Reaction mass of Sodium 2-(2 or 3-{[(chloroacetyl)amino]methyl}-4-{[4-(cyclohexylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}phenoxy)-5-methylbenzenesulfonate and Sodium 2-(3 or 2-{[(chloroacetyl)amino]methyl}-4-{[4-(cyclohexylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}phenoxy)-5-methylbenzenesulfonate

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:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Stability in light: Continuous Light
- pH dependence on stability:
Start of the test
Control: 7.58
Treatment: 7.54- 7.58

End of the test
Control: 8.73- 8.76
Treatment: 8.54- 8.76

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:

50.04 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. From the stock solution, each required test concentration was prepared as follows: 40 µl, 120 µl, 360 µl, 1.08 ml, 3.24 ml and 9.72 ml 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.

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:

None

Hardness:

None

Test temperature:

Start of the test
Control: 22.8
Treatment: 22.8

End of the test
Control: 25.0 - 25.3
Treatment: 25.0 - 25.3

pH:

Start of the test
Control: 7.58
Treatment: 7.54- 7.58

End of the test
Control: 8.73- 8.76
Treatment: 8.54- 8.76

Dissolved oxygen:

None

Salinity:

None

Nominal and measured concentrations:

None

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
- Adjustment of pH: Start of the test
Control : 7.58
Treatment : 7.58-7.53
End of the test
Control : 8.73-8.76
Treatment : 8.56-8.76

- Photoperiod: Continuous Light
- Light intensity and quality: 8120-8420 Lux

TEST CONCENTRATIONS
RANGE FINDING EXPERIMENT:
All the required test concentrations were prepared for 600 ml.
10.02 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. 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.
15.01, 30.01 and 60.04 mg of the test item was dissolved and made upto 600 ml of 20x-AAP medium each to prepare 25, 50, 100 mg/L concentrations respectively. For Control, 20x-AAP medium was used.


- Test concentrations:
All the required test concentrations were prepared for 800 ml.
50.04 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. From the stock solution, each required test concentration was prepared as follows: 40 µl, 120 µl, 360 µl, 1.08 ml, 3.24 ml and 9.72 ml 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.:

7.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

6.64 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.:

2.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond Dry Weight; Yield

Details on results:

None

Results with reference substance (positive control):

The sensitivity of the test system and reliability of the experimental technique was assessed with (3,5-Dichlorophenol) during November-December 2014 (Study No. 4897). This was performed in accordance with the following guideline: OECD Guidelines for the Testing of Chemicals, Section 2. Number 221, “Lemna sp. Growth Inhibition Test” Adopted on 23rd March 2006.
Lemna gibba (Duckweed) were exposed to the test item (3,5-Dichlorophenol) at different test concentrations viz., 0.19, 0.48, 1.19, 2.97 and 7.42 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 a period of ‘9’ 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 on day ‘0’ (pre-culture representative samples) and day ‘7’.
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 7.42 mg/L where root filaments were observed short in all the replicates on 3rd, 5th and 7th day. The doubling time (Td) of frond number in the control group was calculated as 1.62 day -1 .

Frond Numbers:
From the average specific growth rate recorded with the different test concentrations with 0.19, 0.48, 1.19, 2.97 and 7.42 mg/L, the test concentration that inhibits 50% growth (ErC50) was calculated as 4.30 mg/L with upper confidence limits of 4.46 mg/L and lower confidence limit of 4.15 mg/L. The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.19, 0.48, 1.19, 2.97 and 7.42 mg/L, was calculated as 0.11 %, 4.46%, 6.31 %, 28.65 % and 83.00 % respectively. From the yield recorded with the different test concentrations with 0.19, 0.48, 1.19, 2.97 and 7.42 mg/L the concentration that inhibited 50 % yield (EyC50) was calculated as 2.38 mg/L with upper confidence limit of 2.29 mg/L and lower confidence limit of 2.48 mg/L. The lowest observed effect concentration (LOEC) was calculated as 0.48 mg/L and the no observed effect concentration (NOEC) was calculated as 0.19 mg/L respectively.

Frond Dry Weight:
From the average specific growth rate recorded with the test concentrations with, 0.19, 0.48, 1.19, 2.97 and 7.42 mg/L, the concentration that inhibited 50% growth (ErC50) was calculated as 3.02 mg/L with upper confidence limit of 3.16 mg/L and lower confidence limit of 2.89 mg/L. The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.19, 0.48, 1.19, 2.97 and 7.42 mg/L was calculated as 0.74 %, 4.86 %, 7.03 %, 55.76 % and 83.20 % respectively. From the yield recorded with the test concentrations viz., 0.19, 0.48, 1.19, 2.97 and 7.42 mg/L, the concentration that inhibited 50% yield (EyC50) was calculated as 1.53 mg/L with upper confidence limit of 1.59 mg/L and lower confidence limit of 1.46 mg/L

The lowest observed effect concentration (LOEC) was calculated as 0.48 mg/L and no observed effect concentration (NOEC) was calculated as 0.19 mg/L respectively.
Analytical verification of the test item in the exposure medium samples were carried out in the test item concentrations of low (0.19 mg/L), intermediate (1.19 mg/L) and high (7.42 mg/L) on days ‘0’ and ‘7’. The mean recoveries data are given below:
• Day ‘0’ – 91.18 %, 93.06 % and 94.93 % for 0.19, 1.19 and 7.42 mg/L test concentrations respectively.
• Day ‘7’ – 89.44 %, 91.85 % and 93.39 % for 0.19, 1.19 and 7.42 mg/L test concentrations respectively.

Reported statistics and error estimates:

None

Validity criteria fulfilled:

yes

Conclusions:

ErC50 of FAT 20077/D TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 7.84 mg/L with upper confidence limits of 8.23 mg/L and lower confidence limits of 7.47 mg/L.

Executive summary:

Lemna gibba (Duckweed) were exposed to the test item (FAT 20077/D TE) at different test concentrations with 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 concentration except 12.15 mg/L where necrosis observed and root filament were turned blue onday ‘7’.

The doubling time (T_d) 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 with 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the test concentrations that inhibited 50% growth (E_rC₅₀) was calculated as 7.84 mg/L with upper confidence limits of 8.23 mg/L and lower confidence limits of 7.47 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 0.11%, 0.25%, 3.31 %, 6.57 %, 21.55 % and 72.38 % respectively. From the yield recorded with the different test concentrations with 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50 % yield (E_yC₅₀) was calculated as 3.76 mg/L with upper confidence limits of 3.95 mg/L and lower confidence limits of 3.58 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 0.35 %, 0.78 %, 10.01 %, 18.95 %, 50.32 % and 93 % 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.

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 (E_rC₅₀) was calculated as 6.64 mg/L with upper confidence limits of 7.03 mg/L and lower confidence limits of 6.28 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 0.78 %, 1.82 %, 5.85 %, 13.93 %, 21.64 % and 78.48 % respectively. From the yield recorded with the test concentrations with 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L, the concentration that inhibited 50% yield (E_yC₅₀) was calculated as 2.55 mg/L with upper confidence limits of 2.70 mg/L and lower confidence limits of 2.41 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 2.49 %, 6.01 %, 18.07 %, 37.89 %, 52.34 % and 95.92 % 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.

Description of key information

Lemna gibba (Duckweed) were exposed to the test item (FAT 20077/D TE) at different test concentrations with 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 concentration except 12.15 mg/L where necrosis observed and root filament were turned blue onday ‘7’. The doubling time (T_d) 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 with0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the test concentrations that inhibited 50 % growth (E_rC₅₀) was calculated as 7.84 mg/L with upper confidence limits of 8.23 mg/L and lower confidence limits of 7.47 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 0.11 %, 0.25 %, 3.31 %, 6.57 %, 21.55 % and 72.38 % respectively.

From the yield recorded with the different test concentrations with,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50% yield (E_yC₅₀) was calculated as 3.76 mg/L with upper confidence limits of 3.95 mg/L and lower confidence limits of 3.58 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 0.35 %, 0.78 %, 10.01 %, 18.95 %, 50.32 % and 93 % 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.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrations with.,0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 mg/L the concentration that inhibited 50 % growth (E_rC₅₀) was calculated as 6.64 mg/L with upper confidence limits of 7.03 mg/L and lower confidence limits of 6.28 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 0.78 %, 1.82 %, 5.85 %, 13.93 %, 21.64 % and 78.48 % respectively. From the yield 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% yield (E_yC₅₀) was calculated as 2.55 mg/L with upper confidence limits of 2.70 mg/L and lower confidence limits of 2.41 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 2.49 %, 6.01 %, 18.07 %, 37.89 %, 52.34 % and 95.92 % 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.

Key value for chemical safety assessment

EC50 for freshwater plants:

7.84 mg/L