Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Testing was conducted between 11th August 2014 and 4th October 2014

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:

See principles of method if other than guideline

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Principles of method if other than guideline:

Deviation No 1:
The isolation of the gravid adults during the range-finding test occurred approximately 26 hours prior to the young be added to the test solutions and hence exceed the required time frame of 24 hours or less. This oversight was considered to have had no adverse effect on the outcome of the range-finding test as it is likely that the majority if not all of the young used were produced within the 24 hours preceding the test.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 1,3-Propanediol, 2-ethyl-2-(hydroxymethyl)-, oligomeric reaction product with
chloromethyl)oxirane (TK30174)
Physical state/ Appearance: Clear colorless liquid
Batch: AAB1584600
Purity: Not provided
Expiry Date: 31 October 2015
Storage Conditions: Room temperature in the dark
Intended use/ Application: Resin for coating systems

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L.

- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Vehicle:

yes

Details on test solutions:

Reconstituted water:
Reconstituted water (ISO medium) used for both the range-finding and definitive tests.

Ingredient Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 65
KCl 5.8

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °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

Post exposure observation period:

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

The temperature was maintained at 21° C throughout the test.

pH:

The pH was recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter.

The pH was measured to be between 7.7 and 8.1 throughout the test

Dissolved oxygen:

The dissolved oxygen concentrationswas recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter.

The dissolved oxygen concentration was kept between 8.6 and 9.2 mg O2/L.

Salinity:

Not applicable as a fresh water study was conducted

Nominal and measured concentrations:

Range finding study:
Nominal concentrations: 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.

Definitive study:
Nominal concentrations: Based on the results of the range-finding test the following nominal test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Measured 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 % of the Nominal Test Concentration
1.0 0.86 86
3.2 2.0 63
10 6.9 69
32 21 66
100 73 73

Details on test conditions:

TEST SYSTEM
As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 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 at approximately 21 °C with a photoperiod of 16 hours light (546 to 577 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.

EXPERIMENTAL ANALYSIS:
Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in test water 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 the remaining test concentrations of 32, 10, 3.2 and 1.0 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 and 48 hours.


TEST MEDIUM / WATER PARAMETERS:
Standard Elendt M7 medium was used. The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 for approximately 15 seconds after gentle agitation.

RANGE FINDING STUDY:
- Test concentrations: 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.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% confidence limits are between 2.0 mg/L and 6.9 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1*.

* Table 1 can be found in the any other information on results section, under the heading tables.

No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L. However, immobilization was observed at 10 and 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 test concentrations to range from 64% to 73% of nominal. A decline in measured test concentration was observed at 48 hours in the range of 49% to 60% of nominal (67% to 86% of the 0-Hour measured test concentrations) suggesting that the test item was slightly unstable over the test duration. Inspection of the data could find no cause for the lower than accepted measured concentrations obtained at 0 hours.


Definitive Test:
Verification of Test Concentrations:
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 61% to 87% of nominal value. Given the slight variability in results it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Inspection of the data could find no cause for the lower than accepted measured test concentrations obtained at 0 hours. Given that a similar effect was observed in the range-finding test it was considered possible that the test item had limited solubility in the test water.

The geometric mean measured test concentrations were determined to be:

Measured 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 % of the Nominal Test Concentration
1.0 0.86 86
3.2 2.0 63
10 6.9 69
32 21 66
100 73 73

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney, 1971) using the ToxCalc computer software package (ToxCalc, 1999). At 48 hours the EC50 value was calculated using the geometric mean method as follows:

EC50 value = √ C1 x C2

Where:

C1 = concentration showing 0% immobilization
C2 = concentration showing 100% immobilization

Probit analysis is used where two or more partial responses to exposure are shown.

If there is no immobilization between 0% and 100% immobilization, then the geometric mean of the highest test concentration showing no lethality and the lowest test concentration showing 100% lethality is calculated. The concentrations resulting in 0% and 100% immobilization will be the 95% confidence limits.


Geometric Mean Measured Test Concentrations:
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 – R4 pooled:

GM = √C0C1

Where:

GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

  Immobilization Data

Cumulative immobilization data 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 the probit method (Finney, 1971) at 24 hours and the geometric mean method at 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	14	9.1 - 21
48	3.7	2.0 – 6.9

 

The No Observed Effect Concentration after 24 and 48 hours exposure was 2.0 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 6.9 mg/L.

 

The slope and standard error of the response curve at 24 hours were 1.9 (SE = 0.31). Due to the nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.

* Table 2 can be found further down within this section

 Observations on Test Item Solubility

All control and test preparations were observed to be clear colorless solutions throughout the duration of the test.

TABLES:

Table 1     Cumulative Immobilization Data in the Range-finding Test

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	5	6
100	10	10

 

Table 2     Cumulative Immobilization Data in the Definitive Test:

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0	0	0	0	0	0
10	3	0	1	4	8	40	5	5	5	5	20	100
32	5	2	5	2	14	70	5	5	5	5	20	100
100	5	4	4	4	17	85	5	5	5	5	20	100

 

R₁– R₄= Replicates 1 to 4

Tables for Analytical Information:

Table 3     Results for Spiked Recovery Samples:

Nominal Concentra­tion of Test Item cnom	Fortified Concentra­tion of Test Item in the Spiked Sample cfort	Measured Concentra­tion of Test Item in the Sample Vial x	Sample Preparation Factor F	Determined Concentra­tion of Test Item in the Spiked Sample c	Mean Analytical Recovery Rate R	Precision (Relative Standard Deviation of Recovery)
[mg/L]	[mg/L]	[mg/L]		[mg/L]	[%]	[%]
		1.00	1	1.00	99	1.1
		0.999	1	0.999
1.00	0.999	0.978	1	0.978
		0.982	1	0.982
		0.979	1	0.979
Acceptance Target					80-120	<10

 

Table 4     Results for Test Samples:

Time point	Nominal Concentration of Test Item in Test Sample cnom	Measured Concentration  of Test Item in Sample Vial x	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample   c	% of Nominal Concentration
[hours]	[mg/L]	[mg/L]		[mg/L]	[%]
0	Control	<LOQ	1	<LOQ	-
	1.0	0.871	1	0.871	87
	3.2	0.616	3.33	2.05	64
	10	0.707	10	7.07	71
	32	0.661	33.3	22	69
	100	0.752	100	75.2	75
48	Control	<LOQ	1	<LOQ	-
	1.0	0.858	1	0.858	86
	3.2	0.589	3.33	1.96	61
	10	0.682	10	6.82	68
	32	0.631	33.3	21.0	66
	100	0.714	100	71.4	71

 

LOQ                      =             Limit of Quantification

-                              =             not applicable

For reporting purposes, x has been calculated from c retrospectively.

Validity criteria fulfilled:

yes

Conclusions:

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

Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
48 3.7 2.0 - 6.9 2.0 6.9

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, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of approximately 21°C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

 

 

Results:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 61% to 87% of nominal value. Given the slight variability in results it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

 

Exposure of Daphnia magna to the test item 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)
48	3.7	2.0	-	6.9	2.0	6.9

 

 

Description of key information

The 48 hr EC50 for daphnia magna was 3.7 mg/l

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

3.7 mg/L