Toxic effects of the non-steroidal anti-inflammatory drug diclofenac. Part I: histopathological alterations and bioaccumulation in rainbow trout.

Human and veterinary pharmaceuticals have been shown to occur in considerably high amounts in sewage treatment plant (STP) effluents and surface waters. The non-steroidal inflammatory drug diclofenac represents one of the most commonly detected compounds. Information concerning possible ecotoxicological risks of the substance are rather scarce. So far there are no data available on its possible effects in fish after prolonged exposure. In order to evaluate sublethal toxic effects of diclofenac in fish, rainbow trout (Oncorhynchus mykiss) exposed to diclofenac concentrations ranging from 1 microg/L to 500 microg/L over a 28 day period were investigated by histopathological methods. In addition, diclofenac residues in various organs were analyzed by means of gas chromatography/mass spectrometry (GC/MS). The histopathological examinations of diclofenac-exposed fish revealed alterations of the kidney such as an hyaline droplet degeneration of the tubular epithelial cells and the occurrence of an interstitial nephritis. In the gills, the predominant finding consisted in a necrosis of pillar cells leading to damage of the capillary wall within the secondary lamellae. The lowest observed effect concentration (LOEC) at which both renal lesions and alterations of the gills occurred was 5 microg/L. In contrast, the light microscopical examination of the liver, the gastro-intestinal tract, and the spleen did not reveal any histopathological alterations neither in diclofenac-exposed fish nor in solvent controls or control individuals. Chemical analysis showed a concentration-related accumulation of diclofenac in all organs examined. The highest amounts could be detected in the liver, followed by the kidney, the gills and the muscle tissue. Dependent on the diclofenac concentration used, the bioconcentration factors (BCF) were 12-2732 in the liver, 5-971 in the kidney, 3-763 in the gills, and 0.3-69 in the muscle respectively. From the present findings it can be assumed, that prolonged exposure in environmentally relevant concentrations of diclofenac leads to an impairment of the general health condition of fish.

Endocrine effects of environmental pollution on Xenopus laevis and Rana temporaria.

To determine the capacity of sewage treatment work effluents to disrupt the endocrine system under semifield conditions, two amphibian species, Xenopus laevis and Rana temporaria, were exposed to the effluent of a regional sewage treatment plant in South Bavaria during larval development until completion of metamorphosis. Exposure was carried out in river water (Würm) as a reference, and a 1:12-mixture sewage effluent representing the real situation on the spot, and in a higher concentration of sewage using a 1:2 mixture. An accidental impact of industrial wastewater into the reference and dilution medium, Würm, which was caused by a spate in the respective area during the sensitive period of sex differentiation of amphibian larvae, is assumed to be responsible for the relatively high percentage of females observed by histological analysis in all treatment groups. All of these values were higher than those determined in controls exposed to artificial tap water in laboratory experiments conducted in a comparable study design. Sex ratios between species, revealed by the semifield study with decreasing portions of females from control to 1:12 to 1:2, were strongly correlated. Determination of biomarker-mRNA-levels in Xenopus liver using semiquantitative RT-PCR at the end of the experimental phase, when exposure regime has turned into the initially expected situation with the highest load of potential estrogens in the effluent, followed by 1:2 and 1:12 mixture, resulted in a significant increase of Vitellogenin-mRNA in female juveniles exposed to the highest portion of sewage, whereas expression of both androgen and estrogen receptor-mRNA showed no clear differences. The results concerning the induction of estrogenic biomarkers are in accordance with our findings for estrogen receptor binding of sample extracts from the Würm and sewage taken in parallel at the end of the experiment, when sewage extracts possessed a much higher ability to displace [3H]estradiol from the estrogen receptor than the ones extracted from the mixtures.

How estrogenic is nonylphenol? A transgenerational study using rainbow trout (Oncorhynchus mykiss) as a test organism.

The aim of the present study was to evaluate both estrogenic effects in directly NP-exposed sexually mature rainbow trout and possible transgenerational effects in the offspring of exposed fish. Four months prior to spawning, adult rainbow trout of both sexes were exposed intermittently to NP concentrations of 1 and 10 microg/l. At the end of the exposure period, which coincided with the beginning of spawning time, vitellogenin levels in the plasma of adult male rainbow showed a significant increase compared to the control group. After exposure to 10 microg NP/l reproduction was impaired as indicated by significantly reduced hatching rates. Histological examination of the testicular tissue of NP-exposed individuals revealed no morphological differences from the controls. In the offspring, vitellogenin levels of male individuals were not affected, whereas in females they were significantly higher than in the control progeny. The histological examination revealed no alteration in sex ratios. In single cases, intersex occurred in both male and female offspring of exposed fish. The analysis of sex steroid levels revealed a two-fold increase of estradiol in the plasma of male offspring and a 13-fold elevation of testosterone in the plasma of female progeny. The present findings indicate that NP, in an environmentally relevant concentration range, acts as a weak estrogen in directly exposed adult male rainbow trout as indicated by elevated plasma vitellogenin levels. Reproduction success was reduced as indicated by decreased hatching rates. Hormonal imbalances detected in the offspring of exposed fish indicate a transgenerational effect mediated by the endocrine system.

Chronic toxicity of nonylphenol and ethinylestradiol: haematological and histopathological effects in juvenile Common carp (Cyprinus carpio).

In recent ecotoxicological research, there is an increasing concern about alkylphenolic industrial chemicals, such as nonylphenol (NP), because of their estrogenic properties. Data on the general fish toxicity of these wide spread aquatic pollutants are scarce. In order to evaluate sublethal toxic effects of environmentally relevant concentrations of NP, juvenile Common carp (Cyprinus carpio) were exposed to NP concentrations ranging from 1 to 15 microg NP/l over a 70-day period. Classical toxicological endpoints, such as various haematological parameters and histopathological alterations were investigated. In a comprehensive protocol, besides NP-induced effects also alterations due to a treatment with the synthetic estrogen ethinylestradiol (EE2) were evaluated. After both the NP-exposure as well as the EE2-treatment, the predominant haematological finding was a severe anaemia. Histopathological alterations in the kidney, the liver and the spleen occurred exclusively after treatment with EE2, whereas NP-exposed fish did not show any tissue lesions. Depending on the haematological parameter examined, a NOEC between 1 and 5 microg NP/l could be established. From the present findings, it is assumed, that under field conditions, the NP-induced, general toxic effects, might outbalance the relatively weak estrogenic effects of this compound and possibly might disturb ecologically relevant processes such as fish reproduction.