Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03-23 till 2009-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

Com. Reg. (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.4-D, Manometri

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

EU Commission Directive 92/69 EEC, C.4-D, Manometric Respirometry Test, 1992.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 301 F, Ready Biodegradability: Manometric Respirometry Test, 1992.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

EC Number:

266-273-5

EC Name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Cas Number:

66241-11-0

Molecular formula:

Molecular formula is not available.

IUPAC Name:

Reaction products of 2,4-dinitrophenol with polysulfide, leuco derivatives, sodium salt

Details on test material:

- Name of test material : Leuco Sulfur Black 1
- Physical state: thixotrophic, black, wet paste
- Composition: Leuco Sulfur Black 1:52.75 % (w/w) (calculated by amount of by-compounds)
2,4-Dinitrochlorobenzene: < 20 ppm
2,4-Dinitrophenole: < 20 ppm
Sulfate: 970 ppm
Chloride: 750 ppm
Sodium: 1.3 % (w/w)
Water: 45.75 % (w/w) by Karl-Fischer titration
- Lot/batch No.: ESA0007595
- Expiration date of the lot/batch: 2011-07-11
- Stability under test conditions: Stable under storage conditions.
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater
- Laboratory culture:
- Method of cultivation: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. - Storage conditions/length: During the holding period of three days prior to use, the sludge was aerated at room temperature.
- Preparation of inoculum for exposure: Prior to use, the sludge was first thoroughly mixed and then diluted with test water
- Concentration of sludge: Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

140 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

141 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Apparatus
The test flasks (500 mL reaction vessels, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day.
The final test volume was 250ml per test flask

Principle: Electro-chemical analysis process:
The biodegradation process consumes the dissolved oxygen in the test medium and generates CO2. The CO2 is adsorbed by soda lime, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.
Test duration: 28 days

Light conditions: Darkness

Test temperature: 22 °C, maintained with a built-in thermostat and checked once per week.

pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum. At the end of incubation, the pH was measured again in each test flask.

Test water
prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L
The pH of this solution was 7.4.

2) MgSO4 x 7H2O 22.50 g/L
3) CaCl2 x 2H2O 36.40 g/L
4) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter

To obtain the final test water, 10 mL of stock solution No. 1 and 1 mL each of stock solution Nos. 2, 3 and 4 were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.7 to 7.4 with a diluted hydrochloric acid solution.
Control and Blank System
Inoculum blank: yey
Toxicity control: yes
Reference substance: yes

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg.
In the procedure controls, the reference item was degraded by an average of 80% by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 82%.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

ca. 0

St. dev.:

0

Sampling time:

28 d

Remarks on result:

other: St.dev. not applicable

Details on results:

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the COD of the test item and the ThOD of the reference item.
In the toxicity control, the course of biodegradation over the 28-day exposure period was similar to the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation amounted to 50%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 142 mg/L because biodegradation in the toxicity control was >25% within 14 days.
The pH measured in all flasks at the start of the test was 7.4. At the end of exposure (Day 28), pH values of 7.1-7.8 were measured.

BOD5 / COD results

BOD5 / COD

Parameter:

BOD5

Value:

ca. 0 mg O2/g test mat.

Results with reference substance:

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg.
In the procedure controls, the reference item was degraded by an average of 80% by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the
reference item was degraded by an average of 82%.

Any other information on results incl. tables

Biodegradation of the Test Item

The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 0.795 mg O2/mg test item.

The biochemical oxygen demand (BOD) of the test item Leuco Sulfur Black 1 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days.

Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days.

Biodegradation in the Test Flasks

	Percentage Biodegradation1
	Test item		Procedure control		Toxicity control
Time	Replicate No.		Replicate No.		Replicate No.
(days)	1	2	1	2	1
0	0	0	0	0	0
1	0	0	19	21	14
2	---	---	---	---	---
3	---	---	---	---	---
4	-6	-3	63	68	41
5	-4	-3	68	72	44
6	-4	-4	70	74	45
7	-4	-4	72	77	47
8	-5	-4	73	78	48
9	---	---	---	---	---
10	---	---	---	---	---
11	-6	-6	77	80	49
12	-9	-8	76	81	49
13	-9	-8	78	81	50
14	-10	-9	79	81	50
15	---	---	---	---	---
16	---	---	---	---	---
17	---	---	---	---	---
18	-12	-11	80	84	51
19	-13	-11	80	84	51
20	-14	-12	80	84	51
21	-16	-12	78	84	50
22	-16	-12	80	84	51
23	---	---	---	---	---
24	---	---	---	---	---
25	-18	-14	78	84	51
26	-18	-14	80	84	51
27	-19	-15	79	84	51
28	-20	-15	80	84	51
Mean (Day 28)	(-17) *		82		n.a.

1Corrected for the mean oxygen uptake of the inoculum controls (see Section 3.5)

-- Not determined

n.a. not applicable

* Negative value due to higher oxygen consumption in the inoculum controls than in the test flasks with test item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Leuco Sulfur Black 1 is not biodegradable under the test conditions within 28 days.

Executive summary:

The test item Leuco Sulfur Black 1 was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to the EU Commission Directive 92/69 EEC, C.4-D Commission Regulation (EC) No 440/2008, C.4-D and the OECD Guideline for Testing of Chemicals No. 301 F.

The biochemical oxygen demand (BOD) of the test item Leuco Sulfur Black 1 in the test media was in the normal range found for the inoculum controls.

Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days.

In the toxicity control, containing both Leuco Sulfur Black 1 and the reference item sodium benzoate, Leuco Sulfur Black 1 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 142 mg/L.

In the procedure controls, the reference item sodium benzoate was degraded by an average of 80% by Exposure Day 14, and reached an average biodegradation of 82% by the end of the test (Day 28), thus confirming suitability of the activated sludge.