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.

Segment specificity of the cytological response in rainbow trout (Oncorhynchus mykiss) renal tubules following prolonged exposure to sublethal concentrations of atrazine.

Cytopathological alterations in proximal (PS I, PS II) and distal segments (DS) of rainbow trout (Oncorhynchus mykiss) renal tubules following exposure to 0, 10, 20, 40, 80, and 160 micrograms/liter atrazine for 4 weeks were investigated by means of electron microscopy. Cellular responses were clearly dose-dependent with a gradual increase in variability and intensity of effects. Ultrastructural modifications in PS I and II were observed from 10 micrograms/liter atrazine, in DS from 20 micrograms/liter. In PS I, major changes included proliferation of smooth endoplasmic reticulum, atypical mitochondria and lysosomes, as well as gradual alterations of the apical plasmalemma. Typical changes in PS II cells were a proliferation of peroxisomes and ring- and cup-shaped mitochondria, as well as alterations in the basal labyrinth; DS cells were characterized by a proliferation of atypical mitochondria with longitudinally oriented cristae, disorganization of Golgi fields and vacuolization of the cell base. Results document that different segments of the renal tubule in rainbow trout react not only with different levels of sensitivity to atrazine exposure, but also in a segment-specific way. Moreover, comparison of effects induced by atrazine with those resulting from chronic exposure to the herbicide linuron revealed a distinct substance specificity in the reaction of different tubular segments.

[Rainbow trout and zebrafish, two models for continuous toxicity tests: relative sensitivity, species and organ specificity in cytopathologic reaction of liver and intestines to atrazine]. / Regenbogenforelle und Zebrabärbling, zwei Modelle für verlängerte Toxizitätstests: Relative Empfindlichkeit, Art- und Organspezifität in der cytopathologischen Reaktion von Leber und Darm auf Atrazin.

In order to elucidate cytopathological alterations in hepatic and intestinal cells, immature rainbow trout (Oncorhynchus mykiss) were exposed for five weeks to 10, 20, 40, and 160 micrograms/l of the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine; model 1). For comparison, ultrastructural changes in female zebra fish (Brachydanio rerio) liver were studied after exposure to 100, 1,000 and 10,000 micrograms/l atrazine for three months (exposure from egg stage to sexual maturation; model 2). Neither epithelial nor glandular cells in the gastrointestinal tract of rainbow trout reveal cytological modifications following exposure to atrazine. In contrast, hepatocytes of rainbow trout and zebra fish clearly display dose-dependent and species-specific cytopathological effects at 40 and 1000 micrograms/l, respectively. In rainbow trout (model 1), rough endoplasmic reticulum (RER) appears of particular diagnostic value for the effects of atrazine, since it already shows a full spectrum of cytological alterations after 40 micrograms/l, and since in cells without RER modifications no further cytopathological symptoms can be revealed. At 40 micrograms/l atrazine, further changes include disturbance of the intracellular compartmentation, increased heterogeneity of mitochondria (longitudinally arranged cristae, branching, size), formation of myelinated bodies as well as immigration of macrophages and granulocytes along the biliary system and the space of Disse. The separation of peripheral storage areas from the central organelle-containing cytoplasm is no longer evident at 80 micrograms/l, and the phagocytic activity of invading macrophages is drastically increased. Following exposure to 160 micrograms/l atrazine, additional pathological changes comprise clubshaped deformation of mitochondria, formation of myelinated bodies in the intermembranous space of mitochondria, increase of degranulated ER cisternae and lysosomes, as well as perisinusoidal accumulation of lipid droplets. Deformation of the nuclear envelope, elevated mitotic activity and an increased number of nuclei with two or more nucleoli indicate interactions between atrazine and the nucleus. In the liver of female zebra fish (model 2), atrazine-induced alterations are limited to increased parenchymal variability, disturbance of the intracellular compartmentation, partial RER fractionation and vesiculation, club-shaped deformation of mitochondria and an increase in the number of lysosomes, myelinated bodies and invading macrophages at 1000 micrograms/l atrazine. After three months at 10,000 micrograms/l, mortality of zebra fish is increased to 100%. According to cytopathological alterations of hepatocytes following long-term exposure, susceptibility of the test model rainbow trout to atrazine appears higher than that of the model zebra fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphological effects of acute and chronic atrazine exposure in rainbow trout (Oncorhynchus mykiss).

The effects of the herbicide atrazine (2-Chlor-4-ethyl-amino-6-isopropyl-amino-s-triazine) on the kidney of rainbow trout (Oncorhynchus mykiss) were studied by exposing them to sublethal concentrations of 1.4 and 2.8 mg atrazine per liter of water for 96 h (acute exposure) respectively to 5, 10, 20, 40, and 80 micrograms/L for a period of 28 days (chronic exposure). Alterations of the different components of renal corpuscles and of renal tubules, as well as an increase in cells with mitotic figures in renal hemopoietic interstitium were constant features at lower chronic (5, 10, 20, 40 micrograms/L) exposure; necrosis of endothelial cells and renal hemopoietic tissue were prominent at concentrations of 80 micrograms/L, and 1.4 and 2.8 mg/L atrazine.

Lungs of Polypterus and Erpetoichthys.

The wall of the asymmetrical saclike lungs of the fishes Polypterus and Erpetoichthys consists of several functionally different tissue layers. Their lumen is lined by a surface epithelium composed of (1) highly attenuated cells, termed pneumocytes I; (2) pneumocytes II with lamellar bodies, presumably indicating surfactant production; (3) mucous cells; and (4) ciliated cells. Underlying the pneumocytes I is a dense capillary net. The thin continuous endothelium of this net, together with the pneumocytes I, constitute the very thin blood-air barrier. The basement membrane of epithelium and endothelium fuse in the area of the blood-air barrier (thickness 210 m̈m). Secretory and ciliary cells form longitudinal rows in the epithelium. Below the zone with a gas-exchanging tissue, a layer of connective tissue containing collagen and special elastic fibers occurs. The blood vessels that give rise to or drain the superficial capillary plexus are located in this connective tissue. The outermost layer of the lung consists of muscle cells, a narrow inner zone with smooth muscle cells, and an outer, broader zone with cross-striated muscle cells. The lung is innervated by myelinated and nonmyelinated nerve fibers. The morphology of the gas-exchange tissue in the lungs of these primitive bony fish is fundamentally very similar to that of the lungs of tetrapod vertebrates. The morphologic observations are in close agreement with physiologic data, disclosing well-developed respiratory capacities. Structural simplicity can be regarded as a model from which the lungs of the higher vertebrates derived. In addition to respiratory function, the lungs seem also to have hydrostatic tasks.