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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:
07 Apr - 07 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland
Specific details on test material used for the study:
Reference Item:
The reference item and the following information were supplied by Sigma-Aldrich:

Identity: Sodium benzoate
Product No.: 71295
Lot No.: BCBB4145
Purity: 99.7%
Expiration Date (as given by the supplier): November 2015 (date of recommended retest)
Expiration Date (handled at Harlan Laboratories): 30 November 2015 (date of recommended retest)
Storage Conditions (as provided by the supplier): Keep container tightly closed in a dry and wellventilated place. Store in cool place.
Storage Conditions (as handled at Harlan Laboratories): In tightly closed original container, at room temperature at about 20°C.
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): wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland)- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the dry weight of this diluted activated sludge was determined again and defined amounts were added to mineral medium to obtain a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
28 d
Initial conc.:
ca. 50 - ca. 50.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS-
Composition of medium:
The mineral medium was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:

1) KH2PO4: 8.50 g/L, K2HPO4: 21.75 g/L, Na2HPO4 × 2H2O: 33.40 g/L, NH4Cl: 0.50 g/L. The pH of this solution was 7.4.

2) MgSO4 × 7H2O: 22.50 g/L.

3) CaCl2 × 2H2O: 36.40 g/L.

4) FeCl3 × 6H2O: 0.25 g/L, stabilized with one drop of concentrated HCl per liter.

To obtain the final mineral medium, 10 mL of stock solution 1) and 1 mL each of stock solutions 2) to 4) were combined and made up to 1000 mL with purified water. The pH was adjusted from 8.1 to 7.4 with a diluted hydrochloric acid solution.

TEST SYSTEM- Culturing apparatus: 2000-mL Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The final volume was 1000 mL per flask. 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.

- Number of culture flasks/concentration: 2
- Test performed in open system: loosely covered with aluminium foil
- Test temperature: 21-24°C. The inoculated flasks were incubated in a temperature controlled room. The temperature was checked on each sampling date in one of the inoculum controls. Additionally, the room temperature was continuously recorded.
- pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum and found to be pH 7.4. At the end of incubation, the pH was measured again in each test flask.
- Light conditions: The test flasks were incubated in a dark room.

SAMPLING- Sampling frequency: All vessels were sampled at Exposure Day 0, 2, 5, 8, 11, 14, 21 and 28.
- Sampling method: One sample of about 10 mL was taken from each test flask per sampling date. Prior to sampling, water evaporation losses were determined by weighing the flasks and were compensated by adding purified water. Deposits on the test vessels were resuspended.

CONTROL AND BLANK SYSTEM- Inoculum blank: yes (2)
- Toxicity control: yes (1)- Procedure control: yes (2)
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
25
Sampling time:
11 d
Key result
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
14 d
Key result
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
28 d
Results with reference substance:
In the procedure controls, containing the reference item sodium benzoate and activated sludge (inoculum), sodium benzoate was readily biodegraded by an average of 98% within two days of exposure, thus confirming suitability of the activated sludge.

Degradation of the Test Item:

In the test flasks, containing the test item Cyrene™ and activated sludge (inoculum), the initial mean concentration of dissolved organic carbon (DOC) of 28.7 mg/L on Day 0 rapidly decreased after Exposure Day 8, reaching 99% decrease of the initial value within 14 days of exposure. The pass level for ready biodegradability (70% removal of DOC in a 10-day window within a 28-day period) was reached.

Degradation in the Toxicity Control:

In the toxicity control, containing the test item (corresponding to 50.1% of total DOC), the reference item (corresponding to 49.9% of total DOC) and activated sludge (inoculum), the initial DOC concentration of 58.4 mg/L measured on Day 0 decreased to 0.5 mg/L on Day 14. Biodegradation amounted to 99% 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 50.4 mg/L because degradation was > 35% within 14 days.

Measurement of pH and Temperature:

The pH measured in all flasks at the end of exposure (Day 28) was 7.2 to 7.4. The temperature during the test period was in the range 21-24°C.

Table: Dissolved Organic Carbon Concentration (DOC) in the Test Flasks

Time [days]

DOC [mg/L]*

Test Item

Procedure control

Inoculum control

Toxicity control

Replicate No.

Replicate No.

Replicate No.

Replicate No.

1

2

mean#

1

2

mean#

1

2

mean#

1

0

32.4

32.5

28.7

32.5

32.7

28.8

3.7

3.9

3.8

58.4

2

29.9

31.4

28.4

2.8

2.8

0.6

2.1

2.4

2.3

29.4

5

30.4

31.0

28.5

2.8

3.0

0.7

2.2

2.3

2.3

29.1

8

30.4

31.2

28.0

3.2

3.4

0.5

2.9

2.7

2.8

27.8

11

27.9

20.7

21.4

3.2

3.6

0.5

2.9

2.9

2.9

26.3

14

3.7

3.6

0.3

3.3

3.8

0.2

3.5

3.3

3.4

0.5

21

2.9

3.4

0.2

2.9

2.8

-0.1

2.8

3.0

2.9

0.4

28

3.5

3.8

0.3

3.9

3.9

0.6

3.9

2.8

3.4

0.8

* Mean values of at least triplicate measurements per sample.

# Values corrected for the mean inoculum control.

Table: Percentage Biodegradation in the Test Flasks

Time [days]

Percentage Biodegradation*

Test Item

Procedure control

Toxicity control

Replicate No.

Replicate No.

Replicate No.

1

2

mean

1

2

mean

1

0

0

0

0

0

0

0

0

2

3

-2

1

98

98

98

50

5

2

0

1

98

97

98

50

8

3

1

2

99

98

98

52

11

13

38

25

99

98

98

55

14

99

99

99

100

98

99

99

21

100

98

99

100

100

100

99

28

99

98

99

98

98

98

99

* Corrected for the inoculum controls.

Table: pH Values at the Start and at the End of the Test

Replicate No.

Identification

pH

pH

 

 

Start

End

1

Test item

7.4

7.4

2

Test item

7.4

7.4

1

Procedure control

7.4

7.3

2

Procedure control

7.4

7.3

1

Inoculum control

7.4

7.2

2

Inoculum control

7.4

7.2

1

Toxicity control

7.4

7.3

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
A biodegradation of 99% in 28 days (DOC removal) was determined for the substance using a relevant test method and in compliance with GLP. The result is considered to be reliable.

Description of key information

Biodegradation in water (Cyrene™/Gem Diol): 99% in 28 days (DOC removal, OECD 301A)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A biodegradation value of 99% in 28 days (DOC removal) was determined for the substance in accordance with OECD Test Guideline 301A and in compliance with GLP. The result is considered to be reliable.