Environmental Fate

Biodegradation studies

OECD 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Study Purpose and Introduction:

The inhibitory effect of the test item at the particular concentration is expressed as percentage of the mean respiration rate of the blank controls. Additionally reference controls (for checking the sensitivity of each batch of activated sludge), abiotic control (to measure the abiotic oxygen consumption), and nitrification control (containing N-allylthiourea for checking the nitrification potential of activated sludge) are included in the test series. The test method of application and the test system comply with all relevant test guidelines.

Guidelines and literature:

OECD Guidelines for the Testing of Chemicals, Section 2, No. 209, “Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)” (link: https://www.oecd-ilibrary.org/environment/test-no-209-activated-sludge-respiration-inhibition-test_9789264070080-en)

OECD 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Study Purpose and Introduction:

The purpose of the test is to evaluate the influence of the test item on the activity of activated sludge (originated from domestic waste water plant) by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage are measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test is typically used to determine the ECx (e.g. EC₅₀) of the test item and/or the no-observed effect concentration (NOEC).
The inhibitory effect of the test item at the particular concentration is expressed as percentage of the mean respiration rate of the blank controls. Additionally reference controls (for checking the sensitivity of each batch of activated sludge), abiotic control (to measure the abiotic oxygen consumption), and nitrification control (containing N-allylthiourea for checking the nitrification potential of activated sludge) are included in the test series. The test method of application and the test system comply with all relevant test guidelines.

Guidelines and literature:

OECD 216: Soil Microorganisms: Nitrogen Transformation Test

Study Purpose and Introduction:

Microorganisms play an important role in break-down and transformation of organic matter in fertile soils with many species contributing to different aspects of soil fertility. Any long-term interference with these biochemical processes could potentially interfere with nutrient cycling and this could alter soil fertility. Transformation of nitrogen occurs in all fertile soils. Although the microbial communities responsible for these processes differ from soil to soil, the pathways of transformation are essentially the same. In order to estimate the role and effect of an agrochemical on soil microflora, it is necessary to determine the effects on nitrogen transformation in the test soil samples and compare them to nitrogen transformation in control soil samples. This is a test method using natural soil samples to develop data on toxicity of test item to microbial population indigenous to the soil.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 2, No. 216, “Soil Microorganisms: Nitrogen Transformation Test” (link: https://www.oecd-ilibrary.org/environment/test-no-216-soil-microorganisms-nitrogen-transformation-test_9789264070226-en)

OECD 217: Soil Microorganisms: Carbon Transformation Test

Study Purpose and Introduction:

The purpose of this study is to determine the effect of the test item on carbon transformation of soil microflora. Microorganisms play an important role in break-down and transformation of organic matter in fertile soils with many species contributing to different aspects of soil fertility. Any long-term interference with these biochemical processes could potentially interfere with nutrient cycling and this could alter soil fertility. Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities responsible for these processes differ from soil to soil, the pathways of transformation are essentially the same. In order to estimate the role and effect of a chemical on soil microflora, it is necessary to determine the effects on carbon transformation in the test soil samples and compare them to carbon transformation in control soil samples. This is a test method using natural soil samples to develop data on toxicity of test item to microbial population indigenous to the soil.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 2, No. 217, “Soil Microorganisms: Carbon Transformation Test” (link: https://www.oecd-ilibrary.org/environment/test-no-217-soil-microorganisms-carbon-transformation-test_9789264070240-en)

OECD 301: Ready Biodegradability

OECD 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Study Purpose and Introduction:

Guidelines and literature:

OECD 301B: A CO2 Evolution Test (Modified Strum Test)

Study Purpose and Introduction:

The purpose of this study is to determine the ready biodegradability of the test item. Defined volume of inoculated mineral medium, containing a known concentration of the test item (in the range of 10-20 mg/L dissolved organic carbon, DOC) as the nominal sole source of organic carbon is aerated by carbon dioxide-free air at a controlled rate in the dark. The degradation is followed over 28 days by determining the carbon dioxide produced. The CO₂is trapped in barium hydroxide and is measured by titration of the residual hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test substance (corrected for that derived from the abiotic control and blank inoculum) is expressed as percentage ThCO₂. The degree of biodegradation may also be calculated from the supplemental DOC (Dissolved Organic Carbon) analysis made at the start and end of incubation. A reference item is tested simultaneously under the same conditions as the test item, serving as a procedure control.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 3, No. 301, “Ready Biodegradability” (link: https://www.oecd-ilibrary.org/environment/test-no-301-ready-biodegradability_9789264070349-en)

OECD 301C: A Modifilied M.I.T.I. (Ministry of International trade and Industry, Japan) Test (I)

Study Purpose and Introduction:

The purpose of this study is to determine the ready biodegradability of the test item. The test item (e.g.: 100 mg/L) is exposed to sludge (e.g.: 30 mg/L) cultured in the testing laboratory, and originated from ten different places of the country (industrial and domestic treatment works, rivers, lakes) for 28 days. The biodegradation is followed by the oxygen uptake of the microorganisms during exposure. In the modified MITI (I) (Ministry of International Trade and Industry, Japan) ready biodegradability test, the daily oxygen uptake (e.g. with BOD meter) by a stirred suspension of the test item in a mineral medium, inoculated with cultured not pre-adapted microorganisms, is measured. Additionally for assessment of biodegradation, the test item concentration is determined using specific analytical methods. A reference item is tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

OECD 301 D: A Closed Bottle Test

Study Purpose and Introduction:

The purpose of this study is to determine the ready biodegradability of the test item. The test item is exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles, in the dark and at a controlled temperature for 28 days. The biodegradation is followed by the oxygen uptake of the microorganisms during exposure. A reference item is tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

OECD 301 F: Manometric Respirometry Test

Study Purpose and Introduction:

The purpose of this study is to determine the ready biodegradability of the test item. The test item as the nominal sole source of organic carbon in mineral medium is stirred and exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in respirometer bottle at constant, controlled temperature for 28 days. The oxygen consumption is determined by measuring of decrease in pressure within the respirometer bottle. Evolved carbon dioxide is absorbed in a solution of potassium hydroxide. The amount of oxygen taken up by the microbial population (not pre-adapted microorganisms) during biodegradation of the test item (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD. A reference item is tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

OECD 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Study Purpose and Introduction:

Guidelines and literature:

OECD 302C: Inherent Biodegradability : Modified MITI Test (II)

Study Purpose and Introduction:

The purpose of this modified MITI (Ministry of International Trade and Industry, Japan) test is to determine the inherent biodegradability of the test item. The test item (e.g.: 30 mg/L) as the nominal sole source of organic carbon in basal culture medium (mineral medium) in a closed respirometer bottle is stirred and exposed to cultured, mixed microorganisms inoculum (e.g.: 100 mg/L), originated from at least ten different places of the country, at constant, controlled temperature for 28 days, in the dark. The biodegradation is followed by the oxygen uptake of the microorganisms during exposure. The oxygen consumption is determined by measuring of decrease in pressure within the respirometer bottle. Evolved carbon dioxide is absorbed in solution of potassium hydroxide. The amount of oxygen taken up by the microbial population during the biodegradation process of the test item (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD and/or COD (Chemical Oxygen Demand). Additionally, for assessment of biodegradation, the test item concentration is determined in water and in mineral medium using specific analytical method. A reference item is tested simultaneously under the same conditions as the test item, and functions as reference control.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 3, No. 302B, “Inherent Biodegradability : Zahn-Wellens/ EVPA Test” (link: https://www.oecd-ilibrary.org/environment/test-no-302b-inherent-biodegradability-zahn-wellens-evpa-test_9789264070387-en)

OECD 307: Aerobic and Anaerobic Transformation in Soil

Study Purpose and Introduction:

The purpose of this test is to measure the time course of biodegradation of a test substance in soil under aerobic and anaerobic conditions and to quantify the observations in the form of kinetic rate expressions.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 3, No. 307, “Aerobic and Anaerobic Transformation in Soil” (link: https://www.oecd-ilibrary.org/environment/test-no-307-aerobic-and-anaerobic-transformation-in-soil_9789264070509-en)

OECD 308: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems

Study Purpose and Introduction:

The purpose of this test is to measure the time course of biodegradation of a test substance in aquatic sediment systems and to quantify the observations in the form of kinetic rate expressions.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 3, No. 308, “Aerobic and Anaerobic Transformation in Aquatic Sediment Systems” (link: https://www.oecd-ilibrary.org/environment/test-no-308-aerobic-and-anaerobic-transformation-in-aquatic-sediment-systems_9789264070523-en)

OECD 309: Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test

Study Purpose and Introduction:
The purpose of this test is to measure the time course of biodegradation of a test substance at low concentration in aerobic natural water and to quantify the observations in the form of kinetic rate expressions.

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 3, No. 309, “Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test” (link: https://www.oecd-ilibrary.org/environment/test-no-309-aerobic-mineralisation-in-surface-water-simulation-biodegradation-test_9789264070547-en)

Bioaccumulation and Bioconcentration

OECD 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Study Purpose and Introduction:

Guidelines and literature:

OECD 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure

Study Purpose and Introduction:

The purpose of this study is characterisation of the bioaccumulation potential of the test item in fish under flow-through test conditions.

Parameters which characterise the bioaccumulation potential include the uptake rate constant (k₁), loss rate constants including the depuration rate constant (k₂), the steady-state bioconcentration factor (BCF_SS), the kinetic bioconcentration factor (BCF_K) and the dietary biomagnification factor (BMF).

The test consists of two phases: the exposure (uptake) and post-exposure (depuration) phases. During the uptake phase, a group of fish of one species is exposed to the test substance at one or more chosen concentrations, depending on the properties of the test substance. They are then transferred to a medium free of the test substance for the depuration phase. The depuration phase is always necessary unless uptake of the test substance during the uptake phase has been insignificant. The concentration of the test substance in/on the fish is followed through both phases. In addition to the exposed group(s), a control group of fish is held under identical conditions except for the absence of the test substance.

In the aqueous exposure the bioconcentration factor is calculated preferably both as the ratio of concentration in the fish (C_f) and in the water (C_w) at steady-state (BCF_SS) and as a kinetic bioconcentration factor (BCF_K) which is estimated as the ratio of the rate constants of uptake (k₁) and depuration (k₂) assuming first order kinetics. If a steady-state is not achieved or can only be achieved within an impractically long uptake phase, the kinetic approach is preferred. Alternatively, for highly hydrophobic substances the conduction of a dietary study should be considered. The dietary exposure is used for substances where the aqueous exposure methodology is not practicable (for example because stable, measurable water concentrations cannot be maintained, or adequate body burdens cannot be achieved within 60 days of exposure). The endpoint from the this test is a dietary biomagnification factor (BMF) rather than a bioconcentration factor (BCF).

Guidelines and literature:
OECD Guidelines for the Testing of Chemicals, Section 3, No. 305, “Bioaccumulation in Fish: Aqueous and Dietary Exposure” (link: https://www.oecd-ilibrary.org/environment/test-no-305-bioaccumulation-in-fish-aqueous-and-dietary-exposure_9789264185296-en)