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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008/09/08-2008/12/05

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:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI1999/3106 as amended by SI 2004/09994)).

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:
6.25, 12.5, 25, 50 and 100 mg/l

- Sampling method:
Not stated.

- Sample storage conditions before analysis:
Not stated.

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 (100mg) was dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 50 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock colution to give further stock solutions of 100, 50, 25 and 12.5 mg/l. An aliquot (250 ml) of each of the stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours
- Eluate:
None.
- Differential loading:
Not applicable.
- Controls:
Yes.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Not stated.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Not stated.
- Strain:
CCAP 276/20
- Source (laboratory, culture collection):
Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
Not stated.
- Method of cultivation:
The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 deg C.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1C until the algal cell density was approximately 10^4 - 10^5 cells/ml.



ACCLIMATION
- Acclimation period:
Not applicable.
- Culturing media and conditions (same as test or not):
Same as test.
- Any deformed or abnormal cells observed:
Culture Medium
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period stated.

Test conditions

Hardness:

Not stated.

Test temperature:

The pH values of each test and control flask are given in Table 3. Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

The pH values of the control cultures (see Table 3) were observed to increase from pH 7.3 at 0 hours to pH 7.8 – 7.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not stated.

Salinity:

Not applicable.

Nominal and measured concentrations:

Please see attached document.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
Closed
- Material, size, headspace, fill volume:
250 ml glass conical flasks were used, each containing 100 ml of the test preparation and plugged with polyurethane foam plugs to reduce evaporation.
- Aeration:
Not stated.

- Type of flow-through (e.g. peristaltic or proportional diluter):
Not applicable.
- Renewal rate of test solution (frequency/flow rate):
Not applicable.
- Initial cells density:
Mean cell density of control at 0 hours : 4.74x10^3 cells per ml.
- Control end cells density:
Mean cell density of control at 72 hours : 2.32x10^5 cells per ml.
- No. of organisms per vessel:
See Table 1 below.
- No. of vessels per concentration (replicates):
3 flasks each containing 100ml were used for each treatment group (5)
- No. of vessels per control (replicates):
6 flasks each containing 100ml of test preparation were used for control
- No. of vessels per vehicle control (replicates):
Not applicable.



GROWTH MEDIUM
- Standard medium used:
Yes
- Detailed composition if non-standard medium was used:
Not applicable.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reverse osmosis purified deionised water prepared using an Elga Ptima 15+ or Elga Purelab Option R-15 BP.
- Total organic carbon:
None.
- Particulate matter:
None.
- Metals:
Not stated.
- Pesticides:
None.
- Chlorine:
None.
- Alkalinity:
Not stated.
- Ca/mg ratio:
See attached.
- Conductivity:
Not stated.
- Culture medium different from test medium:
No.
- Intervals of water quality measurement:
Not applicable.


OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
Yes.
- Adjustment of pH:
Yes. 0.1N NaOH or HCL was used to adjust pH of the test medium to 7.5 ± 0.1
- Photoperiod:
72 hours (continuous).
- Light intensity and quality:
Intensity approximately 7000 lux provided by warm white lighting (380-730nm).
- Salinity (for marine algae):
Not applicable.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
Cell concentration was measured at and 72 hours using a Coulter(R) Multisizer Particle Counter.
- Chlorophyll measurement:
Not stated.
- Other:
None.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
2
- Justification for using less concentrations than requested by guideline:
Not applicable.
- Range finding study
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test material.
The results of the initial range-finding test showed a relatively flat response in terms of inhibition of growth rate occurred and so a second range-finding test was conducted in an identical manner to the initial test to confirm the results obtained.
Exposure conditions in the second range-finding test were the same as those in the initial test.
At the start of the range-finding tests a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
- Test concentrations:
0.1, 1.0, 10, 100 mg/l
- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test:6.25, 12.5, 25, 50 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

-Range-finding Tests
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding tests are given in Table 1 and Table 2.
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/l. However, growth was observed to be slightly reduced at 100 mg/l.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l were selected for the definitive test to ensure that a No Observed Effect Concentration (NOEC) was obtained.

-Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5.

- Exponential growth in the control (for algal test):
Yes, from the data given in Tables 3 and 5, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at nominal test concentrations of up to 100 mg/l over the 72-Hour exposure period.
Whilst slight inhibition of yield and biomass integral was observed at the test concentration of 100 mg/l in both range-finding tests conducted, no significant effect was observed at this concentration in the definitive test. This was considered to be due to slight biological variation.
Accordingly the following results were determined from the data:
Inhibition of growth rate
ErC10 (0 - 72 h) : > 100 mg/l
ErC20 (0 - 72 h) : > 100 mg/l
ErC50 (0 - 72 h) : > 100 mg/l
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences (P0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/l.
Inhibition of yield
EyC10 (0 - 72 h) : > 100 mg/l
EyC20 (0 - 72 h) : > 100 mg/l
EyC50 (0 - 72 h) : > 100 mg/l
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences (P0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/l.
Inhibition of biomass integral
EbC10 (0 - 72 h) : > 100 mg/l
EbC20 (0 - 72 h) : > 100 mg/l
EbC50 (0 - 72 h) : > 100 mg/l
where EbCx is the test concentration that reduced biomass integral (area under the growth curve) by x%.
Statistical analysis of the biomass integral data was carried out as in Section 5.2.2.1. There were no statistically significant differences (P0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 100 mg/l.
Verification of test concentrations
Analysis of the test preparations at 0 hours (see Appendix 4) showed measured test concentrations to range from 89% to 105% of nominal. Analysis of the test preparations at 72 hours (see Appendix 4) showed measured test concentration to be near nominal with the exception of the 6.25 and 12.5 mg/l test concentrations which showed measured test concentrations of 70% and 78% of nominal respectively. However, given that these test concentrations were below the No Observed Effect Concentration (NOEC) it was considered appropriate to calculate the results based on nominal test concentrations only.

- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Unusual cell shape: Not stated
- Colour differences: Not stated
- Flocculation: Not stated
- Adherence to test vessels: Not stated
- Aggregation of algal cells: Not stated
- Other: None.
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None stated.
- Effect concentrations exceeding solubility of substance in test medium:
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1066) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:
ErC50 (0 – 72 h) : 0.52 mg/l, 95% confidence limits 0.43 – 0.62 mg/l
EyC50 (0 – 72 h) : 0.29 mg/l, 95% confidence limits 0.25 – 0.33 mg/l
EbC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.26 – 0.34 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on yield rate: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral: 0.625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield rate: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral:0.125 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Any other information on results incl. tables

Table1              Cell Densities and Percentage Inhibition of Growth from the Initial Range-finding Test

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	4.24E+03	9.37E+05
	R2	4.20E+03	6.56E+05	-	-
	Mean	4.22E+03	7.97E+05
0.10	R1	4.32E+03	1.12E+06
	R2	4.17E+03	8.65E+05	[4]	[25]
	Mean	4.25E+03	9.91E+05
1.0	R1	4.26E+03	8.84E+05
	R2	4.18E+03	1.02E+06	[3]	[20]
	Mean	4.22E+03	9.53E+05
10	R1	4.10E+03	6.91E+05
	R2	4.20E+03	8.77E+05	0	2
	Mean	4.15E+03	7.84E+05
100	R1	4.24E+03	6.20E+05
	R2	4.06E+03	4.64E+05	7	32
	Mean	4.15E+03	5.42E+05

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Table 2              Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	2.81E+03	2.62E+05
	R2	3.62E+03	2.85E+05	-	-
	Mean	3.21E+03	2.74E+05
0.10	R1	4.30E+03	2.62E+05
	R2	4.69E+03	2.68E+05	8	4
	Mean	4.49E+03	2.65E+05
1.0	R1	4.12E+03	2.56E+05
	R2	4.10E+03	2.88E+05	6	1
	Mean	4.11E+03	2.72E+05
10	R1	4.14E+03	2.72E+05
	R2	4.12E+03	2.88E+05	5	[2]
	Mean	4.13E+03	2.80E+05
100	R1	3.68E+03	2.39E+05
	R2	3.57E+03	1.75E+05	10	25
	Mean	3.62E+03	2.07E+05

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table 3              Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.3	4.32E+03	9.22E+03	4.16E+04	2.22E+05	7.9
	R2	7.3	4.73E+03	1.25E+04	3.64E+04	2.40E+05	7.9
	R3	7.3	5.14E+03	1.13E+04	3.71E+04	2.57E+05	7.8
	R4	7.3	4.74E+03	1.09E+04	4.14E+04	1.64E+05	7.8
	R5	7.3	4.79E+03	1.10E+04	3.76E+04	2.99E+05	7.8
	R6	7.3	4.71E+03	7.02E+03	4.16E+04	2.08E+05	7.8
	Mean		4.74E+03	1.03E+04	3.93E+04	2.32E+05
6.25	R1	7.3	4.91E+03	8.78E+03	4.17E+04	2.32E+05	7.8
	R2	7.3	4.56E+03	9.46E+03	4.22E+04	2.84E+05	7.8
	R3	7.3	4.99E+03	1.04E+04	3.98E+04	2.99E+05	7.8
	Mean		4.82E+03	9.56E+03	4.12E+04	2.72E+05
12.5	R1	7.3	5.38E+03	1.36E+04	3.43E+04	2.01E+05	7.8
	R2	7.3	4.32E+03	6.91E+03	2.83E+04	2.03E+05	7.7
	R3	7.3	4.31E+03	1.04E+04	2.15E+04	2.28E+05	7.7
	Mean		4.67E+03	1.03E+04	2.80E+04	2.11E+05
25	R1	7.3	5.89E+03	8.54E+03	3.41E+04	3.11E+05	7.8
	R2	7.3	4.87E+03	9.66E+03	4.16E+04	2.89E+05	7.7
	R3	7.3	4.26E+03	1.01E+04	3.31E+04	3.01E+05	7.8
	Mean		5.01E+03	9.42E+03	3.63E+04	3.00E+05
50	R1	7.3	4.83E+03	8.99E+03	3.59E+04	3.24E+05	7.8
	R2	7.3	4.62E+03	1.00E+04	2.94E+04	2.73E+05	7.8
	R3	7.3	4.74E+03	1.29E+04	3.09E+04	2.35E+05	7.7
	Mean		4.73E+03	1.06E+04	3.21E+04	2.78E+05
100	R1	7.3	5.30E+03	9.10E+03	2.41E+04	2.96E+05	7.6
	R2	7.3	5.06E+03	7.06E+03	1.88E+04	1.97E+05	7.7
	R3	7.3	5.22E+03	1.07E+04	2.88E+04	2.36E+05	7.7
	Mean		5.19E+03	8.94E+03	2.39E+04	2.43E+05

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 4              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.035	0.063	0.070
	R2	0.048	0.044	0.079
	R3	0.043	0.049	0.081
	R4	0.042	0.056	0.057
	R5	0.042	0.051	0.086
	R6	0.023	0.074	0.067
	Mean	0.039	0.056	0.073

R₁- R₆= Replicates 1 to 6

See attachments for Table 5.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validation criteria were met.

Conclusions:

Exposure of Desmodesmus subspicatus to the test material gave EC50 values of greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

Executive summary:

Introduction.

A study was perford to assess the effect of the test material on the growth of the green algaDesmodesmus subspicatus. Thethod followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Methods. 

Following preliminary range-finding tests,Desmodesmus subspicatuswas exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results. 

Exposure ofDesmodesmus subspicatusto the test material gave EC₅₀values of greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 89% to 105% of nominal. Analysis of the test preparations at 72 hours showed measured test concentrations to be near nominal with the exception of the 6.25 and 12.5 mg/l test concentrations which showed measured test concentrations of 70% and 78% of nominal respectively. However, given that these test concentrations were below the No Observed Effect Concentration (NOEC) it was considered appropriate to calculate the results based on nominal test concentrations only.