Technical report 123

APPENDIX E: MEASUREMENT OF BIODEGRADATION

OECD 301 – Ready biodegradation (OECD, 1992b)

The Ready Biodegradation test is the first step or tier in biodegradation screening. It utilises estringent’ (low biomass) test conditions where positive test results (pass) of ereadily biodegradability’ one may infer the chemical will undergo rapid and complete mineralisation (CO2 evolution). The Ready Biodegradation test is available in several standard options (A-F) that accommodates DOC, DO, CO2 evolution and O2 uptake as endpoints and for chemicals of differing solubility, volatility and sorptive characteristics. Biosolid levels are usually at . 30 mg/l; test concentration range of 2 to 100 mg/l and may use chemical specific analysis as needed. Pass criteria for the test are generally noted as 70% DOC removal (A, E); 60% ThCO2 (B); 60% ThOD (C, D, F) within a 10-day window of the 28-day test.

The main output of the test is for classification purposes. Passing this test, however, supports action criteria that ‘no further testing’ is required as found in most if not all regulatory environmental assessment frameworks. A chemical not passing the test does not mean the chemical will persist in the environment. This result typically indicates further testing is needed at one of the higher tiers, such as the OECD 302 Inherent test, or one of the simulation tests, such as OECD 314, 303, 308 or 309 depending on what compartment is of interest.

The test is applicable to most chemical substances and is not uniquely sensitive or insensitive to ionic substances as such. The test is often not selected for chemicals that typically fail the ‘Ready Biodegradation’ test, such as pharmaceuticals, in lieu of initiating testing at a higher tier. This strategy is often employed as such to better characterise the emissions of compounds whose main route of entry into the environment is the wastewater treatment plant.

OECD 302 – Inherent biodegradation (OECD, 1981b,c, 1991, 1992a)

The inherent biodegradation tests (OECD 302 A-C) constitute the second step or tier in screening. The tests are not as stringent as the ready biodegradation series, typically using biomass concentrations ranging from 100 mg/l up to STP realistic concentrations of 1,000 to 2,000 mg/l. Chemical specific analysis is employed to monitor loss of parent substance (primary degradation), along with other general endpoints such as DOC or O2 uptake. Biodegradation above 20% theoretical may be evidence of inherent primary biodegradation; biodegradation above 70% of theoretical may be evidence of inherent ultimate biodegradation.

As an intermediate level test, it does not provide the assurance of rapid and complete mineralisation as ‘passing’ a ready biodegradation test, nor provide the accurate kinetics of a simulation test for refining a predicted environmental concentration. The inherent tests may eliminate the need for additional simulation testing if conducted longer than 28 days in the Modified SCAS test; and may extrapolate kinetics for STP removal when the chemical passes 70% degradation in a Zahn-Wellens test within 7 day period.

As with the ‘ready biodegradation’ tests, there are no specific limitations to ionisable substances. Inherent tests may not be selected for those compounds that are not readily biodegradation in lieu of a simulation test. For those tests not utilising radiolabel 14C- test material one should take care in interpreting degradation losses where volatility or non-specific adsorption may occur in the test system. Without the use of chemical specific analyses the test is of little value when the main route of depletion is primary rather than ultimate degradation.

OECD 303A – Simulation of aerobic sewage treatment: Activated sludge (OECD, 2001)

OPPTS 835.3220 – Activated sludge (EPA, 2008a) or OPPTS 835.3220 – Porous pot test (EPA, 1998)

The OECD 303 Sewage Treatment Plant (STP) simulation is designed to provide aerobic removal kinetic data to simulate those results of a typical treatment plant either with activated sludge units or biofilters. The test systems are designed to be continuous, with various options available for settling sludge in the aerobic sludge units (porous candle, settling vessel, etc.) to simulate STP clarifier. Biomass and test concentrations are at more realistic conditions than ready or inherent tests; i.e. biomass around 2000 mg/l and test material concentrations ranging from ìg/l to mg/l levels. Specific analysis for test material, along with general endpoints of DOC and COD may be measured on influent and effluent. The system is run with hydraulic and solids retention times of typical public sewage treatment plants of 6 hours and 6-10 days respectively. The study may be conducted with control units to assess the potential inhibitory effects of the test material, or potential problems with settleability; or potential abiotic depletion mechanisms. Use of radiolabel 14C-test material would allow for mass balance determination and assessment of mineralisation.

The main output of the test is the removal kinetics, first order decay elimination rate for assessing removal during a typical 6-hour hydraulic retention time. The kinetic data is essential to revising predicted environmental surface water concentration as a result of sewage treatment.

There are no specific limitations to ionisable substances. The study does allow for the measurement of test material sorbed to the wasted sludge, allowing for a direct measurement of removal on wasted sludge. As the test is conducted at realistic solids levels, any impact of sorption to the availability of the test material to biodegradation may be realistically evaluated. This may be of particular interest for ionisable substances, given the variety of ionic mechanisms of sorption prevalent for many of these substances. It is essential that activated sludge collected in the test is collected when the STP is operating normally without any upsets (poor settleability) or when COD removal is diminished. Information as to whether any ionic surfactants have been used in the STP for control of flocculation is very helpful in the assessment, as that may also influence sorption. It is not well characterised as to what extent synthetic feed influences the microbial diversity over time and to what extent the original microbial population changes. It may in fact be surmised (by the authors) that it helps standardise the sludge microbial population from one test to the next.

OECD 303B – Simulation of aerobic sewage treatment: Biofilms (OECD, 2001) or OPPTS 835.3260 (EPA, 2006)

In biofilms, synthetic or domestic sewage, and the test substance, in admixture or alone, are applied to the internal surface of a slowly rotating inclined tube. A layer of microorganisms is built up on the internal surface. Effluent from the tube is collected and either settled and/or filtered before analysis for DOC and/or the test substance by a specific method. Control units are operated in parallel under the same conditions.

The difference between the concentrations of DOC/COD in the effluent from the test and control units is assumed to be due to the test substance although specific compound analysis is often carried out before and after treatment. This difference is compared with the concentration of the added test substance to calculate the elimination of the test substance. Biodegradation may normally be distinguished from bio-adsorption by careful examination of the elimination-time curve.

OECD 314 – Simulation of chemicals discharged in wastewater: Mixing zone and river die-away (OECD, 2008)

The OECD 314 series of simulation tests are designed to provide the extent and kinetics of degradation (primary and ultimate) for 5 potential scenarios related to those chemicals discharged through a sewage treatment plant. Individual test guidance is provided for wastewater during sewer transit, activated sludge, anaerobic sludge, effluent in mixing zone and untreated effluent directly discharged to surface water. The test methods are either open batch or closed gas flow through batch systems allowing for the use of 14C-labeled test materials. Like the OECD 303 simulation, biomass and test concentrations are at more realistic conditions than ready or inherent tests; i.e. biomass for activated sludge around 2000 mg/l and test material concentrations ranging from ìg/l to mg/l levels. Specific analysis for test material, along with general endpoints of DOC and COD may be measured on influent and effluent. The simulations are conducted as a die-away test with no additional synthetic feed used as with the OECD 303. Sewage and river inocula are used within 24 hours of collection. As no additional feed is provided other than the COD that is present in those samples, the study should be initiated within that 24 hour period and continued no longer than required for the particular protocol.

The main output of the test is the first order decay elimination rate for assessing removal. The kinetic data is essential to revising predicted environmental concentrations for the various environmental scenarios outlined in the protocols.

There are no specific limitations to ionisable substances. The study conducted as batch studies provides easier maintenance of test systems compared to those of the OECD 303 thereby minimising some of the associated study costs. Otherwise most comments noted in the OECD 303 are applicable here as well.

OECD 308 – Simulation in aquatic sediment system (OECD, 2002)

The OECD 308 Aerobic and Anaerobic Water-Sediment Biodegradation test is designed to provide kinetic degradation rates for the water phase, sediment phase and the total test system, ultimate kinetic degradation rate for mineralisation of total test system, mass balance, distribution of activity between the phases and identification of transformation products as needed. It is designed to use 14C-labeled test material to follow the complete fate of the test material in the test system. Originally to represent over spray of pesticides/biocides in irrigation ditches, the method over time has been adopted by veterinary and human medicine regulatory frameworks as well to represent fate of pharmaceuticals in surface waters and sediments. The test uses 2 water-sediment samples in a ratio of 3:1 representing high and low organic content. Test concentration applied to the water phase is typically approximately 1 mg/l to allow for sufficient quantification of 14C-test material in all phases throughout the study period.

The main output of the test is the first order decay elimination rate of the total test system (primary degradation); along with mass balance, distribution of residues between the phases, metabolite ID if > 10% dose and dissipation of residues from the aqueous phase. Determination of the kinetic degradation data from the sediment phase alone is often not achievable due to the concurrent processes of sorption and degradation that occur, especially for those compounds that become highly bound to sediment, such as cationic compounds. This is true for most pharmaceuticals. The anaerobic phase of the test is typically not required for human pharmaceuticals as its been found the outcomes are similar to the aerobic phase except the degradation rates are slower with no new observation of transformation products than what is seen in the aerobic study13. For human pharmaceuticals it is a standalone test and primarily used to note the occurrence of transformation products. Kinetic data is not used to revise PEC estimates for water or sediment. For veterinary medicines and pesticides the kinetic data for total system half-life may be used in surface water models (EXPRESS; FOCUS; EUSES) to revise PEC surface water. For these products both aerobic and anaerobic studies are required by their respective regulatory authorities.

There are no specific limitations to ionic compounds, though the outcome is highly influenced by the type of ionic species present in these water-sediment systems. Cations in particular are shown to highly bind to sediments when compared to neutral and anionic compounds when evaluating 36 pharmaceuticals (Ericson, 2007); 55% irreversibly bound compared to 33% and 29% respectively. Similarly, cations typically show less biotransformation than neutral and anionic compounds, potentially inferring that cations are less bioavailable for degradation; half-life of 87 days compared to 29 and 30 days respectively. It is not clear whether these half-lives and the amount found to be bound are truly representative of fresh water-sediment systems as found downstream of sewage treatment plants.

OECD 309 – Simulation in surface waters: River die-away (OECD, 2004a)

The OECD 309 is an aerobic surface water biodegradation test and provides the kinetic degradation data for primary and ultimate degradation. It follows the OECD 314 river die-away test in that it is designed as a batch test to use 14C-labeled test material, run at relatively low concentrations in the range of 1 to 100 ìg/l range and may be run with surface water as is (pelagic) or amended at low solids levels of 0.01 but up to 1 g/l to represent a suspended sediment system. Unlike the OECD 314 river die-way, it is run at multiple concentrations. Test duration may be run for an extended period out to 90 days only if degradation has started within the first 60 days. Should the test run for an extended period, the test system may be renewed with a portion of the water or suspension allowing for a semi-continuous operation.

The main output of the test is the first order decay elimination rate for assessing removal. The kinetic data is essential to revising predicted environmental concentrations for the surface water scenarios outlined in the protocol.

The test is applicable to most test substances and has no specific limitations to ionisable substances.

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