## 4.3.1. Bioaccumulation testing

Author: Kees van Gestel

Reviewers: Joop Hermens, Michiel Kraak, Susana Loureiro

Learning objectives:

You should be able to

- describe methods for determining the bioaccumulation of chemicals in terrestrial and aquatic organisms
- describe a test design suitable for assessing the bioaccumulation kinetics of chemicals in organisms
- mention the pros and cons of static and dynamic bioaccumulation tests

Keywords: bioconcentration, bioaccumulation, uptake and elimination kinetics, test methods, soil, water

Bioaccumulation is defined as the uptake of chemicals in organisms from the environment. The degree of bioaccumulation is usually indicated by the bioconcentration factor (BCF) in case the exposure is via water, or the biota-to-soil/sediment accumulation factor (BSAF) for exposure in soil or sediment (see section on Bioaccumulation).

Because of the potential risk for food-chain transfer, experimental determination of the bioaccumulation potential of chemicals is usually required in case of a high lipophilicity (log Kow > 3), unless the chemical has a very low persistency. For very persistent chemicals, experimental determination of bioaccumulation potential may already be triggered at log Kow > 2. The experimental determination of BCF and BSAF values makes use of static or dynamic exposure systems.

In static tests, the medium is dosed once with the test chemical, and organisms are exposed for a certain period of time after which both the organisms and the test medium are analyzed for the test chemical. The BCF or BSAF are calculated from the measured concentrations. There are a few concerns with this way of bioaccumulation testing.

First, exposure concentrations may decrease during the test, e.g. due to (bio)degradation, volatilization, sorption to the walls of the test container, or uptake of the test compound by the test organisms. As a consequence, the concentration in the test medium measured at the start of the test may not be indicative of the actual exposure during the test. To take this into account, exposure concentrations can be measured at the start and the end of the test and also at some intermediate time points. Body concentrations in the test organisms may then be related to time-weighted-average (TWA) exposure concentrations. Alternatively, to overcome the problem of decreasing concentrations in aquatic test systems, continuous flow systems or passive dosing techniques can be applied. Such methods, however, are not applicable to soil or sediment tests, where repeated transfer of organisms to freshly spiked medium is the only way to guarantee more or less constant exposure concentrations in case of rapidly degrading compounds. To avoid that the uptake of the test chemical in test organisms leads to decreasing exposure concentrations, the amount of biomass per volume or mass of test medium should be sufficiently low.

Second, it is uncertain whether at the end of the exposure period steady state or equilibrium is reached. If this is not the case, the resulting BSAF or BCF values may underestimate the bioaccumulation potential of the chemical. To tackle this problem, a dynamic test may be run to assess the uptake and elimination rate constants to derive a BSAF or BCF values using uptake and elimination rate constants (see below).

Such uncertainties also apply to BCF and BSAF values obtained by analyzing organisms collected from the field and comparing body concentrations with exposure levels in the environment. Using data from field-exposed organisms on one hand have large uncertainty as it remains unclear whether equilibrium was reached, on the other hand they to do reflect exposure over time under fluctuating but realistic exposure conditions.

Dynamic tests, also indicated as uptake/elimination or toxicokinetic tests, may overcome some, but not all, of the disadvantages of static tests. In dynamic tests, organisms are exposed for a certain period of time in spiked medium to assess the uptake of the chemical, after which they are transferred to clean medium for determining the elimination of the chemical. During both the uptake and the elimination phase, at different points in time, organisms are sampled and analyzed for the test chemical. The medium is also sampled frequently to check for a possible decline of the exposure concentration during the uptake phase. Also in dynamic tests, keeping exposure concentrations constant as much as possible is a major challenge, requiring frequent renewal (see above).

Toxicokinetic tests should also include controls, consisting of test organisms incubated in the clean medium and transferred to clean medium at the same time the organisms from the treated medium are transferred. Such controls may help identifying possible irregularities in the test, such as poor health of the test organisms or unexpected (cross)contamination occurring during the test.

The concentrations of the chemical measured in the test organisms are plotted against the exposure time, and a first-order one-compartment model is fitted to the data to estimate the uptake and elimination rate constants. The (dynamic) BSAF or BCF value is then determined as the ratio of the uptake and elimination rate constants (see section on Bioconcentration and kinetic models).

In a toxicokinetics test, usually replicate samples are taken at each point in time, both during the uptake and the elimination phase. The frequency of sampling may be higher at the beginning than at the end of both phases: a typical sampling scheme is shown in Figure 1. Since the analysis of toxicokinetics data using the one-compartment model is regression based, it is generally preferred to have more points in time rather than having many replicates per sampling time. From that perspective, often no more than 3-4 replicates are used per sampling time, and 5-6 sampling times for the uptake and elimination phases each.

Figure 1. Sampling scheme of a toxicokinetics test for assessing the uptake and elimination kinetics of chemicals in earthworms. During the 21-day uptake phase, the earthworms are individually exposed to a test chemical in soil, and at regular intervals three earthworms are sampled. After 21 days, the remaining earthworms are transferred to clean soil for the 21-day elimination period, in which again three replicate earthworms are sampled at regular points in time for measuring the body concentrations of the chemical. Also the soil is analyzed at different points in time (marked with X in the Medium row). Drawn by the author.

Preferably, replicates are independent, so destructively sampled at a specific sampling point. Especially in aquatic ecotoxicology, mass exposures are sometimes used, having all test organisms in one or few replicate test containers. In this case, at each sampling time some replicate organisms are taken from the test container(s), and at the end of the uptake phase all organisms are transferred to (a) container(s) with clean medium.

Figure 2 shows the result of a test on the uptake and elimination kinetics of molybdenum in the earthworm Eisenia andrei. From the ratio of the uptake rate constant (k1) and elimination rate constant (k2) a BSAF of approx. 1.0 could be calculated, suggesting a low bioaccumulation potential of Mo in earthworms in the soil tested.

Figure 2. Uptake and elimination kinetics of molybdenum in Eisenia andrei exposed in an artificial soil spiked with a nominal Mo concentration of 10 µg g-1 dry soil. Dots represent measured internal Mo concentrations. Curves were estimated by a one-compartment model (see section on Bioconcentration and kinetic models). Parameters: k1 = uptake rate constant [gsoil/gworm/d], k2 = elimination rate constant [d-1]. Adapted from Diez-Ortiz et al. (2010).

Another way of assessing the bioaccumulation potential of chemicals in organisms includes the use of radiolabeled chemicals, which may facilitate easy detection of the test chemical. The use of radiolabeled chemicals may however, overestimate bioaccumulation potential when no distinction is made between the parent compound and potential metabolites. In case of metals, stable isotopes may also offer an opportunity to assess bioaccumulation potential. Such an approach was also applied to distinguish the role of dissolved (ionic) Zn in the bioaccumulation of Zn in earthworms from ZnO nanoparticles. Earthworms were exposed to soils spiked with mixtures of 64ZnCl2 and 68ZnO nanoparticles. The results showed that dissolution of the nanoparticles was fast and that the earthworms mainly accumulated Zn present in ionic form in the soil solution (Laycock et al., 2017).

Standard test guidelines for assessing the bioaccumulation (kinetics) of chemicals have been published by the Organization for Economic Cooperation and Development (OECD) for sediment-dwelling oligochaetes (OECD, 2008), for earthworms/enchytraeids in soil (OECD, 2010) and for fish (OECD, 2012).

References

Diez-Ortiz, M., Giska, I., Groot, M., Borgman, E.M., Van Gestel, C.A.M. (2010). Influence of soil properties on molybdenum uptake and elimination kinetics in the earthworm Eisenia andrei. Chemosphere 80, 1036-1043.

Laycock, A., Romero-Freire, A., Najorka, J., Svendsen, C., Van Gestel, C.A.M., Rehkämper, M. (2017). Novel multi-isotope tracer approach to test ZnO nanoparticle and soluble Zn bioavailability in joint soil exposures. Environmental Science and Technology 51, 12756−12763.

OECD (2008). Guidelines for the testing of chemicals No. 315: Bioaccumulation in Sediment-dwelling Benthic Oligochaetes. Organization for Economic Cooperation and Development, Paris.

OECD (2010). Guidelines for the testing of chemicals No. 317: Bioaccumulation in Terrestrial Oligochaetes. Organization for Economic Cooperation and Development, Paris.

OECD (2012). Guidelines for the testing of chemicals No. 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure. Organization for Economic Cooperation and Development, Paris.