Reaction mass of Disodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](hydroxy)chromate(2-) and Trisodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](dihydroxy)chromate(3-)

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:

16 December 2020 to 05 May 2021

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: FAT 20044/C
Physical appearance (Aspect): Black powder
LC purity: 99.0 % (a/a) at 254 nm
Batch No.: SI/May 2020/CR/001 (India)
Manufactured date: 08 June 2020
Expiry date: 07 June 2025
Recommended storage condition: Ambient (+15 to +25 °C)

Analytical monitoring:

no

Details on sampling:

Test Sample

Vehicle:

yes

Details on test solutions:

To prepare test concentrations of 5, 15, 45, 135 and 405 mg/L, 5, 15, 45, 135 and 405 mg of test item was mixed with test medium followed by making up to 1 L in volumetric flask using test medium. For control (G1), 1 L of test medium was used.
The test was performed on a black, non-reflecting surface due to coloured nature of test medium as per the OECD Guidance Document No. 23.

Test organisms (species):

Lemna minor

Details on test organisms:

Lemna minor was used as the test system as it is a recommended Lemna species in the guideline. Lemna obtained from the following source was used in this test and stock culture maintained as below:
Eurofins Agroscience Services Ecotox GmbH Aquatic Toxicology, Eutinger Strasse 24 D-75223 Niefern Oschelbronn, Germany

Monocultures (culture with one plant species) of Lemna plants were maintained aseptically between 22 and 26 °C under a continuous illumination (of light intensity 6500 to 10000 Lux). Periodically (7 - 10 days), a number of young, light-green plants were sub-cultured using glass beakers containing fresh medium at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

23.0 to 24.9 °C

pH:

6.80 to 7.78

Nominal and measured concentrations:

5, 15, 45, 135 and 405 mg/L

Details on test conditions:

Glass beakers (at least 250 mL capacity), crystallising dishes or glass petri dishes of appropriate dimensions will be used as test vessels. Test vessels will be covered with watch glass or petri plate cover to minimise evaporation and accidental contamination, while allowing necessary air exchange. Test vessels and particularly covers of test vessels will avoid shadowing or changes in the spectral characteristics of light. Random repositioning of the test vessels will be made when observations are made (or repositioning more frequently). At least 100 mL of prepared test solutions will be dispensed for each replicate and Lemna colonies will be transferred on the test medium. Colonies consisting of 2 to 4 visible fronds will be transferred from the inoculum culture (pre-culture) and randomly (as well as impartially) assigned to the test vessels under aseptic conditions. Each test vessel will contain a total of 9 to 12 fronds. The number of fronds and colonies will be the same in each test vessel using at least three replicates per treatment, with each replicate containing 9 to 12 fronds initially. The test is terminated 7 days after the plants are transferred into the test vessels.

Reference substance (positive control):

yes

Remarks:

3,5 Dichloro phenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

119.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

119.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

210.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

135 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

164.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Parameter
Endpoint At 7 days ; EC value (mg test item/L); Fiducial limits at 95 %
Inhibition in specific growth rate in frond number
ErC50 234.3 229.5 - 239.2
ErC20 114.4 110.9 -118.0
ErC10 75.21 72.00 - 78.49
LOEC 45 NA
NOEC 15 NA

Inhibition in yield rate in frond number EyC50 119.3 117.1 - 121.4
EyC20 63.89 61.86 - 65.99
EyC10 44.35 42.38 - 46.41
LOEC 45 NA
NOEC 15 NA

Inhibition in dry weight- growth rate ErC50 210.1 upper limits 240.7
ErC20 154.9 139.0 - 179.5
ErC10 129.7 113.0 - 153.0
LOEC 405 NA
NOEC 135 NA

Inhibition in dry weight - yield EyC50 164.4 123.3 - 220.3
EyC20 110.7 56.32 -147.8
EyC10 87.88 33.25 lower limits
LOEC 45 NA
NOEC 15 NA

Results with reference substance (positive control):

The calculated EC value for growth rate and yield (for frond numbers and dry weight) on the basis of nominal concentration:

Endpoint At 7 days EC values (mg/L) Fiducial limits at 95 %

Frond numbers NOECr 1 Not applicable
ErC10 2.437 0.6265 – 0.6265<
ErC50 8.247 4.943 – 13.46
NOECy 1 Not applicable
EyC10 0.9272 0.3599 – 0.3599<
EyC50 3.283 2.200 – 5.099

Dry weight NOECr 1 Not applicable
ErC10 2.402 0.3734 – 0.3734<
ErC50 8.785 4.398 – 15.83
NOECy 1 Not applicable
EyC10 0.8331 0.2063 – 0.2063<
EyC50 3.137 1.823 – 5.835

Reported statistics and error estimates:

The statistical analysis of the growth rate and yield data was evaluated using licensed copies of SYSTAT Statistical package version 12.0. The growth rate and yield for frond number and dry weight was tested for normality (Shapiro-Wilk test) and homogeneity of variance before performing further analysis. The growth rate and yield for frond number and dry weight data were found to be normal and homogeneous and performed two sample t-test (for NOEC evaluation).
The inhibition of growth will be determined by calculating the EC10, EC20 and EC50 (the index “Ir” refers to inhibition in specific growth rate and the index “Iy” refers to inhibition in yield) by Probit analysis of Finney (1978) using a validated computer program.
The unit of expression of the endpoints is mg/L

Range Finding Test

The Fronds were found to be normal in appearance in control and all test item treated groups at the end of the test. Test medium was clear and deep blue to violet colour tinge in treatment groups. There was colour gradient across the test concentrations from lower to higher. Test medium was similar in appearance on days 3, 5 and 7 as compared to the start of the exposure.

 

Definitive Test

Fronds were found to be normal in appearance in control and test item treated group at the end of the test. Test medium was clear in control and increase in the intensity of color gradient from deep blue to violet colour tinge preparation with no precipitation of solids in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared to the start of the exposure.

 

VALIDITY OF THE TEST

The Lemna growth inhibition test fulfils the validity criteria of the OECD guideline 221. The factor of frond number increase, measured in the control between day 0 and day 7, was 19.5 corresponding to an average specific growth rate for frond number in the control of 0.424 per day and to a doubling time of 1.634 days (validity criterion: growth rate 2: 0.275 per day, doubling time <2.5 days).

Validity criteria fulfilled:

yes

Conclusions:

EC 50 and NOEC of FAT 20044/C for inhibition in specific growth rate in frond number was 243.3 and 15 mg/L; EC 50 and NOEC for inhibition in yield rate in frond number was 119.3 and 15 mg/L, EC 50 and NOEC for inhibition in dry weight- growth rate was 210.1 and 135 mg/L while inhibition in dry weight – yield was 164.4 and 15 mg/L respectively.

Executive summary:

In GLP-compliant study, the effect of FAT 20044/C was tested on the growth of freshwater vascular aquatic plant, Lemna minor carried out according to OECD guideline 221. Based on the results of the range finding test, the definitive test was carried out using the test concentrations of 5, 15, 45, 135 and 405 mg/L; along with control. Each test item group had three replicate and control had six replicates. Lemna was exposed in semi-static mode with renewal of test medium on days 3 and 5. Frond observations were made on days 3, 5 and 7. Dry weight was determined at the start (day 0) and at the end of the test (day 7). At measured test concentrations of 5, 15, 45, 135 and 405 mg/L, the observed growth rate and yield for frond numbers and dry weight resulted in toxic effects on growth of Lemna when compared with the control during the test period fronds were found to be normal in appearance in control and test item treated group at the end of the test. Test medium was clear in control and deep blue to violet color tinge in test item treated groups. Test medium was similar in appearance on days 3, 5 and 7 as compared to the start of the exposure. The test item was recoverable using the analytical method at nominal concentrations of 1.022 and 601 mg/L in the test medium. The stability test results concluded that the test item was found to be stable in the test medium at the test conditions up to 4 days at nominal concentrations of 1.022 and 601 mg/L. The results of active ingredient analysis of 5, 15, 45, 135 and 405 mg/L showed that overall percent agreement was from 91.039 to 118.927 % (with % RSD 0.878 to 14.774) at the start of renewals; 81.797 to 119.097% (with % RSD 1.124 to 16.988) at the end of renewals indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20 %). No measurable concentration of test item was recorded in the control samples. The end point values for growth rate and yield for frond numbers and dry weight at end of the test are presented below based on nominal concentration.

 

Parameter	End point (mg test item/L)
	EC value		95 % Fiducial limits
Inhibition in specific growth rate in frond number	ErC50	234.3	229.5 - 239.2
	ErC20	114.4	110.9 -118.0
	ErC10	75.21	72.00 - 78.49
	LOEC	45	NA
	NOEC	15	NA
Inhibition in yield rate in frond number	EyC50	119.3	117.1 - 121.4
	EyC20	63.89	61.86 - 65.99
	EyC10	44.35	42.38 - 46.41
	LOEC	45	NA
	NOEC	15	NA
Inhibition in dry weight- growth rate	ErC50	210.1	upper limits 240.7
	ErC20	154.9	139.0 - 179.5
	ErC10	129.7	113.0 - 153.0
	LOEC	405	NA
	NOEC	135	NA
Inhibition in dry weight - yield	EyC50	164.4	123.3 - 220.3
	EyC20	110.7	56.32 -147.8
	EyC10	87.88	33.25 lower limits
	LOEC	45	NA
	NOEC	15	NA

Description of key information

EC 50 and NOEC of test substance for inhibition in specific growth rate in frond number was 243.3 and 15 mg/L; EC 50 and NOEC for inhibition in yield rate in frond number was 119.3 and 15 mg/L, EC 50 and NOEC for inhibition in dry weight- growth rate was 210.1 and 135 mg/L while inhibition in dry weight – yield was 164.4 and 15 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater plants:

119.3 mg/L

EC10 or NOEC for freshwater plants:

15 mg/L

Additional information

In GLP-compliant study, the effect of FAT 20044/C was tested on the growth of freshwater vascular aquatic plant, Lemna minor carried out according to OECD guideline 221. Based on the results of the range finding test, the definitive test was carried out using the test concentrations of 5, 15, 45, 135 and 405 mg/L; along with control. Each test item group had three replicate and control had six replicates. Lemna was exposed in semi-static mode with renewal of test medium on days 3 and 5. Frond observations were made on days 3, 5 and 7. Dry weight was determined at the start (day 0) and at the end of the test (day 7). At measured test concentrations of 5, 15, 45, 135 and 405 mg/L, the observed growth rate and yield for frond numbers and dry weight resulted in toxic effects on growth of Lemna when compared with the control during the test period Fronds were found to be normal in appearance in control and test item treated group at the end of the test. Test medium was clear in control and deep blue to violet color tinge in test item treated groups. Test medium was similar in appearance on days 3, 5 and 7 as compared to the start of the exposure. The test item was recoverable using the analytical method at nominal concentrations of 1.022 and 601 mg/L in the test medium. The stability test results concluded that the test item was found to be stable in the test medium at the test conditions up to 4 days at nominal concentrations of 1.022 and 601 mg/L. The results of active ingredient analysis of 5, 15, 45, 135 and 405 mg/L showed that overall percent agreement was from 91.039 to 118.927 % (with % RSD 0.878 to 14.774) at the start of renewals; 81.797 to 119.097% (with % RSD 1.124 to 16.988) at the end of renewals indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of <20 %). No measurable concentration of test item was recorded in the control samples. The endpoint values for growth rate and yield for frond numbers and dry weight at end of the test are presented below based on nominal concentration.

 

Parameter	Endpoint (mg test item/L)
	EC value		95% Fiducial limits
Inhibition in specific growth rate in frond number	ErC50	234.3	229.5 - 239.2
	ErC20	114.4	110.9 -118.0
	ErC10	75.21	72.00 - 78.49
	LOEC	45	NA
	NOEC	15	NA
Inhibition in yield rate in frond number	EyC50	119.3	117.1 - 121.4
	EyC20	63.89	61.86 - 65.99
	EyC10	44.35	42.38 - 46.41
	LOEC	45	NA
	NOEC	15	NA
Inhibition in dry weight- growth rate	ErC50	210.1	upper limits 240.7
	ErC20	154.9	139.0 - 179.5
	ErC10	129.7	113.0 - 153.0
	LOEC	405	NA
	NOEC	135	NA
Inhibition in dry weight - yield	EyC50	164.4	123.3 - 220.3
	EyC20	110.7	56.32 -147.8
	EyC10	87.88	33.25 lower limits
	LOEC	45	NA
	NOEC	15	NA