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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:
key study
Study period:
From 20th April 1999 to 29th July 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge from the sewage plant at Waldems- Esch was used.
- Preparation of inoculum for exposure: The sample of activated sludge was washed twice with the mineral nutrient solution used in this test to eliminate organic components and carbonates from the sludge. After resuspension in the mineral nutrient medium the sludge was aerated by means of compressed humidified air for about four hours. Before use as an inoculum for the CO2 evolution test, the sludge was homogenized in a "Waring Blender" at low speed for 2 minutes and then filtered through a cotton filter which had been carefully rinsed with deionized water. The first 200 mL of filtrate were discarded. The filtrate was used as inoculum (1% of the final volume of the test solution) on the same day of preparation.
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
other: TOC
Initial conc.:
16 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: As described in the Guideline.
- Test temperature: 18.3-21.3 ºC

TEST SYSTEM
- Culturing apparatus: 5-litre amber carboys served as test vessels
- Number of culture flasks/concentration: two flasks for the test substance
- Method used to create aerobic conditions: Before starting, the mineral nutrient solution with inoculum but without any test component were aerated for 24 hours with CO2-free compressed air.
- Measuring equipment: The gas outlet (exit air line) was connected to three CO2 absorber bottles each filled with 100 mL 0.025 N Ba(OH)2 and bubbling of CO2 free compressed air through the solution was continued.
- Test performed in closed vessels: The vessels were closed with stoppers with tubing for gas inlet and outlet.
- Details of trap for CO2 and volatile organics if used: CO2 generated by the test substance was trapped by 0.025 N Ba(OH)2 in a trap system described in the Guideline. Remaining Ba(OH)2 (0.025 N) in the CO2 trap removed from the trap system was titrated with HCl (0.05 N) using phenolphthalein as an indicator.

SAMPLING
- Sampling frequency: Titrations were performed at day 2, 5, 9, 13, 16, 23, 28 and 30.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Two test solutions were prepared in the same way as the test solutions with the test substance, but without the addition of any test or control substance.
- Toxicity control: One test solution containing 120 mg Na-benzoate plus 88 mg test substance/3.5 L of test solution.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
59.4
Sampling time:
28 d
Remarks on result:
other: Test solution with 15 mg C/L
Parameter:
% degradation (CO2 evolution)
Value:
63.3
Sampling time:
28 d
Remarks on result:
other: Test solution with 16 mg C/L
Details on results:
The final degradation value after the test period of 28 days was 61% (mean value of two parallel test solutions).
The toxicity control was degraded 70% within 28 days and shows that no toxicity of the test substance has reduced the activity of the inoculum.
The CO2 evolution measured in the Blank was in the required range.
The reference substance was degraded 88% within 28 days. The threshold of 60% was met within 5 days.

Table 1: Results of testing biodegradability: Test item 15 mg C/L

 

Time

mg CO2 generated by the test substance, cumulative net values after deduction of the blank values

% TCO2

(% biodegradation)

2d

10.31

5.4

5d

45.85

23.9

9d

72.94

38.0

13d

79.83

41.5

16d

87.84

45.7

23d

101.73

52.9

28d

114.08

59.4

30d

123.69

64.4

 

Table 2: Results of testing biodegradability: Test item 16 mg C/L

 

Time

mg CO2 generated by the test substance, cumulative net values after deduction of the blank values

% TCO2

(% biodegradation)

2d

10.60

5.2

5d

48.79

24.1

9d

77.82

38.4

13d

87.39

43.1

16d

99.74

49.2

23d

114.85

56.6

28d

128.45

63.3

30d

140.81

69.4

Validity criteria fulfilled:
yes
Remarks:
(Total CO2 evolution in blank was in the required range; degradation of reference substance reached pass level by day 5; the toxicity control was degraded 70% within 28 days; difference of extremes of replicate values of test item was < 20 %).
Interpretation of results:
readily biodegradable
Conclusions:
Calculated from the organic content of the test substance and the measured CO2 generation 59% and 63% of the theoretical CO2 (ThCO2) had been generated by the test substance within 28 days. From these data obtained the test substance is regarded as "readily biodegradable".
Executive summary:

The test substance was tested for ready biodegradability according to OECD 301B, CO2 evolution Test. Calculated from the organic content of the test substance and the measured CO2 generation 59% and 63% of the theoretical CO2 (ThCO2) had been generated by the test substance within 28 days. From these data obtained the test substance is regarded as "readily biodegradable".

The control substance Na-benzoate was degraded by 88% within 28 days. The threshold of "ready biodegradability" of 60% was met within 5 days of incubation (66%). Therefore, the test is considered to be valid.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO 11734
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 311 (Anaerobic Biodegradability of Organic Compounds in Digested Sludge: Measurement of Gas Production)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
anaerobic
Inoculum or test system:
anaerobic sludge
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Anaerobic digester of the municipal waste water treatment plant of the city of D-31137 Hildesheim
- Pretreatment: Homogenisation, centrifugation and washing with dilution medium according to ISO 11734. The prepared sludge was stored 2 days until the start of the test at 35 ºC for pre-digestion. The content of total solids was 2.5 g/L.
Duration of test (contact time):
57 d
Initial conc.:
173 mg/L
Based on:
test mat.
Initial conc.:
100 other: mgC/L
Based on:
other: C
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
CH4 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral nutrient solution according to ISO 11734 mixed with anaerobic sludge. Reagent grade chemicals were used for preparations.
- Test temperature: Nominally 35 ± 2 ºC, constantly kept in a climate chamber.
- Suspended solids concentration: The content of total solids was adjusted to 2.41-2.55 g/L.

TEST SYSTEM
- Culturing apparatus: Erlenmeyer flasks (vol. 1000 mL) with scaled glass tubes for measuring of gas volume (volume 50 mL, scale 1 ± 0.1 mL).
- Number of culture flasks/concentration: Three test item replicates
- Method used to create anaerobic conditions: After application of the test and reference item the headspace was flushed with nitrogen to remove atmospheric oxygen.
- Measuring equipment: Carbon analyser
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: The intervals of biogas measurement depended on the course of the biogas production. Daily measurements were carried out when maximum gas production was observed. The TOC of the test medium was carried out at test begin. The determination of TIC in the test medium was carried out with a carbon analyser at end of the study. Total solids and pH value of the sludge were determined at the beginning and at the end of the study.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Six replicates. Control replicates with anaerobic sludge and nutrient solution according to ISO 11734 were used.
- Toxicity control: Two inhibition control replicates receiving both test and reference item to check for test item related toxic effects.
Reference substance:
other: Polyethylene glycol 400 (CAS number 25322-68-3)
Parameter:
other: CO2 and CH4 evolution
Value:
60 - 70
Sampling time:
57 d
Details on results:
The gas production started within one day after application. After 30 days the plateau phase was reached. The anaerobic degradation of the test item reached a level of 60-70% after 57 days.

In the inhibition control containing both test and reference item 60-70% degradation occurred within 57 days. Since the gas production curve of the inhibition control was comparable to the functional control it is considered that the degradation of the reference item is not inhibited by the test item. Only one replicate was included in calculations because the scaled glass tube of the second replicate was leaking.
Results with reference substance:
In order to check the activity of the anaerobic sludge polyethylene glycole 400 was used as functional control. The reference item was degraded in a range of 80-90% after 57 days. A typical degradation curve was observed.

Table 1: Total biodegradation after 57 days – Carbon of the headspace and the liquid

(mean values: test item: three replicates; control: six replicates; inhibition control: one replicate)

 

 

Theoretical carbon content CV(mmol)

Net carbon in the headspace CH

(mmol)

Net carbon content in the liquid CL (mmol)

Total gasified carbon CT (mmol)

Total biodegradation

 

 

DTvalue (%)

Range (%)

Reference item

100 mgC/L

8.33

4.62

2.87

7.49

89

80-90

Test item

100 mgC/L

8.33

3.93

1.95

5.88

70

60-70

Inhibition control

200 mgC/L

16.67

6.03

4.58

10.61

63

60-70

Validity criteria fulfilled:
yes
Remarks:
(all replicates were under anaerobic conditions; the percentage degradation of reference item was > 60%; pH at the end of test was in the acceptable range; gas production of the test and reference item replicates was not lower than that of the control)
Interpretation of results:
other: biodegradable under anaerobic conditions
Conclusions:
In this study, the test substance was found to be biodegradable under anaerobic conditions in the range of 60-70% in a period of 57 days. In the tested concentration of 100 mgC/L (corresponding to 173 mg/L) the test item was not toxic to anaerobic sludge under test conditions.
Executive summary:

The ultimate anaerobic biodegradability was determined for the test item over a period of 57 days. The study was conducted according to ISO 11734.

Anaerobic not adapted sludge was used as test system. Content of total solids was 2.5 g/L. Three replicates were tested for test item and functional control and six replicates for inoculum control. Additionally, two inhibition control replicates were tested. Each replicate was tested with a volume of 1000 mL.

The test item concentration was 100 mgC/L. The test item was clearly dissolved at the test concentration. The test vessels were incubated at 35 ± 2 ºC.

The biodegradation was followed by the gas production of carbon dioxide and methane. The gas production was measured volumetrically.

In order to check the activity of the anaerobic sludge polyethylene glycole 400 was used as functional control at a concentration level of 100 mgC/L. It was degraded to a level of 80 -90% after 57 days.

In the inhibition control containing both test and reference item 60 -70% degradation occurred within 57 days. The degradation of the reference item seemed not to be inhibited by the test item.

The anaerobic degradation of the test item reached a level of 60 -70% after 57 days.

The study met the validity criteria of the test guideline.

Description of key information

Key study: Experimental results: OECD guideline 301B. GLP study.
Calculated from the organic content of the test substance and the measured CO2 generation 59% and 63% of the theoretical CO2 (ThCO2) had been generated by the test substance within 28 days. From these data obtained the test substance is regarded as "readily biodegradable".

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Key study: Experimental results: OECD guideline 301B. GLP study.

Calculated from the organic content of the test substance and the measured CO2 generation 59% and 63% of the theoretical CO2 (ThCO2) had been generated by the test substance within 28 days. From these data obtained the test substance is regarded as "readily biodegradable".