Sodium bis[N-(2-chlorophenyl)-2-[[2-hydroxy-5-(N-methylsulphamoyl)phenyl]azo]-3-oxobutyramidato(2-)]cobaltate(1-)

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:

12 October 2015 to 28 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:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: AT-0032668200
- Expiration date of the lot/batch: January 29, 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under storage conditions: stable

Analytical monitoring:

yes

Details on sampling:

0.0001 g of the test item was added to 1L of 20X- AAP medium in a 1L standard flask, ultrasonicated for 15 minutes and then the test dispersion was kept on the magnetic stirrer up to 5 days. The test dispersion that was kept on the magnetic stirrer was removed and allowed to settle down for approximately one hour. Then the test dispersion was filtered through the filter paper and the filtrate was used for the dose formulation, which is should be 203 mg/L concentration as per the previous analytical results.
20X- AAP medium was selected as vehicle based on the solubility check of the test item.

Vehicle:

yes

Details on test solutions:

For Main experiment 1.0003 g of the test item was added to 1L of 20X- AAP medium in a 1L standard flask, ultrasonicated for 15 minutes and then the test dispersion was kept on the magnetic stirrer up to 5 days. The test dispersion that was kept on the magnetic stirrer was removed and allowed to settle down for approximately one hour. Then the test dispersion was filtered through the filter paper and the filtrate was used for the dose formulation, which is should be 203 mg/L concentration as per the previous analytical results. The concentrations selected were 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L which is equivalent to 6.3mg/625 ml, 9mg/600ml, 15.3mg/600 ml, 21.09mg/625 ml, 30.38 mg/600ml, 45.56 mg/600 ml and 71.19 mg/625 ml respectively.
From the stock solution, each required test concentration was prepared as follows: 31.03 ml, 44.33 ml, 75.37 ml, 103.89 ml, 149.66 ml, 224.43 ml and 350.69 ml were taken and made up to 600 ml except 6.3, 21.09 and 71.19 mg/625 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).

Hardness:

None

Test temperature:

Start of the test
Control: 22.9
Treatment: 22.9
End of the test
Control: 24.7-24.9
Treatment: 24.8-25.5

pH:

Start of the test
Control: 7.50
Treatment: 7.38-7.51
End of the test
Control: 8.75-8.79
Treatment: 8.59-8.77

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): 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

TEST CONCENTRATIONS
-For range finding experiment the concentrations selected were 0.01, 0.1, 1, 10, 50 and 100 mg/L
-The concentrations selected were 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

86.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

51.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

87.38 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.:

51.08 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.:

15 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.

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 20035/E based upon the frond numbers (growth rate) observed for a period of ‘7’ days for Lemna gibba was calculated as 86.90 mg/L with upper confidence limits of 89.83 mg/L and lower confidence limits of 84.06 mg/L.
EyC50 of FAT 20035/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 51.84 mg/L with upper confidence limits of 53.04 mg/L and lower confidence limits of 50.68 mg/L.
ErC50 of FAT 20035/E based upon the frond dry weight (growth rate) observed for a period of ‘7’ days for Lemna gibba was calculated as 87.38 mg/L with upper confidence limits of 90.33 mg/L and lower confidence limits of 84.52 mg/L.
EyC50 of FAT 20035/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 51.08 mg/L with upper confidence limits of 52.24 mg/L and lower confidence limits of 49.95 mg/L.

Executive summary:

Following OECD guideline 221, Lemna gibba (Duckweed) were exposed to the test item (FAT 20035/E TE) at different test concentrations viz.,10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 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. 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 113.91 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.,10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 the test concentration that inhibited 50 % growth (E_rC₅₀) was calculated as 86.90 mg/L with upper confidence limits of 89.83 mg/L and lower confidence limits of 84.06 mg/L.

The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 was calculated as 0.00 %, 0.23 %, 4.59 %, 7.34 %, 20.92 %, 41.38 and 66.13 %, respectively.

From the yield recorded with the different test concentrationsviz.,10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L, the concentration that inhibited 50 % yield (E_yC₅₀) was calculated as 51.84 mg/L with upper confidence limits of 53.04 mg/L and lower confidence limits of 50.68 mg/L.

The percent inhibition on the yield on day ‘7’ for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L was calculated as 0.00 %, 0.72 %, 13.59 %, 20.89 %, 49.21 %, 74.96 %, and 90.84 %, respectively.

 

The no observed effect concentration (NOEC) was calculated as 15 mg/L and the low observed effect concentration (LOEC) was calculated as 25.5 mg/L respectively.

 

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrationsviz.,10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L, the concentration that inhibited 50 % growth (E_rC₅₀) was calculated as 87.38 mg/L with upper confidence limits of 90.33 mg/L and lower confidence limits of 84.52 mg/L.

The percent inhibition on the average growth rate on day ‘7’ for the concentration of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L was calculated as 0.12 %, 0.31 %, 4.13 %, 6.15 %, 17.2 3%, 53.41 %, and 59.12 %, respectively. 

From the yield recorded with the test concentrations viz.,10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L the concentration that inhibited 50 % yield (E_yC₅₀) was calculated as 51.08 mg/L with upper confidence limits of 52.24 mg/L and lower confidence limits of 49.95 mg/L.

The percent inhibition on the yield on day ‘7’ for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/L was calculated as 0.44 %, 1.06 %, 13.16 %, 18.99 %, 44.86 %, 85.87 %, and 88.96 %, respectively.

 

The no observed effect concentration (NOEC) was calculated as 15 mg/L and the low observed effect concentration (LOEC) was calculated as 25.5 mg/L respectively.

 

E_rC₅₀of FAT 20035/E based upon the frond numbers (growth rate) observed for a period of ‘7’ days for Lemna gibba was calculated as 86.90 mg/L with upper confidence limits of 89.83 mg/L and lower confidence limits of 84.06 mg/L.

E_yC₅₀ of FAT 20035/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 51.84 mg/L with upper confidence limits of 53.04 mg/L and lower confidence limits of 50.68 mg/L.

E_rC₅₀ of FAT 20035/E based upon the frond dry weight (growth rate) observed for a period of ‘7’ days for Lemna gibba was calculated as 87.38 mg/L with upper confidence limits of 90.33 mg/L and lower confidence limits of 84.52 mg/L.

E_yC₅₀of FAT 20035/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 51.08 mg/L with upper confidence limits of 52.24 mg/L and lower confidence limits of 49.95 mg/L.

 

Description of key information

7 day ErC50 of test substance based upon the frond number (growth rate) is 86.90 mg/L (89.83 - 84.06 mg/L) while,
7 day EyC50 based upon the frond number is 51.84 mg/L (53.04 - 50.68 mg/L).
7 day ErC50 based upon the frond dry weight (growth rate) is 87.38 mg/L (90.33 - 84.52 mg/L).
7 day EyC50 based upon the frond dry weight is 51.08 mg/L (52.24 - 49.95 mg/L).

Key value for chemical safety assessment

EC50 for freshwater plants:

51.08 mg/L

EC10 or NOEC for freshwater plants:

15 mg/L

Additional information

In a GLP-compliant OECD 221 guideline study Lemna gibba (Duckweed) were exposed to the test item FAT 20035/E TE at different test concentrations viz.,10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 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 86.90 mg/L (89.83 - 84.06 mg/L).

7 day EyC50 based upon the frond number is 51.84 mg/L (53.04 - 50.68 mg/L).

7 day ErC50 based upon the frond dry weight is 87.38 mg/L (90.33 - 84.52 mg/L).

7 day EyC50 based upon the frond dry weight is 51.08 mg/L (52.24 - 49.95 mg/L).

Considering frond number the no observed effect concentration (NOEC) was calculated as 15 mg/L and the low observed effect concentration (LOEC) was calculated as 25.5 mg/L respectively. Considering frond dry weight the no observed effect concentration (NOEC) was calculated as 15 mg/L and the low observed effect concentration (LOEC) was calculated as 25.5 mg/L, respectively.