Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

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:

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:

Identification: FAT 20010/E
Description: Solid
Appearance: Powder
Colour: Acid Orange
Batch number: 59015 (India)
CASNo: 56819-40-0
Purity: 70.9 %
Molecular weight: 851.61 g/mol
Storage conditions: Room temperature in the dark

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

15.02 mg of the test item was weighed and made up to 15 ml using 20X-AAP growth medium to make a stock concentration of 1mg/ml. From the stock solution, each required test concentration was prepared as follows: 8 µl, 18 µl, 60 µl, 264 µl, 630 µl, 2016 µl and 8592 µl was taken and made up to 600 ml except for 0.008, 0.264 and 8.592 mg/800 ml with 20X-AAP medium. 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)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Post exposure observation period:

Frond number in each replicate was counted on days ‘0’, ‘3’, ‘5’ and ‘7’. A representative sample (approximately 12 fronds) of pre-culture was checked for its average dry weight on day ‘0’ of exposure and dry weight of plant material from each test and control vessel on day ‘7’ of exposure. Observation was performed on day ‘0’, ‘3’, ‘5’ and ‘7’ for the abnormal appearance or any developmental changes due to the effect of the test item (if any).

Test temperature:

Start of the test
Control: 23.5 °C
Treatment: 23.5 °C

End of the test
Control: 24.5 - 24.8 °C
Treatment: 24.6 - 25.8 °C

pH:

Start of the test
Control: 7.54 °C
Treatment: 7.52 - 7.54 °C

End of the test
Control: 8.83 - 8.86
Treatment: 8.59 - 8.85

Nominal and measured concentrations:

0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L which is equivalent to 0.008 mg/800mL, 0.018 mg/600mL, 0.06 mg/600mL, 0.264 mg/800mL, 0.63 mg/600mL, 2.016 mg/600mL and 8.592 mg/800mL respectively.

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: 8440 - 8580 Lux

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

8.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

8.15 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.:

0.72 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.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and dry weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and dry weight

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.

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 20010/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 8.03 mg/L.
EyC50 of FAT 20010/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 1.33 mg/L.
ErC50 of FAT 20010/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 8.15 mg/L.
EyC50 of FAT 20010/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 0.72 mg/L.

Executive summary:

Following OECD guideline 221,Lemna gibba (Duckweed) were exposed to the test item (FAT 20010/E TE) at different test concentrations viz.,0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L. Prior to exposure, Lemna gibba was pre-cultured in the 20x-AAP medium for period of ‘8’ days and the pre-culture was found healthy. After exposure, Lemna gibba was observed for frond count on days ‘3’, ‘5’ and ‘7’. Dry weight of the fronds was recorded at start (pre-culture representative samples) and at the end of the test. All fronds were found normal and healthy in all replicates of control and test concentrations except 10.74 mg/L where necrosis and root filaments were turned brown colour on day ‘7’.

Frond Numbers:

From the average specific growth rate recorded with the different test concentrations viz.,0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L the test concentration that inhibited 50% growth (ErC50) was calculated as 8.03 mg/L with upper confidence limits of 8.93 mg/L and lower confidence limits of 7.23 mg/L. The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L, was calculated as 0.00 %, 0.61 %, 7.77 %, 8.83 %, 20.45 %, 24.53 % and 59.85 %, respectively. From the yield recorded with the different test concentrations viz.,0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L the concentration that inhibited 50 % yield (EyC50) was calculated as 1.33 mg/L with upper confidence limits of 1.45 mg/L and lower confidence limits of 1.22 mg/L. The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L was calculated as 0.00%, 1.90 %, 21.87 % 24.49 %, 48.25 %, 54.81 % and 87.76 %, respectively. The no observed effect concentration (NOEC) was calculated as 0.01 mg/L and the low observed effect concentration (LOEC) was calculated as 0.03 mg/L, respectively.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrations viz.,0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L, the concentration that inhibited 50 % growth (ErC50) was calculated as 8.15 mg/L with upper confidence limits of 9.33 mg/L and lower confidence limits of 7.12 mg/L. The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L was calculated as 0.03 %, 2.09 %, 13.42 %, 18.17 %, 21.73 %, 27.74 % and 58.31 %, respectively. From the yield recorded with the test concentrationsviz.,0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L the concentration that inhibited 50% yield (EyC50) was calculated as 0.72 mg/L with upper confidence limits of 0.78 mg/L and lower confidence limits of 0.66 mg/L. The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/L was calculated as 0.09 %, 6.75 %, 36.52 %, 46.14 %, 52.41 %, 61.53 % and 88.24 %, respectively. The no observed effect concentration (NOEC) was calculated as 0.01 mg/L and the low observed effect concentration (LOEC) was calculated as 0.03 mg/L, respectively.

ErC50 of FAT 20010/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 8.03 mg/L.

EyC50 of FAT 20010/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 1.33 mg/L.

ErC50 of FAT 20010/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 8.15 mg/L.

EyC50 of FAT 20010/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 0.72 mg/L.

Description of key information

7 day ErC50 based upon the frond number is 8.03 mg/L (8.93 - 7.23 mg/L).
7 day EyC50 based upon the frond numbers is 1.33 mg/L (1.45 - 1.22 mg/L).
7 day ErC50 based upon the frond dry weight is 8.15 mg/L (9.33 - 7.12 mg/L).
7 day EyC50 based upon the frond dry weight is 0.72 mg/L (0.79 - 0.66 mg/L).

Key value for chemical safety assessment

EC50 for freshwater plants:

8.03 mg/L

EC10 or NOEC for freshwater plants:

0.01 mg/L

Additional information

In a GLP-compliant OECD 221 guideline study Lemna gibba (Duckweed) were exposed to the test item FAT 20010/E at different test concentrations viz., 0.01, 0.03, 0.1, 0.33, 1.05, 3.36 and 10.74 mg/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:

 

7-day ErC50 based upon the frond number is 8.03 mg/L (8.93 - 7.23 mg/L).

7-day EyC50 based upon the frond number is 1.33 mg/L (1.45 - 1.22 mg/L).

7-day ErC50 based upon the frond dry weight is 8.15 mg/L (9.33 - 7.12 mg/L).

7-day EyC50 based upon the frond dry weight is 0.72 mg/L (0.79 - 0.66 mg/L).

 

Considering frond number, the no observed effect concentration (NOEC) was calculated as 0.01 mg/L and the low observed effect concentration (LOEC) was calculated as 0.03 mg/L, respectively. Considering frond dry weight, the no observed effect concentration (NOEC) was calculated as 0.15 mg/L and the low observed effect concentration (LOEC) was calculated as 0.45 mg/L, respectively.