## 4.4.5. Chronic toxicity - Eco

Author: Michiel Kraak

Reviewers: Kees van Gestel and Lieven Bervoets

Learning objectives:

You should be able to

- explain the concepts involved in chronic toxicity testing, including the Acute to Chronic Ratio (ACR).
- design chronic toxicity experiments and to solve the challenges involved in chronic toxicity testing.
- interpret the results of chronic toxicity experiments and to mention the types of effects of toxicants that cannot be determined in acute toxicity experiments.

Key words: Chronic toxicity, chronic sublethal endpoints, Acute to Chronic Ratio, mode of action.

Introduction

Most toxicity tests performed are short-term high-dose experiments, acute tests in which mortality is often the only endpoint. This is in sharp contrast with the field situation, where organisms are often exposed to relatively low levels of contaminants for their entire life span. The shorter the life cycle of the organism, the more realistic this scenario becomes. Hence, there is an urgent need for chronic toxicity testing. It should be realized though, that the terms acute and chronic have to be considered in relation to the length of the life cycle of the organism. A short-term exposure of four days is acute for fish, but chronic for algae, comprising already four generations.

From acute to chronic toxicity testing

The reason for the bias towards acute toxicity testing is obviously the higher costs involved in chronic toxicity testing, simply caused by the much longer duration of the test. Yet, chronic toxicity testing is challenging for several other reasons as well. First of all, during prolonged exposure organisms have to be fed. Although unavoidable, especially in aquatic toxicity testing, this will definitely influence the partitioning and the bioavailability of the test compound. Especially lipophilic compounds will strongly bind to the food, making toxicant uptake via the food more important than for hydrophilic compounds, thus causing compound specific changes in exposure routes. For chronic aquatic toxicity tests, especially for sediment testing, it may be challenging to maintain sufficiently high oxygen concentrations throughout the entire experiment (Figure 1).

Figure 1. Experimental design of a chronic sediment toxicity experiment, showing the experimental units and the aeration system.

Obvious choices to be made include the duration of the exposure and the endpoints of the test. Generally it is aimed at including at least one reproductive event or the completion of an entire life cycle of the organism within the test duration. To ensure this, validity criteria are set to the different test guidelines, such as:

- the mean number of living offspring produced per control parent daphnid surviving till the end of the test should be above 60 (OECD, 2012).

- 85% of the adult control chironomid midges from the control treatment should emerge between 12 and 23 days after the start of the experiment (OECD, 2010).

- the mean number of juveniles produced by 10 control collembolans should be at least 100 (OECD, 2016a).

Chronic toxicity

Generally toxicity increases with increasing exposure time, often expressed as the acute-to-chronic ratio (ACR), which is defined as the LC50 from an acute test divided by the door NOEC of EC10 from the chronic test. Alternatively, as shown in Figure 2, the acute LC50 can be divided by the chronic LC50. If compounds exhibit a strong direct lethal effect, the ACR will be low, but for compounds that slowly build up lethal body burdens (see section on Critical body concentrations) it can be very high. Hence, there is a relationship between the mode of action of a compound and the ACR. Yet, if chronic toxicity has to be extrapolated from acute toxicity data and the mode of action of the compound is unknown, an ACR of 10 is generally considered. It should be realized though that this number is chosen quite arbitrarily, potentially leading to under- as well as over estimation of the actual ACR.

Figure 2. Average mobility (% of initial animals) of Daphnia magna (n = 15) exposed to a concentration range of the flame retardant ALPI (mg L−1) in Elendt medium after 48 h (± s.e. in x and y, n = 4 × 5 individuals per concentration) and after 21 days (± s.e. in x, n = 15 individuals per concentration). The toxicity increases with increasing exposure time with an Acute Chronic Ratio (ACR) of 5.6. Redrawn from Waaijers at al. (2013) by Wilma IJzerman.

Since reproductive events and the completion of life cycles are involved, chronic toxicity tests allow an array of sublethal endpoints to be assessed, including growth and reproduction, as well as species specific endpoints like emergence (time) of chironomids. Consequently, compounds with different modes of action may cause very diverse sublethal effects on the test organisms during chronic exposure (Figure 3). The polycyclic aromatic compound (PAC) phenanthrene did not affect the completion of the life cycle of the midges, but above a certain exposure concentration the larvae died and no emergence was observed at all, suggesting a non-specific mode of action (narcosis). In contrast, the PAC acridone caused no mortality but delayed adult emergence significantly over a wide range of test concentrations, suggesting a specific mode of action affecting life cycle parameters of the midges (Leon Paumen et al., 2008). This clearly demonstrates that specific effects on life cycle parameters of compounds with different modes of action need time to become expressed.

Figure 3. Effect of two polycyclic aromatic compounds (PACs) on the emergence time of Chironomus riparius males from spiked sediments. X-axis: actual concentrations of the compounds measured in the sediment. Y-axis: 50% male emergence time (EMt50, days, average plus standard deviations). †Concentrations with no emerging midges. *EMt50 value significantly different from control value (p < 0.05). Redrawn from Leon Paumen et al. (2008) by Wilma IJzerman.

Chronic toxicity tests are single species tests, but if the effects of toxicants are assessed on all relevant life-cycle parameters, these can be integrated into effects on population growth rate (r). For the 21-day daphnid test this is achieved by the integration of age-specific data on the probability of survival and fecundity. The population growth rates calculated from chronic toxicity data are obviously not related to natural population growth rates in the field, but they do allow to construct dose-response relationships for the effects of toxicants on r, the ultimate endpoint in chronic toxicity testing (Figure 4; Waaijers et al., 2013).

Figure 4: Population growth rate (d−1) of Daphnia magna (n = 15) exposed to a concentration range of the flame retardant DOPO (mg L−1) in Elendt medium after 21 days. The average population growth rate rate (◊) is shown (s.e. in x and y are smaller than the data points and therefore omitted). The EC50 is plotted as ● (s.e. smaller than data point) and the logistic curve represents the fitted concentration−response relationship. Redrawn from Waaijers et al. (2013) by Wilma IJzerman.

Chronic toxicity testing in practice

Several protocols for standardized chronic toxicity tests are available, although less numerous than for acute toxicity testing. For water, the most common test is the 21 day Daphnia reproduction test (OECD, 2012), for sediment 28-day test guidelines are available for the midge Chironomus riparius (OECD, 2010) and for the worm Lumbriculus variegatus (OECD, 2007). For terrestrial soil, the springtail Folsomia candida (OECD, 2016a) and the earthworm Eisenia fetida (OECD, 2016b) are the most common test species, but also for enchytraeids a reproduction toxicity test guideline is available (OECD, 2016c). For a complete overview see (https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-effects-on-biotic-systems_20745761/datedesc#collectionsort).

References

Leon Paumen, M., Borgman, E., Kraak, M.H.S., Van Gestel, C.A.M., Admiraal, W. (2008). Life cycle responses of the midge Chironomus riparius to polycyclic aromatic compound exposure. Environmental Pollution 152, 225-232.

OECD (2007). OECD Guideline for Testing of Chemicals. Test No. 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment. Section 2: Effects on Biotic Systems; Organization for Economic Co-operation and Development: Paris, 2007.

OECD (2010). OECD Guideline for Testing of Chemicals. Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment. Section 2: Effects on Biotic Systems; Organization for Economic Co-operation and Development: Paris, 2010.

OECD (2012). OECD Guideline for Testing of Chemicals. Test No. 211. Daphnia magna Reproduction Test No. 211. Section 2: Effects on Biotic Systems; Organization for Economic Co-operation and Development: Paris, 2012.

OECD (2016a). OECD Guideline for Testing of Chemicals. Test No. 232. Collembolan Reproduction Test in Soil. Section 2: Effects on Biotic Systems; Organization for Economic Co-operation and Development: Paris, 2016.

OECD (2016b). OECD Guideline for Testing of Chemicals. Test No. 222. Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei). Section 2: Effects on Biotic Systems; Organization for Economic Co-operation and Development: Paris, 2016.

OECD (2016c). OECD Guideline for Testing of Chemicals. Test No. 220: Enchytraeid Reproduction Test. Section 2: Effects on Biotic Systems; Organization for Economic Co-operation and Development: Paris, 2016.

Waaijers, S.L., Bleyenberg, T.E., Dits, A., Schoorl, M., Schütt, J., Kools, S.A.E., De Voogt, P., Admiraal, W., Parsons, J.R., Kraak, M.H.S. (2013). Daphnid life cycle responses to new generation flame retardants. Environmental Science and Technology 47, 13798-13803.