3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

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

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

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 stock solution, from the test media of all test concentrations and from 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).
All samples were taken from the approximate center of the aquaria without mixing of the test media, and were deep-frozen (at about -20 °C) immediately after sampling.
The concentrations of BLUE GS 5664.80 were analyzed in all test medium samples of the two highest test concentrations of nominal 46 and 100 mg/L from the sampling times at 0, 48 and 96 hours. The lowest test concentrations of 4.6 to 22 mg/L were not analyzed, since they were below the 96-hour NOEC, determined in this test.
From the control samples only one of the duplicate samples was analyzed from the start and the end of the test.

Vehicle:

no

Details on test solutions:

The test media were prepared as follows: A concentrated stock solution of nominal 500 mg/L was freshly prepared by dissolving 2999 mg of the test item completely in 6 liters 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 test concentrations were based on the results of a range-finding test (without GLP). However, concentrations in excess of nominal 100 mg/L have not been tested according to the guidelines.

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): 5.7 ± 0.3 cm
- Weight at study initiation (mean and range, SD): 1.8 ± 0.3 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 the last two weeks prior to the test the mortality rate in the test fish batch was lower than 5% 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:

14-16 °C

pH:

Between 7.6 and 8.0

Dissolved oxygen:

Between 8.0 and 9.6 mg/L in test media and control

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:

Nominal: 4.6, 10, 22, 46, 100 mg/L
Concurrent with the sample analysis, recoveries of spiked test water samples in the relevant concentrations (49.8 and 99.6 mg/L of the test item) were performed. The average concentrations were found to be 104 % and 104 % of the spiked values, with an overall mean of 104 % (n = 4). Therefore, no correction for possible losses during the analytical procedure is necessary.

Details on test conditions:

TEST SYSTEM
- Test vessel: One 20 liter glass aquarium with 15 liters test medium was used for each test concentration and the control.
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. 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 media were prepared as follows: A concentrated stock solution of nominal 500 mg/L was freshly prepared by dissolving 2999 mg of the test item completely in 6 liters 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 morality)

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 50 to 500 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:

LC50

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:

LC0

Effect conc.:

46 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.:

46 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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Abnormalities:
The following visible abnormalities were monitored during the study but not found: Strongly extended gills, Apathy, Fish mainly at the water surface, Mucus secretion, Distended abdomen, Fish mainly at the bottom of the aquarium, Exophthalmus, Convulsions, Fish lying on side or back on the bottom, Strong ventilation, Tumbling during swimming and Changed body color.

Note that, due to the intense blue coloration of the test medium, a halogene lamp was used for observation of visible abnormalities.

Results with reference substance (positive control):

Positive control has not been used.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item BLUE GS 5664.80 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 item BLUE GS 5664.80 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.

During the test period the measured test item concentrations in the analyzed test media were in the range from 106 to 110% of the nominal values. Under the test conditions, the concentrations of the test item BLUE GS 5664.80 were constant during the test period of 96 hours.

All reported results are related to the nominal concentrations of the test item.

The biological test results after 96 hours test duration:

- 96-hour LC50: > 100 mg/L (95% confidence interval not calculable)

- 96-hour LCO: 46 mg/L

- 96-hour LC 100: > 100 mg/L

- 96-hour NOEC: 46 mg/L

- 96-hour LOEC: 100 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L