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:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Definitions to explain the test parameters chosen:
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.
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.
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Conformity All procedures according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- State of aggregation: contains polymer which is not soluble after dessication
- Particle size distribution: not appropriate (liquid)
- Mass median aerodynamic diameter (MMAD): not appropriate
- Geometric standard deviation (GSD): not appropriate
- Shape of particles: not appropriate
- Surface area of particles: not appropriate
- Crystal structure: not appropriate
- Coating: not appropriate
- Surface properties: surface tension see appropriate section of IUCLID
- Density: see appropriate section of IUCLID
- Moisture content: see above
- Residual solvent: see above
- Activation: not appropriate
- Stabilisation: not required as long as treated properly and instructions of manufacturer are followed
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At the beginning and at the end of the experiment, the DOC in the test solution is analytically determined.
The following instruments and devices will be used in the test. Specifications will be stated in the final report.
• Data logger for temperature
• Analytical scales
• Luxmeter
• Adjustable pipettes with one-way tips
• pH-Meter
• Cell Counter
• Microscope
• Autoclave
• Orbital shaker
• Carbon analyser
Standard laboratory equipment will also be used.
Usage and, if applicable, calibration of all instruments following the corresponding SOP in the current edition.

Test solutions

Vehicle:
no
Details on test solutions:
5 concentrations ranging from 1.0 to 100 mg/L nominal concentration and geometric mean
in the 2 highest concentrations, the measured concentration was in a range of 89 % - 98 %
The tabulated test solutions are reported in the tabulatable text feields.
The weighted mineral concentrations may vary within ± 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):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
Origin and Culture
The culture of Desmodesmus subspicatus was obtained from MBM Sciencebridge 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 prepared.
Pre-culture
3 or 4 days before the start of each experiment, an aliquot of the permanent culture is brought into nutrient medium. The mixture is incubated for 72 - 96 hours at 21.0 –24.0 °C and 4440 - 8880 Lux. The resulting pre-culture is growing exponentially.
Before usage, the culture will be checked for the absence of cell aggregates.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
72 ± 3 hours

Test conditions

Hardness:
Composition of the Solutions (all solutions were used sterilised):
3.4.1 Stock Solution I
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL
3.4.2 Stock Solution II
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL
3.4.3 Stock Solution III
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL
3.4.4 Stock Solution IV
NaHCO3 50 g
H2O deionised ad 1000 mL
3.4.5 Algal medium
stock solution I 10.0 mL
stock solution II 1.0 mL
stock solution III 1.0 mL
stock solution IV 1.0 mL
H2O deionised ad 1000 mL

The weighted mineral concentrations may vary within ± 5 %.
Test temperature:
21.0 – 24.0 °C during 4440 – 8880 Lux
pH:
pH 7.6 at start of incubation, pH 7.5 - 8.4 after 72 h of incubation, pH decreasing with increasing test item content
Dissolved oxygen:
not measured
Salinity:
not measured (see hardness)
Conductivity:
not measured
Nominal and measured concentrations:
5 concentrations ranging from 0.046 to 1 mg/L (nominal).
The concentrations tested were based on non-GLP pre-tests
Treatments: 3 vessels, each filled with 45 ± 1 mL test solution and algae
Blank Control: 6 vessels, each filled with 45 ± 1 mL algal medium and algae
Details on test conditions:
For each treatment, 200 mL of the respective test item solution was mixed with the neces-sary amount of algal pre-culture (0.349 mL) to achieve a cell concentration of 2.5 *103 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item so-lution and mixed with the necessary amount of algal pre-culture (0.611 mL).
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
From these mixtures samples for the analytical determination were taken, afterwards the pH-value was measured.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of the test.
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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
86.94 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
45.51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Details on results:
Abbreviations
NOEC Highest concentration of the test item causing no significant inhibition of the respective parameter
LOEC Lowest concentration of the test item causing significant inhibition of the respective parameter
72h-ErC50 Concentration of the test item inhibiting the growth rate by 50% after an exposition time of 72 h.
72h-EyC50 Concentration of the test item inhibiting the yield by 50% after an exposition time of 72 h.
72h-ErC10 Concentration of the test item inhibiting the growth rate by 10% after an exposition time of 72 h.
72h-EyC10 Concentration of the test item inhibiting the yield by 10% after an exposition time of 72 h.

Any other information on results incl. tables

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables.

Daily Growth Rates of the blank controls

Growth rates

days 0 – 1

days 1 – 2

days 2 – 3

CV of sectional daily growth rates

days 0 – 3

Replicate 1

1.339

1.882

1.836

18%

1.686

Replicate 2

1.360

1.701

1.894

16%

1.652

Replicate 3

1.491

1.487

1.886

14%

1.621

Replicate 4

1.501

1.668

1.809

9%

1.659

Replicate 5

1.532

1.725

1.797

8%

1.685

Replicate 6

1.397

1.897

1.811

16%

1.702

Mean

1.437

1.727

1.839

14%

1.667

Standard deviation

0.082

0.151

0.042

 

0.029

CV

6%

9%

2%

 

2%

CV = Coefficient of Variation

In the following table, the appearance of the algae at the end of the test is stated:

Microscopical Observations

Nominal Concentrationin mg/L

Normal and Healthy Appearance of the Algae

Blank control

Yes

1

Yes

3.2

Yes

10

Yes

32

Yes

100

Yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Significant inhibition of algal growth was observed at the following concentrations: 3.2 – 100 mg/L, 72 h EC50 groth rate > 100 mg/L, yield 86.94 mg/L
Executive summary:

The study was performed according to OECD 201 resp. EU C.3 using 5 concentrations ranging from 1 to 100 mg/L of the test item. Incubation time of Desmodesmus 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 were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed in concentrations >= 3.2 mg/L.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser with the measured concentrtion range at the highest test concentrations within 89 -98 % of nominal.

The 72h-EC50s of potassium dichromate (K2Cr2O7) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Results of the test item based on nominal concentrations

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1.00 mg/L

3.20 mg/L

45.51 mg/L

> 100 mg/L

Yield

1.00 mg/L

3.20 mg/L

3.20 mg/L

86.94 mg/L