Estrogenicity of agricultural runoff: A rainfall simulation study of worst-case scenarios using fresh layer and roaster litter, and farrowing swine manure.

Scientists have correlated land application of animal wastes as fertilizer with the feminization of fish. Two questions were asked. 1) Under a worst case scenario when animal waste (layer and roaster litter, or farrowing swine slurry) is applied and tilled in 24 h prior to a surface-runoff producing rainfall, will estrogenic equivalents exceed the Lowest Observable Effect Concentration (LOEC) for fish (10 ng/L)? 2) Can calcium concentrations in runoff, measured using a rapid meter-based method, be used as a sentinel of elevated estrogenic activity? In a 3-yr study wastes were surface-applied and incorporated and 24 h later, 1.5 by 3 m plots were subjected to simulated rainfall and again 1 wk. and 3 wk. later. Nutrients in runoff were also measured, and in year 1 total coliforms and E. coli. were assessed. Except for an initial preliminary test run, runoff from all plots and years never exceeded 10 ng/L E2Eq equivalent. Calcium concentrations in runoff were not related to estrogenicity, negating its use as a sentinel marker. Specific estrogens in animal waste and runoff were identified by mass spectrometry with concentrations in runoff dependant on manure source and timing of rainfall. As expected, total coliform and E. coli concentrations in runoff were increased by the application of layer litter. Concentrations of nutrients in runoff would not be expected to result in surface water concentrations higher than guidelines for protection of aquatic species. Animal wastes applied in quantities appropriate for crop nutrient requirements, tilled into the soil surface, in observance of regulations avoiding application within 24 h of a predicted rain event, should not result in estrogen levels of environmental concern.

Estrogenic activity and nutrient losses in surface runoff after winter manure application to small watersheds.

Confined Animal Feeding Operations generate large amounts of wastes that are land-applied to provide nutrients for crop production and return organic matter to the soil. Production practices and storage limitations often necessitate that wastes be applied to frozen and snow-covered soil. Use of application setbacks have reduced concerns related to nutrient losses in surface runoff from manure, but the estrogenic activity of runoff under these conditions has not been evaluated. Therefore, we measured and sampled surface runoff when manure was applied in the winter at a rate to meet crop N needs and measured estradiol equivalents (E2Eqs) using E-Screen. In year one, six small watersheds used to produce corn were evaluated, treatments: 2 no-manure controls, 2 liquid swine manure with 30-m setbacks, and 2 turkey litter with 30-m setbacks. In addition, beef manure was applied to six frozen plots of forage. For years 2 and 3, applications were repeated on the swine manure watersheds and one control watershed. E2Eqs and nutrient concentrations generally peaked in the first runoff event after application. The highest measured E2Eq (5.6 ng L(-1)) was in the first event after swine manure application and was less than the 8.9 ng L(-1) Lowest Observable Effect Concentration (LOEC) for aquatic species and well below the concentrations measured in other studies using ELISAs to measure hormone concentrations. No runoff occurred from plots planted with forage, indicating low risk for environmental impact, and therefore plots were discontinued from study. In years 2 and 3, estrogenic activity never exceeded the Predicted No Effect Concentrations for E2 of 2 ng L(-1). When post-application runoff contained high estrogenic activity, strong correlations (R(2) 0.86 to 0.96) of E2Eq to Ca(2+), Mg(2+), and K(+) concentrations were observed, indicating under some condition these cations might be useful surrogates for E2Eq measurements.

Bioassay of estrogenicity and chemical analyses of estrogens in streams across the United States associated with livestock operations.

Animal manures, used as a nitrogen source for crop production, are often associated with negative impacts on nutrient levels in surface water. The concentrations of estrogens in streams from these manures also are of concern due to potential endocrine disruption in aquatic species. Streams associated with livestock operations were sampled by discrete samples (n = 38) or by time-integrated polar organic chemical integrative samplers (POCIS, n = 19). Samples were analyzed for estrogens by gas chromatography-tandem mass spectrometry (GC-MS(2)) and estrogenic activity was assessed by three bioassays: Yeast Estrogen Screen (YES), T47D-KBluc Assay, MCF-7 Estrogenicity Screen (E-Screen). Samples were collected from 19 streams within small (≈ 1-30 km(2)) watersheds in 12 U.S. states representing a range of hydrogeologic conditions, dominated by: dairy (3), grazing beef (3), feedlot cattle (1); swine (5); poultry (3); and 4 areas where no livestock were raised or manure was applied. Water samples were consistently below the United Kingdom proposed Lowest Observable Effect Concentration for 17ß-estradiol in fish (10 ng/L) in all watersheds, regardless of land use. Estrogenic activity was often higher in samples during runoff conditions following a period of manure application. Estrone was the most commonly detected estrogen (13 of 38 water samples, mean 1.9, maximum 8.3 ng/L). Because of the T47D-KBluc assay's sensitivity towards estrone (1.4 times 17ß-estradiol) it was the most sensitive method for detecting estrogens, followed by the E-Screen, GC-MS(2), and YES. POCIS resulted in more frequent detections of estrogens than discrete water samples across all sites, even when applying the less-sensitive YES bioassay to the POCIS extracts.

Effects of clenbuterol on body stores of polychlorinated dibenzofurans (PCDF) and dibenzo-p-dioxins (PCDD) in rats.

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF), persistent pollutants that accumulate in the food chain, pose a risk to humans through consumption of tainted livestock. Clenbuterol, a leanness-enhancing agent, was tested for usefulness in PCDD/F body store reduction through body fat reduction (the predominant site of accumulation). To mimic the situation of contaminated animals, rats were given feed with or without a mixture of PCDD/F (0.6 to 2.7 ng/congener per day) for 10 d, followed by 16 d of feed with or without dietary clenbuterol (2 mg/kg feed). Clenbuterol reduced body fat by 28% (P < 0.05), increased muscle mass by 25% (P < 0.02), and decreased liver mass by 7% (P < 0.02). Although the concentrations of most PCDD/F per gram of fat were slightly increased after clenbuterol treatment, the total amount of PCDD/F that remained in fat was reduced by approximately 30%. Muscle PCDD/F concentrations and total burden were decreased by clenbuterol. In contrast, clenbuterol tended to increase concentration, but not total burden of PCDD/F in livers. One congener known to be rapidly metabolized and excreted, 2,3,7,8-TCDF, was the exception to this increase, decreasing 40% with clenbuterol treatment. This was also the congener that showed the greatest reduction in both fat and muscle. Examination of the ratio of PCDD/F in liver and fat revealed that clenbuterol increased the liver's share of the body burden of PCDD/F, from 38 to 75%. In a remediation/disposal context, these findings would be beneficial if clenbuterol lowered the meat and carcass burden of PCDD/F to safe levels, requiring only livers to be disposed of as hazardous waste.

Multiple receptors on porcine intestinal epithelial cells for the three variants of Escherichia coli K88 fimbrial adhesin.

We evaluated intestinal epithelial membrane preparations from five phenotypes of pigs, distinguished by the variant of K88 fimbrial adhesin (K88ab, K88ac, K88ad) which bind to their intestinal epithelial cells (A-all three variants, B-K88ab and K88ac, C-K88ab and K88ad, D-K88ad, and E-none of the variants), for the presence of K88 adhesin receptors. Intestinal brush border membranes were prepared from 20 animals (four from each phenotype). Brush border proteins, that had been separated using SDS-PAGE and transferred to nitrocellulose membranes, were overlaid with biotinylated K88 adhesin, 35S-labelled K88+ Escherichia coli, or biotinylated K88+ E. coli. Biotinylated K88ab and K88ac fimbrial adhesins and labelled E. coli expressing K88ab or K88ac adhesin bound to 210- and 240-kDa receptors in phenotype A and B, but not phenotype C, D, or E animals. In contrast, no phenotype-specific receptors were identified for the K88ad adhesin. Previously, purified K88ab and K88ac fimbriae were shown to block K88ad binding, but purified K88ad fimbriae were unable to block K88ab or K88ac binding in phenotype A animals. These results point to the existence of three K88 adhesin receptors to account for the observed phenotypes: (1) Receptor bcd binds all three variants and is found in phenotype A pigs, (2) Receptor bc (210- and 240-kDa receptors) binds K88ab and K88ac and is found in phenotype A and B pigs, and (3) Receptor d binds K88ad and is found in phenotype C and D pigs.

Distribution of K88 Escherichia coli-adhesive and nonadhesive phenotypes among pigs of four breeds.

Enterotoxigenic Escherichia coli expressing K88 fimbrial adhesins often cause diarrhea in young pigs. However, some pigs are inherently resistant to colibacillosis, because they lack receptors on their epithelial cell brush borders to which the fimbriae bind. Phenotypic diversity with respect to the binding of E. coli expressing K88 of the three variant types (K88ab, K88ac, and K88ad) was reported by Bijlsma et al. (1982), and binding specificities for each phenotype were described: A (adhesive to all three variants), B (adhesive to K88ab and K88ac), C (adhesive to K88ab and K88ad), D (adhesive to K88ad) and E (nonadhesive). Because brush border adhesiveness has been correlated with disease susceptibility, swine K88 adhesive phenotypes are of significance in the control of enteric disease. To determine the prevalence of the various K88 adhesive phenotypes in the swine population in the Midwestern United States, we tested epithelial cell brush borders of 24 purebred pigs from each of four breeds (Chester White, Duroc, Hampshire and Yorkshire) for adhesiveness to each of the K88 variants. Four, 4-week-old pigs (the largest and smallest healthy female littermates from two litters) were collected from each of 24 farms. Brush border vesicles from the pigs were tested for ability to bind E. coli expressing each K88 variant. The five brush border adherence patterns described for phenotypes A-E were observed. In addition, brush borders from some pigs only bound K88ab + bacteria. Nearly three quarters of the pigs whose brush borders tested, were found to be phenotype A (43%) or phenotype E (28%). These were the most common phenotypes in each breed, except Hampshire, in which case phenotypes C (17%) and D (25%) were more common than E (8%). There appeared to be no relationship between the phenotype of a pig and its weight relative to its littermate.

A three-receptor model for the interaction of the K88 fimbrial adhesin variants of Escherichia coli with porcine intestinal epithelial cells.

Four phenotypes of pigs distinguished by the variant(s) of K88 fimbrial adhesin (K88ab, K88ac, K88ad) that bind to their intestinal epithelial cells (I-none of the variants, II-K88ad, III-K88ab and K88ac, and IV-all three variants) have been identified. We hypothesize that the differences between the phenotypes are defined by the presence or absence of K88 adhesin receptors. We propose a three-receptor model to account for the observed phenotypes: 1) Receptor bed which binds all three variants and is found in phenotype IV, 2) Receptor be which binds K88ab and K88ac and is found in phenotype III and IV, and 3) Receptor d which binds K88ad and is found in phenotype II. We have identified the be receptor activity as a pair of mucin-type sialoglycoproteins (210 and 240 kDa). Although neither the bcd nor d receptor has been identified biochemically, their presence has been established using both blocking and receptor localization studies. Blocking studies using phenotype IV brush borders demonstrated that K88ab and K88ac fimbriae block the binding of E. coli expressing any of the K88 variants, but K88ad fimbriae block only K88ad E. coli binding. These results indicate that two receptors (bcd and bc) exist in the phenotype IV animals. Receptor localization studies on intestinal sections from phenotype IV animals showed that K88ab and K88ac adhesin binding is continuous from the crypt to the tip of the villus. The binding of the K88ad adhesin binding is multifocal in phenotype IV pigs, but continuous from crypt to tip of the villus in sections of phenotype II pigs. These studies verify the presence of two receptors (bcd and bc) in phenotype IV animals, and indicate that the K88ad receptor in phenotype IV animals (bcd) is different than in phenotype II animals (d).