2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

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:

18 October 2017 to 15 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Secondary effluent, microorganisms from a domestic waste water treatment plant, was used in the study. The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).

Duration of test (contact time):

28 d

Initial conc.:

7.1 mg/L

Based on:

test mat.

Details on study design:

TEST CONDITIONS
- Composition of medium: reconstituted water made from the following stock solutions:
a) Solution: KH2PO4 (2.125 g), K2HPO4 (5.4375 g), Na2HPO4 x 12H2O (16.795 g), NH4Cl (0.125 g), Deionised water (ad 250 mL)
b) Solution: MgSO4 x 7 H2O (5.625 g), Deionised water (ad 250 mL)
c) Solution: CaCl2 x 2 H2O (9.10 g), Deionised water (ad 250 mL)
d) Solution: FeCl3 x 6 H2O (0.25 g), Deionised water (ad 1000 mL)
Adequate amount of the stock solutions a) - d) were combined and filled up with deionised water to the appropriate final volume (ratio of composition referring to 1 mL of the stock solutions a) - d) filled up with deionised water to a final volume of 1000 mL according to the guideline). The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature.
- Test temperature: 20.8 - 23.3°C
- pH: 7.32
- pH adjusted: no

TEST SYSTEM
- Culturing apparatus: BOD bottles (300 ml) with special neck and glass stoppers
- Number of culture flasks/concentration:
10 bottles containing the test material and inoculum
10 bottles containing the reference item and inoculum (procedure control)
10 bottles containing only inoculum (inoculum control)
10 bottles containing the test material, reference item and inoculum (toxicity control)
- Measuring equipment: The COD (chemical oxygen demand) of the test material was determined in the analytical department of the test facility using Lovibond® COD Measuring System. The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
- Test performed in closed vessels: yes (flasks were tightly closed with glass stoppers)

PREPARATION OF TEST FLASKS
Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.

CALCULATIONS
The BOD (mg O2 per mg test item) expected after each period was calculated as follows:

mg O /L of T.i. and/or R. i. -mg O /L of i.control / mg T.i. and/or R.i./L in flask

where:
T.i. = test material
R.i. = reference item,
i.control = inoculum control

Calculation of Biodegradation %
The percentage biodegradation of the test item and of the reference item was calculated as follows:

[BOD(mg O2 /mg T.i. or R.i.) / COD (mg O2 /mg T.i.) or ThOD (mg O2 /mg R.i.)] x 100

where:
T.i. = test material
R.i. = reference item,
i.control = inoculum control

Reference substance:

benzoic acid, sodium salt

Remarks:

(3.6 mg/L)

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test material reached a mean of 0.0 % after 28 days based on the COD of the test material.
Therefore the test material is considered not readily biodegradable.
In the toxicity control containing both, the test material and the reference item sodium benzoate, a mean of 35.3 % biodegradation was noted within 14 days and 40.5% biodegradation after 28 days of incubation.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 79.2% after 14 days, and to a mean of 91.7% after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

Table 1: Summary of Results

Series	Duration (Days)	Percent biodegradation (average of two replicates)
Reference substance	14	79.2
	28	91.7
Toxicity control	14	35.3
	28	40.5
Test material	28	0.0

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test material can not be considered to be readily biodegradable.

Executive summary:

The ready biodegradability of the test material was investigated in a GLP study which was conducted in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and US EPA OPPTS 835.3110.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 0.0 % after 28 days based on the COD of the test material.

The reference item sodium benzoate was sufficiently degraded to a mean of 79.2% after 14 days, and to a mean of 91.7 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test material and the reference item sodium benzoate, a mean of 35.3% biodegradation was noted within 14 days and 40.5%

biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 7.1 mg/L on the secondary effluent

microorganisms because degradation was >25 % within 14 days.

The study met the validity criteria.

Based on the results of this study, the test material can not be considered to be readily biodegradable.

Description of key information

Under the conditions of the study, the test material cannot be considered to be 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 a GLP study which was conducted in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and US EPA OPPTS 835.3110. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 0.0 % after 28 days based on the COD of the test material.

The reference item sodium benzoate was sufficiently degraded to a mean of 79.2% after 14 days, and to a mean of 91.7 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test material and the reference item sodium benzoate, a mean of 35.3% biodegradation was noted within 14 days and 40.5%

biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 7.1 mg/L on the secondary effluent

microorganisms because degradation was >25 % within 14 days.

The study met the validity criteria.

Based on the results of this study, the test material can not be considered to be readily biodegradable.