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

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:

Experimental starting date: 04 August 2015; Experimental completion date: 15 October 2015; Study completion date: 28 November 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

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:

not specified

Details on test solutions:

For range finding experiment the concentrations selected were 0.0001, 0.001, 0.01, 0.1, 1 and 3.9 mg/L which is equivalent to 0.00003, 0.0003, 0.003, 0.03, 0.3 and 1.17 mg/300ml, respectively. 10.01 mg of the test item was received and from which 3.9 mg/L was reweighed and dissolved and made up to 1000 ml with test medium (OECD) in standard flask to make a stock concentration of 0.0039 mg/ml. All the required test concentrations were prepared for 300 ml. For 0.00003, 0.0003, 0.003, 0.03, 0.3 and 1.17 mg/300ml, 7.7 µl, 76.9 µl, 769.2 µl, 7.692 ml, 76 ml and 300 ml was taken from stock and made up to 300 ml with OECD medium respectively. For 1.17 mg/300ml 300 ml was taken from stock. For Control, OECD medium was used. For limit Test 10.03 mg of the test item was received and from which 3.9 mg/L was reweighed and dissolved and made up to 1000 ml with test medium (OECD) in standard flask and ultrasonicated for 20 minutes, the test medium was stirred well before transferring 100 ml into each of the replicate conical flask for control group OECD medium was used.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Test species: Unicellular green alga
- Sub-culture: In-house Algae culture, RCC Laboratories India Private Limited.
- Species- Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): ATCC 22662 (Procured from LGC Promochem India Pvt. Ltd, Bangalore 560 058).
- Inoculation:
Range Finding Experiment: 25375 cells/mL
Limit test: 17375 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.5 °C – 23.8 °C

pH:

Limit Test
Start of the test (day '0') in bulk of medium
Control: 8.10
Treatment: 8.06
End of the test (72 hour)
Control: 8.63 – 8.67
Treatment: 8.13 – 8.16

Nominal and measured concentrations:

For range finding experiment:
The concentrations selected were 0.0001, 0.001, 0.01, 0.1, 1 and 3.9 mg/L which is equivalent to 0.00003, 0.0003, 0.003, 0.03, 0.3 and 1.17 mg/300ml, respectively. 10.01 mg of the test item was received and from which 3.9 mg/L was reweighed and dissolved and made up to 1000 ml with test medium (OECD) in standard flask to make a stock concentration of 0.0039 mg/ml.

Limit test: For limit Test 10.03 mg of the test item was received and from which 3.9 mg/L was reweighed and dissolved and made up to 1000 ml with test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flask
- Range finding Initial cells density: 25375 cells/mL
- Limit test: 17375 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: Continuous cool white Light Intensity
Range Finding Experiment: 6720 – 6820 Lux
Limit Test: 6760 – 6880 Lux

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

The ErC50 of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga (Pseudokirchneriella subcapitata) was found to be >3.9 mg/L.
The EyC50 of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga (Pseudokirchneriella subcapitata) was found to be >3.9 mg/L

Results with reference substance (positive control):

The ErC50 of Potassium Dichromate observed for a period of ‘72’ hour for freshwater alga (Pseudokirchneriella subcapitata) was found to be 2.14 mg/L.
The EyC50 of Potassium Dichromate observed for a period of ‘72’ hour for freshwater alga (Pseudokirchneriella subcapitata) was found to be 0.57 mg/L

Reported statistics and error estimates:

Mean coefficient of variation of average specific growth rate in replicate control and treated cultures were calculated. No analysis was done as the study is conducted with the Limit Test.

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga (Pseudokirchneriella subcapitata) was found to be >3.9 mg/L.
The EyC50 of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga (Pseudokirchneriella subcapitata) was found to be >3.9 mg/L

Executive summary:

Growth Inhibition study was conducted with Freshwater green Algae (Pseudokirchneriella subcapitata) in accordance with OECD 201 guideline with FAT 36091/I TE. The test was conducted as limit test with concentration of 3.9 mg/L. Test item was dissolved in OECD medium. Initially on ‘0’day approximately 17,375 cells/mL were inoculated in a conical flask containing OECD medium, the volume of OECD medium was 100 ml each in control and treatment group. Six replicates were maintained for control and the limit test concentration (3.9 mg/L). Prior to the exposure pre-culture was maintained for a period of 3 days. After the exposure on day ‘0’ the algal cells were observed microscopically and the cell count was done at 24, 48 and 72 hours by using Hemocytometer. Microscopic observations revealed that the cells were normal and no abnormal appearance of cells were observed due on exposure to the test item.

 

Average Specific growth Rate:

The concentration that inhibited 50 % growth (ErC50) was calculated as > 3.9 mg/L.

The percent inhibition on the average specific growth rate at the end of 72 hours for the limit test concentration of 3.9 mg/L was calculated as 0.15 %.

The no observed effect concentration (NOEC) was calculated as >3.9mg/L.

 

Yield:

The concentration that inhibited 50 % yield (EyC50) was calculated as > 3.9mg/L. The percent inhibition on the yield at the end of 72 hours for the limit test concentration of 3.9 mg/L was calculated as 0.02 %. The no observed effect concentration (NOEC) was calculated as >3.9 mg/L.

Analytical verification of the test item in the test medium sample was carried out in the test item concentration of 3.9 mg/L. The samples at 0 hour and 72 hour were analyzed. The mean recoveries data are given below:

- 0 Hour – Mean Recovery results: 97.59 % for 3.90 mg/L

- 72 Hours– Mean Recovery results: 96.50 % for 3.90 mg/L

CONCLUSION

Based on the test results,

The ErC50of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga was found to be >3.9 mg/L.

The EyC50of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga was found to be >3.9 mg/L.

Description of key information

A growth Inhibition study was conducted with Freshwater green Algae (Pseudokirchneriella subcapitata) in accordance with OECD 201 guideline with FAT 36091/I TE. The test was conducted as limit test with concentration of 3.9 mg/L. Test item was dissolved in OECD medium. Initially on ‘0’day approximately 17,375 cells/mL were inoculated in a conical flask containing OECD medium, the volume of OECD medium was 100 ml each in control and treatment group. Six replicates were maintained for control and the limit test concentration (3.9 mg/L). Prior to the exposure pre-culture was maintained for a period of 3 days. After the exposure on day ‘0’ the algal cells were observed microscopically and the cell count was done at 24, 48 and 72 hours by using Hemocytometer. Microscopic observations revealed that the cells were normal and no abnormal appearance of cells were observed due on exposure to the test item.

 

Average Specific growth Rate:

The concentration that inhibited 50 % growth (ErC50) was calculated as >3.9 mg/L.

The percent inhibition on the average specific growth rate at the end of 72 hour for the limit test concentration of 3.9 mg/L was calculated as 0.15 %.

The no observed effect concentration (NOEC) was calculated as >3.9 mg/L.

 

Yield:

The concentration that inhibited 50 % yield (EyC50) was calculated as >3.9mg/L. The percent inhibition on the yield at the end of  72 hour for the limit test concentration of 3.9 mg/L was calculated as 0.02 %. The no observed effect concentration (NOEC) was calculated as > 3.9 mg/L. Analytical verification of the test item in the test medium sample was carried out in the test item concentration of 3.9 mg/L. The samples at ‘0’ hour and ‘72’ hour were analyzed. The mean recoveries data are given below:

- 0 Hour – Mean Recovery results: 97.59 % for 3.90 mg/L

- 72 Hours– Mean Recovery results: 96.50 % for 3.90 mg/L

CONCLUSION

Based on the test results,

The ErC50of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga was found to be >3.9 mg/L.

The EyC50of FAT 36091/I TE observed for a period of ‘72’ hour for freshwater alga was found to be >3.9 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.9 mg/L