Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-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:

The study was conducted between 26 January 2015 and 06 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Water samples were taken from the control and 100 mg/L test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.

Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (2000 mg) was dissolved in test water and the volume adjusted to 1 liter to give a 2000 mg/L stock solution. The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 02 December 2014. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 19 January 2015 to 26 January 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.8 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm (sd = 0.29) and a mean weight of 1.60 g (sd = 0.39) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.56 g bodyweight/liter (static volume).

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

approximately 14 °C

pH:

7.2 - 8.0

Dissolved oxygen:

greater than or equal to 9.8 mg O2/L

Salinity:

No data

Nominal and measured concentrations:

Nominal concentration of 100 mg/L

Details on test conditions:

Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

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)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Mortality Data
There were no mortalities in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours.

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 100 mg/L, hence the No Observed Effect Concentration (NOEC) was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.


Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours, however a slight blue staining was observed on the fish exposed to the 100 mg/L test concentration.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

An estimate of the LC50 values was given by inspection of the mortality data.

Sublethal observations / clinical signs:

Verification of Test Concentrations

Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the LC₅₀ values in terms of the nominal test concentrations only.

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O₂/L) in the control and test vessels.

Mortality Data

Inspection of the mortality data gave the following results:

 

Time (h)	LC50(mg/L)
3	>100
6	>100
24	>100
48	>100
72	>100
96	>100

 

Water Quality Criteria

Temperature was maintained at approximately 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

 

Observations on Test Item Solubility

The test solutions were observed to be an extremely dark blue solution throughout the test.

Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0

  Water Quality Measurements

Nominal Concentration (mg/L)	Time (Hours)
	0 Hours (Fresh Media)			24 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.4	11.3	13	7.5	9.9	14
100	7.5	11.2	14	8.0	10.0	14

 

Nominal Concentration (mg/L)	Time (Hours)
	24 Hours (Fresh Media)			48 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.8	11.3	14	7.9	9.6	14
100	7.7	11.3	14	8.0	9.5	14

 

Nominal Concentration (mg/L)	Time (Hours)
	48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.4	10.4	13	7.8	9.5	14
100	7.7	10.8	14	7.9	9.5	14

 

Nominal Concentration (mg/L)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.2	10.5	14	7.6	9.6	14
100	7.4	10.9	13	7.8	9.5	14

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) was >100 mg/L.

Executive summary:

A study according to OECD guideline 203 and EU Method C.1, was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The test was conducted at a single concentration of 100 mg/L to ensure that toxicity was not observed at this concentration. Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours. Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only. No mortality was observed throughout the duration of exposure. Hence, 96 -hour LC50 of the test item to the freshwater rainbow trout (Oncorhynchus mykiss) was determined to be >100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Description of key information

The 96-Hour LC50 of the test item to freshwater rainbow trout was >100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

A study according to OECD guideline 203 and EU Method C.1, was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The test was conducted at a single concentration of 100 mg/L to ensure that toxicity was not observed at this concentration. Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours. Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only. No mortality was observed throughout the duration of exposure. Hence, 96 -hour LC50 of the test item to the freshwater rainbow trout (Oncorhynchus mykiss) was determined to be >100 mg/L. The No Observed Effect Concentration was 100 mg/L.