Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2006 to 14 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, Notification No. 2 (November 13, 2003) of the Manufacturing Industries Bureau, METI & Notification No. 031121002 of the Environmental Health Department, MOE

Version / remarks:

21 November 2003

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: Sewage sludge, surface water and surface soil near the water's edge from 10 locations in Japan.

Details on inoculum:

- Source of inoculum/activated sludge:
Sludge samples were collected from the following 10 sites around Japan.
Fushiko River sewage-treatment plant (Sapporo-shi, Hokkaido)
Fukashiba sewage-treatment plant (Kamisu-shi, Ibaraki Prefecture)
Nakahama sewage-treatment plant (Osaka-shi, Osaka Prefecture)
Ochiai Water Regeneration Center (Shinjuku-ku, Tokyo)
Kitakami River (Ishinomaki-shi, Miyagi Prefecture)
Shinano River (Niigata-shi, Niigata Prefecture)
Yoshino River (Tokushima-shi, Tokushima Prefecture)
Lake Biwa (Otsu-shi, Shiga Prefecture)
Hiroshima Bay (Hiroshima-shi, Hiroshima Prefecture)
Dokai wan (Kitakyushu-shi, Fukuoka Prefecture)

- Sampling method
Sewage treatment plant: Return sludge sampling.
River, lake, and sea: Surface water and surface soil near the water edge in contact with the atmosphere were collected.

- Laboratory culture: After stopping the aeration toward the incubation tank for about 30 minutes, approximately one-third of the whole volume of supernatant was discharged. Dechlorinated tap water was added to the tank to make the total volume 10 L followed by aeration for at least 30 minutes. Then, 50 g/L synthetic sewage was added so that the concentration of synthetic sewage in the dechlorinated tap water added would be 0.1 %. This procedure was repeated once daily, followed by culture to prepare activated sludge. The culture temperature was adjusted to 25 ± 2 °C.
Synthetic sewage: Glucose, peptone, and potassium dihydrogen phosphate were dissolved in purified water to make 50 g/L each, followed by adjusting the pH to 7.0 ± 1.0 with sodium hydroxide.

- Preparation: In order to maintain the homogeneity of activated sludge samples, 5 L of the filtrate of the local sludge mixture collected above and 5 L of the filtrate of activated sludge (for preparation of this activated sludge, 10 L of the filtrate of the local sludge mixture collected above were cultured) cultured for approximately 3 months were mixed to make 10 L, and the pH was adjusted to 7.0 ± 1.0 and subjected to aeration in an incubation tank (outdoor air passed through a pre-filter was used for aeration).
In order to maintain the normal state of activated sludge, the appearance of the supernatant and the conditions of activated sludge formation were observed during incubation, and the settlement characteristics of the activated sludge, pH, temperature, and dissolved oxygen concentration were measured to check that they were within the control standards. The results were stored as raw data. Biota in the activated sludge was observed under a light microscope as appropriate, and the activated sludge samples were subjected to the test after checking that they had no abnormality. Activated sludge 18.5 hours after adding the synthetic sewage was used.
The activity of the activated sludge was checked using a reference material before using the activated sludge for the test.
- Initial cell/biomass concentration: To determine the amount of activated sludge to be added, the concentration of suspended matter was measured. The concentration of suspended matters in the activated sludge was 2920 mg/L.
- Preparation of base culture medium: Purified water (Japanese Pharmacopoeia, Takasugi Pharmaceutical Co., Ltd.) was added to 3 mL each of solutions A, B, C, and D of the composition specified in “Testing Method for Industrial Wastewater, Biochemical Oxygen Demand” (JIS K0102-1998-21) to make 1 L, followed by adjusting pH to 7.0.

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

Details on study design:

TEST CONDITIONS
- Test temperature: 25 ± 1 °C
- Suspended solids concentration: The concentration of suspended matters in the activated sludge was 2920 mg/L.
- Continuous darkness: Yes, under protection from light.
- Carbon dioxide absorbent: Soda lime, No.1 (for carbon dioxide absorption, Wako Pure Chemical Industries, Ltd.).
- Stirring: With a magnetic stirrer.

TEST SYSTEM
- Culturing apparatus: 300 mL culture bottle (modified culture bottle)
- Number of culture flasks/concentration:
Water + test material system (1, test vessel [1])
To make the test material concentration 100 mg/L, 300 mL of purified water and 30 mg of the test material were added to a test vessel. The test material was accurately weighed with an electronic analytical balance and added.
Sludge + test material system (3, test vessels [2] [3] [4])
To make the concentration of the test material 100 mg/L, the basic culture medium [amount obtained by subtracting the amount of activated sludge solution added (3.08 mL) from 300 mL] and 30 mg of the test material were added to the test vessels. The test material was accurately weighed with an electronic analytical balance and added.
Sludge + aniline system (1, test vessel [6])
The basic culture medium [amount obtained by subtracting the amount of activated sludge solution added (3.08 mL) from 300 mL] and 29.5 μL of aniline [amount added, 30 mg = 29.5 μL × 1.022 g/cm³ (density)] were added to the test vessel so that the concentration of aniline was 100 mg/L. Aniline was dispensed using a microsyringe and added.
Sludge blank system (1, test vessel [5])
The basic culture medium [amount obtained by subtracting the amount of activated sludge solution added (3.08 mL) from 300 mL] was added to the test vessel.
- Inoculation of activated sludge: The activated sludge that was prepared under the conditions in 3 was inoculated into the test solutions (b), (c), and (d) to achieve the concentration of the suspended matters at 30 mg/L.
- Environmental factors that may have affected the reliability of test results: No such factors were noted.

SAMPLING
- Sampling frequency: For measurement of BOD, changes in BOD of each test solution were continuously recorded automatically by a data processor during the incubation stage. In-vessel temperature was measured and recorded every day. After the incubation periods, the test material in each test solution was analysed.
- Other: During the incubation period, the test solutions were visually observed every day. In addition, the operation status of the equipment was checked appropriately.

STATISTICAL METHODS:
Calculation method of biodegradability
The biodegradability was calculated based on the following formula and was expressed as an integer by rounding the first digit after the decimal point.

Biodegradability based on BOD

Biodegradability (%) = ((BOD – B) / TOD) x 100

Where:
BOD: Biochemical oxygen demand in the sludge + test material system (measured value, mg)
B: Biochemical oxygen demand in the sludge blank system (measured value, mg)
TOD: Theoretical oxygen demand required for complete oxidation of the test material (calculated value, mg). The purity was considered 100 %, and TOD was calculated from the composition formula which was obtained from the elemental analysis.

Biodegradability of test material

Biodegradability (%) = ((Sw – Ss) / Sw) x 100

Where:
Ss: Residual amount of the test item (mg) in the sludge + test material system (measured value, mg)
Sw: Residual amount of the test item (mg) in the water + test material system (measured value, mg)

Handling of numerical values
Numerical values were rounded in accordance with JIS Z8401: 1999 rule B.

Reference substance:

aniline

Parameter:

other: BOD

Value:

0

Sampling time:

28 d

Remarks on result:

other: Standard deviation not reported

Parameter:

other: Biodegradability of test material (HPLC)

Value:

5

Sampling time:

28 d

Remarks on result:

other: Standard deviation not reported

Details on results:

The analytical results of test solutions on day 28 were as follows: Although intercomponent changes in the test material were found in the sludge + test material system, the theoretical residual amount of the test material almost remained in both the water + test material and sludge + test material systems, and no peaks other than the test material were found in the HPLC chromatograms. No structurally modified new substances appeared to be generated in this test, and no further analysis was therefore performed on these substances.

Based on the results of qualitative analysis with LC-MS, the peak shapes detected on the chromatogram for UV detection (280 nm) were almost identical with those on the HPLC chromatograms obtained in the test material analysis of HPLC samples. The structural analysis based on the mass spectrum of each peak indicated that the major component in the test material corresponded to Peak 3 on the HPLC chromatogram. For the minor components, structures could be estimated for 3 components, but not for the remaining ones.

Conditions of the Test Solutions

	Test solution	Description
At the start of incubation	Water + test material system	The test material was not dissolved
	Sludge + test material system	The test material was not dissolved
At the end of incubation	Water + test material system	Insoluble matters were found
	Sludge + test material system	Insoluble matters other than sludge were found. No sludge growth was found

 

Analytical Results of Test Solutions

		Water + test material system	Sludge + test material system			Theoretical amount
		1	2	3	4
BOD	mg	0.8	0.9	-0.8	0.1	71.7
Residual amount and residual rate of the test material (HPLC)*	mg	30.3	28.7	29.0	28.9	30.0
	%	101	96	97	96	-

The sludge + test material system was expressed by subtracting the value of sludge blank system.

* Calculated using the total area of peaks of the chromatograms. In the sludge + test material system, intercomponent changes in the test material were found.

 

Biodegradability

		Sludge + test material system
		2	3	4	Mean
Biodegradability based on BOD	%	1	-1	0	0
Biodegradability of test material (HPLC)	%	5	4	5	5

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the conditions of the study some test material components were changed to other test material components, but none of the components were degraded by microorganisms. The mean biodegradability based on BOD was 0 %.

Executive summary:

To assess the biodegradability of the test material a study was conducted according to Japanese Guidelines and in compliance with GLP.

100 mg/L of the test material was added to sewage sludge, surface water and surface soil samples from near the water's edge from ten locations in Japan and the BOD was measured after 28 days. The test solutions of the water + test material system, sludge + test material system, and sludge blank system were pre-treated to prepare samples for HPLC to analyse the test material.

Although inter-component changes in the test material were found in the sludge + test material system, the theoretical residual amount of the test material almost remained in both the water + test material and sludge + test material systems, and no peaks other than the test material were found in the HPLC chromatograms. No structurally modified new substances appeared to be generated in this test. Based on the results it is considered that the biodegradability of each peak does not indicate microbial degradation and that the positive biodegradability indicates a decrease in the inter-component change and the negative biodegradability indicates an increase in the inter-component change.

Under the conditions of the study some test material components were changed to other test material components, but none of the components were degraded by microorganisms. The mean biodegradability based on BOD was 0 %.

Description of key information

Under the conditions of the study some test material components were changed to other test material components, but none of the components were degraded by microorganisms. The mean biodegradability based on BOD was 0 %.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

To assess the biodegradability of the test material a study was conducted according to Japanese Guidelines and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

100 mg/L of the test material was added to sewage sludge, surface water and surface soil samples from near the water's edge from ten locations in Japan and the BOD was measured after 28 days. The test solutions of the water + test material system, sludge + test material system, and sludge blank system were pre-treated to prepare samples for HPLC to analyse the test material.

Although inter-component changes in the test material were found in the sludge + test material system, the theoretical residual amount of the test material almost remained in both the water + test material and sludge + test material systems, and no peaks other than the test material were found in the HPLC chromatograms. No structurally modified new substances appeared to be generated in this test. Based on the results it is considered that the biodegradability of each peak does not indicate microbial degradation and that the positive biodegradability indicates a decrease in the inter-component change and the negative biodegradability indicates an increase in the inter-component change.

Under the conditions of the study some test material components were changed to other test material components, but none of the components were degraded by microorganisms. The mean biodegradability based on BOD was 0 %.