Rapid bioconcentration of steroids in the plasma of three-spined stickleback Gasterosteus aculeatus exposed to waterborne testosterone and 17β-oestradiol

The relationship over time between the concentrations of two steroids, singly and in combination, in a static exposure system and in the blood of three-spined stickleback Gasterosteus aculeatus, held within the exposure system was investigated. Groups of three-spined stickleback were exposed (nominally) to either 1000 ng l1 17β-oestradiol (E2), testosterone (T) or E2 and T in combination at the same concentrations for 6 days. Both water and fish were sampled at intervals and steroid concentrations in both compartments were determined. The plasma steroid time profile revealed a rapid bioconcentration within the first 6 h of exposure. The plasma steroid levels attained at this time point (20–90 ng ml1) were up to 50-fold (E2) and 200-fold (T) greater than the actual levels of steroid measured in the exposure water, while levels in the blood of control fish did not exceed 4 ng ml1. The substantial elevation of plasma steroid levels relative to the concentrations of steroid to which the fish were exposed in the ambient water gives scope for delivery of the steroids to target endocrine tissues at levels far in excess of what might be predicted on the basis of passive branchial uptake alone. These results are discussed in relation to endocrine disruption, and in particular the occurrence of effects in fish exposed to levels of endocrine active substances that are seemingly physiologically irrelevant

Overwinter fasting and re-feeding in rainbow trout: plasma growth hormone and cortisol levels in relation to energy mobilization

This study investigated the roles of cortisol and growth hormone during a period of fasting in overwintering salmonid fish. Indices of carbohydrate (plasma glucose, liver glycogen), lipid (plasma free fatty acids) and protein metabolism (plasma protein, total plasma amino acids) were determined, together with plasma growth hormone (GH), cortisol and somatolactin levels (SL) at intervals in three groups of rainbow trout (continuously fed; fasted for 9 weeks then fed; fasted for 17 weeks). In fasted fish, a decline in body weight and condition factor was accompanied by reduced plasma glucose and hepatic glycogen and increased plasma FFA. No consistent elevation of plasma GH occurred until after 8 weeks of fasting when plasma GH levels increased nine-fold. No changes were observed in plasma total protein and AA until between weeks 13 and 17 when both were reduced significantly. When previously fasted fish resumed feeding, plasma glucose and FFA, and hepatic glycogen levels rapidly returned to control values and weight gain resumed. No significant changes in plasma cortisol levels, related to feeding regime, were evident at any point during the study and there was no evidence that SL played an active role in the response to fasting. The results suggest that overwinter fasting may not represent a significant nutritional stressor to rainbow trout and that energy mobilisation during fasting may be achieved without the involvement of GH, cortisol or SL

The Effects of Acute and Chronic Hypoxia on Cortisol, Glucose and Lactate Concentrations in Different Populations of Three-Spined Stickleback

The response of individuals from three different populations of three-spined sticklebacks to acute and chronic periods of hypoxia (4.4 kPa DO, 2.2 mg l-1) were tested using measures of whole-body (WB) cortisol, glucose and lactate. Although there was no evidence of a neuroendocrine stress response to acute hypoxia, fish from the population least likely to experience hypoxia in their native habitat had the largest response to low oxygen, with significant evidence of anaerobic glycolysis after two hours of hypoxia. However, there was no measurable effect of a more prolonged period (seven days) of hypoxia on any of the fish in this study, suggesting that they acclimated to this low level of oxygen over time. Between-population differences in the analytes tested were observed in the control fish of the acute hypoxia trial, which had been in the laboratory for 16 days. However, these differences were not apparent among the control fish in the chronic exposure groups that had been held in the laboratory for 23 days suggesting that these site-specific trends in physiological status were acclimatory. Overall, the results of this study suggest that local environmental conditions may shape sticklebacks’ general physiological profile as well as influencing their response to hypoxia

Divergence in behavioural responses to stress in two strains of rainbow trout (Oncorhynchus mykiss) with contrasting stress responsiveness

The aim of this study was to establish whether two lines of rainbow trout divergent for their plasma cortisol response to a standardized stressor would show consistent differences in their behavioural response to a range of challenging situations. Our results show that the high- and low-responding (HR and LR) lines of rainbow trout did not differ in the aggression shown towards an intruder or in their response to the introduction of a novel object to their home environment. However, there was a difference in behaviour between the two selection lines when they were exposed to two unfamiliar environments. These results suggest that the behaviour of the HR and LR fish differs when they are challenged in unfamiliar environments, while their behaviour does not differ when they are challenged in their home environment. These observations are in agreement with studies on mammals that show that individuals with reactive coping styles perform similarly to proactive animals when they are challenged in a familiar environment, while they show different behaviour when they are challenged in unfamiliar environments. Thus, these results provide further evidence that the HR and LR selection lines of rainbow trout exemplify the two different coping styles described in mammals

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

Behavioral and neuroendocrine correlates of displaced aggression in trout

In humans and other primates, violent actions performed by victims of aggression are often directed toward an individual or object that is not the original source of provocation. This psychological phenomenon is often referred to as displaced aggression. We demonstrate that displaced aggression is either rooted in evolutionarily conserved behavioral and neuroendocrine mechanisms, or represent a convergent pattern that has arisen independently in fish and mammals. Rainbow trout that briefly encountered large, aggressive fish reacted with increased aggression towards smaller individuals. There was a strong negative correlation between received aggression and behavioral change: Individuals subjected to intense aggression were subdued, while moderate assaults induced strong agitation. Patterns of forebrain serotonin turnover and plasma cortisol suggest that the presence of socially subordinate fish had an inhibitory effect on neuroendocrine stress responses. Thus, subordinate individuals may serve as stress reducing means of aggressive outlet, and displaced aggression towards such individuals appears to be a behavioral stress coping strategy in fishes

The influence of sex, parasitism, and ontogeny on the physiological response of European eels (Anguilla anguilla) to an abiotic stressor

Migration of adult European eels (Anguilla anguilla) from freshwater feeding grounds to oceanic spawning grounds is an energetically demanding process and is accompanied by dramatic physiological and behavioral changes. Humans have altered the aquatic environment (e.g., dams) and made an inherently challenging migration even more difficult; human activity is regarded as the primary driver of the collapse in eel populations. The neuroendocrine stress response is central in coping with these challenging conditions, yet little is known about how various biotic factors such as sex, parasites, and ontogeny influence (singly and via interactions) the stress response of eels. In this study, mixed-effects and linear models were used to quantify the influence of sex, parasitism (Anguillicola crassus), life stage (yellow and silver eels), and silvering stage on the stress response of eels when exposed to a standardized handling stressor. The physiological response of eels to a standardized abiotic stressor (netting confinement in air) was quantified through measurements of blood glucose and plasma cortisol. The relationships between biotic factors and the activity of gill Na+/K+-ATPase was also examined. Analyses revealed that in some instances a biotic factor acted alone while in other cases several factors interacted to influence the stress response. Blood glucose concentrations increased after exposure to the standardized stressor and remained elevated after 4 h. Variation in plasma cortisol concentrations after exposure to the stressor were found to be time dependent, which was exacerbated by life stage and parasitism condition. Males and nonparasitized silver eels had the highest Na+/K+-ATPase activity. Silvering stage was strongly positively correlated with Na+/K+-ATPase activity in female eels. Collectively, these findings confirm that the factors mediating stress responsiveness in fish are complicated and that aspects of inherent biotic variation cannot be ignored

Effects of brownification and warming on algal blooms, metabolism and higher trophic levels in productive shallow lake mesocosms

An increase of dissolved organic carbon (DOC) in inland waters has been reported across the northern temperate region but the effects of this on whole lake ecosystems, often combined with other anthropogenic stressors like nutrient inputs and warming, are poorly known. The effects of these changes on different component of the ecosystem were assessed in an experiment using twenty-four large (3000L) outdoor mesocosms simulating shallow lakes. Two different temperature regimes (ambient and ambient +4 °C) combined with three levels of organic matter (OM, added as filtered peaty water), simulating the DOC increase that is predicted to take place over the next 4 to 21 years were used. Neither temperature nor OM had significant effects on net ecosystem production, respiration or gross primary production. Phytoplankton chlorophyll a concentration was not significantly affected by warming, however in summer, autumn and winter it was significantly higher in mesocosms receiving intermediate OM levels (July–Feb DOC concentrations 2–6 mg L−1). Summer cyanobacterial blooms were highest in intermediate, and lowest in the highest OM treatments. OM concentration also influenced total macroinvertebrate abundance which was greater in spring and summer in mesocosms with intermediate and high OM. Fish abundance was not significantly affected by OM concentration, but abundance was greater in ambient (55 fish subsample−1) compared to heated mesocosms (17 fish subsample−1) and maximum abundance occurred two weeks later compared to heated mesocosms. The results suggest that changes in OM may have a greater effect on shallow lakes than temperature and that phytoplankton, especially cyanobacteria, benefit from intermediate OM concentrations, therefore, nuisance algal blooms might increase in relatively clear shallow eutrophic lakes where DOC concentrations increase

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