N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

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:

Study Initiation Date: 04 August, 2015; Study Completion Date: 27 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

Specific details on test material used for the study:

Date of receipt: June 01, 2015
The following information was provided by the Sponsor:
Identification: FAT 36156/D TE
Description: Solid
Appearance: Powder
Colour: Blue
Batch number: 20140804 (China)
Expiry date: August 21, 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

Details on sampling:

During the conduct of the main experiment, 100 ml of sample was collected on the ‘0’ day of exposure and on completion at ‘48’ hour (Pooled samples from the replicates) from control and test concentrations (Low (0.001 mg/L), Intermediate (0.005 mg/L) and High (0.014 mg/L).

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
DOSE FORMULATION
RANGE FINDING EXPERIMENT
1.0010 g of the test item was made upto 1 L with M4 medium. The test dispersion was ultrasonicated for 15 minutes and kept on continuos stirring for 5 days (at room temperature in the dark). After stirring, the dispersion was left to settle for approximately 60 minutes and the same was filtered through a preconditioned filter paper. The undiluted filtrate (WAF) which was expected to be 0.78 mg/L as per previous analytical results was considered as the stock solution and used for dose formulation. From the stock solution, required concentrations viz., 0.0001, 0.001, 0.01, 0.1 and 0.78 mg/L were prepared. All the test concentrations were prepared for 200 ml (0.00002, 0.0002, 0.002, 0.02 and 0.156 mg/200 ml). From the stock solution, 25.6 µl, 256 µl, 2,056 µl, 25.64 ml were taken and made upto 200 ml each to prepare the required concentrations viz., 0.0001, 0.001, 0.01, 0.1 mg/L respectively. For 0.78 mg/L concentration, 200 ml from stock solution was used directlty. From the above prepared concentrations, 50 ml of the test solution was transfered into each replicate beaker. Thus, 4 replicates were maintained for control and treatment.

MAIN EXPERIMENT
1.0007 g of the test item was made upto 1 L with M4 medium. The test dispersion was ultrasonicated for 15 minutes and kept on continuos stirring for 5 days (at room temperature in the dark). After stirring, the dispersion was left to settle for approximately 60 minutes and the same was filtered through a preconditioned filter paper. The undiluted filtrate which was expected to be 0.78 mg/L as per previous analytical results was considered as the stock solution and used for dose formulation. From the stock solution, required concentrations viz., 0.001, 0.002, 0.003, 0.005, 0.008 and 0.014 mg/L were prepared. All the test concentrations were prepared for 300 ml (0.0003, 0.0006, 0.0009, 0.0015, 0.0024 and 0.0042 mg/300 ml). From the stock solution, 385 µl, 769 µl, 1,154 µl, 1,923 µl, 3,077 µl and 5,385 µl were taken and made upto 300 ml each to prepare the required concentrations viz., 0.001, 0.002, 0.003, 0.005, 0.008 and 0.014 mg/L respectively. From the above prepared concentrations, 50 ml of the test solution was transfered into each replicate beaker. Thus, 4 replicates were maintained for control and treatment.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: RCC Ltd. (in house)
- Age at study initiation (mean and range, SD): Young Daphnids, aged less than ‘24’ hours old

IN-LIFE Phase: Experimental start date: 05 August, 2015; Experimental completion date: 16 October, 2015

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

(M4 Medium)
Range Finding Experiment: 220 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 : 20.6 °C
Treatment : 20.6 °C

pH:

Range Finding Experiment
Control : 7.73 - 7.78
Treatment : 7.70 - 7.78

Main Experiment
Control : 7.73 - 7.77
Treatment : 7.72 - 7.77

Dissolved oxygen:

Range Finding Experiment
Control : 7.70 – 7.72 mg/L
Treatment : 7.66 – 7.72 mg/L

Main Experiment
Control : 7.71 – 7.75 mg/L
Treatment : 7.66 – 7.75 mg/L

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

A range finding experiment was conducted to determine the range of concentrations for the main experiment. In range finding experiment, the daphnids were exposed to a series of widely spaced concentrations of the test item viz., 0.0001, 0.001, 0.01, 0.1 and 0.78 mg/L.

Based on the results of the range finding experiment, seven test concentrations viz., 0.001, 0.002, 0.003, 0.005, 0.008 and 0.014 mg/L were fixed.

Details on test conditions:

TEST SYSTEM
Test Method: Static
Test Vessel: 100 ml Glass Beaker
No. of Replicate: Control - 4 replicates
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 darkness
Feeding: Daphnids were not fed during the test
Dilution Water: M4 Medium
Volume: 50 ml
Test Duration: '48' hours

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate Pure

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.003 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.007 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.005 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 and 0.001 mg/L concentrations throughout the experimental period. At the end of 24- hour observation period, no immobility was observed in daphnids exposed to 0.01 mg/L test concentration whereas, 10/40 and 34/40 immobility were observed in daphnids exposed to 0.1 and 0.78 mg/L concentration, respectively. Thus, the percent immobilisation at the end of 24 hour observation period was recorded to be 0 % in control and daphnids exposed to 0.0001, 0.001 and 0.01 mg/L concentration whereas 25 % and 85 % were observed in daphnids exposed to 0.1 and 0.78 mg/L concentration, respectively. At the end of 48 hour observation period, 40/40 immobility was observed in the daphnids exposed to 0.01 mg/L concentration and 40/40 cumulative immobility was observed in daphnids exposed to 0.1 and 0.78 mg/L concentrations. Thus, the percent cumulative immobilisation at the end of 48 hour observation period was recorded to be 0 % in control and daphnids exposed to 0.0001 and 0.001 mg/L test concentration whereas, 100 % was observed in daphnids exposed to 0.01, 0.1 and 0.78 mg/L concentrations.

MAIN EXPERIMENT- IMMOBILIZATION
No immobilization was observed in control and daphnids exposed to 0.001, 0.002 and 0.003 mg/L concentrations throughout the experimental period. At the end of 24 hour observation period, no immobility was observed in daphnids exposed to all the test concentrations. Thus, the percent immobilisation at the end of 24 hour observation period was recorded to be 0 % in control and daphnids exposed to all the test concentrations. At the end of 48 hour observation period, 7/40, 21/40 and 40/40 immobility was recorded in the daphnids exposed to 0.005, 0.008 and 0.014 mg/L concentrations respectively. Thus, the percent cumulative immobilisation at the end of 48 hour observation period was recorded to be 0 % in control and daphnids exposed to 0.001, 0.002 and 0.003 mg/L test concentration whereas, 17.5 %, 52.5 % and 100 % was observed in daphnids exposed to 0.005, 0.008 and 0.014 mg/L concentrations, respectively.

CONCLUSION
Based on the test results, the EC50 of FAT 36156/D TE in Daphnia magna observed for a period of 48 hour was calculated as 0.0073 mg/L.
The no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for 48 hours was calculated as 0.003 mg/L and 0.005 mg/L 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).

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of FAT 36156/D TE in Daphnia magna observed for a period of 48-hours was calculated as 0.0073 mg/L with upper confidence limits of 0.0081 mg/L and lower confidence limits of 0.0066 mg/L.

Executive summary:

Acute Immobilization Toxicity Study in Daphnia magna was conducted in accordance with OECD guideline 202 with FAT 36156/D TE. The test item was not soluble until 1 mg/L in M4 medium. Therefore, based on the sponsor communication, Water Accomodated Fraction (WAF) was prepared and it was analytically recorded that 0.78 mg/L as the limit of solubility of the test item in the M4 medium. The WAF filtrate was used for the dose formulation.

Based on the results of the range finding experiment, a main experiment was conducted with a range of concentrations viz.,0.001, 0.002, 0.003, 0.005, 0.008 and 0.014 mg/L. Forty daphnids with four replicates (10 daphnids in each replicate) for each test concentration and control group was maintained. 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 immobilisation at approximately 24 hour and 48 hour and the respective data were recorded.

No immobilization was observed in control and daphnids exposed to 0.001, 0.002 and 0.003 mg/L concentrations throughout the experimental period. At the end of 24-hour observation period, no immobility was observed in daphnids exposed to all the test concentrations. Thus, the percent immobilisation at the end of 24-hour observation period was recorded to be 0 % in control and daphnids exposed to all the test concentrations. At the end of 48-hour observation period, 7/40, 21/40 and 40/40 immobility was recorded in the daphnids exposed to 0.005, 0.008 and 0.014 mg/L concentrations, respectively. Thus, the percent cumulative immobilisation at the end of 48-hour observation period was recorded to be 0 % in control and daphnids exposed to 0.001, 0.002 and 0.003 mg/L test concentration whereas, 17.5 %, 52.5 % and 100 % was observed in daphnids exposed to 0.005, 0.008 and 0.014 mg/L concentrations, respectively.

Analytical verification of the test item in the dilution water (M4 medium) samples were performed in the test item concentrations of low (0.001 mg/L), intermediate (0.005 mg/L) and high (0.014 mg/L) at 0-hour and 48-hour time point. The mean recoveries data are given below:

·      ‘0’ hour  – Mean Recovery results: BDL, BDL and96.93 % for 0.001mg/L, 0.005 mg/L and 0.014 mg/Lconcentrations respectively.

·      ‘48’ hour  – Mean Recovery results: BDL, BDL and94.90 % for 0.001mg/L, 0.005 mg/L and 0.014 mg/Lconcentrations respectively.

 

CONCLUSION

Based on the test results, the EC₅₀ of FAT 36156/D TE in Daphnia magna observed for a period of 48 hour was calculated as 0.0073 mg/L with upper confidence limits of 0.0081 mg/L and lower confidence limits of 0.0066 mg/L and regression equation Y = 19.9244 + 6.9831 log (x). The no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for 48-hours observation period was calculated as 0.003 mg/L and 0.005 mg/L, respectively.

Description of key information

Acute Immobilization Toxicity Study in Daphnia magna was conducted in accordance with OECD guideline 202 with FAT 36156/D TE. Based on the results of the range finding experiment, a main experiment was conducted with a range of concentrations viz.,0.001, 0.002, 0.003, 0.005, 0.008 and 0.014 mg/L. Forty daphnids with four replicates (10 daphnids in each replicate) for each test concentration and control group was maintained. No immobilization was observed in control and daphnids exposed to 0.001, 0.002 and 0.003 mg/L concentrations throughout the experimental period. At the end of 24-hour test period, no immobility was observed in daphnids exposed to all the test concentrations. Thus, the percent immobilisation at the end of 24-hour test period was recorded to be 0 % in control and daphnids exposed to all the test concentrations. At the end of 48-hour tet period, 7/40, 21/40 and 40/40 immobilities were recorded in the daphnids exposed to 0.005, 0.008 and 0.014 mg/L concentrations, respectively. Thus, the percent cumulative immobilisation at the end of 48 hour observation period was recorded to be 0 % in control and daphnids exposed to 0.001, 0.002 and 0.003 mg/L test concentration whereas, 17.5 %, 52.5 % and 100 % was observed in daphnids exposed to 0.005, 0.008 and 0.014 mg/L concentrations, respectively. Based on the test results, the EC₅₀of FAT 36156/D TE in Daphnia magna observed for a period of 48 hour was calculated as 0.0073 mg/L with upper confidence limits of 0.0081 mg/L and lower confidence limits of 0.0066 mg/L and regression equation Y = 19.9244 + 6.9831 log (x). The no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for 48-hours was calculated as 0.003 mg/L and 0.005 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

0.007 mg/L