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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 October to 04 November 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301D with GLP statement. All validity criteria are fulfilled
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: Chimex / op 193- Physical state: Yellow liquid- Date of receipt: 15 July 1992STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Protected from light and at room temperature (between 17 and 29 °C)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Organisms, strains: a mixture of different strains of micro-organisms found in activated sludge.- Source of inoculum/activated sludge: Activated sludge obtained from a municipal sewage treatment plant receiving little or no industrial effluent (from Pierre-Benite, F - 69310 LYON).- Preparation of inoculum for exposure: The supernatant of the activated sludge was aerated from the time of sampling until use; filtration through coarse filter paper; the first 200 mL were discarded; the inoculum was used on the day of sampling.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
5 mg/L
Based on:
test mat.
Initial conc.:
8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS - Composition of medium: The nutrient medium of the CO2 Evolution Test contained per liter of water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4•2H2O, 1.70 mg NH4Cl, 22.5 mg MgSO4•7H2O, 27.5 mg CaCl2 and 0.25 mg FeCl3•6H2O. - Dilution water: Air saturated distilled water, with 10.2 mg of oxygen per litre at 20 °C, containing very little copper (≤ 0.01 mg/L). The distilled water contained: 0.01 mg Cu per litre. - Test medium: consisting in a mixture of nutrient solutions, inoculum and the test substance at the chosen concentration, except the controls (inoculated control and non-inoculated control) without any substance, and the reference where the test substance was replaced by the reference substance. - Test temperature: 21-22 °C - Aeration of dilution water: Yes - The dissolved oxygen concentration was measured in the dilution water (after aeration) and in the test medium (at the beginning of the study). - Continuous darkness: Yes - Other: Theoretical study concentrations 2, 5 and 8 mg/L corresponding to effective study concentrations (used for the calculation of biodegradation) 2.0, 5.2 and 8.2 mg/LThe concentration of 2 mg/L (standard concentration) and the concentrations of 5 and 8 mg/L were used (the test substance had a low Theoretical Oxygen Demand). TEST SYSTEM - Culturing apparatus: preparation of the study: 5 litre graduated flasks; study: 250 to 300 mL calibrated stoppered test flasks. - Number of culture flasks/concentration: 2 flasks/concentration - The solution has been inoculated with micro-organisms, then the test medium was kept in closed bottles, in the dark and at a constant temperature. - Measuring equipment: Analysis of the dissolved oxygen concentration: by electrochemical electrode (OXI 96) EXPERIMENTAL SYSTEM6 assays were performed: - 3 assays with inoculum and test substance at the concentrations of 2, 5 and 8 mg/L - 1 assay with inoculum and reference substance at the concentration of 10 mg/L - 1 inoculated control assay, without any substance - 1 non-inoculated control assay, without any substance MEASUREMENTS AND EXPERIMENTAL ANALYSIS - Measurement of the dissolved oxygen in the dilution water following aeration and in the study medium at the start of the study. - Measurement of the temperature of the dilution water following aeration. - Analysis of the dissolved oxygen concentration (one flask) from each assay (the test substance medium) at the start of the study (time 0). - Analysis of the dissolved oxygen concentration in 2 flasks from each assay (the non-inoculated control and the inoculated control medium) at the start of the study (time 0) and on the 5, 15 and 28th days. CONTROL AND BLANK SYSTEM - Inoculum blank: 2 flasks/concentration - Reference substance: 2 flasks/concentration - Toxicity control: None - Other: An inoculated control assay and a non-inoculated control assay, were involved in the same experimental conditions, without any test substance.OTHERS - Evaluation and calculation of the biotic degradation of the test substance by comparing the consumption of Oxygen under the experimental conditions with the ThOD (Theoretical Oxygen Demand); it was expressed as a percentage.
Reference substance:
acetic acid, sodium salt
Remarks:
10 mg/L
Preliminary study:
None
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
At the concentration of 2 mg of test substance per litre of test medium
Value:
66.7
Sampling time:
28 d
Remarks on result:
other: Nitrogen was assumed to be eliminated as ammoniac
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
At the concentration of 2 mg of test substance per litre of test medium
Value:
62.7
Sampling time:
28 d
Remarks on result:
other: Nitrogen was assumed to be eliminated as nitrate
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
At the concentration of 5 mg of test substance per litre of test medium
Value:
20
Sampling time:
28 d
Remarks on result:
other: Nitrogen was assumed to be eliminated as ammoniac
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
At the concentration of 5 mg of test substance per litre of test medium
Value:
18.8
Sampling time:
28 d
Remarks on result:
other: Nitrogen was assumed to be eliminated as nitrate
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
At the concentration of 8 mg of test substance per litre of test medium
Value:
11.8
Sampling time:
28 d
Remarks on result:
other: Nitrogen was assumed to be eliminated as ammoniac
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
At the concentration of 8 mg of test substance per litre of test medium
Value:
11.1
Sampling time:
28 d
Remarks on result:
other: Nitrogen was assumed to be eliminated as nitrate
Details on results:
- ThOD of the test substance calculated:with nitrogen oxidation in nitrate > 0.877 mg O2/mg of test substance.without nitrogen oxidation (ammoniac) > 0.825 mg O2/mg of test substance.- Temperature of dilution water following aeration: 20 °C- Concentration of dissolved oxygen in the dilution water following aeration: 10.2 mg O2/Lat the start of the study: between 10.0 and 10.3 mg O2/L- The dissolved oxygen depletion in non-inoculated control medium was of 0.3 mg/L at the 5th study day, and 1.4 mg/L at the 28th day.- The dissolved oxygen depletion in inoculated control medium was of 0.35 mg/L at the 5th study day, and 1.35 mg/L at the 28th day.
Key result
Parameter:
ThOD
Value:
> 0.877 other: mg O2/mg of test mat.
Remarks on result:
other: with nitrogen oxidation in nitrate
Key result
Parameter:
ThOD
Value:
> 0.825 other: mg O2/mg of test mat.
Remarks on result:
other: without nitrogen oxidation (ammoniac)
Results with reference substance:
The biodegradation of the reference substance (sodium acetate) was 61.1 % in 28 days.

Table 5.2.1/1: Dissolved oxygen depletion (mg O2/L)

 

Days

Inoculated control*

Test substance*

Reference substance*

2 mg/L

5 mg/L

8 mg/L

0

10.0

10.2**

10.3**

10.2**

10.0**

5

9.65

9.75

9.75

9.65

9.40

15

8.40

8.35

8.35

8.40

7.25

28

8.70

7.80

8.15

8.10

5.80

* 2 flasks/concentration -> mean values

** 1 flask/concentration (time 0: 2 -5 -8 mg/L and reference assay)

 

Table 5.2.1/2: O2 depletion (mg BOD/L) after 5 - 15 and 28 days

 

Test substance concentration

CHIMEXANE CI

O2 depletion (mg BOD/L) after

5 days

15 days

28 days

2 mg/L

0.10

0.25

1.10

5 mg/L

0.20

0.35

0.85

8 mg/L

0.20

0.20

0.80

 

 

Table 5.2.1/3: Biodegradation (%)

 

Days

Biodegradation (%)

A

B

Test substance concentration : 2 mg/L

5

6.1

5.7

15

15.2

14.3

28

66.7

62.7

Test substance concentration : 5 mg/L

5

4.7

4.4

15

8.2

7.7

28

20.0

18.8

Test substance concentration : 8 mg/L

5

3.0

2.8

15

3.0

2.8

28

11.8

11.1

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Remarks:
2 mg/L
Conclusions:
The degree of biodegradation reached 66.7 % (Nitrogen was assumed to be eliminated as ammoniac) and 62.7% (Nitrogen was assumed to be eliminated as nitrate) at 2 mg/L concentration after 28 days. Therefore, the test substance can be considered as “readily biodegradable”.
Executive summary:

A ready biodegradability test was performed according to the OECD Guideline 301D, to determine the biodegradability of the test substance by the Closed Bottle Test.

The test substance was tested using the following concentrations in test medium: the concentration of 2 mg/L (standard concentration) and the concentrations of 5 and 8 mg/L were used (the test substance had a low Theoretical Oxygen Demand). Activated sludge obtained from a municipal sewage treatment plant receiving little or no industrial effluent was used as inoculum. The solution has been inoculated with micro-organisms, then the test medium was kept in closed bottles, in the dark at 21-22 °C for 28 days. An inoculated control assay and a non-inoculated control assay, were involved in the same experimental conditions, without any test substance. Sodium acetate was used as positive control. The dissolved oxygen concentration was measured from each assay at the start of the study (time 0) then on days 5, 15 and 28. The theoretical oxygen demand (ThOD) of the test substance was calculated. The biodegradation of the test substance was evaluated by comparing the oxygen depletion in the test medium with that in the inoculated control assay, and in relation (%) to the total oxygen depletion the test substance could theoretically induced.   

 

All validity criteria were met. The biodegradation of the reference substance (sodium acetate) was 61.1 % in 28 days.

 

ThOD of the test substance calculated: with nitrogen oxidation in nitrate > 0.877 mg O2/mg of test substance; without nitrogen oxidation (ammoniac) > 0.825 mg O2/mg of test substance.

The dissolved oxygen depletion in non-inoculated control medium was of 0.3 mg/L at the 5th study day, and 1.4 mg/L at the 28th day. The dissolved oxygen depletion in inoculated control medium was of 0.35 mg/L at the 5th study day, and 1.35 mg/L at the 28th day.

 

At 2 mg/L, biodegradation of the test substance was 66.7% (Nitrogen was assumed to be eliminated as ammoniac) and 62.7% (Nitrogen was assumed to be eliminated as nitrate).

At 5 mg/L, biodegradation of the test substance was 20.0% (Nitrogen was assumed to be eliminated as ammoniac) and 18.8% (Nitrogen was assumed to be eliminated as nitrate).

At 8 mg/L, biodegradation of the test substance was 11.8% (Nitrogen was assumed to be eliminated as ammoniac) and 11.1% (Nitrogen was assumed to be eliminated as nitrate).

 

The degree of biodegradation reached 66.7 % (Nitrogen was assumed to be eliminated as ammoniac) and 62.7% (Nitrogen was assumed to be eliminated as nitrate) at 2 mg/L concentration after 28 days. Therefore, the test substance can be considered as “readily biodegradable”.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January to 24 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301B with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss GLP Compliance Programme (inspected on 22-26 March and 12-16 April 2010 / signed on 11 January 2011)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: Chimex / 0141653- Expiration date of the lot/batch: 30 June 2011- Purity test date: 28 September 2010STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature at about 20 °C, in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. - Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
28 d
Initial conc.:
75 mg/L
Based on:
test mat.
Remarks:
15.2 mg TOC/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS - Composition of medium: The nutrient medium of the CO2 Evolution Test contained per liter of deionized water: 10 mL of stock solution (8.5 g/L KH2PO4, 21.75 g/L K2HPO4, 33.4 g/L Na2HPO4•2H2O, 0.5 g/L NH4Cl per litre) and 1 mL each of stock solutions (22.5 g/L MgSO4•7H2O, 36.4 g/L CaCl2•2H2O, and 0.25 g/L FeCl3•6H2O) were added to about 800 mL purified water and made up to 1000 mL with purified water. The pH of the final test water was adjusted from 7.1 to 7.4 with a diluted sodium hydroxide solution. - Test temperature: 21-22 °C. - pH: pH measured in all flasks at the end of exposure (Day 28) was between 7.5 and 7.6. - pH adjusted: yes; prior to test start (Day 0), the pH was measured in each test flask after the addition of test and/or reference item and adjusted to 7.4 with a diluted hydrochloric acid solution. - Preparation of Test Flasks: One day before test start (Day -1), between 2400 and 2910 mL of untreated test medium was filled into 5-liter amber glass flasks. To each flask, 90 mL activated sludge inoculum was added. The test media were aerated overnight with CO2-free air to purge the system of carbon dioxide. On the following day (Day 0), defined amounts of the test item were directly added to the test flasks. No emulsifiers or solvents were used. The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL 0.05 M NaOH and the second one containing 200 mL 0.05 M NaOH, were connected in series to the exit air line of each test flask. - Suspended solids concentration: 30 mg dry material per liter - Continuous darkness: yes; test vessels were incubated in a dark room TEST SYSTEM - Culturing apparatus: 5-liter all-glass amber bottles - Number of culture flasks/concentration: 2 - Details of trap for CO2: Air was led through a bottle containing about 750 mL of a 2 M NaOH solution to trap CO2. The CO2-free air was passed through the test solutions at a rate corresponding to about 30–100 mL/min. - Other: The solubility of the test item in test water at a concentration of 75 mg/L was checked in a pre-test without GLP. Based on the appearance, a clear, foaming solution was obtained. However, subsequent TOC/DOC analysis generated scattering values. Therefore, no IC or TC (inorganic or total carbon) was measured in the flasks with the test item at the start of the test. SAMPLINGSampling dates:Test item and inoculum control: Exposure Day 2, 5, 7, 9, 12, 14, 19, 23, 27, 28 and 29*Procedure control: Exposure Day 2, 7, 14, 28 and 29*Toxicity control: Exposure Day 7, 14, 28 and 29*On each sampling day, an aliquot of 5.0 mL was withdrawn from the absorber flask nearest to the test flask for analysis of inorganic carbon (IC). Additional samples for analysis of IC were withdrawn from the second absorber flask of all test vessels on Exposure Day 14 and at the end of the exposure period on Exposure Day 28 in order to correct for any carry over of CO2.After sampling on Exposure Day 28, the pH was measured in each test flask. Next, 1 mL of concentrated HCl was added to each test flask and the flasks were aerated overnight with CO2-free air to drive off any residual CO2 present. On Day 29, a sample from each absorber flask was withdrawn and analyzed for IC to determine residual CO2 that was present in the test suspensions on Exposure Day 28. In this way, any residual CO2 remaining in the test suspensions was determined as the difference between the amounts of IC found before and after acidification.* Exposure Day 29: after driving off residual CO2 CONTROL AND BLANK SYSTEM - Inoculum blank: Yes; duplicate - Procedure control: Yes; duplicate - Toxicity control: Yes; Single - See Table 5.2.1/1 for details
Reference substance:
benzoic acid, sodium salt
Remarks:
25.7 mg/L
Preliminary study:
None
Key result
Parameter:
% degradation (CO2 evolution)
Value:
40
Sampling time:
28 d
Remarks on result:
other: however, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached
Details on results:
Biodegradation of the Test Item:- The percent biodegradation of the test item was calculated based on a measured carbon content of 0.20 mg C/mg test item.- During the first 12 days of the test, the CO2 formation of the test item Chimexane CL in the test media was below the range of the inoculum controls. Starting on about Exposure Day 14, the CO2 formation of Chimexane CL in the test media significantly increased until test termination after 28 days. At the end of the 28-day exposure period, average biodegradation was 40%.- Consequently, Chimexane CL was found to be biodegradable by 40% under the test conditions within 28 days. However, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached.Biodegradation in the Toxicity Control:- The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the measured carbon content of the test item and the total organic carbon content (TOC) of the reference item.- CO2 formation in the toxicity control showed a similar course over the 28-day exposure period as the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation reached 53%. At the end of the test (Exposure Day 28), biodegradation reached 76%.- Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 75 mg/L (equivalent to 22.6 mg a.i./L) because biodegradation in the toxicity control was ≥25% within 14 days of incubation.Driving Off CO2:No significant difference was found between the absolute amounts of IC measured on Exposure Day 28 and the absolute amounts of IC measured after acidification on Day 29. Consequently, no significant amount of residual CO2 was present in the test solutions or suspensions at the end of the test.
Results with reference substance:
The percent biodegradation of the reference item was calculated based on a total organic carbon content (TOC) of 0.58 mg C/mg sodium benzoate.In the procedure controls, average biodegradation of the reference item was 86% by Exposure Day 14, thus confirming suitability of the activated sludge (≥60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 89%.

Table 5.2.1/2: Biodegradation of the Test Item and the Reference Item

Time (days)

% Degradation

Test item

Reference item

Toxicity control1

Replicate No. 1

Replicate No.

Replicate No.

1

2

Mean

1

2

Mean

2

-4.5

 

-4.6

-4.6

46.6

43.1

44.8

-

5

-8.8

-7.6

-8.2

-

-

-

-

7

-11.8

-9.7

-10.7

72.2

73.3

72.7

29.8

9

-13.5

-10.9

-12.2

-

-

-

-

12

-13.7

-10.4

-12.0

-

-

-

-

14

-1.8

7.8

3.0

84.6

88.2

86.4

53.3

19

28.4

31.5

29.9

-

-

-

-

23

34.8

36.8

35.8

-

-

-

-

27

37.7

41.3

39.5

-

-

-

-

28

37.8

42.9

40.4

87.7

90.6

89.1

75.6

--: No samples taken

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Chimexane CL was found to be biodegradable by 40% under the test conditions within the 28-day exposure period. However, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached. Therefore, the test item Chimexane CL was not readily biodegradable under the test conditions within the 28 days.
Executive summary:

A ready biodegradability test was performed according to the OECD Guideline 301B, to determine the biodegradability of the test substance in a 28-Day CO2 Evolution (Modified Sturm) Test

The test substance concentration of 75.0 mg/L is equivalent to 22.5 mg a.i./L was tested in test medium. Activated sludge was used as inoculum (concentration in the test 30.0 mg dry matter/L). Sodium benzoate was used positive control. The test was left running for 28 days.

In the toxicity control, containing both Chimexane CL and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, Chimexane CL had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 75 mg/L (equivalent to 22.6 mg a.i./L).

 

In the procedure controls, average biodegradation of the reference item was 86% by Exposure Day 14, thus confirming suitability of the activated sludge (≥60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 89%.

No significant difference was found between the absolute amounts of IC measured on Exposure Day 28 and the absolute amounts of IC measured after acidification on Day 29. Consequently, no significant amount of residual CO2 was present in the test solutions or suspensions at the end of the test.

The percent biodegradation of the test item was calculated based on a measured carbon content of 0.20 mg C/mg test item. During the first 12 days of the test, the CO2 formation of the test item Chimexane CL in the test media was below the range of the inoculum controls. Starting on about Exposure Day 14, the CO2 formation of Chimexane CL in the test media significantly increased until test termination after 28 days. At the end of the 28-day exposure period, average biodegradation was 40%.

Chimexane CL was found to be biodegradable by 40% under the test conditions within the 28-day exposure period. However, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached.

Therefore, the test item Chimexane CL was not readily biodegradable under the test conditions within the 28 days.

Description of key information

Two experimental study were available:

- A ready biodegradability test was performedaccording to the OECD Guideline 301B, to determine the biodegradability of the test substance in a 28-Day CO2Evolution (Modified Sturm) Test

- A ready biodegradability test was performedaccording to the OECD Guideline 301D, to determine the biodegradability of the test substance by the Closed Bottle Test.

The substance was found to be readily biodegradable in the 301D study from 1993, and not readily biodegradable in the 301B study from 2011. The most conservative result is taken into account and therefore, the substance is not considered as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information