ACUTE AND SUBLETHAL TOXICITY OF SEEPAGE WATERS FROM GARBAGE DUMPS TO PERMANENT CELL-LINES AND PRIMARY CULTURES OF HEPATOCYTES FROM RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) - A NOVEL-APPROACH TO ENVIRONMENTAL RISK ASSESSMENT FOR CHEMICALS AND CHEMICAL-MIXTURES

In order to evaluate the suitability of cytopathological criteria in i solated fish hepatocytes as endpoints in (eco)toxicological research, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by colla genase perfusion were exposed in vitro for up to 5 days to sublethal d ilutions of two seepage water samples collected from garbage dumps. He patocytes were analysed with respect to acute (lactate dehydrogenase l eakage) and sublethal toxicity (electron microscopy, stereology). In a ddition, acute toxicity (24 h) was tested in the piscine fibrocytic ce ll line R1 by means of crystal violet staining and neutral red retenti on. Acute toxicity in R1 cells and isolated hepatocytes could only be documented for sample I at dilutions of 1:2 and 1:4. This difference i n toxicity could be corroborated by cytological alterations in isolate d hepatocytes, which could be documented for dilutions of 1:100 and 1: 8 in samples I and II, respectively. Ultrastructural changes were time - and dose-dependent and included reduction of hepatocellular volume, disturbance of intracellular compartmentation, modified heterochromati n distribution, transformation of rough endoplasmic reticulum into con centric membrane whorls, proliferation of lysosomes and cytoplasmic va cuoles, as well as reduction of hepatocellular glycogen. Although seve ral hepatocellular reactions were found after exposure to either sampl e, the syndrom of ultrastructural alterations allowed clear differenti ation between the two samples. Results illustrate that cytological eff ects far below macroscopically detectable damage can be discovered not only in intact fish, but also in fish cell culture systems. On the ba sis of the data presented, a multi-tiered test procedure for aquatic t oxicity assessment exclusively based on tests with fish cell culture s ystems is proposed: (1) rapid screening for acute toxicity with perman ent cell lines; (2) short-term tests with more complex, yet more sensi tive systems such as primary hepatocytes with straightforward biochemi cal endpoints; (3) prolonged exposure of isolated hepatocytes in combi nation with ultrastructural and biochemical investigations as a sensit ive tool to detect adverse effects at environmentally relevant toxican t concentrations.