Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published in O.J. L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

The inoculum contained a mixed population of microorganisms obtained from the secondary effluent of the waste water treatment plant of Pardubice processing predominantly municipal sewage. The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using.
The inoculum preparation is in conformity with the recommendations of the test guideline.

Duration of test (contact time):

28 d

Initial conc.:

100.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

SOLUTIONS AND CHEMICALS
The deionized water with conductivity less than 5 µS/cm was used for the preparation of all solutions. The same batch of water was used for each set of the experiments. Analytical grade reagents were used.
Stock solution for the preparation of mineral medium
Solution a)
- Monopotassium dihydrogen phosphate, (KH2PO4): 8.50 g
- Dipotassium monohydrogen phosphate, (K2HPO4): 21.75 g
- Disodium monohydrogen phosphate dodekahydrate, (Na2HPO4·12H2O): 67.21 g
- Ammonium chloride (NH4Cl): 0.50 g
Water up to the volume of 1 000 mL
The right composition of medium was checked by the determination of pH value, which must be 7.4.
Solution b)
- 22.50 g Magnesium sulphate heptahydrate, (MgSO4·7H2O) in 1 000 mL of water
Solution c)
- 27.50 g Calcium chloride, anhydrous, (CaCl2) in 1 000 mL of water
Solution d)
- 0.25 g Iron (III) chloride hexahydrate, (FeCl3·6H2O) in 1 000 mL of water
Preparation of mineral medium:
Preparation of 1 litre of mineral medium: 1 mL of solutions a), b), c) and d) were mixed with approx. 500 mL of water, the mixture was replenished with water up to 1 000 mL. The solution was prepared from aerated water.
Chemicals for the determination of COD:
- sulfuric acid, (H2SO4)
- silver sulfate, (Ag2SO4)
- ammonium iron(II) sulfate hexahydrate, (NH4)2 Fe(SO4)2.6H2O
- potassium dichromate, (K2Cr2O7)
- 1,10-phenanthroline, (Ferroin)
- mercury (II) sulfate, (HgSO4)
- sodium thiosulfate pentahydrate, (Na2S2O3·5H2O)
Chemicals for the determination of BOD:
- potassium dihydrogen phosphate, (KH2PO4)
- dipotassium hydrogen phosphate, (K2HPO4)
- disodium hydrogen phosphate dodecahydrate), (Na2 HPO4.12H2O)
- ammonium chloride, (NH4Cl)
- magnesium sulfate heptahydrate, (MgSO4.7H2O)
- calcium chloride anhydrous, (CaCl2)
- potassium iodide, (KI)
- starch soluble, (C6H10O5)n
- iron (III) chloride hexahydrate, (FeCl3.6H2O)
- N-allylthiourea, (C4H8N2S)
Chemicals for the determination of nitrites:
- phosphoric acid, (H3PO4)
- sulfanilamide, (C6H8N2O2S)
- N-(1-Naphthyl) ethylene-diamine dihydrochloride, (C12H14N2·2HCl)

INSTRUMENTS AND EQUIPMENT
- oxygen vessels with glass stopper, volume of approx. 280 mL
- large glass bottles of 5 and 10 L volume for the preparation and inoculation of medium
- closed thermostat
- pH meter WTW Inolab pH 730
- oximetr WTW Oxi 730 with membrane probe Cell Ox 325
- analytical balance XS 105 DU (Mettler Toledo)
- UV/VIS spectrophotometer Helios Alpha
- equipment for the determination of nitrite
- volumetric flasks, flasks, beakers, pipettes and further common laboratory equipment

PREPARATION OF SOLUTIONS FOR THE TEST
Inoculated medium
The volume of waste water for the inoculation of mineral medium was chosen 2.0 mL per 1 L of medium (COD of waste water: 66 mg/L). 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 60 mL of modified waste water. The pH value of medium: 7.4.
Test substance
The stock solution of the test substance was prepared in concentration 0.1006 g/L of deionized water. From this solution 291.6 mL (48.6 mL/L) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L. The solution prepared in this way contained 4.89 mg/L of the test substance, and had COD 3.40 mg/L. The pH value of solution: 7.4.
Reference substance
The stock solution of sodium benzoate was prepared in concentration 1.0000 g/L of deionized water. From this solution the 12.6 mL (2.1 mL/L) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L. The solution prepared in this way contained 2.10 mg/L of sodium benzoate, and had COD 3.46 mg/L. The pH value of solution: 7.5.
Test substance for the nitrification determination
The stock solution of the test substance for the nitrification determination was prepared in concentration 0.1006 g/L of deionized water. From this solution 194.4 mL (48.6 mL/L) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 4 L. The solution prepared in this way contained 4.89 mg/L of the test substance, and had COD 3.40 mg/L.
Toxicity test
The solution was prepared by dosing 194.4 mL of stock solution of the test substance and 8.4 mL of stock solution of the reference substance into inoculated medium and completed with this medium to the volume of 4 L. The mixture prepared in this way contained 4.89 mg/L of the test substance and 2.10 mg/L of sodium benzoate and had COD 6.86 mg/L.
Control (blank) determination
The inoculated mineral medium without test substance was used for the blank determination.

TEST CONDITIONS
Temperature: 20 ± 1.0°C
Illumination: no illumination, in the dark
Exposition time: 28 days

TEST PROCEDURE
The number of bottles for individual series:
Test substance series: 2 × 9 bottles with test substance and inoculum
Reference substance series: 2 × 9 bottles with reference substance and inoculum
Blank series: 2 × 9 bottles with inoculated medium only
Toxicity test series: 2 × 5 bottles with test substance, reference substance and inoculum for toxicity test
Series for nitrification determination: 1 × 9 bottles with test substance and inoculum
The prepared dosing solutions for each series were filled in two parallel bottle replicates (except series for nitrification determination). The bottles were placed into thermostat.
Measurement
The first two bottles (zero-time) from each series were immediately submitted to analysis for dissolved oxygen by the electrochemically using oximeter device (oximeter WTW OXI 730) and other bottles were placed in the thermostat.
On the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test, two bottles (parallel replicate) from the test substance series, from the reference substance series and from the blank series were taken off and the same determination was performed.
On the 3rd, 7th, 10th, 14th day of the test, two bottles from the toxicity test series were taken off and the same determination was performed.
The determination of nitrite-nitrogen concentration was carried out in parallel bottles in these time intervals by means of spectrometric methods.
The determination of nitrate-nitrogen concentration was carried out by ion chromatography.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Table 1: Dissolved oxygen concentrations in the main test

		Dissolved oxygen (mg/L) after period (days)
		0	3	7	10	14	17	21	24	28
Test substance	1	8.61	8.45	8.20	8.21	8.09	8.09	7.89	7.76	7.50
	2	8.51	8.43	8.26	8.26	8.12	8.07	7.85	7.68	7.59
	average	8.56	8.44	8.23	8.24	8.11	8.08	7.87	7.72	7.55
Reference substance	1	8.65	6.16	5.88	5.87	5.66	5.59	5.27	5.06	5.06
	2	8.65	6.24	5.82	5.83	5.63	5.65	5.28	5.16	4.98
	average	8.65	6.20	5.85	5.85	5.65	5.62	5.28	5.11	5.02
Blank experiment	1	8.59	8.47	8.42	8.40	8.33	8.34	7.93	7.93	7.92
	2	8.66	8.50	8.37	8.41	8.32	8.30	7.93	7.87	7.85
	average	8.63	8.49	8.40	8.41	8.33	8.32	7.93	7.90	7.89

Table 2: Biodegradation in the main test

		Biodegradation (%) after period (days)
		0	3	7	10	14	17	21	24	28
Test substance	1	0	0.6	5.2	5.2	6.1	6.0	-0.7	-4.0	-3.8
	2	0	-1.8	0.5	0.7	2.3	3.6	-2.5	-4.5	-9.4
	average	0	-0.6	2.8	3.0	4.2	4.8	-1.6	-4.2	-6.6
Reference substance	1	0	68.0	73.5	74.1	77.8	79.7	77.7	82.9	82.5
	2	0	65.7	75.2	75.2	78.7	78.0	77.4	80.0	84.8
	average	0	66.8	74.4	74.6	78.3	78.8	77.5	81.4	83.6

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

In this 28-day study of ready biological degradability the degradation of 0 % of the test substance, Acid Brown 235, was attained in the end of study.

Executive summary:

The test substance, Acid Brown 235, was tested for the ready biological degradability in Closed Bottle Test. The test was performed according to Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

The results of biological degradation are related to experimentally determined COD values of the test and reference substance at the beginning of the test. The test substance was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out. Sodium benzoate was used as the reference substance. The dosage was carried out from the stock solution.

COD of the test substance in medium at the beginning of the main test: 0.696 mg/mg

COD of the reference substance in medium at the beginning of the main test: 1.646 mg/mg

In parallel to the main test the toxicity test was performed.

Based on the test substance contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out.

The test was performed at temperature of 20 ± 1°C with the pH values of solutions 7 – 8 at the beginning of the test.

The prescribed validity criteria in the test were fulfilled. The test substance was not inhibiting for the used inoculum. Since all criteria of acceptability were met, this study is considered to be valid.

In this 28-day study of ready biological degradability the degradation of 0 % of the test substance, Acid Brown 235, was attained in the end of study.

Description of key information

The test was performed according toEU Method C.4-E (Determination of the “Ready” Biodegradability - Closed Bottle Test)and according to GLP principles. The test substance is not readily biodegradable (degradation rate is 0 % after 28 days).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed