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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Oct - 18 Dec 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed without any technical deficiencies. However, the result of this study may underestimate the biodegradation of the test item since the substance turned out to be inhibitory to the microbial activity.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyl ascorbic acid
- Physical state: powder
- Expiration date of the lot/batch: 21 Apr 2018
- Stability under test conditions: stable
- Storage condition of test material: in freezer at < -15 °C desiccated

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Obtained from the municipal sewage treatment plant "Waterschap Aa en Maas", s'Hertogenbosch, The Netherlands
- Laboratory culture: no
- Storage conditions: The activated sludge was kept under continuous aeration until further treatment
- Pretreatment: Before use the sludge was allowed to settle (86 min) and the supernatant was used as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
25.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline
- Additional substrate: no
- Test temperature: 21.8 - 22.5 °C
- pH: 7.5 - 7.8
- pH adjusted: no
- Suspended solids concentration: 3.6 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 L glass brown-colored flasks
- Number of culture flasks/concentration: 2 bottles
- Method used to create aerobic conditions: The day before incubation the flasks were filled with mineral medium and inoculum and aerated overnight with CO2-free synthetic air.
- Details of trap for CO2 and volatile organics if used: Three CO2 absorbers were connected in series to the exit air line of the bottles (filled with 0.0125 M Ba(OH)2). The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
- Other:

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 d.
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator. On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 bottles
- Toxicity control: yes, 1 bottle
- Other: reference substance: yes, 1 bottle
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
17
Sampling time:
29 d
Remarks on result:
other: Mean of two replicates

BOD5 / COD results

Results with reference substance:
The reference substance was degraded to 79% after 14 d confirming the suitability of the inoculum.

Any other information on results incl. tables

The substance is not readily biodegradable (17% degradation after 28 d). However, it has to be considered that substance is inhibitory to the inoculum since the toxicity control showed degradation < 25% after 14 d (12% based on ThCO2). Thus, the degradation result is expected to underestimate the biodegradation of the substance. It is likely that the substance shows a higher degradation if the study is performed with a lower concentration of the test item.

Table 1: Biodegradation of the test item during the study

Day

Biodegradation [%]

Bottle A

Bottle B

Mean

1

1

0

1

4

3

2

3

6

5

2

4

8

6

2

4

11

8

3

6

14

11

5

8

18

13

5

9

22

13

7

10

27

15

10

13

29

21

13

17

Table 2: Biodegradation in the toxicity control

Day

Biodegradation [%]

1

0

4

4

6

5

8

6

11

10

14

12

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed