Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 March 2017 and 02 June 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 201 (2006) No 201 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 11-0078
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

Range-finding test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.

Definitive test concentrations: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Two additional samples of each test concentration were incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Vehicle:

no

Details on test solutions:

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 40 mg/L could be obtained using a saturated solution method of preparation.
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 Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
A nominal amount of test item (1100 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 stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (5.5 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation. Two replicate flasks were used for each control and test concentration.

Definitive Test
Experimental Preparation
A nominal amount of test item (1100 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 stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (1800 mL) of each of the stock solutions was separately inoculated with 25.0 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were 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 at 0, 24, 48 and 72 hours.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.5 - 9.8.

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100% v/v saturated solution
Definitive test concentrations: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.62 to 77 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.50 to 74 mg/L at 24 hours, 0.072 to 71 mg/L at 48 hours and from less than the limit of quantification (LOQ), determined to be 0.070 mg/L, to 62 mg/L at 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation. Two replicate flasks were used for each control and test concentration.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.61 x 10^5 cells per mL. Inoculation of 1800 mL of test medium with 25.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

- No. of vessels per concentration (replicates):
2

- No. of vessels per control (replicates):
2

- No. of vessels per vehicle control (replicates):
None

GROWTH MEDIUM
- Standard medium used: yes/no
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive tests, additional sodium bicarbonate (250 mg/L) was added to the prepared culture medium prior to use.


- Photoperiod:
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

- Light intensity and quality:
warm white lighting (380 – 730 nm)

- Salinity (for marine algae):
Not applicable

- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding test, two initial experiments were conducted at concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. In both instances poor growth was observed in the control and/or the test cultures.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

Range-finding Test
The results showed no effect on growth rate at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.089 to 71 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the limit of quantification (LOQ), determined to be 0.070 mg/L, to 87 mg/L indicating that the test item was unstable under the conditions of the test.

Initial Experiments
Based on the results of the range-finding test, two initial experiments were conducted at concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. In both instances poor growth was observed in the control and/or the test cultures.

Definitive Test
Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): 1.2 mg/L
ErC20 (0 - 72 h): 2.0 mg/L
ErC50 (0 - 72 h): 4.6 mg/L; 95% confidence limits 3.5 – 6.1 mg/L

Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the 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, 0.27 and 0.86 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) based on growth rate was 0.86 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.7 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 1.1 mg/L
EyC20 (0 - 72 h): 1.3 mg/L
EyC50 (0 - 72 h): 1.9 mg/L*

* It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control, 0.27 and 0.86 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) based on yield was 0.86 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.7 mg/L.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.27, 0.86 and 3.7 mg/L, however no intact cells were observed in the 16 and 71 mg/L test cultures.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 92 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 4.58 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 20% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.62 to 77 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.50 to 74 mg/L at 24 hours, 0.072 to 71 mg/L at 48 hours and from less than the limit of quantification (LOQ), determined to be 0.070 mg/L, to 62 mg/L at 72 hours.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC₅₀values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.035 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0-Hour Measured Test Concentration
1.0	0.27	44
3.2	0.86	39
10	3.7	50
32	16	65
100	71	93

 Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.27 and 0.86 mg/L test cultures were observed to be green dispersions. The 3.7 mg/L test cultures were observed to be pale green dispersions whilst the 16 and 71 mg/L test cultures were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Remarks:

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Conclusions:

The ErC50, ErC10 and NOEC were 4.6, 1.2 and 0.86 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009. Following a preliminary range-finding test and initial experiments, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test material solutions were prepared by saturated solution method 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.62 to 77 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.50 to 74 mg/L at 24 hours, 0.072 to 71 mg/L at 48 hours and from less than the limit of quantification (LOQ), determined to be 0.070 mg/L, to 62 mg/L at 72 hours.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 and EC10 values based on the geometric mean measured test concentration of 4.6 and 1.2 mg/L respectively. The No Observed Effect Concentration was determined to be 0.86 mg/L.

After 72 hours there were no abnormalities detected in the control or test cultures at 0.27, 0.86 and 3.7 mg/L, however no intact cells were observed in the 16 and 71 mg/L test cultures.

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.Following a preliminary range-finding test and initial experiments,Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test material solutions were prepared bysaturated solution methodwith 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.62 to 77 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.50 to 74 mg/L at 24 hours, 0.072 to 71 mg/L at 48 hours and from less than the limit of quantification (LOQ), determined to be 0.070 mg/L, to 62 mg/L at 72 hours.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 and EC10 values based on the geometric mean measured test concentration of 4.6 and 1.2 mg/L respectively. The No Observed Effect Concentration was determined to be 0.86 mg/L.

After 72 hours there were no abnormalities detected in the control or test cultures at 0.27, 0.86 and 3.7 mg/L, however no intact cells were observed in the 16 and 71 mg/L test cultures.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.6 mg/L

EC10 or NOEC for freshwater algae:

0.86 mg/L