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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
key 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
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
Remarks:
see "Test performance"
Interpretation of results:
other: 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 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:
key 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
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to guideline
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
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 1.7012 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
Remarks:
see "Executive summary"
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:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
february 2005 -august 2005
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test performed under GLP and according to an international standard method. Nevertheless, several deviations are observed: no toxicity control was performed thus an inhibition of the sewage respiration rate by the test substance cannot be excluded. Data are lacking on the characterization of the substance, on the test medium used and on the characterization of the effluent.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
no characterisation of the effluent, no toxicity test sample, only one replicate for blank and reference substance, no data on the medium used, temperature lower than the one recommended
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
other: Secondary sewage effluent
Details on inoculum:
Secondary sewage effluent from Runcorn East Sewage works on 18th february 2005.
No data on adaptation, on biomass concentration or on type of sewage treated (domestic or industrial).
Duration of test (contact time):
28 d
Initial conc.:
6 mg/L
Initial conc.:
7 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: no data
- Test temperature: 18.0 - 19.0°C
- pH: no data
- Aeration of dilution water: no data
- Suspended solids concentration: no data

TEST SYSTEM
- Number of culture flasks/concentration: 2 replicates for each sample day for test substance, 2 replicates for each sample day for reference substance, 3 replicates for each sample day for blank

SAMPLING
- Sampling frequency: sampling performed on days 0, 3, 7, 14, 21 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: no
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No data
Parameter:
% degradation (O2 consumption)
Value:
29.9
Sampling time:
28 d
Remarks on result:
other: test material at 6 mg/L
Parameter:
% degradation (O2 consumption)
Value:
27.9
Sampling time:
28 d
Remarks on result:
other: test material at 7 mg/L
Details on results:
Degradation reached 42.9% on day 14 for test material at 6 mg/L. Degradation reached 30.7% on day 21 for test material at 7 mg/L.

The Chemical Oxygen Demand was calculated as 0.707 mg O2/mg.

The following validity criteria were fulfilled:
* Sodium benzoate attained more than 60% of degradation in 14 days.
* In the blank, less than 1.5 mg/L O2 was consumed at the end of the test (at day 28).

No toxicity and abiotic controls performed.

 

% degradability

Material (mg/L)

Day 3

Day 7

Day 14

Day 21

Day 28

Test item (Cocamidopropylhydroxysultaine)

(6.0)

3.1

33.0

42.9

35.1

29.9

Test item (Cocamidopropylhydroxysultaine)

(7.0)

*

30.1

30.3

30.7

27.9

Sodium Benzoate

(2.5)

57.2

59.6

78.3

77.4

68.7

*Indicates negative degradation

** unsing the calculated Theorical oxygen demand (ThOD) of sodium benzoate as 1.67 mg O2 / mg

Validity criteria fulfilled:
no
Remarks:
see 'rationale for reliability' and 'Details on results'
Interpretation of results:
other: not readily biodegradable
Conclusions:
The test item was investigated for its ready biodegradability according to the OECD Guideline No. 301D (1992) and in compliance with GLP. A mean of 29.9% of biodegradation was observed (based on O2 removal and at a concentration of 6 mg/L) within the test period of 28 days. Therefore, the test item is not considered as readily biodegradable under the test conditions. Nevertheless, several deviations are observed and the test cannot be considered as reliable (reliability 3).
Executive summary:

The test item was investigated for its ready biodegradability according to the OECD Guideline No. 301D (1992) and in compliance with GLP. In the test flasks, containing the test item and microbial inoculum, a mean of 29.9% of biodegradation was observed (based on O2removal and at a concentration of 6 mg/L) within the test period of 28 days. Therefore, the test item is not considered as readily biodegradable under the test conditions.

The following validity criteria were respected:

- The mean percentage degradation in the reference substance was greater than 60% by the 14th day of incubation

- In the blank, less than 1.5 mg/L O2 was consumed at the end of the test (at day 28).

Nevertheless, several deviations of the guideline were observed:

- there are no toxicity and abiotic controls performed and it is not possible to assess a toxicity of the substance on the microbial inoculum

- the test substance is not clearly defined

- there is no characterisation of the effluent,

- there is no data on the test medium

- there is only one replicate for blank and reference substance,

- the temperature is lower than the one recommended

Thus, the test cannot be considered as reliable (reliability 3).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
may 2005 -october 2006
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test performed under GLP and according to an international standard method. Nevertheless, several deviations are observed: the reference substance did not attain 60% of degradation in 14 days in thus it is not possible to confirm the suitability of the test. Moreover, no toxicity control was performed thus an inhibition of the sewage respiration rate by the test substance cannot be excluded. Data are lacking for the characterization of the substance, on the test medium used and on the effluent and results of each replicates are not reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
lack of data on the effluent, no toxicity test sample, only one replicate for blank and reference substance, no data on the medium used
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Activated sludge from Runcorn East Sewage.
The sludge was left to settle for several days and fed test media until the study was ready to commence. A final concentration of 30.0 mg dry weight per litre of test media was attained.
No data on biomass concentration or on type of sewage treated (domestic or industrial).
Duration of test (contact time):
28 d
Initial conc.:
39.4 mg/L
Initial conc.:
78.7 mg/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: no data
- Test temperature: 20 +/- 2°C
- pH: no data
- Aeration of dilution water: no data
- Suspended solids concentration: 30 mg dw/L of test media

TEST SYSTEM
- Number of culture flasks/concentration: 2 replicates for each sample day for test substance, 1 replicates for each sample day for reference substance, 1 replicates for each sample day for blank

SAMPLING
- Sampling frequency: sampling performed on days 1, 4, 6, 8, 12, 15, 19, 22, 25, 28 and 29.
- sampling method: see 'Details on analytical method'

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: no
Reference substance:
benzoic acid, sodium salt
Remarks:
102.9 mg (20 mg C per litre) in 3 litres
Preliminary study:
No data
Test performance:
Reference substance failed to reach the 60% degradation level within 28 days.
Parameter:
% degradation (CO2 evolution)
Value:
14.4
Sampling time:
28 d
Remarks on result:
other: test material at 39.4 mg/L
Parameter:
% degradation (CO2 evolution)
Value:
90.8
Sampling time:
28 d
Remarks on result:
other: test material at 78.7 mg/L
Details on results:
Degradation reached 30.8% on day 22 for test material at 39.4 mg/L. Degradation reached 90.8% on day 28 for test material at 78.7 mg/L.

The following validity criteria was not fulfilled: Sodium benzoate did not attain 60% of degradation in 14 days.
There are no results on each replicates thus it is not possible to verify if the difference of degradation between replicates is less than 20%.

No toxicity and abiotic controls performed.
Results with reference substance:
Sodium benzoate did not attain 60% of degradation in 14 days.
see table below.

 

Cumulative degradation (%)

Day of titration

Sodium benzoate (34.3 mg/L)

Test item

(39.4 mg/L)

Test item

(78.7 mg/L)

1

22.9

*

8.2

4

44.4

13.4

28.3

6

49.7

24.5

57.6

8

50.6

17.6

72.0

12

52.9

14.3

71.8

15

54.7

27.4

73.8

19

50.4

20.0

81.6

22

44.4

30.8

82.2

25

44.8

18.1

80.5

28

51.7

14.4

90.8

29

52.0

0.7

85.5

*Indicates negative degradation

Validity criteria fulfilled:
no
Remarks:
see 'rationale for reliability' and 'Details on results'
Interpretation of results:
other: not possible to conclude as the test is not suitable.
Conclusions:
The test item was investigated for its ready biodegradability according to the OECD Guideline No. 301B (1993) and in compliance with GLP. For the test item at 39.4 mg/L, the final 28 day cumulative % degradation value was determined to be 14.4%. At 78.7 mg/L the final 28 day cumulative % degradation value was 90.8%.
Several deviations are observed: the reference substance did not attain 60% of degradation in 14 days in thus it is not possible to confirm the suitability of the test. Moreover, no toxicity control was performed thus an inhibition of the sewage respiration rate by the test substance cannot be excluded.
Therefore, the test is considered as not reliable (reliability 3) and it is not possible to conclude on the ready biodegradation potential of the test item.
Executive summary:

The test item was investigated for its ready biodegradability according to the OECD Guideline No. 301B (1993) and in compliance with GLP.

In the test flasks, containing the test item at 39.4 mg/L and microbial inoculum, the final 28 day cumulative % degradation value was determined to be 14.4%. At 78.7 mg/L the final 28 day cumulative % degradation value was 90.8%.

Several deviations are observed: the reference substance did not attain 60% of degradation in 14 days in thus it is not possible to confirm the suitability of the test. Moreover, no toxicity control was performed thus an inhibition of the sewage respiration rate by the test substance cannot be excluded. Data are lacking for the characterization of the substance, on the test medium used and on the effluent and results of each replicates are not reported.

Therefore, the test is considered as not reliable (reliability 3) and it is not possible to conclude on the ready biodegradation potential of the test item.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
march 2007 - august 2007
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test performed under GLP and according to an international standard method. Nevertheless, several deviations are observed: the reference substance did not attain 60% of degradation in 14 days in thus it is not possible to confirm the suitability of the test. Moreover, no toxicity control was performed thus an inhibition of the sewage respiration rate by the test substance cannot be excluded. Data are lacking for the characterization of the substance, on the test medium used and on the effluent and results of each replicates are not reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
lack of data on the effluent, no toxicity test sample, only one replicate for blank and reference substance, no data on the medium used
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Activated sludge from Runcorn East Sewage.
The sludge was left to settle for several days and fed test media until the study was ready to commence. A final concentration of 30.0 mg dry weight per litre of test media was attained.
No data on biomass concentration or on type of sewage treated (domestic or industrial).
Duration of test (contact time):
28 d
Initial conc.:
39.4 mg/L
Initial conc.:
78.7 mg/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: no data
- Test temperature: 20 +/- 2°C
- pH: no data
- Aeration of dilution water: no data
- Suspended solids concentration: 30 mg dw/L of test media

TEST SYSTEM
- Number of culture flasks/concentration: 2 replicates for each sample day for test substance, 1 replicates for each sample day for reference substance, 1 replicates for each sample day for blank

SAMPLING
- Sampling frequency: sampling performed on days 1, 4, 6, 8, 10, 13, 16, 21, 23, 27, 28 and 29.
- sampling method: see 'Details on analytical method'

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: no
Reference substance:
benzoic acid, sodium salt
Remarks:
102.9 mg in 3 litres (corresponding to 20 mg C per litre)
Preliminary study:
No data
Parameter:
% degradation (CO2 evolution)
Value:
> 100
Sampling time:
28 d
Remarks on result:
other: test material at 39.4 mg/L
Parameter:
% degradation (CO2 evolution)
Value:
92.3
Sampling time:
28 d
Remarks on result:
other: test material at 78.7 mg/L
Details on results:
Degradation attained 153% at the end of the test for test material at 39.4 mg/L. Degradation reached 92.3% on day 28 for test material at 78.7 mg/L.

Sodium benzoate attained more than 60% of degradation in 28 days but not in 14 days. Thus, the validity criteria is not fulfilled.
There are no results on each replicates thus it is not possible to verify if the difference of degradation between replicates is less than 20%.

No toxicity and abiotic controls performed.
Results with reference substance:
Sodium benzoate did not attain 60% of degradation in 14 days.
see table below.

 

Cumulative degradation (%)

Day of titration

Sodium benzoate (34.3 mg/L)

Test item

(39.4 mg/L)

Test item

(78.7 mg/L)

1

2.2

10.6

10.3

2

5.2

35.5

13.3

4

18.9

46.2

25.5

6

37.0

53.5

37.8

8

40.7

74.3

49.0

10

51.6

73.2

60.7

13

59.7

>100

70.8

16

58.0

94.9

61.5

21

67.5

>100

91.4

23

70.6

>100

92.0

27

70.6

>100

91.2

28

76.9

>100

93.5

29

79.8

>100

92.3
Validity criteria fulfilled:
no
Remarks:
see 'rationale for reliability' and 'Details on results'
Interpretation of results:
other: not possible to conclude as the test is not suitable.
Conclusions:
The test item was investigated for its ready biodegradability according to the OECD Guideline No. 301B (1993) and in compliance with GLP. For the test item at 39.4 mg/L, the final 28 day cumulative % degradation value was determined to be >100%. At 78.7 mg/L the final 28 day cumulative % degradation value was 92.3%. A maximum value of 153% was attained and the blank seems invalid.
Several deviations are observed: the reference substance did not attain 60% of degradation in 14 days thus it is not possible to confirm the suitability of the test. Moreover, no toxicity control was performed and an inhibition of the sewage respiration rate by the test substance cannot be excluded.
Therefore, the test is considered as not reliable (reliability 3) and it is not possible to conclude on the ready biodegradation potential of the test item.
Executive summary:

The test item was investigated for its ready biodegradability according to the OECD Guideline No. 301B (1993) and in compliance with GLP.

In the test flasks, containing the test item at 39.4 mg/L and microbial inoculum, 39.4 mg/L, the final 28 day cumulative % degradation value was determined to be >100%. At 78.7 mg/L the final 28 day cumulative % degradation value was 92.3%. A maximum value of 153% was attained and the blank seems invalid.

Several deviations are observed: the reference substance did not attain 60% of degradation in 14 days thus it is not possible to confirm the suitability of the test. Moreover, no toxicity control was performed and an inhibition of the sewage respiration rate by the test substance cannot be excluded.

Therefore, the test is considered as not reliable (reliability 3) and it is not possible to conclude on the ready biodegradation potential of the test item.

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-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts (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 substance and microbial inoculum, a mean of 63.2% biodegradation 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).

The ultimate aerobic biodegradability in seawater of the substance was otherwise investigated in a GLP-compliant study performed in accordance with OECD Guideline 306 (Biodegradability in Seawater). In the test flasks containing the substance and seawater inoculum, a mean of 57% biodegradation was observed (based on O2 consumption) within the test period of 28 days. Altough the substance was considered as not readily biodegradable in seawater under the test conditions, the result of that test is 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 ultimate aerobic biodegradability of the substance was eventually investigated in three other studies (Rowlands, 2005; Hill, 2006; Manley, 2007) which were not considered in the assessment due to major methodological deficiencies and insufficient documentation.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The ultimate aerobic biodegradability in freshwater and seawater of the substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts (EC 939-455-3) 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 key studies for the endpoint. The three other available studies (Rowlands, 2005; Hill, 2006; Manley, 2007) are considered as not reliable (Klimisch 3) and were disregarded for the endpoint.)