## 3.7.3. Degradation test methods

Authors: John Parsons

Reviewers: Steven Droge, Russell Davenport

Learning objectives:

You should be able to:

- explain the strategy used in standardised biodegradability testing
- describe the most important aspects of standard biodegradability testing protocols
- interpret the results of standardised biodegradability tests

Keywords: Environmental fate, chemical degradation, photochemical degradation, biodegradation, mineralisation, degradation rate

Introduction

Many experimental approaches are possible to measure the environmental degradation of chemicals, ranging from highly controlled laboratory experiments to environmental monitoring studies. While each of these approaches has its advantages and disadvantages, a standardised and relatively straightforward set of protocols has clear advantages such as suitability for a wide range of laboratories, broad scientific and regulatory acceptance and comparability for different chemicals.

The system of OECD test guidelines (see links in the reference list of this chapter) is the most important set of standardised protocols although other test systems may be used in other regulatory contexts. As well as tests covering environmental fate processes, they also cover physical-chemical properties, bioaccumulation, toxicity etc. These guidelines have been developed in an international context and are adopted officially after extensive validation and testing in different laboratories. This ensures their wide acceptance and application in different regulatory contexts for chemical hazard and risk assessment.

Chemical degradation tests

The OECD Guidelines include only two tests specific for chemical degradation. This might seem surprising but it should not be forgotten that chemical degradation could also contribute to the removal observed in biodegradability tests. The OECD Guidelines for chemical degradation are OECD Test 111: Hydrolysis as a Function of pH (OECD 2004) and OECD Test 316: Phototransformation of Chemicals in Water – Direct Photolysis (OECD 2008). If desired, sterilised controls may also be used to determine the contribution of chemical degradation in biodegradability tests.

OECD Test 111 measures hydrolytic transformations of chemicals in aquatic systems at pH values normally found in the environment (pH 4 – 9). Sterile aqueous buffer solutions of different pH values (pH 4, 7 and 9) containing radio-labelled or unlabelled test substance (below saturation) are incubated in the dark at constant temperature and analysed after appropriate time intervals for the test substance and for hydrolysis products. The preliminary test is carried out for 5 days at 50°C and pH 4.0, 7.0 and 9.0, this is known as a first tier test. Further second tier tests study the hydrolysis of unstable substances and the identification of hydrolysis products and may extend for 30 days.

OECD Test 316 measures direct photolysis rate constants using xenon arc lamp capable of simulating natural sunlight in the 290 to 800 nm or natural sunlight, and extrapolated to natural water. If estimated losses are superior or equal to 20%, the transformation pathway and the identities, concentrations, and rate of formation and decline of major transformation products are identified.

Biodegradability tests

Biodegradation is in general considered to be the most important removal process for organic chemicals in the environment and it is therefore no surprise that biodegradability testing plays a key role in the assessing the environmental fate and subsequent exposure risks of chemicals. Biodegradation is an extensively researched area but data from standardised tests are favoured for regulatory purposes as they are assumed to yield reproducible and comparable data. Standardised tests have been developed internationally, most importantly under the auspices of the OECD and are part of the wider range of tests to measure physical-chemical, environmental and toxicological properties of chemicals. An overview of these biodegradability tests is given in Table 1.

The way that biodegradability testing is implemented can vary in detail depending on the regulatory context but in general it is based on a tiered approach with all chemicals being subjected to screening tests to identify chemicals that can be considered to be readily biodegradable and therefore removed rapidly from wastewater treatment plants (WWTPs) and the environment in general. These tests were originally developed for surfactants and often use activated sludge from WWTPs as a source of microorganisms since biodegradation during wastewater treatment is a major conduit of chemical emissions to the environment. The so-called ready biodegradability tests are designed to be stringent with low bacterial concentrations and the test chemical as the only potential source of carbon and energy at high concentrations. The assumption is that chemicals that show rapid biodegradation under these unfavourable conditions will always be degraded rapidly under environmental conditions. Biodegradation is determined as conversion to CO2 (mineralisation), either by directly measuring CO2 produced, or the consumption of oxygen, or removal of dissolved organic carbon, as this is the most desirable outcome of biodegradation. The results that have to be achieved for a chemical to be considered readily biodegradable vary slightly depending on the test, but as an example in the OECD 301D test (OECD 2014), the consumption of oxygen should reach 70% of that theoretically required for complete mineralisation within 28 days.

Table 1. The OECD biodegradability tests

OECD TEST GUIDELINE | PARAMETER MEASURED | REFERENCE
Ready biodegradability tests
301A: DOC Die-away test | DOC | OECD 1992a
301B: CO2 evolution test | CO2 | OECD 1992a
301C: Modified MITI(I) test | O2 | OECD 1992a
301D: Closed bottle test | O2 | OECD 1992a
301E: Modified OECD screening test | DOC | OECD 1992a
301F: Manometric respirometry test | O2 | OECD 1992a
306: Biodegradability in seawater | DOC | OECD 1992c
310: Test No. 310: Ready Biodegradability - CO2 in sealed vessels (Headspace Test). | CO2 | OECD 2014
Inherent biodegradability tests
302A: Modified Semi-continuous Activated Sludge (SCAS) test | DOC | OECD 1981b
302B: Zahn-Wellens test | DOC | OECD 1992b
302C: Modified MITI(II) test | O2 | OECD 2009
Simulation tests
303A: Activated sludge units | DOC | OECD 2001
303B: Biofilms | DOC | OECD 2001
304A: Inherent biodegradability in soil | 14CO2 | OECD 1981a
307: Aerobic and anaerobic transformation in soil | 14CO2/CO2 | OECD 2002a
308: Aerobic and anaerobic transformation in aquatic sediment systems | 14CO2/CO2 | OECD 2002b
309: Aerobic mineralization in surface water | 14CO2/CO2 | OECD 2004b
311: Anaerobic biodegradability of organic compounds in digested sludge: by measurement of gas production | CO2 and CH4 | OECD 2006
314: Simulation tests to assess the biodegradability of chemicals discharged in wastewater | Concentration of chemical, 14CO2/CO2 | OECD 2008a

These test systems are widely applied for regulatory purposes but they do have a number of issues. These include the fact that there are practical difficulties when applied to volatile or poorly soluble chemicals, but probably the most important is that for some chemicals the results can be highly variable. This is usually attributed to the source of the microorganisms used to inoculate the system. For many chemicals, there is a wide variability in how quickly they are degraded by activated sludge from different WWTPs. This is probably the result of different exposure concentrations and exposure periods to the chemicals, and may also be caused by dependence on the ability of small populations of degrading microorganisms, which may not always be included in the sludge samples used in the tests. These issues are not dealt with in any systematic way in biodegradability testing. It has been suggested that a preliminary period of exposure to the chemicals to be tested would allow sludge to adapt to the chemicals and may yield more reproducible test results. Further suggestions include using a higher, more environmentally relevant, concentration of activated sludge as the inoculum.

Failure to comply with the pass criteria in ready biodegradability tests does not necessarily mean that the chemical is persistent in the environment since it is possible that slow biodegradation may occur. These chemicals may therefore be tested further in higher tier tests, for what is referred to as inherent biodegradability in tests performed under more favourable conditions or in simulation tests representing specific compartments, to determine whether biodegradation may contribute significantly to their removal. These tests are also standardised (see Table 1). Simulation tests are designed to represent environmental conditions in specific compartments, such as redox potential, pH, temperature, microbial community, concentration of test substance and occurrence and concentration of other substrates.

The criteria used in classifying the biodegradability of chemicals depend on the regulatory context. Biodegradability tests can be used for different purposes: in the EU this includes 3 distinct purposes; classification and labelling, hazard/persistent assessment, and environmental risk assessment Recently regulatory emphasis has shifted to identifying hazardous chemicals, and therefore those chemicals that are less biodegradable and likely to persist in the environment. Examples for the classification as PBT (persistent, bioaccumulative and toxic) or vPvB (very persistent and very bioaccumulative) chemicals are shown in Table 2. As well as the results of standardised tests, other data such as the results of environmental monitoring data or studies on the microbiology of biodegradation can also be taken into account in evaluations of environmental degradation in a so-called weight of evidence approach.

Table 2. Criteria used to classify chemicals as PBT or vPvB (van Leeuwen & Vermeire 2007)

Property | PBT criteria | vPvB criteria
Persistence | T1/2 >60 days in marine water, or T1/2 >40 days in fresh/estuarine water, or T1/2 >180 days in marine sediment, or T1/2 >120 days in fresh/estuarine sediment, or T1/2 >120 days in soil. | T1/2 >60 days in marine, fresh or estuarine water, or T1/2 >180 days in marine, fresh or estuarine sediment, or T1/2 >180 days in soil
Bioaccumulation | BCF > 2000 L/kg | BCF > 5000 L/kg
Toxicity | - NOEC < 0.01 mg/L for marine or freshwater organisms, or - substance is classified as carcinogenic, mutagenic, or toxic for reproduction, or - there is other evidence of chronic toxicity, according to Directive 67/548/EEC |

The results of biodegradability tests are sometimes also used to derive input data for environmental fate models (see section on Multicompartment modeling). It is however not always straightforward to transfer data measured in what is sometimes a multi-compartment test system into degradation rates in individual compartments as other processes (e.g. partitioning) need to be taken into account.

References

OECD, 1981a. OECD Guidelines for the Testing of Chemicals. Test No. 304A: Inherent Biodegradability in Soil.

OECD, 1981b. OECD Guidelines for the Testing of Chemicals. Test No. 302A: Inherent Biodegradability: Modified SCAS Test.

OECD, 1992a. OECD Guidelines for the Testing of Chemicals. Test No. 301: Ready Biodegradability.

OECD, 1992b. OECD Guidelines for the Testing of Chemicals. Test No. 302B: Inherent Biodegradability: Zahn-Wellens/ EVPA Test.

OECD, 1992c. OECD Guidelines for the Testing of Chemicals. Test No. 306: Biodegradability in Seawater.

OECD, 2001. OECD Guidelines for the Testing of Chemicals. Test No. 303: Simulation Test - Aerobic Sewage Treatment - A: Activated Sludge Units; B: Biofilms.

OECD, 2002a. OECD Guidelines for the Testing of Chemicals. Test No. 307: Aerobic and Anaerobic Transformation in Soil.

OECD, 2002b. OECD Guidelines for the Testing of Chemicals. Test No. 308: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems.

OECD, 2004a. OECD Guidelines for the Testing of Chemicals. Test No. 111: Hydrolysis as a Function of pH.

OECD, 2004b. OECD Guidelines for the Testing of Chemicals. Test No. 309: Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test

OECD, 2006. OECD Guidelines for the Testing of Chemicals. Test No. 311: Anaerobic Biodegradability of Organic Compounds in Digested Sludge: by Measurement of Gas Production.

OECD, 2008a. OECD Guidelines for the Testing of Chemicals. Test No. 314: Simulation Tests to Assess the Biodegradability of Chemicals Discharged in Wastewater

OECD, 2008b. OECD Guidelines for the Testing of Chemicals. Test No. 316: Phototransformation of Chemicals in Water – Direct Photolysis.

OECD, 2009. OECD Guidelines for the Testing of Chemicals. Test No. 302C: Inherent Biodegradability: Modified MITI Test (II).

OECD, 2014. OECD Guidelines for the Testing of Chemicals. Test No. 310: Ready Biodegradability - CO2 in sealed vessels (Headspace Test).

Van Leeuwen, C.J., Vermeire, T.G. (2007). Risk Assessment of Chemicals: An Introduction (2nd ed.), Springer, ISBN 978-1-4020-6101-1