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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 7 sept 2009 to 9 dec 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Qualifier:
according to
Guideline:
EU Method C.9 (Biodegradation: Zahn-Wellens Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Laboratory culture: no, aerobic activated sludge came from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland).
- Method of cultivation: During the holding period of one day prior to use, the sludge was aerated at room temperature.
- Preparation of inoculum for exposure: The sludge was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted. Calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter.
- Concentration of sludge: 300 mg dry material per liter
- Initial biomass concentration: 4 g dry material per liter
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
143 mg/L
Based on:
test mat.
Initial conc.:
ca. 86 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: 10 mL of the stock solution: KH2PO4 (8.50 g/L) + K2HPO4 (21.75 g/L) + Na2HPO4, 2H2O (33.40 g/L) + NH4Cl (0.50 g/L) and 1 mL each of stock solutions: MgSO4, 7H20 (22.50 g/L) - CaCl2, 2H2O (36.40 g/L) - FeCl3, 6H2O (0.25 g/L), were combined and made up to 1000 mL with purified water.
- Test temperature: 21 - 24°C
- pH: 6.7 - 7.5 (before adjustment)
- pH adjusted: yes
- Aeration of dilution water: yes
- Continuous darkness: yes
- Oxygen concentration: 7.6 - 8.3 mg O2/L


TEST SYSTEM
- Culturing apparatus: Erlenmeyer flask
- Method used to create aerobic conditions: Each flask was loosely covered with aluminium foil to allow the exchange of air between the flask and the surrounding atmosphere. The test media were continuously stirred by magnetic stirrers.


SAMPLING
- Sampling frequency: exposure day 0 (0 and 3 hours ± 30 minutes after the addition of the test chemical), 3, 7, 10, 14, 21 and 28
- Sampling method: Prior to sampling, water evaporation losses were compensated by adding purified water up to the mark. Deposits on the test vessels were resuspended in the test vessels. Per sampling interval, one sample of about 10 mL was taken and analyzed for DOC on the day of sampling. Samples were filtered through a 0.45 µm filter. The first 3–4 mL of the filtrate were discarded.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes; DOC of inoculum control: mean 4.1 mg/L (n=2)
- Abiotic sterile control: no
- Toxicity control: yes; DOC of toxicity control: 172.8 mg/L


STATISTICAL METHODS: no
Reference substance
Reference substance:
diethylene glycol

Results and discussion

% Degradation
Key result
Parameter:
% degradation (DOC removal)
Value:
91
Sampling time:
28 d
Details on results:
No DOC removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge.
In the toxicity control, containing the test item, the reference item diethylene glycol and activated sludge (inoculum), the initial DOC decreased by 81% within 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 143.5 mg/L beacause degradation was > 35% within 14 days.
In the test flasks containing the test item and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) continuously decreased from initially 86 mg/L on Day 0 (starting value after 3 hours of exposure) to 7 mg/L on Day 28. The mean biodegradation of 4-tert-butylpyrocatechol (4-TBC), based on DOC decrease, amounted to 91% after 28 days. Consequently, 4-Tert-Butylpyrocatechol (4-TBC) was found to be biodegradable by 91% under the test conditions within 28 days.
No DOC removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge.

BOD5 / COD results

Results with reference substance:
In the procedure control containing the reference item diethylene glycol and activated sludge (inoculum), the reference item was completely degraded within the first seven days of exposure, thus confirming suitability of the activated sludge.

Any other information on results incl. tables

Table 1: Percentage biodegradation of the test item and the reference item

 

Percentage Biodegradation1, 2

 

Test item: 4-TBC2

Procedure control:

Diethylene glycol

Toxicity control

Time

Replicate No.

 

Replicate No.

 

Replicate No.

(days)

1

2

Mean

1

2

Mean

1

0

0

0

0

0

0

0

0

3

62

65

64

36

25

31

--

7

84

77

80

100

102

101

71

10

91

77

84

--

--

--

--

14

89

91

90

101

98

99

81

21

95

92

93

--

--

--

--

28

95

88

91

99

100

100

79

1          Corrected for the inoculum controls

2          0 days = 3 hours ± 30 minutes after the addition of the test item

--         Not determined

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
More that 70% degradation of the test item 4-tert-butylpyrocatechol (4-TBC) occurred within the first seven days of the test, including the lag-phase and the log-phase. The log-phase was lower than 3 days.
Therefore, 4-TBC could be considered as inherent ultimately biodegradable according to OECD (2006).

Executive summary:

In a study (Harlan study, C60372, 2009), the inherent biodegradability of 4 -tert-butylpyrocatechol (4 -TBC) (initial concentration: 143 mg/L), was tested in a Zahn-Wellens test during 28 days, according to OECD guideline for Testing of Chemicals n°302B (1992).

Inoculum used in a study was 300 mg/L of a sludge from a wastewater treatment plant, adapted.

The percentage of biodegradation was estimated by following the DOC removal.

4 -TBC did not adsorb on activated sludge.

4 -TBC was not inhibitory to activated sludge at the tested concentration of 143.5 mg/L because degradation was > 35% within 14 days.

The corresponding percentage of biodegradation obtained was 91% of DOC removal after 28 days.

Under the test conditions, 4 -TBC is considered as inherent ultimately biodegradable.

More that 70% degradation of 4 -TBC occurred within the first seven days of the test, including the lag-phase and the log-phase. The log-phase was lower than 3 days.

This study is classified as acceptable and satisfies the guideline requirements for inherent biodegradability study (OECD 302B).