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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:
25/02/2019-20/05/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Date of Receipt February 15, 2019
RCC Code of Test Item 249/006 (Assigned by RCC Laboratories India Private Limited)
The following information was provided by the sponsor:
Identification Tyzor AC436
Colour & Appearance Yellow to Brownish Clear liquid
Chemical Name µ2– (2-oxido-2-acetato-κ1O-1,3-dicarboxylate-κ2O)-bis[(2-oxidopropane-2-carboxylato-1,3-dicarboxylate)(2- oxidopropane-1,2,3-tricarboxylate- κO)titanium](4+)
Formulation or Composition Tyzor AC 436 Contains > 44 % (W/W) Water
Lot Number 3340011167
Purity ˃99%
Molecular Weight 1048.3 g/mol

Molecular Formula [{Ti2(C6H7O7)( C6H6O7)}( µ2-C6H6O7 )]
Structural Formula


Manufacture Date January, 2019
Expiry Date December, 2020
Stability The product is stable under storage at normal ambient temperature
Storage conditions Room Temperature (20 to 30°C)
Safety Precautions Aprons, masks, caps, gloves and goggles were used to ensure
the health and safety of the personnel
Analytical monitoring:
yes
Details on sampling:
Nominal concentration of the test item (without vehicle) at ‘0’ hour was calculated based on the CoA shared by the sponsor as the method shared by the sponsor was not feasible due to matrix difference
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test system (species) Pseudokirchneriella subcapitata (Unicellular green alga)
Justification Recommended by the test guideline
Subculture In-house Algae culture, RCC Laboratories India Private Limited
Source ATCC 22662 (Procured from LGC Promochem India Pvt. Ltd, Bangalore 560 058)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21.3 °C – 22.1 °C
pH:
7.85 - 8.12
Nominal and measured concentrations:
0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L
Details on test conditions:
Room No. E9, Department of Ecotoxicology
RCC Laboratories India Private Limited
Test Method Static Test
Temperature (°C) 21.3 °C – 22.1 °C
Test Vessel 250 mL Conical flask
Algae Inoculation
Range Finding Experiment 21275 cells/mL
Main Experiment 21950 cells/mL
No. of Replicate
Range Finding Experiment Control - 6 replicates
3 replicates per test concentration
Main Experiment Control - 6 replicates
3 replicates per test concentration
Light Continuous cool white Light Intensity
Range Finding Experiment 6720 - 6820 Lux (Refer Section 13)
Main Experiment 6700 - 6800 Lux (Refer Section 20)
Medium OECD
Pre-culture 3 days under test conditions in shaker incubator at 125-150 rpm
Test Duration 72 hours
pH range
Range finding Experiment
Start of the test (day '0') in bulk of medium
Control : 8.1 (Refer Section 12)
Treatment : 7.85 - 8.12
End of the test ('72' hour) Control : 9.22 - 9.26
Treatment : 8.31 - 9.18
Main Experiment
Start of the test (day '0') in bulk of medium
Control : 8.1 (Refer Section 19)
Treatment : 8.00 - 8.11
End of the test ('72' hour) Control : 9.20 - 9.28
Treatment : 8.72 - 9.04
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.67 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
The percent inhibition on the average growth rate at the end of 72 hour was 0.00 %, 4.55 %, 14.84 %, 19.27 %, 92.71%, 94.80% and 95.44 % in 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L test concentrations respectively.
From the average specific growth rate recorded with the concentrations of 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L, the concentration that inhibited 50% growth (ErC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated as 0.42 mg/L, with lower confidence limit of 0.40 mg/L and the upper confidence limit of 0.44 mg/L.
The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were recorded to be 0.01 mg/L and 0.03 mg/L respectively.
Yield:
The percent inhibition on the yield at the end of 72 hour was 0.00 %, 4.96 %, 15.28 %, 20.05 %, 38.02%, 58.49% and 90.47 % in 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L test concentrations respectively.
From the yield recorded with the concentrations of 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L the concentration that inhibited 50% growth (EyC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated as 1.67 mg/L with lower confidence limit of 1.56 mg/L and the upper confidence limit of 1.79 mg/L.
The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were calculated as 0.01 mg/L and 0.03 mg/L respectively
Validity criteria fulfilled:
yes
Conclusions:
Based on the test results:
The ErC50 of AC 436 observed for test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 0.42 mg/L, with lower confidence limit of 0.40 mg/L and the upper confidence limit of 0.44 mg/L.
The EyC50 of AC 436 observed for a test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 1.67 mg/L with lower confidence limit of 1.56 mg/L and the upper confidence limit of 1.79 mg/L.
Executive summary:

Growth Inhibition study was conducted inFreshwater green Algae(Pseudokirchneriella subcapitata)in accordance withOECD-201guideline withAC 436.Test item was found to be soluble inOECDmedium. The study was not supported with the Analytical measurements (stability analysis andformulation analysis) as the method shared by the sponsor was not feasible due to matrix. Hence the nominal concentration of the test item (without vehicle) at ‘0’ hour wascalculated based on the CoA shared by the sponsor and the nominal concentration was not ensured throughout the test as the test item was already mixed in the vehicle.

Range finding experiment was conducted with sevendifferent concentrations such as 0.0001,0.001,0.01, 1, 10, 50and 100mg/L.The percent inhibition on the average growth rate at the end of 72 hour was 0.00 %, 0.00 %, 0.00 %, 94.21 %, 96.58 %, 100.00 % and 100.00 % in test concentrations0.0001,0.001,0.01,1, 10, 50and 100mg/L respectively.The percent inhibition on the yield at the end of 72 hour was 0.00 %, 0.00 %, 0.00 %, 38.69 %, 91.84 %, 100.70 % and 100.78 % in test concentrations of0.0001,0.001,0.01,1, 10, 50and 100mg/L respectively. Based on the results of range finding experiment, testconcentrationswereselected for main experiment as0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L.

Prior to the exposure,pre-culture was maintained for a period of3days.Initially on ‘0’ day,approximately21,950cells/ml wereinoculated inaconical flask containing OECD medium.The volume of OECD medium was 100 mlin eachcontroland treatmentflask.Sixreplicates were maintained for controlandthreereplicates for eachtestconcentration.

After the exposure on day ‘0’, algal cells were observed microscopically and the cell count was done atapproximatelyat24, 48 and 72 hoursby usingHaemocytometer.Microscopic observationrevealed  thatall the cells were found normal and healthy in control and treatments except in 9.22 mg/L test concentration where swollen cells andaggregates were observed.

Average Specific Growth Rate:

The percent inhibition on the average growth rate at the end of 72 hour was 0.00 %, 4.55 %, 14.84 %, 19.27 %, 92.71%, 94.80% and 95.44 % in 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L test concentrations respectively.

From the average specific growth rate recorded with the concentrations of 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L, the concentration that inhibited 50% growth (ErC50) of Freshwater green Algae (Pseudokirchneriella subcapitata)was calculated as 0.42 mg/L, with lower confidence limit of 0.40 mg/L and the upper confidence limit of 0.44 mg/L.

The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were recorded to be 0.01 mg/L and 0.03 mg/L respectively.

Yield:

The percent inhibition on the yield at the end of 72 hour was 0.00 %, 4.96 %, 15.28 %, 20.05 %, 38.02%, 58.49% and 90.47 % in 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L test concentrations respectively.

From the yield recorded with the concentrations of 0.01, 0.03, 0.10, 0.30, 0.95, 2.96 and 9.22 mg/L the concentration that inhibited 50% growth (EyC50) of Freshwater green Algae (Pseudokirchneriella subcapitata) was calculated as 1.67 mg/L with lower confidence limit of 1.56 mg/L and the upper confidence limit of 1.79 mg/L.

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were calculated as 0.01 mg/L and 0.03 mg/L respectively

Nominal concentration of the test item (without vehicle) at ‘0’ hour was calculated based on the CoA shared by the sponsor and the nominal concentration cannot be ensured throughout the test as the test item is already mixed in the vehicle.

As per the sponsor COA, 100 mg of the test item contain Carbon, mass-% 33.75, Hydrogen, mass-% 3.23, Oxygen, mass-% 49.96 and Titanium, mass-% 13.06 

Nominal concentration of the test item (without vehicle) at ‘0’ hour was calculated. The following concentrations are given below

 

Nominal concentrations (mg/L)

 

Carbon (%)

 

Hydrogen (%)

 

Oxygen  (%)

 

Titanium    (%)

0.01

0.003375

0.000323

0.004996

0.001306

0.03

0.010125

0.000969

0.014988

0.003918

0.10

0.03375

0.00323

0.04996

0.01306

0.30

0.10125

0.00969

0.14988

0.03918

0.95

0.320625

0.030685

0.47462

0.12407

2.96

0.999

0.095608

1.478816

0.386576

  9.22

3.11175

0.297806

4.606312

1.204132

Description of key information

Based on the test results:

The ErC50of AC 436 observed for test period of '72' hour for freshwater alga (Pseudokirchneriella subcapitata) was calculated as 0.42 mg/L, with lower confidence limit of 0.40 mg/L and the upper confidence limit of 0.44 mg/L.

TheEyC50ofAC 436observed for a test period of '72' hour for freshwater alga(Pseudokirchneriella subcapitata)was calculated as1.67 mg/L with lower confidence limit of 1.56 mg/L and the upper confidence limit of 1.79 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.42 mg/L
EC10 or NOEC for freshwater algae:
0.01 mg/L

Additional information