Bioconcentration / Bioaccumulation
Information on accumulation in aquatic organisms is important for understanding the behaviour of a compound in the environment. This information is used for hazard classification and PBT assessment.
In general, a Bioconcentration factor (BCF) is estimated based on various prediction techniques as log Kow, (Q)SARs or other computer models, or, if deemed necessary, derived from experimental data measured with aquatic species, preferably fish.
Since bioaccumulation is the result of absorption, distribution, metabolism and excretion (ADME) processes, information on ADME processes derived with in vitro methods is used to improve the prediction of BCF models.
Bioconcentration/Bioaccumulation testing is stipulated for environmental hazard and risk assessment by the regulatory framework on
- Chemicals (REACH; from 100 t/year onwards)
- Plant protection products
- Biocides - until end of August 2013 Directive 98/8/EC and from 1st September 2013 Regulation 528/2012
- Feed additives
List of EU/OECD test guidelines
OECD test guideline
EU Test method
Bioaccumulation in Fish: Aqueous and Dietary Exposure
1.1 In vitro trout S9 assay
The majority of the models currently used to predict bioaccumulation/bioconcentration in fish neglect the contribution of metabolism as a clearance mechanism and therefore might overestimate the bioaccumulative potential of a compound and, in consequence, might trigger unnecessary in vivo tests. Information from in vitro metabolism can be used to improve these models.
Trout S9 is a subcellular fraction of the liver S9 fractions and has both Phase I and Phase II xenobiotic metabolizing enzymes and can therefore be used to evaluate xenobiotic metabolism. In a first step, compounds are incubated with trout S9, followed by analysis of the parent chemical and calculation of the transformation rate and the prediction of in vivo BCF based on in vitro kmet.
Validation (transferability, reproducibility)
The small-scale study (2008-2010) aimed to assess a) the within- and between-laboratory reproducibility and transferability of the in vitro trout S9 assay, and b) its suitability for predicting in vivo BCF. The study covered the phases of protocol refinement, protocol transfer and protocol performance and a total of 15 compounds were tested, six in the phases of protocol refinement and transfer, and nine in the phase of protocol performance.
For the nine chemicals tested with the standardised protocol, first-order enzyme kinetics and linearity in the response of the metabolic rate could be demonstrated. There was variability in the transformation rate k among some laboratories and good agreement in others when data was compared. Data comparison revealed an overall high degree of within- and between-laboratory variability.
Possible sources of this variability could lay in the intrinsic properties of the compounds tested, i.e. due to their high logKow they are considered to be difficult to be analysed, differences in the analytical methods used by the three laboratories, and differences in the concentrations tested. It became also evident that the model used to conclude from the in vitro kmet on the in vivo kmet need to be refined. For the follow-up of the study and further research in this area, see below.
2.1 Contribution to the ILSI HESI Scientific Committee on Bioaccumulation
The ILSI HESI Scientific Committee on Bioaccumulation (of which EURL ECVAM is a member) has launched several projects on development and standardisation of in vitro methods to be used in bioaccumulation testing (e.g. fish cells and their suitability to determine metabolism and uptake) and improvement of bioaccumulation/bioconcentration.
As a follow-up of the two studies on the in vitro trout S9 assay, the collaborators improved the protocol and the prediction model. The protocol was published in Current Protocols in Toxicology in 2012: Johanning et al, Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction.