2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch: 1609501020
Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the degradant cyclohexanone under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
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 immediate quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-Finding Test
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of overnight magnetic stirring and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (7.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Definitive Test
A nominal amount of test item (100 mg) was dissolved in culture medium with the aid of overnight magnetic stirring and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (1.8 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
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 degradant cyclohexanone in the test preparations was verified by chemical analysis at 0 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 kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

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

pH:

The pH value of the test cultures was observed to increase from pH 7.4 at 0 hours to pH 8.3 at 72 hours.

The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Range-Finding Test.
Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Measured concentrations of cyclohexanone 1.49, 6.22, 53.8 mg/L for nominal concentrations of 1.0, 10 and 100 mg/L respectively.
There was no significant decline in measured concentrations at 72 hours indicating that cyclohexanone was stable under the conditions of the test.

Definitive Test
Nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
Measured concentrations of cyclohexanone 0.303, 1.11, 2.68, 11.9, 34.0 mg/L.
A slight decline in measured concentrations was observed at 72 hours to between 0.30 and 34 mg/L.

Details on test conditions:

Range-Finding Test
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 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
The stock solutions and each of the prepared concentrations 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 Version 2 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.

Definitive Test
Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three 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 1.37 x 106 cells per mL. Inoculation of 1 liter of test medium with 1.8 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 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on parent substance

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on parent substance

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on parent substance

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: cyclohexanone

Basis for effect:

growth rate

Remarks on result:

other: based on hydrolysis product

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: based on parent substance

Details on results:

Range-finding Test
The results showed no effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth rate was observed to be reduced at 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured cyclohexanone concentrations to range from 0.61 to 57 mg/L. There was no significant decline in measured concentrations at 72 hours indicating that cyclohexanone was stable under the conditions of the test.

Definitive Test
Verification of Test Concentrations
The test item was found to hydrolyze rapidly upon addition to water, forming two primary hydrolysis species soluble in solution; cyclohexanone and 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate.
Whilst cyclohexanone undergoes no further reaction in the presence of water, 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate was found to undergo further molecular rearrangement to produce a secondary degradation product N,N-bis(2-hydroxyethyl)-2-methylacrylamide. Despite extensive method development work having been conducted, suitable methods of chemical
analysis could not be developed for the quantification of concentrations of either 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate or N,N-bis(2-hydroxyethyl)-2-methylacrylamide in solution. As such, analysis of the test preparations was conducted for the concentration of the primary degradant cyclohexanone only.
Analysis of the test preparations at 0 hours showed measured cyclohexanone concentrations to range from 0.44 to 39 mg/L. A slight decline in measured concentrations was observed at 72 hours to between 0.30 and 34 mg/L.
Given that toxicity cannot be attributed to any single degradant or combination of degradants, in accordance with the OECD Guidance Document on Aquatic Testing of Difficult Substances and
Mixtures (OECD 2000), it was considered appropriate to calculate the results based on the nominal concentrations of the parent test item only.
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 29 mg/L
ErC20 (0 to 72 hour) : 44 mg/L
ErC50 (0 to 72 hour) : 88 mg/L (It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits) 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 (P ≥ 0.05), between the control, 1.0, 3.2 and 10 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 10 mg/L. Correspondingly the LOEC based on growth rate was 32 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour) : 8.6 mg/L
EyC20 (0 to 72 hour) : 14 mg/L
EyC50 (0 to 72 hour) : 31 mg/L; 95% confidence limits 24 to 40 mg/L
Where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P ≥0.05), between the control, 1.0 and 3.2 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 3.2 mg/L. Correspondingly the LOEC based on yield was 10 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 184 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 : 9.20 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 5% 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 hour) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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 1.0, 3.2, 10 and 32 mg/L, however cell debriswas observed to be present in the test cultures at 100 mg/L.

Water Quality Criteria
Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility
At the start of the test period, all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and 1.0, 3.2, 10 and 32 mg/L test cultures were
green dispersions while the 100 mg/L test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) 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 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration 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.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 hour period and based on the geometric mean measured test concentrations gave the following results:
72h-NOEC = 10 mg/L
72h-ErC10 = 29 mg/L
72h-ErC50 = 88 mg/L
72h-EyC50 = 31 mg/L

Given that toxicity cannot be attributed to any single degradant or combination of degradants, in accordance with the OECD Guidance Document on Aquatic Testing of Difficult Substances and Mixtures (OECD 2000), it was considered appropriate to calculate the results based on the nominal concentrations of the parent test item only.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with 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

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

The test item was found to hydrolyze rapidly upon addition to water, forming two primary hydrolysis species soluble in solution; cyclohexanone and 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate. Whilst cyclohexanone undergoes no further reaction in the presence of water, 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate was found to undergo further molecular rearrangement to produce a secondary degradation product N,N-bis(2-hydroxyethyl)-2-methylacrylamide.

Despite extensive method development work having been conducted, suitable methods of chemical analysis could not be developed for the quantification of concentrations of either 2-[(2 -hydroxyethyl)amino]ethyl 2-methylacrylate or N,N-bis(2-hydroxyethyl)-2-methylacrylamide in solution.

As such, analysis of the test preparations was conducted for the concentration of the primary degradant cyclohexanone only.

Analysis of the test preparations at 0 hours showed measured cyclohexanone concentrations to range from 0.44 to 39 mg/L. A slight decline in measured concentrations was observed at 72 hours to between 0.30 and 34 mg/L.

Given that toxicity cannot be attributed to any single degradant or combination of degradants, in accordance with the OECD Guidance Document on Aquatic Testing of Difficult Substances and Mixtures (OECD 2000), it was considered appropriate to calculate the results based on the nominal

concentrations of the parent test item only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on nominal test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	88		*		10	32
Yield	31	24	-	40	3.2	10

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

The toxicity of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate to freshwater algae was determined in an OECD 201 study. The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 hour period and based on the nominal parent test concentrations gave the following results: 72h-ErC50 = 88 mg/L.

The 100 mg/L nominal test concentration is equal to a geometric mean measured concentration of 36.4 mg/L cyclohexanone (CAS 108 -94 -1). Initial concentration cyclohexanone was 39 mg/L, which is similar to the theoretical maximum amount of 38.7 mg/L that can be formed when 100 mg/L parent substance degrades.

From these results it can be shown that the parent substance was fully hydrolyzed at test start and test animals were exposed to cyclohexanone and 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate (CAS 51706 -72 -0).

The 72 -hErC50 is equal to 60.2 mg/L 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate.

The estimated endpoints in EPIwin are lower, but show that 2 -[(2 -hydroxyethyl)amino]ethyl 2 -methylacrylate would show highest toxicity. The estimated endpiont for CAS 45011 -26 -5 is equal to that of CAS 51706 -72 -0, but this substance is less relevant for the environment (see section 5.1.2).

CAS 108 -94 -1: EC50 = 137 mg/L (neutral organics class)

CAS 51706 -72 -0: EC50 = 1.13 mg/L (methacrylates class)

CAS 45011 -26 -5: EC50 = 1.15 mg/L (acrylamides class)