Reaction products of 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, coupled twice with diazotized 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate, sodium salts

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

adult fish: sub(lethal) effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: December 22, 1997 To: May 25, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

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:

- Concentrations: 100, 50.0, 25.0, 12.5 mg/L
- Sampling method: collected from the middle layer of the test vessels and used as it was or properly diluted
- Sample storage conditions before analysis: -
- Sampling days: Day 0, Day 7 and Day 14

Vehicle:

no

Details on test solutions:

The test substance 100 mg was accurately weighed with an electric analytical balance, and dissolved in distilled water to prepare the test substance solution at the concentration of 1,000 mg/L. The resultant solution was diluted with the dilution water to prepare the test substance solutions at the concentrations of 100 and 5.00 ml/L.

The necessary amount of the test substance was added in the previously cooled purified water, the test substance stock solution at the concentration of 10,000 mg/L was prepared. This test substance stock solution was properly diluted with the purified water to prepare the test stock solutions per each concentration area (100 mg/L area, 1,000 mg/L; 50.0 mg/L area, 500 mg/L; 25.0 mg/L area, 250 mg/L; 12.5 mg/L area, 125 mg/L). During the exposure period, the test stock solution was stored in a refrigerator. The test solutions were prepared by mixing 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:

TEST ORGANISM
- Common name: Himedaka
- Strain: -
- Source: Nakajima fish farm (Daimyojin, Nagasu-machi, Tamana-gun, Kumamoto 869-0123, Japan)
- Date of purchase: December 24, 1997
- Length at study initiation (length definition, mean, range and SD): 1.9 cm (1.8 - 2.1 cm), n = 10
- Weight at study initiation (mean and range, SD): 0.11 g (0.080 - 0.14 g), n = 10
- Feeding during test - For 24 hours before the start of exposure, no feeding was made
- Food type: Tetramin (Tetrawerke)
- Amount: 2 % of the fish weight
- Frequency: daily


ACCLIMATION
At the beginning of acclimatization, medicated bathing with use of Erverge (Ueno Fine Chemicals Industries) was performed for 24 hours under the static water conditions.
- Acclimation period: more than 12 days
- Acclimation conditions (same as test or not): yes
- Type and amount of food: Tetramin (Tetrawerke)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): For the test, healthy and macroscopically normal fish were employed. The mortality for 7 days before commencement of exposure was not more than 5 %.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Hardness:

39.5 mg/L

Test temperature:

23.8 - 24.2 °C

pH:

pH 7.2 - 7.9

Dissolved oxygen:

7.9 - 8.2 mg/L

Salinity:

NA

Nominal and measured concentrations:

Nominal 100, 50.0, 25.0 and 12.5 mg/L
The determined concentrations of the test substance against the nominal values accounted for 84.7 % - 109 % at the start of exposure, 87.8 - 91.4 % at Day 7 and 89.3 - 92.3 % at the time of completion of the exposure.

Details on test conditions:

Test conditions:
- Number of the organism: 10 fish/1 test area
- Exposure period: 14 days
- Exposure method: Flow-through system (The system to supply continuously the test solution)
- Test concentrations: 100, 50.0, 25.0, 12.5 mg/L (Common ratio: 2.0) and a control area
- Repeated number: A replicate per test area
- Test solution volume: 1.8 L per test vessel (a replicate)
- Temperature of the test water: 24  1C
- Lighting: Room light, 16 hours lighting / 8 hours darkness
- Aeration: None

Test apparatus, test vessel and thermostat bath, etc.
1) 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.
2) Thermostat bath: A thermostat bath enabling to maintain the water temperature at 23 °C - 25 °C in the test container by means of warming-cooling machine.
3) Diluting apparatus: Such an apparatus was used to continuously supply the diluted test solution at the required graded concentrations from the test stock solution.
4) Metering pump: A glass micro-metering pump Model GMW-A (Tokyo Rikakikai Co., Ltd.)
5) Refrigerator: This was used to prevent decrease of the test substance concentration of the test stock solution.
6) Thermometer for the water temperature: An officially certified glass-rod type thermometer
7) pH meter: A glass-electrode type hydrogen-ion concentration measuring apparatus Model HM-11P ( Toa Denpa Kogyou)
8) Dissolved oxygen determination apparatus: Dissolved oxygen determination apparatus Model 58 (Yellow Springs Instrument Co., Inc.)

Reference substance (positive control):

not specified

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

adult behaviour

Remarks on result:

other: no effects observed

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

adult behaviour

Remarks on result:

other: no effects observed

Duration:

14 d

Dose descriptor:

LC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

adult mortality

Remarks on result:

other: no deaths occurred

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

adult mortality

Details on results:

- Toxicological symptoms and feed-intake: no effects
Since the test solution was opaque which was responsible for difficult observation, the test fishes were transferred to the test vessels containing only the test water at the end of exposure, followed by confirmation of toxicological symptoms or abnormality. However, the results showed no symptoms.
- Weights of surviving test organisms at the end of exposure: no significant difference compared control group (p = 0.05, Statistical method: One-way analysis of variance)
- Maximum no observed effect concentration: both of the minimum lethal concentration and the minimum effective concentration were >100 mg/L. Even at the highest concentration (100 mg/L) in this test, there was no significant difference in terms of the surviving individual organisms relative to those in the control area. Based on these results, the maximum no observed effect concentration (NOEC) in this test was judged to be not less than 100 mg/L.

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

Weights of surviving test organisms at the end of exposure: p = 0.05, Statistical method: One-way analysis of variance

Validity criteria fulfilled:

yes

Conclusions:

Neither effects on behavior nor deaths were observed at test substance concentrations up to 100 mg/L. Hence, Reactive Black 5 is not toxic to fish.

Executive summary:

This test was conducted in accordance with the OECD guideline for testing of chemicals No. 204 “Fourteen-day subacute toxicity test in fish” (1984).

1)   Test substance

2)   Test organism: Oryzias latipes

3)   Number of the organism: 10 fish/ test area

4)   Exposure period: 14 days

5)   Exposure method: Flow-through system (The system to supply continuously the test solution)

6)   Test concentrations: 100, 50.0, 25.0, 12.5 mg/L (Common ratio: 2.0) and a control area

7)   Repeated number: A 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)  (Day 0, Day 7 and Day 14)

Results

1)   Minimum lethal concentration for 14 days (LC0) >100 mg/L

2)   Minimum effective concentration for 14 days (EC0) >100 mg/L

3)   Maximum no observed effect concentration (NOEC) for 14 days≧100 mg/L

4)   Median lethal concentration for 7 days (LC50) >100 mg/L

5)   Median lethal concentration for 14 days (LC50) >100 mg/L

Description of key information

Chronic LC50 and NOEC to freshwater fish was determined to be >100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

In a GLP-compliant fish prolonged toxicity test, the test substance was assessed according to OECD Guideline 204 (publication from Ministry of the Environment Government of Japan). Freshwater fish, Oryzias latipes, were exposed for 14 days to  100, 50.0, 25.0, 12.5 mg/L concentration of test material. Median lethal concentration for 14 days (LC50) and NOEC was determined to be >100 mg/L.

 

Reactive Black 5 rapidly degrades to its oxy-form in water. It is therefore reasonable to test this main metabolite in aquatic toxicity tests. Data from a read across substance generated by following an OECD 204 guideline in compliance to GLP is taken into consideration here to analyze the toxicity of the test substance. In the fish prolonged toxicity test, freshwater fish, Brachydanio rerio were exposed for 28 days to 0, 10, 33, 100 mg/L concentration of test material. Signs of intoxication were observed at 33 and 100 mg/L. The limit concentration for mortality was 100 mg/L, the limit concentration for adverse effects was 33 mg/L. At 10 mg/L, there were no adverse effects observed.

Considering the results from the above studies, chronic LC50 and NOEC to freshwater fish was determined to be >100 mg/L.