N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June 2002 - 22 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge
The microbial inoculum used in this test was a mixture of activated sludge, secondary effluent, and a soil suspension collected on 6 June 2002. The activated sludge was collected from aeration basin #1 at the Columbia Wastewater Treatment Plant in Columbia, Missouri. Approximately 1.0 L of activated sludge was collected. The secondary effluent was collected from the secondary effluent composite sampler at the Columbia Wastewater Treatment Plant in Columbia, Missouri. The Columbia Wastewater Treatment Plant treats predominately domestic sewage. Approximately 0.5 L of secondary effluent was collected. The soil for the soil suspension was collected from an undisturbed meadow at the testing facility.

- Preparation of inoculum for exposure:
The activated sludge was homogenised in a blender for two minutes. The homogenised sludge was allowed to settle for 30 minutes, filtered through glass wool, and then aerated with compressed air until use. The secondary effluent was filtered through glass wool, and then aerated with compressed air until use. The soil suspension was prepared by diluting 101.58 g of soil to 1.0 L with testing facility reagent water. The suspension was stirred for approximately 30 minutes, allowed to settle for approximately one hour, filtered through glass wool, and then aerated with compressed air until use. 30 mL each of the prepared activated sludge, secondary effluent, and soil suspension were used as the inoculum for each reaction flask.

Duration of test (contact time):

29 d

Initial conc.:

10 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: standard
- Test temperature: 22.0 ± 0.1 °C
- pH: 7.52 to 7.55
- pH adjusted: no
- Aeration of dilution water: Stirring and aeration with CO₂-free air of mineral salts medium and activated sludge took place overnight prior to test initiation to purge-the systems of CO₂ before initiation of the test (dosing on day 0). The remaining mineral salts medium was also aerated with CO₂-free air for this amount of time to prevent absorption of atmospheric carbon by the solution prior to dosing.
- Suspended solids concentration: The total concentration of suspended solids in each reaction flask (30 mL of inoculum to 3000 mL of test medium) was 84 mg/L.

TEST SYSTEM
- Culturing apparatus: 5 L carboys. Each 5-L flask received 2400 mL of mineral salts medium and 30 mL each of the prepared activated sludge, secondary effluent and soil suspension and was aerated overnight. Duplicate test material systems were prepared by adding 300 mL of mineral medium, 210 mL of reagent water, and 79.5 mg of the test material.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: After all additions, each of the reaction flasks was connected to a series of three gas-washing bottles containing 100 mL of 0.2 N KOH. Aeration and stirring of the flasks were continued. Flow meters connected to the test systems were adjusted to facilitate air flow at 50-100 mL/min. The bubbling of air and stirring in each flask, as well as the bubbling in each gas-washing bottle, confirmed the constant aeration.
- Details of trap for CO₂: The CO₂ produced in the test systems was trapped in 0.2 N KOH solutions, which were then analysed for inorganic carbon (IC) content.

SAMPLING
- Initial sampling of test solutions
Approximately one hour after dosing, the pH of each test solution was measured, and 170 mL of each test solution was removed and filtered through 0.45 µm nylon syringe filters. Each filtrate was deposited in a 4 oz. amber bottle and three 14-mL autosampler vials. The bottles and vials were filled leaving no headspace, capped, and stored refrigerated until analysis. Filtrates in the 4 oz. amber bottles were analysed for dissolved organic carbon (DOC) concentration. Filtrates in the first replicate of the three 14-mL vials were analysed for inorganic carbon (IC) concentration; and the other two replicates were held as reserve samples.

- Sampling of 0.2 N KOH trapping solutions
Samples of the KOH solutions were collected for CO₂ analysis on days 3, 5, 7, 10, 14, 19, 24, 28, and 29. For each sample day (except 29), triplicate aliquots of the KOH solution from the gas-washing bottle nearest each flask were placed into appropriately-labelled glass autosampler vials. The vials were filled leaving no headspace, capped using Teflon septa, and stored at room temperature until analysis. The first replicate of each set of triplicate samples was analysed for IC content and the other two were used as reserve samples.

- Termination
After sampling the KOH solutions on day 28, 70 mL of each test solution was removed and 60 mL was filtered through 0.45-µm nylon syringe filters. Each filtrate was deposited in a 2 oz. amber bottle. The bottles and vials were filled leaving no headspace, capped, and stored refrigerated until analysis. Filtrates in the 2 oz. amber bottles were analysed for dissolved organic carbon (DOC) concentration. The remaining 10 mL of each test solution that was removed was used for microbial evaluation, and the pH of each test solution was also measured. After sampling the test solutions, 1 mL of concentrated HCI was added to each test solution to drive carbonates and the remaining CO₂ from solution. The flasks were then re-sealed and allowed to aerate overnight. On day 29, triplicate aliquots of each gas-washing bottle were taken for IC analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate control systems were prepared by adding 300 mL of mineral medium and 210 mL of reagent water to 5 L carboys (final volume 3000 mL).
- Toxicity blank: Bacterial plate counts were performed at initiation for the activated sludge, secondary effluent and soil suspension and on day 28 for each test solution.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0.2

Sampling time:

29 d

Remarks on result:

other: replicate 1

Key result

Parameter:

% degradation (CO2 evolution)

Value:

4

Sampling time:

29 h

Remarks on result:

other: replicate 2

Details on results:

DOC CONCENTRATION
At study initiation, the DOC concentration of the control solutions was 1.72 and 1.47 mg C/L for replicates 1 and 2, respectively, and the average was 1.60 mg C/L. At study termination, the DOC concentration of the control solutions was 0.74 and 0.71 mg C/L for replicates 1 and 2, respectively, and the average was 0.73 mg C/L.
The DOC concentrations of the test material solutions at initiation were 1.92 and 1.78 mg C/L for replicates 1 and 2, respectively. Correcting for the mean DOC concentration measured in the control treatment (1.60 mg C/L), DOC concentrations were 0.33 and 0.19 mg C/L, corresponding to 3 and 2 % of the nominal 20 mg C/L testing concentration. The low recoveries confirm that the water solubility of the test material (0.194 mg/L) was lower than the test concentration (10 mg C/L or 25.9 mg/L). At termination, the DOC concentrations of the test material solutions were 1.29 and 1.03 mg C/L for replicates 1 and 2, respectively. Correcting for the mean DOC concentration measured in the control treatment at termination (0.73 mg C/L), DOC concentrations were 0.57 and 0.31 mg C/L.

The DOC concentration of the reagent water used in this test was 0.81 mg C/L.

IC CONCENTRATION
At study initiation, the IC concentrations of the control solutions were 0.60 and 0.252 mg C/L for replicates 1 and 2, respectively. The IC concentrations of the test material solutions at initiation were 0.218 and 0.167 mg C/L for replicates 1 and 2, respectively, corresponding to 2 % and 2 % of the nominal initial DOC concentration, 10.0 mg C/L, for replicates 1 and 2, respectively. The IC concentrations were within the protocol defined limit (<5 % of the nominal concentration).

MICROBIAL EVALUATION
The bacterial plate counts at study initiation showed that the prepared activated sludge, secondary effluent, and soil suspension contained 3.4 x 10⁶, 1.4 x 10⁵ and 4.7 x 10⁵ CFU/mL, respectively. The results of bacterial plate counts at study termination showed that the control treatment solutions contained 1.5 x 10⁵ and 7.6 x 10⁴ CFU/mL for replicates 1 and 2, respectively. The test material treatment contained 1.1 x 10⁵ and 6.8 x 10⁴ CFU/mL for replicates 1 and 2, respectively, indicating that the test material was not toxic to the microbial inoculum at the testing concentration. The reference material treatment contained 2.2 x 10⁵ CFU/mL. This microbial evaluation data suggests that the microbial populations in each flask remained active and viable during the course of the study.

BIODEGRADATION RESULTS
- Endogenous CO₂ Evolution from the Control Systems
CO₂ evolved from the control systems was 39.8 and 27.3 mg CO₂ for replicates 1 and 2, respectively, by day 29 of the study. These values were corrected for the background CO₂ present in the fresh KOH solutions. The total mg CO₂ evolved from the control flasks (39.8 and 27.3 mg CO₂/flask) was within the limits indicated in the protocol (<70 mg CO₂/L or <210 mg CO₂/flask).

- Biodegradation of the Test Material
The test material exhibited final % ThCO₂ values (after correction for background CO₂ from the control treatment) of 0.2 and 4.0 % for replicates 1 and 2, respectively, through day 29 of the study (Table 1). The % ThCO₂ values for the test material solutions did not reach 60 %ThCO₂. Therefore, the test material cannot be classified as readily biodegradable according to the criteria outlined in the testing guideline (60 % ThCO₂ within a 10-day window after reaching 10 % ThCO₂).
Biodegradation of the test material based on DOC measurements of the reaction solutions at initiation (day 0) and termination (day 28) could not be determined because the water solubility of the test material was less than the testing concentration.

Results with reference substance:

The DOC concentration of the reference material solution at initiation was 27.1 mg C/L. Correcting for the mean DOC concentration measured in the control treatment (1.60 mg C/L), the DOC concentration was 25.5 mg C/L, corresponding to 128 % of the nominal 20 mg C/L testing concentration. The recovery result was higher than the normally acceptable range (70 - 120 %), indicating that the test solution or the sample collected for DOC analysis may have been contaminated or that excess reference material may have been added to the test system. However, the percent biodegradation measured from CO₂ production indicated that the high DOC concentration at initiation was not caused by contamination of the test solution or by addition of excess reference material to the test system. Thus, the contamination appears to have been present only in the aliquot sampled for DOC analyses.
At termination, the DOC concentration of the reference material solution was 0.77 mg C/L. Correcting for the mean DOC concentration measured in the control treatment at termination (0.73 mg C/L), the DOC concentration was 0.05 mg C/L.

The IC concentration of the reference material solution at initiation was 0.526 mg C/L or 3 % of the nominal initial DOC concentration, 20.0 mg C/L.

The reference material, sodium benzoate, exhibited a % ThCO₂ value of 90.9 % by day 29 of the study. The results from day 3 (65.8 % ThCO₂ evolved) indicated greater than 60 % ThCO₂ evolved in the first 3 days of the test. These results indicate that the inoculum was viable according to the criteria outlined in the applicable testing guideline.
Biodegradation of the reference material based on DOC measurements of the reaction solution at initiation (day 0) and termination (day 28) was 100 %, confirming the measured biodegradation front CO₂ evolution.

Table 1: Percent Theoretical CO₂ Produced from the Test Material

Day	Test System Replicate	Net mg C*	Cumulative mg C	% ThCO₂**
3	1 2	ND 0.59	0.00 0.59	0.0 2.1
5	1 2	ND ND	0.00 0.59	0.0 2.1
7	1 2	ND ND	0.00 0.59	0.0 2.1
10	1 2	ND ND	0.00 0.59	0.0 2.1
14	1 2	ND 0.09	0.00 0.68	0.0 2.4
19	1 2	ND 0.24	0.00 0.92	0.0 3.2
24	1 2	ND ND	0.00 0.92	0.0 3.2
28	1 2	ND 0.14	0.00 1.05	0.0 3.7
29 (Trap 1)	1 2	0.05 ND	0.05 1.05	0.2 3.7
29 (Trap 2)	1 2	ND 0.076	0.05 1.13	0.2 4.0
29 (Trap 3)	1 2	ND ND	0.05 1.13	0.2 4.0

* Corrected for the average CO₂ evolution in the control flasks

** % ThCO₂ = (Cumulative mg C / 28.3 mgC) x 100

ND = Not detected - net trapped carbon was less than zero

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The %ThCO₂ produced by the test material was 0.2 and 4.0 % by day 29 of the study. Therefore the test material cannot be classified as readily biodegradable under the conditions of this test.

Executive summary:

A study was conducted to investigate the potential of the test material to undergo biodegradation in accordance with the standardised guideline OECD 301B under GLP conditions.

The primary objective of the study was to measure the extent of biodegradation of the test material (via CO₂ evolution) when exposed to a microbial inoculum in an aerobic, mineral salts medium at an initial test concentration of 10 mg carbon/L. The inoculum was composed of activated sludge, secondary effluent, and a soil suspension. Duplicate control systems, containing the microbial inoculum with no test or reference material, were used to determine the endogenous microbial CO₂ evolution. Duplicate inoculated test material systems, which were dosed with the test material at a nominal concentration of 10 mg C/L, were used to monitor biodegradation of the test material. A reference material system containing readily biodegradable sodium benzoate at a nominal concentration of 20 mg C/L was also tested to verify the viability of the microbial inoculum. All systems were sampled for CO₂ trapped in 0.2 N KOH on days 3, 5, 7, 10, 14, 19, 24, 28, and 29. The 0.2 N KOH trapping solutions were analysed for CO₂ by inorganic carbon analysis on a total organic carbon analyser. The average CO₂ evolved from the control systems was subtracted from the CO₂ evolved in the test and reference material systems.

Biodegradation in the reference material system reached 90.9 % ThCO₂ by day 29 of the study (65.8 % ThCO₂ evolved by day 3), verifying that the microbial inoculum was viable and active.

The test material systems yielded theoretical CO₂ (ThCO₂) values of 0.2 and 4.0 % for replicates 1 and 2, respectively, by day 29 of the study. Therefore, the test material cannot be classified as readily biodegradable according to the criteria outlined in the testing guideline.

Description of key information

The test material systems yielded theoretical CO₂ (ThCO₂) values of 0.2 and 4.0 % for replicates 1 and 2, respectively, by day 29 of the study and is therefore not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

A study was conducted to investigate the potential of the test material to undergo biodegradation in accordance with the standardised guideline OECD 301B under GLP conditions. The study was awarded a reliability score of 1 in line with the criteria set forth by Klimisch et al. (1997). 

The primary objective of the study was to measure the extent of biodegradation of the test material (via CO₂ evolution) when exposed to a microbial inoculum in an aerobic, mineral salts medium at an initial test concentration of 10 mg carbon/L. The inoculum was composed of activated sludge, secondary effluent, and a soil suspension. Duplicate control systems, containing the microbial inoculum with no test or reference material, were used to determine the endogenous microbial CO₂ evolution. Duplicate inoculated test material systems, which were dosed with the test material at a nominal concentration of 10 mg C/L, were used to monitor biodegradation of the test material. A reference material system containing readily biodegradable sodium benzoate at a nominal concentration of 20 mg C/L was also tested to verify the viability of the microbial inoculum. All systems were sampled for CO₂ trapped in 0.2 N KOH on days 3, 5, 7, 10, 14, 19, 24, 28, and 29. The 0.2 N KOH trapping solutions were analysed for CO₂ by inorganic carbon analysis on a total organic carbon analyser. The average CO₂ evolved from the control systems was subtracted from the CO₂ evolved in the test and reference material systems.

Biodegradation in the reference material system reached 90.9 % ThCO₂ by day 29 of the study (65.8 % ThCO₂ evolved by day 3), verifying that the microbial inoculum was viable and active.

The test material systems yielded theoretical CO₂ (ThCO₂) values of 0.2 and 4.0 % for replicates 1 and 2, respectively, by day 29 of the study. Therefore, the test material cannot be classified as readily biodegradable according to the criteria outlined in the testing guideline.