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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:
March, 2018 - April, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20170710
- Expiration date of the lot/batch: 24/08/2018
- Purity test date: Not provided

RADIOLABELLING INFORMATION (if applicable) not applicable
- Radiochemical purity: -
- Specific activity: -
- Locations of the label: -
- Expiration date of radiochemical substance: -

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under test conditions: one year; The test item should be considered stable, because the organic carbon values at the end of the test
remain in the required range (80-120%) respect to the initial nominal values.
- Solubility and stability of the test substance in the solvent/vehicle: Stability of the test item in the definitive test has been performed using TOC analyser
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not provided

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material) not applicable

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) not applicable

OTHER SPECIFICS: no data
Analytical monitoring:
not required
Details on sampling:
- Concentrations:
- Sampling method:
- Sample storage conditions before analysis:
Vehicle:
yes
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): laboratory
- Age of inoculum (at test initiation): not specified
- Method of cultivation: culture medium in sterile flasks

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): The algal suspension in breeding in Eurofins Biolab has been kept in culture medium in sterile 250 ml Erlenmeyer flasks in the following conditions until the beginning of the test:
Temperature: 23°C ± 2°C
Lighting: lighting greater than 4300 lux
Stirring: Steady with electrical stirrer
- Any deformed or abnormal cells observed: not specified
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
CELL OBSERVATION TIMES (hours): 0-24-48-72
Post exposure observation period:
At the end of the test the quality of algae appearance has been observed at microscopy (1 h)
Hardness:
not specified
Test temperature:
23 ± 2°C (Temperature of the ambient has been recorded every day)
pH:
At the beginning and at the end of the test (72 hours) pH has been measured in all the vessels: 7,37-7,85
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
Range finding test
The test item has been prepared at 1 g/l in culture medium. 5 different concentrations have been prepared and tested with ten-fold dilution. Tested concentrations: from 1000 mg/l to 0.01 mg/l.
Definitive test
After the range finding test the test item has been prepared at 1 g/l in culture medium. The test has
been performed on the diluted nominal solutions. The total volume of the culture medium and the concentration of the algae inoculum added were the same in all vessels.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density:
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality:
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.334 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.53 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.44 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test): A normal and healthy appearance of the algae at the end of the test was observed at microscopic for control and treated group.
- Colour differences: In range finding test at 1000 mg/l and 100 mg/l tested solutions had a brown coloration but at the Burker chamber algae have been observed and counted as for the other tested solutions.
- Unusual cell shape: no data
- Flocculation: no data
- Adherence to test vessels: no data
- Aggregation of algal cells: no data
- Other: no data
- Any stimulation of growth found in any treatment: no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
EC50 was evaluated using EPA Probit Analysis Program Version 1.5.
NOEC LOEC values have been calculated by PCS Pharma Program in function of the tested concentrations in this study.
Validity criteria fulfilled:
yes
Conclusions:
The obtained results, in compliance with test acceptability criteria, can be summarised as follow:
EC50 (72 h): 4.334 mg/l
NOEC: 1.53 mg/l
LOEC: 2.44 mg/l.
Executive summary:

On the extract of the test item pyrogallol the growth inhibition test on a culture of algae Pseudokirchneriella subcapitata was performed on a culture in exponential growth phase.

This approach, according to OECD 201, allowed to calculate the growth inhibition (effect) concentration

to define a limit not toxic threshold.

A Lowest Observed Effect Concentration (LOEC) and a No Observed Effect Concentration (NOEC) have been calculated using a statistical software.

During the range finding test the algae have been exposed to 5 different dilutions of the test item for 72 hours. After the results of the range finding test an EC50 test has been performed. The algae have been exposed to 5 dilutions of the test item for 72 hours. Algae have been measured using a Burker chamber.

At the end of the test, microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.

At the beginning and at the end of the test (72 hours) pH has been measured in all the vessels.

Temperature of the ambient has been recorded every day.

Stability of the test item in the definitive test has been performed using TOC analyser. Organic carbon concentration has been measured at the beginning and at the end of the test in a pool derived from vessels at the higher, intermediate and lower tested concentration.

The obtained results, in compliance with test acceptability criteria, can be summarised as follow:

EC50 (72 h) of the test item: 4.334 mg/l

NOEC of the test item: 1.53 mg/l

LOEC of the test item: 2.44 mg/l

Description of key information

On the extract of the test item pyrogallol the growth inhibition test on a culture of algae Pseudokirchneriella subcapitata was performed on a culture in exponential growth phase.

This approach, according to OECD 201, allowed to calculate the growth inhibition (effect) concentration to define a limit not toxic threshold.

A Lowest Observed Effect Concentration (LOEC) and a No Observed Effect Concentration (NOEC) have been calculated using a statistical software.

During the range finding test the algae have been exposed to 5 different dilutions of the test item for 72 hours. After the results of the range finding test an EC50 test has been performed. The algae have been exposed to 5 dilutions of the test item for 72 hours. Algae have been measured using a Burker chamber.

At the end of the test, microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.

At the beginning and at the end of the test (72 hours) pH has been measured in all the vessels.

Temperature of the ambient has been recorded every day.

Stability of the test item in the definitive test has been performed using TOC analyser. Organic carbon concentration has been measured at the beginning and at the end of the test in a pool derived from vessels at the higher, intermediate and lower tested concentration.

The obtained results, in compliance with test acceptability criteria, are summirized below.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.334 mg/L
EC10 or NOEC for freshwater algae:
1.53 mg/L

Additional information

LOEC (fresh water): 2.44 mg/L