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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:
2002-09-12 to 2002-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Qualifier:
according to guideline
Guideline:
other: Testing Methods for New Chemical Substances (July 13 1974, Kanpoygo No.5, Planning and Coordination Bureau, Environment Agency, No. 615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and No 392, Basic Industries Bureau. Japan
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Sludge sampling sites and date:
sampling site: On-site sampling was carried out at the following 10 locations in Japan:
- Fushikogawa city sewage plant (Sapporo-shi Hokkaido)
- Fukashiba industry sewage plant (Kashima-gun Ibaragi)
- Nakahama city sewage plant (Osaka-shi Osaka)
- Ochiai city sewage plant (Shinjuku-ku Tokyo)
- Kitakami river (Ishinomaki-shi Miyagi)
- Shinano river (Nishikanbara-gun Niigata)
- Yoshino river (Tokushima-shi Tokushima)
- Lake Biwa (Otsu-shi Shiga)
-Hiroshima bay (Hiroshima-shi Hiroshima)
- Dookai bay (Kitakyushu-shi Fukoka)

Date: June 2002

Sludge sampling method:
City sewage: Return sludges from sewage plants were collected
Rivers, lake and sea: Surface water and surface soil which are in contact with the atmosphere were collected

Preparation of activated sludge:
The filtrate (5 L) of the supernatant of the activated sludge cultivated for about 3 months was mixed mixed with the filtrate (5 L) of the supernatant of a sludge collected newly at each location. The mixed filtrate (10 L) was aerated after the pH value of the mixture was adjusted to 7.0±1.0

Cultivation:
Approximately 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about one-third of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10 L. This mixture was aerated for 30 minutes or more and then a predetermined amount of synthetic sewage was added to the mixture so that the concentration of the synthetic sewage was 0.1 (w/v)% in the volume of the dechlorinated water added. This procedure was repeated once every day. The cultivation was carried out at 25±2°C.

Synthetic sewage:
Glucose, peptone and potassium dihydrogenphosphate were dissolved in dechlorinated water to obtain 50 g/l of the solution for each component. The pH of the solution was adjusted to 7.0±1.0 with sodium hydroxide.

Control and use:
During cultivation, the appearance of the supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. Microflora in the activated sludge was microscopically observed and the sludge with no abnormal symptoms was used for the test.

Inspection of activity and date of initiation of use of activated sludge:
Inspection of activity:
Activity of the sludge was assessed by the use of a standard sludge.

Date of initiation of use: 2002-07-16




Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
Preparation for the test:

Measurement of concentration of suspended solid: The concentration of the suspended solid in the activated sludge was measured on July 3rd 1995 to be 4800 mg/l

Preparation of basal culture medium: Each 3 ml of solution A, B, C and D as prescribed in the Japanese Industrial Standards (JIS) K 0102-1993-21, were made up to 1000 ml with purified water (Takasugi Seiyaku Co., Ltd.) and then the pH of the solution was adjusted to 7.0.

Reference substance:
Aniline (guaranteed reagent, Showa Chemicals Inc., Lot No. SE-31230) was used as a reference substance.

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

1. Addition of test substance or aniline
a) The test solution (water + test substance) (n = 1, Vessel No. 1): In one test vessel, 30 mg of the test substance was added to 300 ml of purified water while stirring contents with a magnetic stirrer, so that the concentration of the test item reached 100 mg/l and the pH of the solution was measured.

b) The test solution (sludge + test substance) (n = 3, vessel No. 2, 3 and 4): In each test vessel, 30 mg of the test substance was added to the basal culture medium (the volume was less than 300 ml by the volume (1.96 mL) of the activated sludge inoculum).

c) The test solution (sludge + aniline) (n = 1, vessel No. 6): In one test vessel, 29.5 µl (30.0 mg = 29.5 µl * 1.022 g/cm3 (density of aniline)) of aniline was added into the basal culture medium (the volume was less than 300 ml by the volume (1.96 mL) of the activated sludge inoculum).

d) The test solution (control blank) (n = 1, vessel No. 5). In one test vessel, nothing was added to the basal culture medium (the volume was less than 300 ml by the volume (1.96 mL) of the activated sludge inoculum).

- Instrument for cultivation:
Closed system oxygen consumption measuring apparatus (Temperature controlled bath and measuring unit: Ohkura Electric Co., Ltd.); (Data sampler: Asahi Instrument Industries Co., Ltd)
Vessel: 300 ml volume (improved type)
Absorbent for carbon dioxide: Soda lime No.1 (for absorption of carbon dioxide, Wako Pure Chemical Industries, Ltd.)
Stirring method: Each test solution was stirred by a magnetic stirrer

- Conditions of cultivation:
Cultivation temperature: 25±1°C
Cultivation duration: 28 days
Room: Apparatus room No. 511 of Kurume Research Laboratories

2. Inoculation of activated sludge:
The activated sludge was added to each test vessel (b), (c) and (d) so that the concentration of the suspended solid reached 30 mg/l

Observation of test solution:
During the cultivation, the appearance of the test solution was observed once a day and conditions of the instrument were checked properly.

Measurement of Biochemical Oxygen Demand (BOD):
During the cultivation period, the change in BOD of the test solution was measured by auto-recording using a data sampler. Cultivation temperature was measured and recorded once a day.

Analysis of test solution:
After the termination of the cultivation, dissolved organic carbon, converted product (methanol) which were expected to form in the test solutions from results of preliminary test and water soluble silicon were determined. The test item was not determined because it was immediately hydrolysed in the test solution and a direct analysis of it was difficult. Then, the qualitative analysis of residual components in the test solution was performed. The pH of the test solution (water + test item) and the test solutions (sludge + test item) was measured.

Pretreatment of test solution for analysis:
After the termination of the cultivation, the test solution (water + test substance), the test solutions (sludge + test substance) and the test solution (control blank) were pretreated for the total organic carbon (TOC) analysis on dissolved organic carbon (DOC), Gas chromatography (GC) analysis of methanol, atomic absorption spectrometry (AAS) analysis of water-soluble silicon and liquid chromatography-mass spectrometry (LC-MS) analysis of residual components.
Reference substance:
aniline
Preliminary study:
In the preliminary study, it was confirmed that direct analysis of the test item is difficult because of rapid hydrolysis of the test substance. Therefore, direct analysis of the test item was not performed as part of this study.
Key result
Parameter:
% degradation (TOC removal)
Value:
20
Sampling time:
28 d
Key result
Parameter:
other: BOD
Value:
19
Sampling time:
28 d
Details on results:
BOD (test substance): 19% degradation after 28 d
Results with reference substance:
59% degradation after 7 d
75% degradation after 14 d
76% degradation after 21 d
77% degradation after 28 d

Results:

Appearances of test solution: The appearances of the test media in cultivation vessels were as follows:

 

Test solution

Appearance

pH

At the start of cultivation

Water + test item

The test item was dissolved

Vessel 1: 9.8

Sludge + test item

The test item was dissolved

Vessel 2: 8.1

Vessel 3: 8.1

Vessel 4: 8.1

At the end of cultivation

Water + test item

Insoluble compound was not observed

Vessel 1: 9.8

 

Sludge + test item

Insoluble compound except for the sludge was not observed. Growth of the sludge was observed.

Vessel 2: 7.2

Vessel 3: 7.2

Vessel 4: 7.2

 

Analytical results of test solutions: Analytical results of the test solution after 28 days were as follows:

 

 

Water + test item

Sludge + test item

Sludge + test item

Sludge + test item

Theoretical amount

 

 

Vessel 1

Vessel 2

Vessel 3

Vessel 4

 

BOD*

mg

0.3

13.7

14.6

11.9

71.1**

Residual amount and % residue* of DOC

mgC

15.5

12.5

12.4

12.4

17.0**

 

%

91

73

73

73

-

Produced amount and % production of methanol (GC)

mg

8.9

0

0

0

8.9***

 

%

100

0

0

0

-

Detected amount and % detection of water-soluble silicon (AA)

mg

2.7

3.1

2.9

3.0

2.9**

 

%

94

105

100

102

-

*The value pf control blank was subtracted from the values of the test solutions (sludge + test item)

**TOD was calculated from the empirical formula (C15.32H27.92N2.24O3.12Si1.12) which was calculated considering the component composition of the test item

***Each theoretical amount was calculated assuming that 3 moles of methanol is produced from 1 mole of the empirical formula (C15.32H27.92N2.24O3.12Si1.12)

Results of the qualitative analysis of residual components:

As the result of qualitative analysis by LC-MS, one peak corresponding to the residual component was detected on the total ion chromatography (TIC) in each of the test solutions (water + test item) and (sludge + test item). The residual component was presumed as 3-aminomethylbenzylaminopropyltrihydroxysilane by analysis of mass spectrum of the peak.

Percentage (%) biodegradation:

% biodegradation by TOC was considered to be calculated from DOC of the hydrolysate of the test item because the test item was immediately hydrolysed in the test solutions.

Table 1: Calculation table for the percentage biodegradation by BOD

Test number: 13925

Cultivating duration: 28 days

vessel

7th Day

14th Day

21st day

28th Day

Mean deg (%)

BOD (mg)

Deg. (%)

BOD (mg)

Deg. (%)

BOD (mg)

Deg. (%)

BOD (mg)

Deg. (%)

[6] Sludge + aniline

54.7

59

69.9

75

72.7

76

74.6

77

 

[5] Control blank

1.7

-

2.4

-

3.9

-

4.7

-

 

[2] Sludge + test substance

13.9

17

15.8

19

17.6

19

18.4

19

19

[3] Sludge + test substance

14.6

18

16.8

20

18.7

21

19.3

21

 

[4] Sludge + test substance

13.0

16

14.4

17

16.0

17

16.6

17

 

[1] Water + test substance

0.3

-

0.3

-

0.3

-

0.3

-

 

 

Table 2: Calculation table for percentage biodegradation by TOC

Sample description

Measured value (mgC/L)

Amount of DOC

% Biodegradation

% Biodegradation

Average % biodegradation

Water blank

not determined

 

 

 

 

[1] Water + test item

51.50

15.5

91

 

 

[2] Sludge + test item

42.78

12.5

73

19

 

[3] Sludge + test item

42.39

12.4

73

20

20

[4] Sludge + test item

42.41

12.4

73

20

 

[5] Control blank

1.22

 

 

 

 

 

Table 3: summary of biodegradation results

Method

% Biodegradation

% Biodegradation

% Biodegradation

Average % biodegradation

Table

[2] Sludge + test item

[3] Sludge + test item

[4] Sludge + test item

BOD

19

21

17

19

1

TOC

19

20

20

20

2

 

In this test, methanol and water-soluble silicon were determined and the residual components except methanol were analysed qualitatively by LC-MS.

From the GC analysis result, the theoretical amount of methanol was detected in the test solution (water + test item) but methanol was not detected in the test solutions (sludge + test item).

From the result of the qualitative analysis of the residual components by LC-MS, 3-aminomethylbenzylaminopropyltrihydroxysilane (triol) which is the hydrolysis product of the test item was presumed as the water-soluble converted product in the test solutions (water + test item) and (sludge + test item). No peak corresponding to other converted product was detected on TIC.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the substance has been determined using a relevant test method and in compliance with GLP. The result is considered to be reliable.

Description of key information

Biodegradation in water, screening tests: 19% BOD in 28 days; 20% TOC in 28 days (OECD 301C). No significant biodegradation is expected for the silanol hydrolysis products.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Biodegradation of 19% BOD in 28 days; 20% TOC in 28 days (OECD 301C) were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

In contact with water, the constituents of the submission substance hydrolyse within the timescale of the ready biodegradation study to N-[3-(trihydroxysilyl)propyl]-1,3-benzenedimethanamine and methanol. The biodegradation observed in the study is attributable to the biodegradation of the methanol hydrolysis product.

Methanol is readily biodegradable (OECD, 2004).

No significant biodegradation is expected for the silanol hydrolysis products.

 

Reference:

OECD (2004): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1.