Reaction mass of Cobaltate(1-), bis[6-(amino-κN)-5-[2-[2-(hydroxy-κO)-4-nitrophenyl]diazenyl-κN1]-N-methyl-2-naphthalenesulfonamidato(2-)]-, sodium (1:1) and disodium [6-amino-5-[(2-hydroxy-4-nitrophenyl)azo]-N-methylnaphthalene-2-sulphonamidato(2-)][6-amino-5-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-2-sulphonato(3-)]cobaltate(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:

August 04, 2015 - November 25, 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 (incl. QA statement)

Specific details on test material used for the study:

Identification : FAT 20036/G TE
Description : Solid
Appearance : Powder
Colour : Blue
Batch number : AT-0033439900
CASNo. : 70236-59-8; 75314-27-1
Purity : > 55%
Molecular weight : 880.68 g/mol ; 889.62 g/mol
Expiry date : March 24, 2020
Stability of test item : Stable.
Storage conditions : Room temperature in the dark.
Safety precautions : Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel.

Analytical monitoring:

yes

Details on sampling:

DOSE FORMULATION
Based on the solubility of the test item, 20X- AAP medium was selected as the vehicle for dose formulation.

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.
15.05, 30.05 and 60.02 mg of the test item was added to 600 ml of 20X-AAP medium each to prepare 25, 50, 100 mg/L concentrations respectively. For Control, 20X-AAP medium was used.

MAIN EXPERIMENT
All the required test concentrations were prepared for 800 ml.
100.05 mg of the test item was weighed and made up to 100 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: 240 µl, 696 µl, 2.016 ml, 5.856 ml, 16.976 ml and 49.224 ml were taken and made up to 800 ml with 20X-AAP medium to obtain 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L concentrations respectively. For Control, 20X-AAP medium was used.

Vehicle:

yes

Details on test solutions:

LINEARITY
Accurately weighed 10.3 mg of FAT 20036/G TE reference standard [Batch No: AT-0033439900 Purity >55%] into a 25 ml volumetric flask and 10 mL of Methanol was added to dissolve the content and sonicated and made upto the mark with the same solvent. The concentration of solution was 226.6mg/L solution. A series of calibration solutions were then prepared by diluting the appropriate volume of stock solution into different 10 mL volumetric flasks and bringing to volume with Methanol as per table given below.

The prepared calibration solutions were 0.01 mg/L, 0.1 mg/L, 1.0 mg/L, 5.0 mg/L, 10.0 mg/L and 50.0 mg/L were analyzed by HPLC at 254 nm. A linear curve was plotted for the concentration of standard versus observed peak area and the correlation coefficient was determined.

PREPARATION OF SAMPLE SOLUTION :
100 ml of Day ‘0’, Day ‘4’ and Day ‘7’ stability samples (100 mg/L) were collected from the Ecotoxicology department and the samples relevant were kept in the refrigerator and during the analysis, samples were taken out to attain room temperature. After attaining the room temperature the samples were shaken vigorously and then 0.5 ml of the solution was taken into a 25 ml volumetric flask and made upto the mark with control sample and the dilution factor was 50 and then the samples were injected into the HPLC under conditions.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- 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:

Not available

Hardness:

Not available

Test temperature:

Temperature in test vessel (°C)
Control : 23.6
Treatment : 23.6
End of the test
Control : 25.1 - 25.4
Treatment : 25.3 - 25.6

pH:

Medium pH range
Control : 7.51
Treatment : 7.35- 7.51
End of the test
Control : 8.60- 8.64
Treatment : 8.31- 8.68

Dissolved oxygen:

Not available

Salinity:

Not applicable

Nominal and measured concentrations:

0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Glass Beaker
- Light : Continuous Light (8320-8460 Lux)
- Medium : 200 ml (20X- AAP medium)/ Replicate

Reference substance (positive control):

yes

Remarks:

The test item was used as the reference item for the study.

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

28.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: with upper confidence limits of 29.51 mg/L and lower confidence limits of 27.16 mg/L.

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

14.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: with upper confidence limits of 15.39 mg/L and lower confidence limits of 14.01 mg/L.

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

24.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond dry weight

Remarks on result:

other: with upper confidence limits of 25.24 mg/L and lower confidence limits of 23.24 mg/L.

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

11.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond dry weight

Remarks on result:

other: with upper confidence limits of 12.51 mg/L and lower confidence limits of 11.38 mg/L.

Details on results:

DOUBLING TIME (Td)
The doubling time (Td) of frond number in the control group was 1.62 day-I.

FROND NUMBER
Total numbers of fronds were counted for each replicates in the control and all the test concentrations on days ' 0', '3', '5' and '7'. Every frond visibly projecting beyond the edge of the parent frond was counted. Frond appearance was observed and recorded on day ' 0', '3', '5' and '7' respectively.

FROND DRY WEIGHT
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 fronds from each test and control vessel on day '7' of exposure.

PERCENT INHIBITION OF AVERAGE GROWTH RATE

RANGE FINDING EXPERIMENT
BASED ON FROND NUMBERS:
The percent inhibition on the average specific growth rate on day '7' for the test concentrations of 1, 10, 25, 50 and 100 mg/L was calculated as 4.99%, 7.68%, 34.28%, 77.89% and 90.46% respectively.

BASED ON FROND DRY WEIGHT:
The percent inhibition on the average growth rate on day '7' for the concentration of 1, 10, 25, 50 and 100 mg/L was calculated as 4.27%, 13.68%, 36.32%, 79.91% and 96.37% respectively.

MAIN EXPERIMENT
BASED ON FROND NUMBERS: The percent inhibition on the average specific growth rate on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.02%, 0.02%, 5.94%, 7.34%, 31.37% and 90.46% respectively.

BASED ON FROND DRY WEIGHT: The percent inhibition on the average growth rate on day '7' for the concentration of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.21%, 0.21%, 4.93%, 13.06%, 34.48% and 96.15% respectively.


AVERAGE SPECIFIC GROWTH RATE
MAIN EXPERIMENT

BASED ON FROND NUMBERS:
From the average specific growth rate recorded with the different test concentrationsviz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L the concentrations that inhibited 50% growth (ErC50) was calculated as 28.31 mg/L with upper confidence limits of 29.51 mg/L and lower confidence limits of 27.16 mg/L.

BASED ON FROND DRY WEIGHT:
From the average specific growth rate recorded with the different test concentrationsviz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L the concentration that inhibited 50% growth (ErC50) ofLemna gibbawas calculated as 24.22 mg/L with upper confidence limits of 25.24 mg/L and lower confidence limits of 2324 mg/L.

PERCENT GROWTH INHIBITION OF YIELD
RANGE FINDING EXPERIMENT
BASED ON FROND NUMBERS: The percent inhibition on the yield on day '7' for the test concentrations of 1, 10, 25, 50 and 100 mg/L was calculated as 14.53%, 21.66%, 67.59%, 95.06% and 98.26% respectively.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the yield on day '7' for the test concentrations of 1, 10, 25, 50 and 100 mg/L
was calculated as 13.09%, 37.59%, 72.18%, 96.36% and 99.48% respectively.

MAIN EXPERIMENT
BASED ON FROND NUMBERS: The percent inhibition on the yield on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.07%, 0.07%, 17.19%, 20.81%, 64.18% and 98.26% respectively.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the yield on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.29 %, 0.29 %, 15.34 %, 35.83 %, 70.17 % and 99.48 % respectively.

YIELD
MAIN EXPERIMENT
BASED ON FROND NUMBERS:
From the yield recorded with the different test concentrationsviz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L the concentration that inhibited 50% yield (EyC50) was calculated as 14.68 mg/L with upper confidence limits of 15.39 mg/L and lower confidence limits of 14.01 mg/L.
BASED ON FROND DRY WEIGHT:
From the yield recorded with the test concentrationsviz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L, the concentration that inhibited 50% yield (EyC50) was calculated as 11.93 mg/L with upper confidence limits of 12.51 mg/L and lower confidence limits of 11.38 mg/L.


LOEC AND NOEC ESTIMATION

MAIN EXPERIMENT BASED ON FROND NUMBERS: The no observed effect concentration (NOEC) was calculated as 0.87 mg/L and the low observed effect concentration (LOEC) was calculated as 2.52 mg/L.

BASED ON FROND DRY WEIGHT:
The no observed effect concentration (NOEC) was calculated as 0.87 mg/L and the low observed effect concentration (LOEC) was calculated as 2.52 mg/L.

ANALYTICAL MEASUREMENTS (MAIN EXPERIMENT)
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 (7.32 mg/L) and high (61.53 mg/L) on days '0' and 7. The mean recoveries data are given below:
- Day '0' — Mean Recovery results: 96.46%, 97.35% and 98.27% for 0.3 mg/L, 7.32 mg/L and 61.53 mg/L concentrations respectively
- Day '7' — Mean Recovery results: 93.11%, 94.82% and 95.54% for 0.3 mg/L, 7.32 mg/L and 61.53 mg/L concentrations respectively

Results with reference substance (positive control):

Same as that of test item

Reported statistics and error estimates:

ErC50, NOEC, LOEC based on the average specific growth rate, EyC50 , NOEC, LOEC based on the yield each of which in turn based upon frond number and frond dry weight were calculated using the statistical package, TOX-STAT 3.5 Version. Mean coefficient of variation of average specific growth rate in replicate control and treated cultures were calculated. Concentration/effect relationship was presented graphically.

STATISTICAL ANALYSIS (MAIN EXPERIMENT)

Percent inhibition of Growth rate (Based on Frond Number)
ErC50	28.31 mg/L
Upper confidence limit	29.51 mg/L
Lower confidence limit	27.16 mg/L
Percent inhibition of yield (Based on Frond Number)
EyC50	14.68 mg/L
Upper confidence limit	15.39 mg/L
Lower confidence limit	14.01 mg/L

 

 

Percent inhibition of Growth rate (Based on Frond Dry Weight)
ErC50	24.22 mg/L
Upper confidence limit	25.24 mg/L
Lower confidence limit	23.24 mg/L
Percent inhibition of yield (Based on Frond Dry weight)
EyC50	11.93 mg/L
Upper confidence limit	12.51 mg/L
Lower confidence limit	11.38 mg/L

                                                                       

NOEC AND LOEC (Based on Frond Number)
NOEC	0.87 mg/L
LOEC	2.52 mg/L

                                                                       

NOEC AND LOEC (Based on Frond Dry weight)
NOEC	0.87 mg/L
LOEC	2.52 mg/L

Response variable	Measurable variable	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Average specific growth rate	Frond number	28.31	0.87	2.52
	Dry weight	24.22
Yield	Frond number	14.68	0.87	2.52
	Dry weight	11.93

Validity criteria fulfilled:

yes

Conclusions:

ErC50 of FAT 20036/G TE based upon the frond numbers observed for a period of '7' days for Lemna gibba was calculated as 28.31 mg/L with upper confidence limits of 29.51 mg/L and lower confidence limits of 27.16 mg/L.

EyC50 of FAT 20036/G TE based upon the frond numbers observed for a period of '7' days for Lemna gibba was calculated as 14.68 mg/L with upper confidence limits of 15.39 mg/L and lower confidence limits of 14.01 mg/L.

ErC50 of FAT 20036/G TE based upon the frond dry weight observed for a period of '7' days for Lemna gibba was calculated as 24.22 mg/L with upper confidence limits of 25.24 mg/L and lower confidence limits of 23.24 mg/L.

EyC50 of FAT 20036/G TE based upon the frond dry weight observed for a period of '7' days for Lemna gibba was calculated as 11.93 mg/L with upper confidence limits of 12.51 mg/L and lower confidence limits of 11.38 mg/L.

Executive summary:

Lemna gibba (Duckweed) was exposed to the test item (FAT 20036/G TE) at different test concentrations viz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L according to OECD Test 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 '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 21.22 and 61.53 mg/L where necrosis observed in fronds exposed to 61.53 mg/L concentration on day '5' and in addition to necrosis, root filaments were turned blue in 21.22 and 61.53 concentrations on day '7'.

The doubling time (T_d) of frond number in the control group was calculated as 1.62 day.

Frond Numbers:

From the average specific growth rate recorded with the different test concentrations viz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L, the test concentrations that inhibited 50% growth(E_rC50) was calculated as 28.31 mg/L with upper confidence limits of 29.51 mg/L and lower confidence limits of 27.16 mg/L.

The percent inhibition on the average specific growth rate on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.02%, 0.02%, 5.94%, 7.34%, 31.37% and 90.46% respectively.

From the yield recorded with the different test concentrations viz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 the concentration that inhibited 50% yield (E_yC₅₀) was calculated as 14.68 mg/L with upper confidence limits of 15.39 mg/L and lower confidence limits of 14.01 mg/L.

The percent inhibition on the yield on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.07%, 0.07%, 17.19%, 20.81%, 64.18% and 98.26% respectively.

The no observed effect concentration (NOEC) was calculated as 0.87 mg/L and the low observed effect concentration (LOEC) was calculated as 2.52 mg/L.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrations viz.,0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L the concentration that inhibited 50% growth (E,C₅₀) was calculated as 24.22 mg/L with upper confidence limits of 25.24 mg/L and lower confidence limits of 23.24 mg/L.

The percent inhibition on the average growth rate on day '7' for the concentration of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.21%, 0.21%, 4.93%, 13.06%, 34.48 and 96.15% respectively.

From the yield recorded with the test concentrations viz., 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L, the concentration that inhibited 50% yield (E_yC₅₀) was calculated as 11.93 mg/L with upper confidence limits of 12.51 mg/L and lower confidence limits of 11.38 mg/L.

The percent inhibition on the yield on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.29%, 0.29%, 15.34%, 35.83%, 70.17% and 99.48% respectively.

The no observed effect concentration (NOEC) was calculated as 0.87 mg/L and the low observed effect concentration (LOEC) was calculated as 2.52 mg/L.

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 (7.32 mg/L) and high (61.53 mg/L) on days '0' and '7'. The mean recoveries data are given below:

· Day 0- Mean Recovery results: 96.46%, 97.35 % and 98.27 % for 0.3 mg/L, 7.32 mg/L and 61.53 mg/L concentrations respectively

·  Day '7' - Mean Recovery results: 93.11%, 94.82 % and 95.54 % for 0.3 mg/L, 7.32 mg/L and 61.53 mg/L concentrations respectively

Description of key information

Lemna gibba (Duckweed) was exposed to the test item (FAT 20036/G TE) at different test concentrations (0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L) according to OECD Test guideline 221.

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 21.22 and 61.53 mg/L where necrosis observed in fronds exposed to 61.53 mg/L concentration on day '5' and in addition to necrosis, root filaments were turned blue in 21.22 and 61.53 concentrations on day '7'.

The doubling time (Td) of frond number in the control group was calculated as 1.62 day.

Frond Numbers:

From the average specific growth rate recorded with the different test concentrations (0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L), the test concentrations that inhibited 50 % growth (ErC50) was calculated as 28.31 mg/L with upper confidence limits of 29.51 mg/L and lower confidence limits of 27.16 mg/L.

The percent inhibition on the average specific growth rate on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated to 0.02%, 0.02 %, 5.94 %, 7.34 %, 31.37 % and 90.46 % respectively.

From the yield recorded with the different test concentrations (0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L) the concentration that inhibited 50 % yield (EyC50) was calculated as 14.68 mg/L with upper confidence limits of 15.39 mg/L and lower confidence limits of 14.01 mg/L.

The percent inhibition on the yield on day '7' for the test concentrations of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.07 %, 0.07 %, 17.19 %, 20.81 %, 64.18 % and 98.26 % respectively.

The no observed effect concentration (NOEC) was calculated as 0.87 mg/L and the low observed effect concentration (LOEC) was calculated as 2.52 mg/L.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentrations (0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L) the concentration that inhibited 50 % growth (E,C50) was calculated as 24.22 mg/L with upper confidence limits of 25.24 mg/L and lower confidence limits of 23.24 mg/L.

The percent inhibition on the average growth rate on day '7' for the concentration of 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L was calculated as 0.21 %, 0.21 %, 4.93 %, 13.06 %, 34.48 % and 96.15 % respectively.

From the yield recorded with the test concentrations ( 0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L), the concentration that inhibited 50 % yield (EyC50) was calculated as 11.93 mg/L with upper confidence limits of 12.51 mg/L and lower confidence limits of 11.38 mg/L.

The percent inhibition on the yield on day '7' for the test concentrations of (0.3, 0.87, 2.52, 7.32, 21.22 and 61.53 mg/L) was calculated as 0.29 %, 0.29 %, 15.34 %, 35.83 %, 70.17 % and 99.48 % respectively.

 

The no observed effect concentration (NOEC) was calculated as 0.87 mg/L and the low observed effect concentration (LOEC) was calculated as 2.52 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

28.31 mg/L