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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:
2018-08-21 to 2018-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
adopted March 23, 2006; corrected 26 September 2014.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Source:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment:
The freshly obtained sludge was kept under continuous aeration with synthetic air (CO2 < 1 ppm; a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%)) until further treatment.
The concentration of suspended solids was determined to be 4.0 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (45 minutes) and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
6 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
See section "Any other information on materials and methods incl. tables" for details.
Reference substance:
other: 1-Octanol
Remarks:
Purity 99.7%
Preliminary study:
No
Test performance:
See section "Any other information on results incl. tables" below.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
71
St. dev.:
6
Sampling time:
28 d
Remarks on result:
other: Test item
Remarks:
based on 5 replicates
Parameter:
% degradation (CO2 evolution)
Value:
80
St. dev.:
10
Sampling time:
14 d
Remarks on result:
other: Reference item 1-octanol
Remarks:
based on 5 replicates
Parameter:
% degradation (CO2 evolution)
Value:
103
St. dev.:
13
Sampling time:
14 d
Remarks on result:
other: Toxicity control (1-octanol plus test item)
Remarks:
based on 5 replicates
Details on results:
See section "Any other information on results incl. tables" below.

Acceptability of the Test

1.    The reference item was biodegraded by at least 60% (80%) within 14 days.

2.    The mean amount of TIC present in the inoculum blanks at the end of the test was < 3 mg C/L (2 mg C/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Biodegradation

Results of inorganic carbon measurement and percentages of biodegradation are listed in Table 5 and Table 6, respectively. An overview of the mean group % biodegradation is listed in Table 3. The curves for biodegradation are shown in the attached illustration.

The relative biodegradation values calculated from the measurements performed during the test period revealed 71% biodegradation of the test item (mean value). Since the test item was a mixture of structurally similar chemicals, the 10-day window was not applied. No significant inorganic carbon production was observed in the abiotic control (8%).

In the toxicity control more than 25% biodegradation occurred within 14 days (103%, based on ThIC). Therefore, the test item was assumed not to inhibit microbial activity.

Table 3: Mean Biodegradation in Percentage of the Theoretical maximum IC Production (ThIC)

Nominal day

Biodegradation in percentage of the ThIC

Degradation

Reference Item

Test Item

Toxicity Control

Abiotic Control

1

1

2

0

0

7

54

9

82

-

14

80

46

103

-

21

-

65

-

-

28

-

71

-

8

Monitoring of Temperature and pH

The temperature, recorded in a vessel with Milli-RO water in the same room, varied between 20 and 21°C, and complied with the requirements as laid down in the study plan.The pH values of the different test media are presented in Table 4.

Table 4: pH Values of Different Test Media

Test medium:

pH at the start of the test:

Inoculum blank

7.5

Procedure control

7.5

Test item

7.5

Toxicity control

7.5

Abiotic control

7.5

Table 5: Inorganic Carbon (IC) concentrations (mg/L) per Vessel

Nominal

day

Individual measurements of inorganic carbon (IC)

Vessel A

Vessel B

Vessel C

Vessel D

Vessel E

Mean

Inoculum Blank

1

1.1

1.1

1.1

n.a.

n.a.

1.1

7

1.1

1.2

1.0

n.a.

n.a.

1.1

14

1.8

1.9

1.9

2.1

1.7

1.9

22

1.1

1.3

1.4

n.a.

n.a.

1.3

28

1.7

1.8

1.7

1.7

1.8

1.7

Procedure Control (Reference Item)

 

1

1.5

1.2

1.2

n.a.

n.a.

 

7

14

14

7.1

n.a.

n.a.

 

14

14

20

18

19

18

 

Test Item

 

1

1.1

1.2

1.3

n.a.

n.a.

 

7

1.6

1.7

1.5

n.a.

n.a.

 

14

4.1

4.9

5.0

n.a.

n.a.

 

22

4.2

6.1

5.2

n.a.

n.a.

 

28

6.3

5.4

6.3

6.0

6.0

 

Toxicity Control (Test Item + Reference Item)

 

1

1.3

1.1

1.1

n.a.

n.a.

 

7

23

22

22

n.a.

n.a.

 

14

25

32

28

32

27

 

Abiotic Control

 

1

1.0

0.9

0.7

n.a.

n.a.

 

28

1.4

1.4

2.41)

1.4

1.3

 

n.a.  = not applicable

1) = According to Dixon’s Q-test this value was an outlier and was excluded from further analysis.

Table 6: Biodegradation as Percentage of ThIC per Vessel

Nominal Day

% Biodegradation

A

B

C

D

E

Mean

Std. Dev.

CV (%)

Procedure Control (Reference Item)

1

2

1

0

n.a.

n.a.

1

n.a.

n.a.

7

64

67

30

n.a.

n.a.

54

n.a.

n.a.

14

62

90

82

84

82

80

10

13

Test Item

1

0

3

4

n.a.

n.a.

2

n.a.

n.a.

7

9

10

7

n.a.

n.a.

9

n.a.

n.a.

14

37

49

52

n.a.

n.a.

46

n.a.

n.a.

21

49

80

65

n.a.

n.a.

65

n.a.

n.a.

28

76

61

76

71

70

71

6

9

Toxicity Control (Test Item + Reference Item)

1

1

0

0

n.a.

n.a.

0

n.a.

n.a.

7

84

81

81

n.a.

n.a.

82

n.a.

n.a.

14

89

117

99

117

96

103

13

12

Abiotic Control

1

2

0

-2

n.a.

n.a.

0

n.a.

n.a.

28

9

9

-1)

9

7

8

0.8

10

n.a.  = not applicable

1)        = According to Dixon’s Q-test the IC value was an outlier and was excluded from further analysis.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The submission substance is readily biodegradable (OECD 310, Headspace Test): 71% biodegradation within 28 days based on ThCO2 evolution
Executive summary:

In a reliable study performed compliant with GLP according to OECD 310 (adopted March 23, 2006, corrected 26 September 2014) the submission substance was tested for ready biodegradability in an aerobic, aqueous inoculated medium in sealed vessels (Headspace Test).

The test item was added to the mineral medium to give a final organic carbon concentration of 6 mg C/L. Silica gel (0.75 g) was added to each vessel in order to aid dispersion and increase availability of the test item to the micro-organisms. Due to volatility of some test item constituents it was not possible to determine the organic carbon content using TOC analysis. Therefore, the organic carbon content was based on calculations. The organic carbon content was calculated to be 68.16%.

Since the test item was a liquid that could be pipetted, aliquots of 1 µL (equalling 0.95 mg) were added directly to the test vessels containing 0.75 g of silica gel, followed by addition of 107 mL medium with microbial organisms and mineral components.

The test was performed in sealed vessels with a headspace of air (headspace to liquid ratio of 1:2). The test consisted of five groups:

1.    Inoculum blank: containing inoculated medium.

2.    Procedure control: containing inoculated medium and reference item.

3.    Test item: containing inoculated medium and test item.

4.    Toxicity control: containing inoculated medium, reference item and test item.

5.    Abiotic control: containing untreated medium, test item and sterilising agent.

The CO2 evolution resulting from the aerobic biodegradation of the test item was determined by determining the inorganic carbon (IC) produced in the test vessels in excess of that produced in inoculum blank. Biodegradation was expressed as a percentage of the ThIC, based on the quantity of test item initially added.

The relative biodegradation values calculated from the measurements performed during the test period revealed 71% biodegradation of the submission substance (mean value). Since the test item was a mixture of structurally similar chemicals, the 10-day window was not applied. No significant inorganic carbon production was observed in the abiotic control (8%).

In the toxicity control more than 25% biodegradation occurred within 14 days (103%, based on ThIC). Therefore, the test item was assumed not to inhibit microbial activity.

Since all acceptability criteria of the test were met, this study was considered to be valid.

In conclusion, the test item is designated as readily biodegradable.

Description of key information

The submission substance Step 2 catalyst is readily biodegradable (OECD 310, Headspace Test): 71% biodegradation within 28 days based on ThCO2 evolution

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

In a reliable study performed compliant with GLP according to OECD 310 (adopted March 23, 2006, corrected 26 September 2014) the submission substance (Step 2 catalyst) was tested for ready biodegradability in an aerobic, aqueous inoculated medium in sealed vessels (Headspace Test).

The test item was added to the mineral medium to give a final organic carbon concentration of 6 mg C/L. Silica gel (0.75 g) was added to each vessel in order to aid dispersion and increase availability of the test item to the micro-organisms. Microbial inoculum was derived from a municipal sewage treatment plant receiving predominantly domestic sewage.

The test was performed in sealed vessels with a headspace of air (headspace to liquid ratio of 1:2). The test consisted of five groups:

1.    Inoculum blank: containing inoculated medium.

2.    Procedure control: containing inoculated medium and reference item.

3.    Test item: containing inoculated medium and test item.

4.    Toxicity control: containing inoculated medium, reference item and test item.

5.    Abiotic control: containing untreated medium, test item and sterilising agent.

The CO2 evolution resulting from the aerobic biodegradation of the test item was determined by determining the inorganic carbon (IC) produced in the test vessels in excess of that produced in inoculum blank. Biodegradation was expressed as a percentage of the ThIC, based on the quantity of test item initially added.

Results:

Test item; 6 mg OC/L

Reference item 1-octanol; 20 mg OC/L

Toxicity control (20 mg OC/L 1-octanol; 6 mg OC/L test item)

Toxicity control - expected biodegradation extent [%]
from test item / reference item results, considering relative TOC contribution

% biodegradation

SD (n=5)

% biodegradation

SD (n=5)

% biodegradation

SD (n=5)

Day 7

9

--

54

--

82

--

43.6

Day 14

46

--

80

10

103

13

72.1

Day 21

65

--

--

--

--

--

--

Day 28

71

6

--

--

--

--

--

The relative biodegradation values calculated from the measurements performed during the test period revealed 71% biodegradation of the submission substance (mean value). Since the test item was a mixture of structurally similar chemicals, the 10-day window was not applied. In the last interval tested (i.e. between 21 and 28 days) still a significant increase in biodegradation extent was observed and thus the plateau phase was probably not yet reached at the end of the test.

It is remarkable that in the toxicity control a stimulation of biodegradation was observed relative to the procedure control. At day 7, 1-octanol alone was biodegraded to 54% while at the same octanol concentration with the test item present in addition 82% biodegradation was achieved (n=3; 84, 81, and 81%). At day 14 the procedure control (1-octanol alone) achieved 80% biodegradation while degradation in the toxicity control was complete (103%). This comes unexpected considering the higher total organic carbon content present in the toxicity control relative to both, test item and procedure control alone. In the last column of the table the expected biodegradation extent for the toxicity control is calculated based on degradation extents observed for test item and reference item alone, considering their relative TOC contribution in the toxicity control (23.1% and 76.9%, respectively). Obviously, microbial activity was stimulated considerably in presence of both substances together, which may indicate some form of co-metabolism. It may thus be assumed that upon simultaneous presence of readily biodegradable substances in STPs or in the environment Step 2 catalyst will be degraded even faster than observed in the experiment without any other substance present in addition.

No significant inorganic carbon production was observed in the abiotic control (8%). All validity criteria of OECD 310 were met.

In conclusion, Step 2 catalyst was identified to be readily biodegradable.