Trimanganese tetraoxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-10 to 2010-03-18

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.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

(see principles of method if other than guideline)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(see principles of method if other than guideline)

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess of test material in culture medium for a period of 48 hours prior to removing any undissolved test material present by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sampling method: The test samples were analysed following addition of nitric acid (1 mL per 20 mL of sample or equivalent).
- Sample storage conditions before analysis: Samples were stored at approximately 4°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20ºC for further analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (200 mg) was dispersed in 2 litres of culture medium with the aid of magnetic stirring at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (24.1 mL) to give the required test concentration of 100% v/v saturated solution.

- Controls: A positive control (Harlan Laboratories Ltd Project Number: 0039/1127) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 12 January 2010 and 15 January 2010.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: Strain CCAP 276/20
- Source: Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°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 10E3 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 10E4 - 10E5 cells/mL.


ACCLIMATION
- Acclimation period:Not recorded.
- Culturing media and conditions:The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the test.

pH:

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.3 – 7.4 at 0 hours to pH 7.7 – 7.8 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

Nominal and measured concentrations:

Range-finding: The 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% v/v saturated solution for a period of 72 hours.
Definitive test: Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type : closed (flasks were plugged with polyurethane foam bungs)
- Fill volume: 100 mL
- Aeration: No aeration
- Renewal rate of test solution: No renewal
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.65 x 10E5 cells per mL. Inoculation of 1litre of test medium with 24.1 mL of this algal suspension gave an initial nominal cell density of 4 x 10E3 cells per mL and had no significant dilution effect on the final test concentration.
- Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.

GROWTH MEDIUM
- Standard medium used: yes - the culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


OTHER TEST CONDITIONS
- Adjustment of pH: The culture medium pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod and light intensity: The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C 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.


EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Test concentrations: Due to the low aqueous solubility and high purity of the test material the test concentrations used in the range-finding test were prepared by diluting (with culture medium) a saturated solution prepared from an initial test material dispersion at a concentration of 100 mg/L.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The 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% v/v saturated solution for a period of 72 hours.
An amount of test material (200 mg) was dispersed in 2 litres of culture medium with the aid of magnetic stirring at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (200 mL) of each of the stock solutions was separately inoculated with algal suspension (4.7 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
A sample of the uninoculated control and 100% v/v saturated solution were taken for chemical analysis at 0 hours to determine the concentration of manganese, and hence the dissolved test material concentration present in the saturated solution (see Appendix 4).
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 control group was maintained under identical conditions but not exposed to the test material.
At the start of the range-finding test 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 ± 1°C 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.

- Results used to determine the conditions for the definitive study:
Based on the results of the pre-study media preparation trial and range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RANGE-FINDING TEST
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.

DEFINITIVE TEST
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (see attached background material for figures 1-3).

VALIDATION CRITERIA
The following data show that the cell concentration of the control cultures increased by a factor of 42 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 4.28 x 10E3 cells per mL
Mean cell density of control at 72 hours : 1.78 x 10E5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 32% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

GROWTH DATA
From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72 hour exposure period.
Accordingly the following results were determined from the data expressed in terms of % v/v saturated solution:

Inhibition of growth rate:
ErC10 (0 - 72 h) : 56% v/v saturated solution
ErC20 (0 - 72 h) : 81% v/v saturated solution
ErC50 (0 - 72 h) : >100% v/v saturated solution*

*It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate
where ErCx is the test concentration that reduced growth rate by x%.


EyC10 (0 - 72 h) : 41% v/v saturated solution
EyC20 (0 - 72 h) : 51% v/v saturated solution
EyC50 (0 - 72 h) : 81% v/v saturated solution
where EyCx is the test concentration that reduced yield by x%.


Observations on cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility:
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, 1.0, 3.2 and 10% v/v saturated solution test cultures were observed to be pale green dispersions. The 32% v/v saturated solution test cultures were observed to be very pale green dispersions whilst the 100% v/v saturated solution test cultures were observed to be clear colourless solutions.

Physico-chemical measurements:
The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 2) were observed to increase from pH 7.2 at 0 hours to pH 7.5 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.

Verification of test concentrations:
The test material contained a theoretical manganese content of 77% w/w. The test samples were analysed for manganese only. Analysis of the test preparations at 0 hours (see Appendix 4) showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method employed to 1.3 mg/L as test material. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ to 1.3 mg/L as test material.
Given that no decline in measured concentration was observed over the test period, it was considered appropriate to determine the theoretical measured concentrations of the 1.0, 3.2 and 10% v/v saturated solution test concentrations based on the mean measured test concentrations obtained for the 32 and 100% v/v saturated solution test preparations. The theoretical measured test concentrations were determined to be:

Nominal Test Concentration (% v/v saturated solution) Theoretical/Mean Measured Test Item Concentration (mg/L)
1.0 0.013
3.2 0.041
10 0.13
32 0.41
100 1.3

Accordingly the following results were determined from the data based on the theoretical/mean measured test concentrations:
Growth rate:
ErC10 (0 - 72 h) : 0.73 mg/L
ErC20 (0 - 72 h) : 1.1 mg/L
ErC50 (0 - 72 h) : >1.3 mg/L*
* It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.

No Observed Effect Concentration (NOEC) = 0.41 mg/L
Lowest Observed Effect Concentration (LOEC) = 1.3 mg/L

Yield:
EyC10 (0 - 72 h) : 0.53 mg/L
EyC20 (0 - 72 h) : 0.66 mg/L
EyC50 (0 - 72 h) : 1.1 mg/L
No Observed Effect Concentration (NOEC) = 0.41 mg/L
Lowest Observed Effect Concentration (LOEC) = 1.3 mg/L

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item 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 item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/L*
EyC50 (0 – 72 h) : 0.18 mg/L, 95% confidence limits 0.16 – 0.21 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

*It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

Inhibition of growth rate: 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 between the control, 1.0, 3.2, 10 and 32% v/v saturated solution test concentrations (P≥0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32% v/v saturated solution.

Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of yield: Statistical analysis of the yield data was carried out as described above. There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32% v/v saturated solution test concentrations (P≥0.05), however the 100% v/v saturated solution test concentration was significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 32% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100% v/v saturated solution.

Table 1: Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.06E+03	1.20E+05
	R2	4.34E+03	1.23E+05	-	-
	Mean	4.20E+03	1.21E+05
0.10	R1	4.54E+03	1.08E+05
	R2	4.67E+03	1.38E+05	2	[1]
	Mean	4.60E+03	1.23E+05
1.0	R1	4.50E+03	1.06E+05
	R2	4.42E+03	1.39E+05	2	[1]
	Mean	4.46E+03	1.22E+05
10	R1	4.54E+03	1.50E+05
	R2	4.35E+03	1.15E+05	0	[10]
	Mean	4.45E+03	1.33E+05
100	R1	4.48E+03	1.06E+05
	R2	4.30E+03	1.42E+05	2	[2]
	Mean	4.39E+03	1.24E+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 pH Values in the DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.4	4.74E+03	1.43E+04	4.87E+04	2.48E+05	7.7
	R2	7.4	4.54E+03	9.78E+03	3.16E+04	2.56E+05	7.8
	R3	7.4	4.20E+03	8.91E+03	4.43E+04	2.62E+05	7.7
	R4	7.4	4.18E+03	1.19E+04	5.06E+04	2.62E+05	7.8
	R5	7.4	4.39E+03	1.14E+04	4.02E+04	2.48E+05	7.8
	R6	7.3	4.15E+03	1.23E+04	4.47E+04	2.78E+05	7.8
	Mean		4.37E+03	1.14E+04	4.33E+04	2.59E+05
100	R1	7.2	4.24E+03	1.21E+04	6.23E+04	2.82E+05	7.8
	R2	7.2	4.43E+03	9.58E+03	3.23E+04	2.80E+05	7.8
	R3	7.2	4.26E+03	9.58E+03	2.94E+04	2.78E+05	7.8
	R4	7.2	4.34E+03	8.10E+03	4.48E+04	2.94E+05	7.7
	R5	7.2	4.42E+03	8.57E+03	4.55E+04	1.92E+05	7.7
	R6	7.2	4.13E+03	1.11E+04	3.90E+04	1.96E+05	7.7
	Mean		4.30E+03	9.83E+03	4.22E+04	2.54E+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 3: 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	0.053	0.051	0.068
	R2	0.037	0.049	0.087
	R3	0.033	0.067	0.074
	R4	0.045	0.060	0.069
	R5	0.044	0.052	0.076
	R6	0.047	0.054	0.076
	Mean	0.043	0.056	0.075

R₁- R₆= Replicates 1 to 6

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.057		2.43E+05
	R2	0.058		2.52E+05
	R3	0.058		2.58E+05
	R4	0.058	-	2.58E+05	-
	R5	0.057		2.43E+05
	R6	0.059		2.74E+05
	Mean	0.058		2.55E+05
	SD	0.001		1.15E+04
100	R1	0.059	[2]	2.78E+05
	R2	0.059	[2]	2.76E+05
	R3	0.059	[2]	2.74E+05
	R4	0.060	[3]	2.90E+05
	R5	0.054	7	1.88E+05
	R6	0.054	7	1.92E+05
	Mean	0.058	1	2.50E+05	2
	SD	0.003		4.64E+04

*In accordance with the OECD test guideline only thean value for yield is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

     

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. This study showed that there were no toxic effects at the limit of solubility of the test material in the test medium.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and EU Method C.3.

Since the test material is considered to be poorly soluble in water a saturated solution of test material was prepared for use in the study.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a nominal test concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess of test material in culture medium via magnetic stirrer at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm filter to produce a 100% v/v saturated solution of the test item.

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

Exposure of Desmodesmus subspicatus to the test material gave EC₅₀values of greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. This study therefore showed that there were no toxic effects at the limit of solubility of the test material in the test medium.

 

Description of key information

No toxic effects up to the limit of solubility of the test material in the test medium, OECD 201, EU Method C.3, Vryenhoef & Mullee (2010)

Additional information

The toxicity of the test material to aquatic algae was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and EU Method C.3.

Since the test material is considered to be poorly soluble in water a saturated solution of test material was prepared for use in the study.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a nominal test concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess of test material in culture medium via magnetic stirrer at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm filter to produce a 100% v/v saturated solution of the test item.

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

Exposure of Desmodesmus subspicatus to the test material gave EC₅₀values of greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. This study therefore showed that there were no toxic effects at the limit of solubility of the test material in the test medium.