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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-08 till 2009-05-14

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.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (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.2,

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute Immobilisation Test, 2004.

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:

no

Details on sampling:

No analytical work was performed due to the specific properties of the test item (insolubility in water).

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low water solubility of the test item, the test medium was prepared prior to the start of the test and prior to the test medium renewals as follows: a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
Tth asphnids were not fed during the test

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium
dichromate is tested as a positive control twice a year. The result of the latest positive control test
in Februar 2009 (48-hour EC50: 0.71 mg/L, study C36915) indicated that the sensitivity of the
test organisms was within the internal historical range (48-hour EC50 from 1996 to 2009:
0.53-1.1 mg/L).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

2.5 mmol/L resp. 250mg/L as CaCO3

Test temperature:

Water temperature: 20°C

pH:

7.6 - 8.0 (in test medium and control)

Dissolved oxygen:

7.9 - 8.3 mg/L (in test medium and control)

Salinity:

see table in section "Details on test conditions"

Nominal and measured concentrations:

nominal 100mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: The test was performed in 100-mL glass beakers filled with 50 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstitutes test water
Reconstituted Test Water
Ingredients / Properties Concentration
mmol/L mg/L
CaCl2 × 2H2O 2.0 294
MgSO4 × 7H2O 0.5 123
NaHCO3 0.75 65
KCl 0.075 5.8
Water Hardness 2.5 250 as CaCO3
Alkalinity 0.8 ---
The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity

OTHER TEST CONDITIONS
A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was approximately between 520 and 680 Lux.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The daphnids were observed for immobility after 24 and 48 hours of exposure (daphnids not being able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobilized).


TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test, 100mg/L nominal
- Range finding study: The limit test was based on the results of a rangefinding test (non-GLP).

Reference substance (positive control):

yes

Remarks:

potassium dichromate, see "results with reference substance

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none
- In the control and at the loading rate of 100 mg/L no immobilized daphnids were determined during the test period of 48 hours.

Results with reference substance (positive control):

Reference Substance
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in Februar 2009 (48-hour EC50: 0.71 mg/L, study C36915) indicated that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 1996 to 2009: 0.53-1.1 mg/L).

Reported statistics and error estimates:

no statistical analysis

Any other information on results incl. tables

Effect of Leuco Sulfur Black 1 on the Mobility ofDaphnia magna

Loading rate (mg/L)	No. of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
		No.	%	No.	%
Control	20	0	0	0	0
100	20	0	0	0	0

Measurements / Observations

	Loading rate	Exposure time
		0 h	24 h		48 h
		new	old	new	old
Appearance (1)	Control	0	0	0	0
	100	0	0	0	0
pH (2)	Control	7.8	7.8	8.0	7.9
	100	7.6	7.6	8.0	8.0
Oxygen (3)	Control	8.3	8.4	7.9	8.0
	100	8.0	8.1	8.0	8.1
Temperature (4)	Control	20	20	20	20
	100	20	20	20	20

(1) Appearance of the Freshly Prepared and Old Test Medium

(2) pH in the Freshly Prepared and Old Test Medium and in the Control

(3) Oxygen Concentrations (mg/L) in the Freshly Prepared and Old Test Medium and in the Control

(4) Temperatures (°C) in the Freshly Prepared and Old Test Medium and in the Control

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In the control no immobilized daphnids were determined during the test period of 48 hours; Oxygen content >7.9mg/L.

Conclusions:

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna up to its solubility limit in the test water under the present test conditions (loading rate 100mg/L nominal).

Executive summary:

The acute toxicity of the test item Leuco Sulfur Black 1 to Daphnia magna was determined in a 48-hour semi-static test according to the EU Commission Directive 92/69/EEC, Part C.2, Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

A limit test with a test medium renewal after 24 hours was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water.

Due to the low water solubility of the test item, the test medium was prepared prior to the start of the test and prior to the test medium renewal as follows: a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substa Thences and Mixtures, 2000.

No remarkable observations were made concerning the appearance of the test medium. It was clear throughout the entire test duration.

No analytical work was performed due to the specific properties of the test item (insolubility in water).

At the beginning and end of the test medium renewal periods, the pH values of the test medium were between 7.6 and 8.0.

The dissolved oxygen concentrations in the test medium and control were at least 7.9 mg/L.

The water temperature was 20 °C during the test.

The biological results were based on the loading rate of the test item of 100 mg/L.

In the control and at the loading rate of 100 mg/L no immobilized daphnids were determined during the test period of 48 hours.

The biological test results (based on the loading rate) were as follows:

Loading rate (mg/L)

– 24-hour EC50: >100

– 24-hour EC0: ≥100

– 24-hour EC100: >100

– 48 -hour EC50: >100

– 48 -hour EC0: ≥100

– 48 -hour NOEC: ≥100

– 48 -hour EC100: >100

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna up to its solubility limit in the test water under the present test conditions.