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Environmental fate & pathways

Biodegradation in soil

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biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
The overlying water in the Biomass sample water was analyzed by Agvise Laboratories for TOC at termination, and degradation products >5% were analyzed by LC-MS/MS not by co-chromatography. This deviation had no effect on the study.
according to guideline
EPA OPPTS 835.4200 (Anaerobic Soil Metabolism)
The overlying water in the Biomass sample water was analyzed by Agvise Laboratories for TOC at termination, and degradation products >5% were analyzed by LC-MS/MS not by co-chromatography. This deviation had no effect on the study.
according to guideline
other: SETAC Procedures for Assessing Environmental Fate and Ecotoxicity of Pesticides (March 1995)
The overlying water in the Biomass sample water was analyzed by Agvise Laboratories for TOC at termination, and degradation products >5% were analyzed by LC-MS/MS not by co-chromatography. This deviation had no effect on the study.
GLP compliance:
Test type:
[phenyl(U)-14C] (specific activity: 51.48 μCi/mg; radiochemical purity: 98.9%), [pyridine-3-14C] (specific activity: 53.58 μCi/mg; radiochemical purity: 99.2%)
Oxygen conditions:
Soil classification:
USDA (US Department of Agriculture)
Nambsheim (Sandy Loam)
Soil no.:
Soil type:
sandy loam
% Clay:
% Silt:
% Sand:
% Org. C:
8.8 meq/100 g soil d.w.
Bulk density (g/cm³):
Details on soil characteristics:
- Geographic location: Nambsheim, Alsace, France
- Storage conditions: The soil was stored at approximately 4°C in the dark in closed bags when not in use
- Soil preparation: Prior to use, the test soil was sieved through an approximately 2-mm mesh sieve

- Moisture at 0 bar (%): 58.6
- Moisture at 1/10 bar (%): 15.4
- Moisture at 1/3 bar (%): 12.0
- Moisture at 15 bar (%): 6.1
- Organic Matter (%): 2.2
- Nitrogen, Total (%): 0.15
- Soluble Salts (mmhos/cm): 0.30
- Calcium (ppm): 1480
- Magnesium (ppm): 53
- Sodium (ppm): 9
- Potassium (ppm): 91
- Hydrogen (ppm): 7
- Phosphorus (ppm): 26
- Initial biomass (μg/g): 254.8
- Biomass at termination (μg/g): 207.4
Soil No.:
144 d
Soil No.:
Initial conc.:
0.245 other: μg/g dry soil
Based on:
act. ingr.
Soil No.:
Initial conc.:
0.253 other: μg/g dry soil
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
radiochem. meas.
Soil No.:
approximately 20 ± 2°C
Microbial biomass:
207.4 µg/g
Details on experimental conditions:
- Soil (g/replicate): 50 g (dry weight) for main test samples and 250 g (dry weight) for microbial biomass samples
- No. of replication biomass: 3 samples (1 initiation, 1 termination, and 1 reserve)
- No. of replication treatments: 11 samples per radiolabel: single samples for nine schedule samplings and two reserves
- Test apparatus: 250-mL (main study) or 1-L (biomass samples) Nalgene bottles
- Details of traps for CO2 and organic volatile: Volatiles were only collected and analyzed from the main study. Air/nitrogen exiting the test apparatus flowed through a Supelco ORBO™-100 adsorbent tube, an empty trap for overflow, then through two traps containing 1 N KOH for collection of any volatile organics and 14CO2 generated from the breakdown of the radiolabeled test substance. A flow rate that sustained a gentle stream of bubbles through the entire trapping series was maintained.
- Acclimation of the test system: The moisture in the samples was appropriately adjusted, and the main study and biomass samples were placed into incubation at 20°C, to acclimate to the test temperature and achieve aerobic conditions prior to study initiation. The main test and biomass samples were incubated for nine days before application of test substance.
- Evaporation procedures: The dose solution was prepared in ACN, but no special evaporation procedures were necessary as the dosing volume was minimal compared to the weight of the soil and the amount of water added during moisture adjustments.
- Identity of co-solvent: ACN

Test material application
- Volume of test solution used/treatment: 53 μL (Pyridine), 50 μL (Phenyl)
- Application method: 10–100-μL Rainin positive displacement pipetter
- Evaporation of application solvent: ACN

Any indication of the test material adsorbing to the walls of the test apparatus: None (verified with successful mass balance at Day 0)

Experimental conditions
- Moisture level verifications: Typically biweekly
- Continuous darkness: Yes (except during sampling and maintenance)

- Methods used to create the anaerobic conditions: the air flow was terminated, and the test systems were flooded with water and subjected to a continuous, gentle steam of humidified nitrogen until the end of the study.

- Sampling intervals: Day 0, 10, 24, 26, 40, 61, 90, 119, and 144
- Collection of CO2 and volatile organics: All sampling intervals excluding Day 0
- Sample extraction: Soil samples were extracted immediately. Extracts stored frozen until analysis.
- Sample storage before analysis: Soil extracts were generally analyzed within approximately one week
Soil No.:
% Recovery:
St. dev.:
Key result
Soil No.:
41.4 d
other: first-order rate constants
20 °C
Transformation products:
Details on transformation products:
The major transformation products (i.e., ≥10% AR) were IN-QDY62, reaching a maximum concentration of 53.0% AR (Day 144); IN-QDY50 (only found in the pyridine label), reaching a maximum concentration of 12.1% AR (Day 40); Non-Extractable Residue (NER), reaching a maximum concentration of 13.3% AR (Day 26); and CO2, reaching a maximum concentration of 12.4% AR (Day 144). The total unassigned radioactivity was ≤3.6% AR throughout the study.

Degradation products were identified by comparison of the chromatographic retention times to those of authentic standards and verified by mass spectrometry.
Details on results:
Distribution and Composition of Residues:
- Nambsheim Soil: The percentage of the applied radioactivity in the extractable fraction decreased from Day 0 to Day 144. Mean extractability values ranged from 97.4% (Day 0) to 68.4% AR (Day 119), and then increased slightly to 71.4% AR (Day 144). The mean level of unextractable residue increased and reached a maximum of 13.3% AR at Day 26, then decreased to 9.5% AR (Day 144). The mean amount of 14CO2 captured in the KOH traps reached a maximum of 12.4% AR (Day 144).
- Confirmation of Volatile Organics and 14CO2: Confirmation of 14CO2 in the KOH was performed as CO2 levels reached >10% AR. A barium chloride test performed on this system confirmed that the activity trapped was due to the presence of 14CO2. The ORBO tubes were analyzed at all time points and showed minimal activity present so no further analysis was performed.

Nature of the Applied Radioactivity in the Soil Extract:
The mean amount of [14C]test substance in the Day 0 (aerobic) extracts was 95.4%. During the anaerobic phase of the study (beginning on Day 24), the amount of [14C]test substance decreased from an average of 55.8% to 8.9% AR by Day 144.
[Pyridine-3-14C] and [Phenyl-14C(U)]test substance degrade slowly under anaerobic conditions
Executive summary:

The anaerobic biotransformation of radiolabeled test substance was studied in one soil system under aerobic conditions for 24 days (approximate aerobic soil half-life), and then the soil was flooded to promote anaerobic conditions. The soil was a sandy loam from Nambsheim, Alsace, France (Nambsheim). The organic matter content (Walkley-Black method) for this soil was 2.2%, while the pH (1:2 soil:0.01M CaCl2) was 7.4.

The test soil was treated with [Pyridine-3-14C] or [Phenyl-14C(U)]test substance at a concentration of 0.25 μg a.i./g dry weight soil and incubated in darkness at approximately 20 ± 2°C. Aerobic conditions were maintained for 24 days by passing a steady stream of humidified air through the test apparatus. After the addition of the water phase, anaerobic conditions were promoted by passing a steady stream of humidified nitrogen through the test apparatus. The flow-through systems were designed to trap evolved carbon dioxide (CO2) and volatile organic compounds. Samples were taken at 0, 10, 24, 26/2 (aerobic/anaerobic), 40/16, 61/37, 90/66, 119/95, and 144/120 days after treatment and the soils extracted and analyzed for [14C]test substance and transformation products via HPLC.

Mean mass balance was calculated as the percent of applied radioactivity (% AR) and was maintained (90.6% to 97.6% AR) throughout the study. The mean extractability value was at 97.4% at Day 0 in the soil, and then it decreased to a minimum of 68.4% AR (Day 119). The level of non-extractable residues (NER) increased in the soil during the aerobic phase of the study and then slightly decreased under anaerobic conditions. At Day 26, the mean was 13.3%, and the NER decreased to 9.5% by Day 144.

The amount of 14CO2 collected increased slightly with time. The level of 14CO2 at Day 144 was 6.8 and 17.9% AR for [Pyridine-3-14C] and [Phenyl-14C(U)]test substance, respectively.

The amount of [Pyridine-3-14C]- and [Phenyl-14C(U)]-test substance in the Day 0 (aerobic) extracts was 96.9 and 93.8% AR, respectively.

During the anaerobic phase of the study (beginning on Day 24), the amount of [Pyridine-3-14C]test substance decreased from 58.5% AR to 8.4% AR by Day 144. The [Phenyl-14C(U)]test substance decreased from 53.1% AR to 9.4% AR by Day 144. Day 144 of the study is equivalent to Day 120 of anaerobic testing (the system was incubated under aerobic conditions for the first 24 days).

The DT50 and DT90 values for test substance were calculated by the fitting the data with a single first order (SFO) model and a first-order multiple compartment (FOMC) model using ModelMaker® 4.0. The SFO provided both a good visual and statistical fit for the soil, and the FOMC model did not provide a better fit. The SFO model first-order rate constants (kp), DT50, and DT90 values for the anaerobic degradation of the test substance in the soil are summarized in the below table:

Soil type

first-order rate constant (per day)

DT50 (days)

DT90 (days)






Description of key information

Half-life of the test substance in soil was 41.4 days.

Key value for chemical safety assessment

Half-life in soil:
41.4 d
at the temperature of:
20 °C

Additional information