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 aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 22. Dec. 1997 To: 24. July 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study Deficiencies: The tested substance is a precursor of Reactive Black 5 bis-vinyl and hydrolyses in aqueous solution from the bis-ester to the bis-vinyl form. The study was performed by the MOE with test substance of unknown origin. The EC50 in this study was found to be 15.7 mg/L, which is not in accordance with the data from studies with Reactive Black 5 performed with our own test compound. From comparative data for acute toxicity studies with Daphnia magna with Reactive Black 5 from Hoechst and from Aldrich, we know that the EC50 is strongly dependent on the supplier of the material. Whereas with the Hoechst substance the EC50 was >800 mg/L, the EC50 was between 25 and 50 mg/L with the Aldrich substance, and the immobile daphnids were found on the surface of the test vessels. This is most likely due to the surface active properties of the test substance. In addition, in the MOE study, the control group showed a mortality rate of 20 %, which raised doubts on the validity of the study. The NOEC of 1.25 mg/L is due to an effect on reproduction. The study was performed at concentrations of 20.0, 10.0, 5.00, 2.50 and 1.25 mg/L. At 5 and 2.5 mg/L, the reproduction rate was statistically significantly increased, therefore, the NOEC was set to 1.25 mg/L. However, this effect could be caused by the same surface active effect as the higher mortality rate with the Aldrich substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 20.0, 10.0, 5.00, 2.50 and 1.25 mg/L
- Sampling days: 0, 1, 11, 12, 18 and 19
- Sampling: from one replicate per concentration: three times before and three times after water exchange
- Sample storage conditions before analysis: -

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The necessary amount of the test substance was added and stirred in the dilution water, and a stock solution at a concentration of 200 mg/L was prepared. The test solutions were prepared by mixing the necessary amount of the stock solution with the dilution water for respective concentration areas, and then divided into each test vessel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas (Daphnia magna)
- Strain/clone: -
- Justification for species other than prescribed by test guideline: -
- Source: Sheffield University in United Kingdom (Sheffield S10 2UQ, United Kingdom)
- Age of parental stock: 2 - 4 weeks
- Feeding during test
- Food type: Chlorella vulgaris
- Amount: 0.1 - 0.2 mgC (organic carbon content)/day per flea
- Frequency: daily



REARING METHOD of Daphnia magna to obtain the juvenile daphnia for this study
Subcultured fleas at 2 - 4 weeks of age were used as the parents for the test organisms. The juvenile fleas were removed at least the previous day of the study when the fleas were matured to give birth. Batches with high mortality, with resting eggs or in which males were observed were not used.
- Water for rearing: Diluted water
- Rearing density: 10 – 30 fleas/800 mL of rearing water
- Water temperature: 19 to 21 °C
- Lighting: Room light, 16-hour lighting/8-hour darkness
- Feed: Single cell green algae (Chlorella vulgaris); Green algae culture solution was centrifuged, followed by substitution to diluted water to feed the fleas.
- Feeding amount: Chlorella vulgaris at 0.1 - 0.2 mgC (organic carbon content)/day per flea was given. According to the growth stages of fleas, the feed amount was changed within this range.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

June 18, 1998 - July 9, 1998

Post exposure observation period:

-

Hardness:

27.6 - 38.2 mgCaCO3/L

Test temperature:

20.3 - 20.7 °C

pH:

7.4 - 7.8

Dissolved oxygen:

7.6 - 8.7 mg/L

Salinity:

NA

Nominal and measured concentrations:

Nominal: 20.0, 10.0, 5.00, 2.50 and 1.25 mg/L (common ratio: 2.0) and a control area
At the time of preparation of the test solution, the ratio of the measured concentrations of the test substance to nominal values accounted for 85.2 - 95.7 % whereas the counterpart one day later (before water exchange) was 58.3 - 76.0 %.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: 100 mL beaker
- Fill volume: 80.0 mL per vessel
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS (see attachment)
- Source/preparation of dilution water: Dechlorinated tap water (Kurume city water treated with active carbon to remove residual chlorine, etc with sufficient aeration) was used. When the dilution water was employed, it was confirmed that the residual chlorine concentration was not more than 0.02 mg/L by the ortho-tolidine method
- Metals: see attachment "Dilution water parameters"
- Pesticides: see attachment "Dilution water parameters"
- Chlorine: < 0.01
- Alkalinity: 28.5 mg/L
- Conductivity: 142 µs/cm
- Culture medium different from test medium: -
- Water quality measurement: The water temperature, dissolved oxygen concentration, pH and hardness were determined in all the test areas (per test vessel) six times (three times before and after water exchange) during the exposure period.


OTHER TEST CONDITIONS
- Adjustment of pH: -
- Photoperiod: 16-hour lighting/8-hour darkness
- Light intensity: not more than 1,200 lux
- Temperature of the test water: 19 to 21 °C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observation of Daphnia:
- Parent Daphnia: The survival numbers, the immobilization numbers, the size and the conditions were macroscopically observed compared to the control area every day. The dead parent Daphnia was removed after counting.
- Reproduced juvenile: The number of live or dead juvenile as well as the occurrence of dormant eggs were observed every day. After counting, the juvenile was removed. The date of the first reproduction of juvenile was recorded.


ESTABLISHMENT OF THE TEST CONCENTRATION
Based on the 48-hour EiC50 (>1,000 mg/L) obtained by the acute immobilization test and the result of preliminary test conducted prior to this study, the test concentration was determined to be 20.0 mg/L as the maximum concentration, with 5 concentration areas (20.0, 10.0, 5.00, 2.50 and 1.25 mg/L) with the common ratio of 2.0. For the control, a control area with use of only the dilution water was prepared.

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on increased number of juveniles

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

15.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 10.5 - 38.9 mg/L

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on increased number of juveniles

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals (cummulative): no statistically significant difference from control
- control: 20 %
- 1.25 and 2.50 mg/L: no deaths
- 5.00 mg/L: 10%
- 10.0 mg/L: 30%
- 20.0 mg/L: 60%
- Mean cumulative number of juveniles:
- control: 176
- 1.25 mg/L: 204
- 2.50 mg/L: 231 - statistically significant difference
- 5.00 mg/L: 234 - statistically significant difference
- 10.0 mg/L: 180
- 20.0 mg/L: 175
- Size and conditions of parent Daphnia:
- 10.0 and 20.0 mg/L: reproduction inhibition
- 20 mg/L: immobilization, attachment of materials on the fleas
- Date of initial deliver:
- Control, 1.25 - 5.00 mg/L: Day 8
- 10.0 mg/L: Day 8.2
- 20.0 mg/L: Day 8.7 - statistically significant difference
- Other biological observations:
- no dormant egg was noted
- dropped eggs were observed in all test areas
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

Number of dead parent Daphnia and the mortality: Kruskal-Wallis rank sum test and Dunnett multiple comparison test
Date of initial delivery: Kruskal-Wallis rank-sum test and Scheffé’s multiple comparison test: p=0.05
Mean cumulative number of juvenile: One-way layout ANOVA and Scheffé’s multiple comparison test: p = 0.01
Effect concentrations: One-way layout ANOVA and Scheffé’s multiple comparison test): p = 0.01

Validity criteria fulfilled:

yes

Conclusions:

1)   50 % lethal concentration for adult daphnids for 21 days (LC50) = 15.7 mg/L (95 % confidence limit: 10.5 - 38.9 mg/L)
2)   50 % reproduction inhibition concentration for 21 days (ErC50) > 20.0 mg/L
3)   Maximum no observed effect concentration (NOECr) = 1.25 mg/L
4)   Lowest observed effect concentration (LOECr) = 2.50 mg/L

Executive summary:

This test was conducted in accordance with the OECD guideline for testing of chemicals No. 211 “Daphnia magnaReproduction Test” (proposed in April 1997) which is a revised edition of the same guidelines No.202 “Daphnia magna, acute immobilization test and reproduction test” (adopted in April, 1984).

The following results were obtained:

1)   50 % lethal concentration for adult daphnids for 21 days (LC50) = 15.7 mg/L (95 % confidence limit: 10.5 - 38.9 mg/L)

2)   50 % reproduction inhibition concentration for 21 days (ErC50) > 20.0 mg/L

3)   Maximum no observed effect concentration (NOECr) = 1.25 mg/L

4)   Lowest observed effect concentration (LOECr) = 2.50 mg/L

Description of key information

50% lethal concentration for adult daphnids for 21 days (LC50) = 15.7 mg/L. 50 % reproduction inhibition concentration for 21 days (EC50) >20.0 mg/L. Maximum no observed effect concentration (NOEC) = 1.25 mg/L. Lowest observed effect concentration (LOEC) = 2.50 mg/L 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.25 mg/L

Additional information

In a GLP-compliant Daphnia magna reproduction test, the test substance was assessed according to OECD Guideline 211 (publication from The Environmental Agency of Japan). Daphnia magna were exposed for 21 days to 20.0, 10.0, 5.00, 2.50 and 1.25 mg/L concentration of the test material. The results observed after 21 days were as follows:

1)   50 % lethal concentration for adult daphnids for 21 days (LC50) = 15.7 mg/L

2)   50 % reproduction inhibition concentration for 21 days (EC50) >20.0 mg/L

3)   Maximum no observed effect concentration (NOEC) = 1.25 mg/L

4)   Lowest observed effect concentration (LOEC) = 2.50 mg/L