Aerobic/anaerobic sediment systems, coming from different sources, are assembled in a biometer or a gas flow-through apparatus, incubated in the dark under controlled conditions of temperature. For aerobic studies 2 sediments are normally used differing in organic carbon content and texture.
The aerobic test simulates an aerobic water column over an aerobic sediment layer that is underlain with an anaerobic gradient. For the anerobic studies 2 sediments are sampled along with their waters. The anaerobic test simulates  a complitely anaerobic water-sediment system.
The test substance is generally applied as aqueous solution on water phase.
After appropriate time intervals, the vessels containing the water-sediment systems are removed.
Sediment and its overlying water are extracted and analysed separately in order to determine the parent substance and the (known) transformation products/metabolites contents in the 2 phases. The test should normally ended 100 days after the test substance application. A plot of the test substance concentrations versus time allows an estimation of its transformation half-life/DT₅₀. Major transformation products have to be identified and their concentrations versus time plotted to show their rates of formation and decline.

STUDY DESIGN
1 concentration of the the test substance is tested in different sediment types
Analytical determination of the parent substance and the transformation products content in sediment and water:
4 replicates (2 replicates for sediment + 2 replicates for water phase)/sampling date for treated system
2 replicates (1 replicate for sediment + 1 replicate for water phase)/sampling date for untreated system
Additional replicates for relevant determinations on the 2 phases:
4 replicates (2 replicates for sediment + 2 replicates for water phase)/sampling date for treated and for untreated system
Additional replicates as spare samples.

Recovery quality check at 2 fortification levels:
2 replicates (1 replicate for sediment + 1 replicate for water phase)/sampling date for fortification level for untreated system.

Repeatability of the anlytical method:
Duplicate analysis of the same extract of water/sediment at proper sampling dates.

LOD (for the test substance and the transformation products) ≤ 0.01 mg/L (water) or 0.01 mg/kg (sediment) or ≤ 1% of the initial amount applied to the test system and LOQ determination.

Analyses performed using a method validated according to SANCO/3029/99 rev. 4 guidance document.
Where relevant, cartridges or traps to determine the active substance volatile fraction or volatile by-products are used.

ENDPOINTS
Kinetics analysis and calculation of DT₅₀ and DT₉₀ values.
Transformation products pattern.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline for Testing of Chemicals, No. 308 (24^th April 2002) – Aerobic and anaerobic transformation in aquatic sediment systems.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.

Sterile aqueous buffer solutions of different pH values (pH = 4, 7 and 9) are treated with the test substance and incubated in the dark under controlled laboratory conditions of temperature. After appropriate time intervals, buffer solutions are analysed for the test substance content and (if required) for hydrolysis products. The test should normally runs until 90% hydrolysis of the test substance is observed or for 30 days whichever comes first. A plot of the test substance concentrations versus time allows an estimation of its hydrolysis half-life/DT₅₀. Major hydrolysis products can be identified and their concentrations versus time plotted to show their rates of formation and decline. Sterility checks are usually planned at the start and at the end of the study in order to ensure that all test phases are carried out in abiotic conditions.

Preliminary test-Tier 1 (if required) is performed at 50 ± 0.5°C and pH 4.0, 7.0 and 9.0. If after 5 days hydrolysis < 10%, the test substance is considered hydrolytically stable and, normally, no additional testing is required.

STUDY DESIGN
1 concentration of the the test substance at different pH and temperature conditions.
Analytical determination of the parent substance and major hydrolysis products content in sterile buffer samples:
Treated sample: 2 replicates for sampling date for each pH/temperature condition
Untreated sample: 1 replicate for each pH/temperature condition at first and last sampling planned
Additional replicates for sterility check:
Untreated sample: 1 replicate for each pH/temperature condition at first and last sampling planned
Additional replicates as spare samples.

Recovery quality check at 2 fortification levels:
1 replicate for sampling for untreated buffer solution

Repeatability of the anlytical method:
Duplicate analysis of the same extract of buffer solution at proper sampling dates.

LOD (for the test substance and the transformation products):
≤ 3% of the initial amount applied to the solutions.

LOQ ≤10% of the initial amount.

Analyses performed using a method validated according to SANCO/3029/99 rev.4 guidance document

ENDPOINTS
Kinetics analysis and calculation of DT₅₀ and DT₉₀ values.
Major hydrolysis identification/quantification (if required).
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline for Testing of Chemicals, No. 111 (13^thApril 2004) – Hydrolysis as a function of pH.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.