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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:
09/02/2018 to 29/03/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Common name: Omnirad EMK
- CAS number: 90-93-7
- Source and lot/batch No.of test material: 177116
- Expiration date of the lot/batch: 16/11/2019
- Purity test date: 99.50%
- Purity: 16/11/2017
- Appearance: white to pale yellow green powder
- Test handling: use amber glassware or wrap container in foil

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

PREPARATION OF STOCK SOLUTIONS
- Method of addition: since the test substance was not sufficiently soluble to alllow preparation of an aqueous solution at a concentration of 1g/L, weighed amounts were added to the 2 litre test vessels containing mineral medium. 31.17, 31.15, 30.88 mg were added to test vessels A, B and the toxicity control bottle.
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): activated sludge freshly obtained from a municipal sewage treatment plant, Waterschap Aa en Maas, s-Hertogenbosch. Treats predominantly domestic sewage.
- Preparation of inoculum for exposure: Sludge was used immediately. Before use, the sludge was washed with mineral medium. Magnetically stirred.
- Concentration of sludge: After treatment the concentration of suspended solids was determined at 3.2 g/L
- Initial cell/biomass concentration: not stated
- Water filtered: not stated
Duration of test (contact time):
28 d
Initial conc.:
16 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium made up using 4 solutions as follows:
Solution A: 8.5g KH2PO4, 21.75g K2HPO4, 67.20g Na2HPO4.12H2O, 0.5g NH4Cl, dissolved in Milli-RO water and made upto 1L, pH 7.4 ± 0.2.
Solution B: 22.50g MgSO4.7H2O dissolved in Milli-RO water and made up to 1L.
Solution C: 36.40g CaCl2.2H2O dissolved in Milli-RO water and made up to 1L
Solution D: 0.25g FeCl3.6H2O dissolved in Milli-RO water and made upto 1L.
Final mineral medium prepared using 10ml solution A, 1ml solution B, C and D. Made upto 1L in Milli-RO water.
- Test temperature: 22 to 23°C
- pH: ranged from 7.5 to 7.8 in all of the test, reference and toxicity control vessels.
- pH adjusted: adjustment carried out at the start of the test in one of the blank control vessels only.
- CEC (meq/100 g): not stated
- Suspended solids concentration: addition of 3ml per litre of mineral medium, resulting in a SS of 9.6 mg/L
- Continuous darkness: glass brown bottles used. Test media exluded from the light.
- Other: the test media was aerated and stirred continuously. Tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon used.

TEST SYSTEM
- Culturing apparatus: 2 litre brown glass bottles
- Number of culture flasks/concentration: 2 flasks, 1 concentration
- Method used to create aerobic conditions: continual aeration and stirring
- Test performed in closed vessels due to significant volatility of test substance: not stated
- Test performed in open system: not stated
- Details of trap for CO2 and volatile organics if used: three CO2 absorbers (bottles filled with 100ml 0.0125 MBa(OH)2 were connected in series to the exit air line of each test bottle.
- Other: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli- RO water. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency: 2, 5, 8, 12, 15, 19, 23, 28 (measured on day 29)
- Sampling method: titrations were made on the sampling day. Each time the CO2 absorber nearest to the test bottle was removed for titration, each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2 absorber was placed at the far end of the series. On the penultimate day, the pH of the respective test suspensions was measured and 1ml of concentrated HCl added to the bottles. The bottles were then aerated overnight to drive off the CO2 present in the test suspension.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles containing inoculum only
- Abiotic sterile control: Not included
- Toxicity control: 1 bottle containing test item, reference item and inoculum.
- Reference control: 1 bottle containing reference item and inoculum.
Reference substance:
other: sodium acetate
Remarks:
see below for further details
Preliminary study:
Not carried out
Test performance:
The performance of the test was valid and met all the criteria as detailed below.
1. The reference item was biodegraded by at least 60% (84%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 8%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (63.6 mg CO2 per 2 litres of medium, corresponding to 31.8 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
other: Bottle A
Key result
Parameter:
% degradation (CO2 evolution)
Value:
8
Sampling time:
28 d
Remarks on result:
other: Bottle B
Details on results:
The average of biodegradation at day 28 was 4%. See Tables 1,3,4,5 and 6 below for further results.
Results with reference substance:
The biodegradation with the reference substance was measured at 12, 34, 50, 61, and 70% at days 2, 5, 8, 12 and 14. See Table 2 for further details.

Table 1: CO2 production in the blank.

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

 

Ba(OH)21)

Blank (mean)

 

2

49.40

46.16

3.24

3.6

3.6

 

5

49.44

43.62

5.82

6.4

10.0

 

8

49.23

41.83

7.40

8.1

18.1

 

12

49.87

42.26

7.62

8.4

26.5

 

15

49.76

43.56

6.19

6.8

33.3

 

19

50.13

43.58

6.55

7.2

40.5

 

23

49.85

44.00

5.85

6.4

46.9

 

292)

52.06

44.30

7.76

8.5

55.5

 

292)

52.27

47.75

4.52

5.0

60.4

 

292)

52.45

49.55

2.91

3.2

63.6

 

1): "Strength" of untreated 0.0125 M Ba(OH)2solution

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

Table 2. CO2 production and percentage biodegradation of the reference item

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Procedure

control

2

46.16

27.14

19.02

20.9

20.9

12

5

43.62

8.19

35.43

39.0

59.9

34

8

41.83

17.19

24.64

27.1

87.0

50

12

42.26

24.18

18.08

19.9

106.9

61

152)

43.56

29.04

14.52

16.0

122.8

70

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 86.3 mg CO2/2L.

2): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

 

Table 3. CO2 production and percentage biodegradation of the test item (bottle A)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Bottle A

2

46.16

46.49

0.00

0.0

0.0

0

5

43.62

44.28

0.00

0.0

0.0

0

8

41.83

44.23

0.00

0.0

0.0

0

12

42.26

43.39

0.00

0.0

0.0

0

15

43.56

44.70

0.00

0.0

0.0

0

19

43.58

44.46

0.00

0.0

0.0

0

23

44.00

44.70

0.00

0.0

0.0

0

292)

44.30

45.43

0.00

0.0

0.0

0

292)

47.75

48.10

0.00

0.0

0.0

0

292)

49.55

50.26

0.00

0.0

0.0

0

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.8 mg CO2/2L.

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table 4. CO2 production and percentage biodegradation of the test item (Bottle B)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Bottle B

2

46.16

46.34

0.00

0.0

0.0

0

5

43.62

43.12

0.50

0.6

0.6

1

8

41.83

42.30

0.00

0.0

0.6

1

12

42.26

43.26

0.00

0.0

0.6

1

15

43.56

43.13

0.43

0.5

1.0

1

19

43.58

41.10

2.48

2.7

3.7

4

23

44.00

42.88

1.12

1.2

5.0

6

292)

44.30

43.64

0.66

0.7

5.7

6

292)

47.75

46.39

1.36

1.5

7.2

8

292)

49.55

49.23

0.32

0.3

7.5

8

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 88.8 mg CO2/2L.

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table 5. CO2 production and percentage biodegradation of the toxicity control.

Day

HCl(0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Toxicity

control

2

46.16

30.34

15.82

17.4

17.4

10

5

43.62

22.21

21.41

23.6

40.9

23

8

41.83

30.73

11.10

12.2

53.2

30

12

42.26

35.49

6.76

7.4

60.6

35

152)

43.56

38.23

5.33

5.9

66.5

38

1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and reference item: 174.3 mg CO2/2L (ThCO2test item: 88.0 mg CO2/2L + ThCO2sodium acetate: 86.3 mg CO2/2L).

2): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Table 6. Comparison of biodegradation of the test item in bottles A and B.

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

2

0

0

0

0

5

0

1

1

1

8

0

1

1

1

12

0

1

1

1

15

0

1

1

1

19

0

4

2

4

23

0

6

3

6

292)

0

6

3

6

292)

0

8

4

8

292)

0

8

4

8

1): Absolute difference in biodegradation between bottles A and B

2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of 4,4’- Bis(diethylamino)benzophenone (0% and 8%, based on ThCO2). The test substance was classifed as not readily biodegradable.
Executive summary:

The biodegradation potential of 4,4’- Bis(diethylamino)benzophenone was assessed using an OECD 301B studydesign. The test was carried out accoring to the guideline and according to GLP. The study is considered a Klimisch 1 study. The biodegradation of the test substance was measured over a 28 day period (final measurement made on day 29, after the reaction was stopped on day 28), using a titration method to assess carbon dioxide production. A reference control, background control and toxicity control were all included. The reference control indicated adequate biodegradation in the reference control vessels, demonstrating the adequate functioning of the inoculum. No toxicity to the bacteria was observed.

After 28 days the percentage biodegradation calculated in the test vessels for 4,4’- Bis(diethylamino)benzophenone was 0 and 8% for the two test vessels respectively. Overall, the test substance was concluded not to be readily biodegradable.

Description of key information

The biodegradation potential of 4,4’- Bis(diethylamino)benzophenone was assessed using an OECD 301B study test design. The test was carried out according to the guideline and GLP. The study is considered a Klimisch 1 study. The biodegradation of the test substance was measured over a 28 day period (final measurement made on day 29, after the reaction was stopped on day 28), using a titration method to assess carbon dioxide production. A reference control, background control and toxicity control were all included. The reference control indicated adequate biodegradation in the reference control vessels, demonstrating the adequate functioning of the inoculum. No toxicity to the bacteria was observed.

After 28 days the percentage biodegradation calculated in the test vessels for 4,4’- Bis(diethylamino)benzophenone was 0 and 8% for the two test vessels, respectively. Overall, the test substance was concluded not to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
other: domestic activated sludge

Additional information