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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
November 21, 2002 to March 27, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP. The study is read across to an analogous substance; refer to image and further information below.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear colourless liquid
Details on test material:
Identification: HATCOL 3331
CAS Number: 144971-1-9
Description: Clear colourless liquid
Batch: D21287
Purity: 97.3%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not indicated
Expiry date: 01 September 2003
Specific Gravity0.969 - 0.976
Stability In vehicle: Not indicated
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):None specified in the study report.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands.Treatment: The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.9 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (30-90 minutes) and the liquid decanted for use as inoculum at the amount of 10 ml/l of mineral medium.Reason for selection: The test has been accepted internationally (EEC, OECD) for determining the 'ready' biodegradability of test substances under aerobic conditions.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
35 other: mg per 2 litres
Based on:
test mat.
Initial conc.:
12 other: mg TC/l
Based on:
other: carbon content
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
PREPARATION OF TEST SOLUTIONSThe batch of HATCOL 3331 tested was a clear colourless liquid and hardly soluble in water.Based on the Total Carbon content (TC) of HATCOL 3331 (68.90%) the test substance was tested in duplicate at 35 mg per 2 litres, corresponding to 12 mg TC/I.Weighed amounts of HATCOL 3331 (test substance botte A and B: 35.1 mg and toxicity control bottle: 35.5 mg) were mixed with 10 ml of mili-RO water. After vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.REFERENCE SUBSTANCE CONCENTRATIONSA solution of sodium acetate (Merck art. 1062680250, batch TA 820068 033) was prepared by dissolving 799.8 mg in 200 ml MiIi-RO water. Amounts of this stock solution corresponding to the 40 mg/l sodium acetate (12 mg TOC/I) were added to the test medium of the positive control bottle and the toxicity control bottle.TEST PROCEDURE AND CONDITIONSTest duration: 28 days (last CO2-measurement on the 29th day). During the test period aeration and stirring took place.Test vessels: 2 litre all-glass brown coloured bottles.MiIi-RO / Milli-Q water: Tap-water purified by reverse osmosis (MiIi-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Mill-Q) (Millpore Corp., Bedford, Mass., USA).Stock solutions of mineral components:A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4CI dissolved in 1 I Mill-Q water, pH 7.4 ± 0.2.B) 22.50 g MgSO4.7H2O dissolved in 1 I Milli-Q water.C) 36.40 g CaCI2.2H2O dissolved in 1 l Mill-Q water.D) 0.25 g FeCl3.6H2O dissolved in 1 I MiIi-Q water.Mineral medium: 11 mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and MiIi-RO water.Barium hydroxide: 0.0125 M, stored in a sealed vessel to prevent absorption of CO2 from the air.CO2-free air: A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).Test concentration: The test substance was tested in duplicate at 35 mg per 2 Iitres, corresponding to 12 mg TC/I. The carbon content was based on TC-analysis.Preparation of bottles:Pre-incubation medium: Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1 % final volume) were added to each bottle. This mixture was aerated with CO2-free air overnight to purge the system of CO2.Type and number of bottles: Test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Positive control: containing reference substance and inoculum (1 bottle). Toxicity control: containing lest substance, reference substance and inoculum (1 bottle).Preparation: The test substance and positive control were added to the bottles.The volumes of suspensions were made up to 2 Iitres with MiIi-RO water, resulting in the mineral medium described before.Three CO2-absorbers (bottles filed with 100 ml 0.0125 M Ba(OH)2 were connected in series to the exit air line of each test bottle.DETERMINATION OF CO2Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount ofCO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCI.Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each lime the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.Phenolphthalein was used as pH-indicalor.On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCI was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.Theoretical CO2 production: Because the theoretical calculation of the CO2 production was not possible and the test substance was insoluble in water, a sample of pure test substance was taken for determination of TC. TC analysis was performed at the Chemical Laboratory "Dr. A. Verwey", Rotterdam, the Netherlands using a Vario-analyzer. (Method: Vario EI AV/29.007, 1999). TC analysis was not performed under GLP conditions.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
No preliminary study detailed in the report
Test performance:
The positive control substance was degraded at least 60% within 14 days (74%).The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (49 mg CO2 per 2 Iitres of medium, this is 25 mg/l).The difference of duplicate values for %-degradation of HATCOL 3331 was always less than 20.Since all criteria for acceptability of the test were met, this study was considered to be valid.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
18 - 22
Sampling time:
28 d
Details on results:
Theoretical CO2 productionThe Total Carbon content (TC) of HATCOL 3331 was determined to be 68.90%.Based on the TC content the Theoretical CO2 production (ThC02) of HATCOL 3331 was calculated to be 2.53 mg CO2/mg.The concentration was 35.1 mg (for both bottles) HATCOL 3331 in 2 litres lest medium. Hence, the theoretical CO2 production following complete degradation was 88.8 mg per 2 litres for both bottles.The positive control contained 80.0 mg sodium acetate (ThC02= 1.07 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 85.6 mg per 2 litres.The toxicity control contained 80.0 mg sodium acetate and 35.5 mg HATCOL 3331 in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of HATCOL 3331 plus sodium acetate was 175.4 mg per 2 litres. BiodegradationThe relative degradation values calculated from the measurements performed during the test period revealed 22 and 18% degradation of HATCOL 3331 for bottle A and B respectively. Since biodegradation of HATCOL 3331 of at least 60% was not reached within 10 days after exceeding 10%, the criterion for ready biodegradability was not met.In the toxicity control more than 25% degradation occurred within 14 days (38%, based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.Monitoring of temperature and pHThe temperature recorded in a vessel with water in the same room varied between 21.6 and 23.4°C.The pH values of the different test media are described in table form, see Any other information.

Any other information on results incl. tables

The pH values of the different test media were:

 

Just before the start of the test:

On day 28:

Blank control (A)

:

7.5

7.3

Blank control (B)

:

7.5

7.3

Positive control

:

7.4

7.6

HATCOL 3331 (A)

:

7.4

7.3

HATCOL 3331 (B)

:

7.3

7.3

Toxicity control

:

7.4

7.6

 

Note: All calculations were performed without rounding off.

 

CO2production and percentage biodegradation of the positive control substance

Day

HCI (0.05 N) titrated (ml)

Produced CO2(ml HCI)

Produced CO2(mg)

Cumulative CO2(mg)

Degradation1)(%)

Blank (mean)

Positive control

0

2

5

7

9

14

19

23

27

29

29

29

-

43.47

43.77

45.27

46.38

45.36

44.58

43.11

42.63

43.77

45.00

45.22

-

40.29

20.96

33.74

38.63

32.80

38.35

39.22

39.69

40.95

44.32

44.75

-

3.18

22.81

11.53

7.75

12.56

6.23

3.89

2.94

2.82

0.68

0.47

-

3.5

25.1

12.7

8.5

13.8

6.9

4.3

3.2

3.1

0.7

0.5

-

3.5

28.6

41.3

49.8

63.6

70.4

74.7

78.0

81.1

81.8

82.3

0

4

33

48

58

74

82

87

91

95

96

96

1) Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.6 mg CO2/2l

 

CO2production and percentage biodegradation of the test substance (bottle A)

Day

HCI (0.05 N) titrated (ml)

Produced CO2(ml HCI)

Produced CO2(mg)

Cumulative CO2(mg)

Degradation1)(%)

Blank (mean)

Positive control

0

2

5

7

9

14

19

23

27

29

29

29

-

43.47

43.77

45.27

46.38

45.36

44.58

43.11

42.63

43.77

45.00

45.22

-

39.89

39.57

41.92

44.62

43.13

43.41

43.35

42.53

42.69

44.34

49.28

-

3.58

4.20

3.35

1.76

2.23

1.17

0.00

0.10

1.08

0.66

0.00

-

3.9

4.6

3.7

1.9

2.4

1.3

0.0

0.1

1.2

0.7

0.0

-

3.9

8.6

12.2

14.2

16.6

17.9

17.9

18.0

19.2

19.9

19.9

0

4

10

14

16

19

20

20

20

22

22

22

1) Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 88.8 mg CO2/2l

 

CO2production and percentage biodegradation of the test substance (bottle B)

Day

HCI (0.05 N) titrated (ml)

Produced CO2(ml HCI)

Produced CO2(mg)

Cumulative CO2(mg)

Degradation1)(%)

Blank (mean)

Positive control

0

2

5

7

9

14

19

23

27

29

29

29

-

43.47

43.77

45.27

46.38

45.36

44.58

43.11

42.63

43.77

45.00

45.22

-

45.03

41.95

44.61

45.11

40.23

41.78

42.02

41.49

43.26

44.81

49.50

-

0.00

1.82

0.66

1.27

5.13

2.80

1.09

1.14

0.51

0.19

0.00

-

0.0

2.0

0.7

1.4

5.6

3.1

1.2

1.3

0.6

0.2

0.0

-

0.0

2.0

2.7

4.1

9.7

12.8

14.0

15.3

15.8

16.0

16.0

0

0

2

3

5

11

14

16

17

18

18

18

1) Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 88.8 mg CO2/2l

 

Comparison of biodegradation of the test substance in bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

0

2

5

7

9

14

19

23

27

29

29

29

0

4

10

14

16

19

20

20

20

22

22

22

0

0

2

3

5

11

14

16

17

18

18

18

0

2

6

8

10

15

17

18

19

20

20

20

0

4

7

11

11

8

6

4

3

4

4

4

1)Absolute difference in biodegradation between bottles A and B.

 

CO2production and percentage biodegradation of the toxicity control

Day

HCI (0.05 N) titrated (ml)

Produced CO2(ml HCI)

Produced CO2(mg)

Cumulative CO2(mg)

Degradation1)(%)

Blank (mean)

Positive control

0

2

5

7

9

14

19

23

27

29

29

29

-

43.47

43.77

45.27

46.38

45.36

44.58

43.11

42.63

43.77

45.00

45.22

-

47.99

19.16

33.29

36.18

31.89

37.74

40.46

40.78

39.99

44.98

49.88

-

0.00

24.61

11.98

10.20

13.47

6.84

2.65

1.85

3.78

0.02

0.00

-

0.0

27.1

13.2

11.2

14.8

7.5

2.9

2.0

4.2

0.0

0.0

-

0.0

27.1

40.2

51.5

66.3

73.8

76.7

78.7

82.9

82.9

82.9

0

0

15

23

29

38

42

44

45

47

47

47

1) Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance and positive control: 175.4 mg CO2/2l

ThCO2test substance: 89.8 mg CO2/2l

ThCO2sodium acetate: 85.6 mg CO2/2l

 

CO2production in the blank

Day

HCI (0.05 N) titrated (ml)

Produced CO2(ml HCI)

Produced CO2(mg)

Cumulative CO2(mg)

Ba(OH)21)

Blank (mean)

0

2

5

7

9

14

19

23

27

29

29

29

-

49.44

49.17

49.43

49.23

49.37

49.66

48.80

47.82

48.55

44.96

46.64

-

43.47

43.77

45.27

46.38

45.36

44.58

43.11

42.63

43.77

45.00

45.22

-

5.97

5.40

4.16

2.85

4.01

5.08

5.69

5.19

4.79

0.00

1.43

-

6.6

5.9

4.6

3.1

4.4

5.6

6.3

5.7

5.3

0.0

1.6

0.0

6.6

12.5

17.1

20.2

24.6

30.2

36.5

42.2

47.4

47.4

49.0

1)“Strength” of untreated 0.0125 M Ba(OH)2solution

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
HATCOL 3331 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with HATCOL 3331.

The study procedure was based on EEC directive 92/69, C4-C, December 1992, and OECD guideline

No. 301 B July 17, 1992.

 

The batch of HATCOL 3331 tested was a clear colourless liquid and hardly soluble in water.

The Total Carbon content (TC) of HATCOL 3331 was determined to be 68.90%. Based on the TC content the Theoretical CO2production (ThCO2) of HATCOL 3331 was calculated to be 2.53 mg CO2/mg. HATCOL 3331 was tested for its ready biodegradability at 35 mg per 2 litres, corresponding to 12 mg TC/I.

 

The study consisted of six bottles:

2 blank controls (no lest material),

2 test bottles (HATCOL 3331, 17.5 mg/l),

1 positive control (sodium acetate, 40 mg/l) and

1 toxicity control (HATCOL 3331, 17.5 mg/l; plus sodium acetate, 40 mg/l).

 

Weighed amounts of HATCOL 3331 were mixed with 10 ml of milli-RO water and after vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

 

The relative degradation values calculated from the measurements performed during the test period revealed 22 and 18% degradation of HATCOL 3331 for bottle A and B respectively.

Since biodegradation of HATCOL 3331 of at least 60% was not reached within 10 days after exceeding 10%, the criterion for ready biodegradability was not met.

In the toxicity control HATCOL 3331 was found not to inhibit microbial activity.

 

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

 

In conclusion, HATCOL 3331 was not readily biodegradable under the conditions of the modified Sturm test presently performed.