Thyroid disruption properties of three indoor dust chemicals tested in Silurana tropicalis tadpoles.

Indoor dust contains a multitude of industrial chemicals, and ingestion of dust is considered an important exposure route to organic contaminants. Some of these contaminants have been shown to interfere with the thyroid system, which may result in significant consequences on public health. The amphibian metamorphosis is a thyroid hormone-dependent process, which can be used as an in vivo model for studies on thyroid hormone-disrupting potency. Three contaminants of indoor dust were tested on metamorphosing Silurana (Xenopus) tropicalis tadpoles. The tested chemicals were Tris (1,3-dichloroisopropyl) phosphate (TDCiPP), tetrabromobisphenol-A (TBBPA) and propylparaben (PrP). Measurements reflecting general growth, development progress and thyroid epithelial cell height were performed on the exposed tadpoles as well as chemical analyses of the exposure water. It was shown that TDCiPP acts as a thyroid hormone-disrupting chemical in metamorphosing tadpoles by causing increased epithelial cell height in thyroid glands after exposure to a nominal concentration of 0.010 mg/L and in higher concentrations. TBBPA caused reductions in general growth of tadpoles at the nominal concentration 0.125 mg/L, and PrP caused acute toxicity at the nominal concentration 12.5 mg/L. However, no evident indications of specific thyroid-disrupting effects caused by TBBPA or PrP were observed.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Naproxen affects multiple organs in fish but is still an environmentally better alternative to diclofenac.

The presence of diclofenac in the aquatic environment and the risks for aquatic wildlife, especially fish, have been raised in several studies. One way to manage risks without enforcing improved wastewater treatment would be to substitute diclofenac (when suitable from a clinical perspective) with another non-steroidal anti-inflammatory drug (NSAID) associated with less environmental risk. While there are many ecotoxicity-studies of different NSAIDs, they vary extensively in set-up, species studied, endpoints and reporting format, making direct comparisons difficult. We previously published a comprehensive study on the effects of diclofenac in the three-spined stickleback (Gasterosteus aculeatus). Our present aim was to generate relevant effect data for another NSAID (naproxen) using a very similar setup, which also allowed direct comparisons with diclofenac regarding hazards and risks. Sticklebacks were therefore exposed to naproxen in flow-through systems for 27 days. Triplicate aquaria with 20 fish per aquarium were used for each concentration (0, 18, 70, 299 or 1232 µg/L). We investigated bioconcentration, hepatic gene expression, jaw lesions, kidney and liver histology. On day 21, mortalities in the highest exposure concentration group unexpectedly reached ≥ 25 % in all three replicate aquaria, leading us to terminate and sample that group the same day. On the last day (day 27), the mortality was also significantly increased in the second highest exposure concentration group. Increased renal hematopoietic hyperplasia was observed in fish exposed to 299 and 1232 µg/L. This represents considerably higher concentrations than those expected in surface waters as a result of naproxen use. Such effects were observed already at 4.6 µg/L in the experiment with diclofenac (lowest tested concentration). Similar to the responses to diclofenac, a concentration-dependent increase in both relative hepatic gene expression of c7 (complement component 7) and jaw lesions were observed, again at concentrations considerably higher than expected in surface waters. Naproxen bioconcentrated less than diclofenac, in line with the observed effect data. An analysis of recent sales data and reported concentrations in treated sewage effluent in Sweden suggest that despite higher dosages used for naproxen, a complete substitution would only be expected to double naproxen emissions. In summary, naproxen and diclofenac produce highly similar effects in fish but the environmental hazards and risks are clearly lower for naproxen. Hence, if there are concerns for environmental risks to fish with diclofenac, a substitution would be advisable when naproxen presents an adequate alternative from a clinical point-of-view.

Uptake and biotransformation of structurally diverse brominated flame retardants in zebrafish (Danio rerio) after dietary exposure.

Zebrafish (Danio rerio) were fed a diet containing a mixture of 11 structurally diverse brominated flame retardants (BFRs) at nominal concentrations of either 1 or 100 nmol/g for up to 42 d, followed by an elimination period of 14 d. Uptake rates and elimination constants for five of the BFRs were calculated from measurements of their concentrations in the male fish during the exposure and elimination phases. Observed uptake efficiencies were highest for 2,4,4'-tribromodiphenyl ether (BDE 28) and 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) and were lowest for decabromodiphenyl ether (BDE 209). Estimated half-lives for TBECH and 2,4,6-tribromophenol were short (<2 d). Four BFR metabolites were identified in the fish: 2,2',3,4',5',6-Hexabromodiphenyl ether (BDE 149), 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE 154), 2,4,6-tribromoanisole, and 1,2,4,5-hexabromobenzene. These metabolites were still present in the zebrafish after the 14-d elimination period. No relationship between the BFR concentrations in the zebrafish and their log octanol-water partition coefficient (Kow) values was found. Generally, low tendencies to bioaccumulate were observed for perbrominated and hydroxylated compounds. The observed accumulation of BFR metabolites in fish, however, shows that low concentration of a BFR does not provide, in isolation, a sound indication that the BFR poses low risks.

Embryotoxicity of ozonated diclofenac, carbamazepine, and oxazepam in zebrafish (Danio rerio).

Pharmaceutical residues are polluting the surface water environments worldwide. Sewage and wastewater treatment, therefore, needs to be improved in order to remove pharmaceutical residues from the effluent. One such treatment improvement is effluent ozonation. Even though ozonation has proven to be very efficient in reducing pharmaceutical parent compound concentrations in wastewater effluents, much remains unclear regarding potentially toxic ozonation by-product (OBP) formation. In this study, we sought to elucidate the aquatic toxicity of ozonated pharmaceuticals in zebrafish (Danio rerio) embryos in a static 144 h post fertilization (hpf) fish embryotoxicity (ZFET) assay. Three pharmaceuticals commonly detected in wastewater effluents, i.e. carbamazepine, diclofenac, and oxazepam, were selected for testing. Toxicity was assessed before and after 1 min ozonation (0.053 mg L-1 peak O3 concentration) and 10 min ozonation (0.147 mg L-1 peak O3 concentration). Chemical analysis showed that carbamazepine and diclofenac were largely removed by ozone (90 ±â€¯11% and 97 ±â€¯3.8%), whereas oxazepam was removed to a lesser extent (19 ±â€¯5.7%). The ZFET assay revealed diverging toxicities. Diclofenac embryotoxicity decreased with increasing ozonation. Oxazepam did not cause embryotoxicity in the ZFET assay either pre- or post ozonation, but larvae swimming activity was affected at 144 hpf. Carbamazepine embryotoxicity, on the other hand, increased with increasing ozonation. Chemical analysis showed the formation of two OBPs (carbamazepine-10,11-epoxide and 10,11-dihydrocarbamazepine), possibly explaining the increased embryotoxicity. The results of this study highlight the importance of new chemical and toxicological knowledge regarding the formation of OBPs in post-ozonated effluents.

Maternal transfer of brominated flame retardants in zebrafish (Danio rerio).

In many species reproduction and embryonic development have been shown to be sensitive to environmental contaminants. Understanding embryonic exposure to environmental contaminants is thus highly important. In this study concentrations of brominated flame retardants (BFRs) were measured in zebrafish eggs after parental exposure for 42 days via the diet. Zebrafish were exposed to two doses of eleven structurally-diverse BFRs. Eight BFRs were detected in the female zebrafish and maternal transfer to eggs was evident for all eight compounds. The highest concentrations in eggs were observed for hexabromocyclododecane (HBCD) and 2,4,4'-tribromodiphenyl ether (BDE 28), followed by 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE 183) and tetrabromobisphenol A 2,3-dibromopropyl ether (TBBPA DBPE). Five potential BFR metabolites were tentatively identified in female fish and maternal transfer was observed also for these compounds. The lipid adjusted concentrations in eggs were significantly higher than the concentrations in female fish for several of the BFRs. Further, the results showed a generally higher transfer in the lower exposure level and also indicated a dependency on the physico-chemical properties of the compounds.

Effects of ozonated sewage effluent on reproduction and behavioral endpoints in zebrafish (Danio rerio).

Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O3/m3) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish.

The impact of the goitrogen 6-propylthiouracil (PTU) on West-African clawed frog (Xenopus tropicalis) exposed during metamorphosis.

This study investigated the suitability of using tadpoles of the West-African clawed frog (Xenopus tropicalis) for studying adverse effects on the thyroid hormone system after chemical exposure. Tadpoles were exposed to the thyroxine synthesis inhibitor 6-propylthiouracil (PTU) at concentrations between 2-75mg/L during 14 days. After 5 and 14 days of exposure the developmental stage, hind limb length, body length and weight were measured. Moreover, histological measurements of the thyroid glands were performed after 14 days of exposure. These measurements included maximum thyroid cross-section area, follicular area and epithelial cell height. Tadpoles in the 75mg/L treatment were less developed and had shorter hind limb length than the control group after 14 days of exposure. No effects were recorded on these parameters at lower PTU concentrations. The histological measurements revealed clear dose-response relationships in both follicular cross-section area and epithelial cell height, with lowest observed effect concentrations (LOECs) recorded at 2 and 5mg/L, respectively. This study shows that X. tropicalis is a suitable species for detection of thyroid disrupting chemicals. Further, histopathological measurements of thyroid glands are more sensitive parameters compared with apical endpoints when studying adverse effects on thyroid hormone system caused by PTU exposure in X. tropicalis.

Distribution of BDE-99 and effects on metamorphosis of BDE-99 and -47 after oral exposure in Xenopus tropicalis.

The high concentrations of polybrominated diphenylethers (PBDEs) in the environment have raised the need for generating more information about the impact of these substances on animals. To study the distribution of (14)C-labelled 2,2',4,4',5-pentabromodiphenyl ether ((14)C-BDE-99) in Xenopus tropicalis (West African clawed frog) (14)C-BDE-99 was administered by dietary exposure to tadpoles at stage 54 or to juvenile frogs at stage 66. Whole-body autoradiography and liquid scintillation counting were used to examine the distribution of the substance at different survival times. Further, X. tropicalis tadpoles were dietarily exposed to the PBDE congeners BDE-47 and BDE-99 to study the effects on metamorphosis process. Measurements like body weight, body length, hind limb length and developmental stage as well as histological measurements on thyroid glands were performed after 14 days of exposure. Autoradiograms revealed high concentrations and long term retention of (14)C-BDE-99 in adipose tissue and melanin in frogs exposed both as tadpoles and juveniles. Further, a difference in uptake was recorded between the exposures at stages 54 and 66, implying that the juvenile frogs have higher uptake and more prolonged retention of the chemical than the tadpoles. Hind limb length was reduced in tadpoles dietarily exposed to 1mg/g feed of both BDE congeners. This was associated with reduced body weight and body length for BDE-47, suggesting general toxicity. Tadpoles exposed to BDE-99 also showed lower developmental stage but no effects on body weight or body length, suggesting possible thyroid hormone disruption. Higher concentrations of both congeners caused increased mortality. Thus, it can be concluded that in the present study, BDE-99 was retained for a longer period in the juvenile frogs than in metamorphosing tadpoles and that BDE-99 had an impact on X. tropicalis metamorphosis that might be of thyroid disrupting origin.

Diclofenac affects kidney histology in the three-spined stickleback (Gasterosteus aculeatus) at low µg/L concentrations.

Diclofenac, a commonly used non-steroidal anti-inflammatory drug, is considered for regulation under the European water framework directive. This is because effects on fish have been reported at concentrations around those regularly found in treated sewage effluents (∼1µg/L). However, a recent publication reports no effects on fish at 320µg/L. In this study, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to 0, 4.6, 22, 82 and 271µg/L diclofenac in flow-through systems for 28days using triplicate aquaria per concentration. At the highest concentration, significant mortalities were observed already after 21days (no mortalities found up to 22µg/L). Histological analysis revealed a significant increase in the proportion of renal hematopoietic tissue (renal hematopoietic hyperplasia) after 28days at the lowest concentration and at all higher concentrations, following a clear dose-response pattern. Skin ulcerations of the jaw were noted by macroscopic observations, primarily at the two highest concentrations. No histological changes were observed in the liver. There was an increase in the relative hepatic mRNA levels of c7 (complement component 7), a gene involved in the innate immune system, at 22µg/L and at all higher concentrations, again following a clear dose-response. The bioconcentration factor was stable across concentrations, but lower than reported for rainbow trout, suggesting lower internal exposure to the drug in the stickleback. In conclusion, this study demonstrates that diclofenac causes histological changes in the three-spined stickleback at low µg/L concentrations, which cause concern for fish populations exposed to treated sewage effluents.

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.

Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals.

Vitellogenin (VTG) protein, VTG mRNA, other egg yolk proteins, vitelline envelope proteins and their mRNAs are produced in the liver of oviparous species by stimulation of endogenous estrogen and exogenous estrogenic chemicals. The VTG assay based on enzyme-linked immunosorbent assay (ELISA) has been widely used for many fish species to screen estrogenic and anti-estrogenic activities of chemicals and sewage effluents using immature fish and/or male fish. In order to reduce the number of fish for screening of estrogenicity and anti-estrogenicity of chemicals, primary cultured fish hepatocytes can be used. In fact, primary cultured hepatocytes have been successfully used for the detection of estrogenic and anti-estrogenic activities of environmental chemicals in selected OECD fish species, e.g., medaka (Oryzias latipes) and rainbow trout (Oncorhynchys mykiss) together with other fish species such as Atlantic salmon (Salmo salar L.), Siberian sturgeon (Acipenser baeri), tilapia (Oreochromis mossambicus), carp (Cyprinus carpio), bream (Abramis brama), Carassius auratus, silver eel (Anguilla anguilla L.), and channel catfish (Ictalurus punctanus). In terms of hepatocyte assays relating to other taxa, these include frogs such as Xenopus laevis and the common green frog (Rana esculenta), chickens (Gallus domesticus) and herring gulls (Larus argentatus). VTG mRNA measurement by quantitative reverse transcription-polymerase chain reaction has also been successfully applied in the primary cultured hepatocytes of various species.

Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17α-ethinylestradiol and 17ß-trenbolone.

Environmental estrogens and androgens can be present simultaneously in aquatic environments and thereby interact to disturb multiple physiological systems in organisms. Studies on interaction effects in fish of androgenic and estrogenic chemicals are limited. Therefore, the aim of the present study was to evaluate feminization and masculinization effects in zebrafish (Danio rerio) exposed to combinations of two synthetic steroid hormones detected in environmental waters: the androgen 17ß-trenbolone (Tb) and the oestrogen 17α-ethinylestradiol (EE2). Juvenile zebrafish were exposed between days 20 and 60 post-hatch to different binary mixtures of Tb (1, 10, and 50 ng/L) and EE2 (2 and 5 ng/L). The endpoints studied were whole-body homogenate vitellogenin concentration at 40 days post-hatch, and sex ratio including gonad maturation at 60 days post-hatch. The feminizing potency of 5 ng/L of EE2, alone as well as in combination with Tb, was clear in the present study, with exposures resulting in almost all-female populations and females being sexually immature. Masculinization effects with male-biased sex ratios were observed when fish were exposed to 2 ng/L of EE2 in combination with Tb concentrations. Intersex fish were observed after exposure to mixtures of 2 ng/L EE2 with 50 ng/L Tb. Sexual maturity generally increased among males at increasing concentrations of Tb. The results of the present study show that exposure to environmentally relevant mixtures of an oestrogen and androgen affects the process of gonad differentiation in zebrafish and lead to sexual disruption.

Delayed effects on plasma concentration of testosterone and testicular morphology by intramuscular low-dose di(2-ethylhexyl)phthalate or oestradiol benzoate in the prepubertal boar.

The immediate and delayed effects of prepubertal exposure to di(2-ethylhexyl)phthalate (DEHP) or oestradiol benzoate on the plasma concentrations of testosterone, oestradiol and LH, as well as testicular morphology were examined in prepubertal boars. In a split litter design experiment, prepubertal boars were intramuscularly exposed to DEHP, oestradiol or vehicle during five weeks, starting at six weeks of age. The dose of DEHP was 50mg/kg of bodyweight twice weekly, which is in the same range as recently used oral doses in rodents. Oestradiol-benzoate was administered at 0.25mg/kg of bodyweight twice weekly. One set of animals was examined immediately after the exposure, and the other set was examined at an age of 7.5 months. During the exposure period concentrations of LH in plasma were lower (p=0.02) in the oestradiol-treated animals than in the control group. In the group exposed to oestradiol, the relative to the body weight of the testicles tended to be lower (p=0.07) than control immediately after five weeks of exposure, and the relative to the body weight of the seminal vesicles tended to be lower (p=0.05) than control at 7.5 months of age. In the DEHP-exposed group an elevated (p=0.005) concentration of testosterone and increased (p=0.04) area of the Leydig cells in the testicles compared to the control group were seen at 7.5 months of age. These data suggest that DEHP early in life causes delayed effects on the reproductive system in the adult.

Tissue uptake, distribution and elimination of (14)C-PFOA in zebrafish (Danio rerio).

Perfluorooctanoic acid (PFOA) is a long-chain perfluorinated chemical that has been shown to be non-degradable and persistent in the environment. Laboratory studies on bioconcentration and compound-specific tissue distribution in fish can be valuable for prediction of the persistence and environmental effects of the chemicals. In the present study male and female zebrafish (Danio rerio) were continuously exposed to 10µg/L of radiolabeled perfluorooctanoic acid ((14)C-PFOA) for 40 days, after which the exposed fish were transferred to fresh clean water for another 80 days wash-out period. At defined periodic intervals during the uptake and wash-out, fish were sampled for liquid scintillation counting and whole body autoradiography to profile the bioconcentration and tissue distribution of PFOA. The steady-state concentration of (14)C-PFOA in the zebrafish was reached within 20-30 days of exposure. The concentration-time course of (14)C-PFOA displayed a bi-exponential decline during washout, with a terminal half-life of approximately 13-14 days. At steady-state the bioconcentration of (14)C-PFOA into whole-body fish was approximately 20-30 times greater than that of the exposure concentration, with no differences between females and males. The bioconcentration factors for liver and intestine were approximately 100-fold of the exposure medium, while in brain, ovary and gall bladder the accumulation factors were in the range 15-20. Whole-body autoradiograms confirmed the highest labeling of PFOA in bile and intestines, which implies enterohepatic circulation of PFOA. The (14)C-PFOA was also observed in maturing vitellogenic oocytes, suggesting chemical accumulation via yolk proteins into oocytes with plausible risk for adverse effects on early embryonic development and offspring health. The bioconcentration at several (14)C-PFOA exposure concentrations were also investigated (0.3-30µg/L). This showed that bioconcentration increased linearly with tank exposure in the present in vivo model under steady-state conditions. From this model tissue concentrations of PFOA can be predicted when the external exposure level is known. The present study has generated experimental data on PFOA kinetics in zebrafish that can be valuable for aquatic environmental risk assessment.

Physiological, biochemical and morphological studies of Baltic salmon yolk-sac fry with an experimental thiamine deficiency: relations to the M74 syndrome.

Sea-run Baltic salmon (Salmo salar) populations are suffering from the M74 syndrome, a reproduction disorder affecting both broodfish and their progeny. The syndrome is usually manifested during the middle part of the yolk-sac fry stage and has been shown to be associated with a thiamine (vitamin B(1)) deficiency. Development of the disease is reversible by thiamine treatments of broodfish or progeny. This study aimed at investigating the ability of the thiamine antagonist pyrithiamine, administered by microinjections 3 days after hatch, to cause M74-like signs i.e. typical clinical symptoms, high mortality rates and histopathological changes. Furthermore, the effects of pyrithiamine on hepatic activities of the thiamine-dependent enzyme transketolase (TK), the glucose-6-phosphate dehydrogenase (G6PDH) and the cytochrome P4501A (CYP1A) were evaluated. Six family groups with differing thiamine status were sampled on three occasions during the yolk-sac fry stage. All pyrithiamine exposed groups, with the exception of the one with the highest thiamine concentration, showed M74-like symptoms and suffered from high mortality. Enzyme activities were not different in pyrithiamine groups as compared with controls. However, the TK-activities were strongly associated with the thiamine concentrations. The G6PDH-activity demonstrated small variations with the highest activities in the M74-groups. The [TK]/[G6PDH]-ratios were considerably lower in the M74-groups than in the healthy controls, indicating an imbalance between the oxidative and the non-oxidative part of the pentose-phosphate shunt due to a deficit in thiamine. The pyrithiamine-injections induced several M74-like symptoms including incoordination, lethargy, whitened liver and yolk-sac precipitates. They also caused high mortality rates, in addition to lowered glycogen levels and increased prevalence of necrotic brain cells. Moreover, the study demonstrates that the TK, G6PDH and CYP1A-activities are associated with the thiamine content.

Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone.

In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.

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).

Impact of PCB on resistance to Flavobacterium psychrophilum after experimental infection of rainbow trout Oncorhynchus mykiss eggs by nanoinjection.

The effects of sublethal exposure of a commercial blend of polychlorinated biphenyls (PCB), i.e. Clophen A50, on disease resistance to the aetiological agent of rainbow trout fry syndrome, Flavobacterium psychrophilum, were investigated. Newly fertilised rainbow trout Oncorhynchus mykiss eggs were nanoinjected with 2 doses of Clophen A50 (0.4 or 2 microg egg(-1)) and/or 100 colony forming units of F. psychrophilum. The mean cumulative mortality in control groups, and groups exposed to the lower dose of Clophen A50 (0.4 microg egg(-1)) was below 5.0%. The mean cumulative mortality in groups exposed to the higher dose of Clophen A50 (2.0 microg egg(-1)) was 5.8%, which was not significantly different from the control groups. In all groups infected with F. psychrophilum, with or without exposure to Clophen A50, significantly higher cumulative mortalities compared with control groups were recorded. No differences in mortality were recorded between groups exposed to bacteria alone or bacteria in combination with the higher dose of Clophen A50 (21.6 and 20.4%, respectively). Decreased disease resistance was recorded in groups exposed to F. psychrophilum and the lower dose of Clophen A50, with a mean cumulative mortality of 56.0%. These results could be due to non dose-dependent effects on the immune system, or toxic effects of PCB or their metabolites on the bacteria in groups exposed to the higher dose of Clophen A50. The present study indicates that maternal transfer of PCB might affect disease resistance to vertically transmitted F. psychrophilum.