Ultrastructural alterations in liver and gills of embryonic and larval zebrafish (Danio rerio) following prolonged exposure to waterborne 0.05, 0.5, and 5 mg/L 4-chloroaniline for up to 31 days as well as after a 14-day regeneration period were investigated by means of light and electron microscopy. Acute toxicity was also tested at 25 and 50 mg/L. Survival of zebrafish embryos and larvae was only impaired from 25 mg/L 4-chloroaniline, but-after a transient stimulation following exposure to 0.5 mg/L-4-chloroaniline hatching was retarded after exposure to >/=5 mg/L, and fish displayed increasing rates of abnormal development and pigmentation. In contrast, hepatocytes displayed a time- and dose-dependent response from 0.05 mg/L 4-chloroaniline, including changes in nuclei, mitochondria, peroxisomes, endoplasmic reticulum, Golgi fields, lysosomes, and hepatic glycogen and lipid stores, as well as invasion of macrophages. In gills, dose-dependent effects were evident from 0.5 mg/L 4-chloroaniline and included deformation of secondary lamellae due to vacuolization and desquamation of respiratory epithelial cells in conjunction with dilation of intercellular spaces. Respiratory epithelial cells displayed progressive mitochondrial changes, induction of cytoplasmic myelinated structures, augmentation of lysosomes, and modifications of Golgi fields. Erythrocytes were severely deformed. A 14-day regeneration period was sufficient for almost complete recovery of pathological symptoms in both liver and gills. Only minor volumetric changes in hepatocellular organelles and a limited number of myelinated bodies, lysosomes, and cytoplasmic vacuoles were reminiscent of prior 4-chloroaniline exposure. In both qualitative and quantitative terms, most effects in hepatocytes after exposure of embryonic and larval zebrafish to waterborne 4-chloroaniline are comparable to the reaction of hepatocytes in adult zebrafish liver after prolonged sublethal exposure as well as in larval zebrafish after microinjection. Morphological changes in erythrocytes indicate disturbance of respiration as an additional mode of action of 4-chloroaniline.

Hepatic and renal cytopathological alterations in fingerl~ng rainbow trout Oncorhynchus mykiss following 5 \vk exposure to 30, 120, and 240 pg I -' linuron [3(3,4-dichloropheny1)-l-methoxy-lmethylurea] were studied by electron microscopy. Ultrastructural alterations were detected in liver and kidney at 230 pg l.', 2 orders of magnitude below conventional LCo. The response suggested a doseresponse relationship with a change from adaptive to degenerative features at 120 pg I-' Hepatocyte changes included: stimulation of mitosis; segmentation of nuclei; partial reorganization of heterochromatin; multiplication of nucleoli; fractionation, vesiculation and transformation of rough endoplasmic reticulum (RER) into myelinated bodies; induction of smooth endoplasmic reticulum; moderate steatosis; apparent proliferation of mitochondria, peroxlsomes, Golgi fields and lysosomal elements; depletion of glycogen; perisinusoidal lipid accumulat~on; elevated rate of hepatocytes in various stages of necrosis; infiltration and increased phagocytic activity of macrophages. Reactions of renal tubular cells were differentiated in different nephron segments. Major alterations by site in kidney were (1) renal corpuscle: lobulation of podocyte nuclei; (2) proximal segment I: elevated heterogeneity of all cell components, increased heterochromatin and nuclear size, rearrangement of RER, proliferation of Golgi fields, novel lysosomal elements, decreased mitochondria and lysosomes at 240 1-19 I-'; (3) proximal segment 11: nuclear lobulation, binucleated cells, proliferation of lysosomes and peroxisomes (lower concentrations), decreased peroxisomes and mitochondria (240 pg I-'), crystalline inclusions in lysosomal matrix, fragmentation, degranulation and circular arrangement of RER; (4) distal segment: induction of giant mitochondria with longitudinal crystalline inclusions, atypical lysosomes with long crystalline matrix inclusions, and augmentation of various lysosomal elements. Comparison of linuroninduced cellular alterations with cytopathological effects by potential linuron breakdown products, namely 4-chloroaniline and 3,4-dichloroaniline, revealed a high degree of s~milarity of cytopathological phenomena, indicating that part of the changes observed after linuron exposure might well be due to the action of linuron metabolites.

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In addition to survival and hatching parameters, cytological alterations in liver and kidney of 4- and 6-d old zebrafish larvae (Brachydanio rerio) following single microinjection of fertilized eggs at the germ-ring stage with 5, 12.5, and 25 ng 4-chloroaniline/egg were investigated by means of electron microscopy. Whereas survival remained unaffected, microinjection with 4-chloroaniline disturbed hatching of larvae. Hatching was delayed by microinjection of 12.5 ng 4-chloroaniline/egg and above when compared to controls. Cytological investigations revealed ultrastructural changes in both liver and kidney in a dose- and time-dependent fashion. In the liver, major cytopathological changes included fenestration, fragmentation, and vesiculation of the rough endoplasmic reticulum, proliferation of atypical mitochondria, and atypical lysosomes. Furthermore, myelin whorls, lipid inclusions, and cholesterol crystals were increased, whereas glycogen stores were reduced. Renal tubular cells displayed altered brush borders, proliferation of nucleoli, atypical mitochondria, fenestrated, fragmented, and vesiculated RER cisternae, as well as giant lysosomes. Most of these effects indicate cellular dysfunction (e.g., disturbance of lipid metabolism in the liver), whereas others illustrate general cellular stress-responses to chemical aggression. Comparisons of results with those of previous studies based on conventional fish exposure prove the suitability and sensitivity of microinjection bioassays with zebrafish eggs as an alternative to conventional early life-stage tests.

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