Tag Archives: Helmut Segner

In Vitro Methodologies in Ecotoxicological Hazard Assessment: The Case of Bioaccumulation Testing for Fish

Helmut Segner

The concerted research efforts undertaken in recent years have
highlighted the potential of in vitro approaches, as part of an
integrated testing strategy, to replace or reduce in vivo bioaccumulation testing in fish

Worldwide programmes for the regulation of chemicals require an assessment of the risks of chemicals to human and environmental health based on three categories of concern: Persistence, Bioaccumulation and Toxicity (PBT). Among these three categories, bioaccumulation refers to the enrichment of environmental chemicals in organisms. It encompasses the absorption, distribution, metabolism and excretion (ADME) of a chemical inside an organism, and ultimately determines the internal toxic dose. For the aquatic environment, the most widely used parameter to estimate the bioaccumulation potential of a chemical is the so-called Bioconcentration Factor (BCF). The BCF represents the ratio of the steadystate chemical concentration in the organism and the chemical concentration in the respiratory medium, i.e. water. For the experimental determination of the BCF, the test procedure as described in OECD Test Guideline 3051 represents the current ‘gold standard’. In this test, fish are exposed to a chemical for 28 days, to reach an equilibrium of chemical concentration between fish and water, followed by a 28-day depuration period to measure the elimination rate. This test, in addition to being lengthy and costly, requires a high number of animals (> 100 fish per test).

Regulatory programmes require bioaccumulation information for chemicals which are lipophilic (for example, those with a log Kow > 3), and which are produced at a certain tonnage (for example, the European Community REACH Regulation requires BCF information for lipophilic chemicals that are produced at > 100 tonnes per year). Experi mentally determined BCF data are not available for the vast majority of existing compounds. For instance,  in a Canadian investigation of 23,000 existing chemicals, it was found that bioaccumulation data existed for less than 4% of them (cf. Nichols et al.2). If the missing BCF data had to be generated by means of the OECD 305 test, this would entail a drastic increase in animal use.3,4 Therefore, there is an urgent need to develop alternative methods to reduce the number of fish used for in vivo bioaccumulation testing. The bioconcentration of chemicals in fish results from the competing rates of chemical uptake via the gills and skin (k1) and chemical elimination via respiratory exchange (k2), faecal egestion (ke) and metabolic biotransformation (km).5 In addition, dilution as a result of growth (kd) can influence bioconcentration.

With the involvement of these different processes, it is clear that non-animal approaches to bioconcentration assessment cannot be based on one single method, but have to rely on an array of methodologies.2,4,6 An initial non-animal based approximation of the bioconcentration potential of an organic chemical in aquatic organisms can be obtained from an in silico hydrophobicity model, which considers bioconcentration as a passive partitioning process resulting from the competing uptake and elimination processes. In this model, bioconcentration can be predicted from the lipophilicity of a chemical, as estimated from its octanol–water partition coefficient, Kow.5 Also, it can actually be measured by using artificial membranes which simulate the passive diffusion processes across the respiratory epithelia.7

The development of in vitro methods

Diffusion-based methodologies have proven instrumental in the prediction of the BCF values of lipophilic chemicals that undergo no endogenous metabolism in the organism. However, as they are not able to take into account chemical loss due to biotransformation (km), they overestimate the BCF values of metabolisable xenobiotics. To correct for the influence of biotransformation on fish BCF values, a possible approach is the use of metabolically competent in vitro assays that show which chemicals are biotransformed, and at what rates. In mammalian toxicology, in vitro assays for the analysis of xenobiotic metabolism largely rely on liver preparations such as subcellular liver fractions (S9, microsomes) and isolated hepatocytes, as the liver is the organ with the highest metabolic activity. Corresponding technologies are also available for fish, and it has been demonstrated that they are suitable for determining biotransformation parameters (see Segner & Cravedi8 and Fitzimmons et al.9). However, their reproducibility and their capability of predicting in vivo BCF values remain to be demonstrated.

In recent years, intensive efforts have been undertaken — largely coordinated by the Health and Environmental Sciences Institute (HESI) — to advance the development of piscine in vitro assays for regulatory purposes. After an initial phase of reviewing the available knowledge and technologies,2,6 in the next step, standardised protocols for liver S9 preparations and isolated hepatocytes from rainbow trout were established.10,11 A major drawback experienced in these studies, particularly with freshly-isolated hepatocyte suspensions, was the between-isolate variability of metabolic capabilities, which is related to factors such as seasonal oscillation, and the nutritional status, gender or genetic background of the donor fishes. Here, a major step forward was the introduction of a cryopreservation method for fish hepatocytes,12 enabling the year-round provision of uniform batches of metabolically characterised hepatocytes to laboratories worldwide. By using a standardised assay protocol, Fay et al.13 recently performed an international ring study with cryopreserved rainbow trout hepatocytes, and were able to demonstrate the good interlaboratory and intra-laboratory reproducibility of the metabolic rate values obtained with the in vitro hepatocyte assay.

To be able to extrapolate from the metabolic rate values measured in the isolated fish hepatocytes to the metabolic rate value (km) in the intact fish, physiologically-based prediction models were developed. 14,15 These models initially scale from the clearance rate of the isolated liver cells to that of the whole liver, and from there to the metabolic transformation rate of the whole fish. The predicted km values are then used to calculate the in vivo BCF value of the test chemical. Currently, the availability of data on BCF values  predicted from in vitro assays is still limited, and it is still too early to come up with a conclusive statement on the predictability of the in vitro approach, partly also because of the variable quality of the in vivo BCF data; however, the existing results look promising.

Looking to the future

There are lessons to be learned from the recent development of in vitro assays as components of alternative integrated testing strategies for the assessment of bioaccumulation in fish. Although a broad spectrum of in vitro assays and methods have been available in ecotoxicology for a while,16 they have never made their way to regulatory implementation. Partly, this is due to the fact that they were considered to be technically not ready nor sufficiently standardised. In the case of the piscine in vitro metabolism assays, this obstacle has been overcome through targeted and internationally concerted research efforts on the standardisation and harmonisation of the assay protocols. Another constraint to the regulatory acceptance of in vitro assays is that they were considered not to be appropriate for the protection goals of ecotoxicology, which are ecological entities such as populations and communities. However, ecotoxicological hazard assessment largely relies on classical toxicity tests for measuring organism-level endpoints such as lethality (cf. Segner17), and these endpoints may well be predictable by in vitro assays, provided that: a) the in vitro assays are rationally selected to represent the critical toxicological processes; b) the assays are standardised; and c) valid extrapolation models are available. These requirements are fulfilled in the case of bioaccumulation assessment — i.e. the in vitro assays measure biotransformation as the critical toxicokinetic process, they are standardised, and there exist physiologically-based models for the scaling of the in vitro metabolic rate values to the in vivo metabolic rates. As ecotoxicology deals with a huge diversity of species, the interspecies scaling of metabolic rates is another critical issue, but this question is also currently under investigation. In conclusion, the concerted research efforts undertaken in recent years have substantially moved the field ahead, and the results obtained highlight the potential of in vitro approaches, as part of an integrated testing strategy,4 to replace or reduce in vivo bioaccumulation testing in fish.


The financial support of Stiftung Forschung 3R, ünsingen (Switzerland) and the Health and Environmental Sciences Institute (HESI) is gratefully acknowledged.

Prof. Dr Helmut Segner
Centre for Fish and Wildlife Health
Department of Infectious Diseases and Pathobiology
Vetsuisse Faculty
University of Bern
PO Box 8466
CH 3012 Bern
E-mail: Helmut.segner@vetsuisse.unibe.ch


1 OECD (2011). OECD Guideline for Testing of Chemicals No. 305. Bioaccumulation in Fish: Aqueous and Dietary Exposure, 72pp. Paris, France: Organisation for Economic Co-operation & Development.
2 Nichols, J.S., Erhardt, S., Dyer, M.J., 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 & Ecological Risk Assessment 13, 1164–1191.
3 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. Integrated Environmental Assessment & Management 3, 3–17.
4 Lombardo, A., Roncaglioni, A., Benfenati, E., Nendza, M., Segner, H., Fernández, A., Kühne, R., Franco, A., Pauné, E. & Schüürmann, G. (2014). Integrated testing strategy (ITS) for bioaccumulation assessment under REACH. Environment International 69, 40–50.
5 Arnot, J.A. & Gobas, F. (2006). A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environmental Reviews 14, 257–330.
6 Weisbrod, A.V., Sahi, J., Segner, H., James, M.O., Nichols, J., Schultz, I., Erhardt, S.,  Cowan-Ellsberry, C., Bonnell, M. & Hoeger, B. (2009). The state of   science for use in bioaccumulation assessments for fish. Environmental Toxicology & Chemistry 28, 86–96.
7 Kwon, J.H. & Escher, B.I. (2008). A modified parallel artificial membrane permeability assay for evaluating bioconcentration of highly hydrophobic chemicals in fish. Environmental Science & Technology 42, 1787–1793.
8 Segner, H. & Cravedi, J.P. (2001). Metabolic activity in primary cultures of fish hepatocytes. ATLA 29, 251–257.
9 Fitzsimmons, P.N., Lien, G.J. & Nichols, J.W. (2007). A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions. Comparative Biochemistry & Physiology 145C, 485–506.
10 Han, X., Nabb, D., Mingoia, R. & Yang, C. (2007). Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environmental Science & Technology 41, 3269–3276.
11 Johanning, K., Hancock, G., Escher, B., Adekola, A., Bernhard, M.J., Cowan-Ellsberry, C., Domodoradzki, J., Dyer, S., Eickhoff, C., Embry, M., Erhardt, S., Fitzsimmons, P., Halder, M., Hill, J., Holden, D., Johnson, R., Rutishauser, S., Segner, H., Schultz, I. & Nichols, J. (2012). Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction. Current Protocols in Toxicology 53, 14.10.1–14.10.28.
12 Mingoia, R.T., Glover, K.P., Nabb, D.L., Yang, C.H., Snajdr, S.I. & Han, X. (2010). Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): A validation study to support their application in bioaccumulation assessment. Environmental Science & Technology 44, 3052–3058.
13 Fay, K.A., Mingoia, R.T., Goeritz, I., Nabb, D.L., Hoffman, A.D., Ferell, B.D., Peterson, H.M., Nichols, J.W., Segner, H. & Han, X. (2014). Intra- and inter-laboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential. Environmental Science & Technology 48, 8170–8178.
14 Nichols, J.W., Schultz, R.I. & Fitzsimmons, P.N. (2006). In vitro–in vivo extrapolation of quantitative hepatic biotransformation data for fish. I. A review of methods, and strategies for incorporating intrinsic clearance estimates into chemical kinetic methods. Aquatic Toxicology 78, 74–90.
15 Cowan-Ellsberry, C.S., Dyer, S., Erhardt, S., Bernhard, M.J., Roe, A., Dowty, M. & Weisbrod, A. (2008). Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates. Chemosphere 70, 1804–1817.
16 Castano, A., Bols, N.C., Braunbeck, T., Dierickx, P., Halder, M., Isomaa, B., Kawahara, K., Lee, L.E.J., Mothersill, C., Pärt, P., Repetto, G., Sintes, J.R., Rufli, H., Smith, R., Wood, C. & Segner, H. (2003). The use of fish cells in ecotoxicology. The report and recommendations of ECVAM workshop 47. ATLA 31, 317–351.
17 Segner, H. (2011). Moving beyond a descriptive aquatic toxicology: The value of biological process and trait information. Aquatic Toxicology 105, 50–55.

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