{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate

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:

2017

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 407940
- Expiration date of the lot/batch: 2024

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light in the tightly closed original container.
- Stability under test conditions: stable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal concentration of 1.25 mg/L of test item and at 0 mg/L for the controls
- Sampling performed from freshly prepared media after 0 and 72 hours and from the corresponding aged test media after 24 and 96 hours in the testede concentration level and the control.
- Sampling method: Sample analyzed without dilution.
- Sample storage conditions before analysis: stored at 6°C +/- 2°C until the start of the analysis if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct addition, the appropriate quantity of test item is weighted and transferred into a test vessel filled with an appropriate volume of dilution water. The test media is mixed with an ultra turrax for 1 min at 17 000 rpm.
- Controls: dilution water without test item
- Evidence of undissolved material: no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin
- Length at study initiation : mean of 2.84 cm
- Weight at study initiation : mean of 0.224 g
- Method of breeding: from a single brood stock from the supplier

ACCLIMATION
- Acclimation period: 12 days at least
- Acclimation conditions : same as the test, except for the photoperiod which is a natural photoperiod during the acclimatation and of 16 hours during the test.
- Type and amount of food during acclimation: Food (Sera Vipan, SERA GmBH, 52518 Heinsberg, Germany). Amount: 4% of the fish body weight per feeding day.
- Feeding frequency during acclimation: 3 times per week. The feeding stopped 24 h before the start of the test.
- Health during acclimation (any mortality observed): no mortality, no disease observed.

FEEDING DURING TEST
- Frequency: no feeding during the test.

Test type:

semi-static

Water media type:

other: Tap water of local origin filtered on activated charcoal and aerated for at least 24 h to remove chlorine

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

total hardness: 63 mg/L as CaCO3/L

Test temperature:

Environmental temperature: 23 °C constant in a range of +/- 1°C during the test
Water temperature: mean of 22.4°C (minimum of 21.8°C - max of 22.9°C).

pH:

pH-value during the test: 7.44 - 7.75

Dissolved oxygen:

Dissolved oxygen concentration during the test: 91% - 100 %

Salinity:

not measured

Conductivity:

Conductivity: 181 µS/cm (recent measurement 2017-01-10)

Nominal and measured concentrations:

Nominal concentrations: 1.25 mg/L
Measured concentrations:
- 0 hour (fresh medium) : mean concentration of 0.852 mg/L
- 24 hours (old medium): mean concentration of 1.05 mg/L
- 72 hours (fresh medium): mean concentration of 1.10 mg/L
- 96 hours (old medium): mean concentration 1.03 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Type: loosely covered with a glass lid
- Material, size, headspace, fill volume: 13 L (25.5 x 18 x 28 cm), filled with ~ 10 L of dilution (depth of water: 20 cm)
- Aeration: none
- Renewal rate of test solution: daily renewal of the test media
- No. of organisms per vessel: 7 fishes
- No. of vessels per concentration : 1 replicate
- No. of vessels per control : 1 replicate


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters:
* total hardness: 10-250 mg CaCO3/L (measured: 63 mg/L as CaCO3)
* pH-value: 6.0-8.3 (measured: 7.4 - 7.75).
* Acidity = 0.2 mmol/L (recent measurement 2017-01-10)
* Acid capacity = 1.0 mmol/L (recent measurement 2017-01-10)
* Conductivity = 181 µS/cm (recent measurement 2017-01-10)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod
- Light intensity: 7-750 lux (light intensity measur at the start of the exposure between 94 to 102 (mean value 98)).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Biological parameters observed: death of the fishes (no visible movement and if touching of the caudal peduncle produced no reaction) and abnormalities (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation, etc.). Observation at 2, 24, 48, 72 and 96 hours post-treatment
- Physico-chemical parameters measured: pH-value, test temperature and oxygen saturation mesured in all test vessels every 24 hours and after renewal of the test media from fresh and old test media. Water temperature recorded once per hour with a data logger. Light intensity at the surface of the test aquaria measured at the start of the exposure. Total hardness measured at the start of the exposure.

TEST CONCENTRATIONS
- Test concentrations: limit test performed at 1.25 mg/L.
- Justification for using less concentrations than requested by guideline: taking into account the results of the aquatic toxicity data of test item in daphnia and alga the test is performed without a range-finding test and the test concentration is set at 1.25 mg/L.
- Results used to determine the conditions for the definitive study: alga ErC50 = 1.25 mg/L.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: none observed
- Observations on body length and weight: none observed
- Mortality of control: no mortality
- Other adverse effects control: none observed

Results with reference substance (positive control):

reference substance not required

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the geometric mean measured concentration of the test item. The threshold concentration of the test item was found to have no effect on the vitality of zebrafish.
In conclusion, the LC0 corresponds to the geometric mean measured concentration of 1.00 mg test item/L.

Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008) from 2017-03-27 to 2017-03-31 at the test facility.

 

An acute toxicity test with the threshold concentration of 1.25 mg/L was conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold concentration and the control.

Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

 

The determination of the test concentrations was carried out via HPLC on a reversed phase column in gradient mode with a Diode Array Detector (DAD). Evaluation was done from freshly prepared media after 0 and 72 hours and from the corresponding aged test media after 24 and 96 hours. The analytical method validation was done according SANCO/3029/99 rev. 4 (2000).

The measured concentrations in freshly prepared media were 68 and 88 % of the nominal value. In corresponding 24 hours aged test media, the measured concentrations were 82 and 84% of the nominal value.Therefore, all effect values are based on the geometric mean measured concentration of the test item.

 

All validity criteria of the test guideline were met.

In conclusion, the LC0 corresponds to the geometric mean measured concentration of 1.00 mg test item/L.

Description of key information

LC₀(96h) for freshwater fish (Danio rerio) > 1 mg/L; limit test (OECD TG 203; semi static).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

75 mg/L

Additional information

Limit test performed at threshold concentration of 1.25 mg/L (lowest concentration in two other taxonomic groups (invertebrates and algae). The LC0 (96 hours) is > 1 mg/L (geometric mean of the measured concentration).