Reaction products of 2,4-bis(2-chlorophenyl)-5-(3,4-dimethoxy phenyl)-1H-imidazole and 2-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: “Testing method for new chemical substances” (Biodegradability test using microorganisms for chemical substances) prescribed in (Environmental Preservation Services, No. 5, Drug No. 615, 49 Base Station, No. 392, June 13, 1974)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 100 wt% as a reaction product

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

Sludge was nationally collected from the ten places in Japan consisting of return sludge from sewage disposal plants and when collected from river, lake, or sea areas samples were obtained from surface soil at the beach in contact with the outermost water layer and the air.

Preparation of activated sludge
Five liters of mixed solution filtrate were added to 5 L of an activated sludge filtrate cultured for about 3 months. The pH was adjusted to 7.0 ± 1.0. The mixture was exposed to the air in a culture tank.

Culture
After stopping the exposure to the culture tank for about 30 min, the supernatant fluid of 1/3 of the entire amount was removed. The total amount was adjusted to 10 L by adding dechlorinated water and re-exposed to the air (30 min or more), and 50 g/L synthetic sewage was added so that the synthetic sewage concentration in the added dechlorinated water is 0.1 wt%. This operation was repeated once a day, and an activated sludge was obtained by culturing. The culture temperature was set to 25 ± 2ºC. Synthetic sewage preparation; glucose, peptone, and calcium dihydrogen phosphate were dissolved in dechlorinated water to reach respectively 50 g/L, and its pH was adjusted to 7.0 ± 1.0 with sodium hydroxide.

Biota in the activated sludge was observed by an appropriate optical microscope, and after confirming that there was no abnormality, the activated sludge was provided to the test.

The suspended substance concentration in the activated sludge was 7,200 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:

O2 consumption

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Six total test vessels were prepared according to items (a) through (d).

(a) (Water + test substance) system (1 replicate, test vessel (5))
300 mL purified water was put into the test vessel, 30 mg sample provided was precisely weighed by an electronic analysis balance so that the test substance concentration was 100 mg/L and then added.

(b) (Sludge + test substance) system (3 replicates, test vessels (1), (2), and (3))
A basic culture medium (an amount in which the amount of activated sludge solution added (1.25 mL) was subtracted from 300 mL) was put into the test vessels, 30 mg providing sample was precisely weighed by the electronic analysis balance so that the test substance concentration was 100 mg/L and then added.

(c) (Sludge + aniline) system (1 replicate, test vessel (6))
A basic culture medium (an amount in which the amount of activated sludge solution added (1.25 mL) was subtracted from 300 mL) was put into the test vessel, 29.5 μL (an amount of addition, 30 mg = 29.5 μL x 1.022 g/cm3 (density)) was sampled by a microsyringe so that the aniline was 100 mg/L and then added.

(d) Sludge blank system (1 replicate, test vessel (4))
A basic culture medium (an amount in which the amount of activated sludge solution added (1.25 mL) was subtracted from 300 mL) was put into the test vessel.


Test solutions in (b), (c), and (d) were inoculated with activated sludge so that the suspended substance concentration was 30 mg/L.

The experiment ran for 28 days with stirring at 25 ± 1ºC.
For the culture period, the change of the BOD of the testing solutions was automatically recorded and measured by a data processor.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Remarks on result:

other: Average of 3 replicates

% Degradation 2

Parameter:

other: HPLC

Value:

0

Sampling time:

28 d

Remarks on result:

other: All 3 replicates

Details on results:

Under these test conditions, the test substance was not biodegraded by microorganisms.
See results table in the Executive Summary.

Testing Solution Status
At the time of culture start, the water + test substance system and sludge + test substance system contained test substance that was not dissolved.
At the time of culture end, the water + test substance system had un-dissolved substances and the sludge + test substance system had un-dissolved substances other than the sludge and multiplication of the sludge was not recognized.


From the HPLC analysis after finishing the culture, it was confirmed that an approximately theoretical amount of test substance remained in any of the testing solutions and had no difference from the chromato pattern obtained from the test system and the standard solution. Since it was anticipated that the test substance was changed by lights, a test culture under the condition (light source for light irradiation: natural lights and fluorescent lights, testing solution culture period: 28 days) in which lights were irradiated was applied to the (water + test substance) system and provided to the HPLC analysis. As a result, it was confirmed that since no difference from the chromato pattern obtained from the test system and the standard solution was recognized, the test substance was not influenced by the lights under the biodegradability test conditions (the state in which the test substance was not dissolved in water but was floated) but was stable.


Additional Analysis Procedure to Evaluate the Effect of Lighting on the Test Substance

In order to confirm the existence of the change of the test substance by the lights, the HPLC analysis was carried out based on the following testing solution culture apparatus and the environmental conditions. The flow scheme and the HPLC analysis conditions were similar to these test conditions.

(1) Testing solution and culture apparatus
Light source for Light irradiation: Natural light and fluorescent light
Irradiation time: 8-12 h for 1 day
Testing solution: (Water + test substance) system
Test vessel: Culture bottle for 300 mL
Amount of testing substance added: 30 mg
Carbonic acid gas absorbent: Soda lime No.1

(2) Environmental conditions
Testing solution culture temperature: Room temperature
Testing solution culture period: 28 days
Stirring method: Rotary stirring by a magnetic stirrer

BOD5 / COD results

Results with reference substance:

Biodegradability after 7 days and 14 days of the aniline obtained from the BOD was respectively 72% and 88%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under these test conditions, the test substance was not biodegraded by microorganisms.
Test results: by BOD 5%, 3%, 0% average 3% and by HPLC 0%, 0%, 0% average 0%.

Executive summary:

The test was conducted according to OECD 301C (Modified MITI) using a test substance concentration of 100 mg/L (300 mL) and 30 mgL of activated sludge. Aniline was used as a reference substance. The experiment ran for 28 days with stirring at 25 ± 1ºC. Biodegradability was analyzed by measurement of the amount of biochemical oxygen demand (BOD) and by HPLC. Test results: by BOD 5%, 3%, 0% average 3% and by HPLC 0%, 0%, 0% average 0%.

Under these test conditions, the test substance was not biodegraded by microorganisms.

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BOD Results:

Test Vessel	Day 7 BOD (mg)	Day 7 Deg %	Day 14 BOD (mg)	Day 14 Deg %	Day 21 BOD (mg)	Day 21 Deg %	Day 28 BOD (mg)	Day 28 Deg %
Sludge + aniline (#6)	65.5	72	80.2	88	81.8	89	82.9	90
Sludge blank (#4)	0.7		1		1		1.7
Sludge + test substance (#1)	2	2	2.5	2	3.4	3	4.9	5
Sludge + test substance (#2)	1.7	1	2.3	2	2.8	3	3.8	3
Sludge + test substance (#3)	1.3	1	1.6	1	1.6	1	1.6	0
Water + test substance (#5)	0		0		0		0

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