alpha-[butyl(dimethylsilyl)]-omega-[3-(2-{[2- (methacryloyloxy)ethyl]carbamoyloxy}ethoxy) propyl]poly[oxy(dimethyl)silylene]

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:

The study was conducted between 18 July 2011 and 12 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, 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 item in cases where the test item 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 (50 mg/l) of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Pre-study media preparation trial
Information provided by the Sponsor indicated the water solubility of the test item to be less than 6.89 x 10-8 g/l. Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions (see analytical methods section).

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test
The results obtained from the pre-study media preparation trial conducted indicated that a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was most appropriate for this test item.

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 Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres 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 and 1.0% v/v saturated solution. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (5.3 ml) to give the required test concentrations of 1.0, 10 and 100% v/v saturated solution.
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 item.

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 ± 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.
Samples were taken for chemical analysis from each test concentration at 0 and 72 hours in order to determine the stability of the test item under test conditions.

All 0-Hour samples were stored at approximately -20°C prior to analysis.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name:
Algae


- Strain:
Strain CCAP 278/4

- Source:
Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:

Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2)( see below). The master cultures were maintained in the laboratory under constant aeration and 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 (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.


- Any deformed or abnormal cells observed:
None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v
saturated solution.

Details on test conditions:

Experimental Preparation
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres 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 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (1 litre) of each of the stock solutions was separately inoculated with 9.2 ml of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
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 item in the test solutions were verified by chemical analysis at 0 and 72 hours (see Appendix 5)(see in attached section).

Exposure conditions
As in the range-finding test 250 ml glass conical flasks were used. 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.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 5.43 x 10e5 cells per ml. Inoculation of 1 litre of test medium with 9.2 ml of this algal suspension gave an initial nominal cell density of 5 x 10e3 cells per ml and had no significant dilution effect on the final test concentration.

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.

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

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 other: %

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: %

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 Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1. (see in other information on results and tables section).
The results showed no effect on growth at the test concentrations of 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 10 and 100% v/v saturated solution test samples at 0 and 72 hours (see Appendix 5 in attached section) showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.17 mg/l.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (see in other information on results and tables section). Daily specific growth rates for the control cultures are given in Table 3 (see in other information on results and tables section). Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.(see in other information on results and tables section).
The mean cell densities versus time for the definitive test are presented in Figure 1. (see in attached background section).
The differences in inhibition observed between the range-finding and definitive test was considered to be due to possible contamination of the saturated solution prepared for use in the range-finding test from an unknown source.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 117 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 : 5.33 x 10e3 cells per ml
Mean cell density of control at 72 hours : 6.24 x 10e5 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 6% 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 4% 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, (see in other information on results and tables section) it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not significantly affected by the presence of the test item over the 72 Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErC10 (0 - 72 h) : >100% v/v saturated solution
ErC20 (0 - 72 h) : >100% v/v saturated solution
ErC50 (0 - 72 h) : >100% v/v saturated solution
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 (P0.05), between the control, 6.25, 12.5, 25, 50 and 100% v/v saturated solution test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of yield
EyC10 (0 - 72 h) : 94% v/v saturated solution
EyC20 (0 - 72 h) : 100% v/v saturated solution
EyC50 (0 - 72 h) : >100% v/v saturated solution

where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out. There were no statistically significant differences ((P greater or equal to 0.05), , between the control, 6.25, 12.5, 25, 50 and 100% v/v saturated solution test groups and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.

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 and test cultures were observed to be green dispersions.

Physico-chemical measurements
The pH values of the control and each test preparation are given in Table 2 (see in other information on results and tables section). Temperature was maintained at 24 ± 1ºC throughout the test.
The pH value of the control cultures (see Table 2)(see in other information on results and tables section) was observed to increase from pH 7.5 at 0 hours to pH 8.2 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
Analysis of the test preparations at 0 and 72 hours (see Appendix 5)( see in attached background section) showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.17 mg/l. This does not infer that no test item was in solution just that which was present was so at a concentration below 0.17 mg/l.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 41100094) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/l

EyC50 (0 – 72 h) : 0.48 mg/l, 95% confidence limits 0.43 – 0.54 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l

No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l

Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l

Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

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

Reported statistics and error estimates:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table1              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	5.08E+03	5.64E+05
	R2	5.01E+03	5.58E+05	-	-
	Mean	5.04E+03	5.61E+05
1.0	R1	5.09E+03	7.06E+05
	R2	5.02E+03	5.29E+05	[3]	[10]
	Mean	5.05E+03	6.18E+05
10	R1	5.06E+03	5.29E+05
	R2	5.10E+03	6.61E+05	[2]	[6]
	Mean	5.08E+03	5.95E+05
100	R1	5.05E+03	2.30E+05
	R2	5.01E+03	2.04E+05	20	62
	Mean	5.03E+03	2.17E+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]

Table2              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	7.5	5.72E+03	2.50E+04	1.23E+05	5.58E+05	8.2
	R2		5.28E+03	2.45E+04	9.09E+04	4.05E+05
	R3		5.15E+03	2.46E+04	1.25E+05	6.65E+05
	R4		5.71E+03	2.46E+04	1.33E+05	7.23E+05
	R5		5.17E+03	2.38E+04	1.26E+05	7.41E+05
	R6		4.93E+03	2.47E+04	1.42E+05	6.52E+05
	Mean		5.33E+03	2.45E+04	1.23E+05	6.24E+05
6.25	R1	7.5	5.37E+03	2.22E+04	1.38E+05	6.99E+05	8.2
	R2		5.58E+03	2.50E+04	1.23E+05	6.08E+05
	R3		5.10E+03	2.45E+04	1.39E+05	7.21E+05
	Mean		5.35E+03	2.39E+04	1.33E+05	6.76E+05
12.5	R1	7.5	5.20E+03	2.48E+04	1.36E+05	5.13E+05	8.1
	R2		5.57E+03	2.75E+04	1.36E+05	6.78E+05
	R3		5.16E+03	2.26E+04	9.83E+04	5.97E+05
	Mean		5.31E+03	2.50E+04	1.23E+05	5.96E+05
25	R1	7.5	5.58E+03	2.26E+04	1.16E+05	6.63E+05	8.1
	R2		5.38E+03	2.08E+04	1.05E+05	6.45E+05
	R3		5.14E+03	2.46E+04	1.36E+05	6.59E+05
	Mean		5.37E+03	2.27E+04	1.19E+05	6.56E+05
50	R1	7.4	5.11E+03	2.22E+04	1.17E+05	6.81E+05	8.1
	R2		5.57E+03	2.58E+04	1.02E+05	6.13E+05
	R3		5.54E+03	2.04E+04	9.91E+04	6.34E+05
	Mean		5.41E+03	2.28E+04	1.06E+05	6.43E+05
100	R1	7.4	5.29E+03	1.51E+04	1.04E+05	5.21E+05	8.1
	R2		4.79E+03	1.23E+04	9.21E+04	4.35E+05
	R3		5.10E+03	1.57E+04	1.12E+05	5.66E+05
	Mean		5.06E+03	1.44E+04	1.03E+05	5.07E+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.

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	0.067	0.067	0.063
	R2	0.066	0.055	0.062
	R3	0.066	0.068	0.070
	R4	0.066	0.070	0.071
	R5	0.065	0.069	0.074
	R6	0.066	0.073	0.064
	Mean	0.066	0.067	0.067

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	0.065		5.52E+05
	R2	0.061		4.00E+05
	R3	0.068		6.60E+05
	R4	0.069	-	7.17E+05	-
	R5	0.069		7.36E+05
	R6	0.068		6.47E+05
	Mean	0.067		6.19E+05
	SD	0.003		1.25E+05
6.25	R1	0.069	[3]	6.94E+05
	R2	0.067	0	6.03E+05
	R3	0.069	[3]	7.16E+05
	Mean	0.068	[2]	6.71E+05	[8]
	SD	0.001		6.00E+04
12.5	R1	0.064	4	5.08E+05
	R2	0.068	[1]	6.73E+05
	R3	0.066	1	5.92E+05
	Mean	0.066	1	5.91E+05	5
	SD	0.002		8.25E+04
25	R1	0.068	[1]	6.58E+05
	R2	0.068	[1]	6.40E+05
	R3	0.068	[1]	6.54E+05
	Mean	0.068	[1]	6.50E+05	[5]
	SD	0.000		9.20E+03
50	R1	0.068	[1]	6.76E+05
	R2	0.067	0	6.07E+05
	R3	0.067	0	6.29E+05
	Mean	0.067	[0]	6.37E+05	[3]
	SD	0.001		3.50E+04
100	R1	0.065	3	5.16E+05
	R2	0.062	7	4.30E+05
	R3	0.066	1	5.61E+05
	Mean	0.064	4	5.02E+05	19
	SD	0.002		6.63E+04

[ ]

 

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

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata 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 saturation.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with 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 761/2009.

Methods

Information provided by the Sponsor indicated that the test item was insoluble in water (water solubility less than 6.89 x 10^-8g/l). Pre-study solubility conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was the most appropriate method of preparation for this test item.

Following a preliminary range-finding test, Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solutions were prepared by stirring an excess (50 mg/l) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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 of Pseudokirchneriella subcapitata to the test item gave EC₅₀values of greater than 100% v/v saturated solution. 

The No Observed Effect Concentration was determined to be 100% v/v saturated solution.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.17 mg/l. This does not infer that no test item was in solution just that which was present was so at a concentration below 0.17 mg/l.

This study showed that there were no toxic effects at saturation.

Conclusion

The effect of the test item on the growth of Pseudokirchneriella subcapitatahas been investigated and gave EC₅₀values 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 saturation.