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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 December 2002 to 15 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 301F. Ready Biodegradability: Manometric Respiration Test. Organisation for Economic Co-operation and Development. Paris (1992).
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
European Commission Directive 92/69/EC, Part C: Ready biodegradability Section V. Manometric Respiration Test (C.4-D). Off. J. Eu. Comm. L383A, 207-211 (1992).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified int he study report.
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands, on 3 December 2002. The oxidation ditch is used to treat domestic waste water. The activated sludge was transported in a plastic flask and aerated until use. The dry weight of the sludge was stated to be 3 .8 g.l-1, Although the use of 30 mg of solids per liter was intended, 8 ml of sludge (wrongly calculated, corresponding with approximately 30 mg.flask-1) was added to 300 ml of mineral medium. This resulted in a concentration of 100 mg of solids per liter.
Relevant data are:
Date of activated sludge collection: 3 December 2002
Date of use: 6 December 2002
Used for inoculation per flask: 8ml
Concentration solids in one test flask: 100 mg (d.w.).l-1
Duration of test (contact time):
28 d
Initial conc.:
23.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Theoretical Oxygen Demand (ThOD) calculation
Based on the empirical formula, the Theoretical Oxygen Demand without nitrification (ThODNID) value of dioctyloxostannane was calculated to be 2.13 mg O2.mg-1. The degree of biodegradation was calculated with this ThODNID value, assuming that the purity of the test substance was 100%.

Biodegradability test
Test substance concentrations
The determination was performed with a single mean concentration of dioctyloxostannane of 23. 7 ± 1.9 mg.l-1, corresponding to a ThODNID of 50.5 ± 4.0 mg O2.l-1, or 15 .2 ± 1.2 mg O2.f1ask-1.

Preparation of the biodegradation test flasks
The test substance sample was thoroughly mixed (homogenization was not possible). Subsequently portions with an average weight of 0.00712 g of the test substance (average of all bottles containing test substance, including the toxicity control bottles) were dosed directly on a glass fibre filters (Whatman GF/C 0 47 mm). Subsequently the filters were placed into the test flasks containing 300 ml mineral medium. This resulted in the final concentration of 23.7 mg per litre.
This series was completed with an inoculum blank, containing mineral medium only and a blank filter.

Preparation of the activity control
The final test concentration of sodium acetate was prepared by dissolving 2.9938 g of the reference substance in 100 ml of ultrapure water. From this stock solution one ml was added to 300 ml mineral medium to give a final nominal test concentration of 100 mg.l-1 sodium acetate, corresponding to a ThOD of 68 mg.l-1.
A blank series containing mineral medium only was included as blank control for the activity control.

Preparation of the toxicity control
The test substance was added to the test medium as described for the biodegradation flasks. In addition one ml of the reference substance stock solution was added.

Test series
The following test series were prepared:
Test substance
-Concentrations: 0 (blank), 23.7 mg.l-1
-Number of replicate flasks: 3
-Oxygen concentration determined: every 5 hours

Inoculum activity control
-Concentration of the reference substance: 0 (blank without filter), 100 mg.l-1
-Number of replicate flasks: 3 and 2
-Oxygen concentration determined: every 5 hours

Toxicity control
-Concentration of reference substance: 100 mg.l-1
-Concentration of test substance: 23.7 mg.l-1
-Number of replicate flasks: 2
-Oxygen concentration determined: every 5 hours

pH measurement
After addition of the test substance and mineral medium, the pH was measured in
each treatment (before inoculation). The pH was also measured in the test media at the end of the test.

Inoculation, incubation and measurements
After pH measurement and adjustment, the mineral medium in each flask was inoculated with 8 ml of the diluted sludge. This resulted in a inoculum concentration of 100 mg (dry weight).l-1 mineral medium. The flasks were closed and placed in the incubator of the Manometric Respirator (Micro-Oxymax).
When all test flasks were placed in the incubator and connected to the MicroOxymax, the incubator was closed, the temperature was set at 20 ± 2°C and the oxygen and temperature measurements were started.

Operation of the Micro-Oxymax
The Micro-Oxymax measures the percentage oxygen in the air of the respective flasks and calculates, based on the earlier measurement, the resulting oxygen consumption in a certain time period. Based on these values the oxygen consumption per flask was derived.

Termination of the experiment
After 24 days of incubation there were problems with the oxygen sensor. It was decided to prolong the test additional weeks. The actual termination was after 39 days (948.5h). The data of the last weeks (680 to 948.5h) again showed that there were problems with the oxygen sensor. Data between 581 and 675h and all data after approximately 755h were therefore considered unreliable and not taken into account. After termination of the test, the pH was measured in each flask.

Calculation of results
Calculations were performed as given in the test Guidelines. The oxygen consumption (in mg O2.l-1) in each test flask was calculated based on the respiration rate (mg O2.flask-1 .hour-1). The oxygen consumption due to the test or control substance at each time was calculated by subtracting the mean cumulative oxygen consumption in the blanks from that in the flask under consideration. These crude values were then converted to values per mg substance (BOD). The percentage biodegradation of the test substance was calculated as BOD/ThOD x 100.
Reference substance:
other: sodium acetate, anhydrous
Test performance:
The OECD Guideline 301F and the EU Test Guideline C.4-D5 list the following five conditions of validity:
-The difference of extreme replicate values of the removal of the test chemical at the plateau at the end of the test or at the end of the 1 0d ( 14d for closed flask tests) window, as appropriate, should be less than 20%.
-The reference substance sodium acetate should reach >60% degradation within 1-2 weeks.
-If less than 25% degradation is found in the toxicity control within 14 days, the test substance is considered to be toxic.
-The oxygen uptake in the inoculum blank should not exceed 60 mg O2.l-1
-If the pH value is outside the range 6.0-8.5 at the end of the test and the oxygen consumption by the test substance is less than 60% the test should be repeated with a lower concentration of test substance.

These conditions of validity have been met as follows.
-The amount of oxygen consumed in the biodegradation test is so low, that this criteria has little relevance. However, the extreme replicate cumulative oxygen consumption values for the inoculum toxicity and activity control vary less than 20 % at the end of the test.
-The reference substance was degraded 99% (ThOD) within 14 days.
-The degradation in the toxicity control was >25% during the test.
-The oxygen consumption in the blanks with filter was 1.21 - 2.96 mg per flask after 28 days of incubation, which is equal to 4.0- 9.9 mg.l-1, which is within the limits of validity.
-The pH remained within the valid range during the test in the blank and control tests.
Thus, the conditions for test validity were met.
Key result
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
28 d
Details on results:
Test conditions
The pH of the medium in the test flasks remained fairly constant during the test, with values in the range 7.2 to 7.8 after 39 days of incubation. The higher pH values were found in the activity and toxicity controls, due to the higher CO2 production.
The pH value was within the limits of the validity criteria.
The average temperature in the flasks was 21.2 ± 0.4 °C during the test (range 20.1-22.3°C, with a negligible number of measurements above 22°C.
The oxygen measurements stopped for short periods of time at day 32 (7 January 2003) to recalibrate the system, due to which the time between two sampling points is longer. In a later stage all data after day 31 have been considered unreliable and were not reported. Between 661 and 675h a data point is missing too, because the measurements were out of range, due to which the MicroOxyrnax stopped measuring and had to be restarted.
Very low negative values of oxygen consumption and high values that are not part of a 'peak' are most likely caused by leakages of the system. These occurred mainly after 755h and between 581 and 675h, and are related in this test to technical failure of the oxygen sensor. Therefore these data have not been taken into account.

Inoculum activity and toxicity control tests
The oxygen consumption in the blanks (with and without filter) was 1.21 to 2.96 mg per flask after 28 days of incubation, which is equal to 4.0-9 .9 mg.1-1. This is within the validity limits given in the guideline.
The reference test was sufficiently degraded (>60%) within 14 days of incubation. The cumulative oxygen consumption in the toxicity control (sodium acetate and test substance) was 21.5 mg O2.flask-1 after 14 days, which was similar than that of the inoculum activity control with sodium acetate only (21.6 mg O2.flask-1). Based on the combined ThOD of both substances, a biodegradation >25% was reached, which, according to the guidelines, means that the test substance is not considered toxic to the inoculum.

Biodegradation test
The average percentage of degradation of dioctyloxostannane after 28 days of incubation was approximately 2% in a manometric respiration test at a concentration of 23.7 mg.l-1, calculated from the ThODNID (2.13 mg O2.mg-1) of the test substance. In two flasks no significant biodegradation was observed and in one flask, approximately 8% biodegradation was determined after 28 days of incubation. The biodegradation of dioctyloxostannane did not exceed the pass level of 60% ThODNID within 28 days and, therefore, is classified as not readily biodegradable.
Results with reference substance:
The reference test was sufficiently degraded (>60%) within 14 days of incubation.

Results of the inoculum activity and toxicity control tests with dioctyloxostannane mean values of the cumulative oxygen consumption (mg O2.flask-1) and biodegradation as percentage of the ThODNH3(mean values) after 14, 28 and 31 days

Time (days)

Inoculum blank6)

mg O2.flask-1

Inoculum blank with filter6)

mg O2.flask-1

Inoculum activity control1), 5)

Toxicity control2), 5)

mg O2.flask-1

Biodegradation ThOD %3)

mg O2.flask-1

Biodegradation ThOD %4)

14 (336 hours)

28 (680 hours)

31 (755 hours)

1.4

1.8

1.7

1.7

2.1

2.0

21.6

23.1

23.0

99

105

105

21.5

23.2

23.1

56.0

59.6

59.6

1)100 mg.l-1sodium acetate

2)100 mg.l-1sodium acetate + 23.7 mg.l-1dioctyloxostannane

3)Corrected for blank without filter

4)Corrected for blank with filter

5)Mean value of duplicate samples

6)Mean value of triplicate samples

 

Biodegradation of dioctyloxostannane (23.7 mg.l-1) expressed as the BOD (mg O2.mg-1) and as percentage of its ThODNH3(mean values, n=3)

Time days

BOD

mg O2.mg-1

% Biodegradation ThODNH3

14 (336 hours)

29 (680 hours)

31 (755 hours)

0.0

0.0

0.0

1.5

1.9

1.9

 

pH measurements

Sample (Chamber MicroOxymax)

pH start (*)

Adjusted? y/n

pH end

Blank 1 (1)

7.4

n

7.2

Blank 2 (8)

7.4

n

7.3

Blank 3 (16)

7.4

n

7.3

Blank with filter 1 (11)

7.4

n

7.3

Blank with filter 2 (18)

7.4

n

7.3

Blank with filter 3 (23)

7.4

n

7.3

Test substance 1 (14)

7.4

n

7.2

Test substance 2 (15)

7.3

n

7.3

Test substance 3 (17)

7.4

n

7.3

Toxicity control 1 (19)

7.4

n

7.7

Toxicity control 2 (20)

7.3

n

7.7

Activity control 1 (21)

7.4

n

7.7

Activity control 2 (22)

7.4

n

7.8

 

Cumulative oxygen consumption in individual flasks

 

Blank * = blank with filter., tc = toxicity control, ref = inoculum activity control.

 

Individual values of the cumulative oxygen consumption (mg O2.flask-1) in the test flasks after 14, 28 and 31 days of incubation

Day

4019/1

4019/2

4019/3

4019tc/1

4019tc/2

Ref/1

Ref/1

14

28

31

2.76

3.30

3.18

1.38

1.67

1.54

1.52

2.10

1.98

20.66

22.22

22.10

22.36

24.17

24.04

21.66

23.25

23.13

21.55

22.98

22.86

 

Day

Blank*/1

Blank*/2

Blank*/3

Blank/1

Blank/2

Blank/3

14

28

31

1.00

1.21

1.09

1.51

2.05

1.93

2.49

2.96

2.84

1.70

2.18

2.06

1.23

1.43

1.31

1.28

1.75

1.63

 

Variation6(%) in the cumulative oxygen consumption between replicates in the test flasks after 14, 28 and 31 days of incubation

 

4019

4019tc

Ref

Day

Average

Variation %

Average

Variation %

Average

Variation %

14

28

31

1.9

2.4

2.2

73.5

69.4

73.2

21.5

23.2

23.1

7.9

8.4

8.4

21.6

23.1

23.0

0.5

1.2

1.2

 

 

Blank*

Blank

Day

Average

Variation %

Average

Variation %

14

28

31

1.7

2.1

2.0

89.7

84.2

89.4

1.4

1.8

1.7

33.7

41.9

44.7

 

6Variation is calculated as: (maximum value – minimum value)/average * 100%

 

Individual values of the biodegradation (%) in the test flasks after 14, 28 and 31 days of incubation

Day

4019/1

4019/2

4019/3

4019tc/1

4019/tc2

Ref/1

Ref/2

14

28

31

7.25

8.10

8.12

-1.91

-2.71

-2.71

-0.98

0.18

0.18

53.59

56.82

56.81

58.38

62.30

62.30

99.64

105.57

105.57

99.09

104.24

104.24

 

BOD (mg O2.mg-1(%) in the test flasks after 14, 28 and 31 days of incubation

Day

4019/1

4019/2

4019/3

Ref/1

Ref/2

14

28

31

0.15

0.17

0.17

-0.04

-0.06

-0.06

-0.02

0.00

0.00

0.68

0.72

0.72

0.67

0.71

0.71

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The average percentage degradation of dioctyloxostannane after 28 days of incubation was approximately 2% in a manometric respiration test at a test substance concentration of 23.7 mg.r1, calculated from the ThODNID (2.13 mg O2.mg-1) of test substance. The study was carried out with three replicate flasks with test substance. In two flasks no significant biodegradation was observed and in one flask, approximately 8% biodegradation was determined after 28 days of incubation. The biodegradation of dioctyloxostannane did not exceed the pass level of 60% ThODNID within 28 days and, therefore, is classified as not readily biodegradable.
Dioctyloxostannane is not toxic to the inoculum, as no inhibition of the degradation of the reference substance was observed in the toxicity control.
Executive summary:

The biodegradability of dioctyloxostannane (CAS # 870-08-6) was determined as described under 'Manometric Respirometry Test' in the OECD Guideline 301F, for testing of chemicals: 'Ready Biodegradability', using oxygen consumption as test criterion in a 31 day test. This method is in agreement with the EU Test Guideline C.4-D. The only deviations of the guidelines were that additional NaNO3 was added to the mineral medium to prevent nitrogen limitation to occur, and a higher suspended solids concentration was used.

The study was carried out in accordance with the OECD Principles of Good Laboratory Practice.

 

The test substance (a white powder) was insoluble in water. Therefore, it was added to the test medium by direct addition of the appropriate amount on a glass fibre filter and placing the filter in the mineral test medium. One concentration of 23.7 mg.l-1 (based on the substance as received) was tested, corresponding to a ThODNID of 50.5 mg O2.l-1. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (100 mg (d.w.).l-1).

 

The oxygen consumption at a target temperature of 20 ± 2°C was monitored with a Columbus Instruments Micro Oxymax respirometer.

 

The test met the conditions of validity given by the guidelines. The inoculum activity appeared to be sufficient; the reference substance sodium acetate reached the 60 % pass level of degradation within 14 days. In a toxicity control test with 23.7 mgS1 of test substance and 100 mg.l-1 sodium acetate, no inhibition of the degradation of sodium acetate was found. The test substance was not toxic according to the definition given by the test guideline at the tested concentration.

 

The test was prolonged to 39 days; however, because of problems with the oxygen sensor, data after 31 days are considered unreliable. The average percentage degradation of dioctyloxostannane after 28 days of incubation was approximately 2%. The study was carried out with three replicate flasks with test substance. In two flasks no significant biodegradation was observed and in one flask, approximately 8% biodegradation was determined after 28 days of incubation. In this study, the 60% degradation criterion after 28 days was not met and therefore the test substance was considered to be not readily biodegradable.

Description of key information

he average percentage degradation of dioctyloxostannane after 28 days of incubation was approximately 2% therefore the test substance was considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The biodegradability of dioctyloxostannane (CAS # 870-08-6) was determined as described under 'Manometric Respirometry Test', using oxygen consumption as test criterion in a 31 day test.

 

The test substance (a white powder) was insoluble in water. Therefore, it was added to the test medium by direct addition of the appropriate amount on a glass fibre filter and placing the filter in the mineral test medium. One concentration of 23.7 mg.l-1 (based on the substance as received) was tested, corresponding to a ThODNID of 50.5 mg O2.l-1. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (100 mg (d.w.).l-1).

 

The test met the conditions of validity given by the guidelines. The inoculum activity appeared to be sufficient; the reference substance sodium acetate reached the 60 % pass level of degradation within 14 days. In a toxicity control test with 23.7 mgS1 of test substance and 100 mg.l-1 sodium acetate, no inhibition of the degradation of sodium acetate was found. The test substance was not toxic according to the definition given by the test guideline at the tested concentration.

 

The test was prolonged to 39 days; however, because of problems with the oxygen sensor, data after 31 days are considered unreliable. The average percentage degradation of dioctyloxostannane after 28 days of incubation was approximately 2%. The study was carried out with three replicate flasks with test substance. In two flasks no significant biodegradation was observed and in one flask, approximately 8% biodegradation was determined after 28 days of incubation. In this study, the 60% degradation criterion after 28 days was not met and therefore the test substance was considered to be not readily biodegradable.