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3R-Project 141-14

In-vitro alternatives to in-vivo bioconcentration-testing in fish: restricted to rainbow trout or broadly applicable ?

Helmut Segner1, Christian Schlechtriem2

1Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, Switzerland

2Fraunhofer Institute of Molecular Biology and Applied Ecology, Schmallenberg, Germany,,

Keywords: carp; rainbow trout; bioaccumulation; pbt-assessment; bioaccumulation; in-vitro metabolism assay

Duration: 2 years Project Completion: 2015

Background and Aim

Worldwide programmes appertaining to the regulation of chemicals call for an assessment of their risks to mankind and the environment in three respects: Persistence, Bioaccumulation and Toxicity (PBT). In aquatic organisms, the bioaccumulation of chemicals is assessed using a standardized in-vivo bioconcentration-test in fish (OECD Test Guideline 305). The OECD-305 test is implemented to determine the bioconcentration factor (BCF), which represents the ratio between the concentration in the fish and its concentration in the animal’s aquatic environment (under steady-state conditions). The test involves exposing many fish (> 100) to the targeted chemical for long periods of time (> 50 day), and is a costly undertaking.

As an alternative to the in-vivo test in fish, an in-silico approach can be implemented to predict the BCF from the lipophilicity of the chemical (Arnot and Gobas 2006, Nichols et al. 2007). Although this approach works well for lipophilic chemicals that undergo no biotransformation in the organism, it overestimates the BCF-values of metabolized xenobiotics (Lombardo et al. 2014). Such overestimates could be corrected using in-vitro biotransformation assays that are able to identify the metabolized compounds. By combining the in-silico methods with the in-vitro metabolism-assay in an Integrated Testing Strategy (ITS), it should be possible to greatly reduce if not to replace altogether in-vivo BCF-testing in fish (de Wolf et al. 2007, Weisbrod et al. 2009, Lombardo et al. 2014). However, as yet the in-vitro methodologies have neither been validated nor implemented.

Previous studies, which have also been funded by the 3R-Research Foundation, have succeeded in standardizing an in-vitro metabolism assay using the isolated hepatocytes of rainbow trout (Fay et al. 2014). In addition, they revealed that the in-vivo BCF-values of chemicals in fish can be reasonably well predicted from the in-vitro metabolic data. The available in-vitro methodology has been developed exclusively for rainbow trout (Oncorhynchus mykiss), whereas in-vivo BCF-testing relies on the use of a broad array of cold- and warm-water species of fish. The nature of the species that is used for testing purposes can depend on the geographic region. In Northern European countries, rainbow trout are preferred, whereas in Asian ones, carp are favoured. Hence, an in-vitro assay that is relevant for only one of the many species that are employed for in-vivo testing would severely restrict the applicability and acceptance of the alternative assay. The proposed project will overcome this limitation by demonstrating that the in-vitro methodology that has been developed for the cold-water species – the rainbow trout – can be instrumented also for the warm-water species, the common carp (Cyprinus carpio), thereby proving that its use is not confined to one specific species of fish. More specifically, the objectives of the project are

-       To ascertain that the in-vitro methodology that has been developed for the hepatocytes of rainbow trout is relevant also for another species of fish (the carp) that is used in the OECD-305 test;

-       To ascertain whether species differences in the metabolism of xenobiotics that exist in-vivo are reflected by the in-vitro hepatocyte assay.

In addition, it will be ascertained whether the in-vitro hepatocyte-assay is useful not only in determining metabolic rates, but also in identifying metabolite patterns.

Method and Results

In a first step, a standardized protocol for the in vitro metabolism assay with carp hepatocytes was developed. It included the implementation of the methodology for isolation of carp hepatocyte isolation (Segner et al. 1993, Vogt and Segner 1997), and the adaptation of the assay protocol. In a second step, metabolism of selected xenobiotics was comparatively tested in both trout and carp hepatocytes. The in vitro metabolic rate values were extrapolated to the intact fish in order to learn whether the in vitro measurements are able to predict species differences in the in vivo metabolism of xenobiotics.  The results of the experiments showed that the in vitro metabolism protocol which has been developed originally for rainbow trout can be successfully transferred to non-salmonid species like carp.

The currently established protocol of the in vitro-metabolism assay using fish hepatocytes assay is intended to predict in vivo-metabolism rates of xenobiotics; it is not intended to identify the metabolites arising from biotransformation of the parent compound. For biocide regulatory testing, however, metabolite data are required. Therefore, the question arised whether the in-vitro metabolism assay may provide an efficient tool for metabolite identification. It is well known that metabolite spectra can differ strongly among fish species, and this can have major implications for chemical toxicity. Thus, we examined the suitability of the in vitro assay with fish hepatocytes to provide information on species-specific metabolite patterns. The results clearly demonstrated that this is the case: differences of metabolite spectra existed between isolated trout and carp hepatocytes, and, importantly, the in-vitro differences well reflected the in vivo differences between the two species.  

Conclusions and Relevance for 3R

The project provided two deliverables. First, it highlighted that interspecies differences of xenobiotic metabolic rates as they exist in vivo, can be predicted from the respective in vitro systems. Interspecies scaling of in vitro metabolism data has been found to be a critical issue in human toxicology, and this question for fish must be addressed in fish as well. Here, the results of the present project represent an important step forward as they point to the transferability of the in vitro assay across species. This information is critical with respect to the acceptance of the in vitro hepatocyte assay for reduction or replacement of the in vivo bioaccumulation test with fish.

Second, the project demonstrated the utility of the in vitro hepatocyte assay to inform on species differences in the metabolite pattern. This information is of relevance for biocide regulations and could open an avenue to introduce in vitro approaches also in that part of chemical regulations.


Arnot JA, Gobas F (2006) A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environ Rev 14:257-330
de Wolf W, Comber M, Douben P, Gimeno S, Holt M, Léonard M, Lillicrap A, Sijm D, van Egmond R, Weisbrod A, Whale G (2007) Animal use replacement, reduction, and refinement: development of an integrated testing strategy for bioconcentration of chemicals in fish. Integr Environ Assessm Mngmt 3: 3-17.
Iwatsubo T, Suzuki H, Sugiyama Y (1997). Prediction of species differences (rats, dogs, humans) in the in vivo metabolic clearance of YM796 by the liver from in vitro data. J Pharmacol Exp Therap 283: 462-469.
Fay KA, Mingoia RT, Goeritz I, Nabb DL, Hoffman AD, Ferell BD, Peterson HM, Nichols JW, Segner H, Han X 82014). Intra- and inter-laboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential. Environ Sci Technol,48:8170-8178. in press
Lombardo A, Roncaglioni A, Nendza M, Segner H, Jeram S, Benfenati E (2014) Integrated testing strategies to optimize the assessment of bioconcentration under REACH framework. Environ Internat 69:40-50.
Nichols JS, Erhardt S, Dyer MJ, Moore M, Plotzke K, Segner H, Schultz I, Thomas K, Vasiluk J, Weisbrod A (2007) Use of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) data in bioaccumulation assessments for fish. Human Ecol Risk Assessm 13: 1164-1191.
Segner H, Böhm R, Kloas W (1993). Binding and bioactivity of insulin in cultured carp, Cyprinus carpio, hepatocytes. Fish Physiol Biochem 11:411-420.
Vogt G, SegnerH (1997). Spontaneous formation of intercellular bile canaliculi and hybrid biliary pancreatic canaliculi in co-culture of hepatocytes and exocrine pancreatic cells from carp. Cell Tissue Res 289:191-194.
Weisbrod AV, Sahi J, Segner H, James MO, Nichols J, Schultz I, Erhardt S, Cowan-Ellsberry C, Bonnell M, Hoeger B (2009). The state of in vitro science for use in bioaccumulation assessments for fish.  Environ Toxicol Chem 28:86-06.

Bischof I, Köster J, Segner H, Schlechtriem C (2016). Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: comparison between in vivo and in vitro and across species. Comparative Biochemistry and Physiology 187C: 62-73.


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