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Toxicity to aquatic plants other than algae

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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:
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:
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 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.

Definitive Test
Samples of the control and 100 mg/L test group were taken from the bulk test preparation on Day 0 (fresh media) and from the pooled replicates (R1-R6) on Day 7 (old media) for quantitative analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary
Vehicle:
yes
Remarks:
Test Medium
Details on test solutions:
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 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.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

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
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:
Not reported
Test temperature:
24 ± 1 ºC
pH:
6.5 - 7.6
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Chemical analysis of the 100 mg/L test preparations on Day 2 (fresh media) and Days 5 and 7 (old media) showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.
Details on test conditions:
Experimental Design and Study Conduct
3.4.1 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 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.
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) on a black non-reflective surface 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 (see Annex 3).

Exposure Conditions
As in the range-finding test 500 mL 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) on a black non-reflective surface 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
Remarks:
Based on Frond Number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Based on Frond Number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Based on Dry Weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Based on Dry Weight
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Based on Frond Number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Based on Frond Number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Based on Dry Weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Based on Dry Weight
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 were determined.
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 Day 2 (fresh media) and Days 5 and 7 (old media) showed near nominal concentrations were 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) showed measured test concentrations to range from 97% to 105% 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 1.73 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: 131

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) =>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 as above. 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 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) =>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 above. There were no statistically significant decreases in dry weight 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 as above. There were no statistically significant decreases in dry weight 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.
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

Results:

 Response Variable  Measurement Variable  EC50 (mg/L)  No Observed Effect Concentration (NOEC) (mg/L)
 Average Specific Growth Rate  Frond Number  >100  100
   Dry Weight  >100  100
 Yield  Frond Number  >100  100
   Dry Weight  >100  100
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 gave the following results:
Response Variable Measurement Variable EC50 (mg/L) No Observed Effect Concentration (NOEC) (mg/L)
Average Specific Growth Rate Frond Number >100 100
Dry Weight >100 100
Yield Frond Number >100 100
Dry Weight >100 100
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 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, 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 to range from 97% to 105% 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:

Response Variable  Measurement Variable  EC50(mg/L)  No Observed Effect Concentration (NOEC) (mg/L)
 Average Specific Growth Rate  Frond Number  >100  100
   Dry Weight  >100  100
 Yield  Frond Number  >100  100
   Dry Weight  >100  100
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and the target substance have very similar physicochemical and (eco)toxicological properties because their chemical structures are nearly identical. An analogue approach has thus been employed. The target substance is the meta-isomer of the dye Reactive Yellow 095, where the sulphonate group is bound at the meta-position of the amino benzene moiety. The source chemical is the reaction mass of both the meta-isomer and the para-isomer of Reactive Yellow 095.
The presence of sulphonate groups make both dyes highly water soluble and therefore less critical for human health and environmental issues. Based on their chemical similarity, similar properties are expected in both humans and the environment.
2. SOURCE AND TARGET CHEMICAL(S)
Source: Reactive Yellow 095 meta/para (CAS# --- / EC# 944-218-2)
Target: Reactive Yellow 095 meta (CAS# 84045-63-6 / EC# 281-865-3)
3. ANALOGUE APPROACH JUSTIFICATION
see attachment under 4.12 Auto flammability
Reason / purpose:
read-across source
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
The 7d EC50 in Lemna minor was determined to be >100 mg/L, based on growth rate and on yield.
Executive summary:

A study was performed to assess the effect of the source substance on the growth of the freshwater plant Lemna minor. The method followed that described in OECD TG 221.

Following a preliminary range-finding test, 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 °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 preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 97% to 105% of nominal and so the results are based on nominal test concentrations only.

Exposure of Lemna minor to the source substance based on nominal concentrations gave the below results. The structurally related target substance will show similar results when tested in Lemna.

Response Variable  Measurement Variable  EC50(mg/L)  No Observed Effect Concentration (NOEC) (mg/L)
 Average Specific Growth Rate  Frond Number  >100  100
   Dry Weight  >100  100
 Yield  Frond Number  >100  100
   Dry Weight  >100  100

Description of key information

The 7d EC50 in Lemna minor was determined to be >100 mg/L, based on growth rate and on yield.

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 the source substance on the growth of the freshwater plant Lemna minor. The method followed that described in OECD TG 221.

Following a preliminary range-finding test, 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 °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 preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 97% to 105% of nominal and so the results are based on nominal test concentrations only.

Exposure of Lemna minor to the source substance based on nominal concentrations gave the below results. The structurally related target substance will show similar results when tested in Lemna.

Response Variable  Measurement Variable  EC50(mg/L)  No Observed Effect Concentration (NOEC) (mg/L)
 Average Specific Growth Rate  Frond Number  >100  100
   Dry Weight  >100  100
 Yield  Frond Number  >100  100
   Dry Weight  >100  100