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.

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.

Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

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.

Improved source apportionment and speciation of low-volume particulate matter samples.

New chemical analysis methods for the characterization of atmospheric particulate matter (PM)* samples were developed and demonstrated in order to expand the number of such methods for use in future health studies involving PM. Three sets of methods were, developed, for the analysis (1) of organic tracer compounds in low-volume personal exposure samples (for source apportionment), (2) of trace metals and other trace elements in low-volume personal exposure samples, and (3) of the speciation of the oxidation states of water-soluble iron (Fe), manganese (Mn), and chromium (Cr) in PM samples. The development of the second set of methods built on previous work by the project team, which had in the past used similar methods in atmospheric source apportionment studies. The principal challenges in adapting these methods to the analysis of personal exposure samples were the improvement of detection limits (DLs) and control of the low-level contamination that can compromise personal exposure samples. A secondary goal of our development efforts was to reduce the cost and complexity of the three sets of methods in order to help facilitate their broader use in future health studies. The goals of the project were achieved, and the ability to integrate the methods into existing health studies was demonstrated by way of conducting two pilot studies. The first study involved analysis of trace elements in size-resolved PM samples that had been collected to represent study subjects' personal exposures along with simultaneous measures of indoor and outdoor PM concentrations. The second study involved analysis of the speciation of organic tracer compounds in personal exposure samples, indoor samples, and outdoor samples in order to understand the diesel PM exposure of study subjects in various job classifications in an occupational setting. Both pilot studies used existing samples from. large multi-year health studies and were intended to demonstrate the feasibility and value of using the new chemical analysis methods to better characterize the personal exposure samples. Analysis of the health data and the broader implications of the exposure assessments were not evaluated as part of the present study, but our pilot-study measurements are expected to contribute to investigators' future analyses in the large multi-year health studies. The methods we developed for the low-cost measurement of the oxidation states of Fe, Mn, and Cr in atmospheric PM samples are extremely sensitive and well suited for use in health studies. To demonstrate the utility of these methods, small-scale studies were conducted to characterize the redox cycling of Fe in PM on the time scale of atmospheric transport from source to personal exposure and to provide preliminary data on the atmospheric concentrations of soluble forms of the target metals in selected urban environments (in order to help focus future research seeking to understand the role of metals in human exposure to PM and its adverse health effects). The present report summarizes the methods that were developed and demonstrated to be suitable for use in health studies and provides pilot-scale data that can be used to develop hypotheses and experimental strategies to further enhance the ability of future health studies to elucidate the role of PM, PM sources, and PM components in the observed associations between atmospheric PM and adverse human health outcomes.

A comparison of summertime secondary organic aerosol source contributions at contrasting urban locations.

Primary and secondary sources contributing to atmospheric organic aerosol during the months of July and August were quantitatively assessed in three North American urban areas: Cleveland, Ohio, and Detroit, Michigan, in the Midwest region and Riverside, California, in the Los Angeles Air Basin. Organic molecular marker species unique to primary aerosol sources and secondarytracers derived from isoprene, alpha-pinene, beta-caryophyllene, and toluene were measured using gas chromatography-mass spectrometry. Source contributions from motor vehicles, biomass burning, vegetative detritus, and secondary organic aerosol (SOA) were estimated using chemical mass balance (CMB) modeling. In Cleveland, primary sources accounted for 37 +/- 2% of ambient organic carbon, measured biogenic and anthropogenic secondary sources contributed 46 +/- 6%, and other unknown sources contributed 17 +/- 4%. Similarly, Detroit aerosol was determined to be 44 +/- 5% primary and 37 +/- 3% secondary, while 19 +/- 7% was unaccounted for by measured sources. In Riverside, 21 +/- 3% of organic carbon came from primary sources, 26 +/- 5% was attributed to measured secondary sources, and 53 +/- 3% came from other sources that were expected to be secondary in nature. The comparison of samples across these two regions demonstrated that summertime SOA in the Midwestern United States was substantially different from the summertime SOA in the Los Angeles Air Basin and indicated the need to exert caution when generalizing about the sources and nature of SOA across different urban areas. Furthermore, the results of this study suggestthatthe contemporary understanding of SOA sources and formation mechanisms is satisfactory to explainthe majority of SOA in the Midwest Additional SOA sources and mechanisms of formation are needed to explain the majority of SOA in the Los Angeles Air Basin.