2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2002 to 21 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: SETAC Part 1, Section 1.1 (March 1995)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Commission Directive 91/414/EEC (as amended by Directive 94/37/EEC)

Deviations:

no

GLP compliance:

yes

Test type:

laboratory

Specific details on test material used for the study:

Purity: 99.6%

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil no.:

#1

Soil type:

clay loam

% Clay:

23

% Silt:

36

% Sand:

41

% Org. C:

1.5

pH:

7.7

CEC:

14.4 meq/100 g soil d.w.

Bulk density (g/cm³):

0.88

Soil no.:

#2

Soil type:

sand

% Clay:

3

% Silt:

6

% Sand:

91

% Org. C:

1.5

pH:

5.6

CEC:

6.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.28

Soil no.:

#3

Soil type:

other: Light clay

% Clay:

26

% Silt:

32

% Sand:

42

% Org. C:

1

pH:

5.8

CEC:

15.1 meq/100 g soil d.w.

Bulk density (g/cm³):

1.08

Soil no.:

#4

Soil type:

loam

% Clay:

14

% Silt:

26

% Sand:

60

% Org. C:

1

pH:

7.7

CEC:

10 meq/100 g soil d.w.

Bulk density (g/cm³):

1.18

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location:
Soil #1: Thessaloniki, Greece (Cropland, weeds)
Soil #2: Bedfordshire, England, UK. OS Map reference TL 14074373 (Oilseed Rape)
Soil #3: Loire Valley, Charentilly France (No-till for 3 years, weed cover)
Soil #4: Nordchein, Germany (Winter wheat, bare soil)

- Pesticide use history at the collection site:
Soil #1: None previous 2 years
Soil #2: 2001/02, Roundup, Butisan, Trifluralin, Genie 25, Poraz, Ceres Promise, Ammonium Sulfate. 2000/01, Nitrogen, Dimethoate, L.I.700, Boxer, Adjust.
Soil #3: Glyphosate past 2 years
Soil #4: No data

- Collection date:
Soil #1: 1 March 2002
Soil #2: 4 March 2002
Soil #3: 26 March 2002
Soil #4: 10 April 2002

- Storage conditions: Refrigerated until samples weighed out for pre-incubation (all soils)

- Storage length:
Soil #1: 2.5 months
Soil #2: 2.5 months
Soil #3: 2 months
Soil #4: 1.5 months

- Soil preparation: 2 mm sieve (all soils)

Duration:

<= 123 d

Initial conc.:

0.16 other: µg/g

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on experimental conditions:

EXPERIMENTAL APPARATUS
Samples were incubated in two-chambered biometer flasks. One side of the biometer, a 250-mL Erlenmeyer flask, contained the moist soil (50 g dry-weight) while the other chamber held 100 mL of 0.2 M NaOH solution for collection of CO₂. The caustic solution also helped to maintain soil moisture during incubation by maintaining a constant relative humidity. Biometers were connected via an expansion bulb in the caustic trap to an oxygen manifold to sustain aerobic conditions during incubation. The soil side of each flask was closed with a ground glass stopper, using vacuum grease to create an airtight seal.
Biometers were also used for the sterile soil samples, including the 100 mL of 0.2 N NaOH solution for the trapping of CO₂. However, the 5 g of (oven-dry weight) moist soil was weighed into a 24-mL glass centrifuge tube. After the soil samples were sterilised, this tube was inserted into the 250-Erleruneyer soil flask.
Desiccators containing approximately 500 g of soil were used to incubate soils until after the study incubation period ended so that the matrix biomass could be determined at experimental completion. The moisture of these soils was not adjusted, as the original biomass determination was conducted on soils with unadjusted moistures. These samples were incubated in the dark at 20 °C for at least 4 months.

STUDY DESIGN
Duplicate flasks of each soil type were prepared for each time point. Extra flasks of each soil type were also prepared. Each flask contained 50 g (oven dry weight) moist soil. HPLC-grade water was used to adjust the soil to 40 % MHC as needed. Since the application solutions were prepared in water, the moisture adjustment took the projected application volume into account. Samples were weighed out 7 days before treatment, modifying the moisture content to 40 % MHC, and pre-incubated in the dark at 20 °C to allow the soil moisture and temperature to equilibrate.
The Parabraun Erde loam was selected to determine the effect of temperature on test material aerobic soil degradation. Additional 50 g samples at 40 % MHC were incubated at 10 and 30 °C for 8 days before treatment.
Additional Parabraun Erde samples were also sterilised before dosing. The sterilisation of some samples allowed for differentiation between microbial and abiotic degradation of the test material in soil. Approximately 5 g (oven-dry weight) of moist soil was weighed into 24-mL glass tubes and the moisture was adjusted to 40 % MHC. The samples were capped with Teflon-lined caps and shipped to Steris (Morton Grove, IL) where the samples were irradiated with gamma irradiation (50-60 kGy) for approximately 350 minutes. The sterilised samples were shipped unopened to the testing facility.
Upon arrival, the caps were removed and the samples were dosed with the test material. The dosed tubes (uncapped) were placed in the 250-mL Erlenmeyer flasks of the biometers and the 125-mL chambers were filled with 100 mL 0.2 N NaOH. While these samples were initially sterilised, the sterility of these samples was not confirmed at sample sacrifice. The primary objective of dosing sterilised samples was to eliminate microbial degradation, not to maintain sterile conditions throughout the study period.

TRAPPING OF CO₂
Each biometer side arm contained 100 mL of 0.2 M NaOH for trapping CO₂. The biometer is a self-contained system, closed to atmosphere to prevent volatile losses.

TEST SOLUTION PREPARATION
Stock solutions were prepared by dissolving the test material in acetone. Stock solutions were stored in a freezer when not in use and always brought to room temperature prior to preparation of the application solutions.
The 20 °C sample application solution was prepared by transferring an aliquot of the stock solution in acetone to a 4 oz. glass jar (120-mL) and adding 80 mL of HPLC-grade water . The dosing solution for the 10 and 30 °C samples was prepared by transferring an aliquot of the stock solution in acetone to a 60 mL glass jar (2 oz.). HPLC-grade water (50 mL) was added. This dosing solution was also used for the 5 g sterile samples.

APPLICATION PROCEDURES
All 50 g samples were dosed with 1 mL of application solution for a nominal soil concentration of 0.16 µg a.i./g. A total of 7.2 or 7.8 µg of test material was added to each 20 °C, or 10 and 30 °C sample flask, respectively. The application solution was applied drop-wise with a syringe uniformly over the soil surface.
The 5 g sterile samples were dosed with 0.1 mL of application solution (nominally 0.16 µg/g) for a total of 0.83 µg in each sample. The application solution was likewise applied drop-wise with a syringe to the soil surface.
The homogeneity and application rates were determined by taking aliquots of the application solutions periodically during treatment. These direct spikes were assayed by LSC.

EXPERIMENTAL CONDITIONS AND MONITORING
Samples of the four soil types were incubated in a darkened incubator set at 20 °C for up to 4 months after treatment. Additional Parabraun Erde samples were incubated at 10 and 30 °C. Aerobic conditions were maintained by connection of the biometer flask to an oxygen manifold via the expansion bulb. Incubator temperatures were monitored with the Camille system. The Temperature Monitoring Coordinator was alerted if any temperature controlled devices were out of the acceptable range for more than 1 hour.
No adjustment of soil moisture was necessary during study incubation because the caustic trap maintained a constant relative humidity in the sealed test system. The sacrificed soil sample was weighed at each time point so any change in soil moisture during incubation could be determined.
Duplicate samples of each soil were taken at each time point. The entire soil sample and caustic trap were removed from the biometer at the time of sample sacrifice. A portion of the caustic trapping solution (approximately 20 mL) was retained for analysis while the remainder was discarded. The entire soil sample was extracted. Caustic traps were assayed by LSC the day of sample sacrifice. Soil samples were extracted with acidified organic solvent, also on the day of sample sacrifice. HPLC analysis of extracts took place within one month and generally within one week of sample sacrifice. The extracted soil pellet was allowed to air-dry in a hood for at least one week prior to combustion analysis.
Samples incubated 92 DAT were selected to characterise the non-extractable residues (NER). These samples all contained at least 10 % of the applied radiocarbon as NER. Non-extractable residues were characterised by partitioning into fulvic acid, humic acid and humin pools.

MAINTENANCE AND COLLECTION OF VOLATILE TRAPS
At each sampling time point (except Time 0) approximately 20 mL of the caustic trapping solution was transferred by aspirator to a glass scintillation vial (the rest was discarded as waste). Triplicate aliquots of the trapping solution were counted by LSC to determine mineralisation to CO₂. Since radioactivity was presumed not present, the Time 0 traps were not assayed.
Samples sacrificed at 92 DAT were selected to confirm the radioactivity in the caustic traps was 14CO₃^-2. Sub-samples (5 mL) of previously assayed caustic trap solutions were mixed briefly in a centrifuge tube with 5 mL of a saturated solution of BaCl₂. The precipitate was separated by centrifugation at 2500 rpm for 15 minutes. An aliquot (approximately 5-mL) of the supernatant was filtered through a 0.45 µm PTFE syringe filter, and three 1-mL aliquots were analysed for 14C by LSC. Carbon dioxide in basic solution is precipitated with BaCl₂ to form insoluble BaCO₃ while presence of radioactivity in solution after precipitation with BaCl₂ is indicative of non-14CO₂ volatiles. When no radioactivity is present in the caustic solution after precipitation with BaCl₂, the radioactivity in the trap is presumed to be 14CO₃^-2.

Key result

Soil No.:

#1

DT50:

67 d

St. dev.:

59

Remarks on result:

other: half lifes ranged from 18 - 143 days

Transformation products:

no

Verification of Chromatographic Procedures

HPLC column recoveries were determined by directly counting an aliquot of each sample analysed by HPLC and comparing to the sum of the radioactivity eluted from the column. HPLC recoveries were generally between 90 and 110 %.

Material Balance

Mass balance values were generally in the 90 - 110 % of applied radioactivity range. Samples with recoveries less than 87 % of the applied radiocarbon were not used to determine aerobic degradation rates of test material in soil.

Distribution and Compositions of Residues

Radiocarbon Distribution

Mineralisation increased steadily over time in all tested soils. At 3 months after treatment, roughly 70 % of applied radiocarbon was recovered in the caustic traps in the Thessaloniki clay loam and the Charentilly light clay soils. At the end of the incubation period approximately 30 and 40 % mineralisation was seen in the Cuckney sand and Parabraun Erde loam soils, respectively.

The Parabraun Erde loam samples incubated at 10 and 30 °C produced 10 and 35 % CO₂ respectively, at 123 DAT. About 3 % of the applied radioactivity was recovered in the caustic traps of the sterile Parabraun Erde samples at the end of the study period.

The amount of extractable radioactivity decreased with time. Greater than 90 % of applied was extractable from all soil types at 0 DAT. At the end of the study period only 1 - 5 % was extracted from the Thessaloniki clay loam and Charentilly light clay samples while approximately 35 and 50 % of the applied radiocarbon was extracted from the Parabraun Erde loam and Cuckney sand, respectively.

The Thessaloniki clay loam produced the greatest amount of NER with about 20 % of the applied radioactivity recovered by combustion after 3 months incubation. Only about 10 % of applied radiocarbon was in the Cuckney sand NER at the 3-month mark. Both the Charentilly light clay and the Parabraun Erde loam produced about 15 % NER at the end of the study period.

Characterisation of the Non-Extractable Residues

Roughly 20 - 50 % of the NER was present in the fulvic acid pool (acid and base soluble). The remaining portion of the NER was fairly equally divided between the humic acid and humin pools, with the exception of the Thessaloniki clay loam, where about 70 % of the NER, or 15 % of applied was present in the humin. No other fraction accounted for more than 10 % of applied in any other soil tested.

Identification and Characterization of Transformation Products

Only parent test material was present in any HPLC chromatogram of the organic extracts.

An average of 99.8 ± 0.1 % of the radioactivity in the caustic traps was confirmed to be 14CO₃^-2 by precipitation with BaCl₂ to form insoluble Ba 14CO₃. Therefore, no degradates other than CO₂ (as CO₃^-2) were present in the caustic trap.

Non-extractable residues were also characterised.

No degradation products other than CO₂ and NER were observed in this study.

Kinetic Analysis of Data

Kinetics of Parent Compound Degradation

First-order, log-transformed half-lives of test material on aerobic soil at 20 °C were calculated for the four soils tested in this study. The half-lives ranged from 18 to 143 days and the average half-life was 67 ± 59 days.

As expected, the half-life of test material at 10 °C was much longer, 409 days vs. 84 days at 20 °C. Less predictably, the half-life at 30 °C was longer than at 20 °C, 115 days vs. 84 days. Several factors could cause this result. For one, the microbes responsible for test material soil degradation could respond less favourably to higher temperatures. Soil is a viable, variable system that does not necessarily respond to temperature changes in a linear fashion, as abiotic systems will.

No half-life on the sterile Parabraun Erde soil could be calculated due to insufficient degradation through 4 months of incubation. Despite the generation of a small amount of CO₂, about 3 % at 122 DAT, the slope of the degradation curve in the sterile samples was negative, indicating that degradation of test material did not occur.

Degradation Pathway

The primary route of test material aerobic soil metabolism is mineralisation to CO₂.

Incorporation to non-extractable soil residues is also a critical component to test material degradation. No degradates, other than CO₂ and bound residues were detected in this study.

Conclusions:

The degradation rate of test material in aerobic soil varied greatly by soil type. On 4 soils from the EU half-lives ranged from 18 to 143 days, with an average half-life of 67 ± 59 days. Only CO2 and non-extractable residues were observed as a result of aerobic degradation of test material in soil. In samples incubated at 20 °C, CO2 accounted for 30 - 70 % of the applied radioactivity by the end of the study period. Up to 20 % of the applied radioactivity was present as NER.
The effect of temperature on the degradation of test material was studied on one soil, the Parabraun Erde loam. While test material degradation was significantly slowed at 10 °C, no temperature dependant degradation could be calculated because degradation at 30 °C was slower than at 20 °C in the Parabraun Erde loam. No degradation of test material took place on Parabraun Erde loam samples sterilised with gamma-irradiation prior to treatment with the test material. No abiotic degradation of test material was observed.

Executive summary:

The rate and route of aerobic degradation of test material in soil was studied on four European soils; a Thessaloniki clay loam from Greece, a Cuckney sand from the UK, a Charentilly light clay from France and a Parabraun Erde loam from Germany. The study was conducted under GLP conditions and in accordance with standardised guidelines, as outlined in SETAC Part 1, Section 1.1 (March 1995) and European Commission Directive 91/414/EEC (as amended by Directive 94/37/EEC).

During the study 14C-test material was applied to 50 g (dry weight) moist soil at a nominal rate of 0.16 µg/g, or approximately equivalent to the seasonal maximum application rate of 120 g a.i./ha (based upon 5 cm depth and 1.5 g/mL bulk density). Samples were incubated.in the dark at 20 °C and 40 % moisture holding capacity (MHC) for up to 123 days after treatment.

No major transformation products other than CO₂ and NER were detected. At the end of the study period, approximately 40 - 70 % of the applied radioactivity was recovered in the CO₂ traps and approximately 15 - 20 % of applied was characterised as NER. None of the radioactivity detected was unidentified-the entire extractable radioactivity was characterised as test material.

Aerobic degradation rates of test material in soil varied depending upon soil type. At 20 °C, half-lives ranged from 18 to 143 days and the average half-life was 67 ± 59 days on the four soils tested in this study.

The effect of temperature on the degradation of test material was studied on one soil, the Parabraun Erde loam. While test material degradation was significantly slowed at 10 °C (half-life of 409 days), no temperature-dependant degradation rate could be calculated because degradation at 30 °C was slower than at 20 °C. The Parabruan Erde half-life was 115 days at 30 °C compared to 84 days at 20 °C.

No degradation of test material took place on Parabraun Erde loam samples sterilised with gamma-irradiation.in this study.

Description of key information

Aerobic degradation rates of test material in soil varied depending upon soil type. At 20 °C, half-lives ranged from 18 to 143 days and the average half-life was 67 ± 59 days on the four soils tested in this study. SETAC Part 1, Section 1.1 (March 1995) and European Commission Directive 91/414/EEC (as amended by Directive 94/37/EEC). Yoder & Smith (2003).

Key value for chemical safety assessment

Half-life in soil:

143 d

at the temperature of:

20 °C

Additional information

The rate and route of aerobic degradation of test material in soil was studied on four European soils; a Thessaloniki clay loam from Greece, a Cuckney sand from the UK, a Charentilly light clay from France and a Parabraun Erde loam from Germany. The study was conducted under GLP conditions and in accordance with standardised guidelines, as outlined in SETAC Part 1, Section 1.1 (March 1995) and European Commission Directive 91/414/EEC (as amended by Directive 94/37/EEC). The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

During the study 14C-test material was applied to 50 g (dry weight) moist soil at a nominal rate of 0.16 µg/g, or approximately equivalent to the seasonal maximum application rate of 120 g a.i./ha (based upon 5 cm depth and 1.5 g/mL bulk density). Samples were incubated.in the dark at 20 °C and 40 % moisture holding capacity (MHC) for up to 123 days after treatment.

No major transformation products other than CO₂ and NER were detected. At the end of the study period, approximately 40 - 70 % of the applied radioactivity was recovered in the CO₂ traps and approximately 15 - 20 % of applied was characterised as NER. None of the radioactivity detected was unidentified-the entire extractable radioactivity was characterised as test material.

Aerobic degradation rates of test material in soil varied depending upon soil type. At 20 °C, half-lives ranged from 18 to 143 days and the average half-life was 67 ± 59 days on the four soils tested in this study.

The effect of temperature on the degradation of test material was studied on one soil, the Parabraun Erde loam. While test material degradation was significantly slowed at 10 °C (half-life of 409 days), no temperature-dependant degradation rate could be calculated because degradation at 30 °C was slower than at 20 °C. The Parabruan Erde half-life was 115 days at 30 °C compared to 84 days at 20 °C.

No degradation of test material took place on Parabraun Erde loam samples sterilised with gamma-irradiation in this study.