2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

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:

March 2017- March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AH03201
- Expiration date of the lot/batch: 01 February 2019
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Analytical monitoring:

yes

Details on sampling:

Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

Verification of Test Concentrations
Samples were taken from the control and each group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to 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:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined as below.
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.

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 20 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, 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 (3 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 by stirring an excess (100 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. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

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 kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C laboratory.
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 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
A positive control used potassium dichromate as the reference item. The positive control was conducted between 06 November 2017 and 09 November 2017.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 +/- 1°C

pH:

7.1 to 9.0 (7.1 to 8.5 in the controls)

Dissolved oxygen:

not applicable

Nominal and measured concentrations:

Nominal Concentrations: 10, 3.2, 10, 32, 100 %v/v saturated solution
Geometric Mean Measured Concentrations: 0.11, 0.24, 1.1, 3.7, 17 mg/L

Details on test conditions:

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 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 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 6.5 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.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and 3 flasks each containing 100 mL 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 6.95 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 6.5 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.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 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.

Test Organism Observations
Samples were taken at 24, 43 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

95% CI:

2.1 - 3.2

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.61 mg/L

95% CI:

0.48 - 0.77

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.33 to 24 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.25 to 23 mg/L at 24 hours, less than the limit of quantification (LOQ), determined to be 0.011 mg/L, to 21 mg/L at 48 hours and from less than the LOQ to 1.5 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 concentrations in order to give a “worst case” analysis of the data.

Reported statistics and error estimates:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determne any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of the green algae, Pseudokirchneriella subcapitata has been investigated over a 72-hr period and based on the geometric mean measured test concentrations, the ErC50 value was estimated to be 2.6 mg/L with a 72-hr NOEC value for growth rate of 0.11 mg.L.

Executive summary:

An algal growth inhibition test was conducted with exposure of MCS-1562 to the green algae, Pseudokirchneriella subcapitata over a 72 -hr exposure period. The study was conducted under GLP and followed OECD 201. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.33 to 24 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.25 to 23 mg/L at 24 hours, less than the limit of quantification (LOQ), determined to be 0.011 mg/L, to 21 mg/L at 48 hours and from less than the LOQ to 1.5 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 concentrations. After 72 -hrs, the geometric mean measured test concentraitons resulted in an ErC50 value of 2.6 mg/L.

Description of key information

An OECD 201 algal growth inhibition test was conducted with exposure of MCS-1562 to the green algae, Pseudokirchneriella subcapitata. The resulting 72 -hr ErC50 value, based on geometric mean measured concentrations was 2.6 mg/L.  The 72-hour NOEC (growth rate) based upon geometric mean measured concentrations was determined to be 0.11 mg/L.  

Key value for chemical safety assessment

EC50 for freshwater algae:

2.6 mg/L

EC10 or NOEC for freshwater algae:

0.11 mg/L