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Environmental fate & pathways

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:
21 January 2020 to 02 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: 2-amino-5-chloro-N,3-dimethylbenzamide (M198)
Batch number: 628-042-00
Date of receipt: November 07, 2019
Packaging: Plastic tube
Internal number: 1906385-001
Production date: October 16, 2019
Expiry date: ctober 17, 2021
Purity: 983.7 ± 2.8 g/kg
Storage: At room temperature without particular attention to light.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Aerobic activated sludge from BRIANZACQUE civil wastewater treatment plant.
- Collected on January 23, 2020.
- A homogenized aliquot of the wet sludge was centrifuged, the supernatant was removed while the solid phase was weighed and dried
- The ratio of wet to dry weight was calculated to be equal to 7.2%.
- Based on this ratio, calculated amounts of wet sludge was centrifuged, the supernatant was removed and the solid phase was suspended in the test medium to get a concentration equivalent to 3 g dry material per litre.
- The prepared sludge inoculum was pre-conditioned to the experimental conditions by aerating it in the dark.
- The concentrated suspension was used as inoculum to give a final concentration of 15 mg dry material per litre into test flasks.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The test medium was prepared according to the OECD Guidelines No. 301 A, 1992: salts of analytical grade were dissolved in purified water to obtain the following stock solutions (up to 1000 mL):
a) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.57
b) CaCl2 x 2H2O 36.40 g/L
or
CaCl2 27.50 g/L
c) MgSO4 x 7H2O 22.50 g/L
d) FeCl3 x 6 H2O 0.25 g/L
- In order to allow the storage of solution, one drop of concentrated HCl was added.
10 mL of stock solution a) and 1 mL of stock solutions b), c) and d) were added to 800 mL of purified water, then made up to 1000 mL with purified water. The pH of test medium was 7.58 (mean value between two blank bottles), so adjustment with a diluted hydrochloric acid or sodium hydroxide solution, before making up to volume, was not needed.

- Test solution: Since the test item was poor soluble in water, the test flask was prepared by weighing proper amount of test item directly in mineral medium (0.0125 g into 250 mL and 0.0075 g into 150 mL).
The volume of mineral medium was dosed in the test flasks to obtain the final concentrations of 50 mg/L of test item.
Moreover, stock solutions (10 g/L) of reference item Sodium Benzoate and sterilizing agent HgCl2 were prepared and an appropriate aliquot of the stock solution was added to the test medium (final volume: 250 mL or 150 mL ) in the test flasks to obtain a final concentration of 100 mg/L.
Activated sludge was added to each flask (with the exception of the abiotic control) before incubation.
Finally, the test flasks, containing stir bars covered with teflon, were kept under continuous stirring by the means of a magnetic stirrer for the 28-days test period.

- Test temperature: The incubation temperature was continuously monitored during the course of the study by means of a data logger.
It was in the range 21.1 – 21.4 °C with a mean value of 21.3 °C and a standard deviation of 0.05 °C.
- pH of test flask: 7.36 – 7.65 at the test start. 7.34 – 8.15 at the test end.
The pH of test item mixtures at the end of test period was 7.53 (mean value), within the provided range by guideline of 6.0 - 8.5.

Determination of BOD: The sensor on the top of the test bottle detects the pressure decrease inside the bottles and gives the measure as BOD, expressed in mg O2/L. The BOD value is registered daily except for not working days.
Determination of ThOD: It was calculated on the basis of the chemical formula and molecular weight of reference item.
Temperature: Test temperature was recorded by the means of a data logger.
pH: At the start of the test, pH values of test solutions were measured taking a small aliquot of solutions from the flasks before the addition of the activated sludge (inoculum). At the end of the incubation period, pH was measured in all test flasks

Light conditions: Darkness
Stirring: Continuous
Test duration: 28 days
Continuous darkness: yes


SAMPLING
The assay on test item was carried out in parallel with the following bottles:
- a known ready biodegradable reference item (one replicate), Sodium Benzoate, at 100 mg/L;
- two inoculum control replicates (blank);
- two test item replicates at 50 mg/L concentration;
- one abiotic control containing the test item at 50 mg/L and a sterilizing agent (HgCl2) at 100 mg/L (without inoculum);
- one toxicity control, containing the test item at 50 mg/L and reference item at 100 mg/L.
BOD flasks of 500 mL capacity, equipped with pressure sensors fitted on the bottle mouth, were used as test units.


NITRIFACATIONS
Nitrate and nitrite concentration were measured at the start and at the test end.
At the start of test, nitrate and nitrite were not observed.
At the end of test (28 days) nitrite was not observed.
The quantity of nitrate in the inoculum control flasks (as mean value) was subtracted from the mean nitrate concentration determined in the test item flasks.
The oxygen consumed in the formation of nitrate was calculated multiplying the increase of nitrate-N concentration by a factor of 4.57, which is the oxygen consumed per gram of nitrogen, as the oxygen taken up by 14 g of nitrogen is 64 g (OECD Guideline No. 301, 1992).
Reference substance:
other: Sodium Benzoate
Remarks:
EC No.: 208-534-8 , CAS No.: 532-32-1
Test performance:
CONDITIONS FOR THE VALIDITY OF THE TEST

The test complies with the validity criteria because:
- The mean oxygen uptake in the inoculum control at the end of test period (28 days) was 27 mg O2/L, not exceeding the maximum value provided by OECD guideline 301F of 60 mg O2/L.
- The difference between duplicate values of test item removal was 5%; this value was within the maximum value provided by OECD guideline 301F of 20 %.
- The reference item exceeded the biodegradation pass level (60%) in 14 days (79.6%), within the guideline fixed range by OECD guideline 301F.
- The toxicity control exceeded the 25 % value (31.2 %), within the first 14 days period as provided by OECD Guideline 301F, showing that no inhibitory effect was due to test item.
- The pH of test item mixtures at the end of test period was 7.53 (mean value), within the provided range by guideline of 6.0 – 8.5.
Key result
Parameter:
% degradation (O2 consumption)
Value:
-7
Sampling time:
28 d
Details on results:
A study to evaluate the ready biodegradability of the test item 2-amino-5-chloro-N,3-dimethylbenzamide, following the “Manometric Respirometry” method, was performed.
Test item concentration should be 100 mg/L or a concentration giving at least 50-100 ThOD or COD/L.
The tested concentration was 50 mg/L (corresponding to 93.5 mg ThOD/L).
All reported results are related to the nominal concentration of test item, since the test concentrations were not measured during the test.
The biodegradation was followed by measuring the oxygen uptake (mg/L) of the test item under defined conditions by the means of a manometric method and calculating from these values the BOD (Biochemical Oxygen Demand).
The ThODNH3 value (Theoretical Oxygen Demand without nitrification process) was calculated to be equal to 1.67 mg O2/mg for reference item, Sodium Benzoate. while ThODNO3 value (Theoretical Oxygen Demand with nitrification process) was assessed to be 1.87 mg O2/mg for the test item.
These values, 1.67 mg O2/mg for reference item and 1.87 mg O2/mg for the test item, were used to calculate per % biodegradation.
Nitrate and nitrite concentration were measured at the start and at the test end.
At the start and test end no nitrite were present.
At the test end nitrate concentration in the blank bottles (10.00 mg/L as mean value) was slightly higher than nitrate concentration found in test item bottles (9.15 mg/L as mean value), therefore the found negative value (-0.219 mg/L) was assumed as equal to zero, so Nitrification wasn’t occurred during test period.
At the end of the test the biodegradation was -7.0% at the end of the test (mean percentage values between two replicates).
Therefore, the test item, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, since the 10-day window wasn’t start and a maximum biodegradation of -7.0% was found at the end of the test, (mean percentage values between two replicates).
No significant degradation was observed in the abiotic control (equal to 0.0% at the test end).
The toxicity control clearly showed that the test item does not cause inhibitory effect on the microorganisms activity at the tested concentration. Actually, the toxicity control exceeded the 25 % pass level within the 14 days of exposure (31.2%).
The reference item exceeded (79.6%) the biodegradation pass level (60%) within the range fixed by OECD Guideline 301F (first 14 days).

BOD value in the test flasks (mg/L):

DATE

TIME (DAYS)

BOD mg O2/L (letture testine)

BLANK 1

BLANK 2

REF. ITEM

TEST ITEM 1

TEST ITEM 2

TOX. CONTROL

ABIOTIC CONTROL

31/01/2020

1

1

0

14

0

1

29

0

01/02/2020

2

4

3

70

3

4

70

0

02/02/2020

3

9

7

98

4

7

67

0

03/02/2020

4

10

9

104

6

7

99

0

04/02/2020

5

14

12

115

9

10

109

0

05/02/2020

6

14

15

125

9

12

122

0

06/02/2020

7

15

17

131

10

14

125

0

07/02/2020

8

17

17

137

10

15

132

0

08/02/2020

9

18

18

140

12

15

135

0

09/02/2020

10

19

20

145

13

14

138

0

10/02/2020

11

20

21

148

14

17

140

0

11/02/2020

12

20

23

150

14

17

145

0

12/02/2020

13

20

24

152

14

16

145

0

13/02/2020

14

20

24

155

15

18

146

0

14/02/2020

15

21

25

156

15

18

147

0

15/02/2020

16

23

25

157

15

18

148

0

16/02/2020

17

23

25

160

15

18

151

0

17/02/2020

18

23

26

162

15

18

151

0

18/02/2020

19

23

26

162

15

18

151

0

19/02/2020

20

23

27

163

15

19

151

0

20/02/2020

21

23

28

164

15

20

151

0

21/02/2020

22

23

28

164

15

20

154

0

22/02/2020

23

23

28

165

17

20

154

0

23/02/2020

24

23

28

165

17

20

154

0

24/02/2020

25

25

29

167

17

20

154

0

25/02/2020

26

25

29

167

18

20

154

0

26/02/2020

27

25

29

167

18

21

154

0

27/02/2020

28

25

29

167

20

21

154

0

*Mean oxygen uptake in the inoculum control at the end of test period (28 days) was equal to 27 mg/L,

according to the maximum value provided by OECD Guideline of 60 mg O2/L.

BOD mg O2/mg of test item:

DATE

BOD mg02/mg test item

REF. ITEM

TEST ITEM 1

TEST ITEM 2

TEST ITEM MEAN

TOXiCITY CONTROL

ABIOTIC CONTROL

31/01/2020

0.1350

-0.0100

0.0100

0.0000

0.2817

0.000

01/02/2020

0.6650

-0.0100

0.0100

0.0000

0.6117

0.000

02/02/2020

0.9000

-0.0800

-0.0200

-0.0500

0.8567

0.000

03/02/2020

0.9450

-0.0700

-0.0500

-0.0600

0.9317

0.000

04/02/2020

1.0200

-0.0800

-0.0600

-0.0700

1.0033

0.000

05/02/2020

1.1050

-0.1100

-0.0500

-0.0800

1.0083

0.000

06/02/2020

1.1500

-0.1200

-0.0400

-0.0800

1.0533

0.000

07/02/2020

1.2000

-0.1400

-0.0400

-0.0900

1.0667

0.000

08/02/2020

1.2200

-0.1200

-0.0600

-0.0900

1.0800

0.000

09/02/2020

1.2550

-0.1300

-0.1100

-0.1200

1.0750

0.000

10/02/2020

1.2750

-0.1300

-0.0700

-0.1000

1.1083

0.000

11/02/2020

1.2850

-0.1500

-0.0900

-0.1200

1.0917

0.000

12/02/2020

1.3000

-0.1600

-0.0800

-0.1200

1.0933

0.000

13/02/2020

1.3300

-0.1400

-0.0800

-0.1100

1.1033

0.000

14/02/2020

1.3300

-0.1600

-0.1000

-0.1300

1.0967

0.000

15/02/2020

1.3300

-0.1800

-0.1200

-0.1500

1.0800

0.000

16/02/2020

1.3600

-0.1800

-0.1200

-0.1500

1.1100

0.000

17/02/2020

1.3750

-0.1900

-0.1300

-0.1600

1.1017

0.000

18/02/2020

1.3750

-0.1900

-0.1300

-0.1600

1.1017

0.000

19/02/2020

1.3800

-0.2000

-0.1200

-0.1600

1.0933

0.000

20/02/2020

1.3850

-0.2100

-0.1100

-0.1600

1.0850

0.000

21/02/2020

1.3850

-0.1700

-0.1100

-0.1400

1.1150

0.000

22/02/2020

1.3950

-0.1700

-0.1100

-0.1400

1.1150

0.000

23/02/2020

1.3950

-0.1700

-0.1100

-0.1400

1.1150

0.000

24/02/2020

1.4000

-0.2000

-0.1400

-0.1700

1.0900

0.000

25/02/2020

1.4000

-0.1800

-0.1400

-0.1600

1.0900

0.000

26/02/2020

1.4000

-0.1800

-0.1200

-0.1500

1.0900

0.000

27/02/2020

1.4000

-0.1400

-0.1200

-0.1300

1.0900

0.000

Oxygen uptake (mg):

DATE

mg UPTAKE 02

BLANK 1

BLANK 2

BLANK

MEAN

REF. ITEM

TEST ITEM 1

TEST ITEM 2

TOX. CONTROL

ABIOTIC CONTROL

31/01/2020

0.25

0.00

0.13

3.50

0.00

0.25

4.35

0.00

01/02/2020

1.00

0.75

0.88

17.50

0.75

1.00

10.05

0.00

02/02/2020

2.25

1.75

2.00

24.50

1.00

1.75

14.85

0.00

03/02/2020

2.50

2.25

2.38

26.00

1.50

1.75

16.35

0.00

04/02/2020

3.50

3.00

3.25

28.75

2.25

2.50

17.40

0.00

05/02/2020

3.50

3.75

3.63

31.25

2.25

3.00

18.30

0.00

06/02/2020

3.75

4.25

4.00

32.75

2.50

3.50

18.75

0.00

07/02/2020

4.25

4.25

4.25

34.25

2.50

3.75

19.80

0.00

08/02/2020

4.50

4.50

4.50

35.00

3.00

3.75

20.25

0.00

09/02/2020

4.75

5.00

4.88

36.25

3.25

3.50

20.70

0.00

10/02/2020

5.00

5.25

5.13

37.00

3.50

4.25

21.00

0.00

11/02/2020

5.00

5.75

5.38

37.50

3.50

4.25

21.75

0.00

12/02/2020

5.00

6.00

5.50

38.00

3.50

4.50

21.75

0.00

13/02/2020

5.00

6.00

5.50

38.75

3.75

4.50

21.90

0.00

14/02/2020

5.25

6.25

5.75

39.00

3.75

4.50

22.05

0.00

15/02/2020

5.75

6.25

6.00

39.25

3.75

4.50

22.20

0.00

16/02/2020

5.75

6.25

6.00

40.00

3.75

4.50

22.20

0.00

17/02/2020

5.75

6.50

6.13

40.50

3.75

4.50

22.65

0.00

18/02/2020

5.75

6.50

6.13

40.50

3.75

4.50

22.65

0.00

19/02/2020

5.75

6.75

6.25

40.75

3.75

4.75

22.65

0.00

20/02/2020

5.75

7.00

6.38

41.00

3.75

5.00

22.65

0.00

21/02/2020

5.75

7.00

6.38

41.00

4.25

5.00

23.10

0.00

22/02/2020

5.75

7.00

6.38

41.25

4.25

5.00

23.10

0.00

23/02/2020

5.75

7.00

6.38

41.25

4.25

5.00

23.10

0.00

24/02/2020

6.25

7.25

6.75

41.75

4.25

5.00

23.10

0.00

25/02/2020

6.25

7.25

6.75

41.75

4.50

5.00

23.10

0.00

26/02/2020

6.25

7.25

6.75

41.75

4.50

5.25

23.10

0.00

27/02/2020

6.25

7.25

6.75

41.75

5.00

5.25

23.10

0.00

Percentage of biodegradation in test item, reference item, toxicity control and abiotic flask:

DATE

% BIODEGRADATION

REF. ITEM

TEST ITEM

Replicate

1

TEST ITEM

Replicate

2

TEST ITEM MEAN

TOXICITY CONTROL

ABIOTIC CONTROL

31/01/2020

8.1

-0.5

0.5

0.0

8.0

0.0

01/02/2020

39.8

-0.5

0.5

0.0

17.3

0.0

02/02/2020

53.9

-4.3

-1.1

-2.7

24.2

0.0

03/02/2020

56.6

-3.7

-2.7

-3.2

26.3

0.0

04/02/2020

61.1

-4.3

-3.2

-3.7

28.3

0.0

05/02/2020

66.2

-5.9

-2.7

-4.3

28.5

0.0

06/02/2020

68.9

-6.4

-2.1

-4.3

29.8

0.0

07/02/2020

71.9

-7.5

-2.1

-4.8

30.1

0.0

08/02/2020

73.1

-6.4

-3.2

-4.8

30.5

0.0

09/02/2020

75.1

-7.0

-5.9

-6.4

30.4

0.0

10/02/2020

76.3

-7.0

-3.7

-5.3

31.3

0.0

11/02/2020

76.9

-8.0

-4.8

-6.4

30.8

0.0

12/02/2020

77.8

-8.6

-4.3

-6.4

30.9

0.0

13/02/2020

79.6

-7.5

-4.3

-5.9

31.2

0.0

14/02/2020

79.6

-8.6

-5.3

-7.0

31.0

0.0

15/02/2020

79.6

-9.6

-6.4

-8.0

30.5

0.0

16/02/2020

81.4

-9.6

-6.4

-8.0

31.4

0.0

17/02/2020

82.3

-10.2

-7.0

-8.6

31.1

0.0

18/02/2020

82.3

-10.2

-7.0

-8.6

31.1

0.0

19/02/2020

82.6

-10.7

-6.4

-8.6

30.9

0.0

20/02/2020

82.9

-11.2

-5.9

-8.6

30.6

0.0

21/02/2020

82.9

-9.1

-5.9

-7.5

31.5

0.0

22/02/2020

83.5

-9.1

-5.9

-7.5

31.5

0.0

23/02/2020

83.5

-9.1

-5.9

-7.5

31.5

0.0

24/02/2020

83.8

-10.7

-7.5

-9.1

30.8

0.0

25/02/2020

83.8

-9.6

-7.5

-8.6

30.8

0.0

26/02/2020

83.8

-9.6

-6.4

-8.0

30.8

0.0

27/02/2020

83.8

-7.5

-6.4

-7.0

30.8

0.0

The difference between duplicate values of test item removal at the end of the test was 5 %; this value

was within the maximum value provided by OECD Guideline of 20 %.

Value of biodegradation at the end of the test is -7.0%

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item, 2-amino-5-chloro-N,3-dimethylbenzamide, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, having showed a maximum biodegradation of -7.0% at the end of the test (mean percentage values between two replicates).
No significant degradation was observed in the abiotic control.
The test item showed no inhibitory effects on the microorganism activity at the tested concentration, as demonstrated by the biodegradation percentage in the toxicity control exceeding the 25% pass level within 14 days of exposure (31.2%).
Executive summary:

For 2 -amino-5 -chloro-N,3 -dimethylbenzamide a 28 days biodegradation test according to OECD 301F (1992) was performed. The study lead to no significant biodagradation measured by oxygen demand.

No significant degradation was observed in the abiotic control (equal to 0.0% at the test end). The toxicity control clearly showed that the test item does not cause inhibitory effect on the microorganisms activity at the tested concentration. Actually, the toxicity control exceeded the 25 % pass level within the 14 days of exposure (31.2%).

The reference item exceeded (79.6%) the biodegradation pass level (60%) within the range fixed by OECD Guideline 301F (first 14 days).

Description of key information

The test item, 2-amino-5-chloro-N,3-dimethylbenzamide, was found to be not ready and completely biodegradable under the conditions applied in a manometric respirometry test, having shown a maximum biodegradation of -7.0% at the end of the test (mean percentage values between two replicates).

Biological results: Maximum biodegradation at the end of the 10-day window: the 10-day window was not started.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information