C-M5

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:

July 4 2007 to August 23 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in compliance with the following GLP principles: (1) "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17,2003, No. 3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry of the Environment) (2) "OECD Principles of Good Laboratory Practice (November 26, 1997, ENV IMCICHEM (98)17)" The final report reflects the raw data accurately and it has been confirmed that the test data is valid.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

Principles of method if other than guideline:

Study performed according to above guideline.

GLP compliance:

not specified

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

3. Activated sludge
3.1 Preparation of activated sludge
Activated sludge used in the present test was prepared as follows.
(I) Sampling sites
On-site sludge sampling was carried out at 10 locations in Japan.

(2) Sampling method
Sewage plant: Return sludge was collected.
Rivers, lake and sea: Surface water and surface soil which was in contact with the atmosphere
were collected.

(3) Sampling date
March,2007

(4) Preparation method of activated sludge
Activated sludge was prepared as follows to maintain its uniformity.
The filtrate (5 L) of the supernatant of the activated sludge cultivated about for 3
months was mixed with the mixed 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.

3.2 Cultivation
Approximately 30 minutes after ceasing aeration of the sludge mixture, supernatant
corresponding to about 1/3 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 % in
the volume of dechlorinated water added. This procedure was repeated once every day.
Cultivation was carried out at 25±2 °C.

* Synthetic sewage
Glucose, peptone and potassium dihydrogenphosphate were dissolved in
purified 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.

3.3 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. It was confirmed that these were
within the scope of the control standard stipulated in the "Testing Methods for New
Chemical Substances", and these results were stored as raw data. Microflora in the
activated sludge was microscopically observed and sludge with no abnormal symptoms
was used for the test. The activated sludge, which was cultivated for 19.5 hours after it
had been added the synthetic sewage, was used.

3.4 Inspection of activity and date of initiation of use of activated sludge
(I) Inspection of activity
Activity of the sludge was assessed using standard items before initiation of use.

-Concentration of activated sludge (as the concentration of suspended solid) = 30 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 estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

other: Percentage biodegradation by BOD

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

other: Percentage biodegradation by DOC

Parameter followed for biodegradation estimation 3

Parameter followed for biodegradation estimation:

other: Determination of test item by HPLC

Details on study design:

4. Performance of biodegradation test

4.1 Preparations for test

(1) Measurement of concentration of suspended solid in activated sludge.
The concentration of suspended solid was measured to determine the amount of
activated sludge to add.
Method: In accordance with Japanese Industrial Standards (JIS) K 0102-1998
Section 14.1
Date: July 2, 2007
Result: Concentration of suspended solid in the activated sludge was 3750
mg/L.

(2) Preparation of basal culture medium
Each 3 mL of solutions A, B, C and D, which are prescribed in JIS K 0 I 02-1998
Section 21, were made up to 1000 mL with purified water (Japanese Pharmacopeia,
Takasugi Pharmaceutical Co., Ltd.), and then the pH of this solution was adjusted to
7.0.

4.2 Preparation of test solutions

The following test solutions were prepared and cultivated under the conditions
described in Section 4.3.

(I) Addition of test item or aniline
(a) Test solution (water + test item) (n= I, Vessel No. I )
In one test vessel, 3 mL of 10.0 g/L of the test item in water was added to 297
mL of purified water, so that the concentration of the test item reached 100 mg/L and
then the pH of the solution was measured. 10.0 g/L of the test item in water was
pretreated as follows. The test sample was accurately weighed and dissolved in
purified water to obtain it.

(b) Test solution (sludge + test item) (n=3, Vessel No.2, 3 and 4)
In each test vessel, 3 mL of 10.0 g/L of the test item in water was added to the
basal culture medium [the volume was less than 297 mL by the volume (2.40 mL) of
activated sludge inoculated], so that the concentration of the test item reached 100
mg/L and then the pH of the solution was measured. 10.0 g/L of the test item in
water was pretreated as follows. The test sample was accurately weighed and
dissolved in purified water to obtain it.

(c) Test solution (sludge + aniline) (n=l, Vessel No.6)
In one test vessel, 29.5 µL [30 mg = 29.5 ilL x 1.022 g/cm3 (density)] of aniline
was taken out by microsyringe and added to [the volume was less than 300 mL by the
volume (2.40 mL) of activated sludge inoculated], so that the concentration of aniline
reached 100 mg/L.

(d) Test solution (control blank) (n=l, 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 (2.40 mL) of activated sludge inoculated].
(2) Inoculation of activated sludge
The activated sludge cultivated was
added to each test vessel, (b), (c) and (d), so that the concentration of the suspended
solid reached 30 mg/L.

4.3 Instruments and conditions of cultivation

(1) Instruments for cultivation:
Closed system oxygen measuring apparatus;
Temperature controlled bath and measuring unit
Vessel: 300 mL in volume (improved type)
Absorbant for carbon dioxide: Soda lime No.1 (for absorption of carbon dioxide).

(2) Conditions of Cultivation
Cultivation temperature: 25°C (+/- 1 °C)
Cultivation duration: 28 days (under conditions of darkness)
Stirring method: Each test solution was stirred by a magnetic stirrer

4.4 Observation and measurement of test conditions
(1) Observation of test solution
During the cultivation, the appearance of the test solution was observed once a day
and conditions of the instruments were checked properly.
(2) Measurement ofbiochemica1 oxygen demand (BOD)
During the cultivation period, the change in BOD of the test solutions was
measured by autorecording using a data sampler. Cultivation temperature was
measured and recorded once a day.

4.5 Analysis of test solution
After the end of the cultivation, dissolved organic carbon and the test item in the test
solutions were determined. The pH of the test solution (water + test item) and the test
solutions (sludge + test item) was measured.

Reference substance

Reference substance:

aniline

Results and discussion

Preliminary study:

Analytical results of test solutions:
Analytical results of the test solutions after 28 days were as follows. In the test solution (water + test item) and the test solutions (sludge + test item),
approximately theoretical amount of the test item remained and no peak except the test item was detected on the HPLC chromatogram. Therefore, any converted products were judged not to be produced and then not analyzed.

Test performance:

See attachment -' validity' in attached background material.
The validity of the test was confirmed.

% Degradation

% Degradation 1

Parameter:

other: Percentage biodegradation by BOD

Value:

2

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: Average from 3 vessels

% Degradation 2

Parameter:

other: Percentage degradation by DOC

Value:

0

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: Average from 3 vessels. The average percentage biodegradation was regarded as 0 % because the calculated average value shown in parentheses was negative.

% Degradation 3

Parameter:

other: Percentage biodegradation of test item (HPLC)

Value:

0

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: Average of 3 vessles

Details on results:

The test item was not biodegraded by microorganisms under the test conditions of this study.

Analytical results of the test solutions after 28 days were as follows.
In the test solution (water + test item) and the test solutions (sludge + test item), approximately theoretical amount of the test item remained and no peak except the test item was detected on the HPLC chromatogram. Therefore, any converted products were judged not to be produced and then not analyzed.

BOD5 / COD results

Results with reference substance:

Percentage biodegradation by BOD of aniline = 79%.

Any other information on results incl. tables

The test item was not biodegraded by microorganisms under the test conditions of this study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

The test item was not biodegraded by microorganisms under the test conditions of this study.

Executive summary:

Biodegradation study of C-M5 by microorganisms.

Conditions of cultivation

(1) Concentration of test item: 100 mg/L

(2) Concentration of activated sludge (as the concentration of suspended solid): 30 mg/L

(3) Volume oftest solution: 300 mL

(4) Cultivation temperature: 25 °C

(5) Cultivation duration (under conditions of darkness): 28 days

Measurement and analysis for calculation of percentage biodegradation:

(1) Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus

(2) Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC)

(3) Determination of test item by high-performance liquid chromatography (HPLC)

Results:

(1) Percentage biodegradation by BOD: 3%, 5%, -1%, Average 2%

(2) Percentage biodegradation by DOC: -4%, -2%, -1%, Average 0% (-2%)*

(3) Percentage biodegradation of test item (HPLC): 0%, 1%, 0%, Average 0%

*The average percentage biodegradation was regarded as 0 % because the calculatedaverage value shown inparentheses was negative.

Conclusion:

The test item was not biodegraded by microorganisms under the test conditions of this study.