Reaction mass of sodium amino-bis{[4-(ethenylsubstituted)phenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-{[4-(ethenylsubstituted)phenyl]diazenyl}-{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-bis{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

None

Vehicle:

yes

Details on test solutions:

None

Test organisms (species):

Lemna gibba

Details on test organisms:

None

Test type:

static

Water media type:

freshwater

Limit test:

no

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).

Hardness:

None

Test temperature:

Temperature at the start (‘0’ Day) of the experiment in control & treatments from the bulk sample
Test Concentration (mg/L) Temperature (°C)
Control 23.1
0.3 23.2
0.96 22.5
3.07 23.1
9.83 22.5
31.46 22.4
100.66 22.5

Temperature at the end (‘7’Day) of the experiment in control & treatments
Test Concentration (mg/L) R1 R2 R3 R4 R5 R6 Range of Temperature (°C)
Control 25.7 25.6 25.4 25.4 25.3 25.3 25.3 - 25.7
0.3 25.4 25.3 25.2 - - - 25.2 - 25.4
0.96 25.3 25.3 25.4 - - - 25.3 - 25.4
3.07 25.4 25.4 25.4 - - - 25.4
9.83 25.6 25.7 25.6 - - - 25.6 - 25.7
31.46 25.7 25.6 25.6 - - - 25.6 - 25.7
100.66 25.6 25.7 25.7 - - - 25.6 - 25.7

pH:

pH at the start (‘0’ Day) of the experiment in control & treatments from the bulk sample
Test Concentration (mg/L) pH Values
Control 7.60
0.3 8.00
0.96 8.01
3.07 8.01
9.83 7.99
31.46 7.98
100.66 7.98

pH at the end (Day ‘7’) of the experiment in control & treatments
Test Concentration (mg/L) R1 R2 R3 R4 R5 R6 Range of pH
Control 8.79 8.80 8.77 8.80 8.71 8.80 8.71 - 8.80
0.3 9.38 9.36 9.36 - - - 9.36 - 9.38
0.96 9.24 9.26 9.26 - - - 9.24 - 9.26
3.07 9.00 9.04 9.04 - - - 9.00 - 9.04
9.83 9.03 9.05 9.05 - - - 9.03 - 9.05
31.46 8.83 8.81 8.72 - - - 8.81 - 8.83
100.66 8.70 8.71 8.72 - - - 8.70 - 8.72

Dissolved oxygen:

None

Salinity:

None

Nominal and measured concentrations:

Analytical verification of the test item in the exposure medium samples were carried out in the test item concentrations of low (0.3 mg/L), intermediate (9.83mg/L) and high (100.66 mg/L) on days ‘0’ and 7. The mean recoveries data are given below:
Day ‘0’ – 90.47 %, 91.36 % and 93.68 % for 0.3 mg/L, 9.83 mg/L and 100.66 mg/L test concentrations respectively.
Day ‘7’ – 89.36 %, 90.22 % and 92.02 % for 0.3 mg/L, 9.83 mg/L and 100.66 mg/L test concentrations respectively.

Details on test conditions:

None

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

25.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

7.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

19.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

5.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

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 May 2014 (Study No. 4521). 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, 0.46, 1.09, 2.63 and 6.30 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 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 6.30 mg/L where root length variation was observed in all the replicates on 3rd, 5th and 7th day.
The doubling time (Td) of frond number in the control group was calculated as 1.59 day-1 .
Frond Numbers:
From the average specific growth rate recorded with the different test concentrations viz., 0.19, 0.46, 1.09, 2.63 and 6.30 mg/L, the test concentration that inhibits 50% growth (ErC50) was calculated as 4.38 mg/L.
The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.19, 0.46, 1.09, 2.63 and 6.30 mg/L was calculated as 0.04%, 1.00%, 2.18%, 14.94% and 82.73 % respectively.
From the yield recorded with the different test concentrations viz., 0.19, 0.46, 1.09, 2.63 and 6.30 mg/L the concentration that inhibited 50% yield (EyC50) was calculated as 3.01 mg/L.
The lowest observed effect concentration (LOEC) was calculated as 1.09 mg/L and the no observed effect concentration (NOEC) was calculated as 0.46 mg/L respectively.

Frond Dry Weight:
From the average specific growth rate recorded with the test concentrations viz., 0.19, 0.46, 1.09, 2.63 and 6.30 mg/L, the concentration that inhibited 50% growth (ErC50) was calculated as 4.72 mg/L.
The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.19, 0.46, 1.09, 2.63 and 6.30 mg/L was calculated as 0.39%, 1.11%, 7.65%, 10.39% and 74.18% respectively.
From the yield recorded with the test concentrations viz., 0.19, 0.46, 1.09, 2.63 and 6.30 mg/L, the concentration that inhibited 50% yield (EyC50) was calculated as 2.92 mg/L.
The lowest observed effect concentration (LOEC) was calculated as 1.09 mg/L and no observed effect concentration (NOEC) was calculated as 0.46 mg/L respectively.

Based on the test results, ErC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 4.38 mg/L with a lower confidence limit of 4.26 mg/L and the upper confidence limit of 4.51 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 3.01 mg/L with a lower confidence limit of 2.92 mg/L and the upper confidence limit of 3.11 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 4.72 mg/L with a lower confidence limit of 4.56 mg/L and the upper confidence limit of 4.88 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 2.92 mg/L with a lower confidence limit of 2.82 mg/L and the upper confidence limit of 3.03 mg/L.

Reported statistics and error estimates:

Percent inhibition of Growth rate (Based on Frond Number)
ErC50 25.03 mg/L
Upper confidence limit 26.80 mg/L
Lower confidence limit 23.37 mg/L
Percent inhibition of yield (Based on Frond Number)
EyC50 7.65 mg/L
Upper confidence limit 8.13 mg/L
Lower confidence limit 7.19 mg/L

Percent inhibition of Growth rate (Based on Frond Dry Weight)
ErC50 19.43 mg/L
Upper confidence limit 21.04 mg/L
Lower confidence limit 17.94 mg/L
Percent inhibition of yield (Based on Frond Dry weight)
EyC50 5.02 mg/L
Upper confidence limit 5.33 mg/L
Lower confidence limit 4.72 mg/L

NOEC AND LOEC (Based on Frond Number)
NOEC 0.3 mg/L
LOEC 0.96 mg/L

NOEC AND LOEC (Based on Frond Dry weight)
NOEC 0.3 mg/L
LOEC 0.96 mg/L

None

Validity criteria fulfilled:

yes

Conclusions:

ErC50 of FAT 40865/A TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 25.03 mg/L with upper confidence limits of 26.80 mg/L and lower confidence limits of 23.37 mg/L.

Executive summary:

Lemna gibba (Duckweed) were exposed to the test item (FAT 40865/A TE) at different test concentrations viz.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L following OECD Guideline 221. 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 ‘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 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 31.46 and 100.66 mg/L where necrosis observed in all the replicates on 5th day and in addition to necrosis, root filaments were turned red in all the replicates on 7th day respectively. 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.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L, the test concentration that inhibited 50% growth (ErC50) was calculated as 25.03 mg/L with upper confidence limits of 26.80 mg/L and lower confidence limits of 23.37 mg/L.

The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 0.23%, 4.57%, 6.42%, 25.84%, 59.09% and 80.99% respectively.

From the yield recorded with the different test concentrationsviz.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L the concentration that inhibited 50% yield (EyC50) was calculated as 7.65 mg/L with upper confidence limits of 8.13 mg/L and lower confidence limits of 7.19 mg/L.

The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 0.73%, 13.50%, 18.43%, 56.75%, 87% and 96% respectively.

The no observed effect concentration (NOEC) was calculated as 0.3 mg/L and the low observed effect concentration (LOEC) was calculated as 0.96 mg/L respectively.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrations viz 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L, the concentration that inhibited 50% growth (ErC50) was calculated as 19.43 mg/L with upper confidence limits of 21.04 mg/L and lower confidence limits of 17.94 mg/L.

The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 2.08, 6.25, 8.33, 50.00, 58.33 and 77.08% respectively.

From the yield recorded with the test concentrations viz.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L, the concentration that inhibited 50% yield (EyC50) was calculated as 5.02 mg/L with upper confidence limits of 5.33 mg/L and lower confidence limits of 4.72 mg/L.

The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 2.21, 16.57, 20.54, 84.35, 88.21 and 95.75% respectively.

The no observed effect concentration (NOEC) was calculated as 0.3 mg/L and the low observed effect concentration (LOEC) was calculated as 0.96 mg/L respectively.

Analytical verification of the test item in the exposure medium samples were carried out in the low (0.3 mg/L), intermediate (9.83 mg/L) and high (100.66 mg/L) concentration groups on days ‘0’ and ‘7’. The mean recoveries data are given below:

Day ‘0’ – 90.47, 91.36  and 93.68 % for 0.3, 9.83 and 100.66 mg/L test concentrations respectively.

Day ‘7’ – 89.36, 90.22 and 92.02 % for 0.3, 9.83 and 100.66 mg/L test concentrations respectively.

Description of key information

ErC50 of FAT 40865/A TE based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 25.03 mg/L with upper confidence limits of 26.80 mg/L and lower confidence limits of 23.37 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

25.03 mg/L

Additional information

Lemna gibba (Duckweed) were exposed to the test item (FAT 40865/A TE) at different test concentrations viz.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L following OECD Guideline 221. 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 ‘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 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 31.46 and 100.66 mg/L where necrosis observed in all the replicates on 5th day and in addition to necrosis, root filaments were turned red in all the replicates on 7th day respectively. 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.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L, the test concentration that inhibited 50% growth (ErC50) was calculated as 25.03 mg/L with upper confidence limits of 26.80 mg/L and lower confidence limits of 23.37 mg/L.

The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 0.23%, 4.57%, 6.42%, 25.84%, 59.09% and 80.99% respectively.

From the yield recorded with the different test concentrationsviz.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L the concentration that inhibited 50% yield (EyC50) was calculated as 7.65 mg/L with upper confidence limits of 8.13 mg/L and lower confidence limits of 7.19 mg/L.

The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 0.73%, 13.50%, 18.43%, 56.75%, 87% and 96% respectively.

The no observed effect concentration (NOEC) was calculated as 0.3 mg/L and the low observed effect concentration (LOEC) was calculated as 0.96 mg/L respectively.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrations viz 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L, the concentration that inhibited 50% growth (ErC50) was calculated as 19.43 mg/L with upper confidence limits of 21.04 mg/L and lower confidence limits of 17.94 mg/L.

The percent inhibition on the average growth rate on day ‘7’ for the concentration of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 2.08, 6.25, 8.33, 50.00, 58.33 and 77.08% respectively.

From the yield recorded with the test concentrations viz.,0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L, the concentration that inhibited 50% yield (EyC50) was calculated as 5.02 mg/L with upper confidence limits of 5.33 mg/L and lower confidence limits of 4.72 mg/L.

The percent inhibition on the yield on day ‘7’ for the test concentrations of 0.3, 0.96, 3.07, 9.83, 31.46 and 100.66 mg/L was calculated as 2.21, 16.57, 20.54, 84.35, 88.21 and 95.75% respectively.

The no observed effect concentration (NOEC) was calculated as 0.3 mg/L and the low observed effect concentration (LOEC) was calculated as 0.96 mg/L respectively.

Analytical verification of the test item in the exposure medium samples were carried out in the low (0.3 mg/L), intermediate (9.83 mg/L) and high (100.66 mg/L) concentration groups on days ‘0’ and ‘7’. The mean recoveries data are given below:

Day ‘0’ – 90.47, 91.36  and 93.68 % for 0.3, 9.83 and 100.66 mg/L test concentrations respectively.

Day ‘7’ – 89.36, 90.22 and 92.02 % for 0.3, 9.83 and 100.66 mg/L test concentrations respectively.