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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation Date: 13 August 2018 and Study Completion Date: 29 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
Adopted by the Council on 17 July 1992. OECD, Paris, France.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: XTJ-785, experimental
Lot No.: 9570-2-6738
CAS No.: Not listed
Purity: >92% C1214 alcohol, propoxylated, aminated, ethyoxylated
Expiry Date: No date established

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste.
- Laboratory culture: Approximately 8 L of activated sludge was collected on 21 August 2018 and transported to Smithers Viscient

Upon arrival at Smithers Viscient, the sludge was passed through a 2-mm sieve and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral medium (SMV No. 21Aug18MM-A), the contents were centrifuged again, and the supernatant was discarded. The moisture content of the activated sludge was determined, using a Sartorius MA 37-1 US automated moisture analyzer, to be 95.89% and the percent solids was determined to be 4.11%. An inoculum solution (SMV No. 21Aug18inoc-A) with 15 mg suspended solids/mL was prepared (73.00 g wet weight sludge brought to 200 mL with mineral medium), stirred with a Teflon magnetic stir bar at approximately 22 ± 2 °C, and aerated until used. The test suspension vessels, the blank vessels, the procedural control vessel, the toxicity control vessel, and the pH/TIC vessel all received 6.0 mL of the inoculum to produce an activated sludge concentration of 30 mg solids/L (Table 2).
Duration of test (contact time):
56 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
ThCO2
Remarks:
ThCO2 per vessel (mg C/L)
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test

Preparation of stock solutions using analytical grade reagents:

*Reagents for solution (a):
- Potassium dihydrogen orthophosphate, KH2PO4: 8.50 g.
- Dipotassium hydrogen orthophosphate, K2HPO4: 21.75 g.
- Disodium hydrogen orthophosphate dihydrate, Na2HPO4 2H2O: 33.40 g.
- Ammonium chloride, NH4Cl: 0.50 g.
Dissolve in water and make up to 1 Liter. The pH of the solution should be 7.4.

*Reagents for solution (b):
- Calcium Chloride dihydrate, CaCl2: 27.50 g.
- OR Calcium chloride dihydrate, CaCl2 2H2O: 36.40 g
Dissolve in water and make up to 1 liter

*Reagents for solution (c):
- Magnesium suplphate heptahydrate, MgSO4 7H2O: 22.50 g.

*Reagents for solution (d):
- Iron (III) Chloride hexahydrate, FeCl3 6H2O: 0.25 g.

Dissolve in water and make up to 1 Liter.
Note: in order to avoid having to prepare this solution immediately before use, add one drop of concentrated HCl per liter.

Preparation of mineral medium: Mix 10 mL of solution (a) with 800 mL water, add 1 mL of solution (b), (c) and (d) and make up to 1 Liter with water.

The aqueous medium for testing provided the essential nutrients, except for carbon, necessary to sustain the inoculum throughout the testing period. Salts with different amounts of hydration were used and weights were adjusted proportionately. High purity reagent grade water, free from inhibitory concentrations of toxic substances (e.g., Cu2+ ions), was used for the preparation of the mineral medium. Only one batch of water, which had been checked by DOC analysis, was used. The water contained no more than 10% of the total carbon content introduced by XTJ-785. The pH of the test medium upon preparation was 7.60 and was adjusted to 7.40 with 1 N hydrochloric acid.

- Test temperature: 20.4 to 21.5 °C
- pH: The pH of the test medium upon preparation was measured to be 7.60 and was adjusted to 7.40. The pH of the pH check vessel on day 0 was measured to be 7.19. The pH values of the test vessels ranged from 7.20 to 7.31 at the end of the study on day 28.
- pH adjusted: yes with concentrated hydrochloric acid
- Aeration of dilution water:
- Continuous darkness: yes

Each test unit consisted of a 4-L glass bottle with a rubber stopper into which one stainless steel needle with a Luer-Lok connection and two pieces of glass tubing were inserted. Prior to test initiation, the test vessels were washed with detergent and rinsed with water. The test vessels were then acid washed with 50% nitric acid and rinsed repeatedly with reagent grade water. The stainless-steel needle was extended through the stopper into the test solution serving as a sampling port for solution samples. A rubber policeman cap was used to cover the top of the sample port. The glass tubing provided the inlet and outlet ports for air exchange. CO2 free air was pumped under positive pressure through a hydration flask before entering the test system. The outlet port of each system was connected to two CO2 effluent gas traps, the first consisting of 200 mL of 0.2 N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2 N KOH. The test vessels were identified with the study number, replicate (A or B), and treatment type. Each test vessel was placed on a magnetic stir plate located in a dark environmental chamber set to maintain a temperature of 22 ± 2 °C.

TEST SYSTEM
- Culturing apparatus: the moisture content of the activated sludge was determined using a Sartorius MA-37-1 US automated moisture analyzer to be 95.89% and the percent solids was determined to be 4.11%.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: not available but all test systems with the exception of the pH/TIC vessel were aerated continuously for 56 days under positive pressure using CO2-free air in order to provide oxygen for the microbes and to capture evolved carbon dioxide.
- Measuring equipment: The amount of evolved CO2 in each trap was determined using a Shimadzu TOC V-CPH Carbon Analyzer
- Test performed in closed vessels due to significant volatility of test substance: no


SAMPLING
- Sampling frequency: On test days 1, 2, 5, 7, 9, 13, 16, 20, 23, 26, 28, and 35, a 7-mL sample was removed from the first KOH carbon dioxide trap on each test system and analyzed for CO2 evolution
- Sample storage before analysis: Aeration was continued overnight to drive any residual inorganic carbon from the test vessels.
After overnight aeration, 7-mL samples for analysis of CO2 evolution were removed from the first and second traps. The amount of evolved CO2 in each trap was determined using a Shimadzu TOC V-CPH Carbon Analyzer.


CONTROL AND BLANK SYSTEM
- Inoculum blank: two
- Positive control (Sodium Benzoate): One
- Toxicity control: one
- Other: An additional vessel was established in the same manner as the test suspension vessels but was only used for pH measurment and sampling for TIC and Total Carbon (TC) on day 0.

Reference substance
Reference substance:
other: Sodium benzoate
Remarks:
batch: SLBT3039, CAS: 532-32-1, purity: 100% and Total organic Carbon was calculated to be 58.29% based on the empirical formula (C7H5O2Na)

Results and discussion

Preliminary study:
n/a
Test performance:
The following table presents the acceptance criteria required by OECD Test Guideline 301 and Test Method 301B (OECD, 1992):

*Acceptability Criterion: Reference substance exhibits ≥60% biodegradation by day 14.
Study performance: Day 13 Net Percent CO2: 62.37%
Criterion Met (Yes/No): Yes

*Acceptability Criterion: Difference between replicates at the plateau and at test termination should be within ±20%.
Study performance: The difference between the replicates at the plateau and at test termination (i.e., day 56) did not exceed 20% variation.
Criterion Met (Yes/No): Yes

*Acceptability Criterion: The evolution of CO2 in the blank controls should be ≤40 mg C/L.
Study performance: Blank Control Mean: 24.45 mg C/L
Criterion Met (Yes/No): Yes

*Acceptability Criterion: Toxicity Control should be ≥25% degradation (based on CO2 evolution data) within 14 days for the test substance to be considered non-toxic.
Study performance: Evolved CO2: 50.60% at day 13
Criterion Met (Yes/No): Yes

*Acceptability Criterion: The inorganic carbon content of the test substance suspension in the mineral medium at the beginning of the test must be <5% of the total carbon.
Study performance: Inorganic carbon content of the test substance suspension in the mineral medium at the beginning of test was 3.5% of the total carbon
Criterion Met (Yes/No): Yes
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
46.13
Sampling time:
56 d
Remarks on result:
other: The test item cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301, Method B since it did not achieve 60% CO2 evolution within a 10-day window of reaching 10% biodegradation.
Remarks:
CO2 evolution reached an average value of 46% by the end of the extended test. This is a significant amount of mineralization and indicates that the test item would not be persistent in the environment.
Details on results:
The mean cumulative net percent CO2 evolved (percent biodegradation) from the aqueous test medium fortified with XTJ-785 at 10 mg C/L was 46.13% on day 56. The toxicity control produced a cumulative net percent CO2 of 50.60% (day 13) within 14 days, demonstrating that the test substance is not toxic to the inoculum as defined by OECD Guideline 301, Method B (i.e., <25% on day 14 is considered toxic).

The cumulative net percent CO2 evolved from the procedural control was 62.37% of theoretical by day 14, thus meeting the “pass” criteria of the test (reaching 60% or greater CO2 evolution by day 14). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

The difference between the replicates at the plateau and at test termination (i.e., day 56) did not exceed 20% variation.

The inorganic carbon content of the test substance vessels in the mineral medium at the beginning of the test was determined to be <5% of the total carbon.

The control blanks did not exceed 30 mg evolved CO2 per liter.

CO2 evolution:
The total inorganic carbon measured in the KOH traps was used to calculate the cumulative CO2 evolved from the test vessels. The mean cumulative CO2 values from the test item, procedural control, toxicity control, and blank control test vessels at day 56 were 41.37, 49.76, 64.53, and 24.45 mg/L, respectively. The mean cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, values for the test item, procedural control, and toxicity control were calculated to be 46.13, 69.01, and 54.64% respectively.

BOD5 / COD results

Results with reference substance:
The cumulative net percent CO2 evolved from the procedural control was 62.37% of theoretical by day 14, thus meeting the “pass” criteria of the test (reaching 60% or greater CO2 evolution by day 14). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Any other information on results incl. tables

Test item Determination of the Biodegradability-Total Inorganic Carbon(mg C/L) Measured in KOH Traps

Vessel

Replicate

Day

1

2

5

7

9

13

16

20

23

26

28

35

56

Trap 1

Trap 2

1

Test

Suspension A

13.92

19.09

52.19

72.87

84.92

113.3

132.9

160.7

176.8

186.4

195.9

218.2

271.8

31.43

2

Test

Suspension B

10.30

14.45

42.75

59.25

71.93

103.6

125.7

145.9

157.7

173.2

181.7

196.7

258.5

30.29

3

Inoculum

Blank A

12.39

20.93

41.19

52.40

59.38

72.83

80.65

89.55

95.05

102.5

106.6

119.8

150.6

29.96

4

Inoculum

Blank B

14.75

20.90

34.45

45.19

50.90

64.57

74.79

83.47

87.72

95.56

98.67

114.1

144.4

27.94

5

Procedural Control

18.51

59.86

120.7

144.4

156.4

182.1

196.0

210.0

220.0

230.0

232.8

278.0

327.2

27.52

6

Toxicity

Control

12.04

51.51

147.8

179.4

201.0

252.7

282.3

301.5

319.4

331.2

340.9

359.4

425.7

34.08



Test item: Determination of the Biodegradability-Cumulative CO2(mg/L) Evolved from the Test Vessels

Vessel

Replicate

Daya

1

2

5

7

9

13

16

20

23

26

28

35

56

1

Test Suspension A

3.40

4.50

11.87

15.94

17.85

22.85

25.67

29.66

31.12

31.21

31.13

32.81

42.38

2

Test Suspension B

2.52

3.41

9.72

12.96

15.12

20.89

24.28

26.93

27.76

29.00

28.87

29.57

40.36

 

Mean:

2.96

3.96

10.79

14.45

16.49

21.87

24.97

28.29

29.44

30.11

30.00

31.19

41.37

 

Std. Dev.:

0.63

0.77

1.52

2.11

1.93

1.38

0.98

1.93

2.38

1.56

1.60

2.29

1.43

3

Inoculum Blank A

3.03

4.94

9.36

11.46

12.48

14.69

15.58

16.53

16.73

17.16

16.94

18.01

25.02

4

Inoculum Blank B

3.61

4.93

7.83

9.89

10.70

13.02

14.44

15.41

15.44

16.00

15.68

17.15

23.89

 

Mean:

3.32

4.93

8.60

10.68

11.59

13.86

15.01

15.97

16.09

16.58

16.31

17.58

24.45

 

Std. Dev.:

0.41

0.01

1.08

1.12

1.26

1.18

0.80

0.79

0.91

0.82

0.89

0.61

0.80

5

Procedural Control

4.53

14.12

27.44

31.59

32.88

36.73

37.85

38.76

38.72

38.52

36.99

41.79

49.76

6

Toxicity Control

2.94

12.15

33.60

39.25

42.26

50.97

54.52

55.65

56.22

55.46

54.17

54.03

64.53

Corrected for removal of 7-mL KOH sample from each trap at each sampling interval beginning on day 1.

NOTES:    The following equation was used to determine evolve CO2mg/L = (TIC value 1sttrap × 3.667)*((Vol. remaining in trap 1 / Vol. originally in trap 1) /
(Vol. of Test Medium/Vol. KOH trap originally))

 For Day 56, the following is added to the value calculated above:
(TIC value 2ndtrap × 3.667)*((Vol. remaining in trap 2/Vol. originally in trap 2) / (Vol. of Test Medium/Vol. KOH trap originally))

 

Example Calculation for XTJ-785, Replicate A (Day 56):

((271.8 mg/L × 3.667) × ((137 mL / 200 mL)/(3000 mL / 200 mL))) +
((31.43 mg/L × 3.667) × ((100 mL / 100 mL)/(3000 mL / 100 mL))) = 42.38 mg/L

 Results were calculated using the actual analytical (unrounded) results and not the rounded values presented in this table.

3.667 is the molecular weight conversion factor for carbon to carbon dioxide.

Test item: Determination of the Biodegradability-Cumulative Net Percent CO2Evolved from the Test Vessels (Ultimate Biodegradation)

Vessel

Replicate

Day

1

2

5

7

9

13

16

20

23

26

28

35

56

1

Test Suspension A

0.23

-1.17

8.91

14.36

17.07

24.53

29.06

37.34

41.00

39.90

40.41

41.51

48.89

2

Test Suspension B

-2.18

-4.16

3.06

6.24

9.63

19.20

25.27

29.89

31.83

33.87

34.26

32.70

43.37

 

Mean:

-0.97

-2.67

5.98

10.30

13.35

21.86

27.17

33.62

36.42

36.88

37.34

37.11

46.13

 

Std. Dev.:

1.71

2.11

4.14

5.75

5.27

3.77

2.68

5.27

6.48

4.26

4.35

6.23

3.91

5

Procedural Control

3.29

25.05

51.39

57.04

58.06

62.37

62.29

62.16

61.74

59.81

56.40

66.02

69.01

6

Toxicity Control

-0.51

9.84

34.09

38.96

41.81

50.60

53.87

54.11

54.72

53.01

51.62

49.70

54.64

Notes: The following equation was used to determine % biodegradation

% Ultimate Biodegradability = (( mg CO2 produced) / ( mg TOC added x 3.667)) x100

Example Calculation for day 56 Test Suspension Mean value

((41.37 mg CO2 - 24.45 mg CO2) / ( 10 mg TOC x 3.667)) x 100 = 46.31%

Results were calculated using the actual analytical (unrounded) results and not the rounded values presented in this table.

All values were corrected for mean inoculum blank CO2 production.


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Based on the extent of CO2 evolution during this study, the test item cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B since it did not achieve 60% CO2 evolution within a 10-day window of reaching 10% biodegradation.
However, CO2 evolution reached an average value of 46% by the end of the extended test (Day 56). This is a significant amount of mineralization and indicates that the test substance would not be persistent in the environment.
Executive summary:

This study was performed to determine the potential for ultimate biodegradation ofthe test item in an aerobic aqueous medium by the carbon dioxide evolution method, OECD Guideline 301, Method B. The amount of carbon dioxide (CO2) released upon biodegradation of the test substance and a reference substance, sodium benzoate, was measured to assess the potential for ultimate biodegradation. Two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance), and a toxicity control (containing inoculum, reference substance, and the tzest item) were established to account for background CO2production, viability of the inoculum, and the toxicity of the test item, respectively. Test flasks were incubated aerobically in the dark for a period of 56 days. 

Results at Day 56:

The mean cumulative net percent CO2evolved (percent biodegradation) from the aqueous test medium fortified with the test item at 10 mg C/L was 46.13% on day 56. The toxicity control produced a cumulative net percent CO2of 50.60% (day 13) within 14 days, demonstrating that the test substance is not toxic to the inoculum as defined by OECD Guideline 301, Method B (i.e., <25% on day 14 is considered toxic).

The cumulative net percent CO2evolved from the procedural control was 62.37% of theoretical by day 14, thus meeting the “pass” criteria of the test (reaching 60% or greater CO2evolution by day 14). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

The difference between the replicates at the plateau and at test termination (i.e., day 56) did not exceed 20% variation.

The inorganic carbon content of the test substance vessels in the mineral medium at the beginning of the test was determined to be <5% of the total carbon.

The control blanks did not exceed 30 mg evolved CO2per liter.

Conclusion at Day 56 :

Based on the extent of CO2evolution during this study, the test substance cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301, Method B since it did notachieve 60% CO2evolution within a 10-day window of reaching 10% biodegradation. However, CO2evolution reached an average value of 46% by the end of the extended test. This is a significant amount of mineralization and indicates that the test substance would not be persistent in the environment.