Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.7.- 26.10.2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: method No. L 49: “Acute Toxicity for Daphnia, immobilization test. Chemical formulations and waste waters”

Deviations:

no

Principles of method if other than guideline:

Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
The sensitivity of the test species is periodically monitored by testing with the reference substance, potassium dichromate.

The sample is dissolved in the dilution water at a concentration of about 0.1 g / L and forms a dark opaque solution. Higher concentrations can not be used for the intense color and the swiming of Daphnia to the surface.

The volume of the test mixture for one dilution is 50 ml.
Preliminary test is carried on with 10 organisms, full test with 10 as well in two parallel rows in which the number of individuals for evaluating is aggregated. Immobilization is monitored at 24 and 48 hours. The test is repeated 2x.
Solutions are not aerated. The temperature of the solution during the test is 21.0 ± 1.0 °C. The oxygen content and pH values are measured in each vessel at the beginning and end of the test.

GLP compliance:

no

Specific details on test material used for the study:

Ostazinová modř H-5R, mixture No. A0 671
No. of sample: 698A/00
Curent name: Reactive Blue 234

Analytical monitoring:

no

Details on test solutions:

The stock solution of the test substance was prepared in the dilution water.
100 mg of the test substance was weighed into 1000 mL of the dilution water for the preliminary test; dissolved sample forms a dark opaque solution. Higher concentrations can not be used for the intense color and the swiming of Daphnia to the surface.
The concentrations of solutions used in test were obtained by dilution of the stock solution with dilution water.

The stock solution for the preliminary test: 0.1 g·L-1
The stock solution for the main test No.1, 2: 0.1038 g·L-1

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus were obtained by aperiodic parthenogenesis from own laboratory breeding. Daphnia were fed by a mixture of algae.
The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
The sensitivity of the test species and correctness of test performance is periodically verified by testing with the reference substance, potassium dichromate.

TEST ORGANISM
- Common name: Daphnia Magna
- Strain: Straus
- Source: own laboratory breeding
- Age at study initiation: young daphnia born in 24 hours
- Method of breeding: aperiodic parthenogenesis
- Feeding during test: no

ACCLIMATION
The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
- Type and amount of food: a mixture of algae
- Health during acclimation: healthy

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

21.0 ± 1.0 °C
Main test No. 1: 20.8 - 21.1 °C
Main test No. 1: 20.0 – 20.9 °C

pH:

Main test No. 1:
pH of the control: 7.84 – 7.90
pH of the tested concentrations: 7.90 – 8.24
Main test No. 2:
pH of the control: 7.86 – 7.91
pH of the tested concentrations: 7.90 – 8.18

Dissolved oxygen:

Main test No. 1:
Oxygen content in the control: 8.7 mg·L-1
Oxygen content in the tested concentrations: 8.5 – 8.7 mg·L-1
Main test No. 2:
Oxygen content in the control: 8.7 – 8.8 mg·L-1
Oxygen content in the tested concentrations: 8.6 – 8.8 mg·L-1

Nominal and measured concentrations:

Preliminary test: 80,70,60,50,40,30,20 mg/L mg/L + control
Full test 1 and 2: 103.8, 101.7, 99.6, 97.6 , 95.5, 93.4, 91.3, 89.3 mg/L + control

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (volume of the test solution is 50ml in each test beaker)
- Type (delete if not applicable): open
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

Preliminary Test: 10 animals
Main test No.1, 2: 10 animals in two parallel rows (20 animals)


Dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D into 1 000 mL of deionized water.
A. 117.6 g CaCl2.2H20 in 1 L of deionized water
B. 49.3 g MgSO4.7H2O in 1 L of deionized water
C. 25.9 g NaHCO3 in 1 L of deionized water
D. 2.3 g KCl in 1 L of deionized water

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

98.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

The test quality criteria

These quality criteria given in the current guideline were fulfilled.

1.      Immobilisation in the controls must not exceed 10 % at the end of the test (there were no immobilisation)

2.      The dissolved oxygen concentrations (throughout the test) should remain above 3 mg·L-1

Validity criteria fulfilled:

yes

Executive summary:

The test substance, Reactive Blue 234, was tested in acute immobilisation test on Daphnia magna. The test was performed as static.

Test result: EC50 (48h) = 98.8 mg·L-1  (measured concentration - arithmetic mean)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

98.8 mg/L

Additional information

An ErC50 value from acute toxicity test on invertebrates is 98.8 mg/l and the substance is not rapidly degradable. These experimental results would led to classification (Aquatic Chronic 3, H412) however the results on other taxonomic species - fish and algae - were above 100 mg/L and the result on daphnids is practically 100 mg/L as well, so it led to the decision not to classified the substance Reactive Blue 234 as toxic for aquatic environment.