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:
From 2018-03-27 to 2018-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
* 0 mg/l (control)
* 1.00 mg/L of test item
* 3.162 mg/L of test item
* 10.0 mg/L of test item
* 31.62 mg/L of test item
* 100 mg/L of test item

- Sampling method: Sampling done at the start of exposure (0 hours) and at the end of exposure (72 hours) with algae.

- Preparation of the samples:
The control and the 3 lowest test item concentrations ( from 1 mg/L to 10 mg/L) were analyzed undiluted.
The two highest test item concentrations (31.62 mg/L and 100 mg/L were diluted with ultra-pure water prior to analysis

- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water. The solution was agitated until it was visually clear.

The four other concentrations 1.00 - 3.162 - 10.0 - 31.62 mg/l were prepared by appropriate dilution of the stock solution.
- Control: Six replicates (without test item)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokierchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): Three days old
- Method of cultivation:
* Fresh stocks are prepared on Z-Agar.
* Light intensity amounted to 2567 - 5130 lux corresponding to 35 - 70 μE ∙ m-2 ∙ s-1 for 24 hours per day.
* Nutrient medium Z according to LÜTTGE et al. (1994)
* Culture apparatus: Climate room

ACCLIMATION
- Acclimation period: No acclimation period as the preculture was incubated under the same conditions as the test replicates.
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
other: OECD TG 201 medium
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21 - 24°C, controlled at ± 2°C
pH:
8.1 ± 0.2
Nominal and measured concentrations:
Nominal concentration : 1.00 - 3.162 - 10.0 - 3.162 - 100 mg/L of test item

Details on test conditions:
TEST SYSTEM
- Test vessel: sterile glass Erlenmeyer flasks (250mL)
- Type : sealed with cotton wool plugs
- Material, size, headspace, fill volume: 250 mL sterile glass Erlenmeyer flasks
- Renewal rate of test solution (frequency/flow rate): no renewal

- Initial cells density: 5523 cells/mL
- Control end cells density: 2370246 cells/mL
- No. of organisms per vessel: 5523 cells/mL

- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates




TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0 and 72h

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter (Microplate Reader with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study : yes
- Test concentrations: 1.00 - 10.0 - 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.162 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.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 7.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 5.28 - 8.68 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 8.38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
CI: 6.55 - 9.22 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
CI: 10.5 - 14.6 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 3.162 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 3.01 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
CI: 2.16 - 3.99 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 3.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
CI: 3.32 - 4.83 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 5.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
CI: 4.84 - 6.62 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: 0.574 mg/L CI: 0.549 - 0.596 mg/L (Growth rate)
- EC50: 0.337 mg/L CI: 0.313 - 0.345 mg/L (Biomass)
Reported statistics and error estimates:
ErC50 (based on growth rate) is determined via the following the Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))

EyC50 (based on yield) is determined via the following the Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))

NOEC/LOEC based growth rate were determined after Wiliams multiple sequential t-test procedure
NOEC/LOEC based on yield were determined after Wiliams multiple sequential t-test procedure

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, the effect values of Additive 8020 on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on nominal test item concentrations):

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 11.0 (10.5 - 14.6) mg/L and 5.70 (4.84 – 6.62) mg/L, respectively. The NOEC for growth rate and yield was 3.162 mg/L and 1.00 mg/L, respectively.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018-06-11 to 2018-06-14 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

 

The test item is a blue powder and has a water solubility of > 100 mg/L. The study was conducted under static conditions with an initial cell density of 5523 cells/mL. A stock solution and four concentration levels were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.162 - 10.0 - 31.62 - 100 mg/L.

Three replicates were tested for each concentration level and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The concentrations of the test item were analytically verified via photometric analysis at a wavelength of 662 nm in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (72 hours) in all concentration levels and in the control.

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were in the range of 94 to 105% of the nominal test item concentrations. At the end of the exposure period (72 hours), the measured test item concentrations in the old media were in the range of 96 to 107% of the test item concentrations.

Since the measured concentration of the test item did remain stable within ± 20% of the nominal concentration throughout the exposure period, the nominal concentrations were used for evaluation.