Structural dynamics of microbial communities in polycyclic aromatic hydrocarbon-contaminated tropical estuarine sediments undergoing simulated aerobic biotreatment.

Coastal sediments contaminated by polycyclic aromatic hydrocarbons (PAHs) can be candidates for remediation via an approach like land farming. Land farming converts naturally anaerobic sediments to aerobic environments, and the response of microbial communities, in terms of community structure alterations and corresponding effects on biodegradative activities, is unknown. A key goal of this study was to determine if different sediments exhibited common patterns in microbial community responses that might serve as indicators of PAH biodegradation. Sediments from three stations in the Lagos Lagoon (Nigeria) were used in microcosms, which were spiked with a mixture of four PAH, then examined for PAH biodegradation and for shifts in microbial community structure by analysis of diversity in PAH degradation genes and Illumina sequencing of 16S rRNA genes. PAH biodegradation was similar in all sediments, yet each exhibited unique microbiological responses and there were no microbial indicators of PAH bioremediation common to all sediments.

Suspended microfluidics.

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (µDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that µDots can also be used as a simple multiplexed 3D cellular growth platform. Using the µDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

Influence of Acidification on the Partitioning of Steroid Hormones among Filtrate, Filter Media, and Retained Particulate Matter.

Hormone contamination of aquatic systems has been shown to have deleterious effects on aquatic biota. However, the assessment of hormone contamination of aquatic environments requires a quantitative evaluation of the potential effects of sample preservation on hormone concentrations. This study investigated the influence of acidification (pH 2) of surface water samples on the partitioning of hormones among filtrate, filter media, and filter-retained particulate matter. Hormones were spiked into unpreserved and sulfuric acid-preserved ultrapure water and surface water runoff samples. The samples were filtered, and hormones were extracted from the filter and filtrate and analyzed by high-performance liquid chromatography. Acidification did not influence the partitioning of hormones onto the filter media. For the majority of the hormones investigated in this study, the partitioning of hormones to the filter-retained particulate matter was not influenced by acidification. Acidification increased the partitioning of progesterone and melengestrol acetate onto the retained particulate matter (about 25% for both analytes). Incorporation of an isotopically labeled internal standard (ISTD) for progesterone accounted for the loss of progesterone to the filter-retained particulates and resulted in accurate concentrations of progesterone in the filtrate. The incorporation of an ISTD for melengestrol acetate, however, was unable to account for the loss of melengestrol acetate to the retained particulates and resulted in underestimations of melengestrol acetate in the filtrate. Our results indicate that the analysis of melengestrol acetate in acid preserved surface runoff samples should be conducted on the filter-retained particulates as well as the filtrate.

Measurement of 25-hydroxyvitamin D(2&3) and 1,25-dihydroxyvitamin D(2&3) by tandem mass spectrometry: A primate multispecies comparison.

Vitamin D metabolites are widely studied for their roles in bone health, immune functions, and other potential physiologic roles in humans. However, the optimal blood levels of vitamin D metabolites are still unclear. Various methods for measuring vitamin D metabolites have been used and recently liquid chromatography tandem mass spectroscopy (LC-MS/MS) has been adopted as the gold standard for vitamin D metabolite measurement. Here, we report the use of LC-MS/MS to measure 25-hydroxyvitamin D (25(OH)D(2&3)), and 1,25-dihydroxyvitamin D (1,25(OH)2D(2&3)), in three laboratory nonhuman primate species: common marmoset (Callithrix jacchus), rhesus macaque (Macaca mulatta), and cynomolgus macaque (Macaca fascicularis), and compare them to humans using the same technique. The nonhuman primates showed blood levels for 25(OH)D3 and 1,25(OH)2D3 significantly higher than human values with marmosets having the highest levels. Marmoset samples showed significantly more variability among individuals than those from macaques for both metabolites, but all three nonhuman primate species exhibited large variation within species for both 25(OH)D(2&3) and 1,25(OH)2D(2&3). Marmoset females had significantly lower values than the males for 25(OH)D3, while rhesus males showed a significant decrease in 25(OH)D3 with age. The most striking finding is the variation within species for vitamin D levels even in laboratory primates that have a controlled diet, UV exposure, and in some cases, genetic constraints. Similar variation in 25(OH)D responses to a fixed dose of oral vitamin D supplementation has been reported in humans. We suggest that these species can provide primate models for examining the factors influencing variation in the levels of vitamin D necessary for human and nonhuman primate health.

Circulating serum xenoestrogens and mammographic breast density.

INTRODUCTION: Humans are widely exposed to estrogenically active phthalates, parabens, and phenols, raising concerns about potential effects on breast tissue and breast cancer risk. We sought to determine the association of circulating serum levels of these chemicals (reflecting recent exposure) with mammographic breast density (a marker of breast cancer risk). METHODS: We recruited postmenopausal women aged 55 to 70 years from mammography clinics in Madison, Wisconsin (N = 264). Subjects completed a questionnaire and provided a blood sample that was analyzed for mono-ethyl phthalate, mono-butyl phthalate, mono-benzyl phthalate, butyl paraben, propyl paraben, octylphenol, nonylphenol, and bisphenol A (BPA). Percentage breast density was measured from mammograms by using a computer-assisted thresholding method. RESULTS: Serum BPA was positively associated with mammographic breast density after adjusting for age, body mass index, and other potentially confounding factors. Mean percentage density was 12.6% (95% confidence interval (CI), 11.4 to 14.0) among the 193 women with nondetectable BPA levels, 13.7% (95% CI, 10.7 to 17.1) among the 35 women with detectable levels below the median (<0.55 ng/ml), and 17.6% (95% CI, 14.1 to 21.5) among the 34 women with detectable levels above the median (>0.55 ng/ml; Ptrend = 0.01). Percentage breast density was also elevated (18.2%; 95% CI, 13.4 to 23.7) among the 18 women with serum mono-ethyl phthalate above the median detected level (>3.77 ng/ml) compared with women with nondetectable BPA levels (13.1%; 95% CI, 11.9 to 14.3; Ptrend = 0.07). No other chemicals demonstrated associations with percentage breast density. CONCLUSIONS: Postmenopausal women with high serum levels of BPA and mono-ethyl phthalate had elevated breast density. Further investigation of the impact of BPA and mono-ethyl phthalate on breast cancer risk by using repeated serum measurements or other markers of xenoestrogen exposure are needed.

Mammographic breast density and serum phytoestrogen levels.

Some forms of estrogen are associated with breast cancer risk as well as with mammographic density (MD), a strong marker of breast cancer risk. Whether phytoestrogen intake affects breast density, however, remains unclear. We evaluated the association between serum levels of phytoestrogens and MD in postmenopausal women. We enrolled 269 women, ages 55-70 yr, who received a screening mammogram and had no history of postmenopausal hormone use. Subjects completed a survey on diet and factors related to MD and provided a blood sample for analysis of 3 phytoestrogens: genistein, daidzein, and coumestrol. We examined whether mean percent MD was related to serum level of phytoestrogens, adjusting for age and body mass index. Genistein and daidzein levels correlated with self-reported soy consumption. Mean percent MD did not differ across women with different phytoestrogen levels. For example, women with nondetectable genistein levels had mean density of 11.0% [95% confidence intervals (CI) = 9.9-12.4], compared to 10.5% (95% CI = 8.0-13.7) and 11.2% (95% CI = 8.7-14.6) for < and ≥ median detectable levels, respectively. In a population with relatively low soy intake, serum phytoestrogens were not associated with mammographic density. Additional studies are needed to determine effects of higher levels, particularly given patterns of increasing phytoestrogen intake.

Hydroxycarboxylic acid-derived organosulfates: synthesis, stability, and quantification in ambient aerosol.

Organosulfates have been proposed as contributors to aerosol growth and have been detected in both chamber and atmospheric aerosol samples. We present a simple method for the synthesis of quantitative analytical standards of two small hydroxycarboxylic acid-derived organosulfates, glycolic and lactic acid sulfate. Additionally, we discuss the stability of hydroxycarboxylic acid-derived organosulfates and their previously proposed sulfate hemiacetal isomers in commonly used solvents for filter extraction. The hydroxycarboxylic acid-derived organosulfates were found to be stable under acidic conditions comparable to those found in ambient aerosol. By using synthesized standards, quantitative organosulfate concentrations were measured from ambient particulate matter (PM(2.5)) collected in urban locations in the United States, Mexico City, and Pakistan. Lactic acid sulfate and glycolic acid sulfate concentrations ranged 0.4-3.8 ng/m(3) and 1.9-11.3 ng/m(3), respectively. We propose that glycolic acid sulfate represents an important tracer for atmospheric processes that form organosulfates in ambient particulate matter.

Occurrence of estrogens, androgens and progestogens and estrogenic activity in surface water runoff from beef and dairy manure amended crop fields.

Hormone contamination of aquatic systems has been shown to cause reproductive impairment of aquatic organisms. To assess to what extent beef and dairy farms represent a source of hormones to the aquatic environment, surface water runoff samples from three beef and dairy farms that utilize best manure management practices were evaluated for hormone concentrations (estrogens, androgens, progestogens) and estrogenic activity. Runoff was collected from weirs at the edge of each of six study fields from March 2008 to March 2010 and was analyzed for hormone concentrations using liquid chromatography with tandem mass spectrometry and for estrogenic activity using the E-screen bioassay. The majority of runoff events occurred in February and March when the soil was frozen. Progesterone and 4-androstenedione were the most frequently detected hormones (63% and 50%, respectively) and occurred at event loads up to 49,000 µg/ha and 26,000 µg/ha, respectively. Progesterone, 4-androstenedione, 17α-estradiol had the highest event load concentrations and were found at the field that sustained dairy cattle grazing during the winter and were likely due to application of excreta on frozen soil. The high progesterone event loads could lead to concentrations in receiving streams that exceed the lowest observable effects concentrations for fish. There was a consistent association with the elevated zearalenone presence and corn production. The synthetic hormones, 17α-trenbolone and 17ß-trenbolone, were not detected in runoff from the beef farm that utilized trenbolone acetate implants, which is likely due to their short half lives. Estrogenic activity in runoff samples ranged from 0.09 to 133 ng/L estradiol equivalents, with 39% of runoff events exceeding the 2 ng/L predicted-no-effect-concentration for fish. These results indicate that grazing cattle and application of manure to frozen fields present the greatest risk to elevated hormones in runoff and that progesterone is the primary hormone of concern from beef and dairy operations.

Removal of organic wastewater contaminants in septic systems using advanced treatment technologies.

The detection of pharmaceuticals and other organic wastewater contaminants (OWCs) in ground water and surface-water bodies has raised concerns about the possible ecological impacts of these compounds on nontarget organisms. On-site wastewater treatment systems represent a potentially significant route of entry for organic contaminants to the environment. In this study, effluent samples were collected and analyzed from conventional septic systems and from systems using advanced treatment technologies. Six of 13 target compounds were detected in effluent from at least one septic system. Caffeine, paraxanthine, and acetaminophen were the most frequently detected compounds, and estrogenic activity was detected in 14 of 15 systems. The OWC concentrations were significantly lower in effluent after sand filtration (p < 0.01) or aerobic treatment (p < 0.05) as compared with effluent that had not undergone advanced treatment. In general, concentrations in conventional systems were comparable to those measured in previous studies of municipal wastewater treatment plant (WWTP) influent, and concentrations in systems after advanced treatment were comparable to previously measured concentrations in WWTP effluent. These data indicate that septic systems using advanced treatment can reduce OWCs in treated effluent to similar concentrations as municipal WWTPs.

New measurements of cyanobacterial toxins in natural waters using high performance liquid chromatography coupled to tandem mass spectrometry.

The presence and levels of the cyanobacterial toxins microcystin-LR, anatoxin-a, and cylindrospermopsin were measured in various Wisconsin waters where algal nuisance or bloom conditions were noted. Out of 74 samples analyzed, 36 had detectable levels of microcystin-LR (49%), and four had detectable levels of anatoxin-a (5%). Cylindrospermopsin, the toxin produced by Cylindrospermopsis (a warm water species that has been moving its range northward, including to Wisconsin), was not detected in the field samples tested. Concentrations of microcystin-LR ranged from 1.2 to 7600 microg L(-1). Anatoxin-a ranged from 0.68 to 1750 microg L(-1), which is the highest concentration reported from around the world. Cyanobacterial toxins, because of their high potency, deserve continued scrutiny by resource managers and public health officials responsible for recreational waters.

Aminoflavone-loaded EGFR-targeted unimolecular micelle nanoparticles exhibit anti-cancer effects in triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer for which there is no available targeted therapy. TNBC cases contribute disproportionately to breast cancer-related mortality, thus the need for novel and effective therapeutic methods is urgent. We have previously shown that a National Cancer Institute (NCI) investigational drug aminoflavone (AF) exhibits strong growth inhibitory effects in TNBC cells. However, in vivo pulmonary toxicity resulted in withdrawal or termination of several human clinical trials for AF. Herein we report the in vivo efficacy of a nanoformulation of AF that enhances the therapeutic index of AF in TNBC. We engineered a unique unimolecular micelle nanoparticle (NP) loaded with AF and conjugated with GE11, a 12 amino acid peptide targeting epidermal growth factor receptor (EGFR), since EGFR amplification is frequently observed in TNBC tumors. These unimolecular micelles possessed excellent stability and preferentially released drug payload at endosomal pH levels rather than blood pH levels. Use of the GE11 targeting peptide resulted in enhanced cellular uptake and strong growth inhibitory effects in TNBC cells. Further, AF-loaded, GE11-conjugated (targeted) unimolecular micelle NPs significantly inhibit orthotopic TNBC tumor growth in a xenograft model, compared to treatment with AF-loaded, GE11-lacking (non-targeted) unimolecular micelle NPs or free AF. Interestingly, the animals treated with AF-loaded, targeted NPs had the highest plasma and tumor level of AF among different treatment groups yet exhibited no increase in plasma aspartate aminotransferase (AST) activity level or observable tissue damage at the time of sacrifice. Together, these results highlight AF-loaded, EGFR-targeted unimolecular micelle NPs as an effective therapeutic option for EGFR-overexpressing TNBC.

Acute Influences of Bisphenol A Exposure on Hypothalamic Release of Gonadotropin-Releasing Hormone and Kisspeptin in Female Rhesus Monkeys.

Bisphenol A (BPA) is an industrial compound with pervasive distribution in the environments of industrialized countries. The U.S. Centers for Disease Control recently found that greater than 90% of Americans carry detectable levels of BPA, raising concern over the direct influences of this compound on human physiology. Epidemiologic evidence links elevated BPA serum concentrations to human reproductive dysfunction, although controlled studies on the acute effect of BPA exposure on reproductive function are limited, particularly in primates. We evaluated the effect of direct BPA exposure on female primate hypothalamic peptide release. Specifically, using a microdialysis method, we examined the effects of BPA (0.1, 1, and 10nM) directly infused to the stalk-median eminence on the release of GnRH and kisspeptin (KP) in mid to late pubertal ovarian intact female rhesus monkeys. We found that the highest level of BPA exposure (10nM) suppressed both GnRH and KP release, whereas BPA at lower concentrations (0.1 and 1nM) had no apparent effects. In addition, we measured BPA in plasma and hypothalamic dialysates after an iv bolus injection of BPA (100 µg/kg). We found a relatively stable distribution of BPA between the blood and brain (plasma:brain ≅ 5:1) persists across a wide range of blood BPA concentrations (1-620 ng/mL). Findings of this study suggest that persistent, high-level exposures to BPA could impair female reproductive function by directly influencing hypothalamic neuroendocrine function.

Steroidogenic factor 1 promotes aggressive growth of castration-resistant prostate cancer cells by stimulating steroid synthesis and cell proliferation.

The dependence of prostate cancer on androgens provides a targeted means of treating advanced disease. Unfortunately, androgen deprivation therapies eventually become ineffective, leading to deadly castration-resistant prostate cancer (CRPC). One of many factors implicated in the transition to CRPC is the onset of de novo steroidogenesis. Although reactivation of steroid receptors likely plays a pivotal role in aggressive CRPC, little is understood regarding the mechanisms whereby prostate cancer cells initiate and maintain steroidogenesis. We hypothesize that steroidogenic factor 1 (SF1, NR5A1, AD4BP), a key regulator of steroidogenesis in normal endocrine tissues, is expressed in CRPC where it stimulates aberrant steroidogenesis and fuels aggressive growth. Notably, SF1 is not expressed in normal prostate tissue. Our results indicated that SF1 was absent in benign cells but present in aggressive prostate cancer cell lines. Introduction of ectopic SF1 expression in benign human prostate epithelial cells (BPH-1) stimulated increased steroidogenic enzyme expression, steroid synthesis, and cell proliferation. In contrast, data from an aggressive human prostate cancer cell line (BCaPT10) demonstrated that SF1 was required for steroid-mediated cell growth because BCaPT10 cell growth was diminished by abiraterone treatment and short hairpin RNA-mediated knockdown of SF1 (shSF1). SF1-depleted cells also exhibited defective centrosome homeostasis. Finally, whereas xenograft experiments in castrated hosts with BCaPT10 control transplants grew large, invasive tumors, BCaPT10-shSF1 knockdown transplants failed to grow. Therefore, we conclude that SF1 stimulates steroid accumulation and controls centrosome homeostasis to mediate aggressive prostate cancer cell growth within a castrate environment. These findings present a new molecular mechanism and therapeutic target for deadly CRPC.

Development of a sensitive LC/MS/MS method for vitamin D metabolites: 1,25 Dihydroxyvitamin D2&3 measurement using a novel derivatization agent.

Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3, whereas liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and -D3 ranges (Amplifex and RIA, pc=0.97; Amplifex and PTAD, pc=0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc=0.81; Amplifex and PTAD, pc=0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume.

Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern.

The monitoring of pharmaceuticals and personal care products (PPCPs) has focused on the distribution in rivers and small lakes, but data regarding their occurrence and effects in large lake systems, such as the Great Lakes, are sparse. Wastewater treatment processes have not been optimized to remove influent PPCPs and are a major source of PPCPs in the environment. Furthermore, PPCPs are not currently regulated in wastewater effluent. In this experiment we evaluated the concentration, and corresponding risk, of PPCPs from a wastewater effluent source at varying distances in Lake Michigan. Fifty-four PPCPs and hormones were assessed on six different dates over a two-year period from surface water and sediment samples up to 3.2 km from a wastewater treatment plant and at two sites within a harbor. Thirty-two PPCPs were detected in Lake Michigan and 30 were detected in the sediment, with numerous PPCPs being detected up to 3.2 km away from the shoreline. The most frequently detected PPCPs in Lake Michigan were metformin, caffeine, sulfamethoxazole, and triclosan. To determine the ecological risk, the maximum measured environmental concentrations were compared to the predicted no-effect concentration and 14 PPCPs were found to be of medium or high ecological risk. The environmental risk of PPCPs in large lake systems, such as the Great Lakes, has been questioned due to high dilution; however, the concentrations found in this study, and their corresponding risk quotient, indicate a significant threat by PPCPs to the health of the Great Lakes, particularly near shore organisms.

Serum factors and clinical characteristics associated with serum E-screen activity.

BACKGROUND: The E-Screen bioassay can measure the mitogenicity of human serum and thus may be useful as a biomarker in epidemiologic studies of breast cancer. While the assay's MCF-7 cells are known to proliferate in response to estrogen, the specific determinants of variation in E-Screen activity in human serum samples are poorly understood. We sought to identify serum molecules and patient characteristics associated with serum E-Screen activity among postmenopausal women. METHODS: Postmenopausal women (N = 219) aged 55 to 70 years with no history of postmenopausal hormone use or breast cancer completed a questionnaire and provided a blood sample. Serum was analyzed for E-Screen activity and a variety of molecules including sex hormones, growth factors, and environmental chemicals. Stepwise selection procedures were used to identify correlates of E-Screen activity. RESULTS: Serum samples from all women had detectable E-Screen activity, with a median estradiol equivalents value of 0.027 ng/mL and interquartile range of 0.018-0.036 ng/mL. In the final multivariable-adjusted model, serum E-Screen activity was positively associated with serum estradiol, estrone, insulin-like growth factor-binding protein (IGFBP)-3, and testosterone levels (all P < 0.05), as well as body mass index (P = 0.03). Serum E-Screen activity was lower among women with higher SHBG (P < 0.0001) and progesterone levels (P = 0.03). CONCLUSION: Serum E-Screen activity varies according to levels of endogenous estrogens and other serum molecules. Obesity appears to confer additional serum mitogenicity beyond its impact on the measured hormones and growth factors. IMPACT: By capturing mitogenicity due to a variety of patient and serum factors, the E-Screen may provide advantages for use as a biomarker in breast cancer studies.

Evaluation of a model for the removal of pharmaceuticals, personal care products, and hormones from wastewater.

Current wastewater treatment processes are insufficient at removing many pharmaceutical and personal care products (PPCPs) from wastewater and it is necessary to identify the chemical characteristics that determine their fate. Models that predict the fate of various chemicals lack verification using in situ data, particularly for PPCPs. BIOWIN4 is a quantitative structure-activity relationship (QSAR) model that has been proposed to estimate the removal of PPCPs from wastewater, but data verifying the accuracy of its predictions is limited. In this study, the in situ soluble and suspended solid concentrations were assessed from raw influent, primary effluent, secondary effluent, and final effluent for 54 PPCPs and hormones over six dates. When assessing the removal efficiency across the different stages of the WWTP, the majority of the removal occurred across the secondary treatment process for the majority of the compounds. The primary treatment and disinfection process had limited impacts on the removal of most PPCPs. Sorption to solids was found to influence the removal for compounds with a log octanol-water partitioning coefficient greater than 4.5 across the secondary treatment process. For other compounds, the removal of PPCPs across the secondary treatment process was significantly correlated with the biodegradation predicted by BIOWIN4. Removal efficiencies across the aerobic secondary treatment process were predicted by integrating BIOWIN4 into pseudo-first order kinetics of PPCPs and these predicted values were compared to the in situ data. This study determines that under a certain set of operating conditions, two chemical characteristics - the expected hydrophobic interaction and the modeled biological degradation from BIOWIN4 - were found to predict the removal of highly degradable and recalcitrant PPCPs from a wastewater secondary treatment process.

Effects of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas).

High concentrations (375 ng/L) of the steroid hormone progesterone (P4) were measured in snowmelt runoff associated with large livestock-feeding operations in Wisconsin. To gain insight into the potential endocrine-disrupting effects of P4 in fish, experiments were conducted to evaluate the effects of short-term exposure to environmentally relevant concentrations of P4 on reproduction and embryonic development in the fathead minnow (Pimephales promelas). For the reproduction assay, groups of reproductively mature fish were exposed for 21 d to nominal concentrations of 0, 10, 100, and 1,000 ng/L P4 in a flow-through system, and various key reproductive endpoints (e.g., egg number, fertilization success) were quantified throughout the exposure period. The embryonic development assay consisted of incubating fathead minnow eggs in static culture to quantify the effects of P4 on early development and hatching success. Progesterone caused dose-dependent decreases in fecundity and fertility and significantly reduced gonadosomatic index and vitellogenin gene expression in females. There were no effects of P4 on early embryonic development or hatching success. Progesterone may be a significant endocrine-disrupting chemical in fish.

Stability, preservation, and quantification of hormones and estrogenic and androgenic activities in surface water runoff.

Degradation of hormones that may occur during storage of surface water samples can lead to underestimations in estrogenic and androgenic activities and inaccuracies in hormone concentrations. The current study investigated the use of sodium azide, hydrochloric acid (HCl), and sulfuric acid (H2SO4) to inhibit the degradation of hormones and estrogenic and androgenic activities in samples of surface water runoff from cattle manure-amended fields during storage at 4°C. Hormones and hormone metabolites were extracted using solid-phase extraction and analyzed using high-performance liquid chromatography (HPLC) with tandem MS. Estrogenic and androgenic activities were assessed by E-screen and A-screen, respectively. Results of the current study indicate significant degradation of estrogenic, androgenic, and progestogenic hormones and activities, which is likely attributable to microbial activity, within hours of sample collection. The inclusion of internal standards provides a means to account for hormone losses caused by extraction inefficiency and to some extent degradation. However, internal standards are unable to adequately account for significant losses and are not available for E-screen and A-screen. Sodium azide did not adequately inhibit androgen degradation at the concentration used (1 g/L). Acid preservation (HCl or H2SO4, pH 2) stabilized the estrogenic and androgenic activities, and coupling acid preservation with the use of internal standards resulted in reliable and accurate recovery of a suite of androgens, estrogens, and progestogens for up to 14 d of storage at 4°C.