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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1st december 2008 - 26 may 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
One deviation was observed on the medium composition without affecting the results or the validity of the test. Lack of data on individual biodegradation of each replicate.
Qualifier:
according to
Guideline:
OECD Guideline 306 (Biodegradability in Seawater)
Deviations:
yes
Remarks:
one deviation was observed on the medium composition without affecting the results or the validity of the test
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: natural seawater
Details on inoculum:
The raw seawater was supplied by a submersible pump situated on Sutherland's pier on the west side of Flotta in Scapa Flow. The raw seawater is pumped continuously from a depth of two meter below low water spring tide level, before passing up to 1.8 kilometers of plastic pipes to a storage tank. Two smaller pumps move the water to three settlement tanks situated nine meters above floor level.
The seawater temperature varies between 6°C in the winter and 14°C in the summer.
The salinity is between 34 parts per thousand and 37 parts per thousand.
Five to seven days before test commencement, raw seawater passes by gravity through a 45 µm filter to the ageing tank stored in darkness.
Inoculum collected on 1 december 2008, seven days before test and at 10.6°C (température throughout ageing: 10.6 to 21.8°C)
Duration of test (contact time):
28 d
Initial conc.:
8.29 mg/L
Initial conc.:
0.627 other: mg (O2)/mg
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to OECD guideline (except for Na2HPO4, 2H2O at 29.92 g/Linstead of 33.30 g and for CaCl2 at 31.84 g/L instead of 27.50 g/L)
- Test temperature: 20 °C +/- 1°C

TEST SYSTEM
- Culturing apparatus: 270-276 ml glass BOD bottles containing 180-184 ml test medium
- Number of culture flasks:
Test material: 3 replicates per timepoint, minimum of two replicates per timepoint for data processing
Oxygen blank, reference, control, RBO: 3 replicates per timepoint, minimum of two replicates per timepoint for data processing

SAMPLING
- Sampling frequency: dissolved oxygen was measured at 7 day intervals during 28 days.
Reference substance:
benzoic acid, sodium salt
Test performance:
The test was considered as valid. All the validity criteria are fulfilled:
- Sodium benzoate attained more than 60% of degradation in 14 days (see table 4)
- The blank respiration did not exceed 30% of the oxygen in the test bottle after 28 days (table 5)
- BOD of the mixture of reference and test substances is less than the sum of the BOD of the separate solutions of the two substances (table 1)
Key result
Parameter:
% degradation (O2 consumption)
Value:
57
Sampling time:
28 d
Details on results:
The test item attained 57% degradation after 28 days.
According to the OECD 306 Guideline, if the result is positive (>70% DOC removal; >60% ThOD - theoretical oxygen demand), it may be concluded that there is a potential for biodegradation in the marine environment. Hence, the test item can't be considered to be biodegradable in seawater under the conditions of the test.
The results obtained from the toxicity control show an inhibition of 9% to seawater bacteria.. The toxicity control attained more than 60% of degradation after 14 days and the BOD of the mixture of reference and test substances is less than the sum of the BOD of the separate solutions of the two substances.
The reference substance, Sodium benzoate, attained 79% degradation after 14 days and 95% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Results with reference substance:
The mean percentage degradation of the reference substance is 95% (greater than 60%) in 28 days of incubation. This result confirms the suitability of the inoculum and test conditions (see table 4).

Table 1 - Average net oxygen consumption (BOD, mg O2.l-1)

 

Day

0

7

14

21

28

Oxygen consumption blank

7.27

7.09

6.87

6.90

6.79

Sodium benzoate

7.33

5.35

4.91

4.79

4.42

Test item

7.31

4.69

4.35

4.11

3.80

Test item + Sodium benzoate

7.24

3.12

2.60

2.39

1.92

Table 2 - Percentage degradation of the test item

Material

100% BOD

(mg/L)

Measured BOD (mg/L)

% degradation

7

14

21

28

7

14

21

28

Test item

5.2

2.40

2.52

2.79

2.98

46

49

54

57

Test item + Sodium benzoate

7.7

3.98

4.27

4.51

4.87

52

56

59

63

Table 3 - Inhibition due to test item

Day

Test item

 

BOD

(mg/L)

Sodium benzoate

 

BOD

(mg/L)

Sum of separate

 

BODs

(mg/L)

Test item + Sodium benzoate

BOD

(mg/L)

Percentage inhibition

%

7

2.40

1.75

4.15

3.98

4

14

2.52

1.96

4.45

4.27

4

21

2.79

2.11

4.90

4.51

8

28

2.98

2.36

5.35

4.87

9

Table 4 - Reference substance degradation

Material

100% BOD

(mg/L)

Measured BOD (mg/L)

Percentage degradation

%

7

14

21

28

7

14

21

28

Sodium benzoate

2.5

1.75

1.96

2.11

2.36

70

78

85

95

Table 5 - Blank Oxygen demand

Day

Mean Dissolved Oxygen

 (mg/L)

Mean BOD

(mg/L)

BOD

 (%)

0

7.27

-

-

7

7.09

0.19

3

14

6.87

0.42

6

21

6.90

0.39

5

28

6.79

0.50

7

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Based on O2 consumption, the biodegradation of the test item was 57% within 28 days incubation in seawater. Based on these results, the test item cannot be considered as biodegradable in seawater in the conditions of the test.
Executive summary:

The ultimate aerobic biodegradability in seawater of the test item (= substance EC 939-455-3 in aqueous commercial product) was investigated in a GLP-compliant study performed in accordance with OECD Guideline 306 (Biodegradability in Seawater). In the test flasks containing the test item and seawater inoculum, a mean of 57% biodegradation was observed (based on O2 consumption) within the test period of 28 days. The test item was thus considered as not readily biodegradable in seawater under the test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 30 march 2010 to 30 may 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: All the criteria fulfilled but the study was not performed on GLP principles
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 14593
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was collected from the aeration tank of a full-scale wasrewater treatment plant which treats predominantly domectic sewage (ETE Samambaia / Campinas-SP/Brazil).

The activated sludge was treated by allowing to settle three times for 30 minutes. For each setting the supernatant was rejected and the solids was redispersed in the mineral medium. In order to lower carbon organic content and to reduce the blanck CO2 evolution, the activated sludge was aerated overnight.

The final concentration of the teated activated sludge was adjusted to 1000 mg/L dry solids.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Mineral medium : As precribed in the 310 OECD guideline. The mineral medium was checked for pH, which was 7.51.
- Additional substrate : no
- Solubilising agent : no
- Test temperature : 22 +/- 2°C
- Continuous darkness : yes
- Agitation : the sealed test vessels were maintained in a rotary shaker, between 166-170 rpm (sufficient to keep the bottle contents well mixed and in suspension).

TEST SOLUTIONS
- Test substance :
A stock solution of the test solution containing 2000 mg C/l was prepared by adding 0.8670 g of the test substance into volumetric flask and completing the final volume to 100 ml with distilled water. One to one hunfred dilution of the test substance stock solution in the mineral medium was analyzed (apparatus : Shimadzu Model TOC 5000 carbon analyzer) and indicated a maean concentration of 21.12 mg C/L.
Sufficient test substance solution was prepared immediately before use by diluting the stock solution with the inoculum medium in order to give a final TOC concentration of 20 mg/L and 4 mg/L activated sludge dry solids. The measured pH was 7.55.

- Reference substance :
A stock solution of the reference substance containing 2000 mg C/l was prepared by adding 0.8569 g of the reference substance into a volumetric flask and completing the final volume to 250 ml with distilled water.
Sufficient reference substance solution was prepared immediately before use by diluting the stock solution with the inoculum medium in order to give a final TOC concentration of 20 mg/L .

INOCULATED MEDIUM
Sufficient inoculated medium was prepared immediately before by diluting the treated activated sludge (see details on inoculum) with mineral medium in order to achieve a concentration of 4 mg/L activated sludge dry solids.

TEST SYSTEM
- Culturing apparatus: Bottles of 120 ml capacity sealed with butyl rubber septa and aluminium crimp seals. The recommended headspace to liquid volume ratio of 1:2 (80 ml liquid volume to 120 ml-capacity bottles) was used.
- Number of culture flasks : 43 serum bottles were used for the study and were designated as follows :
- 13 blank controls : containing the inoculated medium (80 ml)
- 13 tests vessels : containing the test substance (at 21.12 mg/l of TOC) and the inoculated medium (80 ml)
- 13 reference vessels : containing reference substance (sodium benzoate) at 20 mg/l of TOC, the inoculum and the mineral medium (80 ml) (3)
- 4 inhibition controls : containing the test substance (at 21.12 mg /l of TOC) and reference substance (at 20 mg C/l of TOC) and the inoculated test medium

SAMPLING
SAMPLING FREQUENCY :
- Bottles were sacrified for Inorganic carbon (IC) analysis on the following times : days 3, 7, 14, 21 and 28
-Assay for CO2 production was conducted for duplicate bottles on days 3, 7, 14, 21 and five replicate bottles on day 28

SAMPLING METHOD :
The samples were prepared by injecting 1.0 ml of sodium hydroxide solution at 280 g/l through the septum of each bottle sampled, shaking on the incubation rotary shaker for 1h at the test temperature, removing the bottles from the shaker and allowing to settle. Suitable volume of the liquid phase were sampled and injected into a Shimatzu Model TOC 5000 inorganic carbon analyzer, which was calibrated using appropriate standards, and the concentrations of IC were recorded.

Reference substance:
benzoic acid, sodium salt
Remarks:
purity : min. 99.5%; carbon content : 58.35%
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
65.28
Sampling time:
28 d
Details on results:
Mean net CO2 values from the test substance vessels on days 3, 7, 14, 21 and 28 were 23.58, 46.95, 59.73, 63.87 and 65.28% respectively, of the theoretical amount, based on Inorganic Carbon analysis.
Results with reference substance:
The mean percentage degradation of the reference substance was 96.55% (greater than 60%) by the 14th day of incubation.

Table 1 - Cumulative (mg/L) Inorganic Carbon Evolved

Cumulative (mg/L) Inorganic Carbon Evolved

Vessels contents

3 days

7 days

14 days

21 days

28 days

Test

-

-

-

-

16.94 

Test

-

-

-

-

 16.58

Test

-

-

-

-

 16.32

Test

7.10

11.78

15.57

16.20

17.17

Test

8.04

13.71

15.20

16.53

16.73

Mean

7.57

12.75

15.39

16.37

16.75

Reference

-

-

-

-

22.45

Reference

-

-

-

-

22.73

Reference

-

-

-

 -

22.48

Reference

19.02

21.82

22.17

22.27

22.08

Reference

18.41

21.71

21.99

22.09

22.52

Mean

18.72

21.77

22.08

22.18

22.45

Blank

-

-

-

-

2.92

Blank

-

-

-

-

2.89

Blank

-

-

-

-

3.03

Blank

2.77

3.09

2.75

2.83

2.91

Blank

2.41

2.57

2.79

2.92

3.05

Mean

 2.59

 2.83

 2.77

 2.88

2.96

Inhibition

19.67

-

-

-

39.19

Inhibition

19.23

-

-

-

39.05

Mean

19.45

-

-

-

39.12

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
In the test flasks containing the test item and microbial inoculum, a mean of 65.3% of biodegradation was observed (based on the IC analysis) within the test period of 28 days. Therefore, the test item is considered as readily biodegradable (10 -day window does not apply to complex substances) under the test conditions.
Executive summary:

The ultimate aerobic biodegradability in freshwater of the test item (= substance EC 939-457-4 in aqueous commercial product) was investigated in a non-GLP compliant study (De Nadai, 2005) performed in accordance with OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test) and ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)). In the test flasks containing the test item and microbial inoculum, a mean of 65.3% biodegradation was observed (based on IC analysis) within the test period of 28 days. The test item was therefore considered as readily biodegradable under the test conditions (10-day window does not apply to complex substances).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 25 february 2010 to 20 aprile 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: All the criteria fulfilled but the study was not performed on GLP principles
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 14593
Deviations:
no
GLP compliance:
no
Remarks:
study performed in internal facilities without GLP-certification
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was collected from the aeration tank of a full-scale wasrewater treatment plant which treats predominantly domectic sewage (ETE Samambaia / Campinas-SP/Brazil).

The activated sludge was treated by allowing to settle three times for 30 minutes. For each setting the supernatant was rejected and the solids was redispersed in the mineral medium. In order to lower carbon organic content and to reduce the blanck CO2 evolution, the activated sludge was aerated overnight.

The final concentration of the teated activated sludge was adjusted to 1000 mg/L dry solids.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Mineral medium : As precribed in the 310 OECD guideline. The mineral medium was checked for pH, which was 7.67.
- Additional substrate : no
- Solubilising agent : no
- Test temperature : 22 +/- 2°C
- Continuous darkness : yes
- Agitation : the sealed test vessels were maintained in a rotary shaker, between 166-170 rpm (sufficient to keep the bottle contents well mixed and in suspension).

TEST SOLUTIONS
- Test substance :
A stock solution of the test solution containing 2000 mg C/L was prepared by adding 1.7025 g of the test substance into volumetric flask and completing the final volume to 200 ml with distilled water. One to one hunfred dilution of the test substance stock solution in the mineral medium was analyzed (apparatus : Shimadzu Model TOC 5000 carbon analyzer) and indicated a maean concentration of 20.07 mg C/L.
Sufficient test substance solution was prepared immediately before use by diluting the stock solution with the mineral medium and with the treated activated sludge in order to give a final TOC concentration of 20 mg/L and 4 mg/L activated sludge dry solids. The measured pH was 7.64

- Reference substance :
A stock solution of the reference substance containing 2000 mg C/L was prepared by adding 0.6855 g of the reference substance into a volumetric flask and completing the final volume to 200 ml with distilled water.
Sufficient reference substance solution was prepared immediately before use by diluting the stock solution with the imineral medium and with the treated activated sludge in order to give a final TOC concentration of 20 mg/l .

INOCULUM MEDIUM
Sufficient inoculated medium was prepared immediately before use by diluting the treated activated sludge (see details on inoculum) with mineral medium in order to achieve a concentration of 4 mg/L activated sludge dry solids.
To reduce the carbon dioxide production of the blanks, the inoculated medium was preconditioned by aeration for 7 days at the test temperature. Water loss by evaporation was corrected during the aeration and especially before use

TEST SYSTEM
- Culturing apparatus: Bottles of 120 ml capacity sealed with butyl rubber septa and aluminium crimp seals. The recommended headspace to liquid volume ratio of 1:2 (80 ml liquid volume to 120 ml-capacity bottles) was used.
- Number of culture flasks : 43 serum bottles were used for the study and were designated as follows :
- 13 blank controls : containing the inoculated medium (80 ml)
- 13 tests vessels : containing the test substance (at 20.07 mg/l of TOC) and the inoculated medium (80 ml)
- 13 reference vessels : containing reference substance (sodium benzoate) at 20 mg/l of TOC, the inoculum and the mineral medium (80 ml)
- 4 inhibition controls : containing the test substance (at 20.07 mg /l of TOC) and reference substance (at 20 mg C/l of TOC) and the inoculated test medium

SAMPLING
SAMPLING FREQUENCY :
- Bottles were sacrified for Inorganic carbon (IC) analysis on the following times : days 4, 7, 14, 21 and 28
-Assay for CO2 production was conducted for duplicate bottles on days 4, 7, 14, 21 and five replicate bottles on day 28

SAMPLING METHOD :
The samples were prepared by injecting 1.0 ml of sodium hydroxide solution at 280 g/l through the septum of each bottle sampled, shaking on the incubation rotary shaker for 1h at the test temperature, removing the bottles from the shaker and allowing to settle. Suitable volume of the liquid phase were sampled and injected into a Shimatzu Model TOC 5000 inorganic carbon analyzer, which was calibrated using appropriate standards, and the concentrations of IC were recorded.

Reference substance:
benzoic acid, sodium salt
Remarks:
purity : min. 99.5%; carbon content : 58.35%
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
62.59
Sampling time:
28 d
Details on results:
Mean net CO2 values from the test substance vessels on days 4, 7, 14, 21 and 28 were 27.38, 40.22, 53.31, 60.84 and 62.59% respectively, of the theoretical amount, based on Inorganic Carbon analysis.
Results with reference substance:
The mean percentage degradation of the reference substance was 96.77% (greater than 60%) by the 14th day of incubation.

Table 1 - Cumulative (mg/L) Inorganic Carbon Evolved

Cumulative (mg/L) Inorganic Carbon Evolved

Vessels contents

4 days

7 days

14 days

21 days

28 days

Test

-

-

-

-

15.79

Test

-

-

-

-

15.33

Test

-

-

-

-

15.89

Test

7.40

10.33

13.34

15.39

15.91

Test

7.13

10.88

13.72

15.23

15.55

Mean

7.26

10.61

13.53

15.31

15.69

Reference

-

-

-

-

22.76

Reference

-

-

-

-

22.34

Reference

-

-

-

 

22.45

Reference

17.41

21.47

22.26

22.59

23.18

Reference

17.67

21.27

22.44

22.71

22.84

Mean

17.54

21.37

22.35

22.65

22.71

Blank

-

-

-

-

3.11

Blank

-

-

-

-

2.97

Blank

-

-

-

-

3.13

Blank

1.63

2.68

2.92

3.04

3.22

Blank

1.91

2.39

2.74

3.16

3.23

Mean

1.77

2.53

2.83

3.10

3.13

Inhibition

19.86

-

-

-

35.16

Inhibition

18.75

-

-

-

36.21

Mean

19.31

-

-

-

35.69

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
In the test flasks containing the test item and microbial inoculum, a mean of 62.6% of biodegradation was observed (based on the IC analysis) within the test period of 28 days. Therefore, the test item is considered as readily biodegradable (10 -day window does not apply to complex substances) under the test conditions.
Executive summary:

The ultimate aerobic biodegradability in freshwater of the test item (= substance EC 939-457-4 in aqueous commercial product) was investigated in a non-GLP compliant study (De Nadai, 2010) performed in accordance with OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test) and ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)). In the test flasks containing the test item and microbial inoculum, a mean of 62.6% biodegradation was observed (based on IC analysis) within the test period of 28 days. The test item was therefore considered as readily biodegradable under the test conditions (10-day window does not apply to complex substances).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 25 february 2010 to 14 april 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non-GLP study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was collected from the aeration tank of a full-scale wasrewater treatment plant which treats predominantly domectic sewage (ETE Samambaia / Campinas-SP/Brazil).

The activated sludge was treated by allowing to settle three times for 30 minutes. For each setting the supernatant was rejected and the solids was redispersed in the mineral medium. In order to lower carbon organic content and to reduce the blanck CO2 evolution, the activated sludge was aerated overnight.

The final concentration of the teated activated sludge was adjusted to 1000 mg/L dry solids.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Mineral medium : As precribed in the 310 OECD guideline. The mineral medium was checked for pH, which was 7.67.
- Additional substrate : no
- Solubilising agent : no
- Test temperature : 22 +/- 2°C
- Continuous darkness : yes
- Agitation : the sealed test vessels were maintained in a rotary shaker, between 166-170 rpm (sufficient to keep the bottle contents well mixed and in suspension).

TEST SOLUTIONS
- Test substance :
A stock solution of the test solution containing 2000 mg C/l was prepared by adding 1.7012 g of the test substance into volumetric flask and completing the final volume to 200 ml with distilled water. One to one hunfred dilution of the test substance stock solution in the mineral medium was analyzed (apparatus : Shimadzu Model TOC 5000 carbon analyzer) and indicated a maean concentration of 19.56 mg C/L.
Sufficient test substance solution was prepared immediately before use by diluting the stock solution with the inoculum medium in order to give a final TOC concentration of 20 mg/L and 4 mg/L activated sludge dry solids. The measured pH was 7.68.

- Reference substance :
A stock solution of the reference substance containing 2000 mg C/l was prepared by adding 0.6855 g of the reference substance into a volumetric flask and completing the final volume to 200 ml with distilled water.
Sufficient reference substance solution was prepared immediately before use by diluting the stock solution with the inoculum medium in order to give a final TOC concentration of 20 mg/L .

INOCULATED MEDIUM
Sufficient inoculated medium was prepared immediately before by diluting the treated activated sludge (see details on inoculum) with mineral medium in order to achieve a concentration of 4 mg/L activated sludge dry solids.

TEST SYSTEM
- Culturing apparatus: Bottles of 120 ml capacity sealed with butyl rubber septa and aluminium crimp seals. The recommended headspace to liquid volume ratio of 1:2 (80 ml liquid volume to 120 ml-capacity bottles) was used.
- Number of culture flasks : 43 serum bottles were used for the study and were designated as follows :
- 13 blank controls : containing the inoculated medium (80 ml)
- 13 tests vessels : containing the test substance (at 19.56 mg/l of TOC) and the inoculated medium (80 ml)
- 13 reference vessels : containing reference substance (sodium benzoate) at 20 mg/l of TOC, the inoculum and the mineral medium (80 ml) (3)
- 4 inhibition controls : containing the test substance (at 19.56 mg /l of TOC) and reference substance (at 20 mg C/l of TOC) and the inoculated test medium

SAMPLING
SAMPLING FREQUENCY :
- Bottles were sacrified for Inorganic carbon (IC) analysis on the following times : days 4, 7, 14, 21 and 28
-Assay for CO2 production was conducted for duplicate bottles on days 4, 7, 14, 21 and five replicate bottles on day 28

SAMPLING METHOD :
The samples were prepared by injecting 1.0 ml of sodium hydroxide solution at 280 g/l through the septum of each bottle sampled, shaking on the incubation rotary shaker for 1h at the test temperature, removing the bottles from the shaker and allowing to settle. Suitable volume of the liquid phase were sampled and injected into a Shimatzu Model TOC 5000 inorganic carbon analyzer, which was calibrated using appropriate standards, and the concentrations of IC were recorded.

Reference substance:
benzoic acid, sodium salt
Remarks:
purity : min. 99.5%; carbon content : 58.35%
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
63.2
Sampling time:
28 d
Details on results:
Mean net CO2 values from the test substance vessels on days 4, 7, 14, 21 and 28 were 26.05, 40.36, 52.81, 60.94 and 63.16% respectively, of the theoretical amount, based on Inorganic Carbon analysis.
Results with reference substance:
The mean percentage degradation of the reference substance was 96.77% (greater than 60%) by the 14th day of incubation.

Table 1 - Cumulative (mg/L) Inorganic Carbon Evolved

Cumulative (mg/L) Inorganic Carbon Evolved

Vessels contents

4 days

7 days

14 days

21 days

28 days

Test

-

-

-

-

 

Test

-

-

-

-

 

Test

-

-

-

-

 

Test

6.67

10.52

13.37

15.15

15.97

Test

7.06

10.34

12.95

14.89

15.36

Mean

6.86

10.43

13.16

15.02

15.49

Reference

-

-

-

-

22.76

Reference

-

-

-

-

22.34

Reference

-

-

-

 

22.45

Reference

17.41

21.47

22.26

22.59

23.18

Reference

17.67

21.27

22.44

22.71

22.84

Mean

17.54

21.37

22.35

22.65

22.71

Blank

-

-

-

-

3.11

Blank

-

-

-

-

2.97

Blank

-

-

-

-

3.13

Blank

1.63

2.68

2.92

3.04

3.22

Blank

1.91

2.39

2.74

3.16

3.23

Mean

 

 

 

 

3.13

Inhibition

19.27

-

-

-

35.37

Inhibition

18.86

-

-

-

35.11

Mean

19.07

-

-

-

35.24

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the IC analysis results of the study, the biodegradation of the test item was 63.2 % within 28 days incubation. Based on these results, the test item is considered as readily biodegradable (10-day window does not apply to complex substances) in the conditions of the test.
Executive summary:

The ultimate aerobic biodegradability in freshwater of the test item (= substance EC 939-455-3 in aqueous commercial product) was investigated in a non-GLP compliant study (De Nadai, 2010) performed in accordance with OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test) and ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)). In the test flasks containing the test item and microbial inoculum, a mean of 63.2% biodegradation was observed (based on IC analysis) within the test period of 28 days. The test item was therefore considered as readily biodegradable under the test conditions (10-day window does not apply to complex substances).

.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4).

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX
See "Documentation and scientific justification of the read-across approach" in section 13.2.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Parameter:
% degradation (O2 consumption)
Value:
57
Sampling time:
28 d
Remarks on result:
other: OECD 306 (seawater)
Remarks:
read-across from EC 939-455-3
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
63.2
Sampling time:
28 d
Remarks on result:
other: OECD 310 (freshwater)
Remarks:
read-across from EC 939-455-3

Description of key information

The ultimate aerobic biodegradability in freshwater of the substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C12-18(even numbered) acyl) derivs., hydroxides, inner salts (EC 939-457-4) was investigated in two non-GLP compliant studies (De Nadai, 2005, 2010) performed in accordance with OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test) and ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)). In the test flasks containing the substance and microbial inoculum, a mean biodegradation in the range 62.6-65.3% was observed (based on IC analysis) within the test period of 28 days. The substance was therefore considered as readily biodegradable under the test conditions (10-day window does not apply to complex substances).

Above results are consistent with freshwater and seawater biodegradability data obtained on similar substances:

- the ultimate aerobic biodegradability in freshwater of the analogue substance EC 939-455-3 was investigated in a non-GLP compliant study (De Nadai, 2010) performed in accordance with OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test) and ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)). In the test flasks containing the analogue substance and microbial inoculum, a mean of 63.2% biodegradation was observed (based on IC analysis) within the test period of 28 days;

- the ultimate aerobic biodegradability in seawater of the analogue substance EC 939-455-3 was investigated in a GLP-compliant study performed in accordance with OECD Guideline 306 (Biodegradability in Seawater). In the test flasks containing the analogue substance and seawater inoculum, a mean of 57% biodegradation was observed (based on O2 consumption) within the test period of 28 days. Altough the analogue substance was considered as not readily biodegradable in seawater under the test conditions, the result of that test was consistent with the one from the ready biodegradability test (De Nadai, 2010) with a 28-day mineralization level very close to (but just below) the 60% mineralization threshold.

The alkyl C-chain distribution of the source substance EC 939-455-3 significantly overlaps with the one of the target substance EC 939-457-7, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ultimate aerobic biodegradability in freshwater of the substance EC 939-457-4 (in aqueous commercial product) was investigated in two studies (De Nadaï, 2005, 2009) performed in accordance with standard methods, without deviations. Both studies are considered as reliable with restrictions (Klimisch 2) and were selected as key studies for the endpoint. The ultimate aerobic biodegradability in freshwater and seawater of a structural analogue (= substance EC 939-455-3 in aqueous commercial product) was investigated in two studies (De Nadaï, 2010, Finn, 2009) performed in accordance with standard methods, with no or minor deviations. Both studies are considered as reliable with restrictions (Klimisch 2) and were selected as supporting studies for the endpoint.