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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:
05 August, 2016 to 01 November, 2016
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)
Version / remarks:
March 2006
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Identification: FAT 20202/H TE
Physical state/Appearance: Dark brown solid
Batch: 111 (Thailand)
Purity: 65.2 %
Expiry Date: 02 August 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 a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 32, 10, 3.2 and 1.0 mg/L.. The concentrations were mixed to ensure adequate mixing and homogeneity.

Samples were taken from the control and each test group from the bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
Vehicle:
yes
Remarks:
Culture medium
Details on test solutions:
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 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 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.

The control test item was the culture meium alone.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 32, 10, 3.2 and 1.0 mg/L.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

The control test item was the culture meium 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:
no
Total exposure duration:
7 d
Remarks on exposure duration:
Lemna minor was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 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.
Post exposure observation period:
None
Hardness:
Not tested.
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
pH:
Control:
Day 0: 7.2-7.3
Day 7: 7.5

0.86 mg/L
Day 0: 7.1-7.2
Day 7: 7.5-7.6

2.5 mg/L
Day 0: 7.2
Day 7: 7.5

9.1 mg/L
Day 0: 7.2
Day 7: 7.5

30 mg/L
Day 0: 7.2
Day 7: 7.5

99 mg/L
Day 0: 7.2
Day 7: 7.4
Dissolved oxygen:
Not measured.
Salinity:
Not measured.
Conductivity:
Not measured.
Nominal and measured concentrations:
Range finding Test
Control
Nominal concentation: 1.0 mg/L
Nominal concentration: 10 mg/L
Nominal concentration: 100 mg/L


Definitive test
Control:
Nominal concentration: 0 mg/L
Mean measured concentration: 0 mg/L

Nominal concentation: 1.0 mg/L
Mean measured concentation: 0.86 mg/L

Nominal concentration: 3.2 mg/L
Mean measured concentration: 2.5 mg/L

Nominal concentration: 10 mg/L
Mean measured concentration: 9.1 mg/L

Nominal concentration: 32 mg/L
Mean measured concentation: 30 mg/L

Nominal concentration: 100 mg/L
Mean measured concentration: 99 mg/L
Details on test conditions:
Range-finding Test
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) 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: 1.0, 3.2, 10, 32 and 100 mg/L.
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) 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 at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Growth rate
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC20
Remarks:
Growth rate
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Growth rate
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Yield
Effect conc.:
0.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC20
Remarks:
Yield
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Yield
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other:
Remarks:
95% confidence limits 3.1 – 6.2 mg/L
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Growth rate
Effect conc.:
0.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Remarks:
Growth rate
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Growth rate
Effect conc.:
41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Yield
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Remarks:
Yield
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Yield
Effect conc.:
3.6 mg/L
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 concentration of 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 85 % to 100 % of nominal. Near nominal concentrations were obtained from the test preparations on Day 7 with the exception of the 1.0 and 3.2 mg/L test samples which saw a decline in measured concentrations to 76 % and 70 % of nominal, respectively. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Chemical analysis of the test preparations on Days 0 (fresh media) and Days 3 and 7 (old media) showed that with the exception of the 1.0 mg/L test sample on Day 3, near nominal concentrations were obtained indicating that the test item was stable under test conditions. The geometric mean merasured concentrations were determined to be:

Nominal Test conc (mg/L) Geometric Mean Measured conc (mg/L) Expressed as % of Day 0 measured test conc.
1.0 0.86 86
3.2 2.5 77
10 9.1 91
32 30 94
100 99 99

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: 89

Growth Data Based on Frond Number
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) = 0.94 mg/L
ErC20 (frond number) = 3.2 mg/L
ErC50 (frond number) = 26 mg/L*
* It was not possible to calculate 95 % confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

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 and 0.86 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 average specific growth rates calculated from frond numbers was 0.86 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.5 mg/L.

Yield
EyC10 (frond number) = 0.72 mg/L
EyC20 (frond number) = 1.4 mg/L
EyC50 (frond number) = 4.4 mg/L; 95% confidence limits 3.1 – 6.2 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. There were no statistically significant differences between the control and 0.86 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 0.86 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.5 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) = 0.89 mg/L
ErC20 (dry weight) = 3.6 mg/L
ErC50 (dry weight) = 41 mg/L*
* It was not possible to calculate 95 % confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

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. There were no statistically significant differences between the control, 0.86 and 2.5 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 2.5 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 9.1 mg/L.

Yield
EyC10 (dry weight) = 0.94 mg/L
EyC20 (dry weight) = 1.2 mg/L
EyC50 (dry weight) = 3.6 mg/L; 95 % confidence limits 2.3 – 5.5 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. There were no statistically significant differences between the control and 0.86 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 dry weight was 0.86 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be

Observations
All test and control cultures were inspected on days 0, 2, 5 and 7.

Water Quality Criteria
The pH values of each test and control flask are given below:
pH:
Control:
Day 0: 7.2-7.3
Day 7: 7.5

0.86 mg/L
Day 0: 7.1-7.2
Day 7: 7.5-7.6

2.5 mg/L
Day 0: 7.2
Day 7: 7.5

9.1 mg/L
Day 0: 7.2
Day 7: 7.5

30 mg/L
Day 0: 7.2
Day 7: 7.5

99 mg/L
Day 0: 7.2
Day 7: 7.4

Temperature was maintained at 24 ± 1 ºC throughout the test.
Results with reference substance (positive control):
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: See below
The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.
Reported statistics and error estimates:
Statistical analysis was carried out for the control and all test concentrations.
Average Specific Growth Rate - frond number
There were no statistically significant differences between the control and 0.86 mg/L test concentration (P<0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC in terms of inhibition of average specific growth rates calculated from frond numbers was 0.86 mg/L. Correspondingly the LOEC was determined to be 2.5 mg/L.
Yield - frond number
There were no statistically significant differences between the control and 0.86 mg/L test concentration (P<0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC in terms of inhibition of yield calculated from frond numbers was 0.86 mg/L. Correspondingly the LOEC was determined to be 2.5 mg/L.
Average Specific Growth Rate - dry weight
There were no statistically significant differences between the control, 0.86 and 2.5 mg/L test concentrations (P<0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC in terms of inhibition of average specific growth rate calculated from dry weight was 2.5 mg/L. Correspondingly the LOEC was determined to be 9.1 mg/L.
Yield - dryweight
There were no statistically significant differences between the control and 0.86 mg/L test concentration (P<0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC in terms of inhibition of yield calculated from dry weight was 0.86 mg/L. Correspondingly the LOEC as determined to be 2.5 mg/L.

Positive Control 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

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

 

Validity criteria fulfilled:
yes
Remarks:
Mean frond number in control cultures at day 0 :9; Mean frond number in control cultures at day 7: 89
Conclusions:
The EC50 (7d) based on average specific growth rate for frond numbers was 26 mg/L.
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)”. Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 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 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) showed measured test concentrations to range from 85% to 100% of nominal. Near nominal concentrations were obtained from the test preparations on Day 7 with the exception of the 1.0 and 3.2 mg/L test samples which saw a decline in measured concentrations to 76 % and 70 % of nominal respectively. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

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

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

26

 

*

 

0.86

2.5

Dry Weight

41

 

*

 

2.5

9.1

Yield

Frond Number

4.4

3.1

-

6.2

0.86

2.5

Dry Weight

3.6

2.3

-

5.5

0.86

2.5


*It was not possible to calculate 95 % confidence limits for these EC50 values as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

The EC50 (7d) based on average specific growth rate for frond numbers was 26 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:
26 mg/L
EC10 or NOEC for freshwater plants:
0.86 mg/L

Additional information

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 spGrowth Inhibition Test (March 2006)”. Following a preliminary range-finding test, Lemna minor  was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 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 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) showed measured test concentrations to range from 85 % to 100 % of nominal. Near nominal concentrations were obtained from the test preparations on Day 7 with the exception of the 1.0 and 3.2 mg/L test samples which saw a decline in measured concentrations to 76 % and 70 % of nominal respectively. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. Exposure of Lemna minor to the test item gave the following results based on the geometric mean measured test concentrations:


 




















































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



26



 



*



 



0.86



2.5



Dry Weight



41



 



*



 



2.5



9.1



Yield



Frond Number



4.4



3.1



-



6.2



0.86



2.5



Dry Weight



3.6



2.3



-



5.5



0.86



2.5






*It was not possible to calculate 95 % confidence limits for these EC50 values as the data generated did not fit the models available for the calculation of confidence limits.