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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August 2013 - 26 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to EC C.4-D and OECD 301F. GLP study.
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw, Poland. The coarse particles were removed by filtration. Such prepared sludge was washed in the medium and was placed in laboratory-scale unit where the aerobic conditions were maintained by means of an intense aeration with compressor and aerators. Inoculum was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge (in mineral medium) at the test temperature of 22ºC. For the test the activated sludge, inoculum, was taken after 2 days from this pre-conditioning.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The double-distilled water was taken from redistillation set. The mineral medium was in accordance with the guideline. Since the test item is poorly water soluble, CNSL was added directly to the mineral medium at the flask preparation stage on a weight basis.
- Test temperature: 21.8 ± 0.2 ºC
- pH: 6.91-7.08
- pH adjusted: no
- Suspended solids concentration: 30 mg/L (concentration on inoculum)

TEST SYSTEM
- Culturing apparatus: Thermo-cabinet WTW TS 606 CZ-G/3-VAR
- Number of culture flasks/concentration: 3
- Measuring equipment:
Closed WTW OxiTop OC 110 respirometer for BOD determination.
Spectrophotometer Hach DR 3900 and 45600 reactor COD for COD determination (potassium dichromate 0-1000 mg/l), according to ISO 15705.
The consumption of oxygen was determined from the change in pressure in the apparatus. The carbon dioxide, evolved during test item degradation, was absorbed in a solution of potassium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of determined COD of the test item.

SAMPLING
The oxygen uptake from the readings taken at regular and frequent intervals was calculated, using the methods given by the manufacturer of the equipment. The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recoded and stored in the measuring heads of the sample bottles.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 replicates, containing only inoculum 30 mg/l SS.
- Procedure control: 3 replicates, containing reference item (sodium acetate 100 mg/l) ) and inoculum 30 mg/SS
- Toxicity control: 3 replicates, containing test item (100 mg/L) and inoculum 30 mg/l SS

STATISTICAL METHODS:
These collected data, using the controller, were infrared read out from the heads and stored in the controller, to be then transferred to a computer. The calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
83.8
Sampling time:
28 d
Details on results:
The recorded measurement temperature didn’t exceed - by 0.7 °C - the expected 22 °C.
The time averaged temperature equals 21.8 ± 0.2 °C.
At the 28th day of the test the aerobic biodegradation of the testing:
- the test item attained 83.8% of biodegradability,
- the reference item reached 92.6% of biodegradability,
- in the toxicity test the biodegradation was equal to 69.3%,
- the oxygen uptake of the inoculum blank was equal to 38.9 mg/l in 28 days,
- the final pH values of all flasks were inside the range 6.91-7.08.
Results with reference substance:
The reference item reached 92.6% of biodegradability on day 28.

Sample oxygen uptake: biodegradability:

Average: 3 flasks

Time (days)

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2 uptate (mg/L)

71.2

113.8

159.2

188.3

213.5

224.9

233.4

239.8

248.1

252.7

256.7

262.8

Blank test O2 uptake (mg/L)

19.1

23.4

25.6

28.4

29.8

32.0

33.0

32.8

34.7

34.6

36.5

38.9

Reference item O2 uptake (mg/L)

65.0

82.0

90.2

95.0

98.9

102.1

105.0

106.8

108.6

108.4

109.4

111.2

Toxicity control O2 uptake (mg/L)

66.8

118.2

148.9

172.4

205.5

226.4

238.8

251.9

263.1

268.1

272.0

278.2

Corrected test item O2 uptake (mg/L)

52.1

90.4

133.6

159.8

183.8

192.9

200.4

207.0

213.4

218.1

219.6

223.8

Reference item % degradation*

58.9

75.2

82.8

85.4

88.6

89.9

92.2

94.9

94.7

94.6

93.4

92.6

Test item % degradation**

19.5

33.8

50.0

59.9

68.8

72.2

75.1

77.5

79.9

81.7

82.3

83.8

Toxicity control % degradation***

14.8

27.5

35.7

41.7

50.9

56.3

59.6

63.5

66.2

67.7

68.3

69.3

*BOD/CODx100, where COD = 0.78 mgO2/L

** BOD/CODx100, where COD = 2.67 mgO2/L

*** BOD/CODx100, where COD = 345 mgO2/L

Validity criteria fulfilled:
yes
Remarks:
(differences between replicates < 20% or slightly higher, reference item reached pass level (60%) on day 4, biodegradation % in toxicity test was 56.3% on day 14, oxygen uptake of inoculum blank was 38.9 mg O2/L on day 28, the pH values were 6.91-7.08)
Interpretation of results:
readily biodegradable
Conclusions:
The biodegradability of the substance attained 83.8% on day 28. The test item was determined to be ready biodegradable under aerobic conditions according to the OECD 301 criteria.
Executive summary:

The ready biodegradability of the test substance was assessed according to OECD guideline 301F and EU method C.4 -D. A measured, stirred volume of a mineral solution containing 100 mg/l of test item was inoculated with microorganisms (30 mg/L SS of inoculum) and incubated under aerobic conditions in a closed respirometer flask at constant temperature of 22 ± 2°C for 28 days. The blank tests were run in parallel with only inoculum but without test item. A reference item (sodium acetate) was run in parallel to check the operation of the procedures. The degradation was followed by the determination of oxygen uptake and measurements were taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. To check the possible inhibitory effect of the test item the toxicity test was run in parallel. The solutions containing 100 mg/l, of both the test and a reference item, in the mineral medium, were inoculated. The consumption of oxygen was determined from the change in pressure in the apparatus. The carbon dioxide, evolved during test item degradation, was absorbed in a solution of potassium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of determined COD of the test item. The test item biodegradability was 83.8 % after 28 days. The test item was determined to be ready biodegradable under aerobic conditions according to the OECD 301 criteria.

Description of key information

Key study: Test method OECD 301F and EC C.4-D. GLP study. The test item biodegradability (based on O2 consumption) was 83.8 % after 28 days. The test item was determined to be ready biodegradable under aerobic conditions according to the OECD 301 criteria.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Key study: The ready biodegradability of the substance was assessed according to OECD 301F and EC C.4 -D. 100 mg/l of test item was inoculated with microorganisms (30 mg/L SS of inoculum) and incubated under aerobic conditions in a closed respirometer flask at constant temperature of 22 ± 2°C for 28 days. The test item biodegradability (based on O2 consumption) was 83.8 % after 28 days. The test item was determined to be ready biodegradable under aerobic conditions according to the OECD 301 criteria.