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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 14 2003 - August 26 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Referred to: Kanhogyo No 5, Yakuhatsu No. 615, 49Kikyoku No. 392, July 13, 1974 and Guideline 301C, July 17, 1992 stipulated in OECD Guideline for Testing of Chemicals.
Deviations:
yes
Remarks:
Direct analysis was not conducted on the test substance indicating a partial deviation from the test method
Principles of method if other than guideline:
From the sponsor, we received the information that the test substance is hydrolysed by the moisture of the atmospheric air. In the preliminary investigation, the test substance was stirred for 30 minutes in either purified water or the basal cultivation medium. As the results, it was varified that almost theoretical amount of diethylamine as the product was produced. Viewed together, the test substance would be promptly hydrolysed in the test solution, thereby direct analysis of the test substance was not conducted.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Remarks:
Out-door air was passed through the prefilter and used for aeration
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):

On-site sludge sampling was carried out at the following 10 locations in Japan.
Fushikogawa sewage plant (Sapporo-city, Hokkaido)
Fukashiba sewage plant (Kashima-gun, Ibaraki)
Nakahama sewage plant (Osaka-city, Osaka)
Ochiai city sewage plant (Shinjuku-ku, Tokyo)
Kitakami River (Ishinomaki-city, Miyagi)
Shinano River (Niigata-shi, Niigata)
Yoshino River (Tokushima-city, Tokushima)
Lake Biwa (Otsu-city, Shiga)
Hiroshima Bay (Hiroshima-city, Hiroshima)
Dookai Bay (Kitakyushu-city, Fukuoka)

Sampling timings June 2003

(1) Sewage plant: Return sludge from sewage plants
(2) River, lake and sea: Surface water and surface soil (water’s edge) in direct contact with the atmosphere

- Laboratory culture:

- Method of cultivation:
After discontinuation of aeration to the cultivation tank for about 30 minutes, the supernatant corresponding to about 1/3 of the whole volume was removed. For the remaining portion, dechlorinated water was added to make 10 L in total and then, again aerated (for 30 minutes or more). Subsequently, the 50 g/L synthetic sewage*4 was added to make the synthetic sewage concentration 0.1wt% in the added dechlorinated water. This procedure was repeated once every day, followed by cultivation to prepare the activated sludge. Cultivation temperature was 25 ± 2 deg C.

*4 Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to make the concentration of each component 50 g/L. The pH of the solution was adjusted to 7.0 ± 1.0 with sodium hydroxide.

- Storage conditions:
The test substance was placed in a tightly closed container filled with nitrogen gas, and was stored in a cool and dark place.

- Storage length:
Approx 3 months

- Preparation of inoculum for exposure:
Activated sludge was prepared as follows to maintain its uniformity.
The filtrate (5 L) obtained from mixture of respective sludge samples collected at the above-stated locations was mixed with the filtrate (5L) of the activated sludge*2 which was previously cultivated for about 3 months to make 10 L in total. After adjusting its pH to 7.0 ± 1.0, the filtrate mixture was aerated*3 in the cultivation tank.

*2 As directed in the above procedures, this activated sludge was obtained by cultivation of the filtrate mixture (10 L) which was prepared by mixing respective sludge at various locations as stated above.

*3 Out-door air was passed through the prefilter and used for aeration.

- Pretreatment:
Please refer to the attached flow diagram (attachment 1).

- Concentration of sludge:
In order to decide the amount of the activated sludge to add, the concentration of the suspended substance was measured.

Method -
As directed in "Method for industrial wastewater test, suspended substance" (JIS L 0102-1998, section 14.1)

Date -
July 15 2003

Results -
Concentration of the suspended substance in the activated sludge was 3200 mg/L
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
other: Change in BOD
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium:
In accordance with the stipulated provision in "Method for industrial wastewater test, Biochemical oxygen consumption amount" (JIS K 0102-1998 section 21), purified water (Takasugi Seiyaku Co., Ltd. Japanese Phatmacopeia) was added to each 3 mL of solution A, B, C and D to make 1 L in total.

- Additional substrate:
None recorded

- Test temperature:
25 +/- 1 deg C

- pH:
At the start of cultivation the pH was 8.1

- pH adjusted:
yes, the pH was adjusted to 7.0

- Aeration of dilution water:
Out-door air was passed through the prefilter and used for aeration

TEST SYSTEM
- Culturing apparatus:
Instruments for cultivation -
Closed system oxygen consumption measuring apparatus
Temperature controlled bath and measuring unit: Ohkura Electric Co., Ltd
Data processor unit: Asahi Techneion Co., Ltd
Test vessel: Culture bottle for 300 mL (improved type)
Absorbent for CO2: Soda lime No.1 (for absorption of CO2, manufactured by Wako Pure Chemical Industries, Ltd)

- Number of culture flasks/concentration:
3 test vessels

- Method used to create aerobic conditions:
Out-door air was passed through the prefilter and used for aeration

- Method used to create anaerobic conditions:
Not applicable

- Measuring equipment:
During the cultivation period, the appearance of the test solution was macroscopically observed every day. Working conditions of the instruments were investigathed appropriately.

- Test performed in closed vessels due to significant volatility of test substance:
Closed system oxygen consumption measuring apparatus

- Details of trap for CO2 and volatile organics if used:
Soda lime No.1 (for absorption of CO2, manufactured by Wako Pure Chemical Industries, Ltd)

PREPARATION OF TEST SOLUTION
The following test solutions were prepared in 6 test vessels according to the following methods.
These test solutions were cultured under the conditions as described previously.

1) Addition of the test substance and aniline (reference substance)
a) Test solution (water + test substance) (n = 1, Vessel No. 1)
Purified water (300 mL) was placed into the test vessel, while 30 mg of the supplied test sample was accurately weighed with the use of electric analytical balance and added to make the concentration of the test substance 100 mg/L.

b) Test solution (sludge + test substance)(n = 3, Vessel No. 2, 3 and 4)
The basal culture medium (the volume was obtained by reducing the inoculated volume of the activated sludge (2.81 mL) from 300 mL) was placed into each test vessel. Spearately, the 30 mg of the supplied test sample was weighed accurately by using the electric analytical balance to make the concentration of the test substance 100 mg/L, and added to the basal culture medium. The resultant solution was stirred for about 3 hours. After stirring the pH was determined and was adjusted to 7.0 +/- 0.1.

c) Test solution (sludge + aniline)(n = 1, Vessel No. 6)
The basal culture medium (the volume was obtained by reducing the inoculated volume of the activated sludge (2.81 mL) from 300 mL) was placed in the test vessel. Separately, 29.5 micro L of aniline [the added amount 30 mg = 29.5 micro L x 1.022 g/cm3 (density)] was pipetted with a micro syringe and added to the above-stated medium to make aniline 100 mgL

d) Test solution (control blank) (n = 1, Vessel No.5)
The basal culture medium (the volume was obtained by reducing the inoculated volume of the activated sludge (2.81 mL) from 300 mL) was placed in the test vessel

2) Innoculation of the activated sludge
The activated sludge prepared under the conditions described previously was added to each test solution, b), c) and d) so that the concentration of the suspended substance became 30 mg/L.

SAMPLING
During the cultivation period, the change in BOD of the test solutions was determined continuously with self-recording system using a data processor unit. Cultivation temperature in the bath was measured and recorded everyday.

CONTROL AND BLANK SYSTEM
- Inoculum blank:
Test solution (water + test substance) n=1, Vessel No.1)
Purified water (300 mL) was placed into the test vessel, while 30 mg of the supplied test sample was accurately weighed with the use of electric analytical balance and added to make the concentration of the test substance 100 mg/L.

Reference substance:
In order to confirm that the activated sludge exerts adequate activity to conduct the test, aniline (special grade, manufactured by Showa Chemicals Inc. Lot No. HO-2729D) was used as the reference substance. As directed in the test method stipulated in the Law Concerning the Examination and Regulation of Manufacture, etc. of Chemical Substance and in the provision described in the OECD test guidelines, the degadation ratio should be obtained based on BOD 7 days and 14 days after the tests on aniline. When the degradation ratio exceeds 40% and 65% respectively, this study shall be designated to be valid.

STATISTICAL METHODS:
The biodegradation ratio of aniline 7 days and 14 days after treatment obtained by BOD was 64% and 69%, respectively. It was confirmed that the study conditions employed in this study were valid.
Reference substance
Reference substance:
aniline

Results and discussion

Preliminary study:
In the preliminary investigation, the test substance was stirred for 30 minutes in either purified water or the basal cultivation medium. As the results, it was varified that almost theoretical amount of diethylamine as the product was produced.
Test performance:
No unusual observations during test recorded.
% Degradation
Parameter:
% degradation (TOC removal)
Value:
97
Sampling time:
28 d

BOD5 / COD results

BOD5 / COD
Parameter:
BOD5
Value:
83 mg O2/g test mat.
Results with reference substance:
The biodegradation ratio of aniline 7 days and 14 days after treatment obtained by BOD was 64% and 69% respectively. It was confirmed that the study conditions employed in this study were valid.

Any other information on results incl. tables

The analytical results after 28 days were as follows:

 

(Water + test substance) system

(Sludge +test substance) system

Theoretical amount

Table

Fig

1

2

3

4

BOD*

mg

0

56.9

55.7

57.2

68.4

1

1

Residual amount of DOC and residual ration *

mgC

15.6

0.5

0.3

0.5

15.3

2

-

%

102

3

2

3

-

Production amount of diethylamine and production ratio (HPLC)

mg

9.6

0

0

0

9.3

3

5

%

103

0

0

0

-

Production amount of ethanol and production ratio (GC)

mg

16.7

0

0

0

17.6

4

6

%

95

0

0

0

-

Production amount of water-soluble silicon and production ratio (AA)

mg

3.4

3.5

3.3

3.4

3.6

5

7

%

93

97

92

95

-

* For the (sludge + test substance) system, the value was presented after subtracting the values of the sludge blank system.

In this study, analysis was conducted on the products which were expected to be generated (diethylamine*, ethanol^ and water-soluble silicon) (Please refer to attachments 3 and 4 for the changes of the test substance). The chemical structure of water-soluble silicon shown in the figure (attachment 5) is a presumed one.

As the results of analysis of the test solution, almost theoretical amount of all these products were deteched in the (water + test substance) system. On the other hand, in the (sludge + test substance) system, neither diethylamine nor ethanol was detected. Since the degradation ratios by BOD and TOC were 83 and 97% respectively in average and growth of the sludge was observed, it was conceived that resultant diethylamine and ethanol would be biodegraded by microorganisms, with only water-soluble silicon as inorganic material remaining.

Based on the above results, it could be concluded that the test substance was completely hydrolysed, indicating that waivering the direct analysis of the test substance would not cause any influences on the test results.

* Diethylamine : Kampo publications No.:(2)-135 (Good biodegradation)

^ Ethanol : Kampo publications No.: (2)-202 (Good biodegradation).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions for this study, the test substance was biodegraded by microorganisms.
Executive summary:

Title of the study

Biodegradation study of DEATES by microorganisms

Condition of the study

(1) Concentration of the test substance: 100 mg/l

(2) Concentration of activated sludge: 30 mg/l (as the concentration od suspended substance)

(3) Volume of the test solution: 300 mg/l

(4) Cultivation temperature of the test solution: 25 +/- 1 deg C

(5) Cultivation period of the test solution: 28 days

Determination and analysis for calculation of biodegradation degree

(1) Determination of the biochemical oxygen deman (BOD) by closed system oxygen consumption measuring apparatus

(2) Analysis of dissolved organic carbon (DOC) by total organic carbon analytical method (TOC)

Other analysis

(1) Analysis of diethylamine by high-performance liquid chromatography (HPLC)

(2) Analysis of ethanol by gas-chromatography (GC)

(3) Analysis of water-soluble silicon by atomic absorption spectrophotometer (AA)

Test results

(1) Biodegradation ratio by BOD

83%, 81%, 84% - Mean 83%

(2) Biodegradation ratio by TOC

97%, 98%, 97% - Mean 97%

Conclusion

The test substance was biodegraded by microorganisms under this test conditions.