Salts of 9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthenium and 4,4'-bis[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)diazo] biphenyl- 2,2'-disulfonic acid and 5-amino-4-hydroxy-6-[(4-nitrophenyl)diazo]-3-(phenyldiazo)-3,4-dihydronaphthalene-2,7-disulfonic acid (5:3:1)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-10-15 to 2009-04-01

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No.23 (Aquatic Toxicity Testing of Difficult Substances and Mixtures, sec. 3.1.2)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1

IUPAC Name:

Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1

Constituent 2

Reference substance name:

Acid Yellow 42: 6375-55-9

IUPAC Name:

Acid Yellow 42: 6375-55-9

Constituent 3

Reference substance name:

Acid Black 2: reaction product of 8005-03-6 and 68510-98-5

IUPAC Name:

Acid Black 2: reaction product of 8005-03-6 and 68510-98-5

Constituent 4

Reference substance name:

Basic Violet 1: 8004-87-3

IUPAC Name:

Basic Violet 1: 8004-87-3

Details on test material:

- Name of test material: Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1
- Lot/batch No.: 2008041541
- Expiration date of the lot/batch: 2010-01-31
- Stability under test conditions: see expiry date
- Storage condition of test material: store cool and dry
- Analytical purity: 100 % (96.8 %; UVCB substance)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.74; 0.39; 0.17; 0.09; 0.04 and 0.02 mg/L
- Sampling method: For the analysis of the test item concentration, samples from the test media of the three highest test concentrations and the
control were taken at the start and at the end of the test. All test medium samples were taken from the approximate centre of the
test vessels after mixing of the test media.
- Sample storage conditions before analysis: The samples were freeze-dried (25-fold concentrated) before analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAF method (OECD Series on Testing and Assessment No. 23: Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2
- Controls: in the control, test water was used without addition of the test item

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: National Institute of Public Health, Hungary
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: The Daphnia were bred under similar temperature and light conditions as in the test, and in reconstituted water of a similar
quality regarding to pH, components of the main ions and total hardness as the test water used in the test.
The test animals were fed daily with a few mL of centrifuged algae suspension (Pseudokirchneriella subcapitata).


ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not): Brood Daphnids were maintained in ISO medium under test conditions prior to the start of the test.
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):


QUARANTINE (wild caught)
- Duration:
- Health/mortality:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

The hardness (CaCO3) of water was: 142 mg/L.

Test temperature:

19.7 – 20.0 °C

pH:

7.28 - 7.87

Dissolved oxygen:

The dissolved oxygen concentration at the end of the test was ≥ 6.3 mg/L in control and test vessels.

Nominal and measured concentrations:

The following nominal concentrations were used in the main study: S.S./10; S.S./20; S.S./40; S.S./80; S.S./160; S.S./320.
The corresponding calculated test item concentrations were: 0.74; 0.39; 0.17; 0.09; 0.04 and 0.02 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 5

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Results with reference substance (positive control):

- Results with reference substance:
24h-EC50 value: 1.59 mg /L , 95 % confidence limits: 1.29 – 1.96 mg /L
In the test with the reference item the results met the expected toxicity of the Potassium dichromate (the 24h EC50 value of the potassium
dichromate should be within the range 0.6 mg/L to 2.1 mg/L). The results are based on the nominal concentrations.

Reported statistics and error estimates:

The EC50 were calculated based on statistical determination (Probit Analysis, statistical software program SPSS+).
The EC100, EC0, NOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this 48-hour static acute toxicity test to Daphnia magna with F 46 Black the 48 h EC50 value was 0.036 mg/L and the 48 h NOEC was determined to be 0.02 mg/L, based on calculated concentrations.

Executive summary:

A 48-hour static acute toxicity test with Daphnia magna according to EU method C.2 and OECD guideline 202 revealed a 48 h EC50 value of 0.036 mg/L and a 48 h NOEC of 0.02 mg/L.