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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:

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:

Guideline followed

GLP compliance:

yes

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test:
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 0 (fresh media) and Day 3 (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.

Definitive Test
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 2 and 5 (fresh media) and Days 2, 5 and 7 (old media)

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.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.
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 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 50, 25, 12.5 and 6.25 mg/L.
Each of the prepared concentration 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:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

24 ± 1”C

pH:

6.2 - 7.4

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Chemical analysis of the freshly prepared test samples on Days 0, 2 and 5 showed measured test concentrations to range from 3.8 to 122 mg/L. Measured test concentrations obtained from the old or expired media on Days 2, 5 and 7 were observed to range of 3.3 to 131 mg/L. Given that near nominal test concentrations were not obtained for all freshly prepared test samples, and that in some instances a slight decline in measured concentrations occurred in the corresponding old or expired test samples, it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations in order to give a “worst case” analysis of the data.

Details on test conditions:

Experimental Design and Study Conduct
Range-finding Test
The test concentrations to be used in the definitive test were 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 control group was maintained under identical conditions but not exposed to the test item. Data from the control group was shared with similar concurrent studies.
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.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.

Experimental Preparation
Exposure Conditions
As in the range-finding test glass conical flasks were used. Three flasks each containing 250 mL of solution were prepared for the control and each 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 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.
On days 2 and 5 the test solutions were renewed.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 125 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 125 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

120 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

69 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

RESULTS
Range-finding Test
The results showed no significant effect on growth at the test concentrations of 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 10 mg/L test preparations on Day 0 (fresh media) and Days 3 and 7 (old media) showed measured test concentrations of 73%, 58% and 54% of nominal respectively. Analysis of the 100 mg/L test preparations showed measured concentrations to range from 107% to 117% of nominal. Given that measured concentrations below 80% of nominal were obtained in the 10 mg/L test preparations, and also the decline in measured concentrations from the old test media samples, it was considered appropriate to conduct the definitive test using a semi-static testing regime.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the freshly prepared test samples on Days 0, 2 and 5 showed measured test concentrations to range from 3.8 to 122 mg/L. Measured test concentrations obtained from the old or expired media on Days 2, 5 and 7 were observed to range of 3.3 to 131 mg/L. Given that near nominal test concentrations were not obtained for all freshly prepared test samples, and that in some instances a slight decline in measured concentrations occurred in the corresponding old or expired test samples, it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations in order to give a “worst case” analysis of the data.

Validation Criteria
The following data show that the doubling time of the control cultures was 1.72 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 : 110

Growth Data Based on Frond Number
Numbers of fronds in each flask in the definitive test were determined and the average specific growth rates, yields and percentage inhibition values calculated.
Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the frond number data.

Average Specific Growth Rate
ErC10 (frond number) = 8.9 mg/L
ErC20 (frond number) = 56 mg/L
ErC50 (frond number) = >125 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 all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 4.0 and 9.0 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (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 9.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 22 mg/L.

Yield
EyC10 (frond number) = 1.6 mg/L
EyC20 (frond number) = 5.1 mg/L
EyC50 (frond number) = 120 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 all test concentrations as above. There were no statistically significant differences between the control and 4.0 mg/L test concentration (P.0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 4.0 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) was determined to be 9.0 mg/L.

Growth Data Based on Dry Weight
The dry weight of Lemna minor in each flask in the definitive test was determined and average specific growth rates, yield and percentage inhibition values calculated. Accordingly 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) = 6.4 mg/L
ErC20 (dry weight) = 29 mg/L
ErC50 (dry weight) = >125 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 all test concentrations as above. There were no statistically significant differences between the control, 4.0 and 9.0 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (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 9.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 22 mg/L.

Yield
EyC10 (dry weight) = 1.8 mg/L
EyC20 (dry weight) = 4.4 mg/L
EyC50 (dry weight) = 69 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 all test concentrations as in Section 6.2.4. There were no statistically significant differences between the control, 4.0 and 9.0 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 9.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 22 mg/L.

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

Response Variable Measurement Variable EC50 (mg/L) 95% Confidence Limits NOEC (mg/L) LOEC (mg/L)
Average Specific Growth Rate Frond Number 3.4 3.1 - 3.8 0.625 1.25
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
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.

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>125	9.0	22
	Dry Weight	>125	9.0	22
Yield	Frond Number	120	4.0	9.0
	Dry Weight	69	9.0	22

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and based on the time-weighted mean measured test concentrations gave the following results.
 
Response Variable Measurement Variable  EC50 (mg/L)  NOEC (mg/L)  LOEC (mg/L)
 Average Specific Growth Rate Frond Number  >125  9.0  22 
  Dry Weight  >125  9.0  22
 Yield  Frond Number  120  4.0  9.0 
  Dry Weight  69  9.0  22

Executive summary:

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

Methods

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The test solutions were renewed on days 2 and 5.

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 freshly prepared test samples on Days 0, 2 and 5 showed measured test concentrations to range from 3.8 to 122 mg/L. Measured test concentrations obtained from the old or expired media on Days 2, 5 and 7 were observed to range of 3.3 to 131 mg/L. Given that near nominal test concentrations were not obtained for all freshly prepared test samples, and that in some instances a slight decline in measured concentrations occurred in the corresponding old or expired test samples, it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations in order to give a “worst case” analysis of the data.

Exposure of Lemna minor to the test item based on the time-weighted mean measured test concentrations gave the following results:

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>125	9.0	22
	Dry Weight	>125	9.0	22
Yield	Frond Number	120	4.0	9.0
	Dry Weight	69	9.0	22

Description of key information

EC50 to Lemna minor taking into account the avearge specific growth rate based on the frond number was >125 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

125 mg/L

Additional information

Introduction

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

Methods

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The test solutions were renewed on days 2 and 5. 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 freshly prepared test samples on Days 0, 2 and 5 showed measured test concentrations to range from 3.8 to 122 mg/L. Measured test concentrations obtained from the old or expired media on Days 2, 5 and 7 were observed to range of 3.3 to 131 mg/L. Given that near nominal test concentrations were not obtained for all freshly prepared test samples, and that in some instances a slight decline in measured concentrations occurred in the corresponding old or expired test samples, it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations in order to give a “worst case” analysis of the data.

Exposure of Lemna minor to the test item based on the time-weighted mean measured test concentrations gave the following results:

Response Variable	Measurement Variable	EC50(mg/L)	95% Confidence Limits			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>125		-		9.0	22
	Dry Weight	>125		-		9.0	22
Yield	Frond Number	120	-	-	-	4.0	9.0
	Dry Weight	69	-	-	-	9.0	22