AVITERA ROSE SE

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 24, 2021 to August 03, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Date of Receipt: February 03, 2021
The following information was provided by the Sponsor:
Identification: FAT 40879/B TE
Appearance: dark red powder
Batch No.: BOP 05-20 (MC-0065114700/800 1:1)
Purity: 80.5 %
Manufacture Date: June 26, 2020
Expiry Date: September 08, 2025
Storage Conditions: Freezer (-15 to -20 °C)

Analytical monitoring:

yes

Details on sampling:

STABILITY ANALYSIS
100.02 mg of the test item was dissolved and made up to 1000 mL with OECD medium in a 1000 mL volumetric flask. This test solution was checked for its stability at '0', '24', '48' and '72' hour. Based on the analytical results, the test item was found to be unstable under test conditions for '72' hours. Therefore, additional replicates of each test concentration were maintained (under identical conditions) for analysis at 24 hour intervals during exposure period.

ONCENTRATION VERIFICATION ANALYSIS
50 mL of sample was collected from bulk preparation of control and all test concentrations at the beginning of the test. As the test item is unstable, additional replicates of each test concentration and control were maintained and same replicate were sent to the Analytical Chemistry Department at 24 h intervals.

Vehicle:

yes

Remarks:

OECD MEDIUM

Details on test solutions:

RANGE FINDING EXPERIMENT
100.02 mg of the test item was dissolved and made up to 100 mL with OECD medium in a 100 mL standard flask to prepare the stock (concentration of 1 mg/mL). The stock concentration 1 mg/mL, was used to prepare the required concentrations. All the concentrations were prepared for 300 mL ( 0.3, 3, 7.5, 15 and 30 mg/300 mL) From the stock solution 300 µL, 3 mL, 7.5 mL, 15 mL and 30 mL was taken and made up to 300 mL each to prepare the required concentrations viz., 1, 10, 25, 50 and 100 mg/L respectively. For Control, OECD medium was used. From the above prepared concentrations, 100 mL of test solution was transferred into each replicate. On inoculation, control and treatment flasks were kept in the orbital shaker under test conditions.

MAIN EXPERIMENT
100.02 mg of the test item was dissolved and made up to 100 mL with OECD medium in a 100 mL standard flask to prepare the stock (concentration of 1 mg/mL). The stock was used to prepare the required concentrations. All the concentrations were prepared for 600 mL (6.00, 8.40, 11.76, 16.464, 23.052 and 32.268 mg/600 mL) From the stock solution 6 mL, 8.4 mL, 11.760 mL, 16.464 mL, 23.052 and 32.268 mL was taken and made up to 600 mL each to prepare the required concentrations viz., 10.00, 14.00, 19.60, 27.44, 38.42 and 53.78 mg/L respectively. For Control, OECD medium was used. From the above prepared concentrations, 100 mL of test solution was transferred into each replicate. On inoculation, control and treatment flasks were kept in the orbital shaker under test conditions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST SYSTEM
Test System (species): Unicellular green alga (Raphidocelis subcapitata)
Justification: Recommended by the test guideline
Subculture: In-house Algae culture, RCC Laboratories India Private Limited
Source: ATCC 22662 (Procured from LGC Promochem India Pvi. Ltd, Bangalore 560 058)

Test type:

static

Water media type:

other: OECD MEDIUM

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature (°C): 22.6 °C to 23.8 °C

pH:

Main Experiment Start of the test (day '0') in bulk of medium
Control: 8.1
Treatment:7.48 to 7.84
End of the test ('72' hour)
Control: 8.78 to 8.84
Treatment: 8.14 to 8.58

Nominal and measured concentrations:

10.00, 14.00, 19.60, 27.44, 38.42 and 53.78 mg/L ( Nominal concentrations )
12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L (Geometric mean recovered concentrations)

Details on test conditions:

TEST SYSTEM
- Test Method: Static
- Temperature: 22.6 °C to 23.8 °C
- Test Vessel: 250 mL Conical flask

Algae Inoculation
- Range Finding Experiment: 21275 cells/mL
- Main Experiment: 20025 cells/mL

No. of Replicate
- Range Finding Experiment: Control - 6 replicates; 3 replicates per test concentration

- Main Experiment: Control - 9 replicates (6 replicates per test concentration 3 replicates per concentration verification)
3 replicates per test concentration
3 replicates per concentration verification

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD MEDIUM

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes ( 8.1)
- Photoperiod: Continuous cool white Light Intensity
- Light intensity and quality: Main Experiment: 6680 to 6780 Lux

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: 1, 10, 25, 50 and 100mg/L
- Results used to determine the conditions for the definitive study: 0, 24, 48 and 72 hour Cell Count

Reference substance (positive control):

yes

Remarks:

Potassium dichromate ( Conducted twice in a year and the results of the recent run is presented in the report )

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.58 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.:

12.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Average Specific Growth Rate:
The percent inhibition on the average growth rate at the end of 72 hour was 0.15 %, 3.91 %, 26.64 %, 41.15 %, 72.91 % and 96.40 % in geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L respectively.
From the average specific growth rate recorded with the geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L, the concentration that inhibited 50 % growth (ErC50) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 32.58 mg/L, with lower confidence limit of 32.15 mg/L and the upper confidence limit of 33.02 mg/L.
The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were recorded in geometric mean recovered concentrations of 12.62 and 17.37 mg/L respectively.

Yield:
The percent inhibition on the yield at the end of 72 hour was 0.09 %, 3.41 %, 25.28 %, 40.10 %, 70.76% and 95.08 % in geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L respectively.
From the yield recorded with the geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L the concentration that inhibited 50 % growth (EyC50) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 33.18 mg/L with lower confidence limit of 32.74 mg/L and the upper confidence limit of 33.63 mg/L.
The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were recorded in geometric mean recovered concentrations of 12.62 and 17.37 mg/L respectively.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: NA
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: NA
- Any stimulation of growth found in any treatment: Yes
- 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

Growth Inhibition study (Study No. 10812 ) was performed in Freshwater green Algae (Pseudokirchneriella subcapitata) in accordance with guideline OECD-201 with Potassium dichromate. The concentrations used were 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L.
Initially on ‘0’ day approximately 21500 algal cells/mL were inoculated in conical flask containing OECD medium. The volume of OECD medium was 100 mL each in control and treatment groups. 6 replicates were maintained for control and 3 replicates for each concentration. Prior to exposure, pre-culture was maintained for a period of 3 days.
On day of exposure algal cells were observed microscopically and cell count was carried out approximately at ‘24’, ‘48’ and ‘72’ hours after exposure with Haemocytometer. Microscopic observations revealed that cells were normal and no abnormal appearance of the algal cell was observed on exposure to test item.
Average Specific Growth Rate:
The percent inhibition on the average growth rate at the end of ‘72’ hour was 0.09%, 15.47%, 70.77%, 90.25% and 100.00% in concentrations of 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L respectively.
From the average specific growth rate recorded with the different concentrations viz., 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L the concentration that inhibits 50% growth (ErC50) of freshwater green algae (Pseudokirchneriella subcapitata) was calculated to be 0.90 mg/L, with lower confidence limit of 0.87 mg/L and upper confidence limit of 0.93 mg/L.
The lowest observed effect concentration (LOEC) was calculated as 0.5 mg/L and the no observed effect concentration (NOEC) was recorded as 0.2 mg/L respectively.
Yield:
The percent inhibition on the yield at the end of ‘72’ hour was 0.00%, 9.14%, 66.88%, 89.27% and 97.23% in concentrations of 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L respectively.
From the yield recorded with the different concentrations viz., 0.2, 0.5, 1.1, 2.4 and 5.6 mg/L, the concentration that inhibits 50% growth (EyC50) of freshwater green algae (Pseudokirchneriella subcapitata) was recorded as 0.99 mg/L, with lower confidence limit of 0.96 mg/L and upper confidence limit of 1.03 mg/L.
The lowest observed effect concentration (LOEC) was calculated as 0.5 mg/L and the no observed effect concentration (NOEC) was recorded as 0.2 mg/L respectively.

CONCLUSION
Based on the test results,
The ErC50 of Potassium dichromate observed for a period of ‘72’ hour in freshwater alga (Pseudokirchneriella subcapitata) was calculated to be 0.90 mg/L, with lower confidence limit of 0.87 mg/L and upper confidence limit of 0.93 mg/L.
The EyC50 of Potassium dichromate observed for a period of ‘72’ hour in freshwater alga (Pseudokirchneriella subcapitata) was calculated as 0.99 mg/L, with lower confidence limit of 0.96 mg/L and upper confidence limit of 1.03 mg/L.
Based on the above findings the sensitivity of the test system (Pseudokirchneriella subcapitata) and reliability of the experimental technique is proved.

Reported statistics and error estimates:

- ErC50 : The ErC50 of FAT 40879/B TE observed for test period of '72' hour for freshwater alga (Raphidocelis subcapitata) was calculated as 32.58 mg/L, with lower confidence limit of 32.15 mg/L and the upper confidence limit of 33.02 mg/L.
- EyC50 : The EyC50 of FAT 40879/B TE observed for a test period of '72' hour for freshwater alga (Raphidocelis subcapitata) was calculated as 33.18 mg/L with lower confidence limit of 32.74 mg/L and the upper confidence limit of 33.63 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 of FAT 40879/B TE observed for test period of '72' hour for freshwater alga (Raphidocelis subcapitata) was calculated as 32.58 mg/L, with lower confidence limit of 32.15 mg/L and the upper confidence limit of 33.02 mg/L.

Executive summary:

An Algal Growth Inhibition study was conducted in Freshwater green Algae (Raphidocelis subcapitata) in accordance with OECD-201 guideline with FAT 40879/B TE. Test item was soluble in OECD medium.

Stability of the test item in the OECD medium was performed at 0 hour, 24 hour, 48 hour and 72 hour. The mean recoveries data are given below:

The mean recoveries data are for

0 Hour 100.61 mg/L  (100.59 %)

24 Hour 74.86 mg/L (74.58%)

48 Hour 76.31 mg/L (76.29 %)

72 Hour 71.72 mg/L (71.70 %)

Stability analysis of test item revealed that the test item is unstable in OECD medium. As the test item is unstable in OECD medium, additional 3 replicates were maintained for control and each treatment. Additional replicate of each test concentration and control were sent for analysis at 24 hours interval for main experiment.

Analytical verification of the test item (concentration) in the exposure medium sample was carried out in the test item concentrations of 10.00, 14.00, 19.60, 27.44, 38.42 and 53.78 mg/L. The samples at '0' hour, '24' hour, '48' hour and '72' hour were analyzed. The mean recoveries data are given below:

Geometric mean recovered concentration (%) of 10.00 mg/L at 0, 24, 48 and 72 hour are 12.78, 12.44, 12.57 and 12.93 mg/L respectively and the Geometric mean recovered concentration  is 12.62 mg/L.

Geometric mean recovered concentration (%) of 14.00 mg/L at 0, 24, 48 and 72 hour are 17.00, 17.39, 16.56 and 17.66 mg/L respectively and the Geometric mean recovered concentration is 17.37 mg/L.

Geometric mean recovered concentration (%) of 19.60 mg/L at 0, 24, 48 and 72 hour are 22.46, 22.68, 23.14 and 22.93 mg/L respectively and the Geometric mean recovered concentration is 22.68 mg/L.

Geometric mean recovered concentration (%) of 27.44 mg/L at 0, 24, 48 and 72 hour are 33.02, 32.76, 31.49 and 33.25 mg/L respectively and the Geometric mean recovered concentration is 32.89 mg/L.

Geometric mean recovered concentration (%) of 38.42 mg/L at 0, 24, 48 and 72 hour are 37.81, 36.40, 37.11 and 36.71 mg/L respectively and the Geometric mean recovered concentration is 36.68 mg/L.

Geometric mean recovered concentration (%) of 53.78 mg/L at 0, 24, 48 and 72 hour are 54.76, 50.51, 51.90 and 51.24 mg/L respectively and the Geometric mean recovered concentration is 51.31 mg/L.

Geometric mean recovered concentration was calculated and the same was used for probit analysis and represented for the interpretation of the results of main experiment.

Prior to the exposure of range finding and main experiment pre-culture was maintained for a period of 3 days. After the exposure on day ‘0’, algal cells were observed microscopically and the cell count was performed approximately at 24, 48 and 72 hours using Haemocytometer. Microscopic observation revealed that the cells were normal and no abnormal appearance of the algal cell was observed on exposure to the test item.

Initially, range finding experiment was conducted with five different concentrations such as 1, 10, 25, 50 and 100 mg/L. The percent inhibition on the average growth rate at the end of 72 hour was 0.00 %, 0.14 %, 0.21 %, 38.82%, 90.23% and 99.80 % in test concentrations 1, 10, 25, 50 and 100 mg/L respectively. The percent inhibition on the yield at the end of 72 hour was 0.00 %, 0.03 %, 0.06 %, 35.13 %, 90.05 % and 97.07 % in test concentrations of 1, 10, 25, 50 and 100 mg/L respectively. Based on the results of range finding experiment the test concentrations selected for main experiment were 10.00, 14.00, 19.60, 27.44, 38.42 and 53.78 mg/L.

Prior to the exposure of main experiment, pre-culture was maintained for a period of 3 days. Initially on ‘0’ day, approximately 20025 cells/ml were inoculated in a conical flask containing OECD medium. The volume of OECD medium was 100 mL in each control and treatment flask. Nine replicates were maintained for control and six replicates for each test concentration. For control Replicate 1 to Replicate 6 (R1 to R6) were used for cell count whereas, Replicate 7 to Replicate 9 (R7 to R9) were used for concentration verification. For each test concentration,  Replicate 1 to Replicate 3 (R1 to R3) were used for cell count whereas, Replicate 4 to Replicate 6 (R4 to R6) were used for concentration verification.

After the exposure on day ‘0’, algal cells were observed microscopically and the cell count was performed approximately at 24, 48 and 72 hours by using Haemocytometer. Microscopic observation revealed that cells were normal and no abnormal appearance of the algal cells was observed on exposure to the test item.

Average Specific Growth Rate:

The percent inhibition on the average growth rate at the end of 72 hour was 0.15 %, 3.91 %, 26.64 %, 41.15 %, 72.91 % and 96.40 % in geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L respectively.

From the average specific growth rate recorded with the geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L, the concentration that inhibited 50 % growth (E_rC₅₀) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 32.58 mg/L, with lower confidence limit of 32.15 mg/L and the upper confidence limit of 33.02 mg/L.

The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were recorded in geometric mean recovered concentrations of 12.62 and 17.37 mg/L respectively.

 

Yield:

The percent inhibition on the yield at the end of 72 hour was 0.09 %, 3.41 %, 25.28 %, 40.10 %, 70.76% and 95.08 % in geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L respectively.

From the yield recorded with the geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L the concentration that inhibited 50 % growth (E_yC₅₀) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 33.18 mg/L with lower confidence limit of 32.74 mg/L and the upper confidence limit of  33.63 mg/L.

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were recorded in geometric mean recovered concentrations of 12.62 and 17.37 mg/L respectively.

Description of key information

The concentration that inhibited 50 % growth (E_rC₅₀) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 32.58 mg/L, with lower confidence limit of 32.15 mg/L and the upper confidence limit of 33.02 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

32.58 mg/L

EC10 or NOEC for freshwater algae:

12.62 mg/L

Additional information

An Algal Growth Inhibition study was conducted in Freshwater green Algae (Raphidocelis subcapitata) in accordance with OECD 201 guideline with FAT 40879/B. Test item was soluble in OECD medium. However, it was found to unstable till 72 hours, hence, geometric mean recovered concentration were calculated and the same was used for probit analysis and represented for the interpretation of the results of main experiment.

 

Average Specific Growth Rate:

The percent inhibition on the average growth rate at the end of 72 hour was 0.15 %, 3.91 %, 26.64 %, 41.15 %, 72.91 % and 96.40 % in geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L, respectively. From the average specific growth rate recorded with the geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L, the concentration that inhibited 50 % growth (E_rC₅₀) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 32.58 mg/L, with lower confidence limit of 32.15 mg/L and the upper confidence limit of 33.02 mg/L. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were recorded in geometric mean recovered concentrations of 12.62 and 17.37 mg/L, respectively.

 

Yield:

The percent inhibition on the yield at the end of 72 hour was 0.09 %, 3.41 %, 25.28 %, 40.10 %, 70.76% and 95.08 % in geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L, respectively. From the yield recorded with the geometric mean recovered concentrations of 12.62, 17.37, 22.68, 32.89, 36.68 and 51.31 mg/L the concentration that inhibited 50 % growth (E_yC₅₀) of Freshwater green Algae (Raphidocelis subcapitata) was calculated as 33.18 mg/L with lower confidence limit of 32.74 mg/L and the upper confidence limit of  33.63 mg/L. The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were recorded in geometric mean recovered concentrations of 12.62 and 17.37 mg/L, respectively.