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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:
2017-10-10 to November 2017-11-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted on 17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: S7101247
- Expiration date of the lot/batch: 31 August 2020 (Minimum shelf life)
- Molecular Weight: 187.03 g/mol
- Purity (GC area %): 100.0 % (a/a)




Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge, microorganisms from a domestic waste water treatment plant in Balatonfüred, Hungary, on 03 October 2017 (seven days before the main test)
- Preparation of inoculum for exposure: Washed by centrifugation, supernatant liquid phase decanted, solid material re-suspended in isotonic saline solution and again centrifuged (procedure repeated twice); aliquot of final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined; aliquots of washed sludge suspension mixed with test water (mineral medium), then aerated under test conditions (for 7 days) until use; pH of activated sludge inoculum after preparation was 7.39, just before use: 7.24; pH adjustment of activated sludge inoculum was not performed; after preparation the sludge was filtered through cotton wool.
- Pre-conditioning (03-10 October 2017): Aerating (2 L/minute) activated sludge (in mineral medium) for 7 days at test temperature
- Concentration of sludge: 5 g dry material per litre
- Initial cell/biomass concentration: 104 - 106 cells/L
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: In water (purified deionised) analytical grade salts were added to prepare the following stock solutions

a)
KH2PO4, 8.50 g
K2HPO4, 21.75 g
Na2HPO4 x 12H2O 67.16 g1
NH4Cl 0.50 g
Water ad. 1000 mL

b)
CaCl2 x 2H2O 36.40 g
Water ad. 1000 mL

c)
MgSO4 x 7 H2O 22.50 g
Water ad. 1000 mL

d)
FeCl3 x 6H2O 0.125 g
Water ad. 500 mL2

1 This concentration corresponds to 33.4 g of Na2HPO4 x 2H2O per liter as stated in the guideline.
2 The volume of the solution d) was 500 mL, instead of the guideline proposal: 1000 mL; however the composition of these solutions was the same as given in the referred guideline, therefore the smaller prepared volumes (for technical reason) have no effect on the results of the study.
3 The batch number of mineral medium used in this study was: 171009. It was prepared in the testing laboratory on 09 October 2017. This batch was used for the test on 10 October 2017.

- Additional substrate: No
- Test temperature: 20.0 - 20.1 °C
- pH: 7.38 at start of the test
- pH adjusted: No
- Aeration of dilution water: No, but aeration of mineral medium
- Continuous darkness: Yes
- Oxygen concentration of the test water: 8 - 9 mg/L

TEST SYSTEM
- Culturing apparatus: Temperature controlled (22 ± 2 oC) environment room and incubator with thermometer with exclusion of light
- Number of culture flasks/concentration: 16 (+2 reserve)
- Measuring equipment: Oxygen and pH meter with appropriate O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method] and pH electrode, built-in thermometer
- Test performed in closed vessels due to significant volatility of test substance: Yes, tightly closed closed with glass stoppers
- Test performed in open system: No


SAMPLING
- Sampling frequency: Oxygen measurements were performed in all duplicate bottles in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28.
- Sampling method: O2 electrode

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 (+ 2 reserve) bottles
- Procedure conrol: 16 (+ 2 reserve) bottles containing the reference item and inoculum
- Toxicity control: 16 (+ 2 reserve) bottles containing the test item, reference item and inoculum
Preliminary study:
The pre-experiment on solubility of the test item was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance in the final report. The test item solubility was investigated in ultrapure water. At the concentration of 400 mg/L the test item was completely dissolved and a clear solution was obtained after 15 min. ultrasonic treatment.
Test performance:
VALIDITY OF THE STUDY

- Inoculum Control: The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.36 mg O2/L in average.
- Oxygen Concentration: The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 3.12 mg O2/L, it was measured on the 28th day in the procedure control.)
Parallels: The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %. The highest difference (13.7 %) between the duplicate values from the 7th to the 28th day of the test (this period was taken into consideration as biodegradation plateau) or at the end of the test (test item, procedure control, and toxicity control groups) for degradation was calculated in the procedure control group, it was observed on the 7th day of the test.
- Reference Item: The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 84.0 % on the 14th day.)

The test is valid
Key result
Parameter:
% degradation (O2 consumption)
Value:
33.8
Sampling time:
28 d
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7% after 28 days of incubation, based on its ThODNH4. The biodegradability reached its plateau on about the 14th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Table 1: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

[mg/L]

Flask

mg O2/L after n days of exposure

No.

0

2

5

7

12

14

21

28

Test item

4.0

1a

9.14

8.22

6.88

6.08

5.96

5.98

5.92

5.91

1b

9.24

8.58

6.99

6.35

6.23

6.20

6.13

6.05

mean

9.19

8.40

6.94

6.22

6.10

6.09

6.03

5.98

Reference item

3.0

2a

9.27

6.43

4.90

4.69

3.88

3.65

3.57

3.12

2b

9.29

6.18

5.14

4.23

3.95

3.78

3.71

3.65

mean

9.28

6.31

5.02

4.46

3.92

3.72

3.64

3.39

Inoculum control

3a

9.21

8.78

8.37

7.96

7.91

7.96

7.91

7.81

3b

9.24

8.76

8.26

7.88

7.92

7.76

7.96

7.92

mean

9.23

8.77

8.32

7.92

7.92

7.86

7.94

7.87

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

9.30

6.11

4.90

3.89

3.69

3.61

3.49

3.44

4b

9.31

5.57

4.98

3.79

3.76

3.60

3.58

3.38

mean

9.31

5.84

4.94

3.84

3.73

3.61

3.54

3.41

Table 2: Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

mg O2/L after n days of exposure

[mg/L]

No.

2

5

7

12

14

21

28

Test item

4.0

1a

0.46

1.35

1.76

1.87

1.80

1.93

1.87

1b

0.20

1.34

1.59

1.70

1.68

1.82

1.83

Reference item

3.0

2a

2.39

3.46

3.28

4.08

4.26

4.41

4.79

2b

2.66

3.24

3.76

4.03

4.15

4.29

4.28

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

2.74

3.49

4.11

4.30

4.33

4.52

4.50

4b

3.29

3.42

4.22

4.24

4.35

4.44

4.57

oxygen depletion : (mt0- mtx) - (mbo- mbx), where:

 

mt0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

mtx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 1)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 1)

Table 3: BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

[mg/L]

Flask

BOD after n days of exposure

No.

2

5

7

12

14

21

28

Test item

4.0

1a

0.12

0.34

0.44

0.47

0.45

0.48

0.47

1b

0.05

0.34

0.40

0.43

0.42

0.46

0.46

Reference item

3.0

2a

0.79

1.15

1.09

1.36

1.42

1.47

1.60

2b

0.88

1.08

1.25

1.34

1.38

1.43

1.43

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

0.39

0.50

0.59

0.61

0.62

0.65

0.64

4b

0.47

0.49

0.60

0.61

0.62

0.63

0.65

Table 4: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

[mg/L]

Flask

Percent of biodegradation after n days of exposure

No.

2

5

7

12

14

21

28

Test item

4.0

1a

8.5

24.7

32.1

34.2

32.8

35.3

34.2

1b

3.7

24.5

28.9

31.0

30.6

33.2

33.4

mean

6.1

24.6

30.5

32.6

31.7

34.2

33.8

Reference item

3.0

2a

47.7

69.2

65.5

81.6

85.1

88.2

95.8

2b

53.1

64.8

75.1

80.6

82.9

85.8

85.6

mean

50.4

67.0

70.3

81.1

84.0

87.0

90.7

Toxicity control

Test item: 4.0
Reference item: 3.0

4a

25.7

32.9

38.6

40.5

40.7

42.5

42.4

4b

30.9

32.2

39.7

39.9

40.9

41.8

43.0

mean

28.3

32.5

39.2

40.2

40.8

42.2

42.7

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item is considered to be not readily biodegradable, since the percentage biodegradation of 4-Bromobenzyl alcohol reached a mean of 33.8 % after 28 days based on its ThODNH4.
Executive summary:

To determine the ready biodegradability of the test item, a GLP-compliant Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4 -E and EPA Guideline OPPTS 835.3110 was conducted. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item 4-Bromobenzyl alcohol was investigated at the concentration of 4.0 mg/L. The test item concentration was chosen based on its theoretical oxygen demand of 1.37mg O2/mg. In parallel, positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. In result, the percentage biodegradation of the test item reached a mean of 33.8 % after 28 days based on its ThODNH4. The test item biodegradation reached a plateau on about the 7th day of the experiment. From this day the slight changes were considered as being within the biological variability range of the applied test system. The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 40.8 % biodegradation was noted within 14 days and 42.7 % biodegradation after 28 days of incubation. In conclusion, the test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines, the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days. All validity criteria of the guidelines were fulfilled.

Description of key information

The substance is not readily biodegradable but inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

To determine the ready biodegradability of the test item, a GLP-compliant Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4 -E and EPA Guideline OPPTS 835.3110 was conducted. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item 4-Bromobenzyl alcohol was investigated at the concentration of 4.0 mg/L. In parallel, positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. In result, the percentage biodegradation of the test item reached a mean of 33.8 % after 28 days based on its ThODNH4. In conclusion, the test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines, the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days. All validity criteria of the guidelines were fulfilled (reference 5.2.1-1).