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

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:

26 April 2002 to 13 September 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

Specific details on test material used for the study:

Purity: 94.5%

Oxygen conditions:

aerobic

Inoculum or test system:

other: mixture of activated sludge, secondary effluent, and a soil suspension

Details on inoculum:

- Microbial Inoculum
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 the Microbial Inoculum
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 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. Thirty millilitres 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.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: test solution comprised of mineral medium, microbial inoculum, reagent water, and the appropriate test or reference material additions.
- Test temperature: The temperature of the environmental chamber ranged from 21.8 to 22.3 °C during the 28-day test. The mean and standard deviation of the temperature was 22.0 ± 0.1 °C.
- pH: The pH of the control solutions was 7.52 and 7.53 at study initiation and 7.50 and 7.52 at termination for replicates 1 and 2, respectively. The pH of the test material solutions was 7.51 and 7.49 at study initiation and 7.53 and 7.54 at termination for replicates 1 and 2, respectively. The pH of the reference material system increased from 7.50 at study initiation to 7.66 at study termination.
- pH adjusted: no
- Suspended solids concentration: The suspended solids concentration was 300 mg/L in the prepared activated sludge, 100 mg/L in the secondary effluent and 8000 mg/L in the soil suspension. Therefore, the total concentration of suspended solids in each reaction flask (30 mL of inoculum to 3000 mL of test medium) was 84 mg/L.
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 5-L glass flask (reaction flask) containing a 3-L test solution volume
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: To remove CO₂, the incoming air was passed through a column containing Ascarite followed by a pre-trap containing 500 mL of ~5 N KOH. The air was then passed through 500 mL of reagent water to re-humidify the air, as well as to prevent contamination of the flasks from the KOH pre-trap. The CO₂-free and humidified air was then passed through the reaction flasks.
Air was introduced into each flask by positive pressure, and the flow rates (50 to 100 mL/minute) were measured and adjusted using flow meters.
A Teflon-coated magnetic stir bar was placed in each flask. The flasks were then placed on insulated magnetic stir plates and stirred throughout the duration of the study.
- Details of trap for CO₂: The outlet from each flask was connected to three CO₂ absorber gas-washing traps in series, each filled with 100 mL of 0.2 N KOH solution. These traps captured the CO₂ evolved from the reaction flasks.

SAMPLING - TEST SOLUTION
- Sampling frequency: Approximately one hour after dosing.
- Sampling method and storage before analysis: 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 - 0.2 N KOH TRAPPING SOLUTION
- Sampling frequency: Samples of the KOH solutions were collected for CO₂ analysis on days 3, 5, 7, 10, 14, 19, 24, 28, and 29.
- Sampling method and storage before analysis: 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. The remaining KOH solution in this gas-washing bottle was then discarded and replaced with 100 mL of a fresh 0.2 N KOH solution. The refilled gas-washing bottle was then rotated to the position farthest from the flask, and the other two gas-washing bottles were moved forward (nearer to the flask) one position.

CONTROL AND BLANK SYSTEM
- Control: Duplicate control systems, containing the microbial inoculum with no test or reference material were used to determine the endogenous microbial CO₂ evolution.
- Toxicity control: A reference substance 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.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3.2

Sampling time:

29 d

Remarks on result:

other: Replicate 1

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1.3

Sampling time:

29 d

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 24.7 and 23.4 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 23.1 and 21.8 mg C/L, corresponding to 116 and 109 % of the nominal 20 mg C/L, testing concentration. Acceptable recoveries (70 - 120 %) indicate that those solutions were appropriately prepared. At termination, the DOC concentrations of the test material solutions were 19.3 and 18.4 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 18.6 and 17.7 mg C/L.
The DOC concentration of the reference substance 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 substance 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 substance to the test system. Thus, the contamination appears to have been present only in the aliquot sampled for DOC analysis.

IC CONCENTRATION
At study initiation, the IC concentrations of the control solution 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 0.191 and 0.91 mg C/L for replicates 1 and 2, respectively, corresponding to 1 and 5 % of the nominal initial DOC concentration, 20.0 mg C/L, for replicates 1 and 2, respectively. The IC concentration of the reference substance solution at initiation was 0.526 mg C/L or 3 % of the nominal initial DOC concentration, 20.0 mg C/L. The IC concentrations were within the protocol defined limit (<5 % of the nominal concentration).

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 goal of the control systems was to provide the background CO₂ values from the endogenous CO₂ evolution from the microbial inoculum. 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 3.2 and 1.3 % for replicates 1 and 2, respectively, through day 29 of the study. 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) was 20 and 19 % for replicates 1 and 2, respectively, confirming that the test material was not substantially biodegraded.

MICROBIAL EVALUATION
The microbial evaluation data suggests that the microbial populations in each flask remained active and viable during the course of the study.

Results with reference substance:

The reference substance, 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 substance based on DOC measurements of the reaction solution at initiation (day 0) and termination (day 28) was 100 %, confirming the measured biodegradation from CO2 evolution.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The percent theoretical CO2 produced by the test material was 3.2 and 1.3 % by day 29 of the study for replicates 1 and 2, respectively. Therefore, the test material cannot be classified as readily biodegradable under the conditions of this test. Based on DOC analysis, the percentage biodegradability was 20 and 19 % for replicated 1 and 2, respectively, confirming that the test material was not readily biodegradable. The percent theoretical CO2 produced by the reference substance, sodium benzoate, was 65.8 % ThCO2 by day 3 (90.9 % ThCO2 by day 29), proving that the inoculum was viable.

Executive summary:

The ready biodegradability of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EU Method 301 B.

The primary objective of this study was to measure the extent of biodegradation of the test material exposed to a microbial inoculum in an aerobic, mineral salts medium at an initial test concentration of 20 mg carbon/litre (mg C/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 20 mg C/L, were used to monitor biodegradation of the test material. A reference substance 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 substance systems.

The percent theoretical CO₂ produced by the test material was 3.2 and 1.3 % by day 29 of the study for replicates 1 and 2, respectively. Therefore, the test material cannot be classified as readily biodegradable under the conditions of this test. Based on DOC analysis, the percentage biodegradability was 20 and 19 % for replicated 1 and 2, respectively, confirming that the test material was not readily biodegradable. The percent theoretical CO₂ produced by the reference substance, sodium benzoate, was 65.8 % ThCO₂ by day 3 (90.9 % ThCO₂ by day 29), proving that the inoculum was viable.

Description of key information

Not readily biodegradable, EU Method 301 B, Heim et al. (2002)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EU Method 301 B. The study was awarded a reliability score of 1 in accordance with the criteria of Klimisch et al. (1997).

The primary objective of this study was to measure the extent of biodegradation of the test material exposed to a microbial inoculum in an aerobic, mineral salts medium at an initial test concentration of 20 mg carbon/litre (mg C/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 CO₂evolution. Duplicate inoculated test material systems, which were dosed with the test material at a nominal concentration of 20 mg C/L, were used to monitor biodegradation of the test material. A reference substance 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 CO₂trapped 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 CO₂by inorganic carbon analysis on a total organic carbon analyser. The average CO₂evolved from the control systems was subtracted from the CO₂evolved in the test and reference substance systems.

The percent theoretical CO₂ produced by the test material was 3.2 and 1.3 % by day 29 of the study for replicates 1 and 2, respectively. Therefore, the test material cannot be classified as readily biodegradable under the conditions of this test. Based on DOC analysis, the percentage biodegradability was 20 and 19 % for replicated 1 and 2, respectively, confirming that the test material was not readily biodegradable. The percent theoretical CO₂ produced by the reference substance, sodium benzoate, was 65.8 % ThCO₂ by day 3 (90.9 % ThCO₂ by day 29), proving that the inoculum was viable.