disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of Experiments : November 08, 1996 - Completion of Experiments: November 22, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted July 17, 1992

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

dated December 29, 1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

PREPARATION OF SPIKED TEST MEDIUM SAMPLES

To demonstrate the validity of the method untreated test medium was spiked with the test substance. 23. 7 mg of the test substance were dissolved in 100 ml of test medium to prepare a stock solution of 237 mg test substance/I. Defined volumes of 10 ml of this solution were diluted to 25 ml with test medium to obtain spiked test medium samples with a concentration of 94.8 mg/1. These solutions were subjected to the same treatment as a sample.
In addition test medium without the test substance was analysed (analytical blank).

ANALYSIS OF THE TEST SUBSTANCE CONCENTRATIONS

For the analysis of the test substance concentration, duplicate samples were taken from the test medium of 100 mg test substance/I and the control at the start of the test, after 48 hours and at the end of the test. All test medium samples were taken from the approximate centre of the aquaria without mixing of the test media.
The concentration of the test substance SCARLET RN 1165 was analysed in the test medium samples of all sampling times (0, 48 and 96 hours). From the control samples only one of the duplicate samples was analysed from each of both sampling dates. The analytical procedure and the results are described in the attachment of the study report.
All samples are kept stored at about -20 °C to enable additional analyses. After delivery of the final test report all samples will be discarded.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test medium with the test substance concentration of nominal 100 mg/I was prepared by dissolving the test substance homogeneously in test water by intense stirring. The test medium was freshly prepared just before the start of the test (= introduction of the fish).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Source: obtained from P. Hohler, trout breeding station Zeiningen, CH-4314 Zeiningen, Switzerland
- Length at study initiation (length definition, mean, range and SD) :5.6 ± 0.3 cm (Mean± SD) (10 fish from the acclimated test fish batch were measured at the start of the test)
- Weight at study initiation (mean and range, SD): 2.2 ± 0.4 g (Mean± SD) (10 fish from the acclimated test fish batch were measured at the start of the test)

ACCLIMATION
- Acclimation period: in accordance with the test guidelines the fish were held for more than five weeks in the laboratories of RCC without any medication, and were acclimated for one week prior to the test start to the test water and temperature
- Type and amount of food during acclimation: during acclimation until one day before the start of the test the fish were fed daily with a commercial fish diet (HOKOVIT 502, 1.2 mm, supplied by H.U. Hoffinann Ag, CH-Biitzberg/Switzerland)
- Health during acclimation (any mortality observed): during the last five weeks prior to the test no fish died in the test fish batch and all fish were healthy

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

The fish were not fed during the test.

Post exposure observation period:

None

Hardness:

Water hardness: 2.5 mmol/l (= 250 mg/I) as CaCO3

Test temperature:

Test media temperatures ranged from 15.6 to 15.9 °C

pH:

The pH-values ranged from pH 7.8 to pH 8.1

Dissolved oxygen:

During the test duration the dissolved oxygen concentrations in the test media were always 8.5 mg/1 or higher, and thus > 60 % oxygen saturation

Salinity:

CaCl2x2H20: 2.0 mmol/L (= 294 mg/L)
MgS04X 7H20: 0.5 mmol/L (= 123 mg/L)
NaHCO3: 0.75 mmol/L (= 65 mg/L)
KCI: 0.075 mmol/L (= 5.8 mg/L)

Conductivity:

Not specified

Nominal and measured concentrations:

The only concentration tested was nominal 100 mg test substance/l and a control.
The analytically determined test substance concentration in the test medium of 100 mg test substance/l varied in the range from 89 % to 93 % of the nominal value. In the test medium the test substance SCARLET RN 1165 was sufficiently stable during the test period.
Therefore, all reported biological results are related to the nominal concentration of the test substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: One 30 liter glass aquarium with 15 liters test medium was used for each test concentration and the control.
- Aeration: The test media and the control were slightly aerated during the test period.
- Type of flow-through (e.g. peristaltic or proportional diluter): static test
- Renewal rate of test solution (frequency/flow rate): non-renewal test
- No. of organisms per vessel: 7

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium with the test substance concentration of nominal 100 mg/I was prepared by dissolving the test substance homogeneously in test water by intense stirring. The test medium was freshly prepared just before the start of the test (= introduction of the fish).
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on mortality)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: a 16-hour light to 8-hour darkness photoperiod

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

In the control and in the test medium of 100 mg test substance/l all fish survived until the end of the test and no signs of intoxication were observed. Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of
96 hours), and the 96-hour LC O of SCARLET RN 1165 to Rainbow Trout were determined to be at least 100 mg test substance/I. The NOEC might even be higher than this concentration. The 96-hour LOEC (lowest concentration with toxic effects) and the 96-
hour LC 50 are clearly higher than 100 mg test substance/l. These values could not be quantified, since concentrations in excess of 100 mg/I have not been tested according to the test guideline.

Results with reference substance (positive control):

Positive control has not been used.

Sublethal observations / clinical signs:

Number of dead fish/ number of fish with symptoms ( dead fish are added to the sum of fish with symptoms) and observed symptoms of intoxication after:

Concentration mg/l	Observation time
	2 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

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item SCARLET RN 1165 to rainbow trout (Oncorhynchus mykiss) was determined in a 96-hour static test according to the EU Commission Directive 92/69/EEC, Part C.1 (1992), and the OECD Guideline for Testing of Chemicals No. 203, (1992), resulting in a 96-hour LC50 > 100 mg/L.

Executive summary:

The acute toxicity of the test substance SCARLET RN 1165 to Rainbow Trout (Oncorhynchus mykiss) was determined in a 96-hour static test according to the Commission Directive 92/69/EEC, Annex Part C. l dated December 29, 1992, and the OECD Guideline for Testing of Chemicals No. 203, adopted July 17, 1992.

A limit test was performed in accordance with the Commission Directive 92/69/EEC to demonstrate that the test substance has no toxic effect on the test fish up to the concentration of nominal 100 mg test substance/l. Thus the only concentration tested was nominal 100 mg test substance/l and a control.

The analytically determined test substance concentration in the test medium of 100 mg test substance/l varied in the range from 89 %to 93 % of the nominal value. In the test medium the test substance SCARLET RN 1165 was sufficiently stable during the test period. Therefore, all reported biological results are related to the nominal concentration of the test substance.

The 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), respectively the 96-hour LC 0 of SCARLET RN 1165 to Rainbow Trout was determined to be at least 100 mg test substance/l. The NOEC might even behigher than this concentration. The 96-hour LOEC (lowest concentration with toxic effects)and the 96-hour LC 50 were clearly higher than100 mg test substance/l. These values could not be quantified, since the test substance had no toxic effect on the test organisms up to the tested concentration of 100 mg test substance/l and concentrations in excess of 100 mg test substance/l have not been tested according to the Commission Directive 92/69/EEC.

Description of key information

The acute toxicity of the test item SCARLET RN 1165 to rainbow trout (Oncorhynchus mykiss) was determined in a 96-hour static test according to the EU Commission Directive 92/69/EEC, Part C.1 (1992), and the OECD Guideline for Testing of Chemicals No. 203, (1992), resulting in a 96-hour LC50 > 100 mg/L.