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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-09 till 2009-05-15

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.1 (Acute Toxicity for Fish)

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.1:

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Commission Directive 92/69/EEC, C.1: Acute Toxicity for Fish, 1992.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity 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.

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):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish (Brachydanio rerio)
- Source: breeding culture at Harlan Laboratories
- Length at study initiation (length definition, mean, range and SD): 10 fish were measured at the start of the test. The mean body length of the fish was 2.6 ± 0.05 cm (Mean ± SD),
- Weight at study initiation (mean and range, SD): 10 fish were measured at the start of the test. The mean body wet weight was 0.11 ± 0.02 g (Mean ± SD).
- Feeding during test: not feed during test


ACCLIMATION
- Acclimation period: one week
- Acclimation conditions (same as test or not): same as test (test water and temperature)
- Type and amount of food, Feeding frequency: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany)
- Health during acclimation (any mortality observed):During the last two weeks prior to the test, no fish died in the test fish batch and all fish were healthy
- No medication was applied during holding and
acclimatization.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Test conditions

Hardness:

1.25 mmol/L resp. 125 mg/L as CaCO3

Test temperature:

The water temperature was 20-22 °C during the test

pH:

The pH values in the test medium and the control ranged from 7.5 to 7.7

Dissolved oxygen:

The oxygen concentration was always 8.1 mg/L or higher, and thus higher than 60% oxygen saturation.

Salinity:

see table in section "Details on test conditions"

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:One glass test vessel with 5 liters of test medium was used for each treatment.
- Aeration: The test vessels were slightly aerated during the test period.
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.16g fish wet weight per liter test medium


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water


Reconstituted Test Water
Ingredients / Properties Concentration
mmol/L mg/L
CaCl2 × 2H2O 1.0 147
MgSO4 × 7H2O 0.25 61.5
NaHCO3 0.38 32.5
KCl 0.038 2.9
Water Hardness 1.25 125 as CaCO3
Alkalinity 0.4 ---



OTHER TEST CONDITIONS
- Photoperiod, Light intensity: A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period was used (light intensity during the light period was approximately within the range of 100 to 600 Lux).



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and Visible Abnormalities, at 3, 24, 48. 72 and 96 hours


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

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 5

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Behavioural abnormalities:
In the control and in the test medium with the loading rate of 100 mg/L no visible abnormalities were observed at the test fish.

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

no statistical analysis

Any other information on results incl. tables

Sublethal observations / clinical signs:

Mortality and Visible Abnormalities Observed at the Test

Loading rate	Number of abnormal and dead fish / number of dead fishType of visible abnormalities
	Observation time
(mg/L)	3 hours	24 hours	48 hours	72 hours	96 hours
Control	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
100	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
LC50	> 100 mg/L based on the loading rate

FishNumber of fish tested at the single concentration and the control: 7

Measurements / Observations

	Loading rate	Exposure time
		0 h	24 h		48 h		72 h		96 h
		new	old	new	old	new	old	new	old
Appearance  (1)	100	0	0	0	0	0	0	0	0
pH (2)	Control	7.6	7.7	7.6	7.6	7.6	7.5	7.6	7.5
	100	7.6	7.7	7.6	7.6	7.6	7.5	7.6	7.6
Oxygen (3)	Control	8.6	8.8	8.9	8.7	8.7	8.7	8.5	8.6
	100	8.1	8.8	8.3	8.8	8.5	8.6	8.6	8.5
Temperature (4)	Control	21	21	21	20	21	22	21	22
	100	21	21	21	20	21	22	21	21

(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

Conclusions:

In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of
100 mg/L.

Executive summary:

The acute toxicity of the test item Leuco Sulfur Black 1 to zebra fish (Brachydanio rerio) was determined in a 96-hour semi-static test with a daily test medium renewal according to the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

A limit test with a daily test medium renewal was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L.

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. 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 Substances and Mixtures, 2000.

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

The biological results were based on the loading rate of the test item.

In the control and at the loading rate of the test item of 100 mg/L no mortality or other visible abnormalities were determined during the test period of 96 hours.

The pH values in the test medium and the control ranged from 7.5 to 7.7.

The oxygen concentration was always 8.1 mg/L or higher, and thus higher than 60% oxygen saturation.

The water temperature was 20-22 °C during the test.

The summary of the biological results is as follows:

Loading rate (mg/L)

– 96-hour LC50: >100

– 96-hour LC0: ≥100

– 96-hour LC100: >100

– 96-hour NOEC: ≥100

– 96-hour LOEC: >100

In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L.