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.

Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows (Pimephales promelas).

Laboratory studies were conducted to investigate potential adverse effects on development, growth, reproduction and biomarker responses (vitellogenin [VTG] and gonad histology) in fathead minnows (Pimephales promelas) exposed to tamoxifen citrate. Based on the results of a partial life cycle study (nominal [mean measured] concentrations ranged from 0.18 [0.11] to 18 [15.74] microg/L), a 284-d fish full life-cycle (FFLC) flow-through study was conducted using newly fertilized embryos (<24 h postfertilization) exposed to nominal (mean measured) concentrations of 14C-tamoxifen citrate that ranged from 0.01 (0.007) to 5.12 (4.08) microg/L. Triethylene glycol (2.0 microl/L) was used as a solvent carrier, with 17beta-estradiol (E2) as a positive control (nominal 0.1 microg/L). Among the biomarkers measured, significant effects on VTG and gonad histology were observed, although these results required care in their interpretation. Among important population-relevant endpoints, no effects on reproduction were observed at nominal concentrations < or = 5.12 microg/L. Effects on growth (length and weight) were observed in some treatments; however, some of these showed irregular concentration-response relationships, which made interpretation uncertain, or were deemed transient in nature (e.g., reduction in growth of F1 28-d posthatch larval fish at nominal concentrations of 0.08, 0.64, and 5.12 microg/L) and judged not to be biologically significant. Interpretation of results from fish chronic studies is challenging and frequently calls for scientific judgement about statistical and biological significance and what constitutes an adverse effect. Using the principles used in mammalian toxicology studies, data from partial and FFLC studies were evaluated from both statistical and biological perspectives in order to determine no-observed-adverse effect concentrations (expressed as (adverse)NOEC) for use in environmental risk assessment. Careful consideration of both biological and statistical outcomes from these studies suggested overall (adverse)NOEC concentration and lowest-observed-effect concentration ((adverse)LOEC) values for tamoxifen citrate of 5.12 microg/L and 5.6 microg/L, respectively.

Qualitative and quantitative histomorphologic assessment of fathead minnow Pimephales promelas gonads as an endpoint for evaluating endocrine-active compounds: a pilot methodology study.

Although histopathology is routinely employed as a tool for the detection and assessment of xenobiotic-mediated effects in mammals, it is less frequently applied to fish. In part, this is due to a lack of method standardization regarding study design, tissue preservation, tissue sectioning, histopathological evaluation, reporting, and statistical analysis. The objectives of the present study were: (1) to test and refine a method for the microsurgical excision of fathead minnow (FHM) Pimephales promelas gonads for the purpose of histopathologic examination; (2) to determine the optimal combination of fixation and embedding procedures for the histopathologic and morphometric analysis of FHM gonads following exposure to a known estrogenic compound, 17beta-estradiol (E2); and (3) to provide a method for the categorization and quantification of cell types in FHM gonads by manually counting cells in digitized images using image analysis software. The light microscopic evaluation of individual gametogenic cells was greatly facilitated by specimen preparation techniques that included the excision of gonads via microdissection and by optimized fixation and embedding procedures.

Assessing chronic toxicity of bisphenol A to larvae of the African clawed frog (Xenopus laevis) in a flow-through exposure system.

A number of currently used industrial chemicals are estrogenic, and therefore have potential to disrupt sexual differentiation in vertebrate wildlife during critical developmental windows. We assessed the effect of larval exposure to bisphenol A (BPA) on growth, development and sexual differentiation of the gonad in the African Clawed frog, Xenopus laevis. Larvae were maintained in flow-through conditions at 22 +/- 1 degrees C and exposed to BPA at mean measured concentrations of 0.83, 2.1, 9.5, 23.8, 100, and 497 microg/l, from developmental stages 43/45-66 (completion of metamorphosis). Each test concentration, plus dilution water control (DWC) and positive control (17beta-estradiol (E2), 2.7 microg/l) employed four replicate test vessels with 40 larvae per tank. Individual froglets were removed from test vessels upon reaching stage 66, and the study was terminated at 90 days. Froglets were dissected and sex was determined by inspection of gross gonadal morphology. Test concentrations of BPA had no effect on survival, growth, developmental stage distributions at exposure days 32 and 62, or mean time to completion of metamorphosis, compared to DWC. Analysis of post-metamorphic sex ratio, determined by gross gonadal morphology, indicated no significant deviations from expected (50:50) sex ratio, in DWC or any BPA test concentration. In contrast, exposure of larvae to (E2) resulted in feminisation, with sex ratio deviating significantly (31% male, replicates pooled). Exposure to BPA in the concentration range 0.83-497 microg/l in flow-through conditions had no observable effect on larval growth, development or sexual differentiation (as determined by gross gonadal morphology) in this study.