2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 17 July 1992

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Inoculum: of activated sludge: activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not pre-conditioned.
- Source: ARA Werdhölzli, CH-8048 Zürich, 20 August 2002; 9.30 a.m.
- Concentration of sludge: 0.2 g/l dry matter in the final mixture.
- Preparation of inoculum for exposure: the activated sludge was used immediately after sampling from the treatment plant without adaptation. Prior to the test the sludge was washed twice with tap water.
- Initial DOC: the test material was diluted with mineral salts medium to give a final DOC concentration of about 50 mg/l.

Duration of test (contact time):

28 d

Initial conc.:

119 mg/L

Based on:

test mat.

Initial conc.:

50.9 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
- Vessel: 1200 ml closed glass bottle containing a total volume of test solution of 600 ml.
- Composition of medium: aerobic mineral salts medium prepared with double distilled water (conductivity: <1.5 µS/cm; DOC: <0.3 mg/l).
- Test temperature: temperature-controlled dark room (22 ± 0.5 °C).
- Aeration of dilution water: aerated with CO2 free air.
- Oxygen: during the test the oxygen concentration was > 6 mg/l.

TEST SYSTEM
- Number of culture flasks/concentration: 2 replicates, containing activated sludge + test medium + test substance.
- Details of trap for CO2 and volatile organics if used: flasks were fitted to gas-absorption bottles containing 120 ml of 0.5 M NaOH.

CONTROL
- Inoculum blank: 2 replicas, containing activated sludge + test medium.
- Positive control: 1 replicate: containing activated sludge + test medium + diethyleneglycol as ready biodegradable reference compound.

Reference substance:

diethylene glycol

Remarks:

48.8 mg/l as DOC

Parameter:

% degradation (DOC removal)

Value:

28

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

-29

Sampling time:

28 d

Details on results:

Based on these results test item is not biodegradable under the conditions of the OECD guideline no. 302, since less than 20 % degradation was attained aller 28 days of contact time.
At the applied test substance concentration of 119 mg/l significant inhibition of biodegradation was observed. However, more than 25 % of the DOC could be eliminated by adsorption processes within 7 days of incubation.

DOC DETERMINATION
Based on the data of the individual DOC determinations the substance reached a biodegradation/bioelimination of 28 % after 28 days.
The main elimination process occurred during the first 7 days, when more than 25 % of the initial DOC was removed. Maximum biodegradation was attained after 27 days of incubation. No significant adaptation period (lag phase) was observed. No significant 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.

CO2 EVOLUTION
The biodegradation of test item based on CO2 evolution and calculated as % ThCO2 reached -29 % after 28 days of incubation, indicating significant inhibition of the activated sludge microorganisms. Therefore, the observed elimination was only due adsorption.

REFERENCE COMPOUND
The positive control, diethyleneglycol, showed 100 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70 % within 14 days of incubation.
The extent of mineralization of the procedure control with diethyleneglycol was 92 % based on ThCO2.

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

Time (d)	lnoculum blank (B) *	Procedure control with iethylene glycol (R)			Test suspension with test material(T) *
	DOC (mg/l)	DOC (mg/l)	DOC net. (mg/l)	Degrad. (%)	DOC (mg/l)	DOC net. (mg/l)	Degrad. (%)
0	0.7	49.5	48.8	-	48.7	48.0	-
0.125	1	49.1	48.1	0	51.2	50.2	0
1	1.2	47.7	46.6	3	48.9	47.7	5
3	1.6	41.9	40.3	16	48.5	46.8	7
7	1.9	2.1	0.3	99	38.3	36.4	27
10	2.0	2.0	-0.1	100	38.5	36.5	27
14	2.0	2.0	0.0	100	40.2	38.2	24
17	1.5	1.7	0.2	100	39.7	38.2	24
21	1.9	1.7	-0.2	100	38.8	36.9	26
24	1.8	2.2	0.4	99	38.3	36.4	27
27	1.9	2.0	0.1	100	37.9	36.0	28
28	1.8	1.7	-0.1	100	37.8	36.0	28

* 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 (d)	Test suspension lC (mg/l)	Procedure control lC (mg/l)	Inoculum blank lC (mg/l)	Biodegradation Test suspension (% ThCO2)	Biodegradation Procedure control (%ThCO2)
0	nd	nd	nd	-	-
7	15.7	56.5	21.4	-11	72
14	23.3	74.7	31.1	-15	89
21	25.2	80.8	39.1	-27	85
28	25.8	85.6	40.9	-29	92

nd: not determined

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Not inherently biodegradable.

Executive summary:

The biodegradability of test item exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions. Based on the data of the individual DOC determinations the substance reached a biodegradation/bioelimination of 28 % after 28 days. The main elimination process occurred during the first 7 days, when more than 25 % of the initial DOC was removed. Maximum biodegradation was attained after 27 days of incubation. No significant adaptation period (lag phase) was observed. No significant 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 100 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70 % within 14 days of incubation.

The biodegradation of test item based on CO₂ evolution and calculated as % ThCO₂ reached -29 % after 28 days of incubation, indicating significant inhibition of the activated sludge microorganisms. Therefore, the observed elimination was only due adsorption. The extent of mineralization of the procedure controlwith diethyleneglycolwas 92 % based on ThCO₂.

Based on these results test item is not biodegradable under the conditions of the OECD guideline no. 302, since less than 20 % degradation was attained atter 28 days of contact time. At the applied test substance concentration of 119 mg/l significant inhibition of biodegradation was observed. However, more than 25 % of the DOC could be eliminated by adsorption processes within 7 days of incubation.

Conclusion

Not inherently biodegradable

Description of key information

Neither readily nor inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The biodegradability potential of the test item was assessed following method and procedures outlined into the OECD Guideline 302 B. Based on the data of the individual DOC determinations the substance reached a biodegradation/bioelimination of 28 % after 28 days. The main elimination process occurred during the first 7 days, when more than 25 % of the initial DOC was removed; maximum biodegradation was attained after 27 days of incubation. No significant adaptation period (lag phase) was observed. No significant elimination of the test substance due to adsorption 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 biodegradation of test item based on CO₂ evolution and calculated as % ThCO₂ reached -29 % after 28 days of incubation, indicating significant inhibition of the activated sludge microorganisms. Therefore, the observed elimination was attributed only due adsorption.

Based on the described outcomes, the test item resulted to be not biodegradable under the conditions of the test, since less than 20 % degradation was attained after 28 days of contact time. At the applied test substance concentration of 119 mg/l significant inhibition of biodegradation was observed. However, more than 25 % of the DOC could be eliminated by adsorption processes within 7 days of incubation.