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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:
5 September 2018 (Study Initiation) to 26 February 2019 (Study Completion)
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
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt., Ltd.
- Lot/batch No.of test material: MNST9H122A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as supplied by the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: test item assessed for solubility in OECD medium, acetone, DMSO and DMF. During method evaluation, the test item was found to be less soluble in test medium, therefore Water Accommodation Fraction was used and formulations were prepared at nominal concentrations, referred to as the loading rate.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: The pH ranged between 6.52 and 8.39. Osmolality and the presence of any precipitate not specified.
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: Test solutions were subject to active ingredient analysis using the validated analytical method. For the main study, six replicates for the control and three replicate for treatment groups were prepared. 20 mL of test sample from each replicate were drawn and mixed together for each group. Collected samples were centrifuged at 2000 rpm for 10 minutes to remove algal cells. Representative samples were divided into two equal portions. One portion was sent for test concentration analysis at 0 and
72 h and the second portion was stored at -20 ± 5 ºC until report finalisation. Active ingredient concentration in test media was determined using the validated analytical method

- Sample storage conditions before analysis: Not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A quantity of 31.3, 100 and 100 mg Fatty acids, C16-18 (even numbered), manganese (II) salts were mixed with algal culture media and transferred to a 1000 mL volumetric flask and the volume made up with algal culture media to obtain test concentrations of 31.3 mg/L (stock A), 100 mg/L (Stock B) and 100 mg/L (Stock C). These were kept under continuous (magnetic) stirring for 96 h at room temperature at 1000 rpm. After stirring, test solutions were allowed to re-equilibrate for approximately 1 h. After 1 h, 600 mL of each stock solution was collected from the lower portion using an “L” shaped glass tube without disturbing the solution and used for treatment. Prior to adding test solutions to vessels, test vessels were pre-conditioned with the respective test concentrations to saturate the surface of the respective vessel to prevent loss of test concentration due to absorption into test vessel walls. Volumes of 1.5, 4.65 and 14.7 mL from stock C and 0.4 mL algal culture were taken and diluted to 150 mL with sterile algal culture medium in respective sterile conical flasks of 250 mL capacity to obtain loading rates of 1.0, 3.1 and 9.8 mg Fatty acids, C16-18 (even numbered), manganese (II) salts/L, respectively. 150 mL of stock A and B, and 0.4 mL algal culture were taken in respective sterile conical flasks of 250 mL capacity to obtain loading rates of 31.3 and 100.0 mg Fatty acids, C16-18 (even numbered), manganese (II) salts/L, respectively. For the control group, only 0.4 mL alga culture was transferred using a micropipette and diluted to 150 mL with sterile algal culture medium.

- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none, prepared as a Water Accomodated Fraction

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
- Age of inoculum (at test initiation): The pre-culture was prepared two days prior to the commencement of the study
- Method of cultivation: The pre-culture was incubated under the same conditions as those required for the test and were used once growing exponentially (5 x 10-3 - 10-4 cells/mL). The temperature for pre-culture ranged between 22 and 23 °C.

ACCLIMATION
- Acclimation period: The pre-culture was prepared two days prior to the commencement of the study by transferring 8 mL from the latest subculture into a new culture vessel. Not specified
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Standard OECD duration of exposure for OECD 201
Test temperature:
Temperature range: 21 - 23 °C
pH:
pH range: 6.52 – 8.39
Nominal and measured concentrations:
Main study concentrations:
Nominal loading rates of 0.0 (control), 1.0, 3.1, 9.8, 31.3 and 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL sterile conical flask
- Type (delete if not applicable): open
- Material, fill volume: 150 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 6267 cells/mL (0 h)
- Control end cells density: 849167 cells/mL (72 h)
- No. of organisms per vessel: Initial Mean Cell Count: 6267 at 0 hrs
- No. of vessels per concentration (replicates): 3 replicates for each test concentration
- No. of vessels per control (replicates): 6 replicates for the control

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture medium was prepared as per OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Not specified
- Light intensity and quality: Mean illumination (lux): 6583 – 6707 (± 15% of the mean value)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell concentration of each replicate was determined once, at a regular interval of 24 h, by manual counting using a haemocytometer and a light microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Main test used a geometric factor of 3.2.
- Justification for using less concentrations than requested by guideline: n/a, study performed to guideline requirements
- Range finding study: yes
- Test concentrations: prelim range finding test performed at loading rates of 0.0 (control), 1.0, 10.0, 25.0, 50.0 and 100.0 mg of Fatty acids, C16-18 (even numbered), manganese(II) salts/L.
- Results used to determine the conditions for the definitive study:
The coefficient of variation of average specific growth rates during the test period in replicate control cultures was 2.44%. Thus, the validity criteria was met.
The cell concentration in control cultures increased exponentially by a factor of 135.5 within the test period. Thus, the validity criteria was met.
The mean coefficient of variation for day 0-1, 1-2, and 2-3 in control culture was 4.53%. Thus, the validity criteria was met.



Reference substance (positive control):
yes
Remarks:
Potassium dichromate was the positive control.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.15 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.:
0.17 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.:
0.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: Positive control valueswithin acceptabale limits
Reported statistics and error estimates:
A concentration-effect relationship was observed and therefore statistical analysis was performed to determine effect concentrations.

The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits based on the loading rate were ErL10 = 9.59 (3.70 – 24.80) mg/L, ErL20 = 16.89 (6.17 – 46.23) mg/L and ErL50 = 49.95 (16.43 – 151.86) mg/L, and based on the geometric mean measured concentration were; ErC10 = 0.58 (0.43 – 0.78) mg a.i./L, ErC20 = 1.04 (0.83 – 1.30) mg a.i./L and ErC50 = 3.15 (2.61 – 3.79) mg a.i./L.
The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits based on the loading rate were EbL10 = 3.09 (1.94 – 4.93) mg/L, EbL20 = 5.10 (3.50 – 7.44) mg/L and EbL50 = 13.28 (9.85 – 17.89) mg/L and based on the geometric mean measured concentration were; EbC10 = 0.18 (0.12 – 0.29) mg a.i./L, EbC20 = 0.31 (0.21 – 0.44) mg a.i./L and EbC50 = 0.81 (0.60 – 1.09) mg a.i./L.

The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits based on the loading rate were EyL10 = 3.41 (2.15 – 5.40) mg/L, EyL20 = 5.43 (3.75 – 7.87) mg/L and EyL50 = 13.28 (10.08 – 17.49) mg/L, and based on the geometric mean measured concentration were; EyC10 = 0.20 (0.13 – 0.32) mg a.i./L, EyC20 = 0.33 (0.23 – 0.47) mg a.i./L and EyC50 = 0.81 (0.62 – 1.07) mg a.i./L.

Based on the statistical analysis, the NOEC for growth rate, biomass (cell growth), and yield was 0.17 mg a.i./L (based on the geometric mean measured concentration) and the NOELR for biomass (cell growth), growth rate, and yield was 3.1 mg/L (based on the loading rate). The LOEC for biomass (cell growth), growth rate, and yield was 0.60 mg a.i./L (based on the geometric mean measured concentration) and the LOELR for biomass (cell growth), growth rate, and yield was 9.8 mg/L (based on the loading rate).
Validity criteria fulfilled:
yes
Conclusions:
ErC50 for growth rate inhibition (0 - 72 h): 3.15 mg a.i./L
NOEC for growth rate, biomass and yield: 0.17 mg a.i./L
LOEC for growth rate, biomass and yield: 0.60 mg a.i./L

Description of key information

The 72h ErC50 was found to be 3.15 mg/L confidence limits (2.61 - 3.79)

Key value for chemical safety assessment

EC50 for freshwater algae:
3.15 mg/L
EC10 or NOEC for freshwater algae:
0.17 mg/L

Additional information