Halogenated 17ß-Estradiol Surrogates: Synthesis, Estrogenic Activity, and Initial Investigations of Fate in Soil/Water Systems.

17ß-Estradiol (E2), a natural, endocrine-disrupting, steroid hormone, is excreted by all vertebrates and can enter the environment from domestic animal and wildlife wastes. Multiple field studies using livestock manures as E2 sources suggest significant background concentrations of E2 (e.g., wildlife sources, hydrolysis of E2 conjugates, previous inputs). To accurately understand field fate and transport processes of E2, it is necessary to address the issue of background detections. In this study, two fluorinated and three brominated surrogate compounds of E2 were synthesized and compared to native E2 using soil/water batch experiments and for estrogenic activity. Analytical difficulties presented by the two fluorinated congeners deemed these compounds to be unsuitable surrogates of E2, and further assessment was abandoned. However, the brominated congeners proved promising, with log( ) values that fell within the range previously reported for E2. Batch studies yielded similar relative aqueous concentrations and linear sorption isotherms across time for E2 and 2-bromo-17ß-estradiol; however, the relative aqueous concentrations and linear sorption isotherms of 4-bromo-17ß-estradiol and 2,4-dibromo-17ß-estradiol were different from E2 but similar to one another. All three brominated congeners possessed estrogenic activity by E-Screen assay, albeit three orders of magnitude less than native E2, putatively due to steric interference introduced by the large bromine atom on the phenolic ring, the group that mediates interaction with the estrogen receptor. The data suggest that 2-bromo-17ß-estradiol may serve as a suitable surrogate for E2 in planned field scale tracer studies designed to distinguish between antecedent and de novo inputs.

Consumption of ground beef obtained from cattle that had received steroidal growth promotants does not trigger early onset of estrus in prepubertal pigs.

BACKGROUND: The earlier onset of puberty seen in young American girls has led researchers to question if a causal relation exists between dietary sources of estrogenic compounds and precocious puberty. OBJECTIVE: Using the prepubertal gilt (young female pig) as an animal model, our hypothesis is that feeding beef obtained from cattle receiving growth-promoting steroidal implants postweaning does not alter the onset of puberty or the peripubertal body composition of gilts compared with contemporaries fed nonimplanted "natural" beef or a common meat alternative, tofu. METHOD: The base diet was formulated using canola meal replacing soybean meal to reduce diet estrogenicity. Feed intake was monitored and controlled to ensure similar intake. Gilts were assigned to treatments based on dam and initial body weight (mean: 24.5 ± 3.20 kg) at 61 d of age. The negative control base diet was supplemented with daily feedings of a cooked patty from nonimplanted steers (natural), from steers that had been treated with growth promotants [100 mg trenbolone acetate and 14 mg estradiol (E2) benzoate; implanted], or cooked tofu patty. RESULTS: E2 equivalents (nanogram per kilogram, as fed as analyzed by E-Screen) of the tofu (a soy-based product) supplement were ∼570 times the natural and ∼170 times the implanted supplements. There were no observed differences across treatments in live weight gain (P = 0.90), longissimus muscle area developed at the 10th and 11th rib interface (P = 0.46), and subcutaneous fat deposition (P = 0.41) at the same location over time or in the number of days to reach estrus (P = 0.55). CONCLUSIONS: Consumption of beef from growth implanted or natural steers or tofu at levels similar to those typically consumed by humans did not impact growth or onset of estrus in these prepubertal gilts.

Surveillance of plasticizers, bisphenol A, steroids and caffeine in surface water of River Ganga and Sundarban wetland along the Bay of Bengal: occurrence, sources, estrogenicity screening and ecotoxicological risk assessment.

The transboundary River Ganga serves as a conduit for meltwater from the Himalayas and is a major freshwater source for two thirds of Indian population before emptying into the Sundarban Delta, the largest estuary in the Bay of Bengal. Endocrine disrupting compounds (EDCs) such as phthalic acid esters (PAEs) and bisphenol A (BPA) used as organic plastic additives can pollute the aquatic environment receiving plastic litter. Hence, we have investigated these EDCs in water samples from Ganga and Sundarban wetland of India. Since these compounds exhibit estrogenic potential, we have further measured steroids and evaluated the estrogenic activity (estradiol equivalents, BioE2Eqs) using an in-vitro bioassay (E-Screen). Further BioE2Eqs were compared with the sum of predicted estradiol equivalents based on the chemical concentrations of PAEs and BPA by E-Screen (ChemE2Eq) and YES factors (ChemYES). Caffeine was measured as a marker for anthropogenic wastewater discharge. Results showed that the highest BioE2Eq (below the lowest observable effect of E2 on fish) was associated with sites having sewer outfalls in the middle stretch of the river, and concomitantly coinciding with the elevated concentrations of caffeine. Neither ChemE2Eq nor ChemYES correlated with measured BioE2Eqs. River concentrations of BPA (0.04-4.46 µg/L) and ∑7plasticizers (0.43-7.63 µg/L) were higher than BPA (0.21-2.82 µg/L) and ∑7plasticizers (0.85-2 µg/L) in the Sundarban wetland. The only steroids detected were androgens, found at four sites in Ganga (0.007 µg/L± 0.003, mean ± S.D.). The highest estimated ecotoxicological risk to aquatic insect and fish stemmed from BPA. A secondary effect, and a potential impact on human health could be reflected via fish consumption from the productive fisheries region along the lower stretch of River Ganga. Identification of areas of elevated estrogenicity, plasticizer and steroid concentrations in River Ganga can be used to design and implement interventions for the remediation of such emerging contaminants.

In vitro subcellular characterization of flunixin liver metabolism in heifers, steers, and cows.

The majority of cattle found to have violative liver residues of flunixin (FNX) in the United States are dairy cows. It has been hypothesized that illness of cows decreases the rate of FNX metabolism, resulting in violative residues at slaughter. Another contributing factor might be an age-related decrease in FNX metabolism, as dairy cull cows are typically older at slaughter than cattle raised for beef, rather than milk production. In order to investigate this possibility, subcellular fractions were prepared from liver slices from steers (n = 6) and heifers (n = 5) <30 months of age, and cows (n = 8) >48 mos of age. Cytochrome P450 (P450), NADPH-P450 reductase and glucose-6-phosphate dehydrogenase (G6PDH) activity and rate of 5-hydroxy FNX (5-OH FNX) formation were measured in liver homogenate, cytosolic, microsomal, and S9 fractions. Cows had lower concentrations of P450, NADPH-P450 reductase activity, and 5-OH FNX formation (P ≤ 0. 02), supporting the theory that advanced age may contribute to the higher incidence of violative FNX residues in dairy cows.

An In Vitro Comparison of Estrogenic Equivalents Per Serving Size of Some Common Foods.

The public assumes that some foods, such as milk and ground beef from cattle receiving steroidal implants, are associated with estrogenic hormones, while other foods are presumed "safe" or nonestrogenic. Here, we investigate these assumptions by assessing the relative estrogenic activity of a serving size of four foods: skim milk (8 oz), rice (48 g dry wt) in cooking bag, ground beef patties from steers raised with or without hormone implantation (quarter lb each, 114 g), and tofu burgers (isocaloric to beef burger, 198 g), using an in vitro assay (E-Screen). Mean picogram (pg) estradiol equivalents (E2Eqs) on a serving basis were as follows: skim milk 120; rice 400; rice prepared in cooking bag 370; rice boiling bag alone 4 pg per bag, ground beef burger (obtained from the tissue of cattle that had received no hormone implants) 389, beef burger (obtained from cattle that had received hormone implant) 384, and tofu burger 1,020,000. Rice E2Eqs were highly variable, but the plastic cooking bags provided by the manufacturer added negligible E2Eqs. The source of estrogenic activity in rice may have been due to contamination with the mycotoxin zearalenone. The E-Screen E2Eqs of tofu burger extracts agreed with those predicted based on chemical concentrations of the most estrogenic component times their E2Eq factor. While a tofu burger contained around three times the estrogenic activity of a daily dose of estrogen replacement therapy (125 mg, Premarin®, 303,000 pg); the other foods--a quarter pound ground beef burger at approximately equal calorie count, a serving of milk, or rice, were all at least 750-fold less estrogenic. PRACTICAL APPLICATION: When consuming the recognized serving size of a food, how much estrogenic activity can we expect? While the public assumes that some foods, such as milk and ground beef from cattle receiving steroidal implants, are associated with estrogenic hormones, other foods are presumed "safe" or nonestrogenic. Using one assay, a tofu burger contained three times the estrogenic activity of a dose of hormone replacement therapy commonly prescribed for women after hysterectomy or menopause (Premarin®); while other foods--a quarter pound ground beef burger at approximately equal calorie count, a serving of milk, or rice, were all at least 750-fold less estrogenic.

Reproductive Performance of Mares Fed Dietary Zearalenone.

It is known that zearalenone (ZON) interacts directly with estrogen receptors, and its in vivo effects on reproduction have been well-documented in several species. In contrast, reports of ZON's impact on horse reproduction are conflicting and inconclusive, some studies confounded by the presence of mycotoxins such as deoxynivalenol in the feed. This study assesses the effect of chronic consumption of zearalenone on reproduction in cycling mares fed >95% pure ZON (0, 2, or 8 mg/da; n = 7 mares/treatment) for three estrous cycles, followed by artificial insemination, through 16 days of pregnancy. Animals were on ZON treatment for between 70 and 121 days (average 84) depending on individual cycle patterns. ZON-induced changes in serum concentration of estradiol (E2) and progesterone (P4), and total estrogenicity were measured using RIAs and the E-screen assay, respectively. Effects on reproductive physiology and pregnancy were monitored by ultrasound and clinical parameters. No significant changes were found in reproductive hormone levels of E2, or P4 for mares on ZON treatments compared to controls, although there was a significant (P < 0.01) increase in P4 levels across Cycle number in High ZON (8 mg/da) treated mares. There was also an increasing trend in the interovulatory interval in the High ZON treatment group. The overall estrogenicity was similar across treatments and over time, not differing from controls or between ZON treatment groups. Adverse uterine and ovarian effects were also not observed, but pregnancy rates were mixed with only 4 of 7 mares on Low ZON becoming pregnant, and only 3 maintaining pregnancy and fetal heartbeat by Day 30, compared to 5 of 6 control mares and all 7 mares on High ZON. Because reproductive efficiency and hormone concentrations are highly variable across individuals, this study did not demonstrate that ZON at 2 or 8 mg/da was detrimental to mares' reproduction. Yet, inferring that ZON treatments were completely without effect is also not appropriate, as the absence of measurable significant differences could be attributed to the limited sample size. Most importantly, there were no extreme signs of toxicology, in contrast to previous reports when ZON was fed at these "doses."

Distribution of Spiked Drugs between Milk Fat, Skim Milk, Whey, Curd, and Milk Protein Fractions: Expansion of Partitioning Models.

The distributions of eight drugs (acetaminophen, acetylsalicylic acid/salicylic acid, ciprofloxacin, clarithromycin, flunixin, phenylbutazone, praziquantel, and thiamphenicol) were determined in milk products (skim milk, milk fat, curd, whey, and whey protein) and used to expand a previous model (from 7 drugs to 15 drugs) for predicting drug distribution. Phenylbutazone and praziquantel were found to distribute with the lipid and curd phases (≥50%). Flunixin distribution was lower but similar in direction (12% in milk fat, 39% in curd). Acetaminophen, ciprofloxacin, and praziquantel preferentially associated with casein proteins, whereas thiamphenicol and clarithromycin associated preferentially to whey proteins. Regression analyses for log [milk fat]/[skim milk] and log [curd]/[whey] had r2 values of 0.63 and 0.67, respectively, with p of <0.001 for 15 drugs (7 previously tested and 8 currently tested). The robustness of the distribution model was enhanced by doubling the number of drugs originally tested.

Distribution of Chemical Residues among Fat, Skim, Curd, Whey, and Protein Fractions in Fortified, Pasteurized Milk.

The distribution of 12 environmental contaminants or metabolites with diverse polarities (2,2',4,4',5-pentabromodiphenyl ether; bisphenol A; estrone; glyphosate; ß-hexabromocyclododecane; imidacloprid; 2,3',4,4',5-pentachlorobiphenyl; 3'-methylsulfone 2,2',4,5,5'-pentachlorobiphenyl; 1,2,7,8-tetrachlorodibenzo-p-dioxin; 2-hydroxy-1,3,7,8-tetrachlorodibenzo-p-dioxin; tetrabromobisphenol A; and triclocarban) among skim milk, fat, curd, whey, whey retentate, and whey permeate was characterized. Analysis of these compounds along with 15 drugs previously studied provided a robust linear model predicting the distribution between skim and fat and the chemical's lipophilicity (log P, r 2 = 0.71; log D, r 2 = 0.79). Similarly, distribution between curd and whey was correlated with lipophilicity (log P, r 2 = 0.63; log D, r 2 = 0.73). Phenolic compounds had less predictable distribution patterns based on their lipophilicities. Within the whey fraction, chemicals with greater lipophilicity are associated with whey proteins more than hydrophilic chemicals. The resultant model could help predict the potential distribution of chemical contaminants among milk products in cow milk, if present.

Tissue residues, metabolism, and excretion of radiolabeled sodium chlorate (Na[36Cl]O3) in rats.

A novel preharvest technology that reduces certain pathogenic bacteria in the gastrointestinal tracts of food animals involves feeding an experimental sodium chlorate-containing product (ECP) to animals 24-72 h prior to slaughter. To determine the metabolism and disposition of the active ingredient in ECP, four male Sprague-Dawley (approximately 350 g) rats received a single oral dose of sodium [36Cl]chlorate (3.0 mg/kg body weight). Urine, feces, and respired air were collected for 72 h. Radiochlorine absorption was 88-95% of the administered dose, and the major excretory route was the urine. Parent chlorate was the major species of radiochlorine present in urine at 6 h (approximately 98%) but declined sharply by 48 h (approximately 10%); chloride was the only other species of radiochlorine detected. Except for carcass remains (4.6% of dose), skin (3.2%), and gastrointestinal tract (1.3%), remaining tissues contained relatively low quantities of radioactivity, and >98% of radiochlorine remaining in the liver, kidney, and skeletal muscle was chloride. Chlorite instability was demonstrated in rat urine and bovine urine. The previously reported presence of chlorite in excreta of chlorate-dosed rats was shown to be an artifact of the analytical methods employed. Results from this study indicate that chlorate is rapidly absorbed and reduced to chloride, but not chlorite, in rats.

Distribution of Animal Drugs among Curd, Whey, and Milk Protein Fractions in Spiked Skim Milk and Whey.

It is important to understand the partitioning of drugs in processed milk and milk products, when drugs are present in raw milk, in order to estimate the potential consumer exposure. Radioisotopically labeled erythromycin, ivermectin, ketoprofen, oxytetracycline, penicillin G, sulfadimethoxine, and thiabendazole were used to evaluate the distribution of animal drugs among rennet curd, whey, and protein fractions from skim cow milk. Our previous work reported the distribution of these same drugs between skim and fat fractions of milk. Drug distribution between curd and whey was significantly correlated (R2 = 0.70) to the drug's lipophilicity (log P), with improved correlation using log D (R2 = 0.95). Distribution of drugs was concentration independent over the range tested (20-2000 nM). With the exception of thiabendazole and ivermectin, more drug was associated with whey protein than casein on a nmol/g protein basis (oxytetracycline experiment not performed). These results provide insights into the distribution of animal drug residues, if present in cow milk, among milk fractions, with possible extrapolation to milk products.

Estrogenic activity in the environment: municipal wastewater effluent, river, ponds, and wetlands.

Estrogenic activity of regional water samples was evaluated. Samples obtained from wetlands and ponds involved in various agricultural land uses, from three river sites over four seasons, and from municipal wastewater effluent held in storage lagoons were evaluated. The estrogen-responsive cell line MCF-7 BOS was used in the E-screen assay to determine 17beta-estradiol equivalents (E2 Eq) of water samples extracted by solid-phase extraction. Estrogenic activity in surrounding wetlands and ponds from different land uses was not different, with 10(-12) M E2 Eq (0.3 ppt). Estrogenic activity of Red River samples was within the same range as wetland-pond samples. The highest activity was found downstream from municipal wastewater treatment effluent discharge sites, in winter when river flow was lowest (approximately 6 x 10(-13) M E2 Eq). Results showed that 7 of 20 wetland-pond samples and 5 of 12 river samples were below the limits of quantitation (approximately 3 x 10(-14) M E2 Eq). Toxicity was found in fall and summer river samples upstream from municipal wastewater release sites. The timing of toxicity did not coincide to the presence of elevated fecal coliforms. Estrogenic activity in wastewater effluent from lagoons decreased over time (approximately 25 to 5 x 10(-13) M E2 Eq) with an apparent half-life of 8 d for one lagoon. The median concentration of detectable estrogenic activity in regional water samples was approximately 50-fold less than the median 17beta-estradiol concentration of estradiol detected in some U.S. streams in previous studies.

Distribution of Animal Drugs between Skim Milk and Milk Fat Fractions in Spiked Whole Milk: Understanding the Potential Impact on Commercial Milk Products.

Seven animal drugs [penicillin G (PENG), sulfadimethoxine (SDMX), oxytetracycline (OTET), erythromycin (ERY), ketoprofen (KETO), thiabendazole (THIA), and ivermectin (IVR)] were used to evaluate the drug distribution between milk fat and skim milk fractions of cow milk. More than 90% of the radioactivity was distributed into the skim milk fraction for ERY, KETO, OTET, PENG, and SDMX, approximately 80% for THIA, and 13% for IVR. The distribution of drug between milk fat and skim milk fractions was significantly correlated to the drug's lipophilicity (partition coefficient, log P, or distribution coefficient, log D, which includes ionization). Data were fit with linear mixed effects models; the best fit was obtained within this data set with log D versus observed drug distribution ratios. These candidate empirical models serve for assisting to predict the distribution and concentration of these drugs in a variety of milk and milk products.

Ergovaline movement across Caco-2 cells.

Ergovaline's role in the direct causation of fescue toxicosis first requires establishment of its dietary absorption. Therefore, ergovaline movement across human intestinal cells was assessed using Caco-2 cells derived from human colon carcinoma. A pre-equilibrated mixture of ergovaline/ergovalinine (60:40 ratio of isomers) was added to the apical compartment, and isomer movements were assessed by high-performance liquid chromatography (HPLC) of extracted media (initial pre-isomerized ergovaline concentrations of 6 and 22 microM, two doses). Mathematical models for ergot alkaloid movement were developed. Rates of movement were not different for the isomers. In the absence of cells, basal accumulation of isomers was essentially linear for 3 h regardless of loading concentration, after which basal accumulation of ergovaline/ergovalinine plateaued. Rates of ergovaline/ergovalinine movement in the presence of cells slowed to about 25% the rate of movement in the absence of cells (22 microM, kt = 0.0133 no cells, 0.0036 with cells, P < 0.05). Mass transfer rate was 7.5 ng x cm(-2) x min(-1) and was similar to that reported for ergovaline using a parabiotic chamber with sheep omasum. After 6 h in the presence of cells, approximately 25 and 40% of the total ergovaline/ergovalinine administered had accumulated in the basal compartment for 6.6 and 22 microM treatments, respectively. Ergovaline and its naturally occurring isomer, ergovalinine, readily crossed intestinal cells intact and at similar rates. Either isomer, or a combination of both, could be involved in the pathogenesis of fescue toxicosis at sites distal to the intestine.

The Effects of Steroid Implant and Dietary Soybean Hulls on Estrogenic Activity of Sera of Steers Grazing Toxic Endophyte-Infected Tall Fescue Pasture.

Soybean hulls (SBHs) have been fed to cattle pasturing on endophyte-infected tall fescue in attempts to increase rate of gain. Literature reports indicated some symptoms associated with fescue toxicosis were ameliorated by the use of steroidal implants containing estradiol (E2) and progesterone [implantation (IMP)], feeding SBHs, or the combination of the two. While the mechanism for amelioration was unclear, the SBHs were postulated as acting as a diluent of the toxic factors of the fescue. Alternatively, estradiol and phytoestrogens of SBHs might be acting through relaxation of the persistent vasoconstriction found in animals ingesting ergot alkaloids of endophyte-infected fescue. If so, estrogenic activity of serum of steers receiving SBHs, IMP, or a combination of the two should be elevated. Using the cellular proliferation assay of estrogenicity (E-Screen), estradiol equivalents (E2Eqs) were determined on both SBHs and the serum of steers from a previously reported study. Range of SBHs was 5.0-8.5 ng Eqs g(-1) DM (mean 6.5, n = 4 from different commercial sources of SBHs). At the rate fed, theoretically calculated blood E2Eq could be physiologically relevant (~80 pg mL(-1), based on 2.3 kg SBHs d(-1), 300 kg steer, 5.7% blood volume, and 10% absorption). Serum E2Eqs did increase in steers (P ≤ 0.05) with steroidal implants or fed SBHs by 56 and 151% over control, respectively, and treatments were additive (211% increase). Serum prolactin was also greatest for the SBH + IMP group (188 ng mL(-1), P < 0.05), concentrations comparable to values reported for steers grazing endophyte-free fescue. Prolactin in the SBH group was higher than IMP or control groups (146 versus 76 and 60 ng mL(-1), respectively). Still unknown is if additional E2Eqs from dietary phytoestrogens or exogenous sources of estradiol can further reduce symptoms of fescue toxicosis. The E-Screen assay was an effective tool in monitoring serum for estrogenic effects of dietary supplementation with SBHs or estrogenic implants.

Comparative metabolism of chrysene and 5-methylchrysene by rat and rainbow trout liver microsomes.

We have investigated the metabolism of chrysene (CHR) and 5-methychyrsene (5-MeCHR) by Shasta rainbow trout (Oncorhyncus mykiss) and Long Evans rat liver microsomes to assess the effect of a non-benzo ring methyl substituent on the reactions involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). Trout as well as rat liver microsomes metabolized both CHR and 5-MeCHR at essentially similar rates, indicating that the methyl substituent does not alter the substrate specificity of the cytochrome P450(s) involved in the metabolism of the two PAHs. Dihydrodiols were the major CHR metabolites formed by both trout and rat liver microsomes, whereas the trout liver microsomes formed a considerably higher proportion of 5-MeCHR phenols compared to diols, indicating that 5-methyl substitution alters the substrate specificity of trout microsomal epoxide hydrolase for 5-MeCHR epoxides. Unlike trout liver microsomes, rat liver microsomes formed a much greater proportion of 5-MeCHR diols compared to 5-MeCHR phenols, suggesting that 5-MeCHR epoxides are better substrates for the microsomal epoxide hydrolase present in rat liver than for the enzyme in trout liver. Both trout and rat liver microsomes are more efficient at attacking the bay-region bond versus the non-bay-region double bond in chrysene. In contrast the reverse is true in the case of 5-MeCHR, indicating that a non-benzo ring methyl substituent alters the regioselectivity of the enzymes involved in the oxidative metabolism of PAHs.

Ergovaline toxicity on Caco-2 cells as assessed by MTT, alamarBlue, and DNA assays.

The exact mechanisms of fescue toxicity in animals have yet to be established, but it has been associated with an inability to thrive. Ergovaline is the major ergopeptine alkaloid associated with fungal infections of tall fescue. Gastrointestinal (GI) toxicity of ergovaline (10(-11) to 10(-4) M) was evaluated in Caco-2 cells (mimicking the GI epithelium) beginning on days 1, 8, and 18 of culture. Acute and chronic toxicity was assessed after 24 and 72 h of exposure. Treatment periods were chosen to study undifferentiated, semidifferentiated, and completely differentiated cells. Cell loss and metabolic activity were assessed by thiazolyl blue reduction (3-(4,5-dimethylthiozole-2-yl)-2,5,-biphenyl tetrazolium bromide [MTT], mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and deoxyribonucleic acid (DNA) quantitation. Undifferentiated cells were sensitive to 1 x 10(-4) M ergovaline after acute exposure (from 52 to 74% of control values depending on assay). After 72 h of exposure to 1 x 10(-4) M ergovaline, in all three assays, treatment means were reduced to approximately 10% of the control means. By day 11 in culture, ergovaline toxicity to cells had decreased. With 24 h exposure, an apparent paradoxical increase in MTT was seen at some concentrations. This increase in MTT was also found in fully differentiated cells (day 21), whereas alamarBlue activity decreased. No change in DNA was found until 72 h of exposure, when DNA was reduced approximately 12% over most concentrations. These findings indicate differentiation state-dependent sensitivity of Caco-2 cells to ergovaline, potential problems of the MTT assay as an indicator of cellular toxicity, and usefulness of alamarBlue assay over DNA assay for toxicity assessment.

Potential bioactivity and association of 17ß-estradiol with the dissolved and colloidal fractions of manure and soil.

The dissolved (DF) and colloidal fractions (CF) of soil and manure play an important role in the environmental fate and transport of steroidal estrogens. The first objective of this study was to quantify the association of 17ß-estradiol (E2) with the DF and CF isolated from (i) liquid swine manure (LSM), (ii) a soil:water mixture (soil), and (iii) a LSM:soil:water mixture (Soil+LSM). The appropriate CF and DF size fractions of the Soil, Soil+LSM, and LSM media were obtained by first filtering through a 0.45 µm filter, which provided the combined DF and CF (DF/CF). The DF/CF from the three media was spiked with carbon-14 ([(14)C]) radiolabeled E2 ([(14)C]-E2), and then ultrafiltered to isolate the CF (<0.45 µm and >1 kDa) from the DF (<1 kDa). The average recoveries of the [(14)C] associated with the DF were 67%-72%, 67%-79%, and 76%-78% for the Soil, Soil+LSM and LSM, respectively. For the CF that was retained on the 1 kDa filter, organic carbon and [(14)C]-E2 were dislodged with subsequent water rinses the Soil+LSM and LSM, but not the Soil. The second objective was to evaluate whether the E2 associated with the various fractions of the different media could still bind the estrogen receptor using an E2 receptor (17ß-ER) competitor assay, which allowed E2 equivalent concentrations to be determined. The estrogen receptor assay results indicated that E2 present in the DF of the Soil and Soil+LSM solutions could still bind the estrogen receptor. Results from this study indicated that E2 preferentially associated with the DF of soil and manure, which may enhance its dissolved advective transport in surface and subsurface water. Furthermore, this study indicated that E2 associated with DF solutions in the environment could potentially induce endocrine responses through its interactions with estrogen receptor.

Egg wash wastewater: estrogenic risk or environmental asset?

Commercial production of eggs and egg products requires the washing of eggs to remove urinary-fecal material and broken egg residue. In the case of one Ohio farming facility, 1.6 million birds produce 1.4 million eggs per day, using approximately 50 mL of wash water/egg or approximately 70,000 L per day. The aqueous waste stream was evaluated for estrogenicity to determine if potential for endocrine disruption would result from agricultural application of such wastewater. Samples collected the Fall (October) of 2010 included: water from 2 egg washers operating in series, inlet pipe to the treatment lagoon, a lagoon composite, and products used within the facility in the cleaning of equipment and treatment of the waste. In February 2011, the treatment lagoon was fitted with an extensive aeration system and subsequent sample sets were collected on 3 consecutive days in May and November. Samples were extracted by solid phase extraction and assayed for estrogenic activity using the in vitro E-Screen assay. Raw untreated wastewater from the egg washers contained 17ß-estradiol equivalents (E2 Eqs) ranging from 9 to 18 ng/L, pipe grab samples entering into the treatment lagoon ranged from <0.14 to 4.4 ng/L (variability related to time of emptying of egg wash tanks), whereas treatment lagoon water contained 0.3 to 4.0 ng/L E2 Eq. Addition of an aeration system to the treatment lagoon eliminated surface "frothing," reduced noxious odor emission, and E2 Eqs were lower than the pre-aeration concentrations (4 ng/L [n = 1, no statistical comparison possible] vs 0.3 to 1.4 ng/L in 2011). Because of matrix effects, estrogens were not quantifiable by LC-MS2 in even egg washwater extracts, at concentrations in which internal deuterated estrogen standards were quantifiable. Estrone and E2 parent ions were detected in egg washwater samples only, and confirmatory ion fragments were detected in only one of these samples. Estrogenicity of the wastewater from the treatment lagoon was already at the proposed aquatic no effect concentration for 17ß-E2 and would be expected to decrease further as wastewater passes through 2 consecutive storage ponds before application on field crops for irrigation. The original project plan was to follow the wastewater as it was applied by aerial irrigation and concomitant surface runoff, but based on the consistent and extremely low concentration of estrogenic activity of the wastewater from the treatment lagoon, it was concluded that activity would be below limits of quantitation by E-Screen in water used for irrigation from the storage ponds. Use of egg wash wastewater--or gray water--to irrigate crops removes the cost and burden of wastewater treatment by the local wastewater plant, poses little to no potential threat of estrogenic endocrine disruption, and supports the conservation of water resources through the use of wastewater irrigation.