Benzoic acid, 2-hydroxy-5-[[4-[[4-[[8-hydroxy-7-[[4-[(8-hydroxy-3,6-disulfo-1-naphthalenyl)azo]-2-methoxy-5-methylphenyl]azo]-3,6-disulfo-1-naphthalenyl]amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]phenyl]azo]-, pentasodium salt

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:

weight of evidence

Study period:

From March 29 to April 26, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

no

Remarks:

according to ISO/IEC 17025

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: ARA Werdhölzli, CH-8048 Zürich, 28 March 2007, 11.00 p.m.
- Concentration of sludge: 0.2 g/l dry matter in the final mixture
- Storage length: used immediately after sampling from the treatment plant without adaptation
- Preparation of inoculum for exposure: washed twice with tap water

Duration of test (contact time):

28 d

Initial conc.:

50 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral salts medium prepared with double distilled water (conductivity < 1.5 µD/cm, DOC < 0.3 mg/l)
- Test temperature: 22 ± 0.5 °C
- Aeration of dilution water: yes, with synthetic CO2-free air
- pH: 6.5-8.0, adjusted with NaOH or HCl, if necessary
- Oxygen concentration: > 6 mg/l

TEST SYSTEM
- Number of culture flasks/concentration: 2 for test substance
- Test performed in closed vessels: 1200 ml closed glass bottle
- Details of trap for CO2 and volatile organics if used: gas absorption bottles filled with NaOH

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Positive control: yes, 1 replicate

Reference substance:

diethylene glycol

Remarks:

49.7 mg/l as DOC

Parameter:

% degradation (DOC removal)

Value:

30

Sampling time:

28 d

Remarks on result:

other: adsoprtion after 3 h is 0 %

Details on results:

Based on DOC determinations, test substance reached a biodegradation of 30 % after 28 days.
The main degradation process occurred during the first 12 days, when 30 % of the initial DOC was removed. Maximum biodegradation was attained after 12 days of incubation. No significant adaption period was observed. No siginificant elimination 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.
Biodegradation of test substance based on CO2 evolution and calculated as ThCO2 reached 12 % after 28 days of incubation, showing that only part of the organic carbon of the test material was transformed into CO2.

Results with reference substance:

Diethylenglycole showed 99 % biodegradation as DOC removal after 14 days of incubation, thus confirming suitability of inoculum and test conditions.
The extent of mineralization of diethylenglycole was 96 % based on ThCO2.

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

day	inoculum blank*	reference substance*			substance*
	DOC mg/l	DOC (mg/l)	corrected DOC (mg/l)	reduction %	DOC (mg/l)	corrected DOC (mg/l)	reduction %
0	0.6	49.6	49.0	-	46.8	46.2	-
0.125 (3 h)	1.1	50.5	49.4	0	44.8	43.7	0
1	1.3	45.2	43.9	11	38.5	37.2	15
4	2.1	18.9	16.9	66	35.7	33.7	23
7	2.0	2.1	0.1	100	35.0	33.0	25
12	1.6	2.3	0.7	99	32.2	30.6	30
14	1.8	2.3	0.5	99	32.9	31.1	29
18	1.8	2.1	0.3	99	33.5	31.8	27
21	1.9	2.3	0.3	99	33.5	31.6	28
25	1.8	2.5	0.7	99	32.2	30.4	31
27	1.6	2.2	0.7	99	32.6	31.0	29
28	1.6	2.2	0.6	99	32.2	30.6	30

*mean of 2 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	n.d.	n.d.	n.d.	-	-
7	19.1	57.6	19.3	0	77
14	29.4	71.2	27.2	5	88
21	32.9	75.3	28.4	10	94
28	33.5	76.1	28.3	12	96

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

Test substance resulted as inherently biodegradable, based on percentage of biodegradation in terms of DOC removal above 20 % after 28 days of contact time.

Executive summary:

Method

Inherent biodegradability was determined in a 28 days Zahn-Wellens assay according to OECD guideline 302B. Test substance was tested in concentration of 50 mg/l DOC. Diethylenglycol was used as reference substance in a concentration of 49.7 mg/l as DOC.

Additionally the degree of biodegradation was also estimated as % of ThCO₂.

Results

After 28 days, biodegradation based on DOC removal and corrected by blank control was 30 % as average of 2 tests running in parallel. Adsorption after 3 hours resulted as 0 %.

Biodegradation based on CO₂ evolution and calculated as % ThCO₂ was 12 % after 28 days.

Biodegradation of reference substance was 99 % as DOC removal and 96 % as ThCO₂ evolution within the experimental period.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 5 to February 2, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

yes

Remarks:

sludge concentration in test was 1040 mg/l instead of 200-1000 mg/l

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: sludge

Details on inoculum:

- Source of inoculum: biological sewage treatment plant (ARA-Therwil) collected on 04.01.94
- Concentration of sludge: 1040 mg/l

Duration of test (contact time):

28 d

Initial conc.:

150 mg/L

Based on:

DOC

Remarks:

nominal

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: 22 ± 3 °C
- Lighting: indirect light

TEST SYSTEM
- Number of culture flasks: 2
- Measuring equipment: Shimadzu TOC analyzer

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

Reference substance:

diethylene glycol

Parameter:

% degradation (DOC removal)

Value:

19.5

Sampling time:

28 d

Remarks on result:

other: adsorption after 3 h: 1 %

Results with reference substance:

Bioelimination, corrected by blank control a measured as DOC (mg/l) is 98.8 % after 16 days.

Mean values of double determination

time	reference substance			substance
	reference DOC (mg/l)	blank DOC (mg/l)	reduction %	substance DOC (mg/l)	blank DOC (mg/l)	reduction %
-1	156.4			154.0
0 h	157.1	6.4	3.6	156.7	6,.4	2.4
3 h	156.7	5.2	3.1	157.7	5.2	1.0
2 d	154.5	6.9	5.6	152.4	6.9	5.5
5 d	143.5	11.5	15.6	141.5	11.5	15.6
7 d	137.5	12.8	20.3	138.4	12.8	18.4
9 d	73.4	10.6	59.8	136.0	10.6	18.6
12 d	7.9	5.5	98.5	136.8	5.5	14.7
13 d	7.0	5.1	98.8	-	-	-
14 d	8.3	4.0	97.3	145.4	4.0	8.2
15 d	7.7	5.2	98.4	-	-	-
16 d	6.1	4.2	98.8	142.8	4.2	10.0
19 d	-	-	-	141.7	3.4	10.2
21 d	-	-	-	139.0	4.5	12.7
23 d	-	-	-	135.5	3.6	14.4
26 d	-	-	-	132.0	3.4	16.5
28 d	-	-	-	127.7	3.7	19.5

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Test substance resulted as non inherently biodegradable, based on degradation percentage below 20 % after 28 days.

Executive summary:

Method

Inherent biodegradability was determined in a 28 days Zahn-Wellens assay according to OECD guideline 302B. Test substance was tested in concentration of 150 mg/l DOC. Diethylenglycol was used as reference substance in a concentration of 150 mg/l.

Results

After 28 days, bioelimination corrected by blank control was 19.5 % as average of 2 tests running in parallel. Adsorption after 3 hours resulted to as 1 %.

Biodegradation of reference substance after 16 days was 98.8 %.

Description of key information

Not ready biodegradable.

Additional information

Biodegradablility potential of the substance was based on a weight of evidence approach using available experimental data, in particular inherent biodegradability data and BOD5/COD values.

Inherent biodegradability studies were carried out following OECD guideline 302B, using test substances with high purity.

In the study dated 1994, biodegradability based on DOC removal was below the threshold value of 20 % after 28 days, thus the substance was considered as non inherently biodegradable.

In the study from 2007, biodegradability based on DOC removal was 30 %, after 28 days, thus the substance resulted as inherently biodegradable.

In both studies, blank control and positive control (diethylenglycole) were run and were considered as valid.

In addition, BOD5/COD values of ca. 0 were available on samples with purities ranging from 70 to 82 %, but no details on test procedures and results were available.

Based on available experimental data, the substance was considered as non ready biodegradable. Differently, no definite conclusion could be reached on the inherent biodegradability potential, due to biodegradability values overlapping the threshold value of 20 %.