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

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

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 221 (Lemna sp. Growth Inhibition 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:

In Limit test, 100 ml of sample was collected from prepared bulk on the 0 day of exposure from control and limit test concentration (0.73 mg/L) whereas, on the day ‘7’, samples were collected from the pooled replicates of control and limit test concentration (0.73 mg/L) and sent to the Analytical Chemistry Department at RCC Laboratories.

Vehicle:

yes

Details on test solutions:

DOSE FORMULATION
WAF was prepared with 20X-AAP medium and the undiluted filtrate was used for dose formulation.

RANGE FINDING EXPERIMENT
1.0002 g of the test item was made up to 1 L with 20X-AAP medium. The test dispersion was ultrasonicated for 15 minutes and kept on continuous 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 0.73 mg/L as the limit of solubility of the test item was considered as the stock solution and used for dose formulation.
All the required test concentrations were prepared for 600 ml. From the stock solution, each required test concentration was prepared as follows: 822 µl, 8,219 µl, 82,192 µl and 410,959 µl were taken and made up to 600 ml each with 20X-AAP medium to prepare 0.001, 0.01, 0.1 and 0.5 mg/L concentrations respectively. For 0.73 mg/L concentration, stock solution was used directly. For Control, 20X-AAP medium was used.

LIMIT TEST
2.0006 g of the test item was made upto 2 L with 20X-AAP medium. The test dispersion was ultrasonicated for 15 minutes and kept on continuous 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 0.73 mg/L as the limit of solubility of the test item was considered as the stock solution and used for dose formulation.
For 100 mg/L concentration, the WAF filtrate (147 mg/L) was considered as the stock from which 100 mg/L was prepared. For Control, 20X-AAP medium was used.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Strain: Lemna gibba (Duckweed)
- Source (laboratory, culture collection): In-house culture, RCC Laboratories India Private Limited (Originally procured by Department of Plant Biology & Plant Biotechnology Madras Christian College, Chennai)

IN LIFE phase: Experimental start date: 05 August, 2015; Experimental completion date: 28 October, 2015

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Hardness:

None

Test temperature:

Start of the test
Control: 23.1
Treatment: 23.1
End of the test
Control: 24.5 - 24.6
Treatment: 24.8 - 25.0

pH:

Start of the test
Control: 7.51
Treatment: 7.44
End of the test
Control: 8.64 - 8.66
Treatment: 8.52 - 8.53

Nominal and measured concentrations:

Range finding test: 0.001, 0.01, 0.1, 0.5 and 0.73 mg/L
Limit test concentration: 0.73 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Glass Beaker
- No. of organisms per vessel: 12 fronds (3 plants)
- No. of vessels per concentration (replicates): Limit test - 6 replicates
- No. of vessels per control (replicates): Control - 6 Replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Start of the test

- Photoperiod: Continuous Light
- Light intensity and quality: 8520 - 8640 Lux

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond Dry Weight; Growth

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond Dry Weight; Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 0.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and dry weight

Details on results:

PERCENT INHIBITION OF AVERAGE GROWTH RATE
RANGE FINDING EXPERIMENT
BASED ON FROND NUMBERS:
The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.001, 0.01, 0.1, 0.5 and 0.73 mg/L was calculated as 0.02 %, 0.02 %, 0.02 %, 0.07 % and 0.07 %, respectively.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the average growth rate on day ‘7’ was calculated as 0 % in 0.001 mg/L and 0.64 % in all the other tested concentrations viz., 0.01, 0.1, 0.5 and 0.73 mg/L (Refer Section 11).

LIMIT TEST
BASED ON FROND NUMBERS:
The percent inhibition on the average specific growth rate on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0.16 %.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the average specific growth rate on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0 % (Refer Section 17).

AVERAGE SPECIFIC GROWTH RATE
LIMIT TEST
BASED ON FROND NUMBERS
The concentration that inhibited 50 % growth (ErC50) was calculated as >0.73mg/L.
BASED ON FROND DRY WEIGHT:
The concentration that inhibited 50 % growth (ErC50) was calculated as >0.73mg/L.

PERCENT GROWTH INHIBITION OF YIELD
RANGE FINDING EXPERIMENT
BASED ON FROND NUMBERS:
The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.001, 0.01, 0.1, 0.5 and 0.73 mg/L was calculated as 0.07 %, 0.07 %, 0.07 %, 0.22 % and 0.22 %, respectively.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.001, 0.01, 0.1, 0.5 and 0.73 mg/L was calculated as 0.20 %, 2.51 %, 2.34 %, 2.25 % and 2.11 %, respectively.

LIMIT TEST
BASED ON FROND NUMBERS:
The percent inhibition on the yield on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0.50 %.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the yield on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0.03 %.

YIELD
LIMIT TEST
BASED ON FROND NUMBERS:
The concentration that inhibited 50 % yield (EyC50) was calculated as >0.73 mg/L.
BASED ON FROND DRY WEIGHT:
The concentration that inhibited 50 % yield (EyC50) was calculated as >0.73 mg/L.

NOEC ESTIMATION
LIMIT TEST
BASED ON FROND NUMBERS:
The no observed effect concentration (NOEC) was calculated as >0.73mg/L.
BASED ON FROND DRY WEIGHT:
The no observed effect concentration (NOEC) was calculated as >0.73mg/L.

ANALYTICAL MEASUREMENTS (LIMIT TEST)
Analytical verification of the test item in the exposure medium samples were carried out in the limit test item concentrations (0.73 mg/L) days ‘0’ and 7. The mean recoveries data are given below:
• Day ‘0’ – Mean Recovery results: 97.40 % for 0.73 mg/L.
• Day ‘7’ – Mean Recovery results: 96.28 % for 0.73 mg/L.

Results with reference substance (positive control):

ErC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 4.30 mg/L with a lower confidence limit of 4.15 mg/L and the upper confidence limit of 4.46 mg/L.
EyC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 2.38 mg/L with a lower confidence limit of 2.29 mg/L and the upper confidence limit of 2.48 mg/L.
ErC50 of 3,5-Dichlorophenol based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 3.02 mg/L with a lower confidence limit of 2.89 mg/L and the upper confidence limit of 3.16 mg/L.
EyC50 of 3,5-Dichlorophenol based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 1.53 mg/L with a lower confidence limit of 1.46 mg/L and the upper confidence limit of 1.59 mg/L.
Based on the above findings the sensitivity of the test system (Lemna gibba) and reliability of the experimental techniques is proved.

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 concluded with the Limit Test.

Validity criteria fulfilled:

yes

Conclusions:

ErC50 of FAT 36156/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as >0.73 mg/, while the NOEC was estimated as 0.73 mg/L.

Executive summary:

Lemna gibba (Duckweed) were exposed to the test item (FAT 36156/D TE) according to OECD guideline 221, at a limit test concentration of 0.73 mg/L. The test item was not soluble until 1mg/L in 20X-AAP medium. Therefore, based on the sponsor communication, Water Accommodated Fraction (WAF) method was prepared and it was analytically recorded that 0.73 mg/L as the limit of solubility of the test item in the 20X-AAP medium. The WAF filtrate was used for the dose formulation. Initially on day ‘0’, 12 fronds were inoculated in beaker containing 200 ml of test medium (20X-AAP medium). Six replicates for limit test concentration and six replicates for control were maintained. The test item was formulated in 20X-AAP medium. After exposure,Lemna gibba was observed for frond count on days ‘3’, ‘5’ and ‘7’. Dry weight of the fronds were recorded at start (pre-culture representative samples) and at the end of the test. Fronds were observed for their appearance on days ‘3’, ‘5’ and ‘7’. All fronds were found normal and healthy in all replicates of control and limit test concentration. The doubling time (T_d) of frond number in the control group was calculated as 1.61day^-1.

Frond Numbers:

The concentration that inhibited 50 % growth (E_rC₅₀) was calculated as >0.73 mg/L.

The percent inhibition on the average specific growth rate on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0.16 %.

The concentration that inhibited 50 % yield (E_yC₅₀) was calculated as >0.73 mg/L.

The percent inhibition on the yield on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0.50 %.

The no observed effect concentration (NOEC) was calculated as 0.73 mg/L.

 

Frond Dry Weight:

The concentration that inhibited 50 % growth (E_rC₅₀) was calculated as >0.73 mg/L.

The percent inhibition on the average specific growth rate on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0 %.

The concentration that inhibited 50 % yield (E_yC₅₀) was calculated as >0.73 mg/L.

The percent inhibition on the yield on day ‘7’ for the limit test concentration of 0.73 mg/L was calculated as 0.03 %.

The no observed effect concentration (NOEC) was calculated as 0.73 mg/L.

 

Description of key information

In a GLP-compliant OECD 221 guideline study, FAT 36156/D was evaluated for growth inhibition when exposure Lemna gibba. Based on the outcome of the range finding study, a limit test was conducted at 0.73mg/L.Post exposure, Lemna gibba was observed for frond count on days 3, 5 and 7.

The following observations were made till the end of day 7:

ErC50 of FAT 36156/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as > 0.73 mg/L.

EyC50 of FAT 36156/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as > 0.73 mg/L.

ErC50 of FAT 36156/D TE based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba calculated as > 0.73 mg/L.

EyC50 of FAT 36156/D TE based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba calculated as > 0.73 mg/L.

Considering frond number the no observed effect concentration (NOEC) was calculated as 0.73 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

0.73 mg/L

EC10 or NOEC for freshwater plants:

0.73 mg/L