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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:
11 July 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The study was conducted in accordance with OECD Guideline 301B, with a noted exception. The test substance was not sufficiently soluble for measurement of inorganic carbon (IC) of the test substance suspension in mineral medium at test initiation. Measured of the IC content of the concentrated mineral medium and inoculum proved unreliable, as the unfiltered samples were stored refrigerated for 20 days before analysis and were therefore considered to be compromised. This deviation did not adversely affect the validity of the test, as the certificate of analysis suggests that the majority of the carbon in this compound is organic.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
-Purity: Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
-Description: Dark Green Liquid
- Carbon Content: 48.5%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A sample of activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic waste-water catchment. The sample was aerated using a compressed air supply. The suspended solids concentration of the activated sludge was determined to be 4.59 g/L. The activated sludge used in this study was not acclimatised or adapted to the test substance prior to exposure under the test conditions.
Duration of test (contact time):
28 d
Initial conc.:
15 other: mg C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Buffered synthetic, mineral salts medium prepared according to OECD guideline 301B
- Additional substrate: None.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 ± 2°C
- pH: Measurements of pH were made in the blank control and reference substance vessels at the start of incubation and in all vessels at the end of the test prior to the addition of the hydrochloric acid.
- pH adjusted: no.
- Aeration of dilution water: yes.
- Suspended solids concentration: The concentration of suspended solids was calculated to be 4.59 g/L. The medium was inoculated with activated sludge to give a suspended solids concentration of 30 mg/L in each control or test vessel.
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: Test substance vessels were prepared in duplicate (15 mg C/L).
- Method used to create aerobic conditions: continuously sparged with a supply of CO2 free air (ca. 50 mL per minute).
- Measuring equipment: titration (see details on analytical methods).
- Details of trap for CO2 and volatile organics if used: The exhaust air from each vessel was passed through a series of three traps containing a barium hydroxide solution.

SAMPLING
- Sampling frequency: Days 2, 5, 7, 9, 12, 14, 16, 19, 23, 26, 28 and on Day 29 (following acidification and overnight aeration).

- Sampling method: Traps were removed from the series and their contents titrated with hydrochloric acid to determine the quantity of CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that may have remained in solution. Titration of the remaining traps on Day 29 was performed following overnight aeration.

- Sterility check if applicable: not applicable
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank:
Two blank control vessels containing inoculated medium only were prepared to assess the validity of the test and to correct for baseline CO2 evolved.

- Procedure control:
Two procedure control vessels containing a reference substance, sodium benzoate (15 mg C/L), were prepared to assess the performance of the inoculum.

- Toxicity control:
An additional vessel containing a combination of the test and reference substances (15 mg C/L) served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration.

- Abiotic sterile control:
Not applicable.

STATISTICAL METHODS
Not applicable.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
In a preliminary solubility trial conducted in reverse osmosis (RO) water, the test substance did not appear to be in solution with or without sonicated for 5 minutes or stirring for 5 minutes, as evidenced by globules at the surface and the bottom of the test vessel. The test substance was deemed insufficiently soluble for addition as an aqueous stock solution and was therefore administered to the test vessels by direct addition.
Test performance:
The validity criteria were satisfied (degradation of reference material, CO2 production from blank controls), with the exception of the inorganic carbon content (< 8.8%) over the target mineral media of the total carbon content at the beginning of the test, which has been deemed scientifically justified, as having no impact upon the outcome of the study, the results are therefore considered to be valid.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
28 d
Details on results:
Measured pH values ranged from 7.44 to 7.47 on Day 0 and from 7.31 to 7.50 on Day 28.

The mean total CO2 production in the blank control vessels was 40.9 mg/L at the end of the test, satisfying the validity criterion of < 70 mg/L.

Mean biodegradation of the test substance reached 11% of the theoretical carbon dioxide yield after 28 days. The test substance therefore cannot be considered readily biodegradable. Percent biodegradation values at each sampling interval, for the two replicates containing the test substance did not vary by more than 15%, satisfying the validity criterion of less than 20% difference.

Biodegradation of the reference substance vessel (R1) exceeded 60% by Day 9 (64%) and had reached a maximum of 83% by the end of the test. Biodegradation of the reference substance in the presence of the test substance in the toxicity control (61% at Day 12, 73% at the end of the incubation phase on Day 28 and 75% by end of test on Day 29) was similar to that of the reference substance alone, suggesting that the test substance did not have an inhibitory effect on the inoculum under the test conditions, and satisfying the validity criterion.

One of the procedure control vessels (R2) was excluded from this test as it presented a leak at the beginning of the test and failed to meet the validity criterion. The one remaining procedure control vessel met the requirements of the OECD test guideline and exclusion of the leaking vessel was not considered to have adversely impacted the integrity of the study.
Results with reference substance:
No inhibitory effect was observed on the biodegradation of the reference substance in the presence of the test substance.

Table 1: Biodegradation as a Percentage of Theoretical CO2 Yield

 

Biodegradation (%)

 

Day 2

Day 5

Day 7

Day 9

Day 12

Day 14

Day 16

Day 19

Day 23

Day 26

Day 28

Day 29**

Test Substance R1

5

9

12

13

13

14

14

15

16

16

17

18

18

18

Test Substance R2

0

0

0

0

0

0

0

0

0

0

1

2

3

3

Mean

2

4

6

6

7

7

7

8

8

8

9

10

10

11

Reference Substance R1

30

41

55

64

70

72

74

76

79

80

82

83

83

83

Reference Substance R2***

11

27

36

41

43

45

46

48

51

54

56

57

57

57

Toxicity Control*

20

42

53

58

61

63

64

65

69

71

73

74

75

75

 R: Vessel replicate

Note: Numbers are rounded for presentation. Consequently, the displayed mean may not be the calculated mean of the rounded individual values shown.
* Toxicity control values are corrected for mean test substance degradation.

** Day 29 refers to the day that titrations of trap content from acidified vessels were performed. Actual acidification was performed on Day 28.

*** Results for Reference Substance R2 are presented in the table, but were excluded from the test conclusions due to an observed leak at the beginning of the test; biodegradation in this replicate did not meet the validity criterion for the procedure control.

Validity criteria fulfilled:
yes
Remarks:
Exception: The inorganic carbon (IC) content of the test medium was 6.21 mg C/L (or, 13.8%). There was no indication of suppression from increased IC content based on toxicity control and inoculum blanks. The results were therefore considered valid.
Interpretation of results:
not readily biodegradable
Conclusions:
Mean biodegradation of the test substance reached 11% of the theoretical carbon dioxide yield after 28 days. The level of biodegradation did not meet the requirements for ready biodegradability and the test substance, therefore, cannot be classified as readily biodegradable.
Executive summary:

The ready biodegradability of the test substance was assessed by measurement of carbon dioxide (CO2) evolution under standard conditions according to OECD Test Guideline 301B. The test substance was administered to the test system by direct addition. A buffered synthetic, mineral salts medium prepared according to OECD guideline 301B was added to give a test substance concentration equivalent to 15 mg C/L. The concentration of suspended solids was calculated to be 4.59 g/L. The medium was inoculated from a sample of non-adapted activated sludge to give a suspended solids concentration of 30 mg/L in each control or test vessel. Test vessels were incubated in darkness at 22 ± 2°C for 28 days and their contents continuously sparged with a supply of CO2 free air (ca. 50 mL per minute). The exhaust air from each vessel was passed through a series of traps containing a barium hydroxide solution to trap evolved CO2.

On Days 2, 5, 7, 9, 12, 14, 16, 19, 23, 26, 28, traps were removed from the series and their contents titrated with hydrochloric acid to determine the quantity of CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that may have remained in solution. Titration of the remaining traps on Day 29 was performed following overnight aeration.Measurements of pH were made in the blank control and reference substance vessels at the start of incubation and in all vessels at the end of the test prior to the addition of the hydrochloric acid.

The performance of the inoculum was checked by measuring the CO2 evolved from procedure control vessels containing a reference substance, sodium benzoate (15 mg C/L). An additional vessel containing a combination of the test and reference substances (15 mg C/L) served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration. Two blank control vessels were also prepared containing inoculated medium only to check the validity of the test and to correct for baseline CO2 evolved. Duplicate vessels were prepared for the test substance, reference substance and blank control groups. A single vessel was prepared for the toxicity control.

Mean biodegradation of the test substance reached 11% of the theoretical carbon dioxide yield after 28 days. The test substance therefore cannot be considered readily biodegradable.Percent biodegradation values at each sampling interval, for the two replicates containing the test substance did not vary by more than 15%, satisfying the validity criterion of less than 20% difference.

The mean total CO2 production in the blank control vessels was 40.9 mg/L at the end of the test, satisfying the validity criterion of < 70 mg/L.

Biodegradation of the reference substance vessel (R1) exceeded 60% by Day 9 (64%) and had reached a maximum of 83% by the end of the test. Biodegradation of the reference substance in the presence of the test substance in the toxicity control (61% at Day 12, 73% at the end of the incubation phase on Day 28 and 75% by end of test on Day 29) was similar to that of the reference substance alone, suggesting that the test substance did not have an inhibitory effect on the inoculum under the test conditions, and satisfying the validity criterion.

Description of key information

The test item attained 11% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
other: Buffered synthetic, mineral salts medium

Additional information

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