Daniel C. Rearick

Phytoestrogens in the environment, I: Occurrence and exposure effects on fathead minnows

Naturally occurring phytoestrogens may mimic biogenic estrogens and modulate endocrine action in vertebrates. Little is known, however, about their temporal and spatial variability in the environment and the biological effects associated with exposures. The present study assessed the environmental presence of phytoestrogens in human-impacted and relatively pristine areas. The response in larval and sexually mature fathead minnows to environmentally relevant concentrations of 3 common phytoestrogens (genistein, daidzein, and formononetin), both singly and in mixture, was also quantified. Phytoestrogens were only present in the human-impacted surface waters. When detected, mean concentrations were low (± standard deviation) in an urban lake: 1.4 ± 0.5 ng/L, 1.6 ± 0.7 ng/L, and 1.1 ± 0.2 ng/L for genistein, daidzein, and formononetin, respectively, and in treated wastewater effluent: 1.6 ± 0.4 ng/L, 1.8 ± 1.3 ng/L, and 2.0 ng/L. Biochanin A was detected twice, whereas zearalenone and coumestrol were never detected. No clear temporal trends of aqueous phytoestrogen concentration were evident. Larval survival was significantly reduced in genistein, formononetin, and mixture treatments, whereas adult male fish only exhibited subtle changes to their anatomy, physiology, and behavior. Daidzein-exposed adult females produced greater quantities of eggs. The present study indicates that genistein, daidzein, and formononetin are likely attenuated rapidly and are unlikely to cause widespread ecological harm in the absence of other stressors.

Complex mixtures, complex responses: Assessing pharmaceutical mixtures using field and laboratory approaches

Pharmaceuticals are present in low concentrations (<100 ng/L) in most municipal wastewater effluents but may be elevated locally because of factors such as input from pharmaceutical formulation facilities. Using existing concentration data, the authors assessed pharmaceuticals in laboratory exposures of fathead minnows (Pimephales promelas) and added environmental complexity through effluent exposures. In the laboratory, larval and mature minnows were exposed to a simple opioid mixture (hydrocodone, methadone, and oxycodone), an opioid agonist (tramadol), a muscle relaxant (methocarbamol), a simple antidepressant mixture (fluoxetine, paroxetine, venlafaxine), a sleep aid (temazepam), or a complex mixture of all compounds. Larval minnow response to effluent exposure was not consistent. The 2010 exposures resulted in shorter exposed minnow larvae, whereas the larvae exposed in 2012 exhibited altered escape behavior. Mature minnows exhibited altered hepatosomatic indices, with the strongest effects in females and in mixture exposures. In addition, laboratory-exposed, mature male minnows exposed to all pharmaceuticals (except the selective serotonin reuptake inhibitor mixture) defended nest sites less rigorously than fish in the control group. Tramadol or antidepressant mixture exposure resulted in increased splenic T lymphocytes. Only male minnows exposed to whole effluent responded with increased plasma vitellogenin concentrations. Female minnows exposed to pharmaceuticals (except the opioid mixture) had larger livers, likely as a compensatory result of greater prominence of vacuoles in liver hepatocytes. The observed alteration of apical endpoints central to sustaining fish populations confirms that effluents containing waste streams from pharmaceutical formulation facilities can adversely impact fish populations but that the effects may not be temporally consistent. The present study highlights the importance of including diverse biological endpoints spanning levels of biological organization and life stages when assessing contaminant interactions.

Phytoestrogens in the environment, II: Microbiological degradation of phytoestrogens and the response of fathead minnows to degradate exposure

Phytoestrogens are endocrine active compounds derived from plants, including the isoflavones genistein and daidzein, and their methylated derivatives biochanin A and formononetin. These compounds have been detected at the µg/L level in the effluents of plant-processing industries and municipal treatment plants and at the ng/L level in surface waters worldwide. The present study assessed the persistence of genistein and daidzein in natural aquatic systems, specifically riverine samples. Initial concentration, temperature, sample location, and time of sample collection varied. Genistein and daidzein were found to be readily biodegradable at all tested concentrations, at both 10 °C and 20 °C, in samples collected during different seasons, and in samples from 3 different rivers. In addition, organismal responses in larval and sexually mature fathead minnows (Pimephales promelas) were quantified following exposure to microbiologically degraded phytoestrogens (genistein, daidzein, and formononetin). Products of the microbiological degradation of parent phytoestrogens did not affect larval survival, growth, or predator avoidance. Female adult fathead minnows exposed to these degradation products produced significantly fewer eggs than those exposed to a control, but no other morphological, physiological, or behavioral changes were observed with male or female minnows. The present research suggests that although phytoestrogens are not likely to persist in aquatic systems, they may pseudo-persist if discharges are continuous; in addition, caution should be exercised with respect to high-concentration effluents because of the potentially antiestrogenic effects of phytoestrogen degradates.

Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 2. Temperature and food availability.

Fish are subject to constantly changing environmental conditions and food availability, factors that may impact their response to endocrine disruptors (EDs). This may, in part, explain outcome discrepancies between field studies and laboratory exposures to EDs. This study assessed whether standard laboratory conditions for fish exposures adequately represent effects of ED exposure at two environmentally realistic temperatures. The impact of temperature and food availability on male fathead minnow response to estrone (E1) exposure was studied in two experiments (3×2×2 factorial design) with three E1 concentrations (range 0-135ng/L); two temperatures (18°C and 26°C, the latter the prescribed laboratory temperature), and two feeding treatments (full fed vs. 25% of full fed) in a 21-day flow-through system. Morphometric endpoints [including body condition factor, somatic index of gonad (GSI) and liver (HSI), and secondary sex characteristics (SSC)], blood parameters [hematocrit (HCT), blood glucose, cortisol, and vitellogenin (VTG) concentrations], and histology of liver and testis were determined on day 22. High E1 consistently increased VTG, though interactions among E1, temperature and/or food on liver weight, HSI, and HCT were inconsistent between experiments. High temperature impacted the greatest number of parameters, independent of E1 treatment. Three sex-linked parameters were lower at high temperature (testis weight, GSI and VTG), and in Exp. 2SSC and gonad maturity rating were lower. At 26°C, in Exp. 1 HSI and HCT decreased, and in Exp. 2 length, body and liver weight, and body condition factor were lower. Food restriction decreased GSI in Exp. 1, and blood glucose and liver weight in Exp. 2. At 26°C several parameters were altered independent of E1 exposure, including three out of four measurements of sperm differentiation. Concordance between laboratory and field investigations of the biological effects of EDs may improve if environmentally-relevant exposure conditions, especially temperature, are employed.

Sorption of isoflavones to river sediment and model sorbents and outcomes for larval fish exposed to contaminated sediment

Isoflavones are compounds whose presence in the aquatic environment is increasingly recognized and may be of concern due to their potential to act as endocrine disruptors. Sorption to particles may be a relevant removal mechanism for isoflavones. This work investigated the influence of pH, ionic strength, and sediment composition on sorption of genistein and daidzein, two key isoflavones, using sorption isotherms and edges. The effect of sorbed isoflavones on the survival, growth, and predator avoidance performance of larval fathead minnows (Pimephales promelas) was assessed. Sorption to goethite and kaolinite was pH-dependent, with a maximum near pH 7 for both compounds. Sorption to montmorillonite was ionic-strength dependent but largely pH-independent. Overall, sorption to sediments is likely to sequester less than 5% of isoflavones in a discharge. No statistically significant effects were observed for larvae exposed to sorbed isoflavones, suggesting that sorption to sediments reduces exposure to isoflavones.

Phytoestrogens in the environment, II: Microbiological degradation of phytoestrogens and the response of fathead minnows to degradate exposure: Biodegradation of phytoestrogens and biological effects

Phytoestrogens are endocrine active compounds derived from plants, including the isoflavones genistein and daidzein, and their methylated derivatives biochanin A and formononetin. These compounds have been detected at the µg/L level in the effluents of plant-processing industries and municipal treatment plants and at the ng/L level in surface waters worldwide. The present study assessed the persistence of genistein and daidzein in natural aquatic systems, specifically riverine samples. Initial concentration, temperature, sample location, and time of sample collection varied. Genistein and daidzein were found to be readily biodegradable at all tested concentrations, at both 10 °C and 20 °C, in samples collected during different seasons, and in samples from 3 different rivers. In addition, organismal responses in larval and sexually mature fathead minnows (Pimephales promelas) were quantified following exposure to microbiologically degraded phytoestrogens (genistein, daidzein, and formononetin). Products of the microbiological degradation of parent phytoestrogens did not affect larval survival, growth, or predator avoidance. Female adult fathead minnows exposed to these degradation products produced significantly fewer eggs than those exposed to a control, but no other morphological, physiological, or behavioral changes were observed with male or female minnows. The present research suggests that although phytoestrogens are not likely to persist in aquatic systems, they may pseudo-persist if discharges are continuous; in addition, caution should be exercised with respect to high-concentration effluents because of the potentially antiestrogenic effects of phytoestrogen degradates.

Phytoestrogens in the environment, II

Phytoestrogens are endocrine active compounds derived from plants, including the isoflavones genistein and daidzein, and their methylated derivatives biochanin A and formononetin. These compounds have been detected at the µg/L level in the effluents of plant-processing industries and municipal treatment plants and at the ng/L level in surface waters worldwide. The present study assessed the persistence of genistein and daidzein in natural aquatic systems, specifically riverine samples. Initial concentration, temperature, sample location, and time of sample collection varied. Genistein and daidzein were found to be readily biodegradable at all tested concentrations, at both 10 °C and 20 °C, in samples collected during different seasons, and in samples from 3 different rivers. In addition, organismal responses in larval and sexually mature fathead minnows (Pimephales promelas) were quantified following exposure to microbiologically degraded phytoestrogens (genistein, daidzein, and formononetin). Products of the microbiological degradation of parent phytoestrogens did not affect larval survival, growth, or predator avoidance. Female adult fathead minnows exposed to these degradation products produced significantly fewer eggs than those exposed to a control, but no other morphological, physiological, or behavioral changes were observed with male or female minnows. The present research suggests that although phytoestrogens are not likely to persist in aquatic systems, they may pseudo-persist if discharges are continuous; in addition, caution should be exercised with respect to high-concentration effluents because of the potentially antiestrogenic effects of phytoestrogen degradates.