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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

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.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

Com. Reg. (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Reg. (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part C.11, Activated S

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

EU Commission Directive 88/302/EEC, Part C.11, Activated Sludge Respiration Inhibition Test, 1988

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

OECD Guideline for Testing of Chemicals, No. 209, Activated Sludge, Respiration Inhibition Test, 1984.

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.

Sampling and analysis

Analytical monitoring:

not required

Details on sampling:

For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The final nominal concentration tested was 1000 mg/L.

Based on the results of a pre-experiment (without GLP), no stock solution could be prepared due to the low water solubility of the test item under the pre-test conditions.

Therefore, 500.0 mg of the test item was weighed by means of an analytical balance and transferred to the designated test vessel with 284 mL tap water.

The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.

After stirring, the appearance of the test medium was evaluated, and the test concentration was clearly above the water solubility limit of Leuco Sulfur Black 1 under the test conditions. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARAErgolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. 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, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was adjusted from 8.7 to 7.3 with a diluted sulfuric acid solution.
* Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 × 2H2O
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
filled up to a final volume of 1 liter with deionized water

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

none

Test conditions

Hardness:

not dertermined

Test temperature:

20°C

pH:

see table "Remarks on results"

Dissolved oxygen:

see table "Remarks on results"

Salinity:

synthetic waste water

Nominal and measured concentrations:

1000mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000ml glass beakers
- Aeration: During the incubation period of 3 hours the single test medium and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2



TEST MEDIUM / WATER PARAMETERS
- The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. 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, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was adjusted from 8.7 to 7.3 with a diluted sulfuric acid solution.
* Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 × 2H2O
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
filled up to a final volume of 1 liter with deionized water



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate


TEST CONCENTRATIONS
1000mg/L nominal

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Leuco Sulfur Black 1 under the test conditions.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 18 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

no statistical analysis done

Any other information on results incl. tables

Influence of Leuco Sulfur Black 1 (Test Item) and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

Vessel no.	Nominal concentration of test chemical	Oxygen consumption rate	Inhibition	pH values		Oxygen Concentration (mg O2/L)
	(mg/L)	(mg O2 L-1 min-1)	(%)	start*	end*	start*	end*
Control
A B	0 0	1.415 1.423		7.5 7.9	8.4 8.3	7.9 7.9	8.5 8.9
Mean Deviation (%)		1.419 0.6
Test item
5	1000	1.397	1.6	8.1	8.2	7.9	8.7
Reference item
1 2 3	5 16 50	1.520 0.734 0.204	-7.1 48.3 85.6	7.8 8.0 8.0	8.2 8.3 8.4	7.9 8.0 8.2	8.9 8.8 9.2

* Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to control

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour NOEC (EC15) of Leuco Sulfur Black 1 to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested.

Executive summary:

The inhibitory effect of the test item Leuco Sulfur Black 1 on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Leuco Sulfur Black 1 under the test conditions.

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Leuco Sulfur Black 1 under the test conditions) was reached and no inhibitory effect was determined after three hours contact time.

Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L. Page