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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:
2011-10-13 to 2011-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Qualifier:
according to
Guideline:
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes, and inland sea; or return sludges from sewage plants).
- Pretreatment: The activated sludge, which was cultivated for 18.5 hours after the synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0±1.0.
- Concentration of sludge: 30 mg/L (as the concentration of suspended solid)
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: biochemical oxygen demand (BOD)
Details on study design:
Performance of the biodegradability test:

Preparations for the test
a) Measurement of concentration of suspended solid in activated sludge:
The concentration of suspended solid was measured in order to determine the amount of activated sludge to be added.
- Method: In accordance with Japanese Industrial Standards (JIS) K 0102-2010 Section 14.1
- Result: Concentration of suspended solid in the activated sludge was 2980 mg/L.

b) Preparation of basal culture medium
5 L of basal culture medium was prepared by the same method as follows; purified water (Japanese Pharmacopeia, Takasugi Pharmaceutical Co., Ltd.) was added to each 3 mL aliquot of solutions A, B, C and D, which are described in JIS K 0102-2010 Section 21, in order to prepare 1 L of solution. The pH of this solution was then adjusted to 7.0.

c) Validity of activated sludge
Aniline was used as a reference item in order to confirm that the sludge was sufficiently active.

Preparation of test solutions
The following test solutions were prepared and cultured under the conditions described in the following:

a) Addition of test item or aniline
1 ) Test solution (water + test item) (n=1, Vessel No. 1 ): In one test vessel, 30 mg of the test sample was accurately weighed and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L.
2) Test solution (sludge + test item) (n=3, Vessel Nos. 2,3 and 4): In each test vessel, 30 mg of the test sample was accurately weighed and added to the basal culture medium [the volume subtracting the volume (3.02 mL) of activated sludge from 300 mL], so that the concentration of the test item reached 100 mg/L.
3) Test solution (sludge + aniline) (n=1, Vessel No. 6): In one test vessel, 29.5 uL (30 mg) of aniline was removed by using a microsyringe and added to the basal culture medium [the volume subtracting the volume (3.02 mL) of activated sludge from 300 mL], so that the concentration of aniline reached 100 mg/L.
4) Test solution (control blank) (n=1, Vessel No. 5): In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (3.02 mL) of activated sludge from 300 mL].

b) Inoculation of activated sludge
The activated sludge was added to each test vessel described in 2), 3), and 4), so that the concentration of the suspended solid reached 30 mg/L.

Conditions of cultivation:
- Cultivation temperature: 25±1 °C
- Cultivation duration: 28 days (under dark conditions)
- Stirring method: Each test solution was stirred by a stirrer.

Observation and measurement of test oenditions
a) Observation of test solution: During the cultivation period, the appearance of the test solution was observed once a day.
b) Measurement of biochemical oxygen demand (BOD): During the cultivation period, the change in BOD of the test solutions was measured by a closed system oxygen consumption measuring apparatus. The cultivation temperature was measured and recorded once a day.
Reference substance:
aniline
Key result
Parameter:
other: BOD
Value:
91
Sampling time:
28 d
Details on results:
- Percentage biodegradations after 28 days were as follows.
The percentage decrease of the test item, the ratio of decreased amount to the theoretical amount, was calculated instead of the percentage biodegradation of the test item because the percentage residue of the test item in the test solution (water + test item) was less than 90 % (63%). The percentage biodegradation by DOC was not calculated because the test item can not be dissolved in water at the test concentration (100 mg/L) and more.

- Percentage biodegradation by BOD [%] (vessel 2 / vessel 3 / vessel 4 / average): 92 / 91 / 90 / 91
- Percentage decrease of the test item (HPLC) [%] (vessel 2 / vessel 3 / vessel 4 / average): 100 / 100 / 100 / 100

- Results of qualitative analysis of other converted products
The samples for LC-MS analysis -2 (for ethyl acetate-extractable products) and LC-MS analysis -3 (for water-soluble products) were analyzed by LC-MS for the qualitative analysis of other converted products that could not be determined their amounts. As a result, no peak corresponding to other converted product was detected on positive and negative ion total ion current (TIC) chromatograms of the samples for LC-MS analysis -2 and -3. It is considered that other converted products were not produced in the test solution (water + test item) and the test solutions (sludge+test item).


Further deatils:
a) Test solution (water + test item)
The percentage residue of the test item was 63%. Although the test item is insoluble in water, the percentage detection of DOC was 14%. It is considered that some of the test item were converted. Therefore, 2,2-dimethyl-3-oxopropyl dodecanoate, hexamethylenediamine, dodecanoic acid and 2,2-dimethyl-3-hydroxypropionic acid were determined; 2,2-dimethyl-3-oxopropyl dodecanoate and hexamethylenediamine were expected to be produced by the hydrolysis of the test item, and dodecanoic acid and 2,2-dimethyl-3-hydroxypropionic acid were expected to be produced from 2,2-dimethyl-3-oxopropyl dodecanoate. As a result, the percentage productions of 2,2-dimethyl-3-oxopropyl dodecanoate, hexamethylenediamine, dodecanoic acid and 2,2-dimethyl-3-hydroxypropionic acid were 29%, 31 %, 4% and 0%, respectively. Then, the mass balances of hexamethylenediamine part and dodecanoic acid part were 94% and 96%, respectively.
On the other hand, the mass balance of 2,2-dimethyl-3-hydroxypropionic acid part showed a little loss as 92%. Additionally, DOC amount calculated from the produced amount of hexamethylenediamine (water-soluble compound) was 1.1 mg C and the difference between the calculated DOC amount and the measured DOC amount was 2.1 mg C. Therefore, it is considered that other converted products were produced However, other converted products were not detected in the qualitative analysis. These results suggested that other converted products which are converted from 2,2-dimethyl-3-hydroxypropionic acid part and have a low sensitivity on LC-MS were produced
From the above-mentioned results, it is considered that some of the test item were hydrolyzed to 2,2-dimethyl-3-oxopropyl dodecanoate and hexamethylenediamine. Additionally, some of the 2,2-dimethyl-3-oxopropyl dodecanoate were converted to dodecanoic acid and other converted products having a low sensitivity on LC-MS.

b) Test solution (sludge + test item)
The percentage biodegradations by BOD were 90-92% and growth of the sludge was observed at the end of cultivation. Additionally, the percentage residue of the test item and the percentage productions of all converted products were 0%. These results suggested that the test item was completely biodegraded after hydrolysis.
Results with reference substance:
Percentage biodegradation of aniline by BOD after 14 days: 72%

Recovery test:

The recovery rates and their averages are shown below. Though the averages in test solutions of test item were less than 90%, the difference between replicate values in each was within 5%. Therefore, it was considered that the recovery rates of test item could be used as a correction factor, and it does not effect on the reliability and integrity of this test. The averages in other test solutions were 90% and more, and the difference between replicate values in each was within 5%. In addition, no peak exceeding the lowest detectable peak area appeared around the peak of the test item, Aldehyde L and dodecanoic acid on the chromatogram of control blank. Therefore, the validity of the pretreatment applied in this study was confirmed.

The average recovery rates were used as a correction factor, for the determination of the test item, 2,2-dimethyl-3-oxopropyl dodecanoate and dodecanoic acid in the analytical samples. Recovery tests for hexamethylenediamine and 2,2-dimethyl-3-hydroxypropionic acid were not performed because they were soluble in the test solution and the supernatant after centrifugation is used for quantitative analysis of them.

a) Test item

30 mg of the test item was accurately weighed and added in the recovery test.

- Recovery rate in the test solutions (water + test item): 88.8%, 89.2% average 89.0%

- Recovery rate in the test solutions (sludge + test item): 77.7%, 78.6% average 78.1%

b) 2,2-dimethyl-3-oxopropyl dodecanoate

26 mg of Aldehyde L was accurately weighed and added in the recovery test.

- Recovery rate in the test solutions (water+Aldehyde L + dodecanoic acid): 101%, 101% average 101%

- Recovery rate in the test solutions (sludge + Aldehyde L + dodecanoic acid): 100%, 99.5% average 99.8%

c) Dodecanoic acid

18 mg of dodecanoic acid was accurately weighed and added in the recovery test.

- Recovery rate in the test solutions (water+Aldehyde L + dodecanoic acid): 101%, 103%, average 102%

- Recovery rate in the test solutions (sludge + Aldehyde L + dodecanoic acid): 102%, 102%, average 102%

The following interpretation of results are not included in the final report:

The criterion, which is generally used to define the biodegradability, is the 10 -day window. The pass levels for ready biodegradability are 70% removal of DOC and 60% of ThOD or ThCO2 production for respirometric methods. The 10-d window begins when the degree of biodegradation has reached 10% DOC, ThOD or ThCO2 and must end before day 28 of the test.

According to the OECD no. 301 guideline the 10-d window concept does not apply to the MITI method. However, the visual analysis of the diagram of the BOD evolution versus the cultivation time indicates that the 10-day window criterion for defining the readily biodegradability is met, since 60% BOD or 70% BOD, respectively is observed after ca. 6 and ca. 8 days respectively.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions of this study, the test substance Sika Hardener LH was completely hydrolyzed to 2,2-dimethyl-3-oxopropyl dodecanoate and hexamethylenediamine. These converted products were biodegraded. The percentage (average) of biodegradation by BOD was 91% after 28 days. Further, the test substance is considered to be readily biodegradable.
Executive summary:

The biodegradation test with the test substance Sika Hardener LH was performed in accordance with "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5, Manufacturing industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan).

In this study activated sludge (30 mg/L) was used as inoculum. The cultivation period was 28 days. The test substance's concentration was 100 mg/L. Under the test conditions of this study, the test substance was completely hydrolyzed to 2,2-dimethyl-3-oxopropyl dodecanoate and hexamethylenediamine. These converted products were biodegraded. The percentage (average) of biodegradation by BOD was 91% after 28 days. Further, the test substance is considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
2005-05-16 to 2005-09-19
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: This study was disregarded, since a new study according to MITI requirements shows the test substance's biodegradability.
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
- Pre-treatment: The sludge was washed with tap water twice, then filtrated and resuspended in test me-dium. It was then aerated.
- Concentration of sludge: The dry matter was determined after filtration as 14000 mg suspended solids/L and ad-justed to 4120 mg suspended solids/L.
Duration of test (contact time):
28 d
Initial conc.:
68 mg/L
Based on:
test mat.
Initial conc.:
200 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
other: Theoretical Oxygen Demand (ThOD)
Details on study design:
TEST CONDITIONS
- Composition of medium: according to Guideline
- Test temperature: 22 +/- 1°C
- pH: 7.4 +/- 0.1
- pH adjusted: yes
- Suspended solids concentration: 25 +/- 5 mg/L

TEST SYSTEM
- Culturing apparatus: 250 mL glass flasks
- Number of culture flasks/concentration: 2
- Number of reference vessels: 1
- Method used to create aerobic conditions: vessels were connected to the oxygen developers
- Measuring equipment: The oxygen which was consumed by the content of the vessels was measured automatically by the test apparatus (mechanical counters measuring the current intensity needed to electrolytically develop the demanded oxygen).

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: 1, containing no inoculum, but 10 mL 1% HgCl2 solution/L

OTHER:
Since qualitative analysis gave positive results, nitrate and nitrite concentration in each test vessel at the end of the test were determined photometrically: nitrate through measurement after reaction with 2,6-Dimethylphenole at 324 nm, nitrite after reaction with α-naphthylamine and sulfanilic acid through measurement at 530 nm, and ammonium after reaction with Nesslers reagent at 435 nm.

Reference substance:
benzoic acid, sodium salt
Preliminary study:
NA
Test performance:
The oxygen which was consumed by the content of the vessels was measured automati-cally by the test apparatus (mechanical counters measuring the current intensity needed to electrolytically develop the demanded oxygen). The values of the displays were re-corded daily (exception: week-ends).
After 28 days, the test was ended.
Since qualitative analysis gave positive results, nitrate and nitrite concentration in each test vessel at the end of the test were determined photometrically: nitrate through meas-urement after reaction with 2,6-Dimethylphenole at 324 nm, nitrite after reaction with α-naphthylamine and sulfanilic acid through measurement at 530 nm, and ammonium after reaction with Nesslers reagent at 435 nm.
Key result
Parameter:
other: Theoretical Oxygen Demand (ThOD)
Value:
45.1
Sampling time:
28 d
Details on results:
The test item can be considered as „not readily biodegradable“.
The degree of biodegradation reached 45 % after 28 days.
The 10d-window began on day 2, at its end, 38 % were reached, staying below the pass level of 60% given in the OECD guideline.
Results with reference substance:
The reference item reached the pass level on day 5 (criterion <= 14). The degree of biodegradation reached 95.9% after 28 days.

Degradation Table

The calculated percentage degradation values are presented in the following table.

Table: Degradation Values in %

Day

Reference

Test 1

Test 2

Test Mean

Abiotic Control

1

19.7

0.0

0.0

0.0

0.0

2

33.3

15.0

11.2

13.1

0.0

3

43.5

24.1

18.1

21.1

0.0

4

63.6

28.1

21.1

24.6

0.0

5

78.1

29.6

22.1

25.9

0.0

6

82.0

31.2

23.3

27.3

0.0

7

84.5

33.4

25.2

29.3

0.0

8

86.7

35.1

26.6

30.9

0.0

9

87.6

36.0

27.2

31.6

0.0

10

88.6

37.5

28.2

32.9

0.0

11

89.2

39.7

29.8

34.7

0.0

12

90.0

43.1

32.3

37.7

0.0

13

90.6

43.9

33.2

38.6

0.0

15

90.9

44.7

33.6

39.2

0.0

16

91.6

45.8

34.4

40.1

0.0

18

92.1

46.1

34.6

40.3

0.0

20

93.7

47.3

35.5

41.4

0.0

21

94.1

47.5

35.7

41.6

0.0

22

95.0

48.1

36.1

42.1

0.0

24

96.4

49.1

37.2

43.1

0.0

25

97.2

49.6

37.5

43.6

0.0

27

97.9

51.3

38.7

45.0

0.0

28

95.9

50.2

40.0

45.1

-0.3

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Sika Härter LH was tested for ready bipodegradability in the manometric respiratory test according to EU method C.4-D/OECD guideline 301 F.
The test item can be considered as „not readily biodegradable“. The degree of biodegradation reached 45 % after 28 days. The 10d-window began on day 2, at its end, 38 % were reached, staying below the pass level of 60% given in the OECD guideline.
Executive summary:

The test item Sika Härter LH (VP) was tested using a nominal concentration of 68 mg test item/L (equivalent to a theoretical oxygen demand of 200 mg O2/L).Activated sludge was used as inoculum. The test was left running for 28 days. All validity criteria were met. The reference item reached the pass level of 60% on day 5. (criterion: <14).
The following data could be determined for the test item Sika Härter LH (VP):
10-day-window: day 2 – 12

degradation at the end of 10-day-window: 38 %

degradation at the end of the test: 45 %

pass level: 60% at the end of 10-d-window

Therefore Sika Härter LH (VP) is classified as not readily biodegradable following OECD 301F/EU C.4-D.

Description of key information

In a biodegradation study (MITI, 2011), the test substance Sika Hardener LH was completely hydrolyzed to 2,2-dimethyl-3-oxopropyl dodecanoate and hexamethylenediamine. These converted products were biodegraded. The percentage (average) of biodegradation by BOD was 91% after 28 days. Further, the test substance is considered to be readily biodegradable.

 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Key Study

The biodegradation test with the test substance Sika Hardener LH was performed in accordance with "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5, Manufacturing industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan).

In this study activated sludge (30 mg/L) was used as inoculum. The cultivation period was 28 days. The test substance's concentration was 100 mg/L. Under the test conditions of this study, the test substance was completely hydrolyzed to 2,2-dimethyl-3-oxopropyl dodecanoate and hexamethylenediamine. These converted products were biodegraded. The percentage (average) of biodegradation by BOD was 91% after 28 days. Further, the test substance is considered to be readily biodegradable.

Disregarded Study

This study was disregarded, since a new study according to MITI requirements shows the test substance's biodegradability. In accordance with Annex 9 "Guidance on Hazards to the aquatic environment", it is assumed that a positive results in one of the ready biodegadability tests demonstrates that the susbtance will degrade rapidly in the environment.