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

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water and sediment: simulation tests

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2002 to 16 January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Subdivision N Pesticide Guideline 162-3 (Anaerobic Aquatic Metabolism)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC - Europe Procedures for Assessing the Environmental Fate and EcoToxicity of Pesticides, Section 8.1

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Canada PMRA DACO Number 8.2.3.5.6

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.6%

Radiolabelling:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

other: pond water sediment and flooded soil

Details on source and properties of surface water:

Pond water was selected from an area representative of a grassland region of the United States.
- Storage temperature: 20 °C
- pH at time of collection: 7.9
- Electrical conductivity (mmhos/cm): 1.71
- Redox potential (mV) initial/final: -355.4 / -125.7
- Oxygen concentration (ppm) initial/final: 0.10 / -0.21
- Hardness (ppm CaCO₃): 669
- Dissolved organic carbon (ppm): 37.2

Details on source and properties of sediment:

Soil was selected from an area representative of a grassland region of Bedfordshire, England (Cuckney). Soil was collected following ISO 10381-6, Soil quality-Sampling.
- Texture classification: sand
> % sand: 89
> % silt: 8
> % clay: 3
- pH at time of collection: 6.0
- Organic carbon (%): 1.3
- Redox potential (mV) initial/final: -469.2 / -410.3
- CEC (meq/100 g): 5.2
- Bulk density (disturbed) (g/cm³): 1.28
- Biomass (initial / final): 111.4 / 39.5

Sediment was selected from an area representative of a grassland region of the United States in North Dakota.
- Textural classification: sandy loam
> % sand: 57
> % silt: 36
> % clay: 7
- pH at time of collection: 8.1
- Organic carbon (%): 4.9
- Redox potential (mV) initial/final: -430.6 / -261.6
- CEC (meq/100 g): 22.9
- Bulk density (disturbed) (g/cm³): 0.67
- Biomass (initial / final): 42.7 / 54.3

Duration of test (contact time):

120 - 363 d

Initial conc.:

0.08 other: µg/mL

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

EXPERIMENTAL APPARATUS
Samples were incubated in two-chambered biometer flasks. One side of the biometer contained the sediment and pond water or soil and distilled water while the other chamber held 0.2 M NaOH solution for collection of CO₂. The sediment/soil side of each flask was closed with a ground glass stopper, using vacuum grease to create an airtight seal.

EXPERIMENTAL DESIGN
Duplicate flasks of each sediment or soil type were prepared for each time point. For the soil samples, each flask contained 50 g (oven dry weight) of moist soil. Enough HPLC water was added so the total amount of water in the system was 100 mL (1:2 soil:solution ratio). For the sediment samples, each flask contained 30 g (oven dry weight) of moist sediment. Enough pond water was added so the total amount of water in the system was 120 mL (1:4 sediment:solution ratio). Additional flasks of each test system were prepared for a biomass measurement at the end of the study. A total of 43 and 25 flasks were prepared for the sediment and pond water test system and the soil and HPLC-grade water test system, respectively. Prior to treatment, samples were incubated in the dark at 20 °C (soil) or 25 °C (sediment) for at least 30 days to allow the samples to obtain anaerobic conditions.
Each biometer side arm contained 100 mL of 0.2 M NaOH for trapping CO₂. The biometer was a self-contained system, closed to the atmosphere to prevent volatile losses.

PREPARATION OF TEST SOLUTION
A stock solution was first prepared by dissolving the 14C-test material in 3 mL of acetonitrile. The stock solution was stored in a freezer when not in use.
To prepare the dosing solution, the radiolabelled stock solution was removed from the freezer and allowed to warm to room temperature. 2 mL (500 µg) of the stock solution was transferred to a 5-mL volumetric flask. Acetonitrile was added to bring the solution to 5-mL volume. The solution was mixed by inverting the flask repeatedly. The final concentration of the dosing solution was 100 µg ai/mL.

APPLICATION PROCEDURES
Dosing solution (83 µL for soil, 100 µL for sediment) was applied to each sample for a concentration of 0.08 µg ai/mL. A total of 8 µg (soil) or 10 µg (sediment) was added to each sample flask. The dosing solution was applied with a positive displacement pipette over the water surface. The samples were purged with nitrogen to expedite the removal of oxygen entering the test system during dosing.

EXPERIMENTAL CONDITIONS
The sediment samples were incubated for up to 363 days after treatment in a darkened incubator set at 25 °C. The soil samples were incubated for up to 120 days after treatment in a darkened incubator set at 20 °C. Samples were not exposed to oxygen until sample sacrifice. Incubator temperatures were monitored. The Temperature Monitoring Coordinator was alerted if any temperature controlled devices fell out of range for more than 1 hour.
At each time point, the aqueous pH and DO content and the redox potential of the aqueous and sediment/soil layers of each sample were determined.

SAMPLING INTERVALS AND COLLECTION FOR SEDIMENT CO₂
Sediment samples were analysed 0, 10 and 20 days, and 1, 3, 6, 9 and 12 months after treatment. Soil samples were analysed 0, 3, 10 and 20 days, and 1, 2 and 4 months after treatment. Duplicate samples from each test system were sacrificed at each time point. The entire sediment/soil and water sample and caustic trap were removed for analysis at the time of sample sacrifice.
Caustic traps were assayed by LSC, and the pH, DO, and redox potential of both the sediment/soil and water layers were measured the day of sample sacrifice. Separation of the aqueous and sediment/soil layers and extraction of the sediment/soil layer with organic solvent were also conducted the day of sample sacrifice. HPLC analysis of aqueous samples and the organic extracts took place within approximately two weeks of sample sacrifice.

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 no radioactivity was presumed present in the Time 0 traps, they were not assayed.

Key result

Remarks on result:

other: The test material remained stable in the flooded soil test system and the sediment test system under anaerobic conditions

Transformation products:

no

Sediment/ Soil Properties

The anticipated soil/sediment:solution ratio was 1:2. However, the sediment had an average percent moisture greater than 200; therefore, a 1:2 sediment solution ratio could not be achieved. Instead, a 1:4 sediment:solution ratio was chosen.

 

Physical Conditions

Anaerobic conditions were maintained throughout the study. Samples were visibly anaerobic when caustic trapping material was pushed up into the expansion bulb. Measurement of the water DO content and the sediment and water redox potential confirmed anaerobic conditions. Sample temperatures were maintained in the dark at 25.5 ± 1 °C for up to 363 days after treatment for the sediment and 19.5 ± 1 °C for up to 120 days after treatment for the soil.

 

Verification of Extraction Procedures

Originally, both the sediment and soil were extracted with 90:10 acetone:1.0 N HCl; however, this extraction solution did not completely extract the test material residues from the 0 day sediment samples. Therefore, after experimenting with different types of extraction solutions, a 90:10 methanol:1.0 N NaOH solution was chosen for the sediment because it extracted the most test material residues. Then, the extraction efficiency of the method was checked at the 10 DAT sampling point using the 90:10 acetone:1.0N HCl solution for the soil samples and the 90:10 methanol:1.0 N NaOH for the sediment samples. The samples were extracted four times but the results showed that greater than 95 % of the extractable radioactivity was recovered after three extractions; therefore, the study samples were extracted four times.

 

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 between 90 and 110 %.

 

Material Balance

Material balance averaged 95.5 ± 2.8 % (89.9 - 100.5 %) for the soil samples. Material balance averaged 97.9 ± 1.6 % (94.2 - 100.5 %) for the sediment samples.

 

Distribution and Composition of Residues

The distribution of the residues remained fairly constant throughout the study. For the sediment, the mean total recovery was 64.3 ± 2.9 % and 31.5 ± 2.7 % of the applied radioactivity in the water and sediment, respectively. Extractable 14C residues in the sediment increased from 27.4 % at Day 0 to 36.9 % of the applied radioactivity at the end of the incubation period. Non-extractable 14C residues in the sediment increased from 0.9 % at Day 0 to 1.2 % of the applied radioactivity at study termination. At the end of the study 0.6 % of the applied radioactivity was present in the caustic traps.

The concentration of test material in water decreased from 70.6 % at Day 0 to 61.8 % of the applied radioactivity at study termination. The concentration of test material in the sediment increased from 27.3 % at Day 0 to 36.9 % of the applied radioactivity at 363 days.

For the flooded soil system, the mean total recovery was 66.1 ± 4.5 % and 28.4 ± 3.8 % of the applied radioactivity in the water and soil, respectively. Extractable 14C residues in the soil decreased from 30.0 % at Day 0 to 21.5 % of the applied radioactivity at the end of the incubation period. Non-extractable 14C residues in the soil ranged from 0.5 to 1.3 % of the applied radioactivity throughout the study. At the end of the study 0.4 % of the applied radioactivity was present in the caustic traps.

The concentration of test material in water increased from 69.2 % at Day 0 to 71.7 % of the applied radioactivity at study termination. The concentration of test material in the sediment decreased from 30.0 % at Day 0 to 21.5 % of the applied radioactivity at 120 days.

The test material was stable in both the pond water/sediment system and the flooded soil system throughout the study. A t-test showed that the slope of the degradation curve was no different than zero, indicating the test material did not degrade in anaerobic conditions of this study.

For the sediment, the Kd values ranged from 0.44 to 0.65 L/kg, while the Koc values ranged from 9.0 to 13.2 L/kg. For the soil, the Kd values ranged from 0.27 to 0.48 L/kg, while the Koc values ranged from 20.9 to 37.1 L/kg.

 

Identification and Characterisation of Transformation Products

The test material did not degrade in the flooded soil system and the sediment system under anaerobic conditions; no transformation products were detected. NER and CO₂ distributions accounted for less than 5 % of the applied radiocarbon at the end of the incubation period.

 

Kinetic Analysis Data

The test material was stable in the flooded soil system and the sediment system.

 

Decline of Metabolites

No metabolites, other than small amounts of CO₂ and NER, were observed in this study.

 

Degradation Pathway

The test material was stable under the anaerobic conditions of this study. However, small amounts of NER and CO₂ were observed.

Validity criteria fulfilled:

yes

Conclusions:

The test material remained stable in the flooded soil test system and the sediment test system under anaerobic conditions. T-tests showed that the degradation curves' slopes were not significantly different from zero. Anaerobic metabolism is therefore not a major route of degradation for the test material.

Executive summary:

The aquatic degradation of the test material was investigated under anaerobic conditions in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA Subdivision N Pesticide Guideline 162-3, SETAC Section 8.1, and Canada PMRA DACO Number 8.2.3.5.6.

During the study the anaerobic biotransformation of radiolabelled test material was studied in a pond water sediment system (water pH 7.9, dissolved organic carbon 37.2 ppm, sediment texture Sandy Loam, pH 8.1, organic carbon 4.9) from North Dakota, (USA) for 363 days in the dark at 25 °C. Test material was applied at the rate of 0.08 mg a.i./L. The sediment/water ratio used was 1:4.

The anaerobic biotransformation of radiolabelled test material was also studied in a flooded soil system using a Cuckney soil from Bedfordshire (England) (soil texture Sand, pH 6.0, organic carbon 1.3) and HPLC grade water for 120 days in the dark at 20 °C. Test material was applied at the rate of 0.08 mg a.i./L. The soil/water ratio used was 1:2.

The test system consisted of two-chambered biometer flasks with traps for the collection of CO₂. Cuckney soil samples were analysed at 0, 3, 10, 20, 30, 59, and 120 days of incubation. North Dakota sediment samples were analysed at 0, 10, 20, 30, 90, 181, 268, and 363 days of incubation.

Aliquots of the water were directly analysed by LSC and HPLC. The Cuckney soil samples were extracted on a horizontal shaker at low speed with an acetone:1.0 N HCl (90:10, v:v) solution. The North Dakota sediment samples were extracted on a horizontal shaker at low speed with a methanol:l.0 N NaOH (90:10, v:v) solution. Test material residues were analysed by LSC and HPLC.

The test conditions were maintained throughout the study. The total material balance in the North Dakota water/sediment system was 97.9 ± 1.6 % of the applied radioactivity. The mean total recovery of the radiolabelled material was 64.3 ± 2.9 % and 31.5 ± 2.7 % of the applied radioactivity in the water and sediment, respectively. Extractable 14C residues in the sediment increased from 27.4 % at Day 0 to 36.9 % of the applied radioactivity at the end of the incubation period. Non-extractable l4C residues in the sediment increased from 0.9 % at Day 0 to 1.2 % of the applied radioactivity at study termination. At the end of the study 0.6 % of the applied radioactivity was present as CO₂.

The concentration of test material in North Dakota water decreased from 70.6 % at Day 0 to 61.8 % of the applied radioactivity at study termination. The concentration of test material in the sediment increased from 27.3 % at Day 0 to 36.9 % of the applied radioactivity at the end of the study period.

The test material was stable in the North Dakota water/sediment system throughout the study. A t-test showed that the slope of the degradation curve was no different than zero; therefore, no further kinetics calculations were performed. For the flooded Cuckney soil system, the total material balance was 95.5 ± 2.8 % of the applied radioactivity. The mean total recovery of the radiolabelled material was 66.1 ± 4.5 % and 28.4 ± 3.8 % of the applied radioactivity in the water and soil, respectively. Extractable 14C residues in the soil decreased from 30.0 % at Day 0 to 21.5 of the applied radioactivity at the end of the incubation period. Non-extractable 14C residues in the soil ranged from 0.5 to 1.3 % of the applied radioactivity throughout the study. At the end of the study 0.4 % of the applied radioactivity was present as CO₂.

The concentration of test material in water increased from 69.2 % at Day 0 to 71.7 % of the applied radioactivity at study termination. The concentration of test material in the sediment decreased from 30.0 % at Day 0 to 21.5 % of the applied radioactivity at the end of the study period.

The test material was stable in the flooded Cuckney soil system throughout the study. A t-test showed that the slope of the degradation curve was no different than zero; therefore, no further kinetics calculations were performed.

Anaerobic metabolism is therefore not a major route of degradation for the test material.

Reference 2

Endpoint:

biodegradation in water and sediment: simulation tests

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2002 to 21 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Subdivision N Pesticide Guideline 162-4 (Aerobic Aquatic Metabolism)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Directive 91/414/EECIBBA Part IV

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Canada PMRA DACO Number 8.2.3.5.4

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.3%

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: sediment and pond water

Details on source and properties of surface water:

> French water
- Storage temperature: 20 °C
- pH: 5.9
- Electrical conductivity (mmhos/cm): 0.11
- Redox potential (mV) initial/final: 237 / 269
- Oxygen concentration (ppm) initial/final: 4.2 / 7.3
- Hardness (mg/L CaCO₃): 10
- Dissolved organic carbon (mg/L): 2.4
- Total suspended solids (mg/L): 24
- Water filtered: yes (through glass wool prior to transfer to sample tubes)

> Italian water
- Storage temperature: 20 °C
- pH: 8.2
- Electrical conductivity (mmhos/cm): 0.36
- Redox potential (mV) initial/final: 19 / 77
- Oxygen concentration (ppm) initial/final: 3.0 / 4.6
- Hardness (mg/L CaCO₃): 220
- Dissolved organic carbon (mg/L): 1.5
- Total suspended solids (mg/L): 4
- Water filtered: yes (through glass wool prior to transfer to sample tubes)

> US water
- Storage temperature: 20 °C
- pH: 7.9
- Electrical conductivity (mmhos/cm): 1.71
- Redox potential (mV) initial/final: -150 / -57
- Oxygen concentration (ppm) initial/final: 0.2 / 3.3
- Hardness (mg/L CaCO₃): 669
- Dissolved organic carbon (mg/L): 37.2
- Total suspended solids (mg/L): 366
- Water filtered: yes (through glass wool prior to transfer to sample tubes)

Details on source and properties of sediment:

The three sediments and their respective waters used in this study were selected as representative of French, Italian and US aquatic systems.
> French Sediment (Haut Languedoc)
- Storage temperature: 20 °C
- Textural classification: sand / sand (USDA / BBA)
- % sand: 91 / 93 (USDA / BBA)
- % silt: 6 / 8 (USDA / BBA)
- % clay: 1 / (USDA / BBA)
- pH at time of collection: 6.1
- Organic carbon (%): 0.8
- CEC (meq/100 g): 3.4
- Bulk density (disturbed) (g/cm³): 1.19
- Biomass (µg/g) initial/final: 83.8 / 207.2
- Sediment samples sieved: yes (through a 2-mm sieve)

> Italian Sediment (Altogarda)
- Storage temperature: 20 °C
- Textural classification: silt loam / sandy silt (USDA / BBA)
- % sand: 41 / 39 (USDA / BBA)
- % silt: 56 / 58 (USDA / BBA)
- % clay: 3 / 3 (USDA / BBA)
- pH at time of collection: 7.9
- Organic carbon (%): 12.0
- CEC (meq/100 g): 11.9
- Bulk density disturbed) (g/cm³): 0.64
- Biomass (µg/g) initial/final: 166.0 / 63.4
- Sediment samples sieved: yes (through a 2-mm sieve)

> US Sediment (North Dakota)
- Storage temperature: 20 °C
- Textural classification: sandy loam / silty sand (USDA / BBA)
- % sand: 57 / 53 (USDA / BBA)
- % silt: 36 / 40 (USDA / BBA)
- % clay: 7 / 7 (USDA / BBA)
- pH at time of collection: 8.1
- Organic carbon (%): 6.2
- CEC (meq/100 g): 16.6
- Bulk density (disturbed) (g/cm³): 0.67
- Biomass (µg/g) initial/final: 42.7 / 47.2
- Sediment samples sieved: yes (through a 2-mm sieve)

Duration of test (contact time):

<= 101 d

Initial conc.:

0.04 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

EXPERIMENTAL APPARATUS
Kinetics samples were incubated in 45 mL glass centrifuge tubes containing 2-2.5 cm sediment and approximately 6 cm of pond water. Each tube was connected, via PEEK tubing, to a second centrifuge tube containing approximately 20 mL of 0.2 M NaOH. A vacuum pump was connected to this caustic trap and pulled CO₂-scrubbed, moist air through the system (ambient air was pulled through a large caustic trap to remove atmospheric CO₂ and to keep the airflow through the system moist).

STUDY DESIGN
Twenty-three sample tubes of each sediment type were prepared for sample dosing. Several of these tubes were used to determine the test system oxygen content and redox potential prior to sample dosing.
Each tube contained roughly 15 g of wet sediment and 25 mL of their respective pond water.
Samples were weighed out 7 days before treatment to allow the water and sediment samples to equilibrate in the dark at 20 °C.
Each sample was connected to a second tube containing 20 mL of 0.2 N NaOH as traps for CO₂. A continuous flow through each sample was maintained with a vacuum pump pulling CO₂-scrubbed moist air through the system. Flow was determined by a visual inspection of the caustic traps - air bubbles were periodically observed in each trap.

TEST SOLUTION PREPARATION
A stock solution was prepared by adding 2 mL of acetone to the test material and mixing well. The stock solution was stored in a freezer when not in use. The stock solution was brought to room temperature prior to preparation of the dosing solution.
The dosing solution was prepared by transferring 260 µL of the above stock solution to a 10-mL volumetric flask. HPLC-grade water was added to volume and the solution well mixed. The dosing solution was stored at room temperature overnight prior to dosing. The nominal concentration of the dosing solution was 8.6 µg/mL.

APPLICATION PROCEDURE
Each sample was treated with 120 µL of dosing solution for nominal aqueous concentration of 40 µg/L. A total of 1 µg of test material was added to each sample. The dosing solution was applied with a syringe to the surface of the water. The homogeneity of the dosing solution and the application rate were determined by taking aliquots of the dosing solution periodically during treatment. These direct spikes were assayed by LSC.

TEST SYSTEM CONDITIONS AND MONITORING
Samples were incubated in a darkened constant temperature room set at 20 °C for up to 101 days after treatment (DAT). Aerobic conditions were maintained by pulling CO₂-scrubbed, moist air through the system continuously. Airflow was visually confirmed by the presence of bubbling intermittently in the caustic traps. Samples were observed carefully at each sacrifice point. If airflow had been interrupted in a sample, and the caustic trapping material had washed back into the sample container, the aqueous layer became very dark as the NaOH extracted the sediment organic material. A contaminated sample also had a higher than typical aqueous pH for that sediment type. Samples contaminated with the caustic trapping material were not analysed and another sample at that time point was sacrificed.
Incubator temperatures were monitored. The Temperature Monitoring Coordinator was alerted if any temperature controlled devices were out of the acceptable range for more than 1 hour.
At each sampling time point (except Time 0) triplicate 1-mL aliquots of the trapping solution were counted by LSC to determine mineralisation to CO₂.

SAMPLING INTERVALS AND COLLECTION FOR SEDIMENT CO₂
Duplicate samples of each sediment type were taken at each time point. Samples were analysed 0, 3, 7, 14, 21, 30, 62 and 101 DAT.
Caustic traps were assayed by LSC the day of sample sacrifice. Sediment extractions with acidified organic solvent were also conducted the day of sample sacrifice, and aliquots were removed for LSC. Aqueous extracts were assayed by LSC and generally analysed by HPLC the day of sample sacrifice. HPLC analysis of organic extracts took place within one week of sample sacrifice. Extracted sediments were allowed to air-dry for at least one week prior to combustion of sub-samples for mass balance determination.

Key result

Compartment:

entire system

DT50:

819 d

Remarks on result:

other: French system

Key result

Compartment:

water

DT50:

127 d

Remarks on result:

other: French system

Key result

Compartment:

entire system

DT50:

458 d

Remarks on result:

other: Italian system

Key result

Compartment:

water

DT50:

243 d

Remarks on result:

other: Italian system

Key result

Compartment:

entire system

DT50:

999 d

Remarks on result:

other: US system

Key result

Compartment:

water

DT50:

447 d

Remarks on result:

other: US system

Transformation products:

no

Physical Conditions

With the exception of a few samples, the French and Italian aquatic layers were maintained under sufficiently aerobic conditions throughout the study period (>2 ppm oxygen). The US sediment samples started out with a lower oxygen content (<1 ppm), but the amount of oxygen increased steadily over the study period to about 3-4 ppm. The redox potential of the aqueous layer suggested aerobic conditions with generally positive or small negative values. The redox potential of the sediment suggested mildly anaerobic to anaerobic conditions, with values in the negative range. Sample temperatures were maintained in the dark at 20 °C for up to 101 days after treatment.

Verification of Extraction Procedures

In the French and US sediments, the amount of non-extractable radioactivity was 3 and 7 % of applied radioactivity, respectively at the end of the study period. The Italian sediment system produced more NER than the French and US systems, with 15 % of applied radioactivity recovered by combustion at 30 DAT through 101 DAT. Since less than 10 % of the applied radioactivity was extractable in two of the three sediment systems, it can be concluded that three sequential extractions was sufficient to remove all the extractable radioactivity.

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

French sample recoveries ranged from 93.7 to 101.3 %, with an average recovery of 98.9 ± 2.1 %. Italian sample recoveries averaged 100.4 ± 1.3 % (98.7 through 102.1) and US sediment sample recoveries ranged from 98.5 to 102.3 % (averaging 100.5 ± 1.2 %).

Distribution and Composition of Residues

The amount of mineralisation was low, less than 4 % of applied radioactivity was recovered from any sample. The decline in radioactivity from the aqueous layer varied depending upon sediment type. The French sediment samples produced the greatest amount of CO₂, where about 3 % of applied radioactivity was recovered in the caustic traps at 101 DAT. Less than 2 % of applied radioactivity was found in the Italian and US sample traps at 101 DAT.

The French samples showed the most dramatic change, where over 90 % of the applied radioactivity was present in the aqueous layer at 0 DAT, dropping to about 50 % of applied at 101 DAT. The Italian and US sediment samples showed a much more moderate decline in radioactivity from the aqueous layer. At 0 DAT, over 95 % of the applied radioactivity was present in the aqueous layer, before declining to 70 and 80 % of applied, respectively in the Italian and US sediments.

Conversely, the amount of radioactivity extracted from the sediment increased in all test systems with time. About 2 % of the applied radioactivity was extracted from the French sediment at 0 DAT, increasing to about 40 % at the end of the study period. About 2 % of the applied radioactivity was also extracted at 0 DAT from the Italian and US sediments. At the end of the study period the amount of extractable radioactivity increased to 15 and 11 %, respectively for the Italian and US sediments.

Non-extractable residues increased with time in all sample types. At 0 DAT, less than 1 % of the applied radioactivity was recovered by combustion of the extracted sediments. At the end of the study period the amount of NER in each sediment type was 3, 15, and 7 %, respectively for the French, Italian and US sediments.

Characterisation of the Non-Extractable Residues

The Italian sediment was the only test system that produced non-extractable residues at concentrations of greater than 10 % of the applied radioactivity, therefore, only representative samples of the Italian sediment were selected for NER characterisation.

The majority of the radioactivity in the NER was present in the fulvic acid pool (acid and base soluble).

Non-Equilibrium Sorption Coefficients

In all three sediment types, the distribution of test material between the aqueous and sediment phases increased with time, from 0.05 L/kg at 0 DAT to about 0.5 - 1 L/kg at 101 DAT.

Identification and Characterisation of Transformation Products

The test material accounted for 95 % or greater of the radioactivity extractable from any sample. No metabolite was seen at greater than 6 % of applied.

The fulvic acid fraction accounted for virtually all of the NER.

Kinetic Analysis of Data

First-order, log-transformed half-lives of test material were calculated for the three sediment and water systems tested here. Half-lives were calculated for both degradation of test material from the entire system. Dissipation of test material from the aquatic phase was also determined. A t-test performed on the data indicated that the slope of the degradation curve was statistically different from 0 for all three sediments tested, indicating that the test material did indeed degrade, albeit slowly under the conditions of this study. Total system half-lives were greater than 450 days. Aquatic dissipation DT 50 values ranged from 127 to 447 days.

Decline of Metabolites

No metabolites were observed in this study.

Degradation Pathway

The test material degraded slowly to form non-extractable residue and several minor degradates(<6 % of applied radiocarbon). Additionally, a slight amount of mineralisation (<4 % at 101 DAT) was observed.

Validity criteria fulfilled:

yes

Conclusions:

The test material was shown to degrade slowly in three sediment and water systems, with half-lives of greater than 450 days. No major transformation products were observed, other than non-extractable residues. Mineralisation to CO2 was negligible over the 101-day study. Distribution coefficients outlining the movement of test material from the aqueous into the sediment layer increased from about 0.05 L/kg at 0 DAT to about 1 L/kg at 101 DAT.
Therefore, degradation in aquatic systems will not be a major route of degradation for the test material from the environment.

Executive summary:

The degradation of the test material under aerobic conditions was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA Subdivision N Pesticide Guideline OPP162-4, EC Directive 91/414/EEC BBA Part IV, and Canada PMRA DACO Number 8.2.3.5.4.

During the study the aerobic biotransformation of the test material was studied in 3 pond water/sediment systems. One sediment/water system was collected from Haut Languedoc, France (water pH 5.9, dissolved organic carbon 2.4 ppm, sediment texture sand (US and BBA), pH 6.1, organic carbon 0.8 %). A second sediment/water system was collected from the Altogarda region of Italy, (water pH 8.2, dissolved organic carbon 1.5 ppm, sediment texture silt loam (US) or sandy silt (BBA), pH 7.9, organic carbon 12.0 %). The third system was collected from North Dakota, USA (water pH 7.9, dissolved organic carbon 37.2 ppm, sediment texture sandy loam (US) or silty sand (BBA), pH 8.1, organic carbon 6.2).

Samples were incubated in the dark at 20 °C for up to 101 days after treatment. Test material was applied at a rate of approximately 0.04 mg a.i./L to the aqueous phase. Approximately 6 cm of water (~25 mL) was added to 2 - 2.5 cm sediment (~15 g wet weight) in a centrifuge tube, resulting in a wet sediment/water ratio of approximately 3:5. Each sample consisted of a 45-mL glass centrifuge tube, containing the sediment/water, attached to a second tube containing approximately 20 mL of 0.2 N NaOH for the collection of 14CO₂. A vacuum pulled CO₂-scrubbed moist air through the test system to maintain aerobic conditions. Samples were analysed at 0, 3, 7, 14, 21, 30, 62 and 101 days of incubation. The water samples were analysed directly and the sediment samples were extracted with 90:10 acetone:1.0 N HCl. A portion of the organic extracts were concentrated before HPLC analysis. Test material residues were assayed by LSC and analysed by HPLC. No metabolite identification efforts were utilised.

The total material balance in the French water/sediment system was 98.9 ± 2.1 % of the applied amount. The amount of radioactivity in the water phase decreased from over 90 % at 0 DAT to about 50 % at 101 DAT. Extractable 14C residues in the sediment increased from about 2.5 % at o DAT to about 40 % of the applied at the end of the incubation period. Non-extractable residues the sediment increased from 0 % at 0 DAT to 3 % of the applied amount at study termination. At the end of the study less than 4 % of the applied radioactivity was present as CO₂.

The concentration of test material in water decreased from over 90 % at 0 DAT to about 50 % of the applied amount at study termination. The concentration of test material in the sediment increased from about 2 % at 0 DAT to about 40 % of the applied amount at study termination. The distribution coefficient of test material between the water and sediment phases was less than 0.05 mL/g at 0 DAT and increased to about 1 mL/g at study termination.

No major transformation products were detected. The test material accounted for greater than 95 % of the radioactivity present in most HPLC chromatograms. Unidentified radioactivity during the study was less than 6 % of the applied amount.

The half-life of the test material in the entire French system was 819 days and the dissipation DT50 from the water phase was 127 days. No dissipation rate from the sediment could be determined because the concentration of test material in the sediment had not declined at study termination.

The average material balance in the Italian water/sediment system was 100.4 ± 1.3 % of the applied amount. The amount of radioactivity in the water phase decreased from 100 % at 0 DAT to about 70 % at 101 DAT. Extractable 14C residues in the sediment increased from 1.6 % at 0 DAT to about 15 % of the applied at the end of the incubation period. Non-extractable residues in the sediment increased from 1 % at 0 DAT to 15 % of the applied amount at study termination. At the end of the study less than 2% of the applied radioactivity was present as CO₂.

The concentration of test material in water decreased from 100 % at 0 DAT to about 70 % of the applied amount at study termination. The concentration of test material in the sediment increased from less than 2 % at 0 DAT to about 15 % of the applied amount at study termination. The distribution coefficient of the test material between the water and sediment phases was less than 0.05 mL/g at 0 DAT and increased to about 0.5 mL/g at study termination.

Non-extractable residues were the only major transformation products detected. The test material accounted for more than 97 % of the radioactivity present in each HPLC chromatogram. Unidentified radioactivity during the study was less than 3 % of the applied amount.

The half-life of the test material in the entire Italian system was 458 days and the dissipation DT50 from the water phase was 243 days. No dissipation rate from the sediment could be determined because the concentration of test material in the sediment had not declined at study termination.

The total material balance in the North Dakota water/sediment system was 100.5 ± 1.2 % of the applied amount. The amount of radioactivity in the water phase decreased from 97% at 0 DAT to about 80% at 101 DAT. Extractable 14C residues in the sediment increased from 2 % at 0 DAT to about 12 % of the applied at the end of the incubation period. Non-extractable residues in the sediment increased from 1 % at 0 DAT to 7 % of the applied amount at study termination. At the end of the study less than 2 % of the applied radioactivity was present at CO₂.

The concentration of test material in water decreased from 95 % at 0 DAT to about 80 % of the applied amount at study termination. The concentration of test material in the sediment increased from 2 % at 0 DAT to about 12 % of the applied amount at study termination. The distribution coefficient of the test material between the water and sediment phases was about 0.05 mL/g at 0 DAT and increased to about 0.4 mL/g at study termination.

No major transformation products were detected. The test material accounted for more than 97 % of the radioactivity present in each HPLC chromatogram. Unidentified radioactivity during the study was less than 3 % of the applied amount.

The half-life of the test material in the entire US system was 999 days and the dissipation DT50 from the water phase was 447 days. No dissipation rate from the sediment could be determined because the concentration of test material in the sediment had not declined at study termination.

Aquatic degradation is not a significant route of degradation for the test material. Metabolism was slow, with half-lives greater than 450 days. A small amount (less than 5%) of CO₂ was generated. Non-extractable residues were the primary degradation products observed. The test material moved slowly from the aqueous to sediment layer. While distribution coefficients between the sediment and water were small, a 10 to 20 fold increase was observed over time.

Description of key information

Anaerobic metabolism is not a major route of degradation for the test material, EPA Subdivision N Pesticide Guideline 162-3, SETAC Section 8.1, Canada PMRA DACO Number 8.2.3.5.6, Rutherford & Meitl (2004).
Aquatic degradation is not a significant route of degradation for the test material. Metabolism was slow, with half-lives greater than 450 days. A small amount (less than 5 %) of CO₂ was generated. Non-extractable residues were the primary degradation products observed. The test material moved slowly from the aqueous to sediment layer. While distribution coefficients between the sediment and water were small, a 10 to 20 fold increase was observed over time, EPA Subdivision N Pesticide Guideline OPP 162-4, EC Directive 91/414/EEC BBA Part IV, Canada PMRA DACO Number 8.2.3.5.4,  Yoder & Smith (2003).

Key value for chemical safety assessment

Half-life in freshwater:

447 d

at the temperature of:

20 °C

Additional information

Two studies investigating the aquatic degradation of the test material are available. Both studies were conducted under GLP conditions and in accordance with standardised guidelines. Both studies were therefore assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The first study (reported by Rutherford & Meitl (2004)) was conducted under anaerobic conditions while the second study (reported by Yoder & Smith (2003)) was conducted under aerobic conditions. Each is summarised below.

In the first study, reported by Rutherford & Meitl (2004), the aquatic degradation of the test material was investigated under anaerobic conditions in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA Subdivision N Pesticide Guideline 162-3, SETAC Section 8.1, and Canada PMRA DACO Number 8.2.3.5.6.

During the study the anaerobic biotransformation of radiolabelled test material was studied in a pond water sediment system (water pH 7.9, dissolved organic carbon 37.2 ppm, sediment texture Sandy Loam, pH 8.1, organic carbon 4.9) from North Dakota, (USA) for 363 days in the dark at 25 °C. Test material was applied at the rate of 0.08 mg a.i./L. The sediment/water ratio used was 1:4.

The anaerobic biotransformation of radiolabelled test material was also studied in a flooded soil system using a Cuckney soil from Bedfordshire (England) (soil texture Sand, pH 6.0, organic carbon 1.3) and HPLC grade water for 120 days in the dark at 20 °C. Test material was applied at the rate of 0.08 mg a.i./L. The soil/water ratio used was 1:2.

The test system consisted of two-chambered biometer flasks with traps for the collection of CO₂. Cuckney soil samples were analysed at 0, 3, 10, 20, 30, 59, and 120 days of incubation. North Dakota sediment samples were analysed at 0, 10, 20, 30, 90, 181, 268, and 363 days of incubation.

Aliquots of the water were directly analysed by LSC and HPLC. The Cuckney soil samples were extracted on a horizontal shaker at low speed with an acetone:1.0 N HCl (90:10, v:v) solution. The North Dakota sediment samples were extracted on a horizontal shaker at low speed with a methanol:l.0 N NaOH (90:10, v:v) solution. Test material residues were analysed by LSC and HPLC.

The test conditions were maintained throughout the study. The total material balance in the North Dakota water/sediment system was 97.9 ± 1.6 % of the applied radioactivity. The mean total recovery of the radiolabelled material was 64.3 ± 2.9 % and 31.5 ± 2.7 % of the applied radioactivity in the water and sediment, respectively. Extractable 14C residues in the sediment increased from 27.4 % at Day 0 to 36.9 % of the applied radioactivity at the end of the incubation period. Non-extractable l4C residues in the sediment increased from 0.9 % at Day 0 to 1.2 % of the applied radioactivity at study termination. At the end of the study 0.6 % of the applied radioactivity was present as CO₂.

The concentration of test material in North Dakota water decreased from 70.6 % at Day 0 to 61.8 % of the applied radioactivity at study termination. The concentration of test material in the sediment increased from 27.3 % at Day 0 to 36.9 % of the applied radioactivity at the end of the study period.

The test material was stable in the North Dakota water/sediment system throughout the study. A t-test showed that the slope of the degradation curve was no different than zero; therefore, no further kinetics calculations were performed. For the flooded Cuckney soil system, the total material balance was 95.5 ± 2.8 % of the applied radioactivity. The mean total recovery of the radiolabelled material was 66.1 ± 4.5 % and 28.4 ± 3.8 % of the applied radioactivity in the water and soil, respectively. Extractable 14C residues in the soil decreased from 30.0 % at Day 0 to 21.5 of the applied radioactivity at the end of the incubation period. Non-extractable 14C residues in the soil ranged from 0.5 to 1.3 % of the applied radioactivity throughout the study. At the end of the study 0.4 % of the applied radioactivity was present as CO₂.

The concentration of test material in water increased from 69.2 % at Day 0 to 71.7 % of the applied radioactivity at study termination. The concentration of test material in the sediment decreased from 30.0 % at Day 0 to 21.5 % of the applied radioactivity at the end of the study period.

The test material was stable in the flooded Cuckney soil system throughout the study. A t-test showed that the slope of the degradation curve was no different than zero; therefore, no further kinetics calculations were performed.

Anaerobic metabolism is therefore not a major route of degradation for the test material.

In the second study, reported by Yoder & Smith (2003), the degradation of the test material under aerobic conditions was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA Subdivision N Pesticide Guideline OPP 162-4, EC Directive 91/414/EEC BBA Part IV, and Canada PMRA DACO Number 8.2.3.5.4.

During the study the aerobic biotransformation of the test material was studied in 3 pond water/sediment systems. One sediment/water system was collected from Haut Languedoc, France (water pH 5.9, dissolved organic carbon 2.4 ppm, sediment texture sand (US and BBA), pH 6.1, organic carbon 0.8 %). A second sediment/water system was collected from the Altogarda region of Italy, (water pH 8.2, dissolved organic carbon 1.5 ppm, sediment texture silt loam (US) or sandy silt (BBA), pH 7.9, organic carbon 12.0 %). The third system was collected from North Dakota, USA (water pH 7.9, dissolved organic carbon 37.2 ppm, sediment texture sandy loam (US) or silty sand (BBA), pH 8.1, organic carbon 6.2).

Samples were incubated in the dark at 20 °C for up to 101 days after treatment. Test material was applied at a rate of approximately 0.04 mg a.i./L to the aqueous phase. Approximately 6 cm of water (~25 mL) was added to 2 - 2.5 cm sediment (~15 g wet weight) in a centrifuge tube, resulting in a wet sediment/water ratio of approximately 3:5. Each sample consisted of a 45-mL glass centrifuge tube, containing the sediment/water, attached to a second tube containing approximately 20 mL of 0.2 N NaOH for the collection of 14CO₂. A vacuum pulled CO₂-scrubbed moist air through the test system to maintain aerobic conditions. Samples were analysed at 0, 3, 7, 14, 21, 30, 62 and 101 days of incubation. The water samples were analysed directly and the sediment samples were extracted with 90:10 acetone:1.0 N HCl. A portion of the organic extracts were concentrated before HPLC analysis. Test material residues were assayed by LSC and analysed by HPLC. No metabolite identification efforts were utilised.

The total material balance in the French water/sediment system was 98.9 ± 2.1 % of the applied amount. The amount of radioactivity in the water phase decreased from over 90 % at 0 DAT to about 50 % at 101 DAT. Extractable 14C residues in the sediment increased from about 2.5 % at o DAT to about 40 % of the applied at the end of the incubation period. Non-extractable residues the sediment increased from 0 % at 0 DAT to 3 % of the applied amount at study termination. At the end of the study less than 4 % of the applied radioactivity was present as CO₂.

The concentration of test material in water decreased from over 90 % at 0 DAT to about 50 % of the applied amount at study termination. The concentration of test material in the sediment increased from about 2 % at 0 DAT to about 40 % of the applied amount at study termination. The distribution coefficient of test material between the water and sediment phases was less than 0.05 mL/g at 0 DAT and increased to about 1 mL/g at study termination.

No major transformation products were detected. The test material accounted for greater than 95 % of the radioactivity present in most HPLC chromatograms. Unidentified radioactivity during the study was less than 6 % of the applied amount.

The half-life of the test material in the entire French system was 819 days and the dissipation DT50 from the water phase was 127 days. No dissipation rate from the sediment could be determined because the concentration of test material in the sediment had not declined at study termination.

The average material balance in the Italian water/sediment system was 100.4 ± 1.3 % of the applied amount. The amount of radioactivity in the water phase decreased from 100 % at 0 DAT to about 70 % at 101 DAT. Extractable 14C residues in the sediment increased from 1.6 % at 0 DAT to about 15 % of the applied at the end of the incubation period. Non-extractable residues in the sediment increased from 1 % at 0 DAT to 15 % of the applied amount at study termination. At the end of the study less than 2% of the applied radioactivity was present as CO₂.

The concentration of test material in water decreased from 100 % at 0 DAT to about 70 % of the applied amount at study termination. The concentration of test material in the sediment increased from less than 2 % at 0 DAT to about 15 % of the applied amount at study termination. The distribution coefficient of the test material between the water and sediment phases was less than 0.05 mL/g at 0 DAT and increased to about 0.5 mL/g at study termination.

Non-extractable residues were the only major transformation products detected. The test material accounted for more than 97 % of the radioactivity present in each HPLC chromatogram. Unidentified radioactivity during the study was less than 3 % of the applied amount.

The half-life of the test material in the entire Italian system was 458 days and the dissipation DT50 from the water phase was 243 days. No dissipation rate from the sediment could be determined because the concentration of test material in the sediment had not declined at study termination.

The total material balance in the North Dakota water/sediment system was 100.5 ± 1.2 % of the applied amount. The amount of radioactivity in the water phase decreased from 97% at 0 DAT to about 80% at 101 DAT. Extractable 14C residues in the sediment increased from 2 % at 0 DAT to about 12 % of the applied at the end of the incubation period. Non-extractable residues in the sediment increased from 1 % at 0 DAT to 7 % of the applied amount at study termination. At the end of the study less than 2 % of the applied radioactivity was present at CO₂.

The concentration of test material in water decreased from 95 % at 0 DAT to about 80 % of the applied amount at study termination. The concentration of test material in the sediment increased from 2 % at 0 DAT to about 12 % of the applied amount at study termination. The distribution coefficient of the test material between the water and sediment phases was about 0.05 mL/g at 0 DAT and increased to about 0.4 mL/g at study termination.

No major transformation products were detected. The test material accounted for more than 97 % of the radioactivity present in each HPLC chromatogram. Unidentified radioactivity during the study was less than 3 % of the applied amount.

The half-life of the test material in the entire US system was 999 days and the dissipation DT50 from the water phase was 447 days. No dissipation rate from the sediment could be determined because the concentration of test material in the sediment had not declined at study termination.

Aquatic degradation is not a significant route of degradation for the test material. Metabolism was slow, with half-lives greater than 450 days. A small amount (less than 5 %) of CO₂ was generated. Non-extractable residues were the primary degradation products observed. The test material moved slowly from the aqueous to sediment layer. While distribution coefficients between the sediment and water were small, a 10 to 20 fold increase was observed over time.