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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jan - 19 Feb 2016
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)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: JA9215
- Expiration date of the lot/batch: 2017.08.21
- Purity test date: 2015.09.02


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-25 °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): Shenyang Northern Sewage Treatment Plant, aeration tank. Activated sludge was collected two days before the test
- Storage conditions: aerated until use
- Preparation of inoculum for exposure: removal of coarse particles by sieve. Sludge was settled, decanted, resuspended in mineral medium and concentrated twice, final resuspension in mineral medium
- Concentration of sludge: 3806 mg/L suspended solids at time of test.
Duration of test (contact time):
28 d
Initial conc.:
86.7 mg/L
Based on:
test mat.
Initial conc.:
50.3 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: standard mineral medium per OECD 301F
- Test temperature: 21.2 - 22.9 °C
- pH: 7.48-7.50 (initial), 7.63-7.96 (final)
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not stated

TEST SYSTEM
- Culturing apparatus: respirometers, 1-L flasks
- Number of culture flasks/concentration: two
- Measuring equipment: pressure sensors in cap of flask with daily automated data storage
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: 4 mL of 10 M NaOH

CONTROL AND BLANK SYSTEM
- Inoculum blank: two
- Toxicity control: one, 86.7 mg/L test substance + 31.4 mg/L reference
Reference substance:
benzoic acid, sodium salt
Remarks:
32.5 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
26.1
Sampling time:
28 d
Details on results:
Toxicity controls reached 58.1% BOD/ThOD by day 14. Substance is not inhibitory to activated sludge.

The reference substance was 66.2% degraded by day 4, 87.2% by day 14, and 89.5% by day 28

Table 1, Oxygen demand and biodegradation of the test substance.

Oxygen uptake (mg O2/L)  Oxygen uptake Oxygen uptake Oxygen uptake

Oxygen uptake

 BOD (mg O2/mg test item

BOD

BOD

% Biodegradation

Day

Inoculum control 1

Inoculum control 2

Average

Test sample 1

Test sample 2

Test sample 1

Test sample 2

Average

Test sample 1

Test sample 2

Average

1

2.29

2.79

2.5

2.58

2.50

0.001

0.000

0.001

0.2%

0.0%

0.1%

2

4.44

5.04

4.7

4.69

4.25

0.000

0.000

0.000

0.0%

0.0%

0.0%

3

6.29

6.85

6.6

6.60

6.16

0.000

0.000

0.000

0.0%

0.0%

0.0%

4

8.06

8.60

8.3

8.35

8.00

0.001

0.000

0.001

0.2%

0.0%

0.1%

5

9.91

10.35

10.1

10.62

9.95

0.006

0.000

0.003

1.0%

0.0%

0.5%

6

11.12

11.54

11.3

12.89

11.77

0.018

0.005

0.012

3.1%

0.9%

2.0%

7

11.45

11.93

11.7

16.56

13.83

0.056

0.025

0.041

9.7%

4.3%

7.0%

8

12.00

12.83

12.4

22.89

17.37

0.121

0.057

0.089

20.9%

9.8%

15.4%

9

12.43

13.24

12.8

23.89

23.03

0.128

0.118

0.123

22.1%

20.3%

21.2%

10

13.04

13.85

13.4

24.51

24.34

0.128

0.126

0.127

22.1%

21.7%

21.9%

11

13.62

14.37

14.0

25.09

24.97

0.128

0.127

0.128

22.1%

21.9%

22.0%

12

14.20

14.89

14.5

25.86

25.70

0.131

0.129

0.130

22.6%

22.2%

22.4%

13

15.20

15.66

15.4

26.89

26.41

0.133

0.127

0.130

22.9%

21.9%

22.4%

14

15.76

16.29

16.0

28.01

27.13

0.139

0.128

0.134

24.0%

22.1%

23.1%

15

16.06

16.45

16.3

28.20

27.53

0.137

0.130

0.134

23.6%

22.4%

23.0%

16

16.33

16.74

16.5

28.84

28.18

0.142

0.135

0.139

24.5%

23.3%

23.9%

17

16.95

17.45

17.2

29.65

29.16

0.144

0.138

0.141

24.8%

23.8%

24.3%

18

17.37

17.93

17.7

30.30

29.90

0.145

0.141

0.143

25.0%

24.3%

24.7%

19

17.64

18.37

18.0

30.78

30.51

0.147

0.144

0.146

25.3%

24.8%

25.1%

20

18.12

18.97

18.5

31.36

31.32

0.148

0.148

0.148

25.5%

25.5%

25.5%

21

18.72

19.60

19.2

31.95

32.03

0.147

0.148

0.148

25.3%

25.5%

25.4%

22

19.16

20.10

19.6

32.38

32.67

0.147

0.151

0.149

25.3%

26.0%

25.7%

23

19.53

20.41

20.0

32.63

33.11

0.146

0.151

0.149

25.2%

26.0%

25.6%

24

19.62

20.66

20.1

32.92

33.82

0.148

0.158

0.153

25.5%

27.2%

26.4%

25

20.62

21.20

20.9

33.72

34.42

0.148

0.156

0.152

25.5%

26.9%

26.2%

26

20.80

21.32

21.1

33.78

34.63

0.146

0.156

0.151

25.2%

26.9%

26.1%

27

21.53

21.78

21.7

34.03

35.30

0.142

0.157

0.150

24.5%

27.1%

25.8%

28

21.87

22.10

22.0

34.26

35.92

0.141

0.161

0.151

24.3%

27.8%

26.1%

Table 2, Oxygen demand and biodegradation in toxicity and positive controls.

Oxygen uptake (mg O2/L) Oxygen uptake Oxygen uptake Oxygen uptake Oxygen uptake

BOD (mg O2/mg test item)

% Biodegradation 

Day

Inoculum control 1

Inoculum control 2

Average

Tox control

Ref sample

Ref sample

Tox control

Ref sample

1

2.29

2.79

2.5

22.22

22.76

0.624

19.2%

37.4%

2

4.44

5.04

4.7

32.61

33.59

0.889

27.2%

53.2%

3

6.29

6.85

6.6

36.92

38.98

0.997

29.5%

59.7%

4

8.06

8.60

8.3

41.13

44.25

1.106

32.0%

66.2%

5

9.91

10.35

10.1

46.88

48.86

1.193

35.8%

71.4%

6

11.12

11.54

11.3

51.85

51.73

1.244

39.5%

74.5%

7

11.45

11.93

11.7

57.89

53.56

1.288

45.0%

77.1%

8

12.00

12.83

12.4

65.16

55.67

1.332

51.4%

79.8%

9

12.43

13.24

12.8

67.66

56.73

1.352

53.4%

81.0%

10

13.04

13.85

13.4

70.08

58.14

1.377

55.2%

82.5%

11

13.62

14.37

14.0

71.89

59.39

1.397

56.4%

83.7%

12

14.20

14.89

14.5

73.43

60.58

1.418

57.4%

84.9%

13

15.20

15.66

15.4

74.93

62.16

1.439

58.0%

86.2%

14

15.76

16.29

16.0

75.68

63.29

1.456

58.1%

87.2%

15

16.06

16.45

16.3

77.30

64.00

1.468

59.4%

87.9%

16

16.33

16.74

16.5

77.89

64.72

1.484

59.8%

88.9%

17

16.95

17.45

17.2

78.89

65.81

1.496

60.1%

89.6%

18

17.37

17.93

17.7

79.53

66.52

1.503

60.2%

90.0%

19

17.64

18.37

18.0

80.16

67.01

1.508

60.5%

90.3%

20

18.12

18.97

18.5

80.84

67.58

1.511

60.7%

90.5%

21

18.72

19.60

19.2

81.63

68.24

1.509

60.8%

90.4%

22

19.16

20.10

19.6

82.15

68.66

1.510

60.9%

90.4%

23

19.53

20.41

20.0

82.63

68.97

1.507

61.0%

90.2%

24

19.62

20.66

20.1

83.45

69.14

1.509

61.7%

90.4%

25

20.62

21.20

20.9

83.78

69.91

1.508

61.2%

90.3%

26

20.80

21.32

21.1

83.99

69.97

1.504

61.2%

90.1%

27

21.53

21.78

21.7

84.55

70.37

1.498

61.2%

89.7%

28

21.87

22.10

22.0

84.95

70.56

1.495

61.3%

89.5%

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
FBSEE diol is not readily biodegradable (26.1% BOD/ThOD in a 28-day OECD 301F test)
Executive summary:

Biodegradability of FBSEE diol was assessed in a OECD 301F assay. Sodium benzoate was used as reference substance and in a toxicity control. The reference bottle achieved a passing level (>60% BOD/ThOD) by day 4. FBSEE diol achieved an average of 26.1% biodegradation in duplicate test substance bottles. The toxicity control containing both the reference and test substances achieved 61.3% BOD/ThOD by the end of the test. FBSEE diol is not readily biodegradable, but does not inhibit biodegradation.

The test was conducted according to internationally accepted guidelines in accord with GLP criteria. It is considered reliable without restrictions and is acceptable for Risk Assessment, Classification & Labeling, and PBT Analysis.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 Nov 2010 – 11 Jan 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 835.3200 (Zahn-Wellens / EMPA Test)
Deviations:
yes
Remarks:
Analyte specific detection only, without BOD or DOC analysis. Sludge was used without washing.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: L-21256, lead registrant internal source. EPA consent order agreement number P-09-0485
- Expiration date of the lot/batch: 26 Nov 2015
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Test substance delivered as 3.37 mg/mL solution in acetone. Test substance was stable.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Remarks:
(mix of domestic, commercial, and industrial wastewater from a large urban center)
Details on inoculum:
- Source of inoculum/activated sludge: Metro Wastewater Treatment Plant, St. Paul, MN USA. Mixed liquor suspended solids were collected from the aeration basin located between primary and secondary treatment. Alternately, material was taken from secondary treatment when the aeration basin could not be accessed. Dry weight of sludge was determined.
- Storage conditions: sludge was allowed to settle overnight before use.
- Preparation of inoculum for exposure: Settled sludge was diluted 1/10x in mineral salts medium per OPPTS 835.3200.
Duration of test (contact time):
63 d
Initial conc.:
5.06 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
other: Degradation product analysis using authentic standards
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral salts medium per test guideline
- Solubilising agent: acetone, 1.5 µL/mL
- Test temperature: 25 ± 5 °C
- pH: initial pH ca. 7.0. All active cultures had acidified to pH 5.0-5.5 by Day 7. This occured in active cultures regardless of addition of test or reference substance.
- pH adjusted: yes, neutrality of medium restored by addition of either phosphate buffer (flask cultures) or 1N NaOH (vial cultures).

TEST SYSTEM
- Culturing apparatus: Two types of vessels were used. The main experiment used loosely capped 125-mL polymethylpentene flasks. A secondary experiment used 42-mL septum-sealed amber glass vials.
- Number of culture flasks/concentration: three for each of eight time points. In addition duplicate samples were made for each time point and spiked with analyte at time of collection (laboratory matrix spikes)
- Method used to create aerobic conditions: Flask cultures were shaken at 150 RPM to maintain aerobic conditions. Headspace in the vial cultures was flushed with pure oxygen to create and maintain aerobic conditions, with additional oxygen injected on day 28 (after detection of hydrogen sulfide in the headspace). Vial cultures were also shaken to keep material suspended.
- Measuring equipment: pH was monitored using pH paper.
- Test performed in closed vessels due to significant volatility of test substance: The secondary test was conducted to evaluate potential volatile transformation products. This phase of the experiment will be referred to as the headspace test henceforth.
- Concentration of sludge: 1.58 g/L, or 31.6 mg/20-mL incubation volume. Additional settled sludge was added on days 14, 28, and 42, adding 700 mg, 748 mg, and 397 mg, respectively, during the main test.

SAMPLING
- Sampling frequency: Days 0, 1, 3, 7, 14, 28, 42, and 63
- Sampling method: Entire test vessels were sacrificed at each sampling interval. Test vessels were placed in a refrigerator to 2-4 °C to temporarily reduce the metabolic activity of the cultures, and designated LMS cultures were fortified with target analytes. Cultures were then frozen until analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate for each time point.
- Abiotic sterile control: Sterile controls were either entirely abiotic (mineral salts medium only), or were set up using autoclave-sterilized sludge in an amount equal to that in the active cultures. Controls containing test substace were set up in triplicate for each time point. Controls without test substance were set up in duplicate for each time point. In addition, duplicate samples with sterilized sludge were set up and spiked with analyte at the beginning of the test (laboratory control samples). Duplicate samples with sterilized sludge were also set up and spiked at the sampling time (additional laboratory matrix samples). Duplicate mineral medium vessels were set up and spiked at the sampling time (additional laboratory matrix samples).
- Toxicity control: Triplicate samples for each time point containing FBSEE diol and sodium dodecyl sulfate (SDS). Toxicity controls were given a fresh spike of SDS as well as additional settled sludge on days 14, 28, and 42 as per the main test.

- Other:
Duplicate positive controls were set up with SDS for each time point, with addition on days 14, 28, and 42 of a secondary injection of fresh sludge.
Duplicate sterilized sludge and mineral medium controls were set up with SDS for each time point.
A negative control, potassium perfluorohexanesulfonate, was also used. Duplicate controls were set up for each time point with active sludge, sterilized sludge, and mineral medium for each time point.

HEADSPACE TEST
The headspace test, using septum-sealed glass vials, followed the same sampling schedule as the main test. Test vessel set-ups were:
- One bioactive blank per time point
- One sterilized sludge blank per time point
- Duplicate laboratory control samples (all analytes spiked at beginning of test into sterilized sludge) per time point
- Duplicate test substance in bioactive sludge per time point
- Duplicate test substance in sterilized sludge per time point
Reference substance:
other: sodium dodecyl sulfate
Key result
Parameter:
% degradation (test mat. analysis)
Value:
99.6
Sampling time:
7 d
Details on results:
SDS concentration was
Concentrations of FBSEE diol did not measurably change within the test interval in abiotic (sterilized sludge or mineral medium) controls.

FBSEE diol was almost entirely depleted within one day in active cultures (Attachments 1-3, Table 1), both by itself and in toxicity controls. The corresponding diacid (Analyte A, 2,2'-{[(Nonafluorobutyl)-sulfonyl]imino}diacetic acid) appeared within the same time period and constitues the predominant degradation product.

A series of minor degradation production were also observed. A mass balance was calculated by determining the molar concentration and volume of cultures at each time point, and comparing the amount of each analyte thus determined with the molar amount of starting material. Mass balances were essentially complete by day 3 (Table 1). It should be noted that the expected intermediates in the biochemical pathway from diol to diacid were not among the analytes and may explain the incomplete mass balance on Days 0 and 1.

Table 1, Mole fraction analytes in the inherent biodegration study (main test, toxicity controls, and headspace test). Analysis by LC/MS except as indicated

Study day

FBSEE

diol³

2,2'-{[(Nona-

fluorobutyl)-sulfonyl]imino}diacetic acid

N-(2-Hydroxyethyl)-perfluorobutane sulfonamide

Perfluorobutane

sulfonamide

Perfluorobutane

sulfinic acid

Perfluorobutane

sulfonic acid

n-1H-

nonafluorobutane

iso-1H-

nonafluorobutane

total moles of analyte

Total mass balance³

Main study

0

91.4%

<LOQ

2.1%

<LOQ

1.6%

<LOQ

0.249

95.2%

1

7.6%

58.4%

<LOQ

1.9%

5.5%

<LOQ

0.192

73.5%

3

5.2%

110%

<LOQ

2.8%

3.5%

<LOQ

0.317

121%

7

1.5%

91.7%

<LOQ

2.4%

2.3%

<LOQ

0.256

98.0%

14¹

<LOQ

85.9%

<LOQ

2.3%

2.1%

1.5%

0.240

91.8%

28¹

1.5%

98.5%

<LOQ

2.4%

2.4%

2.2%

0.280

107%

42¹

<LOQ

86.3%

<LOQ

2.3%

1.4%

2.8%

0.243

92.8%

63

<LOQ

79.0%

<LOQ

1.7%

1.4%

2.3%

0.221

84.4%

Toxicity control

0

76.9%

<LOQ

1.93%

<LOQ

1.39%

<LOQ

0.210

80.2%

1

8.81%

55.0%

<LOQ

1.90%

4.39%

<LOQ

0.183

70.1%

3

2.11%

93.9%

<LOQ

2.86%

2.91%

<LOQ

0.266

102%

7

3.14%

92.5%

<LOQ

2.99%

2.41%

<LOQ

0.264

101%

14¹

<LOQ

99.0%

<LOQ

2.69%

1.93%

1.57%

0.275

105%

28¹

<LOQ

99.1%

<LOQ

3.15%

2.05%

2.97%

0.281

107%

42¹

<LOQ

90.1%

<LOQ

2.74%

1.38%

3.22%

0.255

97.4%

63

<LOQ

104%

<LOQ

2.83%

0.00%

2.92%

0.286

109%

Headspace

test

analysis by GC/MS analysis by GC/MS

0

77.8%

<LOQ

1.52%

<LOQ

2.33%

<LOQ

<LOQ

<LOQ

0.214

81.7%

1

28.4%

40.8%

<LOQ

1.55%

4.84%

<LOQ

<LOQ

<LOQ

0.198

75.6%

3

3.00%

95.6%

<LOQ

2.74%

5.07%

<LOQ

0.019%

0.0083%

0.278

106%

7

<LOQ

112%

<LOQ

3.54%

4.48%

<LOQ

0.024%

0.0086%

0.314

120%

14

<LOQ

121%

<LOQ

3.98%

4.01%

<LOQ

0.029%

0.0085%

0.338

129%

28²

<LOQ

113%

<LOQ

3.24%

4.25%

<LOQ

0.062%

0.0075%

0.316

121%

42

<LOQ

117%

<LOQ

3.89%

3.07%

<LOQ

0.059%

0.0047%

0.324

124%

63

<LOQ

112%

<LOQ

3.54%

3.29%

<LOQ

0.098%

0.0043%

0.312

119%

1, mole fractions were calculated by determining the molar quantity of each analyte and dividing by the intial amount of FBSEE diol (0.2615 mole in test vessel). Total mass balance is calculated by summing all analytes and dividing by the initial amount of FBSEE diol.

2, additional settled sludge injection.

3, additional oxygen injection.

Validity criteria fulfilled:
yes
Remarks:
reference substance degraded within 14 days, no removal in sterilized sludge controls
Interpretation of results:
inherently biodegradable
Remarks:
rapid primary degradation to form persistent metabolites
Conclusions:
FBSEE diol was rapidly converted to a dicarboxylic acid in an inherent biodegradation test (OPPTS835.3200 as modified to identify degradation products). Other minor metabolites were observed.
Executive summary:

FBSEE diol biodegradation and transformation products were examined in an inherent biodegradation test done under test guideline OPPTS835.3200, as modified to optimize for analysis of parent and potential degradation products, with goal of developing a mass balance for transformation products. Sewage sludge was derived from a municipal sewage system receiving mixed domestic and industrial waste. Individual test chambers were assembled in triplicate, with multiple controls. Controls included abiotic controls (both sterilized sludge and mineral medium), toxicity controls with sodium dodecyl sulfate (SDS), positive controls with SDS, and negative controls with potassium perfluorohexanesulfonate. Flasks received additional sludge at specific time points. In addition, a parallel series of cultures were set up in sealed vials to allow examination of volatile materials in the vial headspace. These headspace test cultures had pure oxygen injected into the vial headspace to help maintain aerobic conditions. All cultures were analyzed by HPLC/MS against a series of authentic standards. Headspace test vials were initially assessed using GC/MS before HPLC. FBSEE diol was not removed from abiotic cultures, but was sharply reduced by Day 1 in all active cultures with quantitative removal by Day 14. SDS was quantitatively removed from positive and toxicity controls within one day of incubation. The predominant metabolite of FBSEE diol was 2,2'-{[(Nonafluorobutyl)-sulfonyl]imino}diacetic acid. Other materials detected in the cultures were N-(2-Hydroxyethyl)-perfluorobutane sulfonamide, Perfluorobutane sulfonamide, Perfluorobutane sulfinic acid, and Perfluorobutane sulfonic acid. Perfluorobutanoic acid was examined but not detected. Two volatile products, n-1H-nonafluorobutane and iso-1H-nonafluorobutane, were detected in the headspace test, although Perfluorobutane sulfonic acid was not. Mass balances were essentially complete by Day 3 of the experiment.

The experiment was conducted in accord with national guidelines with acceptable modifications to include analysis of test substance and potential degradation products, and was conducted under GLP criteria. It is considered reliable without restriction and is suitable for Risk Assessment, Classification & Labelling, and PBT Analysis.

Description of key information

FBSEE diol is not readily biodegradable (26.1% BOD/ThOD in a 28-day OECD 301F test), but was rapidly converted to a dicarboxylic acid in an inherent biodegradation test (OPPTS835.3200 as modified to identify degradation products).

Key value for chemical safety assessment

Additional information

Biodegradation of FBSEE diol was examined in two studies. The key study was ready biodegradation test done under OECD Guideline 301F (Manometric respirometry) and in compliance with GLP criteria. FBSEE diol was not inhibitory to degradation of the sodium bonzoate reference, but was not readily biodegradable. To better characterize biodegradability, the inherent biodegradability test guideline OPPTS832.3200 was modified to identify biodegradation products and provide a mass balance for the degradation pathway. FBSEE diol was not removed from abiotic controls, but was sharply reduced by Day 1 in all active cultures with quantitative removal by Day 14. The predominant metabolite of FBSEE diol was 2,2'-{[(Nonafluorobutyl)-sulfonyl]imino}diacetic acid. Other materials detected in the cultures were N-(2-Hydroxyethyl)-perfluorobutane sulfonamide, Perfluorobutane sulfonamide, Perfluorobutane sulfinic acid, and Perfluorobutane sulfonic acid. Perfluorobutanoic acid was examined but not detected. Two volatile products, n-1H-nonafluorobutane and iso-1H-nonafluorobutane, were detected in the headspace test, although Perfluorobutane sulfonic acid was not. Mass balances were essentially complete by Day 3 of the experiment. This test was also done in compliance with GLP criteria and is considered reliable without restrictions. However, because of the optimization for determining degradation products and mass balance, this test does not provide information on fulfilling specific criteria for inherent biodegradability as defined in OECD guidelines.