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A catchment-based study of endocrine disruption in surface waters: multivariate evaluation of the health of a sentinel fish species exposed to sewage treatment works effluent

Summary of the results in the context of EDCAT 5 project aims: 1. By comparing appropriate biomarkers in fish sampled from STW-impacted sites and control sites during the pre-remediation period, to determine whether there was evidence for any effects that might be attributed to the presence of estrogenic (or androgenic, or anti-androgenic/-estrogenic) endocrine disrupting chemicals in the former. This aim was addressed by measuring concentrations of the estrogen-dependent yolk protein precursor vitellogenin, and the androgen-dependent nest glue spiggin in male and female sticklebacks. In addition histological examination of the gonadal structure of fish captured at the impacted and non-impacted sites was employed to seek evidence of overt alterations in reproductive physiology of the fish. For a subset of matched samples from the two rivers, the relative induction of hepatic choriogenin mRNA, a biomarker of estrogen exposure, was measured. Conclusions: Chemistry data provided by EDCAT3&4 showed that estrogenicity of the effluent was low prior to remediation and lower still following installation of the GAC plant. No evidence of overt estrogenic effects was detected in male sticklebacks in the Ray, VTG and ChG levels were similar in males from both rivers. Nor was there any evidence of alterations in spiggin concentrations in the kidneys of males from the Ray compared to the Ock. However, VTG concentrations in female sticklebacks from the Ray were increased following the STW upgrade as were hepatic ChG transcript levels, and kidney spiggin concentrations. No changes in these elements of the reproductive system were observed in females from the Ock across the same time periods. Chemical analysis of the effluent indicated that prior to installation of the GAC plant substantial concentrations of anti-androgenic chemicals were present, together with a wide range of other organics. Concentrations of these were much reduced following the plant upgrade. It is reasonable to suppose that the changes observed in the female reproductive endocrine system following the upgrade were related to the removal of some or all of this complex mixture of chemicals. The absence of effects in males may be related to the balance between exogenous and endogenous signals, or to the specificity of effects exerted by the chemicals present. No intersex fish were detected from either river. A significant bias in favour of females was detected in the stickleback populations in both rivers suggesting a factor associated with life-history of the fish, rather than contaminant burden, was responsible. 2. By comparing appropriate biomarkers in fish sampled from STW-impacted sites and control sites during the pre-remediation period, to determine whether there was evidence for any effects that might be attributed to the presence of “conventionally” toxic chemicals. This was addressed by measurement of the activity of a key Phase I transforming enzyme in the liver of fish, either using direct enzymatic assay (EROD) or by quantifying the levels of expression of the corresponding gene (CYP1A). Conclusions: EROD activity was significantly greater in fish from the Ray than the Ock in two samples collected prior to the installation of the GAC plant (2006, 2007) and this likely reflects the differential contaminant loading in the two rivers. A single sample following the commissioning of the GAC plant (2008) indicated that EROD activity had increased among fish from the Ock while that in fish from the Ray remained unchanged. While a delayed recovery of this biomarker in fish from the Ray may be expected depending on the route of exposure (direct via water or indirect via contaminated food) the reasons for elevated EROD activity in fish from the Ock/Childrey Brook are not immediately evident. Provision of a full data set for Cyp1A expression awaits the repeat of the assay. When this is complete the factors underlying the EROD findings may become clear. 3. To determine whether the adaptive capacity and energetic status of fish varied between the STW-impacted and non-impacted sites. This was addressed by measurement of indicators of stress (whole-body corticosteroid levels), metabolic status (whole-body glucose levels) and anabolic activity (RNA:DNA ratios). Conclusions: The data provide no evidence that the stress response of fish captured in the Ray prior to installation of the GAC plant was modified by exposure to the effluent. However, large variations in whole-body corticosteroid and glucose concentrations in fish from both rivers, with clear trends over time, were closely linked to perturbations in the river flow regime. Whether there was interaction between environmental and chemical factors in determining corticosteroid and glucose status is difficult to discern but it seems likely that variation in these indicators of the stress axis was driven primarily by environmental factors. The RNA:DNA ratios were closely linked with seasonal change in temperature and closely matched observed patterns of weight and length gain in stickleback populations in the two rivers. The longer growth period enjoyed by fish in the Ray was clearly evident. For both rivers, mean anabolic activity was greater during 2008 than 2007 and it seems likely that this is related to adverse effects associated with the periods of extreme flow change observed on both rivers in 2007. 4. To assess whether there were differences in population size and structure between STW-impacted and non-impacted sites. This was addressed by comparison of key somatic measures, in particular frequency distributions for fork length. Conclusions: Because of the extreme patchiness of the distribution of stickleback populations in both rivers accurate abundance estimates were not obtained. However, the catch per unit effort across the life of the project was similar for both rivers. While population size, and age structure (both rivers hosted annual populations), appeared to be similar fish in the Ray were overall larger than those from the Ock, and spawned earlier. The differences in growth and timing of spawning between the rivers were likely to have been associated with the Rodbourne STW effluent. Downstream of the discharge on the Ray water temperatures were consistently 2 – 3oC above those of the Ock. This temperature difference, in combination with the introduction of additional nutrients into the river which is likely to have affected the availaibility of food, probably accounts for the different growth profile among the sticklebacks in the two rivers. However, over and above this difference, there was a significant increase in size of sticklebacks in the Ray between the matched pre- and post-remediation periods in the Ray while no change in size of the fish in the Ock occurred during the same period. Similarly, the RNA:DNA ratio was higher in fish from the Ock during 2007 but greater in fish from the Ray during 2008. Taken together, these observations suggest that there was an improvement in the status of the fish in the Ray following the commissioning of the GAC plant, while the population in the Ock remained relatively stable. It is reasonable to suppose that this may be linked to the reduction of the chemical load entering the Ray at Rodbourne following the installation of the GAC plant. The Ray is “cleaner” now than was the case prior to remediation but remains nutrient rich and several degrees warmer than the Ock, this combination of factors providing fish in the Ray with greater scope for growth relative to populations in the Ock

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk

Myocarditis Concurrent with Sweet Syndrome: A Presentation of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common acute leukemia in American adults and portends a poor prognosis if untreated. Commonly, AML presents with symptoms related to concurrent leukopenia, anemia, or thrombocytopenia; however, due to its ability to affect many organ systems in the body, AML can have a highly varied clinical presentation. One such presentation is myocarditis, which is a rarely reported manifestation of AML. Myocarditis can have a varied clinical picture and often requires exclusion of other causes of cardiac dysfunction. Sweet syndrome, also known as acute febrile neutrophilic dermatosis, is another presentation of AML; however, it is more commonly associated with AML than cardiac involvement. Sweet syndrome can occur in patients with an already established malignancy or can occur de novo in a patient with previously undiagnosed cancer and, interestingly, can also be accompanied by extracutaneous manifestations, one of which is myocarditis. Herein, we report a case of a 45-year-old male with a history of obesity and depression who presented with chest pain, a tender and diffuse rash, and pancytopenia. Heart catheterization performed at outside institution was negative for coronary artery disease. Cardiac MRI images were compatible with myocarditis. Dermal biopsy of the rash was consistent with sweet syndrome. Peripheral blood flow cytometry and bone marrow biopsy confirmed the diagnosis of AML. He was treated with an induction chemotherapy regimen of 7 days of cytarabine and 3 days of daunorubicin with resolution of his chest pain and skin lesions. The patient had persistent leukemia cells on day 14 postinduction bone marrow biopsy and was treated with high-dose cytarabine reinduction treatment. Bone marrow biopsy with count recovery after reinduction therapy revealed complete response (CR)

Identifying the risk to the aquatic environment of endocrine disrupters derived from agriculture

EXECUTIVE SUMMARY 1. The purpose of this project was to review possible inputs to UK headwater streams of steroid hormones originating from livestock, to investigate hormone contamination in some streams in which concentrations were expected to be maximal, and to draw conclusions about possible risks that these hormones may pose to aquatic organisms. 2. The review concluded that although livestock in the UK excretes more steroid sex hormones (oestradiol and testosterone) than the human population, almost all of the material deposited on soil by livestock and by manure/slurry spreading is likely to be adsorbed and/or degraded in soil before reaching surface waters. Concentrations of oestrogens in field drains are unlikely to exceed 1 ng/l (expressed as 17β-oestradiol equivalents), a concentration that is probably harmless. However, it is possible that direct excretion by livestock into unfenced streams, and direct run-off to surface waters from slurry stores and hard-standing in livestock farms, may contribute higher concentrations. In other words, poor farming practice may lead to significant steroid hormone pollution. 3. The review also concluded that surface waters in some other countries are contaminated with oestrogens at potentially active concentrations, so it was considered that a survey of UK headwater streams for hormonal activity was justified. The literature search clearly showed that pregnant cattle are the single most important source of natural oestrogens on livestock farms. 4. The chosen sampling strategy was to focus on a limited number of predominantly dairy farms that were considered to represent worst-case conditions for hormone translocation to small headwater streams. Criteria that contributed towards the choice of field sites included stocking type and density, soil type and slope, access of livestock to the stream, application of manure or slurry to the land, possible direct drainage to the stream of waste from leaking slurry stores and hard-standing areas used by livestock, and access permission from the land-owner. Confounding factors such as upstream inputs of hormonally active material from sewage treatment works, septic tank soak-aways, and industrial discharges, were excluded as far as possible from the study. 5. In order to obtain semi-quantitative, time-integrated samples of hormones in water, locations up- and downstream of livestock activity were sampled on 10 farms using a passive, solid-phase device known as a Polar Organic Chemical Integrative Sampler (POCIS). These were deployed between November 2004 and January 2005 for 3 to 10 weeks (mean = 39 days). At an eleventh site, a field drain issuing from an experimental plot of cracking clay soil treated solely with dairy cow slurry was also sampled with POCIS. At one site, an automatic flow-driven water sampler was deployed alongside the POCIS to capture water soon after heavy rainfall. 6. POCIS and water extracts were assayed for estrogenic and androgenic activity using the in vitro yeast estrogen screen (YES) and yeast androgen screen (YAS), respectively. As part of a separate project, POCIS extracts were also analysed chemically for oestrone (E1), 17β-oestradiol (E2) and 17α-ethinylestradiol (EE2) by the Environment Agency. 7. The flow from only one rainfall event was captured in its entirety by the autosampler, but this revealed a background concentration (E2 equivalents) of 0-0.3 ng/l, rising to a transient peak of 9.4 ng/l. Average E2 activity at this site as determined from the POCIS samplers was 1.8-2.7 ng E2 equiv./litre, which provides confidence that the POCIS results are reliable. 8. Estimated oestrogenic activity across all sites (with one exception) lay in the range zero-26.5 ng E2 equiv./litre (mean = 2.0 ng/l; standard deviation = 5.1), based on the POCIS samples. The outlier was 292 ng/l, but this could not be specifically linked with intensive livestock rearing. 92% of monitoring stations (at least one on each farm) contained some oestrogenic activity. 9. In 5 of 9 livestock farms where upstream/downstream comparisons were possible, the downstream oestrogenic activity was higher than upstream, implying inputs from the farms under study. There was one case (Farm 3) where there were no known confounding factors whatever, with very little upstream contamination, and the farm increased activity by a factor of 7. 10. However, upstream activity was sometimes higher than downstream, suggesting possible inputs from phyto-oestrogens and scattered septic tank overflows, and in-stream adsorption and/or degradation. There was a low background level of oestrogenic activity in all but two locations. 11. The data did not generally permit discrimination between different potential sources on the farms, but it seems likely that the observed oestrogenic activity was mainly caused by a combination of slurry spreading and farmyard runoff, with direct excretion to pasture by livestock probably contributing less. In one case (Farm 7 slurry application experiment), activity in the field drain was directly attributable to dairy slurry alone. 12. On 8 of the 11 surveyed farms, oestrogenic activity in the stream (or field drain in one case) exceeded the Predicted-No-Effect-Concentration for E2 of 1 ng/l. In two cases, activity was probably high enough to damage reproduction in fish, although in neither case was livestock itself likely to have been the primary cause. 13. Although no EE2 was detected analytically in any stream, E1 and E2 were ubiquitous, with E2 equivalents ranging from 0.04 to 3.62 ng/l across all but two sites. Furthermore, concentrations downstream of livestock were generally higher than upstream, more markedly so than for the YES data. The oestrogen concentrations agree well with the YES data and these observations suggest that most of the detected activity was attributable to E1 and E2 derived from livestock. However, the low levels of oestrogenic activity detected by the YES upstream at several stations, and the much higher upstream levels at Farms 11 and 13, could not be explained by E1 or E2, and it is postulated that phytooestrogens may have contributed to this signal. 14. Although all streams were assayed with the YAS for androgenic activity, this was only detectable in two cases, and at levels which are unlikely to pose a threat to fish. However, it should be noted that uptake of testosterone by the POCIS has not yet been calibrated. 15. On the basis of this survey, the possibility that natural oestrogens (from both livestock and other sources) in headwater streams are causing adverse effects in fish cannot be excluded. 16. Recommendations are made for further research to discriminate between sources, and to evaluate the risks to fish

8th Annual Seminar on Legal Issues for Financial Institutions

Outline of speakers\u27 presentations from the 8th Annual Seminar on Legal Issues for Financial Institutions held by UK/CLE on March 11-12, 1988

9th Annual Seminar on Legal Issues for Financial Institutions

Outline of speakers\u27 presentations from the 9th Annual Seminar on Legal Issues for Financial Institutions held by UK/CLE on March 10-11, 1989

Seventh Annual Seminar on Legal Issues for Bank Counsel

Outlines of speakers\u27 presentations from the Seventh Annual Seminar on Legal Issues for Bank Counsel held on March 13-14, 1987

Boldness Predicts Social Status in Zebrafish (Danio rerio)

This study explored if boldness could be used to predict social status. First, boldness was assessed by monitoring individual zebrafish behaviour in (1) an unfamiliar barren environment with no shelter (open field), (2) the same environment when a roof was introduced as a shelter, and (3) when the roof was removed and an unfamiliar object (Lego® brick) was introduced. Next, after a resting period of minimum one week, social status of the fish was determined in a dyadic contest and dominant/subordinate individuals were determined as the winner/loser of two consecutive contests. Multivariate data analyses showed that males were bolder than females and that the behaviours expressed by the fish during the boldness tests could be used to predict which fish would later become dominant and subordinate in the ensuing dyadic contest. We conclude that bold behaviour is positively correlated to dominance in zebrafish and that boldness is not solely a consequence of social dominance