Reaction product of salicylaldehyde and formaldehyde and phenol

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:

04 October 2005 to 01 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances"

Version / remarks:

November 21, 2003; No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 13, 2003, No. 2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry of the Environment

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: On-site sludge sampling was carried out at the following 10 locations in Japan in June 2005: Fushikogawa city sewage plant (Sapporo-shi, Hokkaido), Fukashiba industrial sewage plant (Kashima-gun, Ibaraki), Nakahama city sewage plant (Osaka-shi, Osaka), Ochiai city sewage plant (Shinjuku-ku, Tokyo), Kitakami River (Ishinomaki-shi, Miyagi), Shinano River (Niigata-shi, Niigata), Yoshino River (Tokushima-shi, Tokushima), Lake Biwa (Otsu-shi, Shiga), Hiroshima Bay (Hiroshima-shi, Hiroshima) and Dokai Bay (Kitakyushu-shi, Fukuoka). For sampling of city sewage return sludge was collected and for sampling from rivers, lakes and the sea surface water and surface soil which was in contact with the atmosphere were collected.
- Preparation 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 with pre-filtered open air after the pH value of the mixture was adjusted to 7.0 ± 1.0.
- Method of cultivation: After ceasing aeration of the sludge mixture for approximately 30 minutes, supernatant corresponding to about one third of the whole volume was removed. De-chlorinated 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 de-chlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25 ± 2 °C.
- For the 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.
- 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.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Preparation of basal culture medium: Each 3 mL of solutions A, B, C and D, which are prescribed in JIS K 0102-1998 Section 21, were made up to 1 L with purified water and then the pH of this solution was adjusted to 7.0.
- Test temperature: 25 ± 1 °C
- pH: 7
- Each test solution was stirred by a magnetic stirrer.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 300 mL in volume (improved type)
- Number of culture flasks/concentration: 3.
- In each test vessel, the basal culture medium [the volume subtracting the volume (2.43 mL) of activated sludge from 300 mL] and 30 mg of the test material supplied by the sponsor were added, so that the concentration of the test material reached 100 mg/L. The test material supplied by the sponsor was accurately weighed and added.
- Measuring equipment: During the cultivation period, BOD of the test solutions was measured by auto recording using a data sampler.
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: Soda lime No.1 (for absorption of carbon dioxide)

SAMPLING
- Cultivation temperature was measured and recorded once a day. During the cultivation, the appearance of the test solution was observed once a day and conditions of the instruments were checked properly.

CONTROL AND BLANK SYSTEM
- Control blank: In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (2.43 mL) of activated sludge from 300 mL].
- In one test vessel, 300 mL of purified water and 30 mg of the test material supplied by the sponsor were added, so that the concentration of the test material reached 100 mg/L. The test material supplied by the sponsor was accurately weighed and added.
- Positive control: In one test vessel, the basal culture medium [the volume subtracting the volume (2.43 mL) of activated sludge from 300 mL] and 29.5 µL [30 mg = 29.5 µL x 1.022 g/cm³ (density)] of aniline were added, so that the concentration of aniline reached 100 mg/L. Aniline was taken out by microsyringe and added.

CALCULATION OF PERCENTAGE BIODEGRADATION
The percentage biodegradations were calculated by the following equations and rounded off to the whole number.

- Percentage biodegradation by BOD:
Percentage Biodegradation (%) = [ (BOD – B) / TOD] x 100
Where:
BOD = Biochemical oxygen demand in the test solution (sludge + test material) (experimental) (mg)
B = Biochemical oxygen demand in the test solution (control blank) (experimental) (mg)
TOD = Theoretical oxygen demand required when the test material was completely oxidised (theoretical) (mg)

- Percentage biodegradation of test material:
Percentage biodegradation (%) = [ (Sw - Ss) / Sw] x 100
Where:
Ss = Residual amount of the test material in the test solution (sludge + test material) (experimental) (mg)
Sw = Residual amount of the test material in the test solution (water + test material) (experimental) (mg)

Reference substance:

aniline

Test performance:

VALIDITY OF THE TEST
- Difference between extremes of replicate values of percentage biodegradation: Percentage biodegradation by BOD was 4 % and percentage biodegradation of test material was 1 %, both meeting the criterion of < 20 %.
- Percentage biodegradation of aniline by BOD: The value of percentage biodegradation by aniline was 72 % after 14 days which met the criterion of ≥ 60 %.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

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

Key result

Parameter:

% degradation (test mat. analysis)

Value:

0

Sampling time:

28 d

Details on results:

APPEARANCES OF TEST SOLUTIONS
At the start of cultivation:
- Water + test material: The test material was not dissolved.
- Sludge + test material: The test material was not dissolved.
At the end of cultivation:
- Water + test material: Insoluble compound was observed.
- Sludge + test material: Insoluble compound in addition to the sludge was observed. Growth of the sludge could not be judged.

ANALYTICAL RESULTS OF TEST SOLUTIONS
- In the test solution (water + test material) and the test solutions (sludge + test material), theoretical amount of the test material approximately remained and no peak except the test material was detected on the HPLC chromatogram. Then, it was judged that any converted products were not produced. Therefore, converted products were not analysed.

PERCENTAGE BIODEGRADATION
- The test material was detected as plural peaks on the chromatogram under the analytical conditions. Then, the percentage biodegradation of each peak of the test material was calculated. The percentage biodegradations of each peaks were from -3 to 3 %. Therefore, it was concluded that all of the components of the test material were not biodegraded by microorganisms under the test conditions of this study.

Results with reference substance:

The value of percentage biodegradation by aniline was 72 % after 14 days which met the validity criterion of ≥ 60 %.

Table 1: Analytical Results

		Water + Test Material	Sludge + Test Material			Theoretical Amount
		Vessel 1	Vessel 2	Vessel 3	Vessel 4
BOD	mg	0.3	-3.3	-3.2	-0.9	70.8
Residual Amount and Percentage Residue of Test Material (HPLC)	mg	29.1	29.1	29.2	29.0	30.0
	%	97	97	97	97	-

 

 

Table 2: Percentage Biodegradation of Test Material Peaks

	Sludge + Test Material
	Vessel 2	Vessel 3	Vessel 4	Average
Peak 1 (%)	0	0	1	1
Peak 2 (%)	0	0	0	0
Peak 3 (%)	1	-1	0	0
Peak 4 (%)	0	-1	1	0
Peak 5 (%)	-1	-1	0	-1
Peak 6 (%)	3	-1	1	1
Peak 7 (%)	-3	-3	-1	-2

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test material was not biodegraded by microorganisms.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the Japanese guideline "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances". The testing was performed under GLP conditions.

Activated sludge was collected from 10 different locations in Japan including city sewage and surface water and surface soil from rivers, lakes and the sea. The sludge was exposed to the test material at 100 mg/L for 28 days in continuous darkness. The concentration of suspended solids was 30 mg/L.

To calculate percentage biodegradation, measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus was performed. Analytical determination of the test material by high-performance liquid chromatography (HPLC) was also performed.

The test material was detected as plural peaks on the chromatogram under the analytical conditions. Then, the percentage biodegradation of each peak of the test material was calculated. The percentage biodegradations of each peaks were from -3 to 3 %. Therefore, it was concluded that all of the components of the test material were not biodegraded by microorganisms under the test conditions of this study.

Under the conditions of this study, the test material was not readily biodegradable.

Description of key information

Under the conditions of this study, the test material was not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was investigated in accordance with the Japanese guideline Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances". The testing was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Activated sludge was collected from 10 different locations in Japan including city sewage and surface water and surface soil from rivers, lakes and the sea. The sludge was exposed to the test material at 100 mg/L for 28 days in continuous darkness. The concentration of suspended solids was 30 mg/L.

To calculate percentage biodegradation, measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus was performed. Analytical determination of the test material by high-performance liquid chromatography (HPLC) was also performed.

The test material was detected as plural peaks on the chromatogram under the analytical conditions. Then, the percentage biodegradation of each peak of the test material was calculated. The percentage biodegradations of each peaks were from -3 to 3 %. Therefore, it was concluded that all of the components of the test material were not biodegraded by microorganisms under the test conditions of this study.

Under the conditions of this study, the test material was not readily biodegradable.