Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30/04/2020-16/07/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: wet sticky solid
Specific details on test material used for the study:
Designation in Test Facility: 19101604G
Date of Receipt: 16. Oct. 2019
Condition at Receipt Ambient temperature, in proper conditions

5.1.1 Specification
The following information concerning identity and composition of the test item was provid-ed by the sponsor.
Name zinc chlorohydroxy sulphate and copper sulphide, precipitation
Batch no. 20190601
CAS no. unknown
Composition 2.9 % Cu; 18.0 % Zn; 0.5 % Fe; 0.1 % Pb; 0.7 % Cd; 0.4 % As; 0.3 % Sb; 0.1 % Sn; 0.0 % Bi; 0.0 % Br; 2.7 % Cl; 1.1 % F
Storage room temperature (20 ± 5 °C)
Expiry date 31. Dec. 2025
Stability stable under storage conditions
Appearance blackish-green, wet sticky solid
Purity 100 % (UVCB)
Homogeneity homogeneous
Production date not stated
EC no. unknown
Molecular formula not stated
Molecular weight not stated
Vapour pressure not stated
Solubility in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
A certificate of analysis is provided by the sponsor and attached (in copy) in chapter 16. This analytical report is only for informative purpose. The composition might differ in re-gards of percentage values from batch to batch

5.1.2 Storage
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5 °C).
5.1.3 Preparation
Since the test item is a poorly soluble UVCB the water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10.2 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shak-ing vigorously for 23.5 hours. After a settling time of 1 hour the resulting solution was fil-trated through 0.45 µm nylon filters.
The lower treatments (0.1 / 0.32 / 1 / 3.2 mg/L) were prepared by dilution of this WAF with nutrient medium. This procedure is in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at very low loading rates. In a non GLP pre-test at the loading rate 1 mg/L clear inhibition of algal growth was observed.

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
not specified

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Selection of the test system was made following the proposal of the guidelines.
5.3.1 Specification
Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
5.3.2 Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was pre-pared

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Details on test conditions:
Date: 19. – 22. May 2020
Treatments tested: 0.1 / 0.32 / 1 / 3.2 / 10 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: within the specified range (4440 – 8880 Lux)
Temperature: 20.7 – 22.3 °C
Replicates
Treatments: 3 vessels, filled with 45 ±1 mL test solution and algae
1 vessels, filled with 45 ±1 mL test solution without algae for analytical determination

Blank Control: 6 vessels, each completely filled with algal medium and algae
1 vessel completely filled with algal medium without algae for analytical determination
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
At the start and at the end of the test, the content of Zn as part of the test item in the test solutions was determined using ICP-analysis.
With the help of this determination method it could be proven that the test item was pre-sent in the test solutions. Since the test item is poorly soluble, it is not possible to calculate back from the Zn concentration to the test item concentration. Therefore, the nominal concentrations are used for statistical evaluation.
For statistical evaluation, the lowest concentration is not used because it is not necessary. The next two higher concentrations also show a stimulation of algae growth.

Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in chapter 18.

The following values were determined.
Table 9 a Biological Results of the Test Item
Parameter Value 95 % confidence interval
NOEL (Growth Rate) 72 h 1.0 mg/L --
NOEL (Yield) 72 h 1.0 mg/L --
LOEL (Growth Rate) 72 h 3.2 mg/L --
LOEL (Yield) 72 h 3.2 mg/L --
72h ErL10 3.37 mg/L 3.10 – 3.63 mg/L
72h EyL10 1.94 mg/L 1.81 – 2.05 mg/L
72h ErL50 8.03 mg/L 7.77 – 8.29 mg/L
72h EyL50 3.75 mg/L 3.66 – 3.85 mg/L




Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 0.1 to 10 mg/L (nominal). Incubation time (test systemDesmodesmus subspicatus)was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield[1]were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations:

3.2 – 10 mg/L (nominal) for yield and growth rate

 

At the start and at the end of the test, the content of Zn as part of the test item in the test solutions was determined using ICP-analysis.

With the help of this determination method it could be proven that the test item was present in the test solutions. Since the test item is a poorly soluble UVCB, it is not possible to calculate back from the Zn concentration to the test item concentration. Therefore, the nominal concentrations are used for statistical evaluation.

 

The 72h-EC50values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

 

The following results for the test itemzinc chlorohydroxy sulphate and copper sulphide, precipitationwere determined:

Table 3‑a      Results of the test item

Endpoint

NOEL

LOEL

EL10

EL50

Growth Rate

1 mg/L

3.2 mg/L

3.37 mg/L

8.03 mg/L

Yield

1 mg/L

3.2 mg/L

1.94 mg/L

3.75 mg/L

 


[1]Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. Calculation see under 7.2 Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/L test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used.