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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19/05/2020 to 22/05/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study undertaken to GLP certification, based on OECD guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The stock solutionswere autoclaved for 20 minutes instead of the required 15 minutes Pre-culture was prepared 5 days before the test start. Experimental temperature was 20.7 – 22.3 °C Pre-culture incubation temperature was 20.5 – 22.4 °C
Principles of method if other than guideline:
Guideline study
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
RM1007030 RL750/18
- Expiration date of the lot/batch:
24/10/2020
- Purity test date:
No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Closed vessel at room temperature (17.3 – 23.0 °C).
- Stability under storage conditions:
No data
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Solubility H2O: < 0.1 g/L; EtOH: > 1 g/L; acetone: > 1 g/L; CH3CN: > 1 g/L; DMSO: unknown; Stability: H2O: unknown; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: unknown
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):
No dat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
No data
- Preparation of a nanomaterial dispersion (incl. dilution):
No data
- Final dilution of a dissolved solid, stock liquid or gel:
No data
- Final preparation of a solid:
No data

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
No data
- other information: No data
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal concentrations were 1, 3.2, 10, 32, 100 mg/l
Geometric mean measured concentrations were 0.85, 2.68, 8.86, 27.93, 87.80 mg/l

- Sampling method: The concentration of the test item was measured every 24 hours during the test. To avoid adsorption onto the algae cells, the concentration of the test item was measured in extra replicates without algae cells. Before each measurement of the samples were diluted using the calibration solvent ace-tonitrile/water 50/50 (% v/v) to stay in the calibrated range.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Following OECD (2019)Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, a stock solution containing 100 mg/L test item in algal medium (demineralised water enriched with miner-als but without algae) was prepared. For this, the loading rate of 106.4 µL/L (based on a density of 0.94 g/mL, stated in the SDS) was mixed with algal medium. This solution was treated with an ultrasonic bath for 30 minutes in order to achieve complete dissolution of the test item. The lower treatments were (32 / 10 / 3.2 / 1 mg/L) prepared by dilution of this stock solution with algal medium.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: SAG Strain Number 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen), January 2016
- Age of inoculum (at test initiation): Five days
- Method of cultivation: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 cul-ture, the pre-culture was prepared.

ACCLIMATION
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 5 days under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 20.5 – 22.4 °C). The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.
Test type:
static
Water media type:
freshwater
Remarks:
OECD TG 201 medium
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
No data (OECD TG 201 medium)
Test temperature:
Temperature ranged from 20.7 to 22.3 °C across the test concentrations, replicates and test duration.
pH:
pH ranged from 7.7 (0 hours) and 7.6 (72 hours) in blank controls, and from 7.6 (0 hours) to 8.5 (72 hours) in test conditions
Dissolved oxygen:
No data (OECD TG 201 medium)
Salinity:
Not applicable
Conductivity:
No data (OECD TG 201 medium)
Nominal and measured concentrations:
Nominal concentrations were 1, 3.2, 10, 32, 100 mg/l
Geometric mean measured concentrations were 0.85, 2.68, 8.86, 27.93, 87.80 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL, filled with 45 ± 1 mL of the respective test solution
- Type (delete if not applicable):open (closed with perforated plastic foil)

- Initial cells density: 3.5 *10^3 cells/mL
- Control end cells density: 9.5 *10^3 cells/mL
- No. of vessels per concentration (replicates): Three, plus one without algae for analytical determination
- No. of vessels per control (replicates): Six, plus one without algae for analytical determination

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: constant illumination
- Light intensity and quality: within the specified range (4440 – 8880 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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. 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).


TEST CONCENTRATIONS
- Range finding study was performed intitially
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (CAS No. 7778-50-9)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
62.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.98 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Details on results:
According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.
Results with reference substance (positive control):
The 72h-EC50 values 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).
Reported statistics and error estimates:
Analysis of the NOEC/LOEC (for yield and growth rate) was carried out using the Williams test. Analysis of the EC10 and EC50 (for yield and growth rate) was carried out using the Levenberg-Marquardt test with non-linear regression. All statistical analysis was carried out using the ToxRat Professional computer software package.
Validity criteria fulfilled:
yes
Remarks:
Deviations from validity stated as uncritical by study director
Conclusions:
The reliable OECD 201 Guideline study on algal growth inhibition reported a 72-hr EC50 of 62.87 mg/l and an EC10 of 7.44 mg/L based on growth of Desmodesmus subspicatus (Measured geometric mean).
Executive summary:

The reliable OECD 201 Guideline algal growth inhibition study was undertaken to GLP standards. The study was performed using five concentrations ranging from 1.0 to 100 mg/L (nominal) using the species Desmodesmus subspicatus for 72 hours duration. 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: 10, 32 and 100 mg/L (nominal).


At the start of the test and every 24 hours, the content of the test item in the test solutions was determined using HPLC. The measured concentrations were between 78 % and 95 % of the nominal concentration. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see § 39 OECD 201).


 


The following results for the test item were determined:



























Endpoint



NOEC



LOEC



EC10



EC50



Growth Rate



2.68 mg/L



8.86 mg/L



7.44 mg/L



62.87 mg/L



Yield



2.68 mg/L



8.86 mg/L



1.98 mg/L



8.13 mg/L



Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10which is read from the graph toxicity vs. concentration may lie lower.


[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 8.2 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.



Description of key information

The 72-h EC50 and EC10 was 62.87 mg/L and 7.44 mg/L respectively, based on growth of Desmodesmus subspicatus (Muckle, 2020)

 

Key value for chemical safety assessment

EC50 for freshwater algae:
62.87 mg/L
EC10 or NOEC for freshwater algae:
7.44 mg/L

Additional information