Butanoic acid, 2-ethyl-, magnesium salt (2:1)

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:

2008/09/10-2008/10/31

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:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Scheduyle 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by IS 2004/0994)).

Test material

Test material information

Constituent 1

Reference substance name:

[(2-ethylbutanoyl)oxy]magnesio 2-ethylbutanoate

EC Number:

700-021-1

Cas Number:

79992-76-0

Molecular formula:

C12H22O4Mg

IUPAC Name:

[(2-ethylbutanoyl)oxy]magnesio 2-ethylbutanoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range finding test:
0.1, 1.0 10.0 and 100 mg.l
Definitive test:
10, 18, 32, 56 and 100 mg/l
- Sampling method:
Not stated
- Sample storage conditions before analysis:
Not applicable.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
An amount of test material (200mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100mg/l test concentration from which a series of dilutions was made to give further test concentrations of 56, 32 18 and 100 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogenity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours.
- Eluate:
Reconsituted water.
- Differential loading:
Not applicable.
- Controls:
Blank and positive controls were used.
A positive control (Safepharm Laboratories Project No: 0039/0977) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at 21°C.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution):
Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Not stated.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
Not stated
- Strain:
Daphnia magna
- Source:
In house laboratory cultures.
- Age at study initiation (mean and range, SD):
<24 hours 1st instar.
- Weight at study initiation (mean and range, SD):
Not stated.
- Length at study initiation (length definition, mean, range and SD):
Not stated.
- Valve height at study initiation, for shell deposition study (mean and range, SD):
not stated.
- Peripheral shell growth removed prior to test initiation:
not stated.
- Method of breeding:
parthenogenesis
- Feeding during test
- Food type:
No feeding during test.
- Amount:
Not applicable
- Frequency:
Not applicable


ACCLIMATION
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water 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 suspension of algae (Chlorella sp.). 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None.

Test conditions

Hardness:

Approximate theoretical hardnesss of 250 mg/l CaCO3

Test temperature:

Maintained at 20-21 deg C.
Water temperature was recorded daily through the out the test using a Hanna Instruments HI93510 digital thermometer.
See attached physico-chemical measurements table.

pH:

8.0 ± 0.2
The pH was recorded at the start and termination of the test. The pH was measured using a WTW pH 3401, pH and dissolved O2 meter. There were no treatment related differences.
See attached physico-chemical measurements table.

Dissolved oxygen:

The dissolved oxygen concentration was recorded at the start and termination of the test. The dissolved oxygen concentratio was measured using a WTW pH 3401, pH and dissolved O2 meter. There were no treatment related differences.
See attached physico-chemical measurements table.

Salinity:

Not applicable.

Nominal and measured concentrations:

Range finding test:
Nominal: 0.1, 1.0 10.0 and 100 mg.l
Measured (at 0 hours): not stated.
Definitive test:
Nominal: 10, 18, 32, 56 and 100 mg/l
Measured (at 0 hours): 9.5, 17.5, 31.7, 55.0, 99.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- 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. The test material was dissolved directly in water.
An amount of test material (50 mg) was dissolved in reconstituted water and the volume adjusted to 500 ml to give a 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 at approximately 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 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test material.
- 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.

- Type (delete if not applicable): open / closed
Closed
- Material, size, headspace, fill volume:
250 ml glass jars containing approximately 200 ml of test preparation.
- Aeration:
None.
- Type of flow-through (e.g. peristaltic or proportional diluter):
Non-applicable.
- Renewal rate of test solution (frequency/flow rate):
Test solution not renewed.
- No. of organisms per vessel:
10
- No. of vessels per concentration (replicates):
- range finding:
4
- definitive:
5
- No. of vessels per control (replicates):
1
- No. of vessels per vehicle control (replicates):
Not applicable
- Biomass loading rate:
Not applicable.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Prepared by ELGA Purelab Option R-15 water purification.
- Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. 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.

- Total organic carbon:
None.
- Particulate matter:
None.
- Metals:
MgSO4.7H2O: 4.93 g/l
NaHCO3: 2.59 g/l
KCl: 0.23 g/l
- Pesticides:
None.
- Chlorine:
None.
- Alkalinity:
Not applicable.
- Ca/mg ratio:
CaCO3 250 mg/l
- Conductivity:
<5 microS cm^-1
- Culture medium different from test medium:
No.
- Intervals of water quality measurement:
Not applicable.


OTHER TEST CONDITIONS
- Adjustment of pH:
pH 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
- Photoperiod:
16 hours light
8 hours dark
including 20 minute dawn/dusk transition periods.
- Light intensity:
Not stated.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Effect parameter: Immobilisation
Criteria: Daphnia magna unable to swim for approximately 15 seconds after gentle agitation.
Observation intervals: 24 hours
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of the exposure.
TEST CONCENTRATIONS
- Spacing factor for test concentrations:
10, 18, 32, 56 and 100 mg/l concentrations used.
- Justification for using less concentrations than requested by guideline:
Not applicable.
- Range finding study
- Test concentrations:
0.1, 1.0, 10.0 and 100.0 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: 10, 18, 32, 56 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: 95% CL

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Behavioural abnormalities:
Not stated.
- Observations on body length and weight:
Not stated.
- Other biological observations:
Not stated.
- Mortality of control:
Not stated.
- Other adverse effects control:
Not stated.
- Abnormal responses:
-Range finding test:
Immobilisation:
100 mg/l group.
4 Daphnids considered immobilised at the 48 hour observation.
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1 (see remarks section).
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 mg/l.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.
-Definitive test:
32 mg/l group.
2 Daphnids considered immobilised at the 48 hour observation.
56 mg/l group.
4 Daphnids considered immobilised at the 48 hour observation.
100 mg/l group.
3 Daphnids considered immobilised at the 24 hour observation.
11 Daphnids considered immobilised at the 48 hour observation.
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2 (see remarks section). The relationship between percentage immobilisation and concentration at 48 hours is given in Figure 1 (see attachments).

Analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
24 >100 -
48 93 71 - 160

The No Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 18 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and standard error of the response curve at 48 hours was 3.3 (SE = 0.81). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Not stated.
- Effect concentrations exceeding solubility of substance in test medium:
At the start and throughout the duration of the test the test preparations were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Safepharm Laboratories Project No: 0039/0977) during the positive control are given in Table 3 (see remarks section). The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 2 and 3.
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits
mg/l)
3 > 3.2* -
24 0.97 0.85 - 1.1
48 0.70 0.62 - 0.80

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 immobilisation at this concentration.
The slope and standard error of the response curve at 48 hours was 11 (SE = 2.3). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 24 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.22).

Reported statistics and error estimates:

None.

Any other information on results incl. tables

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (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	4

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0
32	0	0	0	0	0	2	2	10
56	0	0	0	0	2	2	4	20
100	2	1	3	15	5	6	11	55

R₁– R₂= Replicates 1 and 2

Table 3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	2	3	15
1.0	0	0	0	0	6	5	11	55	9	10	19	95
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	2	1	3	15	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 93 mg/l with 95% confidence limits of 71 - 160 mg/l. The No Observed Effect Concentration at 48 hours was 18 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material toDaphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

Methods. 

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 20°C to 21°C under static test conditions. The number of immobilisedDaphniawere recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magnawas exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀for the test material toDaphnia magnabased on nominal test concentrations was 93 mg/l with 95% confidence limits of 71 - 160 mg/l. The No Observed Effect Concentration was 18 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 92% to 111% of nominal value and so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀for the reference material toDaphnia magnabased on nominal concentrations was 0.70 mg/l with 95% confidence limits of 0.62 – 0.80 mg/l. The No Observed Effect Concentration was 0.32 mg/l.