Reaction mass of lithium sodium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxynaphthalene-2,7-disulfonate 2,2'-(methylimino)diethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol

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:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

(2008)

Deviations:

yes

Remarks:

Deviating to SOP 00139 V.1, but according to the demands of the guideline, the test was performed with 7 fish per concentration and control.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(1992)

Deviations:

yes

Remarks:

Deviating to SOP 00139 V.1, but according to the demands of the OEDC guideline 203, the test was performed with 7 fish per concentration and control.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentration: 100 mg/L plus control
- Storage: Only in exceptional cases, they were stored overnight deep frozen and protected from light.

Vehicle:

no

Details on test solutions:

A direct weighing was prepared to produce the only test concentration of 100 mg/L based on the active ingredient (35.25 %). To achieve this 1418.3 mg of the test item were added to 5 litre of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and afterwards stirred for 24 h on a magnetic stirrer.
The pH was measured to be pH 8.2.
Finally 7 fish were given to the only test item concentration and the control.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

- Name : Zebra fish (Danio rerio)
- Source : Aqua KlöGer (Germany)
- Date supplied : 2012-03-22
- Acclimatisation : Stock held since 2012-03-22 and acclimatised to the test conditions since then.
- Temperature : 20 - 24 °C
- Dissolved oxygen : > 5 mg/L
- Feeding : Commercial fish food, daily. Feeding discontinued 24 h prior to test start.
- Mortalities during acclimatisation period : < 5 %
- Medication : none
- Mean standard length (n = 7) : 3.59 cm (S.D. = 0.17 cm)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

14.2 °dH (= 253 mg/L CaCO3)

Test temperature:

21.6 - 22.5 °C measured at each test vessel at the beginning and the end of the test

pH:

7.8 - 8.2 measured at each test vessel at the beginning and the end of the test

Dissolved oxygen:

8.3 - 8.8 mg O2/L with 96 - 100 % saturation measured at each test vessel at the beginning and the end of the test

Nominal and measured concentrations:

100 mg/L (nominal) puls control
Effective concentrations correspond to 97.4 % of nominal values at 0 hours and to 98.4 % of nominal values at 96 hours.

Details on test conditions:

An acute daphnia toxicity rang finding test and an algal growth inhibition rang finding test preceded the fish test. They provided information about the concentration which was used in the fish test.
The following nominal concentrations of the active ingredient of the test item were tested in the range finding test. Acute daphnia toxicity test: 0.1, 1.0, 10 and 100 mg/L, Algal growth inhibition test: 0.001, 0.01, 0.1, 1.0, 10, 100 mg/L.
Pre-treatment of test item and preparation of test item concentrations:
A direct weighing was prepared to produce the only test concentration of 100 mg/L based on the active ingredient (35.25 %). To achieve this 1418.3 mg of the test item were added to 5 litre of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and afterwards stirred for 24 h on a magnetic stirrer.
The pH was measured to be pH 8.2.
Finally 7 fish were given to the only test item concentration and the control.
- Test vessels : glass aquaria holding 5 L of test media covered by glass plates
- Experimental design : 1 test concentration plus 1 control, 7 fish per test concentration, no feeding during the exposure period, static system
- Method of initiation : fish were placed in prepared media
- Loading : 0.71 g body weight (wet weight) per litre
- Photoperiod : 16 h light: 8 h dark
- Temperature : 21.6 to 22.5 °C
- Aeration : gentle aeration via narrow glass tubes
- Test item concentration : 100 mg/L (active ingredient)
- Method of administration : direct weighing
- Medium renewal : none
- Duration of exposure: 96 hours
- Criteria of effects : The criterion of death used in this study was the absence of response to physical stimulation. In addition to observations on mortality at 2, 24, 48, 72 and 96 hours, type and incidence of sub lethal effects compared with control fish were observed.

Reference substance (positive control):

no

Duration:

2 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

2 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

2 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 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:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

No toxic effects against fish were observed at a limit test concentration of 100 mg/L.

The results are expressed in terms of nominal concentrations. Effective concentrations correspond to 97.4 % of nominal values at 0 hours and to 98.4 % of nominal values at 96 hours.

Validity criteria fulfilled:

yes

Remarks:

(-The mortality in the controls did not exceed 10 % by the end of the test. -The dissolved oxygen concentration remained above 60 % of the air-saturation value throughout the exposure period. -The pH did not vary by more than 1 unit.)

Conclusions:

The acute toxicity to fish (danio rerio) was tested in a static test. After 96 hours of exposure no effect against fish were observed at a limit test concentration of 100 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Danio rerio under static conditions. The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.1 'Acute toxicity for Fish' (2008) which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 203 'Fish, Acute Toxicity Test' (1992).

An acute daphnia toxicity rang finding test and an algal growth inhibition rang finding test preceded the fish test. They provided information about the concentration which was used in the fish test.

Because of the anticipated low toxicity of the test item, fish were exposed in the main test to a limit test concentration of nominally 100 mg/L of active ingredient of the substance for a period of 96 hours. As no toxic effects against fish were observed, no statistical analysis was required to determine the LC 50. Additionally any abnormal behaviour or appearance of the fish was reported every 24 hours.

After 96 hours of exposure a LC50 of >100 mg/L (nominal) was determined.

This toxicity study is classified accptable and satisfies the guideline requirements for the acute fish.

Description of key information

The acute toxicity to fish (danio rerio) was tested in a static test. After 96 hours of exposure no effect against fish were observed at a limit test concentration of 100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L

Additional information

In accordance with REACH Regulation, Art. 3, and ECHA "Guidance for identification and naming of substances under REACH and CLP" v1.2, March 2012, substances have to be separated from solvents if this is not affecting the stability of the substance or changing its composition.

In this case, separation of water would only be possible by thermical damaging of the substance. Furthermore, it was not possible to spray dry the pigment solution as it stuck to and partly melted on the conus dryer.