Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 16th to December 14th, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study described was conducted in accordance with SN EN 45001.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Version / remarks:

adopted on 17 July 1992

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not pre-conditioned. ARA Werdhölzli, CH-8048 Zürich, 16 November 2000; 9.30 a.m.
- Adaptation: the activated sludge was used immediately after sampling from the treatment plant without adaptation.
- Preparation of inoculum for exposure: prior to the test the sludge was washed twice with tap water.
- Concentration of sludge: 0.2 g/l dry matter in the final mixture.

Duration of test (contact time):

28 d

Initial conc.:

160 mg/L

Based on:

test mat.

Initial conc.:

54.8 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Vessels: 1200 ml closed glass bottle containing a total volume of test solution of 600 ml.
- Composition of medium: aerobic mineral salts medium prepared with doubte distilled water. 1 ml/l of stock solution 1: Ammonium chloride 0.5 g, Di-sodium hydrogenphosphate 33.40 g, Potassium dihydrogenphosphate 8.50 g, Di-potassium monohydrogenphosphate 21.50 g. 1 ml/l stock solution 2: Calcium chloride 27.50 g. 1 ml/l stock solution 3: Magnesium sulfate 22.50 g. 1 ml/l stock solution 3: lron (lll) chloride 0.25 g.
- Conductivity: <1.5 µS/cm.
- DOC: <0.3 mg/l
- Test temperature: temperature-controlled, 22 ± 0.5 °C).
- Stirring: the test vessels were stirred (100 r.p.m.).
- pH: checked periodically.
- pH adjusted: adjusted to pH 6.5-8.0 with NaOH or H2SO4, if necessary.
- Aeration of dilution water: aerated with CO2 free air. lt was assured that during the test the oxygen concentration was higher than 6 mg/l.
- Continuous darkness: dark room.

TEST SYSTEM
- Test suspension: 2 replicates, containing activated sludge + test medium + test substance.
- Details of trap for CO2 and volatile organics: gas-absorption bottles containing 120 ml of 0.5 M NaOH.

SAMPLING
- Sampling frequency: the elimination of the test material was followed by DOC determinations at regular intervals. First samples were analyzed at the beginning of and 3 h after starting the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates, containing activated sludge + test medium.
- Positive control: containing activated sludge + test medium + diethyleneglycol as ready biodegradable reference compound.

Reference substance:

diethylene glycol

Remarks:

52.0 mg/l (as DOC)

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

Based on the data of the individual DOC determinations the test item reached a bioetimination of 97 % after 28 days.
The main elimination process occurred during the first day, when 93 % of the initial DOC was removed. Maximum elimination was attained after 4 days of incubration.
No significant adaptation period (lag phase) was observed.
Some elimination of the test substance due to adsorption to the activated sludge, on the glass surface or other physico-chemical processes was found as determined by means of DOC measurements 3 h after the start of the test.

No biodegradation of the test item based on CO2 evolution and calculated as % ThCO2 was observed after 28 days of incubation.

Results with reference substance:

The positive control, diethyleneglycol, showed 99 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions.
The extent of mineralization of the procedure control with diethyleneglycolwas 93 % based on ThCO2.

DOC concentrations of test suspension, inoculum blank and procedure control and calculation of degradation data.

Time (days)	Inoculum blank*	Procedure control reference substance (diethylene glycol)			Test suspension*
	DOC (mg/l)	DOC (mg/l)	DOC net. (mg/l)	Degradation (%)	DOC (mg/l)	DOC net. (mg/l)	Degradation (%)
0	0.5	52.2	51.7	-	44.3	43.8	-
0.125	0.4	51.1	50.7	0	41.0	40.6	0
1	1.2	46.4	45.2	11	3.1	2.0	95
4	2.4	28.2	25.8	49	3.5	1.2	97
7	2.7	3.5	0.9	98	4.0	1.4	97
11	2.1	2.1	0.0	100	3.5	1.4	96
14	2.6	3.3	0.7	99	4.2	1.6	96
18	2.3	2.2	-0.1	100	4.1	1.8	96
21	2.0	2.3	0.3	99	3.3	1.3	97
25	2.0	2.5	0.5	99	3.5	1.5	96
27	1.9	2.3	0.4	99	3.7	1.8	96
28	2.1	2.2	0.1	100	3.5	1.4	97

* Mean of two replicates

IC concentrations, calculated from the concentrations in the gas absorption bottles, of test suspension, inoculum blank and procedure control and corresponding degradation data.

Time (days)	Test suspension IC (mg/l)	Procedure control IC (mg/l)	Inoculum blank IC (mg/l)	Biodegradation Test suspension (% ThCO2)	Biodegradation Procedure control (% ThCO2)
0	nd	nd	nd	-	-
7	23.7	63.6	25.3	-3	74.0
14	33.1	81.2	32.9	0	93.0
21	39.8	88.4	40.2	-1	93.0
28	45.2	94.4	46.0	-1	93.0

nd: not determined

Validity criteria fulfilled:

yes

Remarks:

the degradation of the reference compound reached more than 70 % within 14 days of incubation

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item resulted to be not biodegradable under the test conditions.

Executive summary:

The biodegradability of test item exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions. The procedures outlined into the OECD guideline 302 B were followed.

Based on the data of the individual DOC determinations the test item reached a bioelimination of 97 % alter 28 days. The main elimination process occurred during the first day, when 95 % of the initial DOC was removed. Maximum elimination was attained after 4 days of incubation. No significant adaptation period (lag phase) was observed. Some elimination of the test substance due to adsorption to the activated sludge, on the glass surface or other physico-chemical processes was found as determined by means of DOC measurements 3 h after the start of the test.

The positive control, diethyleneglycol, showed 99 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions.

No biodegradation of the test item based on CO₂ evolution and calculated as %ThCO₂ was observed after 28 days of incubation. The extent of mineralization of the procedure control with diethyleneglycol was 93 % based on ThCO₂.

Conclusion

Based on these results, the test item resulted to be not biodegradable under the test conditions, since less than 20 % degradation was attained after 28 days of contact time. However, the test material is almost completely eliminated within 14 days of incubation.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From April 14th, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Version / remarks:

May 12, 1981

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum: microorganisms from a domestic waste water sewage plant (ARA, Sissach/Switzerland).
- Aeration of dilution water: aerated 2.5 l cylindrical reactor flasks.

Duration of test (contact time):

28 d

Initial conc.:

666.5 mg/L

Based on:

test mat.

Initial conc.:

57.8 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: the test medium (2l per flask) was prepared according to the OECD guideline 302B.
- Test temperature: the study was run at 21.5 - 22 °C.
- Light: the study was run protected from light.
- pH: ranged from 7.6 - 8.2.
- pH adjusted: in case the pH was above 7.9, it was adjusted to pH 7.4 - 7.6 with 1N H2SO4.
- Aeration of dilution water: flasks were aerated with a flow rate of about 0.5 - 0.7 l/minute, resulting in an oxygen concentration of 8.6 - 9.5 mg O2 per litre.
- Other: water evaporation losses were compensated by adding bidistilled water.

TEST SYSTEM
- Test concentration: the test article was dissolved in the test medium, heated up to 60 °C.

SAMPLING
- Sampling frequency: one sample of about 30 ml was taken DOC-in duplicate. Samples were taken at day 0 (0 and 3 hours after treatment), 7, 14, 21, and 28 of the incubation period.
- Sampling method: samples were filtered through a washed fluted filter paper. The first 5 ml of the filtrate were replaced into the reactor. The remaining 25 ml were used for DOC-analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: untreated inoculated control.

Reference substance:

aniline

Remarks:

theoretical amount of 70.5 mg DOC/l

Details on results:

The test article was moderately degradable by 20 - 45 % within 21 - 28 days.

Results with reference substance:

The standard compound, aniline, was degraded within 14 days by 97 %.

Carbon determinations - test item

Culture medium	Analyses	Test item conc. (mg/l)	DOC* (mg/l)
			0 h	3 h	7 d	14 d	21 d	28 d
Test item	a1	666.5	56.0	56.5	41.5	41.5	31.0	45.6
	a2	666.5	59.5	62.0	44.0	39.0	34.5	47.8
	Mean	666.5	57.8	59.3	42.8	40.3	32.8	46.7
Blank	a1	-	0.8	1.5	1.9	1.6	1.2	1.2
	a2	-	0.8	1.6	1.1	1.4	1.3	1.2
	Mean	-	0.8	1.6	1.5	1.5	1.3	1.2

* All DOC-values determined after filtration

% DOC-removal after
3 h	7 d	14 d	21 d	28 d
-1	28	32	45	20

Carbon determinations - aniline

Culture medium	Analyses	Test item conc. (mg/l)	DOC* (mg/l)
			0 h	3 h	7 d	14 d	21 d	28 d
Test item	a1	100.5	70.0	72.0	5.6	3.8	1.2	1.2
	a2	100.5	71.0	75.0	5.5	3.6	1.3	1.2
	Mean	100.5	70.5	73.5	5.6	3.7	1.3	1.2
Blank	a1	-	0.8	1.5	1.9	1.6	1.2	1.2
	a2	-	0.8	1.6	1.1	1.4	1.3	1.2
	Mean	-	0.8	1.6	1.5	1.5	1.3	1.2

* All DOC-values determined after filtration

% DOC-removal after
3 h	7 d	14 d	21 d	28 d
-3	94	97	97	98

Interpretation of results:

other: the 20 - 45 % of test material was removed within 21 - 28 days.

Conclusions:

The 20 - 45 % of test material was removed within 21 - 28 days.

Executive summary:

The biodegradation/bioelimination of the test artlcle was followed by exposing to microorganisms from the secondary effluent of a domestic waste-water sewage plant. Results obtained were compared with those obtained with aniline tested simultaneously under the same conditions. The procedures described into the OECD guideline 302 B were followed.

The 20 - 45 % of test material was removed within 21 - 28 days. The standard compound, aniline, was degraded within 14 days by 97 %.

Conclusion

The 20 - 45 % of test material was removed within 21 - 28 days.

Description of key information

Neither readily nor inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The biodegradability of test item was investigated following the testing procedures outlined into the OECD guideline 302 B. The test item resulted to be mainly eliminated during the first day, when 95 % of the initial DOC was removed. Maximum elimination was attained after 4 days of incubation. However, no biodegradation of the test item, based on CO₂evolution and calculated as %ThCO₂, was recorded after 28 days of incubation; based on these results, the test item resulted to be not biodegradable under the test conditions, since less than 20 % degradation was attained after 28 days of contact time.

A second experiment, performed according to OECD 302 B guideline is available. The 20 - 45 % of test material was removed within 21 - 28 day, based on DOC assay.