Tetrasodium 4-amino-5-hydroxy-3,6-bis[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

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:

1997

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

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
NA

Analytical monitoring:

yes

Details on sampling:

At the start of exposure (0 hour) and at the time of completion of the exposure, the test solution was
collected from the middle layer of the test vessels, followed by analysis with HPLC. Upon analysis of
the test solution, determination of the standard solution (concentration 5.00 mg/L) was conducted per
each sample determination and quantitative analysis was performed based on the peak area ratios.

Vehicle:

no

Details on test solutions:

Preparation of the test solution
The necessary amount of the test substance was added in the previously cooled purified water, the
test stock solution at the concentration of 1,000 mg/L was prepared. During the exposure period,
the test stock solution was stored in a refrigerator. The test solutions were prepared by mixing the
necessary amount of the test stock solution with the dilution water by a constant flow rate (the test
stock solution: 2.5 mL/minute, the dilution water: 22.5 mL/minute). The flow rate of the test stock
solution was controlled by using a metered pump, and the flow rate of the dilution water was adjusted
with use of a flow volume adjusting cock and a capillary tube.

Test organisms (species):

Oryzias latipes

Details on test organisms:

General name: Himedaka
Scientific name: Oryzias latipes
Length: 1.8 cm (1.7 - 2.0 cm), n=10
Body weight: 0.084 g (0.065 - 0.11 g), n=10
Lot No.: LNR-980129
Supplier: Nakajima fish farm (Daimyojin, Nagasu-machi, Tamana-gun, Kumamoto 869-0123, Japan)
Date of purchase: December 24, 1997
Acclimatization
Under the same conditions as done for the test (Quality of the water, temperature, etc.), acclimat
ization was performed for more than 12 days. At the beginning of acclimatization, medicated bathing
with use of Erverge (Ueno Fine Chemicals Industries) was performed for 24 hours under the static wat
er conditions. As the feed, the commercially-available Tetramin (Tetrawerke) was given. Twenty-four
hours before the start of exposure, no feeding was made. For the test, healthy and macroscopically
normal fish were employed. The mortality for 7 days before commencement of exposure was not
more than 5%. The 96-hr LC50 of the standard substance [Copper sulfate (II) pentahydrate, Special
grade, Wako Pure Chemicals Industries Ltd.] was 0.657 mg/L.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

NA

Hardness:

39.5 mg/L (converted to CaCO3)

Test temperature:

39.5 mg/L (converted to CaCO3)

pH:

7.1 - 7.6

Dissolved oxygen:

8.0 - 8.3 mg/L

Salinity:

NA

Nominal and measured concentrations:

100 mg/L and a control area

Details on test conditions:

- Test conditions
Exposure method: The medicated bathing method in which the test organisms were exposed to the
test solution containing the test
substance was used. The test was conducted according to the flow through method.
Exposure period: 96 hours
Repeated number: 1 replicate per test area
Number of the living body: 10 organisms/test area
Test solution volume: 1.8 L per test vessel (a replicate)
Water flow volume: 25.0 mL/minute
Water exchange ratio: About 20 times/day
Temperature of the test water: 23-25°C
Lighting: Room light, 16 hours lighting/8 hours darkness
Feeding: no feeding
Aeration: None
Dilution water: Dechlorinated tap water (The Kurume city water was treated with active carbon to re
move residual chlorine, etc. with sufficient aeration ) was used. The major water quality of the dilution
water comprised 39.5 mg/L (converted to CaCO3) as the hardness and pH 7.6. It was confirmed that
the residual chlorine concentration was not more than 0.02 mg/L by the ortho-tolidine method.
- Test apparatus, test vessel and thermostat bath, etc.
Test vessel: A 3.0-L glass-made vessel (16 cm in inner diameter, 17 cm in depth) was used. On the
lateral side of the vessel, a small pore for
water discharge was opened to make the internal volume of about 1.8 L.
Thermostat bath: A thermostat bath enabling to maintain the water temperature at 23-25°C in the test
vessel by means of warming-cooling machine.
Diluting apparatus: Such an apparatus was used to continuously supply the diluted test solution at the
required graded concentrations from the test stock solution.
Metering pump: A glass micro-metering pump Model GMW-A (Tokyo Rikakikai CO., Ltd.
Refrigerator: This was used to prevent decrease of the test substance concentration of the test stock
solution.
Thermometer for the water temperature: An officially certified glass-rod type thermometer
pH meter: A glass-electrode type hydrogen-ion concentration measuring apparatus Model HM-11P
(Toa Denpa Kogyou )
Dissolved oxygen determination apparatus: Dissolved oxygen determination apparatus Model 58 (Y
ellow Springs Instrument Co., Inc.)

Reference substance (positive control):

not required

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:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no effects
- Observations on body length and weight: no effects
- Other biological observations: no effects
- Mortality of control: no effects
- Other adverse effects control: no effects
- Abnormal responses: no effects
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal
values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: due to the staining nature of the TS, the water was blue discolored

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

NA

Validity criteria fulfilled:

yes

Conclusions:

The median lethal concentration (LC50) for 96 hours >100 mg/L, the maximum lethal concentration
(LC0) is >= 100 mg/L. Hence, the test item is not toxic to fish.

Executive summary:

Study method
This test was conducted in accordance with the OECD guideline for testing of chemicals No. 203 “Acute
toxicity test in fish” (1992).
1) Test substance:
2) Test organism: Oryzias latipes
3) Number of the organism: 10 fish/test area
4) Exposure period: 96 hours
5) Exposure method: Flow-through system (The system to supply the test solution continuously.)
6) Test concentrations: 100 mg/L and a control area

7) Repeated number: 1 replicate per test area
8) Test solution volume: 1.8 L per test vessel (a replicate)
9) Temperature of the test water: 24±1°C
10)Lighting: Room light, 16 hours lighting/8 hours darkness
11)Aeration: None
12)Analysis of the test substance in the test solution: High performance liquid chromatography (HPLC)
Results
1) The median lethal concentration (LC50) for 96 hours >100 mg/L
2) 0% lethal maximum concentration >= 100 mg/L
3) 100% lethal lowest concentration > 100 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L