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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
March 2006
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sponsor Lot/Batch number 43 (India)
- Expiration date of the lot/batch: 03 February 2020
- Purity: 84.3 %
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Frozen at approximately -20 °C
Analytical monitoring:
yes
Details on sampling:
In order to determine the stability of the test item under test conditions a sample of each test concentration was taken for chemical analysis on Day 2 (fresh media) and Day 5 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.
Vehicle:
yes
Remarks:
culture medium
Details on test solutions:
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give the 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.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
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:
freshwater
Limit test:
yes
Total exposure duration:
7 d
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
No data
Test temperature:
24 ± 1 °C
pH:
6.8 - 8.9
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Day 0 Nominal concentration = 100 mg/L Measured concentration = 98.8 mg/L i.e. 99 % nominal
Day 7 Nominal concentration = 100 mg/L Measured concentration = 103 mg/L i.e. 103 % nominal
Details on test conditions:
Range-Finding test
The test concentration to be used in the definitive test was determined by a preliminary 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.
The test was conducted in glass conical flasks (500 mL). Two replicate flasks 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 the 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.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.
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) for 7 days.
On Days 2 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 2, 5 and 7.

Definitive Test
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD Test Guidelines, no effect on growth was observed.
Experimental Preparation
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 concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day 7.

Exposure Conditions
As in the range-finding test glass conical flasks were used. Six flasks each containing 250 mL of solution were prepared for the control and 100 mg/L treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Each control and test flask was inoculated with 3 colonies of Lemna minor (total 11 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) for 7 days.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
EC50
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
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
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Details on results:
Range-finding Test
The frond counts and percentage inhibition of growth values from the exposure of Lemna minor to the test item during the range-finding test are given in Table 1.
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 showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Definitive Test

Average Specific Growth Rate
ErC10 (frond number) = >100 mg/L
ErC20 (frond number) = >100 mg/L
ErC50 (frond number) = >100 mg/L
"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
"No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 100 mg/L.

Average Specific Growth Rate dry weight)
ErC10 (dry weight) = >100 mg/L
ErC20 (dry weight) = >100 mg/L
ErC50 (dry weight) = >100 mg/L
"No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 100 mg/L.

Yield (dry weight)
EyC10 (dry weight) = >100 mg/L
EyC20 (dry weight) = >100 mg/L
EyC50 (dry weight) = >100 mg/L
"No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 100 mg/L.



Results with reference substance (positive control):
None
Reported statistics and error estimates:
Statistical analysis
A Students t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the average specific growth rate and yield data at 7 days for the control and 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

Validation Criteria

The data show that the doubling time of the control cultures was 1.77 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: 11

Mean frond number in control cultures at day 7: 120

Statistical analysis

Statistical analysis of data for growth and yield was carried out for the control and 100 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P≥0.05), between the control and 100 mg/L test groups.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Lemna minor to the test item based on nominal concentrations gave the following results:
EC50: >100 mg/L (Average specific growth rate - frond number and dry weight)
EC50: >100 mg/L (Yield:Frond Number, dry weight)

No Observed Effect Concentration (NOEC):
100 mg/L (Average specific growth rate - frond number)
100 mg/L (Yield:Frond Number, dry weight)
Executive summary:

A key study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor according to the OECD Guideline 221.

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for a period of 7 days, under constant illumination at a temperature of 24 ± 2 °C. The number of fronds in each control and treatment group was recorded on Days 0, 3, 5 and 7 along with observations on plant development. Chemical analysis of the test preparation on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 99 % to 102 % of nominal and so the results are based on nominal test concentrations only.

 

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

EC50: >100 mg/L (Average specific growth rate - frond number and dry weight)

EC50: >100 mg/L (Yield:Frond Number, dry weight)

No Observed Effect Concentration (NOEC):

100 mg/L(Average specific growth rate - frond number)

100 mg/L (Yield: Frond Number, dry weight)

Description of key information

Exposure of Lemna minor to the test item based on nominal concentrations gave the following results:
EC50: >100 mg/L (Average specific growth rate - frond number and dry weight)
EC50: >100 mg/L ( Yield:Frond Number, dry weight)

No Observed Effect Concentration (NOEC):
100 mg/L (Average specific growth rate - frond number)
100 mg/L (Yield: Frond Number, dry weight)

Key value for chemical safety assessment

EC50 for freshwater plants:
100 mg/L

Additional information

The key study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor according to the OECD Guideline 221.

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for a period of 7 days, under constant illumination at a temperature of 24 ± 2 °C. The number of fronds in each control and treatment group was recorded on Days 0, 3, 5 and 7 along with observations on plant development. Chemical analysis of the test preparation on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 99 % to 102 % of nominal and so the results are based on nominal test concentrations only.

 

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

EC50: >100 mg/L (Average specific growth rate - frond number and dry weight)

EC50: >100 mg/L (Yield:Frond Number, dry weight)

No Observed Effect Concentration (NOEC):

100 mg/L (Average specific growth rate - frond number)

100 mg/L (Yield:Frond Number, dry weight)