Simulation tests

Simulation tests aim at assessing the rate and extent of biodegradation in a laboratory system designed to represent either the aerobic treatment stage of a WWTP or environmental compartments, such as fresh or marine surface water. These are aerobic and anaerobic tests that provide data for biodegradation under specified environmentally relevant conditions. They use indigenous biomass, media, and relevant solids (i.e. soil, sediment, activated sludge or other surfaces) to allow sorption of the chemical and a typical temperature that represents the particular environment. Therefore, inoculum size may vary from 104-105 to 107-108 cells/L depending on the test. A representative and low concentration of test substance is

21 used in these tests (e.g. less than 1 μg/L to 100 μg/L), which is low enough to ensure that the biodegradation kinetics reflect those expected in the environment being simulated. The fate of chemicals in WWTPs can be studied in the laboratory by using the Simulation Test – Aerobic Sewage Treatment: Activated Sludge Units (OECD 303 A) and Biofilms (OECD 303 B). The removal of the test substance is determined by monitoring the changes in DOC and/or COD. The following tests are used to simulate the biodegradation of organic chemicals under environmentally realistic conditions in soil, sediment or surface water: Aerobic and Anaerobic Transformation in Soil (OECD 307); Aerobic and Anaerobic Transformation in Aquatic Sediment Systems (OECD 308); Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test (OECD 309); and simulation tests to assess the biodegradation of chemicals discharged in wastewater (OECD 314) (Comber and Holt, 2010). A diagram describing OECD 314 is presented in Appendix Figure 0.1.

1.5.2.3.3.1. Aerobic and anaerobic transformation in soil (OECD 307)

OECD 307 was designed for evaluating transformation of chemicals in soil. The experiments are performed to determine the rate of transformation of the test substance and the nature and rates of formation and decline of transformation products to which plants and soil organisms may be exposed. Such studies are required for chemicals which are directly applied to soil or which are likely to reach the soil environment. The results of such laboratory studies can also be used to develop sampling and analysis protocols for related field studies. Test duration should not exceed 120 days due to decrease of soil microbial activity with time. Estimation of test substance transformation half-life, DT50 is performed (OECD Guidelines for testing of chemicals, Test No. 307, 2002).

1.5.2.3.3.2. Aerobic and anaerobic transformation in aquatic sediment systems (OECD 308)

OECD 308 is used to asses transformation of chemicals which can enter shallow or deep surface waters by such routes as direct application, spray drift, run-off, drainage, waste disposal, industrial, domestic or agricultural effluent and

22 atmospheric deposition. As a result half–life, DT50 and if appropriate, DT75 and DT90 of the test substance are calculated and pseudo first–order kinetics are applied to obtain information on the rate of test substance dissipation in the water and sediment (OECD Guidelines for testing of chemicals, Test No. 308, 2002).

1.5.2.3.3.3. Aerobic mineralisation in surface water (OECD 309)

The purpose of OECD 309 is to measure the time course of biodegradation of a test substance at low concentration in aerobic natural water (fresh, brackish or marine) and to quantify the observations in the form of kinetic rate expressions. This simulation test is a laboratory shake flask batch test based on the ISO/DIS 14592-1 and it also includes elements from OECD Guidelines 307 and 308. With long test times, semi-continuous operation replaces batch operation in order to prevent deterioration of the test microcosm. The principal objective of the test is to determine the mineralisation of the test substance in surface water, and mineralisation constitutes the basis for expressing degradation kinetics. An optional, secondary objective of the test is to obtain information on the primary degradation and the formation of major transformation products. Test substance is applied at low concentration (e.g. less than 1 μg/L to 100 μg/L) which is low enough to ensure that the biodegradation kinetics obtained in the test reflect those expected in the environment. First order kinetics are normally expected in this test. However, there may be circumstances where other kinetics are more appropriate (OECD Guidelines for testing of chemicals, Test No. 309, 2004).

1.5.2.3.3.4. Simulation tests to assess the biodegradation of chemicals discharged in wastewater (OECD 314)

OECD 314 guideline describes methods for determining the extent and kinetics of primary and ultimate biodegradation of organic chemicals whose route of entry into the environment begins with their discharge to wastewater. The five tests were designed to assess biodegradation during key phases of wastewater transit as well as treatment and environmental release. They include elements from OECD Test

23 Guidelines 301, 303A, 309, 310 and 311. The principal objectives of the methods are to: (1) measure the rate of primary biodegradation, (2) measure the rate of mineralization, and (3) follow the formation and decay of major transformation products. These tests are intended as higher tier tests for assessing the biodegradation of chemicals which do not biodegrade in OECD screening tests, or for refining biodegradation rates used for an exposure assessment (OECD Guidelines for testing of chemicals, Test No. 314, 2008). Reproducibility and reliability of all the tests were improved consequently with the use of standardized protocols, which list experimental conditions, analytical methods and criteria for whether a chemical is considered to be biodegradable (pass) or non-biodegradable (fail) (Howard et al., 1987).