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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:
21 April, 2016 to 23 June, 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 (incl. QA statement)
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 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.
Vehicle:
yes
Details on test solutions:
Information provided by the Sponsor indicated the water solubility of the test item to be 0.26 mg/L. The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.11 mg/L could be obtained using a saturated solution method of preparation.

Range-finding Test
The range-finding test was conducted at a series of nominal test concentrations of 1.0, 10 and 100 % v/v saturated solution.
A nominal amount of test item (275 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0 % v/v saturated solution.
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: 10, 18, 32, 56 and 100 % v/v saturated solution.

Experimental Preparation
A nominal amount of test item (275 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 56, 32, 18 and 10 % v/v saturated solution.
Each of the prepared concentrations 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.
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:
no
Total exposure duration:
7 d
Hardness:
Not reported
Test temperature:
24±1 °C
pH:
6.5 ± 0.2
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Range-finding test: 1.0, 10 and 100 % v/v saturated solution
Definitive test: 10, 18, 32, 56 and 100 % v/v saturated solution
Details on test conditions:
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. 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. 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 11 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) for 7 days on a black, non-reflective surface.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 other: % Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
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) >100 % v/v saturated solution
ErC20 (frond number) >100 % v/v saturated solution
ErC50 (frond number) >100 % v/v saturated solution
Where:
ErCx = the test concentration that reduced average specific growth rate by x %
There were no statistically significant differences between the control, 10, 18, 32, 56 and 100 % v/v saturated solution test concentrations (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% v/v saturated solution.

Yield
EyC10 (frond number) >100 % v/v saturated solution
EyC20 (frond number) >100 % v/v saturated solution
EyC50 (frond number) >100 % v/v saturated solution
Where:
EyCx = the test concentration that reduced yield by x %.
There were no statistically significant differences between the control, 10, 18, 32, 56 and 100 % v/v saturated solution test concentrations (P≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 100 % v/v saturated solution.

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 % v/v saturated solution
ErC20 (dry weight) >100 % v/v saturated solution
ErC50 (dry weight) >100 % v/v saturated solution
Where:
ErCx = the test concentration that reduced average specific growth rate by x %.
There were no statistically significant differences between the control, 10, 32, 56 and 100 % v/v saturated solution test concentrations (P≥0.05). Whilst the 18% v/v saturated solution test concentration was observed to be significantly different (P≤0.05), as no significant inhibition occurred at the higher test concentrations of 32, 56 and 100 % v/v the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was considered to be 100 % v/v saturated solution.

Yield
EyC10 (dry weight) >100 % v/v saturated solution
EyC20 (dry weight) >100 % v/v saturated solution
EyC50 (dry weight) >100 % v/v saturated solution
Where:
EyCx = the test concentration that reduced yield by x %.
There were no statistically significant differences between the control, 10, 32, 56 and 100 % v/v saturated solution test concentrations (P≥0.05). Whilst the 18 % v/v saturated solution test concentration was observed to be significantly different (P≤0.05), as no significant inhibition occurred at the higher test concentrations of 32, 56 and 100 % v/v the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was considered to be 100 % v/v saturated solution.
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 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
The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.
Validity criteria fulfilled:
yes
Conclusions:
This study showed that there were no toxic effects at saturation.
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

Information provided by the Sponsor indicated the water solubility of the test item to be 0.26 mg/L.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.11 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (25 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. 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.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0069 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. A single measured concentration of 0.0073 mg/L was obtained from the 32% v/v saturated solution test preparation on Day 7.

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

Response Variable  Measurement Variable  EC50 (% v/v Saturated Solution)  No Observed Effect Concentration (NOEC) (% v/v Saturated Solution)
 Average Specific Growth Rate  Frond Number  >100  100
   Dry Weight  >100  100
 Yield  Frond Number  >100  100
   Dry Weight  >100  100

This study showed that there were no toxic effects at saturation.

Description of key information

Exposure of Disperse Blue 077 to Lemna minor did not produce any toxic effects at saturation.

Key value for chemical safety assessment

Additional information

A study was performed to assess the effect of FAT 92504 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 nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solutions (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (25 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. 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 preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0069 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. A single measured concentration of 0.0073 mg/L was obtained from the 32 % v/v saturated solution test preparation on Day 7. Exposure of Lemna minor to the test item however did not have any adverse effects, hence it was concluded that there were no toxic effects at saturation.