Seasonal testosterone UDP-glucuronosyltransferase activity and biliary steroids in Eurasian perch: response to leachate exposure.

In previous studies there was evidence of endocrine disruption in fish in waterways contaminated with leachate from a Swedish landfill. Symptoms included fewer sexually mature (SM) female perch (Perca fluviatilis), distinct sores, reduced gonadosomatic index, and decreased plasma androgen levels. Based on previous findings it was hypothesized that the decrease in plasma androgen concentrations could have been caused by increased steroid excretion. Therefore, testosterone UDP-glucuronosyl transferase (UDPGT) activity and biliary steroids were measured in SM and sexually immature (SIM) female perch during one reproductive cycle. UDPGT activity in exposed fish did not differ from reference fish and was similar in both SM and SIM female perch. Since androgen levels in SIM fish usually are much lower than in SM fish, this indicates expression of testosterone UDPGT irrespective of substrate availability. There was no general increase in biliary steroids in exposed female perch. Biliary steroid levels correlated with corresponding plasma steroid levels. In conclusion, the reduced androgen levels in exposed female perch are most likely not caused by increased steroid excretion.

Basic physiological biomarkers in adult female perch (Perca fluviatilis) in a chronically polluted gradient in the Stockholm recipient (Sweden).

By measuring a battery of basic physiological biomarkers and the concentration of SigmaDDT in adult female perch (Perca fluviatilis), an assumed aquatic pollution gradient was confirmed, with the city of Stockholm (Sweden) as a point source of anthropogenic substances. The investigation included an upstream gradient, westwards through Lake Mälaren (46 km), and a downstream gradient, eastwards through the Stockholm archipelago (84 km). The results indicated a severe pollution situation in central Stockholm, with poor health status of the perch: retarded growth, increased frequency of sexually immature females, low gonadosomatic index, and disturbed visceral fat metabolism. SigmaDDT, measured as a pollution indicator, was 10-28 times higher than the background in perch from the Baltic Proper. Besides the main gradient other sources of pollution also influenced the response pattern of the measured biomarkers. In particular, there were strong indications of pollution coming from the Baltic Sea.

Biochemical biomarkers in adult female perch (Perca fluviatilis) in a chronically polluted gradient in the Stockholm recipient (Sweden).

A battery of biochemical biomarkers and the SigmaPCB concentration in adult female perch (Perca fluviatilis) verified an aquatic pollution gradient with the city of Stockholm (Sweden) as a point source of anthropogenic substances. The investigation included both an upstream gradient, 46 km westwards through Lake Mälaren, and a downstream gradient, 84 km eastwards through the Stockholm archipelago. Besides the main gradient from Stockholm, there were strong indications of pollution coming from the Baltic Sea. The results indicated a severe pollution situation in central Stockholm, with poor health status of the perch, characterised by increased specific EROD activity in the liver, increased liver EROD somatic index, decreased AChE activity in the muscle, increased amount of DNA adducts in the liver, and a high concentration of biliary 1-pyrenol. In addition, laboratory exposure to common EROD inducers elicited an abnormal response, suggestive of chronic intoxication.

Steroid biosynthetic enzyme activities in leachate-exposed female perch (Perca fluviatilis) as biomarkers for endocrine disruption.

Studies have shown that adult female perch in a freshwater lake, Molnbyggen, Sweden, have a reproductive disorder caused by unidentified endocrine disrupting compounds (EDCs) leaching from a local refuse dump. The adverse effects include shallow open sores, low ratio of sexually mature individuals, low gonadosomatic index and low circulating levels of androgens. We hypothesised that the low androgen levels could be a result of impaired production and/or stimulated excretion of androgens by EDCs. From October 2000 to November 2001, at time-points important in the perch reproductive cycle, adult female perch were collected in Molnbyggen and in the reference lake, Djursjön. The activities of three key enzymes in androgen biosynthesis: 17alpha-hydroxylase (17OHlase), 17,20-lyase (lyase) and 17beta-hydroxysteroid dehydrogenase (17betaHSD) were determined in head kidney or ovary. The relationship between enzyme activities and plasma steroid concentrations was examined. Ovarian histopathology and the determination of brain aromatase activity were also included in the study. Similar 17OHlase, 17betaHSD and aromatase activities were found in Molnbyggen females and reference fish throughout the year. Head kidney 17OHlase showed a positive correlation to cortisol levels (r=0.754; p<0.001) but not to androgen levels. Molnbyggen females exhibited lower ovarian lyase activity during vitellogenesis than reference fish. Atretic oocytes were on most occasions more frequent in sexually immature than in sexually mature females. The results suggest that neither 17OHlase, 17betaHSD nor aromatase is the target for EDCs disrupting the androgen homeostasis of exposed female perch. Further investigation is needed to establish the role of decreased ovarian lyase activity in endocrine homeostasis, but the possibility of increased excretion of androgens should also be examined.

Toxicological effects and reproductive impairments in female perch (Perca fluviatilis) exposed to leachate from Swedish refuse dumps.

We have previously found that leachate from a Swedish refuse dump caused toxicological effects, including endocrine disruption and reproductive failures, in feral female perch (Perca fluviatilis) from Molnbyggen and in brook trout (Salvelinus fontinalis) from Vadbäcken. This raised concerns that leachate-induced toxicity might affect fish in other leachate-contaminated lakes. This study looks at female perch from three different regions in Sweden, focusing on toxicological biomarkers (skin lesions, liver-somatic index (LSI), ethoxyresorufin O-deethylase (EROD) activity and DNA adducts) and reproductive biomarkers (number of mature females, gonadosomatic index (GSI), aromatase (P450arom), progesterone, 17alpha-hydroxyprogesterone (17alpha-OHP), testosterone (T) and 17beta-estradiol (E2)). Five out of seven contaminated locations had lower numbers of mature females and most of them exhibited higher frequencies of fin erosion relative to their comparable reference sites. Females from Lake Nedre Vättern exhibited these effects, as well as body sores, high EROD activity, high levels of DNA adducts in the liver and the intestinal mucosa, low GSI and low plasma levels of T and E2, similar to the anti-estrogenic effects earlier found in SM perch from Molnbyggen in 1997 and in brook trout from Vadbäcken. No effects on LSI, GSI, aromatase, or circulating steroids were found in mature females from Molnbyggen in this study. This indicated less leachate-contamination, but low numbers of mature females in Lake Siljan, at the sewage treatment plant which now processes the leachate that earlier contaminated Molnbyggen and Vadbäcken, suggested insufficient sewage treatment at this site. All females in Lake Simshyttsjön were mature, and their high GSI and LSI, low EROD activity, high level of 17alpha-OHP and low level of T, suggest an estrogenic rather than an anti-estrogenic effect. The results show that our earlier findings in Molnbyggen and Vadbäcken are not common, but neither are they unique. This study also shows that refuse dumps should be considered as potential point-sources for environmental pollutants, and that uncontrolled leachate-contamination of lakes and freshwater reservoirs could be a serious environmental hazard for both wildlife and humans.

Reproductive status in female perch (Perca fluviatilis) outside a sewage treatment plant processing leachate from a refuse dump.

Reproductive failure and endocrine disruption in female perch (Perca fluviatilis) and female brook trout (Salvelinus fontinalis), naturally exposed to toxic leachate from a municipal refuse dump, has raised concerns about the reproductive status in female fish outside the sewage treatment plant (STP), which now treats the same leachate. Female perch from the receiving water, Lake Siljan, were therefore sampled in a gradient from the STP in Fornby and Osterviken and compared with reference females from Isunda or Solleron. A high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) technique enabled the analysis of circulating steroids in blood plasma: the sex steroids progesterone (P), 17alpha-hydroxyprogesterone (17alpha-OHP), androstenedione (A), testosterone (T), estrone (E1), and 17beta-oestradiol (E2), the maturation-inducing hormone (MIH) 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), and the glucocorticoids 11-deoxycortisol (11-DC) and cortisol (C). Body and organ measurements, ratio of sexually mature (SM) females, and frequencies of body lesions, were also recorded. The percentages of SM females found in Fornby on Oct 23 (83.8%) and in Osterviken on May 14 (54.0%) were lower compared with Isunda (100%). The lack of differences in gonadosomatic index (GSI) and liver-somatic index (LSI) indicated a normal vitellogenesis in SM females from Fornby and Osterviken. No differences in circulating levels of steroids were observed between the sites, except at the time of spawning, when elevated P and 17alpha-OHP levels in females from Osterviken indicated a steroidogenic shift from the synthesis of sex steroids towards the production of the MIH 17alpha,20beta-P. The significant peak-levels of both 17alpha,20beta-P and 11-DC in female perch from Osterviken at the same time suggest that both are involved in controlling final oocyte maturation in perch. The elevated levels of androgens (A and T) in females caught on the same occasion in Fornby implied that these females were at an earlier stage of oocyte maturation, prior to the steroidogenic shift towards MIH-production. This study confirms the results of an earlier study revealing low numbers of SM female perch outside the STP in Fornby. The lack of associated effects on organ indexes and circulating levels of steroids does not indicate exposure to endocrine disrupting substances (EDSs) from the treated sewage. The consistently high numbers of SM females in the northern part of Siljan suggest, however, that natural factors are not responsible for the reproductive failures in the southern part of the lake.

Gonad development and plasma steroid profiles by HRGC/HRMS during one reproductive cycle in reference and leachate-exposed female perch (Perca fluviatilis).

Endocrine processes were studied in reference female perch (Perca fluviatilis) from Lake Djursjön and in leachate-exposed females from Lake Molnbyggen during one reproductive cycle. A high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) technique enabled the analysis of circulating sex steroids [progesterone (P), 17alpha-hydroxyprogesterone (17alpha-OHP), androstenedione (A), testosterone (T), estrone (E1), and 17beta-estradiol (E2)], the maturation-inducing hormone (MIH) 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), and glucocorticoids [11-deoxycortisol (11-DC) and cortisol]. Body and organ measurements, ratio of sexually mature (SM) females, and frequencies of body lesions, were also recorded. High frequencies of body lesions and a low ratio of SM females were generally found in Molnbyggen. These females also had lower gonadosomatic index (GSI) and liver-somatic index (LSI) in the early stages of vitellogenesis, indicating either a delayed onset of oogenesis or an impaired vitellogenesis. Non-reproducing females from both lakes showed a similar LSI cycle as SM females. Increasing T and A levels in SM females from Djursjön coincided with the GSI peak at the time of spawning. These two androgens were on several occasions lower in SM females from Molnbyggen, possibly the result of a disrupted 17,20-lyase activity of the P450 17alpha enzyme because only minor effects were observed on circulating P and 17alpha-OHP levels. E2 and E1 levels increased during vitellogenesis in females from both lakes, with E1 levels more closely resembling the peaks of A and GSI. Peak levels of 17alpha,20beta-P at the time of spawning confirmed its function as the MIH in perch, while the simultaneous peak of 11-DC suggests that it also may be involved in final maturation of the oocytes.

Altered steroid metabolism in several teleost species exposed to endocrine disrupting substances in refuse dump leachate.

Endocrine disruption associated with reproductive failure has been reported previously in female perch (Perca fluviatilis) and roach (Rutilus rutilus) from Lake Molnbyggen in Sweden and in female brook trout (Salvelinus fontinalis) from Vadbäcken, a stream emptying into Molnbyggen. Both Molnbyggen and Vadbäcken have been contaminated by toxic leachate from a municipal refuse dump. In this study, female perch were caught in Molnbyggen and the reference lake, Lake Djursjön, to further investigate the endocrine mechanism behind the significant numbers of sexually immature (SIM) female perch in Molnbyggen. Blood plasma analysis of progesterone (P), 17alpha-hydroxyprogesterone (17alpha-OHP), testosterone (T), and 17beta-oestradiol (E2), as well as analysis of brain aromatase activity (P450arom), were carried out. The exceptional high numbers of SIM female perch in Molnbyggen was confirmed in February 1999. In July 1999, at an early stage of oogenesis, perch from Molnbyggen showed significantly decreased gonadosomatic index (GSI) and aromatase activity. The presence of aromatase inhibiting substances in lake sediments were therefore tested in vitro. The aromatase activity was dose-dependently inhibited by clotrimazole, reaching 50% inhibition at a concentration of 0.9 microM. Aromatase inhibiting substances were found both in Molnbyggen and reference sediment extracts, indicating that they were naturally occurring substances and not of anthrophogenic origin. The similar decrease in levels of circulating steroids (P, 17alpha-OHP, T, and E2), aromatase, and GSI therefore suggest that the low aromatase activity is due to down-regulation rather than inhibition. To further investigate the steroidogenesis prior to T, P, and 17alpha-OHP were analysed in perch caught in 1997 and 1998 in Lakes Molnbyggen, Kvarntjärn (downstream), Yxen (upstream), and Djursjön, in female roach caught in Molnbyggen and Djursjön in 1997, and in brook trout caught in Vadbäcken and the reference stream Björntjärnsbäcken in 1998. The absence of differences in P and 17alpha-OHP levels, combined with a significantly lower T level in female perch and roach from Molnbyggen in 1997, could be the result of either increased metabolism and excretion of T, or a disruption downstream of 17alpha-OHP formation. The unaffected P levels and significantly lower 17alpha-OHP levels, together with significantly decreased T and E2 levels, found in adult (>45g) female brook trout from Vadbäcken, further indicated that an altered steroidogenesis downstream of P is one possible mechanism underlying the low T levels and thus the high number of SIM female fish, since too low T levels might be insufficient to activate the brain-pituitary-gonadal axis.

Endocrine disruption in brook trout (Salvelinus fontinalis) exposed to leachate from a public refuse dump.

Lake Molnbyggen was previously found to harbour a large number of sexually immature female perch (Perca fluviatilis) suffering from endocrine disruption. In an attempt to pin-point the source of the endocrine-disrupting substance(s) (EDSs), brook trout (Salvelinus fontinalis) from Vadbäcken, a stream contaminated by leachate from a public refuse dump and which empties into Lake Molnbyggen, were investigated. In addition, female perch from Lakes Yxen and Kvarntjärn, located up-stream and down-stream of Lake Molnbyggen, were investigated. Only 16.7% of the adult female brook trout in Vadbäcken were sexually mature, associated with decreased gonadosomatic index, lower brain aromatase activity, and lower circulating levels of testosterone and 17beta-oestradiol, in comparison to female brook trout from the reference stream Björntjärnsbäcken. Male brook trout showed decreased gonadosomatic index, in addition to bile duct hyperplasia in the liver, which was also found in female brook trout livers from Vadbäcken. In Lake Molnbyggen, 57.6% of the female perch were found to be sexually immature with high frequencies of skin lesions, such as sores and fin erosion, significantly decreased gonadosomatic index, lower aromatase activity, and lower levels of testosterone and 17beta-oestradiol. No signs of reproductive disorders or endocrine disruption were seen in female perch from Lakes Yxen and Kvarntjärn compared to female perch from the reference lake, Lake Djursjön. Since brook trout of both sexes from Vadbäcken displayed the same kind of serious adverse impairment of gonad development and endocrine disruption as perch from Lake Molnbyggen, very strong evidence are provided that the refuse dump is the source for the responsible EDS(s), since both Vadbäcken and Lake Molnbyggen are known to be contaminated by leachate from that dump. The low levels of PAHs and PCBs in the surface sediments of Lake Molnbyggen suggest that these pollutants are not the responsible EDS(s).