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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:
key study
Study period:
from 1998-03-04 to 1998-06-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with Column 2 adaptation statement of REACH Annex VIII and IX, adsorption/desorption screening and further studies on adsorption/desorption, information requirements 9.3.1 and 9.3.3, may be omitted since the log Kow value for the test substance is <3.0 (CSR sections 1.3 and 4.2.1) and has low potential for adsorption. The log Pow of Incozol LV was determined to be 1.8 (see IUCLID section 4.7). Thus, no study was conducted.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted:17 Jul 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
cited as: Directive 92/69/EEC
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Burley Menston sewage treatment works (Yorkshire Water)
- Storage conditions: On arrival in the laboratory, the sample was aerated by means of a compressed air supply delivered through a diffuser block.
- Preparation of inoculum for exposure:
The suspended solids concentration was determined by filtering a 25 mL sub-sample through a pre-dried and pre-weighed glass microfibre filter (Whatman GF/C). The filter and retained solids were then dried in a microwave oven, re-weighed and the contribution made by the sludge solids determined by difference.
- Water filtered: yes
Duration of test (contact time):
28 d
Initial conc.:
15 other: mg carbon/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
The test was conducted in a synthetic mineral salts medium based on reverse-osmosis water. A medium concentrate was prepared which contained 10 mL/L solution (a) and 1 mL/L of each of solutions (b), (c) and (d) which were composed as follows:
(a) 8.5000 g potassium dihydrogen phosphate; 21.7500 g dipotassium hydrogen phosphate ; 33.3999 g disodium hydrogen phosphate dihydrate ; 0.5004 g ammonium chloride, all dissolved in and made up to 1 L in reverse-osmosis water;
(b) 36.3999 g calcium chloride dihydrate, dissolved in and made up to 1 L in reverse-osmosis water;
(c) 22.5005 g magnesium sulphate heptahydrate, dissolved in and made up to 1 L in reverse-osmosis water;
(d) 0.2496 g ferric chloride hexahydrate plus one drop concentrated hydrochloric acid, dissolved in and made up to 1 L in reverse-osmosis water.
- Test temperature: 22 ± 2 °C
- pH: 7.31 to 7.81
Measurements of pH were made at the start of incubation in the blank and reference vessels only. The reason for omitting the vessels which contained Incozol LV product (contrary to the instruction given in the OECD Guideline) was the risk that the pH electrode would become coated with the undissolved test substance to the extent that sufficient might be removed when the electrode was withdrawn to prejudice the eventual yield of C02. Final pH readings were made in all vessels on Day 28, immediately before their contents were acidified to release any residual CO2 remaining in the solution.
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Initial coarse control was provided by air taps and the air flow to each vessel regulated by individual needle valves. Measurements were made with a bubble flow meter and stop-watch, at intervals not exceeding three days, of the flow rate exiting each test vessel through its train of scrubbers. Adjustments were made as necessary to maintain a flow rate in the range of 50 to 100 mL per minute. Initial coarse control was provided by air taps and the air flow to each vessel regulated by individual needle valves. Measurements were made with a bubble flow meter and stop-watch, at intervals not exceeding three days, of the flow rate exiting each test vessel through its train of scrubbers. Adjustments were made as necessary to maintain a flow rate in the range of 50 to 100 mL per minute.

SAMPLING
- Sampling frequency: On Days 2, 4, 6, 8, 10, 15, 19, 23 and 28
- Sampling method: At intervals during incubation, scrubbers were detached and their contents titrated with acid to determine the quantity of CO2 purged from the respective test vessels. At the end of incubation, the test vessel contents were acidified to release any residual CO2 which had remained in the solution.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Toxicity control: 1
Assessment of degradation in the toxicity control was confined to the sodium benzoate fraction.
Reference substance:
benzoic acid, sodium salt
Remarks:
15 mg carbon/L
Test performance:
The medium was inoculated with micro-organisms derived from a sample of activated sludge not previously exposed to the test substance. Test vessels were incubated in darkness within a specified temperature range for 28 days and the medium continually sparged with a supply of CO2-free air. The exhaust air from each vessel was passed through a series of dedicated CO2 scrubbers containing a barium hydroxide (Ba(OH)2) solution. At intervals during incubation, scrubbers were detached and their contents titrated with acid to determine the quantity of CO2 purged from the respective test vessels. At the end of incubation, the test vessel contents were acidified to release any residual CO2 which had remained in the solution. After correcting yields for the CO2 generated from a pair of blank vessels containing only inoculated medium, the extent of biodegradation was determined by expressing the cumulative recovered yield as a percentage of the theoretical, calculated from the carbon content of the test substance. The procedure and the activity of the inoculum were checked by a pair of vessels containing a reference substance. An additional vessel contained a combination of the test and reference substances, and served as a toxicity control to assess whether the test substance was inhibitory at the concentration at which it was applied.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
46
Sampling time:
28 d
Details on results:
Based on the CO2 analysis results, the test item cannot be classified as readily biodegradable under the OECD guideline since the test substance degradation during the study (46 %) was less than the guideline criteria of 60 % degradation within 28 days.
Results with reference substance:
Points of degradation plot (reference substance):
39.5 % degradation after 4 d
64 % degradation after 10 d
74.5 % degradation after 19 d
84.5 % degradation after 28 d

Biodegradation as percentage theoretical CO2 yield

Elapsed

time,

days

Incozol LV product

Sodium benzoate

rep. 1

rep. 2

Tox con

rep. 1

rep. 2

2

10

6

26

22

21

4

22

18

52

41

38

6

31

27

76

54

51

8

36

34

92

61

58

10

39

37

99

65

63

15

42

41

105

70

68

19

44

42

109

76

73

23

44

43

111

80

77

28

48

46

136

85

84

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
Based on the CO2 analysis results, the test item cannot be classified as readily biodegradable under the OECD guideline since the test substance degradation during the study (46 %) was less than the guideline criteria of 60 % degradation within 28 days. According to ECHA Guidance IR/CSR chapter R.7B Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability, thus, Incozol LV is regarded as inherent primarily biodegradable.
Executive summary:

A study was conducted according to OECD TG 301B and Directive 92/69/EEC Part C4 to assess the ready biodegradability of Incozol LV by measuring carbon dioxide evolution. Therefore weights of 0.0756, 0.0756 and 0.0763 g Incozol LV product were added, corresponding to between 25.2 and 25.4 mg/L, and were intended to give concentrations of Incozol LV equating to 15 mg organic carbon per litre. A nominal concentration of sodium benzoate corresponding to 15 mg C/L was used as reference. Rapid CO2 generation began immediately and continued until Day 8. The final CO2 yields for the test substance, expressed as a percentage of theoretical, were 48 % in one replicate and 46 % in the other. These data are above the 10 % level generally regarded as representing the threshold of significant degradation, but below the 60 % 'pass' level that represents complete mineralisation. According to ECHA Guidance IR/CSR chapter R.7B Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability, thus Incozol LV is regarded as inherent primary biodegradable. All the validity criteria were met and the results of this study may therefore be considered entirely valid.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 2006-10-09 to 2006-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted 17 Feb 1992
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of sewage sludge micro-organisms was obtained from the final effluent stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK
- Preparation of inoculum for exposure: The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the filtrate maintained on continuous aeration in a temperature controlled room at 21 °C prior to use.
- Water filtered: yes
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
The culture medium used in this study was that recommended in the OECD Guidelines:
Solution a: KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4.2H2O 33.40 g/L; NH4CI 0.50 g/L; pH = 7.4
Solution b: CaCl2 27.50 g/L
Solution c: MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L
An aliquot (1 mL) of each of solutions a to d was added to each litre of aerated reverse osmosis purified and deionised water. The dilution water' was kept in a temperature controlled room, at approximately 21 °C, under gentle aeration for 24 hours prior to use.

- Test temperature: 21 °C
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
The test preparations were prepared and inoculated in 250 - 300 mL Biological Oxygen Demand (BOD) bottles (darkened glass) with ground glass stoppers. The test media were transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test medium at each sampling occasion.
- Number of culture flasks/concentration: 2

SAMPLING
Dissolved oxygen concentrations for each test medium were determined, in duplicate on Days 0, 3, 5, 7, 11, 14, 18, 21, 24 and 28 by means of a YSI 54A dissolved oxygen meter and BOD Probe.

On each sampling occasion, water samples were taken for the analysis of nitrate concentration from the inoculated control, standard material, test material and toxicity control vessels.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1
- Toxicity control: 1
For the purposes of the study a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage treatment micro-organisms used in the study. An aliquot (12 mL) of the 1000 mg/L test material stock solution plus an aliquot (9 mL) of the sodium benzoate stock solution were dispersed in a final volume of 6 litres of culture medium to give a final concentration of 2.0 mg test material/L plus 1.5 mg sodium benzoate/L.
Reference substance:
benzoic acid, sodium salt
Test performance:
The test material, at a concentration of 2.0 mg/L, was exposed to sewage treatment microorganisms with culture medium in sealed culture vessels in the dark at approximately 20 °C for 28 days. The degradation of the test material was assessed by the determination of the amount of oxygen consumed. The following test preparations were prepared and inoculated in 250 - 300 mL Biological Oxygen Demand (BOD) bottles (darkened glass) with ground glass stoppers: A control, consisting of inoculated culture medium; the standard material (sodium benzoate), in inoculated culture medium to give a concentration of 3.0 mg/L; the test material, in inoculated culture medium to give a concentration of 2.0 mg/L, and the test material plus the standard material (1.5 mg/L), in inoculated culture medium to act as a toxicity control. Test media were inoculated with sewage treatment microorganisms at a rate of 1 drop of inoculum per litre. The test media were transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test medium at each sampling occasion. The BOD bottles were incubated in a temperature controlled water bath at approximately 20 °C.
Key result
Parameter:
% degradation (O2 consumption)
Value:
26
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
7 % degradation after 3 d
7 % degradation after 5 d
7 % degradation after 7 d
11 % degradation after 11 d
16 % degradation after 14 d
14 % degradation after 18 d
15 % degradation after 21 d
24 % degradation after 24 d
26 % degradation after 28 d

Nitrate and nitrite analysis of the test solutions at each sampling occasion confirmed that no significant oxygen consumption as a result of nitrification occurred during the study.

The toxicity control attained 30 % degradation after 14 days and 40 % degradation after 28 days, therefore, confirming that the test material was not toxic to the sewage treatment microorganisms used in the study.
Results with reference substance:
Points of degradation plot (reference substance):
33 % degradation after 3 d
34 % degradation after 5 d
34 % degradation after 7 d
56 % degradation after 11 d
61 % degradation after 14 d
60 % degradation after 18 d
63 % degradation after 21 d
63 % degradation after 24 d
64 % degradation after 28 d

Oxygen Depletion and Mean Percentage Biodegradation Values

Test Series

Day

3

5

7

11

14

18

21

24

28

a) culture Medium

with Inoculum Mean

O2 Depletion (mg O2/L)

Mean O2

Depletion

(mg O2/L)

0.030

-0.050

0.055

0.085

0.175

0.330

0.305

0.400

0.415

b) Sodium Benzoate

(3.0 mg/L)

with Inoculum

O2 Depletion

(mg O2/L)

1.590

1.750

1.675

2.795

3.005

2.970

3.135

3.130

3.245

1.650

1.650

1.725

2.735

3.045

3.060

3.175

3.230

3.145

% Degradation

(mean)

33

34

34

56

61

60

63

63

64

c) Test item (2.0 mg/L)

with Inoculum

O2 Depletion

(mg O2/L)

0.300

0.290

0.165

0.445

0.515

0.530

0.545

1.010

0.835

0.200

0.250

0.355

0.425

0.695

0.520

0.585

0.800

1.175

% Degradation

(mean)

7

7

7

11

16

14

15

24

26

d) Test Material (2.0 mg/L)

plus Sodium Benzoate

(1.5 mg/L) with Inoculum

O2 Depletion

(mg O2/L)

1.030

0.900

1.175

1.325

1.625

1.900

1.945

2.400

2.625

1.100

0.950

1.175

1.425

2.175

1.900

1.905

2.270

2.425

% Degradation

(mean)

17

15

18

22

30

30

30

36

40

The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 5.20 mg O2/L or greater in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20 % in all vessels thereby satisfying the validation criteria.

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
The test material attained 26 % degradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD TG 301D. According to ECHA Guidance IR/CSR chapter R.7B Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability, thus, Incozol LV is regarded as inherent primary biodegradable.
Executive summary:

A study was conducted according to OECD TG 301D to investigate the ready biodegradability of the test material when exposed to sewage treatment micro-organisms under aerobic conditions. The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 5.20 mg O2/L or greater in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20 % in all vessels thereby satisfying the validation criteria. Nitrate and nitrite analysis of the test solutions at each sampling occasion confirmed that no significant oxygen consumption as a result of nitrification occurred during the study. The test material attained 26 % degradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD TG 301D. According to ECHA Guidance IR/CSR chapter R.7B Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability, thus, Incozol LV is regarded as inherent primary biodegradable.

Description of key information

Two studies were conducted to assess the biodegradability of the test item Incozol LV. In the CO2 evolution test according to OECD TG 301B and in the closed bottle test according to OECD TG 301 D the test item does not reach the threshold of degradation to fulfil the stringent criteria of OECD TG for ready biodegradability. According to ECHA Guidance IR/CSR chapter R.7B 'Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability'. In the closed bottle test Incozol LV attained 26 % degradation after 28 days and in the CO2 evolution test 47 % degradation. Thus, Incozol LV is regarded as inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria
Type of water:
freshwater

Additional information

Two studies were conducted to assess the biodegradability of the test item Incozol LV. In the CO2 evolution test according to OECD TG 301B and in the closed bottle test according to OECD TG 301 D the test item do not reach the threshold of degradation to fulfil the stringent criteria of OECD TG for readily biodegradation. According to ECHA Guidance IR/CSR chapter R.7B ‘Biodegradation above 20% of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability’. In the closed bottle test Incozol LV attained 26 % degradation after 28 days and in the CO2 evolution test 47 % degradation. Thus, Incozol LV is regarded as inherently biodegradable.

 

CO2 evolution test

A study was conducted according to OECD TG 301B and Directive 92/69/EEC Part C4 to assess the ready biodegradability of Incozol LV by measuring carbon dioxide evolution. Therefore, weights of 0.0756, 0.0756 and 0.0763 g Incozol LV product were added, corresponding to between 25.2 and 25.4 mg/L, and were intended to give concentrations of Incozol LV equating to 15 mg organic carbon per litre. A nominal concentration of sodium benzoate corresponding to 15 mg C/L was used as reference. Rapid CO2 generation began immediately and continued until Day 8. The final CO2 yields for the test substance, expressed as a percentage of theoretical, were 48 % in one replicate and 46 % in the other. These data are above the 10 % level generally regarded as representing the threshold of significant degradation, but below the 60 % 'pass' level that represents complete mineralisation. According to ECHA Guidance IR/CSR chapter R.7B ‘Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability’, thus, Incozol LV is regarded as inherent primarily biodegradable. All the validity criteria were met and the results of this study may, therefore, be considered entirely valid.

 

Closed bottle test

A study was conducted according to OECD TG 301D to investigate the ready biodegradability of the test material when exposed to sewage treatment microorganisms under aerobic conditions. The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 5.20 mg O2/L or greater in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20 % in all vessels thereby satisfying the validation criteria. Nitrate and nitrite analysis of the test solutions at each sampling occasion confirmed that no significant oxygen consumption as a result of nitrification occurred during the study. The test material attained 26 % degradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD TG 301D. According to ECHA Guidance IR/CSR chapter R.7B ‘Biodegradation above 20 % of theoretical (measured as BOD, DOC removal or COD) may be regarded as evidence of inherent, primary biodegradability’, thus, Incozol LV is regarded as inherently biodegradable.