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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 06 June 2017. Experimental completion date 12 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
please see nay other information on materials and methods section
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
please see nay other information on materials and methods section
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch: 1609501020
Purity: 94.5%
Physical state/Appearance: Extremely pale yellow slightly viscous liquid
Expiry Date: 05 October 2018
Storage Conditions: Approximately 4 °C in the dark
Analytical monitoring:
yes
Details on sampling:
Range-finding Test:
Samples for chemical analysis were taken but as results from the Algal Inhibition Range-Finding Test (Envigo Study Number: CY39CW) were available and indicated that the degradant cyclohexanone was stable for a period of 72 hours, it was considered unnecessary to have them analyzed.

Definitive Test
Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-finding Test
A nominal amount of test item (200 mg) was dissolved in test water with the aid of magnetic stirring for 24 hours to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (200 mg) was dissolved in 2 liters of test water with the aid of magnetic stirring for 24 hours to give the 100 mg/L test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
Temperature was maintained at 21 °C to 22 °C throughout the test,
pH:
7.5 - 7.9
There were no treatment related differences for pH
Dissolved oxygen:
8.2 - 8.6 mg O2/L
There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
Range-finding Test
Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Definitive Test
Nominal concentration: 100 mg/L
Analysis of the test preparation at 0 hours (see Annex 5) showed a measured cyclohexanone concentration of 38 mg/L. Analysis of the test preparation at 48 hours showed a measured cyclohexanone concentration of 32 mg/L (84% of its starting concentration).
Details on test conditions:
Range-finding Test
In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.

Definitive Test
Based on the results 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/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, containing the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 34.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: cyclohexanone
Basis for effect:
mobility
Remarks on result:
other: based on hydrolysis product
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 68.4 mg/L
Nominal / measured:
estimated
Conc. based on:
other: 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate
Basis for effect:
mobility
Remarks on result:
other: based on hydrolysis product
Remarks:
theoretical maximum
Details on results:
Range-finding Test
No immobilization was observed throughout the test.
A sub-lethal effect of exposure was observed in the test concentrations of 0.10 and 1.0 mg/L. This response was trapping at surface.
Based on this information, a single test concentration of four 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/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.

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 preparation at 0 hours showed a measured cyclohexanone concentration of 38 mg/L. Analysis of the test preparation at 48 hours showed a measured cyclohexanone concentration of 32 mg/L (84% of its starting concentration).
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.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a nominal test concentration of 100 mg/L for a period of 48 hours
The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Results with reference substance (positive control):
A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

At 24 hours
EC50 mg/L: 0.79
95% Confidence Limits (mg/L): 0.73 - 0.86
NOEC (mg/L): 0.56
LOEC (mg/L): 1.0

At 48 hours
EC50 mg/L: 0.75
95% Confidence Limits (mg/L): 0.56 - 1.0
NOEC (mg/L): 0.56
LOEC (mg/L): 1.0

The NOEC is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Concentration
(mg/L)

Observations (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

R1

R2

R1

R2

R1

R2

R1

R2

Control

0

0

5N

5 N

0

0

5 N

5 N

0.10

0

0

4 N 1T

5 N

0

0

5 N

5 N

1.0

0

0

5 N

4 N 1 T

0

0

5 N

4 N 1 T

10

0

0

5 N

5 N

0

0

5 N

5 N

100

0

0

5 N

5 N

0

0

5 N

5 N

R = Replicate

N = Normal

T = Trapped at surface

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

 

Nominal Concentration
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

R = Replicate

N = Normal

Water Quality Measurements

Nominal Concentration
(mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/L

Temperature
°C

Temperature
°C

pH

mg O2/L

Temperature
°C

Control

R1

7.5

8.2

22

21

7.9

8.9

21

100

R1

7.5

8.2

22

21

7.9

8.6

21

R = Replicate

Validity criteria fulfilled:
yes
Conclusions:
From the results an EC50 value was calculated that is >100 mg/L (nominal). This is equal to a geometric mean measured concentration of 34.6 mg/L cyclohexanone. Initial concentration cyclohexanone was 37.7 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.
100 mg/L parent substance is equal to 68.4 mg/L 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions.

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

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 preparation at 0 hours showed a measured cyclohexanone concentration of 38 mg/L. Analysis of the test preparation at 48 hours showed a measured cyclohexanone concentration of 32 mg/L (84% of its starting concentration).

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.

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Description of key information

The acute toxicity of 2 -(1 -oxa-4 -azaspiro[4.5]dec-4 -yl)ethyl methacrylate to invertebrates was determined in an OECD 202 study. In a limit test with 100 mg/L parent substance no effects were found. From this result an EC50value was calculated that is >100 mg/L (nominal). This is equal to a geometric mean measured concentration of 34.6 mg/L cyclohexanone (CAS 108 -94 -1). Initial concentration cyclohexanone was 37.7 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).

100 mg/L parent substance is equal to 68.4 mg/L 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate.

This is in line with the estimated endpoints in EPIwin:

CAS 108 -94 -1: EC50 = 257 mg/L (neutral organics class)

CAS 51706 -72 -0: EC50 = 87 mg/L (aliphatic amines class)

CAS 45011 -26 -5: EC50 = 579 mg/L (acrylamides class)

Key value for chemical safety assessment

Additional information