coelom, variable numbers of leukocytes, including many macrophages and fewer heterophils and rodlet cells, were admixed with the nanomaterial. These findings raise concern about in vivo administration of hydroxylated fullerenes in experimental drugs and procedures in human medicine, and should be investigated in more detail.     

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.     

Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas)

Prochloraz is a fungicide known to cause endocrine disruption through effects on the hypothalamic-pituitary-gonadal (HPG) axis. To determine the short-term impacts of prochloraz on gene expression and steroid production, adult female fathead minnows (Pimephales promelas) were exposed to the chemical (0 or 300 μg/L) for a time-course of 6, 12 and 24 h. Consistent with inhibition of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19), known molecular targets of prochloraz, plasma 17β-estradiol (E2) was reduced within 6 h. Ex vivo E2 production was significantly reduced at all time-points, while ex vivo testosterone (T) production remained unchanged. Consistent with the decrease in E2 levels, plasma concentrations of the estrogen-responsive protein vitellogenin were significantly reduced at 24 h. Genes coding for CYP19, CYP17, and steroidogenic acute regulatory protein were up-regulated in a compensatory manner in ovaries of the prochloraz-treated fish. In addition to targeted quantitative real-time polymerase chain reaction analyses, a 15k feature fathead minnow microarray was used to determine gene expression profiles in ovaries. From time-point to time-point, the microarray results showed a relatively rapid change in the differentially expressed gene (DEG) profiles associated with the chemical exposure. Functional analysis of the DEGs indicated changes in expression of genes associated with cofactor and coenzyme binding (GO:0048037 and 0050662), fatty acid binding (GO:0005504) and organelle organization and biogenesis (GO:0006996). Overall, the results from this study are consistent with compensation of the fish HPG axis to inhibition of steroidogenesis by prochloraz, and provide further insights into relatively rapid, system-wide, effects of a model chemical stressor on fish.     

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.     

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.     

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.     

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.     

Successful detection of (anti-)androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development

Screening assays have been successfully developed for the detection of (anti-)oestrogenic substances in several fish species, including the fathead minnow (Pimephales promelas). Previous work suggested that pre-spawning adult fathead minnows might be an appropriate life-stage for developing a screen to detect endocrine active substances (EASs). Pre-spawning adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to three reference substances for 21 days, at 25 degrees C. Male and female fish, held in separate tanks, were exposed to dihydrotestosterone (DHT, androgen), flutamide (anti-androgen) and fadrozole (aromatase inhibitor). Nominal (mean measured) concentrations for DHT were 10 (6.0), 32 (6.1) and 100 (8.6) microg l(-1), for flutamide, 100 (95.3), 320 (320.4) and 1000 (938.6) microg l(-1) and for fadrozole, 25 (24.8), 50 (51.7) and 100 (95.5) microg l(-1). After 14 and 21 days exposure, fish were evaluated for growth, secondary sexual characteristics (SSCs, number and prominence of nuptial tubercles), gonadosomatic index (GSI) and plasma vitellogenin (VTG) concentrations. Development of nuptial tubercles was sensitive to both DHT and flutamide exposure. Exposure to DHT significantly increased the number of nuptial tubercles (male characteristic) in both males (more abundant) and females, after 14 days. Flutamide (938.6 microg l(-1), day 21) significantly reduced nuptial tubercle number in male fish. Fadrozole significantly inhibited ovarian growth (lower GSI) and significantly induced testis growth (51.7 and 95.5 microg l(-1)), after 21 days. Plasma VTG concentrations were significantly elevated in male fish (6.1 and 8.6 microg l(-1)), but inhibited in female fish (6.0 microg l(-1)), exposed to DHT. Flutamide had no effect on plasma VTG in male fish, but significantly induced VTG in female fish, after 21 days. Fadrozole significantly inhibited VTG in females and induced VTG synthesis in males, at day 21. These results show that SSCs, GSI and plasma VTG concentrations can be used in pre-spawning adult fathead minnows to screen for a range of classes of EASs. This work complements other published studies in supporting the current OECD effort towards validating a 21 days non-spawning fish screening assay for assessing (anti-)oestrogens, aromatase inhibitors and (anti-)androgens.     

Prediction of the acute toxicity (96-h LC50) of organic compounds to the fathead minnow (Pimephales promelas) using a group contribution method

A group contribution method has been developed to correlate the acute toxicity (96-h LC50) to the fathead minnow (Pimephales promelas) for 397 organic chemicals. Multilinear regression and computational neural networks (CNNs) were used for model building. The models were able to achieve a fairly good correlation of the data (r2 > 0.9). The linear model, which included four specific interaction terms, provided a rapid means of predicting the toxicity of a compound. The CNN model was able to yield virtually the same predictions with or without the four interaction terms that were included in the multilinear model.     

Associations between altered vitellogenin concentrations and adverse health effects in fathead minnow (Pimephales promelas)

Mechanism specific biomarkers are used in ecotoxicology to identify classes of chemicals and to inform on their presence in the environment, but their use in signalling for adverse effects has been limited by a poor understanding of their associated links with health. In this study an experimental analysis was undertaken to investigate how induction or suppression of an estrogen-dependent biomarker, vitellogenin (VTG), related to health effects in fathead minnow (Pimephales promelas, FHM). Exposure to an oestrogen agonist, estradiol (29 and 60 ng/L), resulted in rapid induction of VTG (elevated plasma concentrations within 2 days of exposure) in male FHM that was subsequently slow to clear from the plasma (concentrations remained elevated 70 days after cessation of exposure). The induction of VTG to concentrations of 0.5 mg/mL, however, and its continued presence in the plasma were not associated with any overt adverse health effects to the males. In contrast, induction of higher concentrations of VTG (>1 mg/mL) in reproductively active FHM exposed to estrone (307 and 781 ng/L), were associated with impacts on male survival (>33% male mortality) and an inhibitory effect on egg production in females (>51% decrease in egg number). Exposure of reproductively active FHM to a chemical that disrupts estrogen biosynthesis (an aromatase inhibitor; fenarimol 497 microg/L) also reduced reproductive success (40% decrease in egg number), and this was associated with a reduction in plasma VTG concentrations in females (36% decrease). These findings show that high level induction or suppression (in females) of plasma VTG are associated with alterations in health status and reproductive fitness. VTG, therefore, has the potential to act as a health measure as well as a biomarker for exposure, for chemicals that alter the oestrogen signalling pathway.