4-(phenylazo)benzene-1,3-diamine

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as per mentioned below

Principles of method if other than guideline:

Decrease of dye 2,4-Diaminoazobenzene by the bacterium Pseudomonas cepacia was studied under aerobic/static condition.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Pseudomonas cepacia 13NA

Details on inoculum:

Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Soil samples were gathered from the draining trenches of the dyestuff works in the Hashima district.
- Laboratory culture: An enrichment culture was made for strain Pseudomonas cepacia 13NA and identification was determined using "Bergey's Manual of Systematic Bacteriology, vol 1," (Krieg and Holt 1984)
- Method of cultivation: Three loopful of cells grown overnight at 37 deg C on an agar slant were then inoculated in 100 ml of medium in a 500 ml flask and cultivated on a rotary shaker (5 cm stroke, 200 rpm) at 37 deg C. Each 100 ml of precultivated broth were used as inoculum for five 10 L flasks (medium, 5 L x 5 = 25 L) and were further cultivated.
- Storage conditions: no data
- Storage length:no data
- Preparation of inoculum for exposure: The cells grown in 10 L flasks were harvested by centrifugation at 10,000 rpm for 15 min. The cells were washed twice with 0.03 M phosphate buffer, pH 6.8. The washed cells were suspended in 0.03 M phosphate buffer, pH 6.8 which contained the test compounds
- Pretreatment: no data
- Concentration of sludge: no data
- Initial cell/biomass concentration: three loopful of cells no data
- Water filtered: no data
- Type and size of filter used, if any: no data

Duration of test (contact time):

24 h

Initial conc.:

5 other: ppm

Based on:

not specified

Initial conc.:

10 other: ppm

Based on:

not specified

Initial conc.:

20 other: ppm

Based on:

not specified

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Details on study design
TEST CONDITIONS
- Composition of medium: 1% glucose,0.5 % polypeptone (Eiken), 0.5 % yeast extract, 0.5 % NaCl and 0.2 % K2HPO4
- Additional substrate: Not specified
- Solubilising agent (type and concentration if used): Not specified
- Test temperature: 37 degree C
- pH: 7.0
- pH adjusted: Yes
- CEC (meq/100 g): Not specified
- Aeration of dilution water: Yes
- Suspended solids concentration: Not specified
- Continuous darkness: Not specified
- Other: -

TEST SYSTEM
- Culturing apparatus: 10L flask
- Number of culture flasks/concentration: 5
- Method used to create aerobic conditions: -
- Method used to create anaerobic conditions: -
- Measuring equipment: Not specified
- Test performed in closed vessels due to significant volatility of test substance: Not specified
- Test performed in open system: Not specified
- Details of trap for CO2 and volatile organics if used: Not specified
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: not specified
- Toxicity control: not specified
- Other: -

STATISTICAL METHODS:
Elimination of dye(%) = [absorbance of dye (initial)] - [absorbance of dye (observed)] / [absorbance of dye (initial)] x 100.
In the case of the cultivation tests, cells were removed by centrifugation at 10,000 rpm for 15 min to exclude absorption by the cell itself.

Reference substance:

not specified

Preliminary study:

No data available

Test performance:

No data available

Key result

Parameter:

% degradation (test mat. analysis)

Value:

90

Sampling time:

24 h

Remarks on result:

other: at 5Conc. (ppm)

Key result

Parameter:

% degradation (test mat. analysis)

Value:

91

Sampling time:

24 h

Remarks on result:

other: at 10 Conc. (ppm)

Key result

Parameter:

% degradation (test mat. analysis)

Value:

88

Sampling time:

24 h

Remarks on result:

other: at 20 Conc. (ppm)

Details on results:

No data available

Results with reference substance:

No data available

Influence of dye on the lag phase, growth constant, generation time and dye decrease in strain 13NA¹

Dye	Conc. (ppm)	T^2(h)	T'	K (h^-1)	K'	G	G'	Decrease of dye (%)^3
2,4-Diaminoazobenzene	0	4.0	-	0.69	-	1.0	-	-
	5	4.6	1.2	0.68	1.0	1.0	1.0	90
	10	4.7	1.2	0.56	0.8	1.2	1.2	91
	20	6.0	1.5	0.56	0.8	1.2	1.2	88

¹Incubation was carried out under static conditions at 37 deg C
²T lag phase; T'=T/T (control); K: growth constant; K'=K/K (control); G: generation time; G'=G/G (control). These growth factors are referred to by Tempest (1971)
³After incubation for 24 h.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

The given test material 2,4-Diaminoazobenzene is determined to be readily biodegradable in water.

Executive summary:

Biodegradation of the test chemical 2, 4-Diaminoazobenzene was studied. Soil samples were gathered from the draining trenches of the dyestuff works in the Hashima district. An enrichment culture was made for strain Pseudomonas cepacia 13NA and was included in the study as inoculumn.

Three loopful of grown overnight culture was inoculated in 100 ml of medium in a 500 ml flask and cultivated on a rotary shaker (5 cm stroke, 200 rpm) at 37 deg. C. 100 ml of precultivated broth were used as inoculum for five 10 L flasks. The cells were harvested by centrifugation at 10000 rpm for 15 mins. The cells were washed twice with 0.03 M phosphate buffer, pH 6.8. The washed cells were suspended in 0.03 M phosphate buffer, pH 6.8 which contained the test compounds and incubated at 37 deg. C under static conditions for 24 hrs. The cultural fluid (25 L) was obtained from the washed cell incubation by centrifugation at I0,000 rpm for 15 min. The cultural fluid was adjusted to pH 12.0 with 1 N-NaOH and extracted 3 times in a separating funnel with CH2CI 2 in an amount equal to the cultural fluid by volume. The CH2Cl 2 layer was washed with water and an aqueous solution saturated with NaCl, respectively, and then dehydrated with anhydrous Na2SO 4. After being filtered, the solution was concentrated in vacuo by a rotary evaporator. Preliminary identification of metabolites was made by thin layer chromatography (TLC) of authentic compounds. The concentrated samples were applied to a silica gel plate (2.5 x 7.5 cm; Merck's TLC plate, Silica gel 60 F254 pre-coated) and developed with MeOHCH2Cl2 (5 : 95, v/v). Spots were also detected under U.V. light at 254 nm wavelength. A high performance liquid chromatograph (HPLC) equipped with a double-scanning detector was used for identification of metabolites. Decrease of the substance observed after 24 hrs of incubation were 90%, 91% and 88% for 5, 10 and 20 ppm of concentrations respectively. Thus, it can be stated that under the given test condition, the test material 2, 4 Diaminoazobenzene is readily biodegradable in water.

Description of key information

The given test material 2,4-Diaminoazobenzene is determined to be readily biodegradable in water.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Different experimental studies for the Biodegradation in water endpoint were reviewed for the test substance 4-(phenylazo)benzene-1,3-diamine and are summarised below:

From peer reviewed journal (Eiichi Idaka et al;Bull. Environ. Contam. Toxicol.,1987), Biodegradation of the test chemical 2, 4-Diaminoazobenzene was studied. Soil samples were gathered from the draining trenches of the dyestuff works in the Hashima district. An enrichment culture was made for strainPseudomonas cepacia13NA and was included in the study as inoculumn. Three loopful of grown overnight culture was inoculated in 100 ml of medium in a 500 ml flask and cultivated on a rotary shaker (5 cm stroke, 200 rpm) at 37 deg. C. 100 ml of precultivated broth were used as inoculum for five 10 L flasks. The cells were harvested by centrifugation at 10000 rpm for 15 mins. The cells were washed twice with 0.03 M phosphate buffer, pH 6.8. The washed cells were suspended in 0.03 M phosphate buffer, pH 6.8 which contained the test compounds and incubated at 37 deg. C under static conditions for 24 hrs. The cultural fluid (25 L) was obtained from the washed cell incubation by centrifugation at I0,000 rpm for 15 min. The cultural fluid was adjusted to pH 12.0 with 1 N-NaOH and extracted 3 times in a separating funnel with CH2CI 2 in an amount equal to the cultural fluid by volume. The CH2Cl2 layer was washed with water and an aqueous solution saturated with NaCl, respectively, and then dehydrated with anhydrous Na2SO 4. After being filtered, the solution was concentrated in vacuo by a rotary evaporator. Preliminary identification of metabolites was made by thin layer chromatography (TLC) of authentic compounds. The concentrated samples were applied to a silica gel plate (2.5 x 7.5 cm; Merck's TLC plate, Silica gel 60 F254 pre-coated) and developed with MeOHCH2Cl2 (5 : 95, v/v). Spots were also detected under U.V. light at 254 nm wavelength. A high performance liquid chromatograph (HPLC) equipped with a double-scanning detector was used for identification of metabolites. Decrease of the substance observed after 24 hrs of incubation were 90%, 91% and 88% for 5, 10 and 20 ppm of concentrations respectively.Thus, it can be stated that under the given test condition, the test material 2, 4 Diaminoazobenzene is readily biodegradable in water.

Another supporting peer reviewed journal of the same authors (Eiichi Idaka et al; Society of Dyers and Colourists. Journal; 94: 914, 1978) indicates the specificity of the test substance4-(phenylazo)benzene-1,3-diamineto the bacterium Aeromonas hydrophila var. 24B was examined in biodegradation study. The source of the bacteria was effluent from the draining ditches at dyestuff factories. Effluent samples were taken from the draining ditches of dyestuff factories in the Gifu district. The pH was adjusted to 7.0. 0.1 ml of the sample of effluent and was added to a 500-ml shaken flask containing 100 ml of the screening medium. Rotary shaking culture was carried out for 1 day at 37°C. Agar plates (1.576, wt/vol.) composed of screening medium were then inoculated with a loopful of incubated broth and incubated for 3 days at 37°C and, after growth, colonies on the plate were removed and stocked on agar slants. Identification of the isolated bacteria was carried out by the method described by Buchanan and Gibbons. Each dye was added to a sample of the sterilized medium in a 500-ml flask and the sample was then inoculated with agar slant culture. Each flask was incubated for 1 -2 day at 37"C, either by rotary shaking (200 rev/min) or under static condition. The culture broth was centrifuged at 10,000 rev/min for 10 min to remove cells, and the absorbance of the supernatant fraction was measured with a spectrophotometer, after adjustment of pH. After removal of cells from the static culture broth by continuous centrifuging, it was adjusted to pH 11.0 with I-M NaOH and extracted with dichloromethane. The extract was passed through a silica gel column and the metabolites were then separated by thin-layer chromatography (t.1.c.) on silica gel. Metabolites was identified by means of high-performance liquid chromatography. Extent of elimination of the test substance4-(phenylazo)benzene-1,3-diamine after incubation for 24 h with shaking culture was 51%, and after 48 h with static culture was 91%. Thus it can be considered that the test material 2,4-Diaminoazobenzene is readily biodegradable in water.

35-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test item 4-(phenylazo)benzene-1,3-

diamine, CAS No. 495-54-5. The test system included control, test item and reference item. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The BOD35 value of 4-(phenylazo)benzene-1,3-diamine, CAS No. 495-54-5 was observed to be 0.7 mgO2/mg. ThOD was calculated as 1.80 mgO2/mg. Accordingly, the % degradation of the test item after 35 days of incubation at 20 ± 1°C according to Closed Bottle test was found to be 38.88%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherent primary biodegradable over a period of 35 days.

Thus considering the water solubility of the substance i.e. moderately soluble and for the purpose of classification and chemical safety assessment, the study from peer reviewed journal is considered as key study and the substance 4-(phenylazo)benzene-1,3-diamine is considered to be readily biodegradable in water.