5-[(4-chloro-2-nitrophenyl)azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 07 August 2015; Experiment completion date: 10 October 2015; Study completion date: 24 November 2015.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 36091/I TE
Description: Solid
Appearance: Powder
Colour: Yellow
Batch number: 20131205lab (China)
CAS Number: 70528-90-4
Purity: 94.6 %
Molecular weight: 361.74 g/mol
Expiry date: August 08, 2019
Stability of test item: Stable
Storage conditions: Room temperature in the dark
Safety precautions: Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel.

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

M4 medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Test species: Daphnia magna
Age: Young Daphnids, aged less than 24 hours old
Source: In - house culture, RCC Laboratories India Private Limited (Originally procured from Department of Advanced Zoology & Biotechnology, Ethiraj College for Women, Chennai)

ACCLIMATISATION
Daphnids with full brood chambers from suitable batch were acclimatized one day prior to the experiment in the test area and were fed with algae suspension. Daphnids aged not more than 24 hours were separated on the day of the experiment and used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

-Range Finding Experiment: 210 mg/L
- Main Experiment: 220 mg/L

Test temperature:

- Range Finding Experiment
Control: 20.3 – 20.4 °C
Treatment: 20.3 – 20.4 °C

- Main Experiment
Control : 20.3 – 20.4 °C
Treatment : 20.3 – 20.4 °C

pH:

- Range Finding Experiment
Control: 7.78 - 7.85
Treatment: 7.67 - 7.85

- Main Experiment
Control: 7.79 - 7.84
Treatment: 7.68 - 7.80

Dissolved oxygen:

Range Finding Experiment
Control: 7.70 – 7.78 mg/L
Treatment: 7.55 – 7.77 mg/L

Main Experiment
Control: 7.70 – 7.78 mg/L
Treatment: 7.51 – 7.76 mg/L

Nominal and measured concentrations:

10.02 mg of the test item was received and from which 3.9 mg was re-weighed, dissolved and made up to 1000 ml with M4 medium. The test solution was ultrasonicated for approximately 10 minutes to prepare a stock solution (3.9 mg/L) which was used to prepare the required test concentrations of 0.0001, 0.001, 0.01, 0.1, 1 and 3.9 mg/L.

10.01 mg of the test item was received and from which 3.9 mg was reweighed, dissolved and made up to 1000 ml with M4 medium. The test solution was ultrasonicated for approximately 10 minutes to prepare a stock solution (3.9 mg/L) which was used to prepare the required test concentrations of 0.03, 0.06, 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L

Details on test conditions:

- Test Vessel: 100 ml Glass Beaker
- No. of Replicate:
Control - 4 replicates and for the Range finding: 4 replicates per test concentration
Main experiment: 4 replicates per test concentration
- Number of Daphnids/replicate: 10
- Light: 16 hour light and 8 hour dark
- Feeding: Daphnids were not fed during the test
- Dilution Water: M4 Medium

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

RANGE FINDING EXPERIMENT- IMMOBILIZATION
No immobilization was observed in control and daphnids exposed to 0.0001, 0.001 and 0.01 mg/L test concentrations throughout the experimental period. At the end of 24 hour, no immobility was observed in daphnids exposed to any of the test concentrations. Thus, the percent immobilisation at the end of 24 hour was recorded to be 0 % in control and daphnids exposed to all the test concentrations. At the end of 48 hour, 7/40, 30/40 and 40/40 immobility was recorded in the daphnids exposed to 0.1, 1 and 3.9 mg/L test concentrations respectively. Thus the percent immobilisation at the end of 48 hour was recorded to be 0 % in control and daphnids exposed to 0.0001, 0.001, 0.01 mg/L concentrations whereas, 17.5 %,75 % and 100 % in daphnids exposed to 0.1, 1 and 3.9 mg/L concentrations, respectively.

MAIN EXPERIMENT- IMMOBILIZATION
No immobilization was observed in control and daphnids exposed to 0.03 and 0.06 mg/L test concentrations throughout the experimental period. At the end of 24 hour, no immobility was observed in daphnids exposed to any of the test concentrations. Thus, the percent immobilisation at the end of 24 hour was recorded to be 0 % in control and daphnids exposed to all the test concentrations. At the end of 48 hour, 6/40, 13/40, 21/40 , 33/40 and 40/40 immobility was recorded in the daphnids exposed to 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L concentrations, respectively. Thus the percent immobilisation at the end of 48 hour was recorded to be 0 % in control and daphnids exposed to 0.03 and 0.06 mg/L concentration whereas, 15 %, 25 %, 52.5 %, 82.5 % and 100 % in daphnids exposed to 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L test concentrations, respectively

Results with reference substance (positive control):

Based on the test results, the EC50 of Potassium Dichromate Pure in Daphnia magna observed for a period of ‘24’ hour was calculated as 1.72 mg/L with upper confidence limits of 2.12 mg/L and lower confident limits of 1.40 mg/L and regression equation Y = 4.0957 + 3.8604 log (x). At ‘48’ hour, the EC50 was calculated as 1.33 mg/L with upper confidence limits of 1.63 mg/L and lower confidence limits of 1.08 mg/L and regression equation Y = 4.5239 + 3.8769 log (x).

Reported statistics and error estimates:

EC50 with 95 % confidence limits for 24 and 48 hour was calculated using Finney Probit analysis. Graph was plotted for dose-response curve at 48 hour.

Validity criteria fulfilled:

yes

Conclusions:

Based on the test results, EC50 of FAT 36091/I TE in Daphnia magna observed for a period of 48 hour was calculated as 0.45 mg/L. The no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for 48 hours was calculated as 0.06 mg/L and 0.13 mg/L respectively.

Executive summary:

Acute Immobilization Toxicity Study in Daphnia magna was conducted in accordance with OECD guideline 202 with FAT 36091/I TE. Test item was checked for its solubility in M4 medium and it was noted that 3.9 mg/L is the limit of solubility. Therefore, dose formulation was performed in M4 medium at a limit of 3.9 mg/L. Based on the results of the range finding experiment, main experiment was conducted with a range of concentrations 0.03, 0.06, 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L. Forty daphnids with four replicates (10 daphnids in each replicate) for each test concentration and control group were maintained. The test item was formulated in M4 medium (dilution water). The daphnids with brood chambers were acclimatized one day prior to the exposure in M4 medium. After the exposure on day 0, daphnids were observed for immobilization at approximately 24 hour and 48 hour and the respective data was recorded. No immobilization was observed in control and daphnids exposed to 0.03 and 0.06 mg/L test concentrations throughout the experimental period. At the end of 24 hour, no immobility was observed in daphnids exposed to any of the test concentrations. Thus, the percent immobilization at the end of 24 hour was recorded to be 0% in control and daphnids exposed to all the test concentrations. At the end of 48 hour, 6/40, 13/40, 21/40, 33/40 and 40/40 immobility were recorded in the daphnids exposed to 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L concentrations respectively. Thus, the percent immobilization at the end of 48 hour was recorded to be 0 % in control and daphnids exposed to 0.03 and 0.06 mg/L concentration whereas, 15 %, 25 %, 52.5 %, 82.5 % and 100 % in daphnids exposed to 0.13, 0.28,0.58, and 2.57 mg/L test concentrations, respectively. Analytical verification of the test item in the dilution water (M4 medium) samples were performed in the test item concentrations of low (0.03 mg/L), intermediate (0.28 mg/L) and high (2.57 mg/L) at 0 hour and 48 hour. The mean recoveries data are given below:

0 Hour: 97.81 %, 96.85 % and 95.98 % for 0.03 mg/L, 0.28 mg/L and 2.57 mg/L concentrations, respectively.

48 Hour: 94.86 %, 94.84 % and 94.50 % for 0.03 mg/L, 0.28 mg/L and 2.57 mg/L concentrations, respectively.

 

Based on the test results,EC₅₀ of FAT 36091/I TE in Daphnia magna observed for a period of 48 hour was calculated as 0.45 mg/L with upper confidence limits of 0.56 mg/L and lower confidence limits of 0.37 mg/L and regression equation Y=5.8314+2.4277 log(x). The no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for 48 hours was calculated as 0.06 mg/L and 0.13 mg/L, respectively.

Description of key information

An acute Immobilization Toxicity Study in Daphnia magna was conducted in accordance with OECD guideline 202 with FAT 36091/I TE. Test item was checked for its solubility in M4 medium and it was noted that 3.9 mg/L is the limit of solubility. Therefore, dose formulation was performed in M4 medium at a limit of 3.9 mg/L. Based on the results of the range finding experiment, main experiment was conducted with a range of concentrations viz. 0.03, 0.06, 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L. Forty daphnids with four replicates (10 daphnids in each replicate) for each test concentration and control group were maintained. The test item was formulated in M4 medium (dilution water). The daphnids with brood chambers were acclimatized one day prior to the exposure in M4 medium. After the exposure on day 0, daphnids were observed for immobilization at approximately 24 hour and 48 hour and the respective data was recorded. No immobilization was observed in control and daphnids exposed to 0.03 and 0.06 mg/L test concentrations throughout the experimental period. At the end of 24 hour, no immobility was observed in daphnids exposed to any of the test concentrations. Thus, the percent immobilization at the end of 24 hour was recorded to be 0 % in control and daphnids exposed to all the test concentrations. At the end of 48 hour, 6/40, 13/40, 21/40, 33/40 and 40/40 immobility were recorded in the daphnids exposed to 0.13, 0.28, 0.58, 1.23 and 2.57 mg/L concentrations respectively. Thus, the percent immobilization at the end of 48 hour was recorded to be 0 % in control and daphnids exposed to 0.03 and 0.06 mg/L concentration whereas, 15 %, 25 %, 52.5 %, 82.5 % and 100 % in daphnids exposed to 0.13, 0.28,0.58, and 2.57 mg/L test concentrations respectively. Analytical verification of the test item in the dilution water (M4 medium) samples were performed in the test item concentrations of low (0.03 mg/L), intermediate (0.28 mg/L) and high (2.57 mg/L) at 0 hour and 48 hour. The mean recoveries data are given below:

0 Hour: 97.81 %, 96.85 % and 95.98 % for 0.03 mg/L, 0.28 mg/Land 2.57 mg/L concentrations, respectively.

48 Hour: 94.86 %, 94.84 % and 94.50 % for 0.03 mg/L, 0.28 mg/L and 2.57 mg/L concentrations, respectively.

 

Based on the test results,EC₅₀of FAT 36091/I TE in Daphnia magna observed for a period of 48 hour was calculated as 0.45 mg/L with upper confidence limits of 0.56 mg/L and lower confidence limits of 0.37 mg/L and regression equation Y=5.8314+2.4277 log(x). The no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for 48 hours was calculated as 0.06 mg/L and 0.13 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.45 mg/L