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

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

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 221 (Lemna sp. Growth Inhibition Test)

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 3: 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 17 Annex 3: Copy of Extract of Analytical Report
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 stock solution containing 100.8 mg/L in STEINBERG medium (demineralised water enriched with minerals) was prepared. The lower treatments were prepared by dilution of this stock solution with STEINBERG me-dium. 

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Before measurement the taken samples of treatment 100 mg/L were diluted with dilution water to stay in the calibrated range. Therefore 2 mL of the sample were filled up to 10 mL with STEINBERG test medium.

Test solutions

Vehicle:

no

Details on test solutions:

6.4 Chemicals
Composition of the Solutions:
6.4.1 Stock Solution I
KNO3 17.5 g
KH2PO4 4.5 g
K2HPO4 0.63 g
H2O deionised ad 1000 mL
6.4.2 Stock Solution II
MgSO4 * 7 H2O 5.0 g
H2O deionised ad 1000 mL
6.4.3 Stock Solution III
Ca(NO3)2 * 4 H2O 14.75 g
H2O deionised ad 1000 mL
6.4.4 Stock Solution IV
H3BO3 120.0 mg
ZnSO4 * 7 H2O 180.0 mg
Na2MoO4 * 2 H2O 44.0 mg
MnCl2 * 4 H2O 180.0 mg
H2O deionised ad 1000 mL
6.4.5 Stock Solution V
FeCl3 * 6 H2O 760.0 mg
Na2EDTA * 2 H2O 1500.0 mg
H2O deionised ad 1000 mL
6.4.6 STEINBERG medium
H2O deionised 900 mL
Stock solution I 20 mL
Stock solution II 20 mL
Stock solution III 20 mL
Stock solution IV 1.0 mL
Stock solution V 1.0 mL
H2O deionised ad 1000 mL

Demineralized water was mixed with the necessary amount of stock solutions.
35 µL/L HCl (1M) were added. The pH was measured and adjusted if necessary.
All stock solutions were used non-sterile. After preparation, the medium was sterilized with a 0.2 µm fil-ter.
Deviations from the nominal weighted loads were less than 5%.
The given volumes are exemplary for the composition of the medium. The real volumes depend on the needed final volume and are stated in the raw data.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

Specification
Freshwater plant
Genus Lemna
Species minor
Common Name Duckweed
Origin and Culture
The culture of Lemna minor was obtained from Umweltbundesamt (Federal Environmental Agency) Ber-lin in February 2017. The colonies were kept as a permanent culture under conditions comparable to the test conditions. From an aliquot of the permanent culture, colonies were taken for preparation of the pre culture.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

7 d

Post exposure observation period:

No

Test conditions

Test temperature:

21.5 – 23.1°C

pH:

Nominal Concentration in mg/L
0 d 7 d
Blank control 5.5 6.7
1* 5.6 8.0
3.2 5.6 7.2
10 5.7 7.8
32 5.9 7.7
100 6.2 7.6

Results and discussion

Effect concentrations

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 2.9 mg/L

Nominal / measured:

nominal

Results with reference substance (positive control):

Positive Control
3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5) was tested as positive control in a current reference study. Results can be found in chapter 10.3 Biological Results of the Refer-ence Study (201808R1011).

10.3 Biological Results of the Reference Study (201808R1011)
The results of the last study with the positive control 3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5) are presented in the following table. The study was per-formed under GLP conditions in June 2018.
The values lay within the mentioned range of 1.7 – 5.7 mg/L.
Table 10.3 a Biological Results of C6H4Cl2O based on frond numbers
Parameter Value 95% confidence interval
7d ErC50 (frond number) 3.32 mg/L 2.85 – 3.86 mg/L
7d EyC50 (frond number) 2.43 mg/L 1.59 – 3.68 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed.
The study was performed using 5 concentrations ranging from 1 to 100 mg/L (nominal
concentration). Incubation time (test system Lemna minor) was 7 days. The frond number
of each replicate was determined at the beginning, at day 2 and 5 during the test and at
the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined
at the beginning of the experiment. At the end of the experiment the dry mass of
each replicate was determined. Growth rate μ and the yield were determined from the
frond number and the drymass at the respective observation times.
Due to contamination of the 1 mg/L treatment, this treatment was excluded from evaluation.
Significant inhibition of plant growth was observed at the following concentrations:
10 – 100 mg/L (nominal concentration)
At the start and at the end of the test, the content of the test item in the test solutions was
determined using a photometer.
The measured concentrations lay between 123 % and 133 % of the nominal concentrations
at the beginning of the test and between 62 % and 434 % of the nominal concentrations
at the end of the test. Therefore, the determination of the results was based on the
geometric mean of the measured concentrations (cf. OECD Guideline 221 §47).
434 % of the nominal concentrations was measured in the lowest concentration 1 mg/L.
The test solution was heavily mouldy at the end of the test. Therefore, photometric measurement
was biased. Because no toxicity was observed in the next highest concentration,
treatment 1 mg/L was excluded from evaluation.
The 7d-EC50s of 3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No.
591-35-5) were determined in a separate reference test. The values lay within the desired
range.
The pH of the blank control should not vary by more than 1.5 units. The change was 1.2
units in the blank control.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study
outcome. The result of the test can be considered valid.