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Ecotoxicological information

Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February, 2020 to 22 May, 2020
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:
Physical appearance: Black powder
Purity as per CoA: 99.1 % (w/w)
Batch No.: 1404301
Manufactured date: 20 September 2014
Expiry date: 08 April 2024
Density: 1.2654 g/cm3
pH: 5.0 (susp. of 2 % (w/w) in water)
Recommended storage condition: Ambient (+15 to +25 °C)
Analytical monitoring:
yes
Details on sampling:
The test concentration along with the control and vehicle control was analyzed for the test item concentration at the beginning and at the end of the test. For analysis, single composite sample was drawn from prepared test concentration and from the control and vehicle control.
Vehicle:
yes
Remarks:
Tween 80
Details on test solutions:
To prepare test concentration of 100 mg/L, 100 mg of test item (after grinding with mortar and pestle) was mixed with 100 μL of Tween 80 and test medium along with sonication followed by making up to 1 L in volumetric flask using test medium. For control (C), 1 L of test medium was
used and for vehicle control (VC), 100 μL of Tween 80 was made up to 1 L with test medium.
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 Öschelbronn, 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:
yes
Total exposure duration:
7 d
Test temperature:
23.5 - 24.9
pH:
6.90 - 7.24
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes

GROWTH MEDIUM
- Standard medium used: yes. Swedish Standard (SIS) medium was used as test medium (Lemna medium) as described in the OECD test guideline 221

OTHER TEST CONDITIONS
- Light intensity and quality: 6050 to 7090 lux

EFFECT PARAMETERS MEASURED :
- Determination of frond number: Frond numbers appearing normal or abnormal were determined at the beginning of the test, on days 3, 5 and 7 (at test termination).
- Determination of biomass: Dry weight was determined at the start of the test from a sample of the inoculum culture representative of what is used to begin the test, and at the end of the test with the plant material from each test and control(s) vessel.
- Changes in plant development, e.g. in frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, and in root length and appearance were observed. Significant features of the test medium (e.g. presence of undissolved material, growth of alga in the test vessel) was also noted.
- The measured frond numbers and dry weight in the test cultures and control(s) were tabulated together with the concentrations of the test item
and the times of measurement.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: Range finding test which was performed as a non-GLP test resulted in no effects on growth (as compared to controls) of Lemna at the tested concentrations of 0.1, 1, 10, 50 and 100 mg/L on day 7 of post treatment.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: with both parameters of frond number and dry weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: with both parameters of frond number and dry weight

Test Item Solubility


Test item was tested for its solubility with Tween 80 and test medium and resulted in violet colored suspension (along with sonication) at 10 mg/mL


(performed as a non-GLP test).


 


Range Finding Test


Range finding test results for growth rate and yield inhibition (as compared with that of vehicle control) are presented below for frond numbers and dry weight:




















































Group & Conc. (mg/L)



Frond numbers



Dry weight



% Ir



%IY



% Ir



%IY



T1 & 0.1



-0.3



-0.3



0.5



1.6



T2 & 1



0.0



0.0



0.3



0.6



T3 & 10



-0.3



-0.7



0.3



0.3



T4 & 50



0.0



0.0



0.3



0.8



T5 & 100



-0.5



-1.0



-0.3



-0.4



Note: % Ir: percent inhibition in average specific growth rate; % IY: percent inhibition in yield


Fronds were found to be normal in appearance in control and vehicle control and test item treated groups at the end of the test. Test medium was clear and violet colour with little settleable solids in treatment groups (colour gradient was there across the test concentrations from lower to higher). Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure.


 


Definitive Test


Definitive test results for growth rate and yield inhibition (as compared with that of vehicle control) are presented below for frond numbers and dry weight:
























Group & Conc. (mg/L)



Frond numbers



Dry weight



% Ir



%IY



% Ir



%IY



T1 & 100



-0.3



-0.4



-0.5



-1.1



At measured test concentration of 100 mg/L, the observed growth rate and yield for frond numbers and dry weight of Lemna was comparable with the control and vehicle control during the test period (statistically non-significant effects were observed in two sample t-test). Growth curve for inhibition of growth rate and yield (for frond numbers and dry weight) was not plotted due to absence of test item effects and also the test being the limit test with one test concentration. Fronds were found to be normal in appearance in control, vehicle control and test item treated group at the end of the test. Test medium was clear in control and vehicle control and violet color in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure. The NOEC and EC50 for growth rate and yield (for frond number and dry weight) based on the nominal concentration of the test item was higher than 100 mg test item/L.


 


Analysis of Test Concentrations


The test item was recoverable using the analytical method at nominal concentrations of 50.30 and 150.20 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 2 days at nominal concentrations of 50.30 and 150.20 mg/L. The results of active ingredient analysis of 100 mg/L showed that overall percent agreement was 82.22 and 100.24 % at the start of renewal (days 0 and 5) and 89.51 and 96.34 % at the end of renewal (days 2 and 7) 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 and vehicle control samples.


 


VALIDITY OF THE TEST


The Lemna growth inhibition test fulfills 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 13.4 corresponding to an average specific growth rate for frond number in the control of 0.371 per day and to a doubling time of 1.869 days (validity criterion: growth rate ≥0.275 per day, doubling time <2.5 days). The factor of frond number increase, measured in the vehicle control between day 0 and day 7, was 13.5 corresponding to an average specific growth rate for frond number in the control of 0.371 per day and to a doubling time of 1.867 days (validity criterion: growth rate ≥0.275 per day, doubling time <2.5 days).


 


CONCLUSION


The NOEC and EC50 for growth rate and yield (for frond number and dry weight) based on the nominal concentration of the test item was higher than 100 mg test item/L.

Validity criteria fulfilled:
yes
Conclusions:
The NOEC and EC50 for growth rate and yield (for frond number and dry weight) on the basis of the nominal concentration of the test item was higher than 100 mg test item/L
Executive summary:

The effect of FAT 36038/J TE was tested on the growth of freshwater vascular aquatic plant, Lemna minor according to OECD Guideline 221. Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L; along with control and vehicle control (each group had six replicates). Lemna was exposed in semistatic mode with renewal of test medium on days 2, 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 concentration of 100 mg/L, the observed growth rate and yield for frond numbers and dry weight of Lemna was comparable with the control and vehicle control during the test period (statistically non-significant effects were observed in two sample t-test). Fronds were found to be normal in appearance in control, vehicle control and test item treated group at the end of the test. Test medium was clear in control and vehicle control and violet colour in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure. The test item was recoverable using the analytical method at nominal concentrations of 50.30 and 150.20 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 2 days at nominal concentrations of 50.30 and 150.20 mg/L. The results of active ingredient analysis of 100 mg/L showed that overall percent agreement was 82.22 and 100.24 % at the start of renewal (days 0 and 5) and 89.51 and 96.34 % at the end of renewal (days 2 and 7) 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 and vehicle control samples.

Hence, based on the findings of the study, the NOEC and EC50 for growth rate and yield (for frond number and dry weight) on the basis of the nominal concentration of the test item was higher than 100 mg test item/L

Description of key information

The NOEC and EC50 for growth rate and yield (for frond number and dry weight) on the basis of the nominal concentration of the test item was higher than 100 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater plants:
100 mg/L
EC10 or NOEC for freshwater plants:
100 mg/L

Additional information

The effect of FAT 36038/J was tested on the growth of freshwater vascular aquatic plant, Lemna minor according to OECD Guideline 221. Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L; along with control and vehicle control (each group had six replicates). Lemna was exposed in semistatic mode with renewal of test medium on days 2, 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 concentration of 100 mg/L, the observed growth rate and yield for frond numbers and dry weight of Lemna was comparable with the control and vehicle control during the test period (statistically non-significant effects were observed in two sample t-test). Fronds were found to be normal in appearance in control, vehicle control and test item treated group at the end of the test. Test medium was clear in control and vehicle control and violet colour in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure. The test item was recoverable using the analytical method at nominal concentrations of 50.30 and 150.20 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 2 days at nominal concentrations of 50.30 and 150.20 mg/L. The results of active ingredient analysis of 100 mg/L showed that overall percent agreement was 82.22 and 100.24 % at the start of renewal (days 0 and 5) and 89.51 and 96.34 % at the end of renewal (days 2 and 7) 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 and vehicle control samples. Hence, based on the findings of the study, the NOEC and EC50 for growth rate and yield (for frond number and dry weight) on the basis of the nominal concentration of the test item was higher than 100 mg test item/L

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