Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

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:
2004-10-13 to 2004-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline compliant GLP compliant

Data source

Reference
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
as at 1992
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
as at 1992
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dl-Lactone (Synonms: DL-Pantolactone, RS-Pantolactone)
- Physical state: white crystalline mass
- Analytical purity: 99.6 %
- Purity test date: 2004-09-24
- Lot/batch No.: 451
- Date of manufacture: 2004-09-21
- Expiration date of the lot/batch: 30 September 2005
- Stability under test conditions: Stable under storage conditions, Stability in vehicle (water) at least for 4 h
- Storage condition of test material: In refrigerator in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge, freshly obtained from a
municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands, receiving predominantly domestic sewage
- Laboratory culture: kept under continuous aeration until further treatment, suspended solids was 4.9 g/I in the concentrated sludge (information
obtained from the municipal sewage treatment plant).
- Method of cultivation: not cultivated
- Storage conditions: see above
- Storage length: not stored, freshly obtained sludge was used
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
21.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of media:
- Stock solutions
A) 8.50 g KH2PO4 21.75 g K2HPO4 67.20 g Na2HPO4.12H20
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 L, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H20 dissolved in Milli-Q water and made up to 1 L
C) 36.40 g CaCl2.2H20 dissolved in Milli-Q water and made up to 1 L
D) 0.25 g FeC13.6H20 dissolved in Milli-Q water and made up to 1 L
- Mineral medium
1 L mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

Barium Hydroxide solution for CO2 absorber
0.0125 M Ba(OH)2 (Merck KGaA, Germany or Boom, the Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm)
- mixture of oxygen (21%) and nitrogen (79%)
- passed 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 synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

- Reference substance:
Name Sodium Acetate
Molecular formula C2H3NaO2
Molecular weight 82.04
Description White powder (determined at NOTOX)
CAS number 127-09-3
Batch number TA820068 033
Article number 1.06268.0250
Purity Min 99.0% (taken from label)
Expiry Date 15 November 2005 (determined at NOTOX)
Certified No
Storage conditions At room temperature in the dark
Supplier Merck
- Solubilising agent (type and concentration if used): not used
- Test temperature: between 21.1 and 22.9°C.
- pH: 7.5 - 7.8 (see table 1 for details)
- pH adjusted: no, buffer capacity of mineral medium was sufficient
- Aeration of dilution water: aerated
- Suspended solids concentration:
- amount of the sludge corresponding to approximately 30 mg/L suspended solids was added to the mineral medium
- in the adsorption control an amount of the sludge corresponding to approximately 1000 mg/I suspended solids was used
- Continuous darkness: no, but use of brown test bottles


TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
- Test suspension: containing test substance and inoculum (2 bottles)
- Inoculum blank: containing only inoculum (2 bottles)
- Procedure control: containing reference substance and inoculum (1 bottle)
- Toxicity control: containing test substance, reference substance and inoculum (1 bottle)
- Abiotic control: containing test substance and sterilising agent 1 (1 bottle)
- Adsorption control: containing test substance, inoculum and sterilising agents (1 bottle)
- Additional adsorption control (only used for DOC analyses): containing test substance at 100 mg/L and inoculum at 1000 mg/L (1 bottle, not in
series with the other bottles and not connected to CO2-absorbers)
- Method used to create aerobic conditions: synthetic air sparged through the scrubbing solutions (see above) at a rate of approximately 1-2
bubbles per second (ca. 30-100 ml/min)
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used:
three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) connected in series to the exit air line of each test bottle
- Solution preparation:
- mineral components, Milli-RO water (ca. 80% total volume, composition see above) and inoculum (1% final volume) added to each bottle and aerated with synthetic air overnight to purge the system of CO2
- test substance and positive control added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume)
- volumes of suspensions made up to 2 litres with Milli-RO water, resulting in the mineral medium described above

SAMPLING
- Sampling frequency: 3 h after addition of the test substance and on day 1, 7 (inconclusive results), 14, 16, 21, 28 and 29
- Sampling method: CO2 traps and sampling for DOC as described above under "Details on analytic methods"
- Sterility check if applicable: no
- Sample storage before analysis: directly analysed


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Other: Additional adsorption control (with high amount of inoculum)
for details see above under "Number of culture flasks/concentration: "

STATISTICAL METHODS: no special statuistic used, calculations according to OECD 301 B
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
for details see above under "reference substance"

Results and discussion

Preliminary study:
not applicable
Test performance:
- The positive control substance was degraded by at least 60% (67%) within 14 days.
- The difference of duplicate values for %-degradation of dl-Lactone was always less than 20.
- The total CO2 release in the blank at the end of the test exceeded 40 mg/I, but did not exceeded 70 mg/I (84 mg CO 2 per 2 litres of medium, corresponding to 42 mg/I).
The test is considered valid as all validity criteria were met.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
79
St. dev.:
4.2
Sampling time:
28 d
Details on results:
CO2 determinations:
- 82 and 76% degradation of RS-Pantolactone over 28 d, for the duplicate bottles tested
- more than 60% degradation of RS-Pantolactone was reached within a 10-day window
- more than 25% degradation in the toxicity control within 14 days (56%, based on ThCO2), indicating RS-Pantolactone not to be inhibitory to microbial activity
- no significant degradation of dl-Lactone in the abiotic control and the adsorption control
DOC analyses:
- no significant DOC removal in the abiotic control and the adsorption control (both containing sterilizing agent)
- additional adsorption control (RS-Pantolactone 100 mg/L, inoculum 1000 mg/L ss, no sterilizing agent): 18% DOC removal was observed after 3 hours, resulting from adsorption of RS-Pantolactone by the activated sludge
Since the DOC concentration of day 1 was approximately the same as after 3 hours, no more adsorption of the test item by the activated sludge was observed.
- degradation values in the additional adsorption control, revealed more than 60% biodegradation (79%) within 14 days.
This was in agreement with the biodegradation pattern obtained after CO 2 determinations.

BOD5 / COD results

Results with reference substance:
The positive control substance was degraded by at least 60% (67%) within 14 days.

Any other information on results incl. tables

- Table 1: The pH values of the different test media

Sample

Just before the start of the test:

On day 28:

Blank control (A)

7.5

7.5

Blank control (B)

7.5

7.5

Positive control

7.5

7.8

dl-Lactone (A)

7.5

7.5

dl-Lactone (B)

7.5

7.5

Toxicity control

7.5

7.7

Abiotic control

7.6

7.6

Adsorption control

7.6

7.6

- Table 2: CO2 production and percentage biodegradation of the positive control substance.

Day

HCL (0.05 N) titrated (mL)

Produced
CO2

(mL HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Degradation (1)
(%)

Blank
(mean)

Positive control

0

-

-

-

-

-

0

3

39.89

18.41

21.48

23.6

23.6

27

6

38.72

20.02

18.70

20.6

44.2

51

8

40.96

34.88

6.08

6.7

50.9

59

10

40.63

36.59

4.04

4.4

55.3

64

15

39.93

37.00

2.93

3.2

58.5

68

20

42.19

40.20

1.99

2.2

60.7

70

24

41.99

43.79

0.00

0.0

60.7

70

29

39.87

37.74

2.13

2.3

63.1

73

29

43.92

43.65

0.27

0.3

63.4

73

29

45.82

45.20

0.62

0.7

64.0

74

(1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 86.7 mg CO2/ 2 L

- Table 3: CO2 production and percentage biodegradation of the test substance (bottle A).

Day

HCl (0.05 N) titrated (mL)

Produced
CO2

(mL HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Degradation (1)
(%)

Blank
(mean)

bottle A

0

-

-

-

-

 -

0

3

39.89

39.45

0.44

0.5

0.5

1

6

38.72

36.29

2.43

2.7

3.2

4

8

40.96

33.88

7.08

7.8

10.9

12

10

40.63

23.45

17.18

18.9

29.8

34

15

39.93

21.88

18.05

19.9

49.7

57

20

42.19

30.63

11.56

12.7

62.4

71

24

41.99

36.51

5.48

6.0

68.4

78

29

39.87

37.35

2.52

2.8

71.2

81

29

43.92

43.23

0.69

0.8

72.0

82

29

45.82

45.64

0.18

0.2

72.2

82

(1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 87.9 mg CO2 / 2 L

- Table 4: CO2 production and percentage biodegradation of the test substance (bottle B).

Day

HCl (0.05 N) titrated (mL)

Produced
CO2

(mL HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Degradation (1)
(%)

Blank
(mean)

bottle B

0

-

-

-

-

-

0

3

39.89

39.00

0.00

0.0

0.0

0

6

38.72

38.10

0.62

0.7

0.7

1

8

40.96

33.25

7.71

8.5

9.2

10

10

40.63

30.06

10.57

11.6

20.8

24

15

39.93

19.04

20.89

23.0

43.8

50

20

42.19

27.73

14.46

15.9

59.7

68

24

41.99

37.58

4.41

4.9

64.5

73

29

39.87

37.43

2.44

2.7

67.2

76

29

43.92

44.03

0.00

0.0

67.2

76

29

45.82

45.91

0.00

0.0

67.2

76

(1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 87.9 mg CO2 / 2 L

- Table 5: Comparison of biodegradation of the test substance in bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

Difference between A and B (1)

0

0

0

0

0

3

1

0

0

1

6

4

1

2

3

8

12

10

11

2

10

34

24

29

10

15

57

50

53

7

20

71

68

69

3

24

78

73

76

4

29

81

76

79

5

29

82

76

79

5

29

82

76

79

6

(1): Absolute difference in biodegradation between bottles A and B

- Table 6: CO2 production and percentage biodegradation of the toxicity control.

Day

HCl (0.05 N) titrated (mL)

Produced
CO2

(mL HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Degradation (1)
(%)

Blank (mean)

toxicity control

0

-

-

-

-

-

0

3

39.89

18.38

21.51

23.7

23.7

14

6

38.72

18.08

20.64

22.7

46.4

27

8

40.96

19.58

21.38

23.5

69.9

40

10

40.63

28.79

11.84

13.0

82.9

47

15

39.93

22.92

17.01

18.7

101.6

58

20

42.19

30.30

11.89

13.1

114.7

66

24

41.99

36.54

5.45

6.0

120.7

69

29

39.87

31.56

8.31

9.1

129.8

74

29

43.92

41.50

2.42

2.7

132.5

76

29

45.82

45.24

0.58

0.6

133.1

76

(1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test substance and positive control: 174.6 mg CO2/2 L

ThCO2test substance: 87.9 mg CO2/2 L

ThCO2 sodium acetate: 86.7 mg CO2/2 L

- Table 7: CO2 production and percentage biodegradation of the abiotic control.

Day

HCI (0.05 N) titrated (mL)

Produced
CO2

(ml HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Degradation (1)

(%)

Blank (mean)

Abiotic Control

OS

-

-

-

-

-

0

3

39.89

44.40

0.00

0.00

0.00

0

6

38.72

44.06

0.00

0.00

0.00

0

8

40.96

44.10

0.00

0.00

0.00

0

10

40.63

43.85

0.00

0.00

0.00

0

15

39.93

43.76

0.00

0.00

0.00

0

20

42.19

45.73

0.00

0.00

0.00

0

24

41.99

44.86

0.00

0.00

0.00

0

29

39.87

42.33

0.00

0.00

0.00

0

29

43.92

44.11

0.00

0.00

0.00

0

29

45.82

46.20

0.00

0.00

0.00

0

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 87.9 mg CO2 / 2 I Note: No correction was made for the samples obtained for DOC analyses (8 * 30 ml).

A deviation for this volume, maximally 8% on day 29, had no effect on the final degradation value.

- Table 8: CO2 production and percentage biodegradation of the adsorption control.

Day

HCl (0.05 N) titrated (mL)

Produced
CO2

(mL HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Degradation (1)

(%)

Blank
(mean)

Adsorption
Control

0

-

-

-

-

0

-

3

39.89

45.30

0.00

0.00

0

0.00

6

38.72

44.51

0.00

0.00

0

0.00

8

40.96

45.28

0.00

0.00

0

0.00

10

40.63

43.87

0.00

0.00

0

0.00

15

39.93

42.74

0.00

0.00

0

0.00

20

42.19

45.50

0.00

0.00

0

0.00

24

41.99

44.49

0.00

0.00

0

0.00

29

39.87

41.52

0.00

0.00

0

0.00

29

43.92

43.55

0.37

0.40

0

0.40

29

45.82

46.38

0.00

0.40

0

0.00

(1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 87.9 mg CO2 / 2 L

Note: No correction was made for the samples obtained for DOC analyses (8 * 30 ml).

A deviation for this volume, maximally 8% on day 29, had no effect on the final degradation value.

 

- Table 9: CO2 production in the blank

Day

HCL (0.05 N) titrated (mL)

Produced
CO2

(mL HCl)

Produced
CO2

(mg)

Cumulative
CO2

(mg)

Ba(OH)2 (1)

Blank (mean)

0

-

-

-

-

0.0

3

47.72

39.89

7.83

8.6

8.6

6

48.08

38.72

9.36

10.3

18.9

8

48.31

40.96

7.35

8.1

27.0

10

46.81

40.63

6.18

6.8

33.8

15

47.77

39.93

7.84

8.6

42.4

20

51.51

42.19

9.33

10.3

52.7

24

52.77

41.99

10.78

11.9

64.5

29

49.27

39.87

9.40

10.3

74.9

29

49.07

43.92

5.15

5.7

80.5

29

48.78

45.82

2.96

3.3

83.8

(1): "Strength" of untreated 0.0125 M Ba(OH)2 solution

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
RS-Pantolactone was readily biodegradable under the conditions of the modified Sturm test presently performed (79 ± 4.2 % degradation within 28 d, > 60 %
wihtin 10 d, based on ThCO2).
Furthermore, no significant elimination of RS-Pantolactone (22 mg/L), by abiotic degradation or adsorption by the activated sludge (30 mg/L ss), was
observed.
At a higher concentration of RS-Pantolactone (100 mg/I) and activated sludge (1000 mg/I ss) 18% adsorption by the activated sludge was observed within 3
hours. Thereafter, no more adsorption of RS-Pantolactone by the activated sludge was observed.
The DOC measurements in the additional adsorption control, revealed more than 60% degradation (79%) within 14 days, which was in agreement with the
biodegradation pattern obtained after CO2 determinations.
Executive summary:

RS-Pantolactone (named dl-Lactone in the study report) was tested for it's 'ready' biodegradability using the carbon dioxide (CO2) evolution test (modified Sturm test). The study procedure was based on EEC directive 92/69, C.4-C, December 1992 and OECD guideline No. 301 B July 17, 1992.

By expert judgement it is concluded that L-Pantolactone is readily biodegradable, too.

The Theoretical CO2 production (ThCO2) of RS-Pantolactone was calculated to be 2.03 mg CO2/mg.

The test item was a white crystalline mass with a purity of 99.6%. RS-Pantolactone was tested in duplicate at 43 mg per 2 litres, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. Furthermore, an additional adsorption control was prepared at 100 mg RS-Pantolactone/L.

Test set up:

Type of bottle

Determination of CO2   DOC

Inoculum

RS-Pantolactone
(mg/I)

Sodium acetate
(mg/I)

Sterilizing agent
(HgC12)

 

blank control

X

yes

30 mg/I ss

-

-

-

test bottle

X

yes

30 mg/I ss

22

-

-

positive control

X

yes

30 mg/I ss

-

40

-

toxicity control

X

yes

30 mg/I ss

22

40

-

abiotic control

X        X

no

22

-

yes

adsorption control

X        X

yes

30 mg/I ss

22

-

yes

 

additional adsorption control

X

yes

1000 mg/I ss

100

-

-

 

ss = suspended solids

Since RS-Pantolactone was easily soluble in water the test media were prepared using a stock solution of 1 g/L in milli-RO water. A weighed amount of 1006.7 mg of RS-Pantolactone was dissolved in milli-RO water and made up to 1000 ml. The stock was a clear and colourless solution.

TOC concentration of the stock solution was 542.7 mg/L. This was in the same order of magnitude as the calculated carbon content (55%). Aliquots of 43 ml of the stock solution were added to the test medium, containing the microbial organisms, of test substance bottles A and B, toxicity control, the abiotic control and the adsorption control. An aliquot of 200 ml of the stock solution was added to the test medium of the additional adsorption control, resulting in a final RS-Pantolactone concentration of 100 mg/L. All test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

Results of CO2 determinations:

The relative degradation values calculated from the measurements performed during the test period revealed 82 and 76% degradation of RS-Pantolactone, for the duplicate bottles tested. Furthermore, more than 60% degradation of RS-Pantolactone was reached within a 10-day window. In the toxicity control more than 25% degradation occurred within 14 days (56%, based on ThCO2). Thus, RS-Pantolactone did not inhibit microbial activity. The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of RS-Pantolactone in the abiotic control and the adsorption control.

Results of DOC analyses:

No significant DOC removal was observed in the abiotic control and the adsorption control (both containing sterilizing agent). The degradation values calculated from the DOC measurements in the abiotic control and in the adsorption control, revealed no degradation of RS-Pantolactone (both containing sterilizing agent).

In the additional adsorption control (RS-Pantolactone 100 mg/L, inoculum 1000 mg/L ss, no sterilizing agent) 18% DOC removal was observed after 3 hours. This was the result of adsorption of dl­Lactone by the activated sludge. Since the DOC concentration of day 1 was approximately the same as after 3 hours, no more adsorption of RS-Pantolactone by the activated sludge was observed. The degradation values calculated from the DOC measurements in the additional adsorption control, revealed more than 60% biodegradation (79%) within 14 days. This was in agreement with the biodegradation pattern obtained after CO2 determinations.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, RS-Pantolactone was readily biodegradable under the conditions of the modified Sturm test presently performed.

Furthermore, no significant elimination of RS-Pantolactone (22 mg/L), by abiotic degradation or adsorption by the activated sludge (30 mg/L ss), was observed.

At a higher concentration of RS-Pantolactone (100 mg/L) and activated sludge (1000 mg/L ss) 18% adsorption by the activated sludge was observed within 3 hours. Thereafter, no more adsorption of RS-Pantolactone by the activated sludge was observed.

The DOC measurements in the additional adsorption control, revealed more than 60% biodegradation (79%) within 14 days, which was in agreement with the biodegradation pattern obtained after CO2 determinations.