A study on temporal trends and estimates of fate of Bisphenol A in agricultural soils after sewage sludge amendment.

Temporal concentration trends of BPA in soils were investigated following sewage sludge application to pasture (study 1: short term sludge application; study 2: long term multiple applications over 13 years). The background levels of BPA in control soils were similar, ranging between 0.67-10.57 ng g(-1) (mean: 3.02 ng g(-1)) and 0.51-6.58 ng g(-1) (mean: 3.22 ng g(-1)) for studies 1 and 2, respectively. Concentrations in both treated and control plots increased over the earlier sampling times of the study to a maximum and then decreased over later sampling times, suggesting other sources of BPA to both the treated and control soils over the study period. In study 1 there was a significant treatment effect of sludge application in the autumn (p=0.002) although no significant difference was observed between treatment and control soils in the spring. In study 2 treated soils contained considerably higher BPA concentrations than controls ranging between 12.89-167.9 ng g(-1) (mean: 63.15 ng g(-1)). This and earlier studies indicate the long-term accumulation of multiple contaminants by multiple sewage sludge applications over a prolonged period although the effects of the presence of such contaminant mixtures have not yet been elucidated. Fugacity modelling was undertaken to estimate partitioning of Bisphenol A (soil plus sewage: pore water: soil air partitioning) and potential uptake into a range of food crops. While Bisphenol A sorbs strongly to the sewage-amended soil, 4% by mass was predicted to enter soil pore water resulting in significant uptake by crops particularly leafy vegetables (3.12-75.5 ng g(-1)), but also for root crops (1.28-31.0 ng g(-1)) with much lower uptake into cereal grains (0.62-15.0 ng g(-1)). This work forms part of a larger programme of research aimed at assessing the risks associated with the long-term application of sewage sludge to agricultural soils.

Exposure to chemical cocktails before or after conception--- the effect of timing on ovarian development.

Exposure of female fetuses to environmental chemicals (ECs) during pregnancy results in a disturbed ovarian adult phenotype. We investigated the influence of pre- and/or post-conception exposure to low-level mixtures of ECs on the structure and function of the fetal ovine ovary. We examined ovarian morphology, expression of oocyte and granulosa cell-specific genes and proteome. Female fetuses were collected at day 110 of gestation, from dams exposed continuously until, and after mating, by grazing in pastures treated with sewage sludge as a fertiliser (TT) or in control fields treated with inorganic fertiliser (CC). In addition, in a cross-over design, fetal ovaries were collected from dams maintained on sludge pastures up to the time of mating but then transferred to control pastures (TC) and, reciprocally, those transferred from control to treated pastures at mating (CT). On examination, the proportion of type 1a follicles (activating primordial follicles) was significantly lower in animals from the CT groups compared with CC and TT groups (P<0.05). Of the 23 ovarian gene transcripts studied, 14 were altered in the ovaries of exposed fetuses (CT, TC, and TT) relative to controls, with the largest number of changes observed in cross-exposure pattern groups (CT or TC). Continuous EC exposure (TT) produced fewer transcript alterations and only two genes (INHBA and GSN) presented differential profiles between CC and TT. Fetal ovarian proteome analysis (2-DE gels) showed, across all exposure groups, 86 differentially expressed protein spots compared to controls. Animals in the CT group exhibited the highest number (53) while TC and TT presented the same number of affected protein spots (42). Fetal ovarian proteins with altered expression included MVP (major vault protein) and several members of the heat-shock family (HSPA4L, HSP90AA1 and HSF1). The present findings indicate that continuous maternal EC exposure before and during gestation, are less deleterious for fetal ovarian development than a change in maternal EC exposure between pre and post-conception. The pathways by which the ovary responds to this chemical stress were common in TT, CT, TC exposed foetuses. In addition to the period of pregnancy, the pre-conception period appears also as crucial for conditioning long-term effects of EC exposure on ovarian development and primordial follicle reserve and hence future fertility.

Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis.

BACKGROUND: The aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis. DESIGN AND METHODS: Mature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9 L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry. RESULTS: No significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined. CONCLUSIONS: These data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110 days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic-pituitary changes.

Selective pressurized liquid extraction of estrogenic compounds in soil and analysis by gas chromatography-mass spectrometry.

A selective pressurized liquid extraction (SPLE) method, followed by gas chromatography-mass spectrometry (GC-MS), for the simultaneous extraction and clean-up of estrone (E1), 17ß-estradiol (E2), 17α-ethynylestradiol (EE2), estriol (E3) and bisphenol A (BPA) from soil samples is described. The on-line clean-up of soil by SPLE was achieved using different organic matter retainers, including silica, alumina and Florisil, the most effective being silica. Thus, different amounts of silica, in conjunction with different extraction solvents (acetone, ethyl acetate, isohexane and dichloromethane), either alone or in combination, were used to extract the target chemicals from spiked soil samples. It was shown that 3g silica resulted in satisfactory rates of recovery of target compounds and acetone:dichloromethane (1:3, v/v) was efficient in extracting and eluting estrogenic compounds for SPLE. Variables affecting the SPLE efficiency, including temperature and pressure were studied; the optimum parameters were 60°C and 1500 psi, respectively. The limits of detection (LODs) of the proposed method were 0.02-0.37 ng g(-1) for the different estrogenic chemicals studied. The outputs using the proposed method were linear over the range from 0.1 to 120 ng g(-1) for E1, E2, EE2, 0.2-120 ng g(-1) for E3, and 0.5-120 ng g(-1) for BPA. The optimized method was further verified by performing spiking experiments in natural soil matrices; good rates of recovery and reproducibility were achieved for all selected compounds and the method was successfully applied to soil samples from Northeast Scotland, for the determination of the target compounds.

Pregnant ewes exposed to multiple endocrine disrupting pollutants through sewage sludge-fertilized pasture show an anti-estrogenic effect in their trabecular bone.

Pregnant ewes were maintained on pastures fertilized, twice yearly, with either sewage sludge (2.25tonnes dry matter/ha; Treated; T) or inorganic fertilizer containing equivalent amounts of nitrogen (Control; C), to determine effects on maternal and fetal bone structures, density and mechanical properties of exposure to environmental concentrations of multiple endocrine disrupting compounds (EDCs) and heavy metal pollutants. The ewes were maintained on the respective pastures from the age of about 8months until they were 4-6years of age and they were slaughtered at 110d gestation. Metaphyseal parts of adult ewe femurs exhibited a significantly reduced mean, total cross sectional area (CSA, -4%; p<0.05), lower trabecular bone mineral content (BMC, mg/mm; -18%; p<0.05), trabecular bone mineral density (BMD, mg/cm(3), -8.0%; p<0.05) and trabecular CSA, mm(2), -11.1%; p<0.05) in T compared with C animals. Femurs of T ewes were stronger than those of C ewes but this may reflect greater body weights. At the mid-diaphyseal part of the fetal bones, there was a reduction in endosteal circumference (-6.7%, p<0.05) and marrow cavity area (-13.8%, p<0.05) in the female T fetuses compared with female C fetuses. In the male fetuses the mid-diaphyseal part total bone mineral content was higher (+3.0%, p<0.05) in T than in C animals. No treatment difference in biomechanical bending was detected in the fetuses. It is concluded that ewes grazing pasture fertilized with sewage sludge exhibited an anti-estrogenic effect on their trabecular bone in the form of reduced mineral content and density, despite increased body weight. It is suggested that human exposure to low levels of multiple EDCs may have implications for bone structure and human health.

Exposure to pastures fertilised with sewage sludge disrupts bone tissue homeostasis in sheep.

The femurs of male and female sheep (Ovis aries), aged 18 months, bred on pastures fertilized twice annually with sewage sludge (2.25 tonnes dry matter/ha; Treated; T)) or on pastures treated with inorganic fertilizer (Control; C) were studied, using peripheral Quantitative Computed Tomography (pQCT) and the three-point bending test. Males were maintained on the respective treatments from conception to weaning and then maintained on control pastures while the females were maintained on the respective treatments until slaughter. T rams exhibited increased total bone mineral density (BMD) at the metaphyseal part of femur (+10.5%, p<0.01) compared with C rams but had a reduced total cross sectional area (CSA, -11.5%, p<0.001), trabecular CSA (-17.1%, p<0.01) and periosteal circumference (-5.7%, p<0.001). In the mid-diaphyseal part, T rams had an increased total BMD (+13.8%, p<0.0001) and stiffness (+6.4%, p<0.01) but reduced total CSA (-12.1%, p<0.0001) and marrow cavity (-25.8%, p<0.0001), relative to C rams. In ewes although pQCT analysis of neither the metaphyseal nor the mid-diaphyseal part of the female femur bones showed any significant differences with treatment, the biomechanical method revealed a reduction in load at failure (-17.3%, p<0.01) and stiffness (-10.7%, p<0.05) amongst T ewes. It is concluded that exposure to pollutants present in sewage sludge can perturb bone tissue homeostasis in sheep, but particularly in males.

Maternal undernutrition and the ovine acute phase response to vaccination.

BACKGROUND: The acute phase response is the immediate host response to infection, inflammation and trauma and can be monitored by measuring the acute phase proteins (APP) such as haptoglobin (Hp) or serum amyloid A (SAA). The plane of nutrition during pregnancy is known to affect many mechanisms including the neuroendocrine and neuroimmune systems in neonatal animals but effects on the APP are unknown. To investigate this phenomenon the serum concentration of Hp and SAA was initially determined in non-stimulated lambs from 3 groups (n = 10/group). The dams of the lambs of the respective groups were fed 100% of requirements throughout gestation (High/High; HH); 100% of requirements for the first 65 d of gestation followed by 70% of requirements until 125 d from when they were fed 100% of requirements (High/Low; HL); 65% of liveweight maintenance requirements for the first 65 d gestation followed by 100% of requirements for the remainder of pregnancy (Low/High; LH). The dynamic APP response in the lambs was estimated by measuring the concentration of Hp and SAA following routine vaccination with a multivalent clostridial vaccine with a Pasteurella component, Heptavac Ptrade mark following primary and secondary vaccination. RESULTS: The Hp and SAA concentrations were significantly lower at the time of vaccination (day 8-14) than on the day of birth. Vaccination stimulated the acute phase response in lambs with increases found in both Hp and SAA. Maternal undernutrition led to the SAA response to vaccination being significantly lower in the HL group than in the HH group. The LH group did not differ significantly from either the HH or HL groups. No significant effects of maternal undernutrition were found on the Hp concentrations. A significant reduction was found in all groups in the response of SAA following the second vaccination compared to the response after the primary vaccination but no change occurred in the Hp response. CONCLUSION: Decreased SAA concentrations, post-vaccination, in lambs born to ewes on the HL diet shows that maternal undernutrition prior to parturition affects the innate immune system of the offspring. The differences in response of Hp and SAA to primary and secondary vaccinations indicate that the cytokine driven APP response mechanisms vary with individual APP.

Effects of exposure of ewes to sewage sludge-treated pasture on phthalate and alkyl phenol concentrations in their milk.

Concentrations of endocrine disrupting compounds (EDCs) of two classes, the alkyl phenols (nonyl phenol (NP) and octyl phenol (OP)) and phthalates, in the milk of ewes grazed on pastures fertilised with sewage sludge or with inorganic fertiliser were determined at three stages of lactation. Milk concentrations of these compounds varied greatly between individuals and stages of lactation for both nonyl phenol (NP; < 30-> 1000 microg/kg DM) and total phthalates (< 200-> 20,000 microg/kg DM). Overall, there was no significant effect of sludge treatment on milk concentrations of chemicals of either class. Significant differences between years were recorded in mean log concentrations of both NP (P < 0.001) and total phthalate (P < 0.001) but there were no consistent changes with stage of lactation, ewe body condition or age in mean milk concentrations of either class of compound. Milk concentrations of NP were low, and little higher than environmental concentrations, while phthalate concentrations were approximately two-fold higher than environmental concentrations. Estimated daily intakes of phthalates were considered to be of potential, biological significance with respect to the health of animal and human consumers. It is concluded that the importance of milk as a route of EDC exposure in growing ruminants differs with class of compound and individual animal. Exposure of the offspring to these EDCs may be transiently exacerbated by exposure of their dams to additional EDCs via the application of sewage sludge to their pasture.

Cellular and hormonal disruption of fetal testis development in sheep reared on pasture treated with sewage sludge.

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12-15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of alpha-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood.

Alkyl phenols and diethylhexyl phthalate in tissues of sheep grazing pastures fertilized with sewage sludge or inorganic fertilizer.

We studied selected tissues from ewes and their lambs that were grazing pastures fertilized with either sewage sludge (treated) or inorganic fertilizer (control) and determined concentrations of alkylphenols and phthalates in these tissues. Mean tissue concentrations of alkylphenols were relatively low (< 10-400 microg/kg) in all animals and tissues. Phthalates were detected in tissues of both control and treated animals at relatively high concentrations (> 20,000 microg/kg in many tissue samples). The use of sludge as a fertilizer was not associated with consistently increased concentrations of either alkylphenols or phthalates in the tissues of animals grazing treated pastures relative to levels in control animal tissues. Concentrations of the two classes of chemicals differed but were of a similar order of magnitude in liver and muscle as well as in fat. Concentrations of each class of compound were broadly similar in tissues derived from ewes and lambs. Although there were significant differences (p < 0.01 or p < 0.001) between years (cohorts) in mean tissue concentrations of both nonylphenol (NP) and phthalate in each of the tissues from both ewes and lambs, the differences were not attributable to either the age (6 months or 5 years) of the animal or the duration of exposure to treatments. Octylphenol concentrations were generally undetectable. There was no consistent cumulative outcome of prolonged exposure on the tissue concentrations of either class of pollutant in any ewe tissue. Mean tissue concentrations of phthalate were higher (p < 0.001) in the liver and kidney fat of male compared with female lambs. We suggest that the addition of sewage sludge to pasture is unlikely to cause large increases in tissue concentrations of NP and phthalates in sheep and other animals with broadly similar diets and digestive systems (i.e., domestic ruminants) grazing such pasture.

Phthalate and alkyl phenol concentrations in soil following applications of inorganic fertiliser or sewage sludge to pasture and potential rates of ingestion by grazing ruminants.

Soil concentrations of dioctyl phthalate (DOP) and the alkyl phenols, octyl phenol (OP) and nonyl phenol (NP), after repeated surface applications of sewage sludge to pastures, were investigated. Liquid sludge was applied at a rate of 2.25 tonnes dry matter (DM) per hectare to each of three treated (T) plots on three occasions during the summer and two occasions in the early spring over a period of 2.5 years. Control (C) plots were treated with inorganic fertiliser containing amounts of nitrogen equivalent to those applied to the treated plots. At between 69 and 81 days after the application of sludge, 15 separate soil samples were collected from one half of each of the plots (Experiment 1). Concentrations (microg g(-1)) of DOP were higher (P < 0.001) than those of NP, while those of OP were generally below detectable levels. Mean soil concentrations of DOP were not significantly different in T and C plots [0.233 vs. 0.155 microg g(-1); standard error of the difference (SED) = 0.046; not significant (NS)], partly because there was already a relatively large amount of DOP present. NP concentrations were, however, significantly higher in T than in C plots (0.021 vs. 0.013 microg g(-1) SED = 0.002; P < 0.05). There was no consistent change over time in the mean soil concentrations of these compounds when sampled at intervals of 3-6 months. Concentrations in soil samples collected at monthly intervals following sludge application indicated that the variation in concentrations of these endocrine-disrupting compounds (EDC) was unrelated to time since sludge application. Rates of soil ingestion, expressed as the percentage of DM intake represented by soil, were higher during the winter than the summer (5.40 vs. 1.17; SED = 0.360; P < 0.001) and estimated daily intakes of DOP and NP were up to 150 microg and 8 microg, respectively. It is concluded that the application of sewage sludge to pasture does not increase soil concentrations of phthalate (as DOP) or alkyl phenols. Thus, the risk of increased exposure to these EDC as a result of sludge application is small. However, the small effect of sludge application on soil concentrations may be largely a reflection of the relatively high concentrations of DOP already present in the soil, which may be biologically significant.