Ability of fractionated petroleum refinery effluent to elicit cyto- and photocytotoxic responses and to induce 7-ethoxyresorufin-O-deethylase activity in fish cell lines

The ability of fractionated petroleum refinery effluent to cause cellular r esponses in fish cell lines was evaluated. The cellular responses, which in cluded direct and indirect cytotoxicity, photocytotoxicity and induction of 7-ethoxyresorufin-O-deethylase (EROD) activity, may potentially be linked to sublethal effects observed in effluent-exposed fish and fish larvae. In order to be able to quantify cellular responses rapidly, microtitre plates were used along with fluorescent probes. For the quantification of cyto-and photocytotoxicity, the fluorescent probes were alamar BIue (TM) and carbox yfluorescein diacetate acetoxymethyl ester (CFDA-AM), which monitor metabol ic activity and cell membrane integrity, respectively. EROD activity was me asured as the rate of conversion by EROD of the substrate 7-ethoxyresorufm to its fluorescent product, resorufin. Effluent from an Ontario refinery wa s fractionated into aqueous and particulate phase. As well, a solid phase e xtract (SPE) was used to prepare concentrated effluent for testing in the c ell lines. The effluent was able to elicit all of the responses of interest although significant cyto- and photocytotoxicity required effluent equival ent concentrations above 100% effluent and could only be revealed upon expo sure of cells to the SPE concentrated effluent. Based on their retention on C18, the cytotoxicants are likely to be non-polar to moderately polar chem icals. The presence of polar compounds affecting cellular metabolism was in dicated by the responses of exposed cells to a 90% aqueous phase effluent. In contrast to cyto- and photocytotoxicity, EROD induction occurred at effl uent equivalent concentrations well below 100% effluent and was elicited by the SPE and the particulate fraction thereby suggesting that most EROD-ind ucers were particle-bound. Among other applications, the described techniqu es could help to determine the source of causative agents of sublethal effe cts in the refining process. (C) 2001 Elsevier Science B.V. All rights rese rved.