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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 20, 2017 to November 02, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
The lab temperature slightly exceeded the 24°C upper tolerance limit on Day 28 of the study. However, the study author concluded that this slight deviation did not affect the test outcome.
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment: freshwater
Details on inoculum:
Surface water was collected from Malmesbury Pond, Sandymoor, Runcorn on 15th September 2017 and assigned a batch number of SE17002. It was filtered to remove coarse particles and aerated at 22 ± 2ºC prior to use. The surface water was then added to test media so as to achieve a final concentration of 100mL per litre of test media. The test suspensions were then aerated until the study pre-conditioning period was ready to commence on 20th September 2017.
Duration of test (contact time):
ca. 42 d
Initial conc.:
ca. 14.9 mg/L
Based on:
test mat.
Initial conc.:
ca. 29.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test medium
Test medium was prepared according to the method detailed in Cheshire Eco Solutions SOP III.19.

Test vessels
Conical flasks containing 3000 mL of test media.

Test and reference substances
Test material was added directly to the test vessels after being accurately weighed onto glass cover slips to give a final test concentrations of 14.9 mg/L and 29.9 mg/L. A control vessel was run in parallel, this contained inoculum but no test or reference material. A test vessel containing 102.9 mg (20 mg/L) of the reference material, sodium benzoate (Batch No: 304526) in 3 litres of test media, was also included in the test. In addition a vessel containing 102.9 mg (20 mg/L) of the reference material, sodium benzoate (Batch No: 304526) and test material at 29.9 mg/L in 3 litres of test media, was also included as an inhibition control.

Test conditions
3 Litre volumes of test media were stirred constantly in round, flat bottomed flasks in the dark. Carbon dioxide free air was passed through the test solutions at an estimated rate of 30 to 100mL per minute. This was in turn passed through two dreschel bottles, per test vessel, containing barium hydroxide of known concentration and volume. The tests were carried out in the laboratory at 22 ± 2C (See Comments). Single vessels were prepared for each test material concentration.

Measurements
Measurements were taken after the appearance of a precipitate of barium carbonate, this was on Day 2, 4, 7, 11, 14, 19, 23, 28, 35, 42 and 43 (post-acidification). The contents of each dreschel bottle were titrated against standardised hydrochloric acid to a pH 7 endpoint. Titres were taken for the barium hydroxide at the start and end of each time period.

Analysis of study data
Based on the titration values the quantities of carbon dioxide evolved over each incubation interval were determined based on the relationship of 1mL of titre being equivalent to 0.3 mg carbon. Since 1 mmol of CO2 is produced for every mmol of barium hydroxide reacted to barium chloride and 2 mmol of HCl are needed for the titration of the remaining barium hydroxide, and given that the molecular weight of CO2 is approximately 44 g, the weight of CO2 produced (mg) is calculated by:

0.1 x (Start titre [mL] - HCl titrated [mL]) x 44 / 2 = 2.2 x (Start titre [mL] - HCl titrated [mL]

Thus, the factor to convert volume of 0.1M HCl titrated to mg CO2 is 2.2. The percentage degradation of the test substance was determined from the ratio of total carbon evolved to carbon present, based on the carbon content of test substance of 66.91% w/w. This analysis was conducted by Scymaris Ltd, an outside contract laboratory, who are members of the UK GLP compliance monitoring programme.

The percentage biodegradation is calculated from:
% degradation = mg CO2 produced / (mg TOC added in test x 3.67)

where, 3.67 is the conversion factor (44/12) for carbon to carbon dioxide.


Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg/L
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
14.9 mg/L test substance
Value:
ca. 25.9
Sampling time:
42 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
29.9 mg/L test substance
Value:
ca. 42.8
Sampling time:
42 d
Details on results:
A figure of 60% degradation within 28 d is usually taken as being indicative of a good potential for degradation. According to this guideline, under these test conditions in the OECD 301B Procedure, test substance showed limited potential for degradation at the test concentrations of 14.9 mg/L and 29.9 mg/L. After 42 d test substance also showed limited potential for degradation at the test concentrations of 14.9 mg/L and 29.9 mg/L in this study. The final 42-d cumulative % degradation values were determined to be 25.9% at 14.9 mg/L and 42.8% at 29.9 mg/L.
Results with reference substance:
A degradation figure of 62.9% after 14 d was obtained for the reference material. This demonstrates that the inoculum was biologically active. A degradation figure of 64.3% after 14 d was obtained for the inhibition control. This demonstrates that the inoculum was biologically active in the presence of test substance at the higher test concentration of 29.9 mg/L.

Table 1: Mean titration values (mL) for barium hydroxide samples neutralised with 0.1 M hydrochloric acid in study CES170602

Day of Titration

 

Control

Sodium Benzoate (34.3 mg/L)

Test substance

(14.9 mg/L)

Test substance

(29.9 mg/L)

Inhibition Control*

2

11.7

35.1

17.6

11.2

36.0

4

7.8

19.7

9.9

9.6

21.0

7

6.5

20.4

10.3

15.4

17.9

11

17.7

24.3

15.0

23.2

18.5

14

11.9

18.9

10.8

9.2

26.4

19

7.4

9.6

8.0

12.4

11.4

23

4.3

5.1

8.2

7.3

7.2

28

12.4

13.0

12.5

15.6

14.1

35

7.5

9.8

8.9

13.4

13.1

42

4.8

3.2

4.5

9.8

7.2

43

5.4

5.1

4.4

12.9

8.1

* Inhibition Control contained sodium benzoate at 34.3 mg/L and test substance at 29.9 mg/L in 3 litres of test media.

The Table 1 values are the difference between the titres at the start and end of each time period.

Table 2: Evolved carbon dioxide values (mg) due to test or reference material in study CES170602

 

Period

 

Control

Sodium Benzoate (34.3 mg/L)

Test substance

(14.9 mg/L)

Test substance

(29.9 mg/L)

Inhibition Control*

2

25.7

77.2

38.7

24.6

79.2

4

17.2

43.3

21.8

21.1

46.2

7

14.2

44.9

22.7

33.9

39.4

11

38.8

53.5

33.0

51.0

40.7

14

26.1

41.6

23.8

20.2

58.1

19

16.3

21.1

17.6

27.3

25.1

23

9.5

11.2

18.0

16.1

15.8

28

27.2

28.6

27.5

34.3

31.0

35

16.4

21.6

19.6

29.5

28.8

42

10.6

7.0

9.9

21.6

15.8

43

11.9

11.2

9.7

28.4

17.8

* Inhibition Control contained sodium benzoate at 34.3 mg/L and test substance at 29.9 mg/L in 3 litres of test media.

Table 3: Cumulative biodegradation

Cumulative degradation (%)

Day of Titration

Sodium Benzoate (34.3 mg/L)

Test substance

(14.9 mg/L)

Test substance

(29.9 mg/L)

InhibitionControl**

2

23.4

11.8

*

24.3

4

35.3

16.0

1.3

37.5

7

49.2

23.7

10.2

48.9

11

55.8

18.4

15.8

49.8

14

62.9

16.3

13.1

64.3

19

65.1

17.5

18.1

68.3

23

65.9

25.3

21.1

71.2

28

66.5

25.6

24.4

72.9

35

68.9

28.5

30.3

78.6

42

67.3

27.9

35.3

81.0

43

67.0

25.9

42.8

83.7

* Indicates negative degradation

** Inhibition Control contained 102.9 mg sodium benzoate and test substance at 29.6 mg/L in 3 litres of test media.

Comments

Carbon Dioxide production in the Control vessels was 71.2 mg/L of test media in this study. This is within the acceptable limits defined in the method. The lab temperature slightly exceeded the 24°C upper tolerance limit on Day 28 of this study. However since reference degradation and control carbon dioxide production were both within acceptable limits, it was clear that this slight deviation did not affect the test outcome. The Inorganic Carbon content of the test substance suspension in the mineral medium at the beginning of the test was less than 5% of the Total Carbon for both test concentrations employed in this study. This analysis was carried out by Chemex Environmental, an approved sub-contract laboratory. As this analysis had no direct impact on the final test result, it was carried out as Non - GLP compliant.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, the test substance was determined to be not readily biodegradable, but can be considered to undergo inherent primary biodegradability.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', according to OECD Guideline 301B, using modified sturm test, in compliance with GLP. The substance was assessed for the rate and extent of biodegradation when exposed to freshwater microorganisms over a period of 42 d. It was added directly to the test vessels after being accurately weighed onto glass cover slips to give a final test concentrations of 14.9 mg/L and 29.9 mg/L. A control vessel was run in parallel, this contained inoculum but no test or reference material. A test vessel containing 20 mg/L of the reference material, sodium benzoate in 3 L of test media, was also included in the test. In addition a vessel containing 20 mg/L of the reference material, sodium benzoate and read across substance at 29.9 mg/L in 3 L of test media, was also included as an inhibition control. 3 L volumes of test media were stirred constantly in round, flat bottomed flasks in the dark. Carbon dioxide free air was passed through the test solutions at an estimated rate of 30 to 100 mL per minute. This was in turn passed through two dreschel bottles, per test vessel, containing barium hydroxide of known concentration and volume. The tests were carried out in the laboratory at 22 ± 2ºC. Single vessels were prepared for each read across substance concentration. Measurements were taken after the appearance of a precipitate of barium carbonate, this was on Day 2, 4, 7, 11, 14, 19, 23, 28, 35, 42 and 43 (post-acidification). The contents of each dreschel bottle were titrated against standardised hydrochloric acid to a pH 7 endpoint. Titres were taken for the barium hydroxide at the start and end of each time period. In the study, the read across substance showed limited potential for degradation at test concentrations of 14.9 and 29.9 mg/L after 28 d. The 28-d cumulative % degradation values were determined to be 25.6% at 14.9 mg/L and 24.4% at 29.9 mg/L. This test was extended up to 42 d, after which time read across substance also showed limited potential for degradation at test concentrations of 14.9 mg/L and 29.9 mg/L in this study. The final 42-d cumulative % degradation values were determined to be 25.9% at 14.9 mg/L and 42.8% at 29.9 mg/L. A degradation figure of 62.9% after 14 d was obtained for the reference material. This demonstrates that the inoculum was biologically active. A degradation figure of 64.3% after 14 d was obtained for the inhibition control. This demonstrates that the inoculum was biologically active in the presence of read across substance at the higher test concentration of 29.9 mg/L. Carbon Dioxide production in the Control vessels was 71.2 mg/L of test media in this study. This was within the acceptable limits defined in the method. The study met all the validity criteria (Cheshire, 2018). Given the >20% degradation of the test substance within 28 d, it may be regarded as evidence of inherent primary biodegradability (OECD, 2005). Based on the results of the read across study, a similar biodegradation potential is expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE'.

Description of key information

Based on the results of the read across study, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE' is considered to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

A study was conducted to determine the ready biodegradability of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', according to OECD Guideline 301B, using modified sturm test, in compliance with GLP. The substance was assessed for the rate and extent of biodegradation when exposed to freshwater microorganisms over a period of 42 d. It was added directly to the test vessels after being accurately weighed onto glass cover slips to give a final test concentrations of 14.9 mg/L and 29.9 mg/L. A control vessel was run in parallel, this contained inoculum but no test or reference material. A test vessel containing 20 mg/L of the reference material, sodium benzoate in 3 L of test media, was also included in the test. In addition a vessel containing 20 mg/L of the reference material, sodium benzoate and read across substance at 29.9 mg/L in 3 L of test media, was also included as an inhibition control. 3 L volumes of test media were stirred constantly in round, flat bottomed flasks in the dark. Carbon dioxide free air was passed through the test solutions at an estimated rate of 30 to 100 mL per minute. This was in turn passed through two dreschel bottles, per test vessel, containing barium hydroxide of known concentration and volume. The tests were carried out in the laboratory at 22 ± 2ºC. Single vessels were prepared for each read across substance concentration. Measurements were taken after the appearance of a precipitate of barium carbonate, this was on Day 2, 4, 7, 11, 14, 19, 23, 28, 35, 42 and 43 (post-acidification). The contents of each dreschel bottle were titrated against standardised hydrochloric acid to a pH 7 endpoint. Titres were taken for the barium hydroxide at the start and end of each time period. In the study, the read across substance showed limited potential for degradation at test concentrations of 14.9 and 29.9 mg/L after 28 d. The 28-d cumulative % degradation values were determined to be 25.6% at 14.9 mg/L and 24.4% at 29.9 mg/L. This test was extended up to 42 d, after which time read across substance also showed limited potential for degradation at test concentrations of 14.9 mg/L and 29.9 mg/L in this study. The final 42-d cumulative % degradation values were determined to be 25.9% at 14.9 mg/L and 42.8% at 29.9 mg/L. A degradation figure of 62.9% after 14 d was obtained for the reference material. This demonstrates that the inoculum was biologically active. A degradation figure of 64.3% after 14 d was obtained for the inhibition control. This demonstrates that the inoculum was biologically active in the presence of read across substance at the higher test concentration of 29.9 mg/L. Carbon Dioxide production in the Control vessels was 71.2 mg/L of test media in this study. This was within the acceptable limits defined in the method. The study met all the validity criteria (Cheshire, 2018). Given the >20% degradation of the test substance within 28 d, it may be regarded as evidence of inherent primary biodegradability (OECD, 2005). Based on the results of the read across study, a similar biodegradation potential is expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE'.

[Type of water: freshwater]