User Guide to Thresholds and Classification - Environmental ...

374User Guide for Thresholds and ClassificationsIn general, the test consists of two phases: The exposure (uptake) and post-exposure(depuration) phases. During the uptake phase, separate groups of fish of one species are exposed to atleast two concentrations of the test substance. A 28-day exposure phase is obligatory unless a steady statehas been reached within this period. The time needed for reaching steady-state conditions may be set on thebasis of KOW – k2 correlations (for example, log k2 = 1.47 – 0.41 log KOW (Spacie and Hamelink, 1982) orlog k2 = 1.69 – 0.53 log KOW (Gobas et al, 1989)). The expected time (d) for, for example, 95% steady statemay thus be calculated by -ln(1-0.95)/k2, provided that the bioconcentration follows first order kinetics.During the depuration phase the fish are transferred to a medium free of the test substance. Theconcentration of the test substance in the fish is followed through both phases of the test. The BCF isexpressed as a function of the total wet weight of the fish. As for many organic substances, there is asignificant relationship between the potential for bioconcentration and the lipophilicity, and furthermore, thereis a corresponding relationship between the lipid content of the test fish and the observed bioconcentration ofsuch substances. Therefore, to reduce this source of variability in the test results for the substances withhigh lipophilicity, bioconcentration should be expressed in relation to the lipid content in addition to wholebody weight (OECD 305; ECETOC (1995)). The guidelines mentioned are based on the assumption thatbioconcentration may be approximated by a first-order process (one-compartment model) and thus that BCF= k1/k2 (k1: first-order uptake rate, k2: first-order depuration rate, described by a log-linear approximation). Ifthe depuration follows biphasic kinetics, that is, two distinct depuration rates can be identified, theapproximation k1/k2 may significantly underestimate the BCF. If a second order kinetic has been indicated,the BCF may be estimated from the relation: C Fish /C Water , provided that ‗steady-state‘ for the fish-watersystem has been reached.Together with details of sample preparation and storage, an appropriate analytical method of knownaccuracy, precision, and sensitivity must be available for the quantification of the substance in the testsolution and in the biological material. If these are lacking it is impossible to determine a true BCF. The useof radiolabelled test substance can facilitate the analysis of water and fish samples. However, unlesscombined with a specific analytical method, the total radioactivity measurements potentially reflect thepresence of parent substance, possible metabolite(s), and possible metabolised carbon, which have beenincorporated in the fish tissue in organic molecules. For the determination of a true BCF it is essential toclearly discriminate the parent substance from possible metabolites. If radiolabelled materials are used in thetest, it is possible to analyse for total radio label (that is, parent and metabolites) or the samples may bepurified so that the parent compound can be analysed separately.In the log K OW range above 6, the measured BCF data tend to decrease with increasing log K OW . Conceptualexplanations of non-linearity mainly refer to either biotransformation, reduced membrane permeation kineticsor reduced biotic lipid solubility for large molecules. Other factors consider experimental artefacts, such asequilibrium not being reached, reduced bioavailability due to sorption to organic matter in the aqueousphase, and analytical errors. Moreover, care should be taken when evaluating experimental data on BCF forsubstances with log KOW above 6, as these data will have a much higher level of uncertainty than BCFvalues determined for substances with log K OW below 6.January 2012 EPA0109