Abstract
In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.
Highlights
Aquaculture diets contain an increasing amount of plant derived feed commodities
The feed conversion was high throughout the dosing phase for both species, as reflected by the feed conversion ratio (FCR) of 1.34 and 1.67 for rainbow trout and common carp, respectively
The results from this study confirm the presence of relevant differences in the metabolism of xenobiotics/pesticides between fish species
Summary
Aquaculture diets contain an increasing amount of plant derived feed commodities. Contaminated feed may lead to pesticide residues in the edible tissues of food producing animals leading to a potential risk for consumers (Regulation (EC) No 1107/2009). Metabolism studies on fish are required when a pesticide of moderate to high lipophilicity (N log P 3) is used on crops and may lead to significant residues in fish feed, generally considered to be ≥0.1 mg kg−1 of the total diet. Fish metabolism studies performed as part of the pesticide approval process characterize the chemical nature of residues which may occur in the fillets of fish exposed to pesticides and provide an estimate of the total residues expected. To achieve these objectives, rainbow trout or common carp of marketable size (EU-Commission, 2013b) are fed for up to 14 days a commercial fish feed spiked with a radiolabeled pesticide. Based on the results of the tissue analysis, a decision on the need for further fish feeding studies can be made in order to set maximum residue levels (MRLs)
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