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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Vinyl 2-ethylhexanoate
CAS Number: 94-04-2
EC Number: 202-297-4
Batch: PM9CEH01
Purity: 99.6%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 04 January 2022
Storage Conditions: Room temperature in the dark over silica gel
Analytical monitoring:
yes
Details on test solutions:
The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 105 to 106 cells/mL.
A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Details on test conditions:
Experimental Design and Study Conduct
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore media preparation trials were conducted in order to determine the solubility of the test item under test conditions (see Annex 4).

Range-Finding Test
The results obtained from the preliminary media preparation trials conducted indicated that a dissolved test item concentration of approximately 15 mg/L could be obtained using a saturated solution (shake flask) 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 Raphidocelis 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.
Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels (250 mL glass conical flasks) in order to minimize possible losses due to volatilization.
Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate (250 mg/L) was added to the culture medium to provide a source of carbon dioxide for algal growth. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
Nominal amounts of test item (56.5 mg) were separately dispensed in duplicate under the surface of 565 mL of test water in completely filled test vessels which were sealed immediately. The flasks were shaken at approximately 150 rpm at a temperature of 24 °C for 24 hours. After 24 hours, the contents of the flasks were pooled prior to the removal of any undissolved test item by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. This saturated solution was then further diluted to give further stock solutions of 0.10, 1.0 and 10% v/v saturated solution of the test item. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (3.5 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.
The stock solutions and each prepared concentration were 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 a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
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. All samples were analyzed on the day of sampling. A duplicate set of samples was taken on each occasion and stored frozen for further analysis if required.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 4.5, 10, 22, 48 and 100% v/v saturated solution.

Experimental Preparation
Nominal amounts of test item (56.5 mg) were separately dispensed in septuplets under the surface of 565 mL of test water in completely filled test vessels which were sealed immediately. The flasks were shaken at approximately 150 rpm at a temperature of 24 °C for 24 hours. After 24 hours, the contents of the flasks were pooled prior to the removal of any undissolved test item by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
A series of dilutions were made from this saturated solution to give stock solutions of 4.5, 10, 22 and 48% v/v saturated solution. An aliquot (1850 mL) of each of the stock solutions was separately inoculated with algal suspension (9.7 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.
The stock solutions and each 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 at 0, 24, 48 and 72 hours (see Annex 5).

Exposure Conditions
Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels (250 mL glass conical flasks) in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate (250 mg/L) was added to the culture medium to provide a source of carbon dioxide for algal growth. Six flasks were used for the control and three flasks were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.50 x 105 cells per mL. Inoculation of 1850 mL of test medium with 9.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 4.6 mg/L
95% CI:
>= 1.8 - <= 11
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Inhibition of Growth Rate
ErC10 (0 to 72 hour): 1.9 mg/L*
ErC20 (0 to 72 hour): 2.5 mg/L; 95% confidence limits 0.02 to 4.0 mg/L
ErC50 (0 to 72 hour): 4.6 mg/L; 95% confidence limits 1.8 to 11 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 the Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm incorporating Shapiro-Wilk’s test on Normal Distribution and Levene’s test on Variance Homogeneity. There were no statistically significant differences between the control, 0.54 and 1.3 mg/L test concentration (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 1.3 mg/L. Correspondingly the LOEC based on growth rate was 3.1 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 1.2 mg/L; 95% confidence limits 0.99 to 1.4 mg/L
EyC20 (0 to 72 hour): 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour): 2.3 mg/L; 95% confidence limits 2.1 to 2.5 mg/L
Where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences between the control, 0.54 and 1.3 mg/L test concentration (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and,
* It was not possible to calculate 95% confidence limits for the ErC10 value as the data generated did not fit the models available for the calculation of confidence limits therefore the NOEC based on yield was 1.3 mg/L. Correspondingly the LOEC based on yield was 3.1 mg/L.

Reported statistics and error estimates:
The following data show that the cell concentration of the control cultures increased by a factor of 149 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 103 cells per mL
Mean cell density of control at 72 hours : 7.45 x 105 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% 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 to 72 hours) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Raphidocelis subcapitata to the test item gave the following results based on the 0 hour measured test concentrations:
* It was not possible to calculate 95% confidence limits for the ErC10 value as the data generated did not fit the models available for the calculation of the confidence limits
Endpoint Concentration
(mg/L)
Growth Rate EC10 1.9*
EC20 2.5 (0.02 – 4.0)
EC50 4.6 (1.8 – 11)
No Observed Effect Concentration 1.3
Lowest Observed Effect Concentration 3.1
Yield EC10 1.2 (0.99 – 1.4)
EC20 1.5 (1.3 – 1.7)
EC50 2.3 (2.1 – 2.5)
No Observed Effect Concentration 1.3
Lowest Observed Effect Concentration 3.1
Executive summary:

Introduction
A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as 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.


Methods
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 0.2 mg/L was obtained from a saturated solution method of preparation.
A shake flask method of preparation was, therefore, undertaken due to the lack of test item found during a traditional saturated solution approach (due to the potential volatility of the test item). The shake flask approach achieved a dissolved test item concentration of approximately 15 mg/L, indicating this to be the limit of water solubility of this item under test conditions.
Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.
Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to solutions of the test item at nominal concentrations of 4.5, 10, 22, 48 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 item solutions were prepared in triplicate replicates by shaking in excess (100 mg/L) of the test item in test water at approximately 150 rpm, at a temperature of 24 °C for 24 hours. After the shaking period the contents of the replicate flasks were pooled and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL was discarded in order to pre-condition the filter) to give a 100% v/v saturated solutions of the test item. This saturated solution was then further diluted to give the 48, 22, 10 and 4.5% v/v saturated solutions of the test item.
Samples of the algal populations were removed daily and cell concentrations determined for the control and treatment group, using a Coulter® Multisizer Particle Counter.


Results
Analysis of the test item preparations at 0 hours showed test concentrations to range from 0.54 to 15 mg/L. Analysis of the test preparations at 24 hours showed test concentrations to range from 0.53 to 14 mg/L. Analysis of the test preparations at 48 hours showed test concentrations to range from 0.33 to 11 mg/L and analysis of the test preparations at 72 hours showed test concentrations to range from 0.15 to 13 mg/L. This decline in measured test concentrations from those observed at 0 hours indicated that the test item was unstable under the conditions of the test. Analysis of additional preparations at 72 hours which had been prepared with the omission of algal cells showed test concentrations to range from 0.46 to 14 mg/L.
There was a decline in the test preparations which contained algal cells after 72 hours exposure indicating that the test item was adsorbing to the algal cells present.
Given the slight decline in measured test concentrations for the samples containing algae, and following the recommendations of the test guideline, it was considered justifiable to base the results on the 0-Hour measured test concentrations as it can be considered that whilst the test item was adsorbing to the algal cells, they were still exposed to the levels present.
Exposure of Raphidocelis subcapitata to the test item gave the following results based on the 0 hour measured test concentrations:


Endpoint                                                            Concentration
                                                                            (mg/L)
Growth Rate                        EC10                            1.9*
                                          EC20                 2.5 (0.02 – 4.0)
                                          EC50                 4.6 (1.8 – 11)
                     No Observed Effect Concentration        1.3
               Lowest Observed Effect Concentration        3.1
Yield                                   EC10                 1.2 (0.99 – 1.4)
                                          EC20                 1.5 (1.3 – 1.7)
                                          EC50                 2.3 (2.1 – 2.5)
                    No Observed Effect Concentration         1.3
               Lowest Observed Effect Concentration        3.1

Description of key information

The EC50 value for this study was 2.3 mg.L and the NOEC for this study was 1.3 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:
2.3 mg/L
EC10 or NOEC for freshwater algae:
1.3 mg/L

Additional information