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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25/02/2019 - 17/05/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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
Note: The identity and composition of the test item [Tyzor AC436] is the responsibility of the sponsor. No analysis was performed at RCC Laboratories India Private Limited to confirm it.
Analytical monitoring:
not specified
Details on sampling:
Each test vessel was checked for immobility of daphnids and any abnormal behavior or appearance at 24 and 48 hours. Daphnids that were not able to swim within 15 seconds, after gentle agitation of the test vessel were considered to be immobilised (even if they can still move their antennae). On completion of test, dead daphnids found and all live daphnids (euthanized with MS222 medium) were discarded safely along with the test solutions.
Vehicle:
no
Details on test solutions:
100.02 mg of test item was dissolved and made up to 1000 mL with M4 medium in a 1000 mL standard flask and shaken well to prepare stock A concentration of 0.1 mg/mL. From stock A, 1 mL was taken and made up to 100 mL with M4 medium in a 100 mL standard flask to prepare the stock B concentration of 0.001 mg/mL. From stock B, 1 mL was taken and made up to 100 mL with M4 medium in a 100 mL standard flask to prepare stock C concentration of 0.00001 mg/mL. From stock A, B and C, the following concentrations were prepared and all the test concentrations were prepared for 200 mL.
From the stock solution C, 2 mL and 4 mL was taken and made up to 200 mL with M4 medium each to prepare the required test concentrations of 0.0001 and 0.0002 mg/L which is equivalent to 0.00002 and 0.00004 mg/200 mL respectively.
From the stock solution B, 100 µL, 220 µL, 460 µL, 1.040 mL, 2.260 mL were taken and made up to 200 mL each to prepare the required test concentration of 0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L which is equivalent to 0.0001, 0.00022, 0.00046, 0.00104 and 0.00226 mg /200 mL respectively.
For control, M4 medium was used
From the above prepared test concentrations, 50 mL of the test solution was transfered into each replicate beaker. Thus, 4 replicates were maintained for control and treatment.
‘24’ hour:
Dose formulation performed at 0 hour for 0.0001, 0.0002, 0.0005, 0.0011, 0.0023 and 0.0052 mg/L concentrations was followed for 24 hours with 100.02 mg of the test item.
NOTE: 0.0113 mg/L concentration was not renewed as all the daphnids were found dead at ‘24’ hour observation
Test organisms (species):
Daphnia magna
Details on test organisms:
Test Species Daphnia magna
Age Young Daphnids, aged less than ‘24’ hour old
Justification Recommended by the test guideline
Source In - house culture, RCC Laboratories India Private Limited
(Originally procured from Department of Advanced Zoology & Biotechnology, Ethiraj College for Women, Chennai)
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
220 mg/L
Test temperature:
21 C
pH:
7.22-7.39
Dissolved oxygen:
7.36-7.59 mg/L
Salinity:
not specififed
Conductivity:
not specified
Nominal and measured concentrations:
0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L
Details on test conditions:
Room No. E3, Department of Ecotoxicology,
RCC Laboratories India Private Limited
Test Method Semi Static
Test Vessel 100 ml Glass Beaker
No. of Replicate Control - 4 replicates
Range Finding Experiment 4 replicates per concentration
Main experiment 4 replicates per concentration
Number of Daphnids/replicate 5
Light 16 hour light and 8 hour darkness
Feeding During the test daphnids were not fed
Dilution Water M4 Medium (During the test, medium was not aerated)
Volume 50 ml (10 ml per daphnid)
Test Duration ‘48’ hour
Range finding Experiment
pH Control : 7.36 – 7.41 (Refer Section 12.1)
Treatment : 7.00 – 7.43 (Refer Section 12.1)
Temperature Control : 21.0°C – 21.1°C (Refer Section 12.1)
Treatment : 21.0°C – 21.2°C (Refer Section 12.1)
Hardness Control : 220 mg/L (Refer Section 12.1)
Dissolved oxygen Control : 7.51 – 7.63 mg/L (Refer Section 12.1)
Treatment : 7.20 – 7.61 mg/L (Refer Section 12.1)
Main Experiment
pH Control : 7.38 – 7.45 (Refer Section 12.2)
Treatment : 7.22 – 7.39 (Refer Section 12.2)
Temperature Control : 21.0°C – 21.2°C (Refer Section 12.2)
Treatment : 21.0°C – 21.2°C (Refer Section 12.2)
Hardness Control : 220 mg/L (Refer Section 12.2)
Dissolved oxygen Control : 7.58 – 7.64 mg/L (Refer Section 12.2)
Treatment : 7.36 – 7.59 mg/L (Refer Section 12.2)
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 0.001 - <= 0.002 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
A range finding experiment was conducted with a range of concentrations such as 0.00001, 0.0001, 0.01, 1, 10, 50 and 100 mg/L. After the exposure on day 0, Test item renewal was performed at 24 hour and daphnids were observed for immobilisation at 24 and 48 hour and the respective data was recorded.
No immobilization was observed in control and daphnids exposed to 0.00001 and 0.0001 mg/L concentrations throughout the experimental period. At the end of 24 hour, 20/20, immobility was observed in daphnids exposed to 0.01, 1, 10, 50 and 100 mg/L concentrations respectively. Thus the percent immobilisation at the end of 24 hour was recorded to be 0% in control and daphnids exposed to 0.00001 and 0.0001 mg/L concentrations whereas, 100% immobilisation was recorded in daphnids exposed to 0.01, 1, 10, 50 and 100 mg/L concentrations respectively. (Refer Section 8)
No immobilization was observed in control and daphnids exposed to 0.0001 mg/L concentration throughout the experimental period. At the end of 24 hour, 1/20, 2/20, 3/20, 5/20, 10/20 and 20/20 immobility was observed in daphnids exposed to 0.0002, 0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L test concentrations. Thus the percent immobilisation at the end of 24 hour was recorded to be 0% in control and daphnids exposed to 0.0001 mg/L test concentration whereas, 5%, 10%, 15%, 25%, 50% and 100% immobility was observed in daphnids exposed to 0.0002. 0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L. At the end of 48 hour, 2/20, 5/20, 8/20, 10/20 and 17/20 cumulative immobility was observed in daphnids exposed to 0.0002, 0.0005, 0.0011, 0.0023 and 0.0052 mg/L test concentrations respectively. Thus the percent cumulative immobilisation at the end of 48 hour was recorded to be 0% in control and daphnids exposed to 0.0001 mg/L concentration whereas, 10%, 25%, 40%, 50%, 85% and 100% immobility was observed in daphnids exposed to 0.0002, 0.0005, 0.0011, 0.0023 , 0.0052 and 0.0113 mg/L concentrations repectively. (Refer Section 9)
Validity criteria fulfilled:
yes
Conclusions:
Based on the test results, the EC50 of AC 436 in Daphnia magna observed for a period of 24 hour was calculated as 0.003 mg/L with upper confidence limits of 0.005 mg/L and lower confidence limits of 0.002 mg/L and regression equation Y = 9.5553 + 1.8460 log (x).
Based on the test results, the EC50 of AC 436 in Daphnia magna observed for a period of 48 hour was calculated as 0.001 mg/L with upper confidence limits of 0.002 mg/L and lower confidence limits of 0.001 mg/L and regression equation Y = 10.1604 + 1.8176 log (x).
Executive summary:

Acute Immobilisation Toxicity Study inDaphnia magnawith AC 436 was conducted in accordance with OECD guideline 202.Test item was found to be soluble in M4 medium.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 and during the test item renewal (‘24’ hour) were calculated 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. Semi-static test (test item renewal at 24 hours)
was performed as the stability of the test item in the vehicle was unknown.
 

Range finding experiment was conducted with seven different concentrations such as 0.00001 0.0001, 0.01, 1, 10, 50 and 100 mg/L through semi-static exposure at 24 hour interval. No immobility was observed in the daphnids treated with 0.00001 and 0.0001 mg/L concentrations, whereas 100% immobility was observed in the daphnids exposed to 0.01, 1, 10, 50 and 100 mg/L test concentrations. Therefore, main experiment was conducted with seven different concentrations such as 0.0001, 0.0002, 0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L concentrationsthrough semi-static exposure at 24 hours interval. 

The daphnids with brood chambers were acclimatized one day prior to the exposure in M4 medium. The test item was formulated with M4 Medium. Twenty daphnids with four replicates (5 daphnids in each replicate) for the test concentrations and control group was maintained

After the exposure on day 0, Test item renewal was performed at 24 hour and daphnids were observed for immobilisation at 24 and 48 hour and the respective data was recorded.

No immobilization was observed in control and daphnids exposed to 0.0001 mg/L concentration throughout the experimental period. At the end of 24 hour, 1/20, 2/20, 3/20, 5/20, 10/20 and 20/20 immobility was observed in daphnids exposed to 0.0002, 0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L test concentrations. Thus the percent immobilisation at the end of 24 hour was recorded to be 0% in control and daphnids exposed to 0.0001 mg/L test concentration whereas, 5%, 10%, 15%, 25%, 50% and 100% immobility was observed in daphnids exposed to 0.0002. 0.0005, 0.0011, 0.0023, 0.0052 and 0.0113 mg/L. At the end of 48 hour, 2/20, 5/20, 8/20, 10/20 and 17/20 cumulative immobility was observed in daphnids exposed to 0.0002, 0.0005, 0.0011, 0.0023 and 0.0052 mg/L test concentrations respectively. Thus the percent cumulative immobilisation at the end of 48 hour was recorded to be 0% in control and daphnids exposed to 0.0001 mg/L concentration whereas, 10%, 25%, 40%, 50%, 85% and 100% immobility was observed in daphnids exposed to 0.0002, 0.0005, 0.0011, 0.0023 , 0.0052 and 0.0113  mg/L concentrations repectively.

No toxicity signs were observed in control and daphnids exposed to 0.0001 mg/L concentration throughout the experiment. At the end of 24 hour Lethargy was observed in daphnids exposed 0.0002, 0.0005, 0.0011, 0.0023 and 0.0052 mg/L test concentrations. At the end of 48 hour no toxicity signs were observed in any of the test concentrations.

Water Parameters such aspH, temperature and dissolved oxygen were recorded at the start of the test, on the day of renewal of test medium (in both old and fresh media) and end of the experiment in control and all test concentrations. Hardness was recorded in control at the start of the test and on the day of renewal of test medium.

Nominal concentration of the test item (without vehicle) at ‘0’ hour and during the test item renewal (‘24’ 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.

Description of key information

Based on the test results, the EC50 of AC 436  in Daphnia magna  observed for a period of 24 hour was calculated as 0.003 mg/L with upper confidence limits of 0.005 mg/L and lower confidence limits of  0.002 mg/L  and regression equation Y = 9.5553 + 1.8460 log (x).  

Based on the test results, the EC50 of AC 436  in Daphnia magna  observed for a period of 48 hour was calculated as 0.001 mg/L with upper confidence limits of 0.002 mg/L and lower confidence limits of  0.001  mg/L and regression equation Y =  10.1604 + 1.8176 log (x).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.001 mg/L

Additional information

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