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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:
2013-07-02 to 2013-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
March 2016
Deviations:
no
Qualifier:
according to
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant, 31137 Hildesheim, Germany
- Pretreatment: activated sludge washed twice with chlorine free tap water; settled sludge resuspended in mineral salts medium and maintained in an aerobic condition by aerarion for 2.5 hours; sludge homogenized with blender; supernatant decanted and maintained in an aerobic condition by aeration with CO2-free air for 5 days
- Initial cell concentration in the test solutions: 10^2 -10^5 CFU/mL (CFU = colony forming units)
Duration of test (contact time):
28 d
Initial conc.:
22.4 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS

- Test temperature: 20 +/- 1 °C; actual 19.0-21.0 °C, up to 26.0 °C on day 25
- Photoperiod: low light conditions

TEST SYSTEM
- Number of culture flasks per treatment resp. control group: triplicates (test end 5 replicates)
- Method used to create aerobic conditions: aeration with CO2-free air for 5 days
- Measuring equipment for IC-analysis: carbon analyzer
- Test performed in closed vessels: yes
- Headspace to liquid ratio: 1:2
- Test vessels volume: headspace flasks / 120 mL
- Test medium / volume: mineral salts medium acc. to OECD 310 with inoculum / 80 mL
- Agitation: shaker (150 - 200 rpm)


SAMPLING
- Sampling frequency: analysis was carried out at test start, twice in the first two weeks and thereafter every 7 days.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Functional control with reference item: yes

STATISTICAL METHODS:
The 95 % confidence interval for the mean percentage biodegradation after 28 days was calculated for the test item as well as for the functional and toxicity controls using software SigmaPlot (Windows) rel. 8.02, 2002, SPSS Corporation.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
no
Test performance:
There was no deviation from the above mentioned guidelines, but regarding the test temperature divergence from the study plan is documentated: The temperature exceeded the range of 20 +/- 1 °C on day 25 up to 26 °C due to technical fault of climatisation. The pass level of 60 % degradation had already been reached. Therefore this deviation is considered to have no impact on quality and integrity of the study. All validity criteria were fulfilled. The biodegradation of the functional control passed the level of 60 % after 3 days and after 28 days a biodegradation of 94 % was determined (95 % confidence interval on day 28: 92 - 96 %). No inhibtion of biodegradation by the test item was observed.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
88
Sampling time:
28 d
Details on results:
The maximum amount of TIC present in the inoculum controls until the end of the test was 1.09 mg C/L (validity criterion: TIC < 3mg C/L).

The biodegradation of the reference item was not inhibited by the test item.
Results with reference substance:
The percentage degradation in the functional control reached the pass level of 60% after 3 days and came to 94% after 28 days.

Table 1: Biodegradation and Confidence Interval of the Test Item in Comparison to the Functional Control and the Toxicity Control; Biod. = Biodegradation, CI = confidence interval after 28 days (P = 95 %).

Biodegradation [%]

Replicates

1

2

3

4

5

CI

Test Item

90

86

85

92

85

84-95

Functional Control

94

96

95

93

92

92-96

Toxicity Control

92

88

95

89

92

88-94

Table 2: CO2 -Production and Biodegradation in the Test Item Samples; P1 -5 = vessel with test item, MP = mean values (NaOH corrected), NET MP = netto mean production (corrected for inoculum control values), Degr. = degradation.

Day  P1 P2 P3 P4 P5 MP NET MP Degr. [%]
0 2.72 2.87 2.82  - - 0.80 - -
1 1.46 1.46 1.47  -  - 0.58 0.00 0
3 2.92 3.21 2.74  -  - 1.81 0.59 4
7 12.22 11.80 11.74  -  - 10.82 9.30 61
10 15.13 14.34 14.25  -  - 13.19 11.33 75
14 14.96 15.28 15.32  -  - 14.09 12.65 83
21 15.25 15.33 15.12  -  - 14.20 12.75 84
28 16.39 15.74 15.66 16.67 15.68 15.22 13.33 88
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The ready biodegradability of the test item was investigated according to the guidelines OECD TG 310 and EN ISO 14593 (CO2 Headspace Test). The test item is classified as ready biodegradable as the pass level of 60% degradation was reached in a 10-d-window within the 28 days period of the study (88% biodegradation in 28 days). The toxicity control showed that the test item is not inhibitory at the tested concentration of 22.4 mg/L.
Executive summary:

The study was conducted under GLP-conditions, according to the guidelines OECD TG 310 and EN ISO 14593 (CO2 Headspace Test), in order to check the rate of biodegradation [%] of the test item Citronellyl formate by determination of TIC. The test was carried out over a period of 28 days with the aqueous phase of non-adapted activated sludge from a municipal sewage treatment plant. The activated sludge was pretreated by washing with chlorine free tap water, resuspension in mineral salts medium, aeration for 2.5 hours and homogenization with a blender. After sedimentation of the homogenized sludge, the supernatant was decanted and finally maintained in an aerobic condition by aeration with CO2 -free air for 5 days. 10 mL/L were used for inoculation, whereby the initial cell concentration (amount of colony forming units, CFU) in the test solutions was 102-105 CFU/mL. The test item was tested in triplicates (on day 28: 5 replicates) at a concentration of 22.4 mg/L, which corresponds to a carbon content of 15.2 mg/L. A functional control with the reference substance sodium benzoate and also a toxicity control, containing the test item as well as the reference item, was performed simultaneously.

In result, the test item replicates exceeded the 10 % level on day 4 and passed the level of 60 % on day 7. Futhermore, after 28 days a biodegradation of 88 % was reached (95 % confidence interval on day 28: 84 - 92 %). The biodegradation of the functional control passed the level of 60 % after 3 days and after 28 days a biodegradation of 94 % was determined (95 % confidence interval on day 28: 92 - 96 %). In the toxicity control the biodegradation came to 91 % after 28 days (95 % confidence interval on day 28: 88 - 94 %), thus no inhibtion of biodegradation by the test item was observed.

In conclusion, all validity criteria were fulfilled. The test item is regarded to be in the 10-d-window and after 28 days readily biodegradable.

Description of key information

The test item is classified as readily biodegradable according to OECD 310.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradability of the test substance was investigated by means of a CO2 Headspace Test, according to the guidelines OECD TG 310 (March 2006) and EN ISO 14593 (1999). The biodegradation of the test substance was followed by exposing it to microorganisms of non-adapted activated sludge from a municipal sewage treatment plant. An inoculum control, containing sole pretreated activated sludge, a functional control with the reference item sodium benzoate, as well as a toxicity control, including both test item and reference item, was conducted simultaneously. The test item concentration selected as appropriate was 22.4 mg/L, corresponding to a carbon content of 15.2 mg/L. The flasks were incubated for 28 days under aerobic and dark conditions at 20 +/- 1°C. The biodegradability was derived from TIC analyses of the quantity of CO2 produced within the test period by the respiration of bacteria . In result, the test item reached the 10 % level on day 4 and passed the level > 60 % on day 7. All validity criteria were fulfilled. In conclusion, the test item is regarded to be in the 10-d-window and after 28 days readily biodegradable. As proven by the toxicity control, no inhibitory effects on microorganisms is expected at the tested concentration of 22.4 mg/L.