Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper (2+) pentasodium 2-[2-{2-amino-5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxyphenyl}diazen-1-yl]-5-({3-carboxy-4-[2-{5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl]phenyl}methyl)benzoate and sodium chloride

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:

February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD 202 EU C2

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

EC Number:

948-009-7

Molecular formula:

Not applicable for a multi-constituent substance

IUPAC Name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

Test material form:

solid: particulate/powder

Details on test material:

Acid brown 161 batch no. ID151860 CAS no. 85338-16-5
Composition of the muti-constituent substance is included in the report as an extract of the analytical report.

Specific details on test material used for the study:

Test Item
Designation in Test Facility: 18011801G
Date of Receipt: 18. Jan. 2018
Condition at Receipt Room temperature, in proper conditions

Specification
The following information concerning identity and composition of the test item was provided by the sponsor.
Name Acid Brown 161
Batch no. ID151860
Appearance dark brown powder
Composition see chapter 17 Annex 4: Copy of Extract of Analytical Report
CAS No. 85338-16-5
EC-No. 286-689-0
Molecular formula not stated
Molecular weight not stated
Purity see chapter
Homogeneity homogeneous
Vapour pressure not stated
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Solubility in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Production date Jan. 2017
Expiry date Oct. 2019
Storage Room Temperature (20 ± 5°C)

Storage
The test item was stored in the test facility in a closed vessel dark and dry at 15.8 – 23.0°C.

Preparation
A solution containing 105.0 mg/L in dilution water was prepared.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Sample preparation
Before measurement the taken samples were diluted with dilution water to stay in the calibrated range. Therefore 2 mL of the sample were filled up to 10 mL with dilution water.

Test solutions

Vehicle:

no

Details on test solutions:

Date of performance 13. – 15. February 2019
Treatments 100 mg/L
The concentrations to be tested are based on the result of a non-GLP pre-test.
Temperature 21.5 – 22.1 °C (see chapter 12 Deviations)
Duration 48 hours
Observation times 24 and 48 hours
Medium renewal none
Test vessels glass beakers, nominal volume 50 mL, tall shape
Replicates (Treatments) 4 vessels, each containing 20 ± 5 mL test solution and 5 daphnia
Replicates (Blank control) 4 vessels, each containing 20 ± 5 mL dilution water and 5 daphnia

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

6.3.1 Specification
Species Daphnia magna
Authority STRAUS
Strain Berlin
Sex female
Age between 0 and 24 hours
Origin Umweltbundesamt Berlin
In-house breeding since 27. September 2007
Selection of the test system was made following the proposal of the guidelines.

Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxici-ty tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), version 12 from 02. Feb. 2015.
Vessels preserving glasses, nominal volume 2 L
Medium M4-Medium (recipe of ELENDT)
Food green algae (Desmodesmus subspicatus)
Medium renewal twice a week
Photo period 16/8 hours, using neon tubes
Temperature 20  2 °C

20 hours before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test.
Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detri-mental effects for test daphnia.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Remarks on exposure duration:

Observation of the organims was done at 24 and 48h

Post exposure observation period:

No

Test conditions

Hardness:

Resulting hardness in mmol/L: 2.502
Resulting hardness in mg CaCO3/L: 250

Test temperature:

21.5 – 22.1 °C

pH:

pH
0 h 48 h
blank control 7.6 7.9
100 7.5 8.0

Dissolved oxygen:

0 h 48 h
blank control 8.0 7.8
100 8.0 7.6

Salinity:

Parameter Concentration in mg/L
CaCl2*2H2O 293.80
MgSO4*7H2O 123.30
NaHCO3 64.80
KCl 5.80

Nominal and measured concentrations:

Nominal
Concentration Test Item Measured concentration
In mg/L % of Nominal
mg/L t = 0 h t = 48 h t = 0 h t = 48 h
Blank control 0.20 < LOD -- --
100 126.17 142.73 126 143

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 134.2 mg/L

Nominal / measured:

nominal

Details on results:

The treatment showed 10% immobilisation. None of the animals was immobilised in the blank control. A statistical evaluation was carried out to check whether there was a significant inhibition. There is no statis-tical difference between the treatment and blank control.

Results with reference substance (positive control):

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study to assure that the test conditions are reliable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed.
The study was performed as a limit test at 100 mg/L (nominal concentration). For the test
concentration and the blank control, 20 Daphnia were exposed to the test item for 48
hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted.
The treatment showed 10% immobilisation. None of the animals was immobilised in the
blank control. A statistical evaluation was carried out to check whether there was a significant
inhibition. There is no statistical difference between the treatment and blank control.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study to assure that the test conditions are reliable.
At the beginning and at the end of the test, the content of the test item in the test solutions
was determined using a photometric method. The concentrations determined at the start of
the test were 126 % of the nominal concentration. At the end of the test the determined
concentrations were 143 % of the nominal concentration. Therefore, the determination of
the biological results was based on the geometric mean of the measured concentrations.
At the end of the test a strong increase of the measured test item concentrations was observed.
Very likely photometric measurement was biased by the presence of the test organism
in the test solutions.
No observations were made which might cause doubts concerning the validity of the study
outcome. All validity criteria were met.
The result of the test is considered valid.