2-methylenepropane-1,3-diyl diacetate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 09 February 2017 and 18 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in c ompliance with GLP, without deviations that influence the quality of the results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EC No. 440/2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methylenepropane-1,3-diyl diacetate

EC Number:

223-225-8

EC Name:

2-methylenepropane-1,3-diyl diacetate

Cas Number:

3775-29-9

Molecular formula:

C8H12O4

IUPAC Name:

2-[(acetyloxy)methyl]prop-2-en-1-yl acetate

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: 2-methylene-1, 3-propanediol diacetate (CAS: 3775-29-9)
Physical state/Appearance: Clear colorless liquid
Batch: 20160308 ( -88)
Purity: 99.7%
Expiry Date: 31 March 2019
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item 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
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication for approximately 1 minute and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

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 (see Annex 1) 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Not reported

Test temperature:

22”C

pH:

7.7 - 8.1

Dissolved oxygen:

7.9 - 8.6

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Chemical analysis of the freshly prepared test samples at 0 and 24 hours (see Annex 3) showed measured test concentrations to range from 81% to 99% of nominal. A decline in measured test concentrations was observed in the old or expired test media at 24 and 48 hours in the range of 67% to 91% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.

Details on test conditions:

Experimental Design and Study Conduct
Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication for approximately 1 minute and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 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 with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item 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
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18 and 10 mg/L.
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 and 48 hours (see Annex 3).

Exposure Conditions
In the definitive 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, in 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. Due to the potentially light unstable nature of the test item, testing was conducted shielded from the light. 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.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

Assessments
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The appearance of the test media was recorded daily.

Verification of Test Concentrations
Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Data Evaluation
Statistical Analysis
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Step-down Cochran-Armitage test. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• No more than 10% of the control control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
• The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

Reference substance (positive control):

yes

Remarks:

Potassum dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours (see Annex 3) showed measured test concentrations to range from 99% to 102% of nominal concentrations. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was unstable under test conditions.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the freshly prepared test samples at 0 and 24 hours (see Table 1) showed measured test concentrations to range from 81% to 99% of nominal. A decline in measured test concentrations was observed in the old or expired test media at 24 and 48 hours in the range of 67% to 91% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.
The geometric mean measured test concentrations were determined to be:
Nominal Test Concentration (mg/L) Geometric Mean Measured Test Concentration (mg/L) Expressed as a Percentage of the Nominal Test Concentration
10 8.0 80
18 15 83
32 27 84
56 49 88
100 90 90

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression method at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:
Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 75 64 - 92
48 74 62 - 91

The No Observed Effect Concentration after 24 and 48 hours exposure was 27 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 49 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 6.9 (SE = 0.034) and 6.2 (SE = 0.037) respectively.

Sub-Lethal Effects
A sub-lethal effect of exposure was observed in the 49 mg/L test concentration. This response was reduced mobility (see Table 2).

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.

Water Quality Criteria
Temperature was maintained at 22”C throughout the test, while there were no treatment related differences for oxygen concentration or pH (Table 3)

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):

Positive Control
A positive control (Envigo Study Number XT01HS) 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.
Exposure conditions for the positive control were similar to those in the definitive test, however, throughout the definitive test the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the 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:
Time Point
(Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 1.3 1.0 - 1.8 1.0 1.8
48 1.2 1.1 - 1.3 0.56 1.0
The No Observed Effect Concentration 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*.

Any other information on results incl. tables

Table 2            Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Test 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	5 N	5 N	5 N	5 N
8.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
15	0	0	0	0	0	0	5 N	5 N	5 N	5 N
27	0	0	0	0	0	0	5 N	5 N	5 N	5 N
49	0	0	2	0	2	10	4 N 1 R	5 N	3 N	4 N 1 R
90	4	3	4	3	14	70	1 N	2 N	1 N	2 N

 

Geometric Mean Measured Test 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
8.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
15	0	0	0	0	0	0	5 N	5 N	5 N	5 N
27	0	0	0	0	0	0	5 N	5 N	5 N	5 N
49	0	0	2	1	3	15	4 N 1 R	4 N 1 R	3 N	4 N
90	4	3	4	3	14	70	1 N	2 N	1 N	2 N

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

Table3            Water Quality Measurements

Geometric Mean Measured Test Concentration (mg/L)		0 Hours (Fresh Media)			24 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.8	8.4	22	8.1	8.5	22
8.0	R1	7.8	8.4	22	8.1	8.5	22
15	R1	7.8	8.5	22	8.0	8.4	22
27	R1	7.8	8.5	22	8.0	8.5	22
49	R1	7.9	8.4	22	8.0	8.3	22
90	R1	7.9	8.5	22	7.9	8.0	22

 

Geometric Mean Measured Test Concentration (mg/L)		24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.7	8.5	22	7.8	8.1	22
8.0	R1	7.8	8.5	22	7.8	8.1	22
15	R1	7.8	8.6	22	7.8	8.1	22
27	R1	7.9	8.6	22	7.8	8.0	22
49	R1	7.9	8.6	22	7.8	8.0	22
90	R1	7.8	8.5	22	7.8	7.9	22

R₁= Replicate 1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results based on the geometric mean measured test concentrations:

Time Point 48 (Hours)

EC50 74 (mg/L)

95% Confidence Limits 62-91 (mg/L)

No Observed Effect Concentration (NOEC) 27(mg/L)

Lowest Observed Effect Concentration (LOEC) 47(mg/L)

Executive summary:

A study was performed to assess the acute toxicity of the test item toDaphnia 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, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 22°C under semi-static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours. Due to the potentially light unstable nature of the test item, all testing was conducted shielded from the light.

  Results

Analysis of the freshly prepared test samples at 0 and 24 hours showed measured test concentrations to range from 81% to 99% of nominal. A decline in measured test concentrations was observed in the old or expired test media at 24 and 48 hours in the range of 67% to 91% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.

Exposure ofDaphnia magnato the test item gave the following results based on the geometric mean measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	74	62	-	91	27	49