disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

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:

July 22, 2002 -November 12, 2002

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:

1992

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

ANALYSES OF THE TEST ITEM CONCENTRATIONS
For the analyses of the test item concentrations in this static test, duplicate samples were taken from the freshly prepared test media of all test concentrations and the control just before the start of the test.
To confirm the maintenance of the test item concentrations throughout the test duration, duplicate samples from the test media of all test concentrations and the control were taken after 48 hours and at the end of the test (after 96 hours).
The concentrations of the test item Red Rwa 4565 were analyzed in all test medium samples from the sampling times at 0 and 96 hours. However, the two lowest test concentrations (nominal 4.6 and 10 mg/L) were not analyzed, since they where below the 96-hour NOEC, determined in this test. From the control samples only one of the duplicate samples was analyzed from the corresponding sampling times.

Vehicle:

no

Details on test solutions:

The following concentrations of Red Rwa 4565 were tested: nominal 4.6, 10, 22,46 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item).
The test media were prepared as follows: A concentrated stock solution of nominal 500 mg/L was freshly prepared by dissolving 3.0 g of the test item completely in 6 liter test water by stirring for 15 minutes at room temperature. Adequate volumes of this intensively stirred stock solution were added to the test water in the aquaria and were intensively mixed to prepare the test media with the test concentrations as stated above. The test media were freshly prepared just before introduction of the fish (= start of the test). The actual concentrations of the test item in the test media were analytically determined.
The test concentrations were based on the results of a range-finding test and on the results of a pre-experiment to the solubility of the test item (without GLP).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Oncorhynchus mykiss
- Source: obtained from P. Hohler, trout breeding station Zeiningen, CH-4314 Zeiningen, Switzerland
- Length at study initiation (length definition, mean, range and SD): 5.5 ± 0.37 cm
- Weight at study initiation (mean and range, SD): 1.5 ± 0.27 g

ACCLIMATION
- Acclimation period: In accordance with the test guidelines the fish were held in the laboratories of RCC for more than two weeks without any medication. Prior to test start, they were acclimated for one week to the test water and temperature.
- Type and amount of food during acclimation: During holding and acclimatization until one day before the start of the test the fish were fed with a commercial fish diet (HOKOVIT 502, 1.2 mm, supplied by H.U. Hofmann AG, CH-4922 Butzberg, Switzerland).
- Health during acclimation (any mortality observed): During holding and acclimatization no mortality was observed in the test fish batch and all fish were healthy.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

The fish were not fed during the test.

Post exposure observation period:

None.

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

14-15 °C

pH:

ranged from 7.9 to 8.0

Dissolved oxygen:

The oxygen concentration was always 8.9 mg/L or higher, and thus higher than 60% oxygen saturation

Salinity:

CaCl2 x 2H20: 2.0 mmol/L (= 294 mg/L)
MgS04 X 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:

Nominal: 4.6, 10, 22, 46, 100 mg/L
Concurrent with the sample analysis, recoveries of spiked test water samples in the relevant concentrations (22.2 and 111 mg/L of the test item) were performed in duplicate. The average concentrations were found to be109 % and 102 % of the spiked values, with an overall mean of 105 % (n = 4). Therefore, no correction for possible losses during the analytical procedure is necessary.

Details on test conditions:

TEST SYSTEM
- Test vessel: One 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. The fish were not fed during the test
- 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 media were prepared as follows: A concentrated stock solution of nominal 500 mg/L was freshly prepared by dissolving 3.0 g of the test item completely in 6 liter test water by stirring for 15 minutes at room temperature. Adequate volumes of this intensively stirred stock solution were added to the test water in the aquaria and were intensively mixed to prepare the test media with the test concentrations
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour darkness photoperiod, with a 30 minute transition period
- Light intensity: light intensity at light period approximately within the range of 280 -290 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : pH, oxygen and temperature at 0h, 24h, 48h, 72h and 96h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not exceeding 2.2
- Justification for using less concentrations than requested by guideline: 5 concentrations used as requested by OECD TG 203
- Range finding study: no
- Test concentrations: 4.6, 10, 22, 46 and 100 mg/L
- Results used to determine the conditions for the definitive study: no

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

22 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.:

61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

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.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Abnormalities:
ln the control and at the test concentrations up to and including 22 mg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. At the next higher test concentration of 46 mg/L visible abnormalities were observed at Day 3 (tumbling during swimming and strongly extended gills) and one test flsh was dead at the observation at Day 4. At the highest test concentration of 100 mg/L all fish were dead at the observation after 48 hours without showing any visible abnormalities before. Thus, the concentration-effect relationship was rather steep.

Results with reference substance (positive control):

Positive control has not been used.

Reported statistics and error estimates:

No statistical analysis was performed.

Sublethal observations / clinical signs:

Mortality and visible abnormalities observed at the test fish

Nominal test item concentration (mg/L)	Number of affected fish*/number of dead fish visible abnormalities
	Observation time
	2 hours	24 hours	48 hours	72 hours	96 hours
Control	0/0	0/0	0/0	0/0	0/0
4.6	0/0	0/0	0/0	0/0	0/0
10	0/0	0/0	0/0	0/0	0/0
22	0/0	0/0	0/0	0/0	0/0
46	0/0	0/0	0/0	5/0 TS, AK	7/1 TS, AK
100	0/0	0/0	7/7	*/*	*/*
LC50	>100	>100	68	68	61
95% C.I.	-	-	46-100	46-100	39-98

*: number of dead fish plus number of fish with visible abnormalities

*/*: all fish dead

95% C.I. : 95% confidence interval of the LC50

AK: strongly extended grilles

TS: tumbling during swimming

Validity criteria fulfilled:

yes

Remarks:

Please refer to "Overall remarks, attachments" here below.

Conclusions:

The acute toxicity of the test item Red Rwa 4565 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 of 61 mg/L

Executive summary:

The acute toxicity of the test item Red Rwa 4565 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).

The nominal test item concentrations tested were 4.6, 10, 22, 46 and 100 mg/L, and in parallel a control.

The analytically determined test item concentrations in the test media varied in the range from 109 to 120% of the nominal values at the start and the end of the test. The test item Red Rwa 4565 was stable under the test conditions during the test period of 96 hours.

Therefore, all reported biological results are related to the nominal concentrations of the test item.

The biological test results after 96 hours test duration:

- 96-hour LC50: 61 mg/L (95% confidence interval: 39 -98 mg/L)

- 96-hour LC0: 22 mg/L

- 96-hour LC100: 100 mg/L

- 96-hour NOEC: 22 mg/L

- 96-hour LOEC: 46 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

61 mg/L