Disodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2-)]chromate(2-)

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 21095/D
Description: Solid
Appearance: Powder
Colour: Brown
Batch/ Lot number: 2004 (Thailand)
CAS Number: 52587-68-5
Purity: >85 %
Molecular weight: 835.70 g/mol

Analytical monitoring:

yes

Details on sampling:

In main experiment, 100 ml of sample was collected from prepared bulk on the 0 day of exposure from control, low (0.05 mg/L), intermediate (1.35 mg/L) and high (12.15 mg/L) concentrations whereas on the 7th day, samples were collected from the pooled replicates of control, low (0.05 mg/L), intermediate (1.35 mg/L) and high (12.15 mg/L) concentrations and sent to the Analytical Chemistry Department at RCC Laboratories India Private Limited. The verification of effective test concentration was adopted using a prescribed validated analytical method.

Vehicle:

yes

Details on test solutions:

10.01 mg of the test item was dissolved and made up to 10 ml using 20X-AAP growth medium to make a stock concentration of 1 mg/ml. All the required test concentrations were prepared for 600 ml. For 1 and 10 mg/L concentrations 600 µl and 6 ml was taken from stock and made up to 600 ml with 20X-AAP medium respectively.

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:

In main experiment, 100 ml of sample was collected from prepared bulk on the 0 day of exposure from control, low (0.05 mg/L), intermediate (1.35 mg/L) and high (12.15 mg/L) concentrations whereas on the 7th day, samples were collected from the pooled replicates of control, low (0.05 mg/L), intermediate (1.35 mg/L) and high (12.15 mg/L) concentrations and sent to the Analytical Chemistry Department at RCC Laboratories India Private Limited. The verification of effective test concentration was adopted using a prescribed validated analytical method. 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: 22.7
Treatment: 22.7
End of the test (Refer Section 20)
Control: 25.0 - 25.4
Treatment: 25.0 - 25.4

pH:

Start of the test
Control: 7.58
Treatment: 7.58 - 7.53
End of the test (Refer Section 19)
Control: 8.73 - 8.76
Treatment: 8.56 - 8.76

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): Main Experiment - 3 replicates for each concentration
- No. of vessels per control (replicates): Control - 6 Replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous Light
- Light intensity and quality: 8320-8580 Lux

TEST CONCENTRATIONS
- Range finding study: 10.01 mg of the test item was dissolved and made up to 10 ml using 20X-AAP growth medium to make a stock concentration of 1 mg/ml. All the required test concentrations were prepared for 600 ml. For 1 and 10 mg/L concentrations 600 µl and 6 ml was taken from stock and made up to 600 ml with 20X-AAP medium respectively. 15.03, 30.02 and 60.01 mg of the test item was added to 600 ml of 20X-AAP medium each to prepare the 25, 50, 100 mg/L concentrations respectively. For Control, 20X-AAP medium was used.

- Test concentrations: 50.03 mg of the test item was weighed and made up to 50 ml using 20X-AAP growth medium to make a stock concentration of 1 mg/ml. All the required test concentrations were prepared for 800 ml. From the stock solution, each required test concentration was prepared as follows: 40 µl, 120 µl, 360 µl, 1080 µl, 3240 µl and 9720 µl were taken and made up to 800 ml with 20X-AAP medium to obtain 0.05, 0.15, 0.45, 1.35, 4.05, and 12.15 mg/L concentrations respectively. For Control, 20X-AAP medium was used.

Reference substance (positive control):

yes

Remarks:

3,5- Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

8.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

6.89 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.:

2.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond Dry Weight; Yield

Results with reference substance (positive control):

ErC50of 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/Land 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.

Validity criteria fulfilled:

yes

Conclusions:

ErC50 of FAT 21095/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 8.18 mg/L with upper confidence limits of 8.61mg/L and lower confidence limits of 7.78 mg/L.
EyC50 of FAT 21095/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 3.77 mg/L with upper confidence limits of 3.95 mg/L and lower confidence limits of 3.59 mg/L.
ErC50 of FAT 21095/D TE based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 6.89 mg/L with upper confidence limits of 7.25 mg/L and lower confidence limits of 6.54 mg/L.
EyC50 of FAT 21095/D TE based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 2.91 mg/L with upper confidence limits of 3.08 mg/L and lower confidence limits of 2.76 mg/L.

Executive summary:

A GLP compliant test was performed according to OECD guideline 221 to assess the acute toxicity of FAT 21095/D on aquatic plant Lemna gibba (Duckweed). Lemna gibba were exposed to the test item (FAT 21095/D) at different test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L. Initially on day ‘0’, 12 fronds were inoculated in beaker containing 200 ml of test medium (20X-AAP medium). Three replicates for each test concentration and six replicates for control were maintained. The test item was formulated in 20X-AAP medium. 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 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 test concentrations except 12.15 mg/L where necrosis and root filaments were turned brown colour onday ‘7’. The doubling time (Td) of frond number in the control group was calculated as 1.61 day-1.

 

Frond Numbers: From the average specific growth rate recorded with the different test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the test concentration that inhibited 50 % growth (ErC50) was calculated as 8.18 mg/L with upper confidence limits of 8.61 mg/L and lower confidence limits of 7.78 mg/L The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L, was calculated as 0.09 %, 0.13 %, 2.68 %, 5.66 %, 25.35 % and 67.95 % respectively. From the yield recorded with the different test concentrationsviz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50 % yield (EyC50) was calculated as 3.77 mg/L with upper confidence limits of 3.95 mg/L and lower confidence limits of 3.59 mg/L. The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L was calculated as 0.28 %, 0.42 %, 8.20 % 16.55 %, 56.29 % and 92 % respectively. 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.

 

Frond Dry Weight: From the average specific growth rate recorded with the test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L, the concentration that inhibited 50 % growth (ErC50) was calculated as 6.89 mg/L with upper confidence limits of 7.25 mg/L and lower confidence limits of 6.54 mg/L. The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L was calculated as 0.99 %, 1.43 %, 5.28 %, 7.34 % 22.71 % and 78.67 %, respectively. From the yield recorded with the test concentrationsviz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50 % yield (EyC50) was calculated as 2.91 mg/L with upper confidence limits of 3.08 mg/L and lower confidence limits of 2.76 mg/L. The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L was calculated as 3.38 %, 4.83 %, 16.57 %, 22.30 %, 54.55 % and 96.04 %, respectively. 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.

Description of key information

ErC50 of FAT 21095/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 8.18 mg/L.
EyC50 of FAT 21095/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 3.77  mg/L.
ErC50 of FAT 21095/D TE based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 6.89 mg/L.
EyC50 of FAT 21095/D TE based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 2.91 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

8.18 mg/L

Additional information

A GLP-compliant test was performed according to OECD guideline 221 to assess the acute toxicity of FAT 21095/D on aquatic plantLemna gibba (Duckweed). Lemna gibba were exposed to the test item (FAT 21095/D) at different test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L. Initially on day ‘0’, 12 fronds were inoculated in beaker containing 200 ml of test medium (20X-AAP medium). Three replicates for each test concentration and six replicates for control were maintained. The test item was formulated in 20X-AAP medium. 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 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 test concentrations except 12.15 mg/L where necrosis and root filaments were turned brown colour onday ‘7’. The doubling time (Td) of frond number in the control group was calculated as 1.61day-1.

 

Frond Numbers: From the average specific growth rate recorded with the different test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the test concentration that inhibited 50 % growth (ErC50) was calculated as 8.18 mg/L with upper confidence limits of 8.61 mg/L and lower confidence limits of 7.78 mg/L. The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L, was calculated as 0.09 %, 0.13 %, 2.68 %, 5.66 %, 25.35 % and 67.95 % respectively. From the yield recorded with the different test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50 % yield (EyC50) was calculated as 3.77 mg/L with upper confidence limits of 3.95 mg/L and lower confidence limits of 3.59 mg/L. The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L was calculated as 0.28 %, 0.42 %, 8.20 % 16.55 %, 56.29 % and 92 % respectively. 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.

 

Frond Dry Weight: From the average specific growth rate recorded with the test concentrations viz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L, the concentration that inhibited 50 % growth (ErC50) was calculated as 6.89 mg/L with upper confidence limits of 7.25 mg/L and lower confidence limits of 6.54 mg/L. The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L was calculated as 0.99 %, 1.43 %, 5.28 %, 7.34 % 22.71 % and 78.67 % respectively. From the yield recorded with the test concentrationsviz.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50 % yield (EyC50) was calculated as 2.91mg/L with upper confidence limits of 3.08 mg/L and lower confidence limits of 2.76 mg/L. The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L was calculated as 3.38 %, 4.83 %, 16.57 %, 22.30 %, 54.55 % and 96.04 %, respectively. 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.