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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:
05 August 2016 to 31 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Not required
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
March 2006
Deviations:
no
Principles of method if other than guideline:
Guideline followed
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: FAT 20063/D TE
Physical state/Appearance: Blue solid
Batch: BS-BOP 01-15 (AT-0032199800)
Purity: 47.9%
Expiry Date: 8 March 2020
Storage Conditions: Approximately 4 ºC in the dark
Analytical monitoring:
yes
Details on sampling:
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give the required test concentration of 100 mg/L and mixed to ensure adequate mixing and homegeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day 7.
Vehicle:
yes
Remarks:
Culture medium
Details on test solutions:
Range Finding Test
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L. The test was conducted in glass conical flasks (500 mL). Two replicate flasks each containing 250 mL were prepared for each control and test concentration. The test item was dissolved directly in culture medium.

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L.

The control test item was culture medium alone. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give the required test concentration of 100 mg/L. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The control test item was culture medium alone.
Test organisms (species):
Lemna minor
Details on test organisms:
A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium . The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.
Test type:
static
Water media type:
other: Culture medium in reverse osmosis purified water (Elga Optima 15+ or Elga Purlab Option R-15 BP)
Limit test:
yes
Total exposure duration:
7 d
Remarks on exposure duration:
Lemna minor was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks for a period of 7 days, under constant illumination at a temperature of 24 ± 1 ºC.
Post exposure observation period:
None
Hardness:
Not tested
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
Control :
Day 0: 7.6
Day 7: 8.4 - 8.6

100 mg/L
Day 0: 6.9 - 7.0
Day 7: 7.9 - 8.3
Dissolved oxygen:
Not measured
Salinity:
Not measured
Conductivity:
Not measured
Nominal and measured concentrations:
Day 0
Nominal concentration of Control 0: mg/L
Determined concentration of Control: Nominal concentration of test item: 100 mg/L
Determined concentration of test item: 98.3 mg/L

Day 7
Nominal concentration of Control 0: mg/L
Determined concentration of Control: Nominal concentration of test item: 100 mg/L
Determined concentration of test item: 100 mg
Details on test conditions:
Range Finding Test
The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days.
At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) on a black non-reflective surface for 7 days. On Days 3 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 3, 5 and 7.
A static testing regime was employed.

Definitive Test
Each control and test flask was inoculated with 3 colonies of Lemna minor (total 9 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) on a black non-reflective surface for 7 days.
A static testing regime was employed.
Reference substance (positive control):
yes
Remarks:
A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Duration:
7 d
Dose descriptor:
EC10
Remarks:
frond number
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Remarks:
frond number
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
frond number
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Remarks:
frond number
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Remarks:
frond number
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
frond number
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC10
Remarks:
dry weight
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Remarks:
dry weight
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
dry weight
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
frond number
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
dry weight
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
frond number
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
dry weight
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Range-finding Test

The results showed no significant effect on growth at the test concentrations of 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a “limit test” to confirm that at the maximum test concentration given in the OECD Test Guidelines, no effect on growth was observed.
Chemical analysis of the 100 mg/L test preparations on Days 0 (fresh media) and Days 3 and 7 (old media) (see Annex 3) showed a measured concentration of 99% of nominal was obtained indicating that the test item was stable under test conditions.

Definitive Test

Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) (see Annex 3) showed measured test concentrations to range from 98% to 100% of nominal and so the results are based on nominal test concentrations only.

Validation Criteria
The following data show that the doubling time of the control cultures was 1.81 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:
Mean frond number in control cultures at Day 0 : 9
Mean frond number in control cultures at Day 7 : 88
Growth Data Based on Frond Number

The following results are based on inhibition of average specific growth rate and yield were determined from the frond number data:
Average Specific Growth Rate
ErC10 (frond number) = >100 mg/L
ErC20 (frond number) = >100 mg/L
ErC50 (frond number) = >100 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x%.
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences between the control and 100 mg/L test concentration (P0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 100 mg/L.

Yield
EyC10 (frond number) = >100 mg/L
EyC20 (frond number) = >100 mg/L
EyC50 (frond number) = >100 mg/L
Where:
EyCx = the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥ 0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 100 mg/L.

Growth Data Based on Dry Weight

The following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:

Average Specific Growth Rate
ErC10 (dry weight) = >100 mg/L
ErC20 (dry weight) = >100 mg/L
ErC50 (dry weight) = >100 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x%.
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration as in Section 6.2.4. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 100 mg/L.

Yield
EyC10 (dry weight) = >100 mg/L
EyC20 (dry weight) = >100 mg/L
EyC50 (dry weight) = >100 mg/L
Where:
EyCx = the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 100 mg/L.

Observations
All test and control cultures were inspected on Days 0, 2, 5 and 7.
Results with reference substance (positive control):
A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. The positive control was conducted between 18 May 2016 and 30 May 2016

Exposure of Lemna minor to the reference item gave the following results:

Response variable Measurement variable EC50 (mg/L) 95% Conf limits NOEC (mg/L) LOEC (mg/L)
Average specific growth rate Frond number 3.4 3.1 - 3.8 0.625 1.25
Average specific growth rate dry weight 3.0 2.7 - 3.2 0.625 1.25
Yield Frond number 1.8 1.6 - 2.2 0.625 1.25
Yield Dry weight 1.4 1.2 - 1.7 0.625 1.25

The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.
Reported statistics and error estimates:
Average Specific Growth Rate based on frond numbers
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥0.05) and therefore the NOEC in terms of inhibition of average specific growth rates calculated from frond numbers was 100 mg/L.

Yield based on frond numbers
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥0.05) and therefore the NOEC in terms of inhibition of yield calculated from frond numbers was 100 mg/L.

Average specific growth rate based on dryweight
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥0.05) and therefore the NOEC in terms of inhibition of average specific growth rate calculated from dry weight was 100 mg/L.

Yield based on dry weight
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P≥0.05) and therefore the "No Observed Effect NOEC in terms of inhibition of yield calculated from dry weight was 100 mg/L.

None

Validity criteria fulfilled:
yes
Remarks:
The doubling time of the control cultures was 1.81 days in line with OECD Guideline 221 that states the doubling time should <2.5 days: Mean frond no. in control cultures at Day 0 :9 ; Mean frond no in control cultures at Day 7: 88
Conclusions:
The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and gave the following results:

Response variable                     Measurement variable       EC50 (mg/L)       NOEC (mg/L)
Average specific growth rate    Frond number                     >100                   100                     
Average specific growth rate    Dry weight                          >100                   100                    
Yield                                         Frond number                     >100                   100
Yield                                       Dry weight                          >100                   100             
Executive summary:

Summary

Introduction

A study was performed to assess the effect of the test item on the growth of the freshwater plantLemna minor. The method followed that described in the OECD Guideline No. 221 “Lemnasp.Growth Inhibition Test (March 2006)”.

Methods

Following a preliminary range-finding test,Lemna minorwas exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks for a period of 7 days, under constant illumination at a temperature of 24 ± 1°C. 

The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 98% to 100% of nominal and so the results are based on nominal test concentrations only.

Exposure of Lemna minorto the test item based on nominal concentrations gave the following results:

Response variable

 Measurement variable

EC50

(mg/L)

NOEC

(mg/L)

 Average specific growth rate  Frond number    >100  100
 Average specific growth rate  Dry weight     >100  100
 Yield    Frond number    >100  100
 Yield    Dry weight     >100  100

Description of key information

EC50 (avg specific growth rate based on frond number as well as dry weight) was >100 mg/L. Similarly, EC50 (yield based on frond number and dry weight) was also >100 mg/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

A study was performed to assess the effect of FAT 20063/D on the growth of the freshwater plantLemna minor. The method followed that described in the OECD Guideline No. 221 “Lemnasp.Growth Inhibition Test (March 2006).

Following a preliminary range-finding test,Lemna minorwas exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks for a period of 7 days, under constant illumination at a temperature of 24 ± 1°C. The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development.

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 98% to 100% of nominal and so the results are based on nominal test concentrations only.

Based on the findings of the study, EC50 (avg specific growth rate based on frond number as well as dry weight) was >100 mg/L. Similarly EC50 (yield based on frond number and dry weight) was also >100 mg/L.