2-Propenoic acid, 2-methyl-, hydroxybutyl ester

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:

Experimental Starting Date: 29 October 2012 Experimental Completion Date: 15 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the 100 mg/L test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. All
samples were stored at approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached Appendix 5.

Test solutions

Vehicle:

no

Details on test solutions:

The test concentration to be used in the definitive test was 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. The test item was
dissolved directly in water.

An amount of test item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.
Serial dilutions were prepared in reconstituted water from the 100 mg/L test concentration to give the remaining test concentrations of 0.10, 1.0 and 10 mg/L.

The prepared concentrations were 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 at 20 °C to 21 °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 250 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized
Daphnia magna 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 at approximately -20 °C 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 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.


Experimental Preparation:
For the purpose of the definitive test the test item was dissolved directly in reconstituted water.

An amount of test item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparation was verified by chemical analysis at 0 and 48 hours (see attached Appendix 5).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Species:
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 (see attached Appendix 2) 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 approximately 25.5 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. The young were slightly older
than required in the GSP, however, this is considered not to have had an effect on the outcome of the study.

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the reference item. Details of the positive control are given in
the attched Appendix 3.


Test Water:
Reconstituted water used for both the range-finding and definitive test is defined in the attached Appendix 4.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
See attached Appendix 4.

Test temperature:

The test vessels were covered to reduce evaporation and maintained in a temperature controlled room at 19 °C to 21 °C
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer and recorded daily throughout the test.

pH:

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 pH was measured using a Hach HQ30d Flexi handheld meter at the start and termination of the test.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test.
See Appendix 6 for results.

Salinity:

freshwater used.

Nominal and measured concentrations:

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.

Details on test conditions:

Exposure Conditions
In the definitive 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 19 °C to 21 °C with a photoperiod of 16 hours light
and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity ranging from 542 to 571 lux. 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. 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.


Physico-Chemical Measurements
Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start
and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the
temperature was measured using a Hanna Instruments HI 93510 digital thermometer.


Verification of Test Concentrations
Water samples were taken from the control and the 100 mg/L test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis.
All samples were stored at approximately -20 °C prior to analysis.

Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached Appendix 5.


Evaluation of Data
An estimate of the EC50 values was given by inspection of the immobilization data.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (see Appendix 3)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence limits (mg/L) 0.56 - 1.0

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence limits (mg/L) 0.42 - 0.48

Details on results:

RESULTS

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

No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.

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.

Chemical analysis of the 100 mg/L test preparations at 0 and 48 hours (see Appendix 5) showed that measured concentrations of 111% to 100% of nominal were obtained respectively, indicating that the test item was stable under test conditions.

Definitive Test

Immobilization Data
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EC50 (mg/L)
24 >100
48 >100

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.


Observations on Test Item Solubility
Throughout the duration of the test the test media was observed to be a clear colorless solution.

Physico-Chemical Measurements
The results of the physico-chemical measurements are given in the attached Appendix 6. Temperature was maintained at 19 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the
outcome or integrity of the test as no adverse effects were observed in the control group.


Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours (see attached Appendix 5) showed measured test concentrations of 102% and 96% of nominal
respectively were obtained and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Study Number: 41203341) 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 by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/L) 95% Confidence limits
(mg/L)
24 0.75 0.56 - 1.0
48 0.45 0.42 - 0.48

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is
based upon zero immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect
Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 102% to 96% of nominal value and so the results are based on nominal test concentrations only.

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977 ) at 48 hours.

Any other information on results incl. tables

Tables        

Table1     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	0	0
100	0	0

 

 

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
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
100	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

 

R₁– R₄= Replicates 1 to 4

Analytical Tables

Table1     Preparation of Test Samples          

Nominal Concentration of Test Item		Sample Volume		Final Volume		Sample Preparation Factor		Final Nominal Concentration of Test Item
[mg/L]		[mL]		[mL]		F		[mg/L]
Control		direct		direct		1		-
100		10		100, then 5→ 100		200		0.50

 

-     =     Not Applicable

 

 

Table 2     Linearity Data

Concentration of Test Item	Area
[mg/L]	[counts]
0	0
0.0532	7.490 x 10^4
0.109	1.515 x 10^5
0.266	3.630 x 10^5
0.546	7.320 x 10^5
1.06	1.325 x 10^6
1.64	1.985 x 10^6

 

 

Table 3     Results for Range-Finding Samples

Time point	Nominal Concentration of Test Item in Range-Finding Sample cnom	Measured Concentration  of Test Item in Sample Vial x	Sample Preparation Factor F	Determined Concentration of Test Item in Range-Finding Sample c	% of Nominal Concentration
[hours]	[mg/L]	[mg/L]		[mg/L]	[%]
0	100	0.111	1	111	111
48	100	0.100	1	100	100

 

 

 

Table 4     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	Analytical Recovery Rate R	Precision (Relative Standard Deviation of Recovery)
[mg/L]	[mg/L]	[mg/L]		[mg/L]	[%]	[%]
100	101	0.510	200	102	101	0.38
		0.515	200	103
		0.510	200	102
		0.510	200	102
		0.510	200	102
Acceptance Target					80-120	<10

 

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

 

Table 5     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	200	<LOQ	-
	100	0.510	200	102	102
48	Control	<LOQ	200	<LOQ	-
	100	0.478	200	95.6	96

 

LOQ           =             Limit of Quantitation

-                =             not applicable

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

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than
100 mg/L.
The No Observed Effect Concentration was 100 mg/L.

Executive summary:

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 a concentration of 100 mg/L for 48 hours at a temperature of 19°C to 21°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

 

Results

The 48-Hour EC₅₀ for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 102% to 96% of nominal value and so the results are based on nominal test concentrations only.