Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 August 2017 - 25 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
other: "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white powder
Storage conditions: at room temperature
Specific details on test material used for the study:
Stability in water: stable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: freshly obtained sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: before use, the sludge was allowed to settle for 35 minutes.
- Pre-treatment: the day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Initial concentration of sludge (suspended solids): 3.2 g/L
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12 mg/L
Based on:
TOC
Initial conc.:
16.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium according to OECD 301B
- Test temperature: 22.2 - 23.2°C
- pH:
at the start: 7.6
on day 14 (in the positive control and the toxicity control): 7.9
on day 28 (in the blank controls and the test solutions): 7.7
- pH adjusted: yes, in one of the test bottles the pH was adjusted from 7.7 to 7.6 using 1 M HCl.
- Suspended solids concentration: not indicated; the supernatant liquid of settled sludge was used at the amount of 10 mL/L of mineral medium.
- Continuous darkness: yes
- Other: the test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: synthetic air (CO2 < 1 ppm) sparged through scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Measuring method: produced CO2 reacted with barium hydroxide Ba(OH)2 in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Each time 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 (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, containing only inoculum (2 bottles)
- Abiotic sterile control: no
- Toxicity control: yes, containing test item, reference item and inoculum (1 bottle).
- Other: positive control: containing reference item and inoculum (1 bottle).

STATISTICAL METHODS: no statistics were used

TEST CONCENTRATIONS:
Test substance (added as weighed amounts to the bottles): bottle A: 16.82 mg/L test substance; bottle B: 16.64 mg/L test substance
Reference control: 40 mg/L reference substance
Toxicity control: 16.67 mg/L test substance and 40 mg/L reference substance
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
Purity: 99.5%

Results and discussion

% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
28 d
Remarks on result:
other: Bottle A
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
other: Bottle B
Details on results:
- The TOC content of the test item was determined to be 72.01% by TOC analysis (corresponding to calculations based on the molecular formula).
- The theoretical CO2 production of the test item was calculated to be 2.64 mg CO2/mg and that of the reference substance was calculated to be 1.07 mg CO2/mg.
- The test item degraded for 3% and 0% in the duplicate bottles, respectively.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item sodium acetate, which was biodegraded for 80% in 14 days and showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:
Sodium acetate showed a biodegradation of 80% after 14 days.

Any other information on results incl. tables

Table 1 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*)

Blank (mean)

1

50.48

48.94

1.54

1.7

1.7

4

50.54

46.88

3.66

4.0

5.7

6

50.18

47.26

2.93

3.2

8.9

8

49.95

46.91

3.05

3.3

12.3

11

49.37

46.22

3.15

3.5

15.7

15

49.87

45.49

4.38

4.8

20.6

18

49.98

45.60

4.38

4.8

25.4

22

49.95

45.23

4.72

5.2

30.6

25

49.83

45.28

4.55

5.0

35.6

29#)

51.10

45.67

5.43

6.0

41.5

29 #)

50.72

48.19

2.53

2.8

44.3

29#)

50.10

48.78

1.32

1.5

45.8

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

#): CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

Table 2 CO2 production and percentage biodegradation of the positive control item

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation*

(%)

Blank

(mean)

Positive

control

1

48.94

48.77

0.17

0.2

0.2

0

4

46.88

25.16

21.72

23.9

24.1

28

6

47.26

32.60

14.66

16.1

40.2

47

8

46.91

38.19

8.72

9.6

49.8

58

11

46.22

38.61

7.61

8.4

58.2

68

15#)

45.49

36.28

9.21

10.1

68.3

80

*): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 mg CO2/2L

#): CO2 measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

 

Table 3 CO2 production and percentage biodegradation of the test item (Bottle A)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation*)

(%)

Blank

(mean)

Bottle A

1

48.94

49.31

0.00

0.0

0.0

0

4

46.88

47.54

0.00

0.0

0.0

0

6

47.26

46.78

0.47

0.5

0.5

1

8

46.91

46.31

0.59

0.7

1.2

1

11

46.22

46.67

0.00

0.0

1.2

1

15

45.49

45.45

0.04

0.0

1.2

1

18

45.60

46.30

0.00

0.0

1.2

1

22

45.23

45.06

0.16

0.2

1.4

2

25

45.28

45.75

0.00

0.0

1.4

2

29#)

45.67

45.51

0.16

0.2

1.6

2

29#)

48.19

47.58

0.61

0.7

2.2

3

29#)

48.78

48.41

0.37

0.4

2.6

3

*): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2of the test item: 88.8 mg CO2/2L.

#): CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table 4 CO2 production and percentage biodegradation of the test item (Bottle B)

Day

HCl (0.05 N) titrated (mL)

Produced

CO2

(mL HCl)

Produced

CO2

(mg)

Cumulative

CO2

(mg)

Biodegradation*)

(%)

Blank

(mean)

Bottle B

1

48.94

49.28

0.00

0.0

0.0

0

4

46.88

47.54

0.00

0.0

0.0

0

6

47.26

47.17

0.08

0.1

0.1

0

8

46.91

46.94

0.00

0.0

0.1

0

11

46.22

47.46

0.00

0.0

0.1

0

15

45.49

46.18

0.00

0.0

0.1

0

18

45.60

46.64

0.00

0.0

0.1

0

22

45.23

46.40

0.00

0.0

0.1

0

25

45.28

46.33

0.00

0.0

0.1

0

29#)

45.67

48.55

0.00

0.0

0.1

0

29#)

48.19

48.13

0.05

0.1

0.2

0

29#)

48.78

48.97

0.00

0.0

0.2

0

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

#): CO2 measured on day 29 is actually part of CO2 production of day 28, since microbial activity was ended on day 28 by addition of HCl.

 

Table 5 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)

Biodegradation*)

(%)

Blank

(mean)

Toxicity

control

1

48.94

49.26

0.00

0.0

0.0

0

4

46.88

22.36

24.52

27.0

27.0

16

6

47.26

33.80

13.46

14.8

41.8

24

8

46.91

39.64

7.27

8.0

49.8

29

11

46.22

39.88

6.34

7.0

56.7

33

15#)

45.49

34.33

11.16

12.3

69.0

40

*): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and positive control: 173.6 mg CO2/2L (ThCO2test item: 88.0 mg CO2/2L + ThCO2 sodium acetate: 85.6 mg CO2/2L).

#): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Table 6 Comparison of biodegradation of the test item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B*)

1

0

0

0

0

4

0

0

0

0

6

1

0

1

1

8

1

0

1

1

11

1

0

1

1

15

1

0

1

1

18

1

0

1

1

22

2

0

1

2

25

2

0

1

2

29#)

2

0

1

2

29#)

3

0

2

3

29#)

3

0

2

3

*): Absolute difference in biodegradation between bottles A and B

#): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see 'additional remarks'
Interpretation of results:
not readily biodegradable
Conclusions:
A ready biodegradation test (Modified Sturm test), performed according to OECD 301B and GLP principles, showed that HYDROPIP-NR was biodegraded for 3% and 0% in duplicate bottles in 28 days. Based on these results the test substance is considered to be not readily biodegradable.
Executive summary:

In a Modified Sturm Test, according to OECD guideline 301 B and GLP principles, HYDROPIP-NR was assessed for its ready biodegradability. Initially, the TOC of the test substance was determined by TOC analysis to be 72.01%, whicih corresponded with calculations based on the molecular formula. The test substance was tested in duplicate at a target concentration of 16.5 mg/L, corresponding to 12 mg TOC/L. The exposure period was 28 days and two inoculum blanks, a positive control (sodium acetate) and a toxicity control were included. The test substance was not sufficiently soluble to prepare an aqueous solution of 1 g/L. Therefore, weighed amounts were added to weighing bottles containing microbial organisms and subsequently 10 mL Milli-RO water was added. After vigorous mixing, the suspension was added to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. The test substance was found not to inhibit microbial activity and all validity criteria were met, thus the study was considered to be valid.

CO2 measurements showed that the test substances was biodegraded for 3% and 0% after 28 days, in duplicate bottles. Since the pass level of 60% was not reached, HYDROPIP-NR was considered to be not readily biodegradable.