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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 25th march 2010 to 20th april 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
not specified
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:
not specified
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 wastewater 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 prescribed in the OECD guideline 310. 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 1.6365 g of the test substance into volumetric flask and completing the final volume to 200 ml with distilled water. One to one hundred dilution of the test substance stock solution in the mineral medium was analyzed (apparatus: Shimadzu Model TOC 5000 carbon analyzer) and indicated a mean concentration of 20.11 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 7.66
- 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.

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 20.11 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 20.11 mg /l of TOC) and reference substance (at 20 mg C/l of TOC) and the inoculated test medium

SAMPLING
SAMPLING FREQUENCY:
- Bottles were sacrificed 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 analysed for CO2 production by measuring the increase in the concentration of Inorganic Carbon in the headspace.
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:
62.26
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 24.97, 40.02, 51.79, 59.34 and 62.26% respectively, of the theoretical amount, based on Inorganic Carbon analysis. The mean cumulative net CO2 evolved from the inhibition control was 80.61% in 28 days. This value indicate that the test substance is not toxic to the inoculum.

The following validity criteria were fulfilled:
- the mean percentage degradation in the reference test was greater than 60% by the 14th day of incubation;
- the mean percentage degradation in the inhibition control was greater than 25% at the en of the test;
- the mean amount of TIC present in the blank controls at the end of the test was < 15% of the organic carbon added initially as the test substance.
Results with reference substance:
The mean percentage degradation of the reference substance was 97.09% (greater than 60%) in 28 days of incubation. These results confirm the suitability of the inoculum and test conditions.

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

-

-

-

-

15.71

Test

-

-

-

-

15.63

Test

-

-

-

-

15.52

Test

6.88

10.54

13.44

14.74

15.97

Test

6.70

10.62

13.05

15.33

15.43

Mean

6.79

10.58

13.25

15.04

15.65

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

18.20

-

-

-

35.54

Inhibition

18.13

-

-

-

35.39

Mean

18.17

-

-

-

35.47

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Based on the IC analysis, the biodegradation extent of the test item reached 62.2% in 28 days.
Executive summary:

The ready biodegradability of the REACh substance (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide (EC 242-893-1) was investigated in a non-GLP compliant study performed in accordance with OECD Guideline 310 and ISO 14593. A mean biodegradation extent of 62.2% was observed in 28 days (based on IC analysis) but the 10-day window criteria was not fulfilled.

Endpoint:
biodegradation in water: screening test, other
Remarks:
biodegradability in seawater
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From December 1st 2008 to February 4th 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 306 (Biodegradability in Seawater)
Deviations:
not specified
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: marine
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. Inoculum collected on 1 december 2008, seven days before test and at 10.6°C (temperature throughout ageing: 10.6 to 21.8°C).
Duration of test (contact time):
28 d
Initial conc.:
7.867 mg/L
Based on:
test mat.
Initial conc.:
0.661 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/L and for CaCl2 at 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
Key result
Parameter:
% degradation (O2 consumption)
Value:
61
Sampling time:
28 d
Details on results:
The test item reached 61% degradation after 28 days. The results obtained from the toxicity control show no inhibitory effects. The toxicity control attained 57% 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, reached 79% degradation after 14 days and 95% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The test was considered valid. All the validity criteria were fulfilled:
- sodium benzoate reached more than 60% of degradation in 14 days;
- the blank respiration did not exceed 30% of the oxygen in the test bottle after 28 days;
- 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.
Results with reference substance:
The mean percentage degradation of the reference substance was 95% (greater than 60%) after 28 days of incubation. This result confirms the suitability of the inoculum and test conditions.

Average net oxygen consumption (BOD, mg O2/L)

 

Day

7

14

21

28

Oxygen consumption blank

0.20

0.43

0.40

0.51

Sodium benzoate

 

1.75

1.96

2.11

2.36

Mackam LSB-50

 

2.48

2.63

2.88

3.16

Mackam LSB-50 + sodium benzoate

4.10

4.41

4.34

4.32

Percentage degradation

Material

100% BOD (mg/L)

Day

7

14

21

28

Mackam

LSB-50

5.2

48

51

56

61

Mackam

LSB-50 +

Na benzoate

7.7

53

57

57

56

Na Benzoate

2.5

70

79

85

95

Inhibition due to test substance

Day

Mackam LSB-

50 BOD (mg/L)

Na benzoate

BOD(mg/L)

Sum of

separate

BODs

(mg/L)

Mackam LSB-

50 + Na

benzoate

BOD(mg/L)

Percentage

inhibition

7

2.48

1.75

4.22

4.10

3

14

2.63

1.96

4.59

4.41

4

21

2.88

2.11

4.99

4.34

13

28

3.16

2.36

5.53

4.32

22

 

Validity criteria fulfilled:
yes
Interpretation of results:
other: rapidly degradable
Conclusions:
Based on O2 consumption, the biodegradation extent of the test item reached 61% in 28 days.
Executive summary:

The biodegradability in seawater of the REACh substance (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide (EC 242-893-1) was investigated in a GLP-compliant study performed in accordance with OECD Guideline 306. A mean biodegradation extent of 61% was observed in 28 days (based on O2 consumption)

Description of key information

The ready biodegradability of the REACh substance (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide (EC 242-893-1) was investigated in a non-GLP compliant study (De Nadai, 2010) performed in accordance with OECD Guideline 310 and ISO 14593. A mean biodegradation extent of 62.2% was observed in 28 days (based on IC analysis) but the 10-day window criteria was not fulfilled. The biodegradability in seawater of the REACh substance was otherwise investigated in a GLP-compliant study (Finn, 2009) performed in accordance with OECD Guideline 306. A mean biodegradation extent of 61% was observed in 28 days (based on O2 consumption).

It is acknowledged that biodegradation in seawater is generally slower than in freshwater. As a result, >60% ThOD in 28 days in a seawater biodegradability test (OECD 306) is an indication of rapid degradation for classification and labelling (source: Guidance on Information Requirements and Chemical Safety Assessment - Chapter R.7b: Endpoint specific guidance, Version 4.0, June 2017). Considering the biodegradation result obtained with the REACh substance in a seawater biodegradability test (>60% ThOD in 28 days), the substance can be regarded as rapidly degradable in the environment. Although the substance failed the 10-day window criterion in a ready biodegradability test, the substance is regarded a readily biodegradable for the chemical safety assessment, based on a weight-of-evidence approach (i.e. using the results of the ready biodegradability test and seawater biodegradability test all together).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The biodegrability of the substance EC 242-893-1 was investigated in two studies (De Nadai, 2010; Finn, 2009) performed in accordance with standard methods, with no or minor deviations. These studies are considered as reliable (Klimisch 2) and were used in a weight-of-evidence approach to fill in the endpoint.