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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-04 to 2016-03-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted 1992-07-17
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-12-16

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical state: pale white to white crystals
- Storage condition of test material: dry, 10 to 30 °C (as provided by the sponsor); at room temperature at approximately 20 °C (as handled at the laboratory)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland)(treats predominantly domestic sewage)

- Preparation of inoculum for exposure: aerobic activated sewage sludge was washed twice with tap water by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.

- Concentration of sludge: based on the determination of the dry weight of the suspended solids, calculated amounts of wet sludge were suspended in mineral medium to obtain a concentration equivalent to 4 g dry material/L.
Prior to use, the sludge was diluted with mineral medium to a concentration of approximately 1 g dry material/L. Day -1: between 2400 and 2910 mL of untreated mineral medium was filled into the 5-liter test vessels. To each vessel 90 mL activated sludge inoculum were added to give 30 mg dry material per liter in the final volume of 3000 mL. Aeration of test media overnight with CO2-free air to purge the system of CO2. Day 0: defined amounts of test item were directly added to test vessels. No emulsifiers or solvents were used.

- Storage length/ storage conditions: during the holding period of one day prior to use, the sludge was aerated at room temperature.

Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
20.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: KH2PO4 (8.50 g/L), K2HPO4 (21.75 g/L), Na2HPO4 × 2H2O (33.40 g/L), NH4Cl (0.50 g/L), CaCl2 × 2H2O (36.40 g/L), MgSO4 × 7H2O (22.50 g/L), and FeCl3 × 6H2O (0.25 g/L; stabilized with one drop of concentrated HCl per liter).
- Test temperature: 21 °C (except for the first five days: 20 to 24 °C)
- pH: start of test: 7.4; end of test: 7.3 - 7.4
- pH adjusted: no
- Aeration of medium: CO2-free air was bubbled through the solution in each test vessel at a rate of 30-100 mL/min.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5-liter amber glass bottles fitted with an aeration glass tube reaching nearly the bottom of the vessel and an outlet were used
- Number of culture flasks/concentration: 2 culture flasks for test item, procedure control, and inoculum control each; one culture flask for toxicity control
- Method used to create aerobic conditions: medium was aerated
- Details of trap for CO2 and volatile organics if used: Two absorber flasks; CO2 evolved from the test vessels were collected in 2 x 500 mL Drechsel bottles (gas-absorbtion flasks) containing 300 and 200 mL of 0.05 M NaOH (1st and 2nd bottle, respectively). The air leeding connections between the test vessel and the absorption flasks were done by glass tubes. CO2-absorber flasks were periodically sampled for IC Analysis
- Test vessels: 3 L mineral medium (purged with CO2-free air).
- Incubation: 28 days in temperature-controlled dark room

SAMPLING
- Sampling frequency: exposure days 2, 5, 7, 9, 12, 14, 19, 21, 28 (before acidification) and 29 (after driving off residual CO2)

Sampling method:
- exposure Day 0: inorganic carbon (IC) content of the 0.05 M NaOH solution used to fill all the absorber flasks was measured.
- on each sampling day: an aliquot of 10.0 mL was withdrawn from the absorber flask nearest to the test vessel 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: pH was measured in each test vessel. Next, 1 mL of concentrated HCl was added to each test vessel and the vessels were aerated overnight with CO2-free air to drive off any residual CO2 present.
- 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. Any residual CO2 remaining in the test suspensions was determined as the difference between the amounts of IC found before and after acidification.
- at the start of the test (Day 0): inorganic and total carbon (IC and TC, respectively) were measured in the test item and inoculum vessels.
- samples were analysed using a TOC infrared gas analyzer (i.e. vario TOC cube from Elementar Analysensysteme GmbH, Hanau, Germany) equipped with an automatic sampler.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (2 replicates); prepared in a similar manner without test and reference items.
- Abiotic sterile control: no
- Toxicity control: yes (1 culture flask; 20.1 mg/l test item included)
- Procedure control: yes (2 replicates); sodium benzoate (reference substance) was used at a concentrationof 25.7 mg/L
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium benzoate (procedural control) was tested simultaneousy under same test conditions. Stock solution: 7.71 g sodium benzoate/L mineral medium (purged with CO2-free air); 10 mL aliquots of stock solution were added to respective test vessels.

Results and discussion

Preliminary study:
Prior to test start, the solubility of the test item in water was checked, and the test item was found to be not soluble at a concentration of 100.5 mg/L. Therefore, a test item stock solution could not be prepared and the test item was added directly to the test vessels.
Test performance:
An unusual observations during the test or any other information potentially affecting the test was not reported. Thus, test performance was guideline-conform.
% Degradation
Parameter:
% degradation (inorg. C analysis)
Value:
2.6
Sampling time:
28 d
Details on results:
RESULTS
- Toxicity control: the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 20.1 mg/L because biodegradation in the toxicity control was ≥ 25 % within 14 days of incubation.

- Inorganic carbon/total carbon ratio (IC/TC ratio): the test item was not soluble at the tested concentration of 20.2 mg/L, therefore, in order to calculate the IC/TC ratio, the nominal total carbon contribution of the test item (15.1 mg C/L), was added to the total carbon concentration measured in the test item vessel before the test item addition. The inorganic carbon content in the test item vessels was less than 5 % of the total carbon content, therefore fulfilling this validity criterion.

- Driving off CO2: A significant difference was not found between the absolute amounts of inorganic carbon measured on exposure day 28 and the absolute amounts of inorganic carbon measured after acidification on Day 29. Thus, a significant amount of residual CO2 was not present in the test solutions or suspensions at the end of the test.

VALIDITY OF THE TEST
The results are considered to be valid since the following criteria are met:
- the percentage degradation of the reference item reached the level for ready biodegradability (>60 % in a 10-day window) by Exposure Day 5 (criterion: at least 60 % in a 10-day window by Day 14).
- the total CO2 evolution in the inoculum controls at the end of the test was 24 and 25 mg CO2/L and thus below the criterion of 40 mg CO2/L.
- the inorganic carbon content in both test item vessels was 0 % of the total carbon (criterion: less than 5 % of the total carbon).
Since the test item was not degraded, other validity criteria are not applicable.

BOD5 / COD results

Results with reference substance:
Average biodegradation of the reference item was 75.1 % and 90.3 % by exposure day 5 and 14, respectively, 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 93.9 %.

Any other information on results incl. tables

Table 1: Biodegradation of the test item and the reference item

 

% Degradation*

Time

(days)

Test item

Reference item

Toxicity

control

Replicate No.

Replicate No.

Replicate No.

1

2

Mean

1

2

Mean

1

2

1.8

2.8

2.3

50.9

61.5

56.2

28.7

5

2.1

3.1

2.6

72.4

77.8

75.1

38.7

7

2.2

3.8

3.0

78.4

83.4

80.9

42.1

9

2.1

4.1

3.1

83.6

85.8

84.7

44.0

12

2.6

4.6

3.6

87.9

90.1

89.0

46.8

14

2.5

4.6

3.6

89.5

91.0

90.3

47.3

19

1.5

4.1

2.8

92.7

93.4

93.1

48.4

21

0.9

4.3

2.6

93.9

94.0

93.9

48.3

28

1.4

3.8

2.6

93.1

94.8

93.9

48.3

* corrected for the inoculum controls

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance was not biodegradable after 28 days of exposure under the conditions of the conducted CO2 Evolution (Modified Sturm) Test.