Ammonium sulphamidate

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 3 June 2011 and 14 July 2011.

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:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

of Commission Regulation (EC) No 440/2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium Sulphamate

IUPAC Name:

Sodium Sulphamate

Details on test material:

Sponsor's identification: Sodium Sulphamate
CAS number : 13845-18-6
Identifier : TIS I0442
Description : white solid
Purity : 99.67%
Batch number : LE12568
Date received : 6 May 2011
Expiry date : 6 May 2013
Storage conditions: room temperature over silica gel in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
1.0, 3.2, 10, 32 and 100 mg/l testing groups (definitive test)

- Sampling method:
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.

- Sample storage conditions before analysis:
All 0-Hour samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test:
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 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 item was dissolved directly in culture medium.

An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/l. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (5.7 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 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 from the range-finding test preparations at 0 and 72 hours in order to determine the stability of the test item under test conditions (see Appendix 4 - attached background material)). All samples were stored at approximately 20°C prior to analysis.

Definitive test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.

Experimental Preparation
For the purpose of the definitive test, the test item was dissolved directly in culture medium.

Amounts of test item (100 and 32 mg) were separately dissolved in culture medium and the volumes adjusted to 1 litre to give 100 and 32 mg/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (3.8 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 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 item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 4 - attached background materia).


- Controls:
A positive control used potassium dichromate as the reference item.

Observations on cultures:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/l, however cell debris was observed to be present in the test cultures at 100 mg/l.

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 and 3.2 mg/ test cultures were observed to be pale green dispersions whilst the 10, 32 and 100 mg/l test cultures were observed to be clear colourless solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test Species
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. 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. 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 illumination at 21 ± 1ºC.
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 10E+03 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 ± 1ºC until the algal cell density was approximately 10E+04 - 10E+05 cells/ml.


Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined below.


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.
* Elga Optima 15+ or Elga Purelab Option R-15 BP

Study design

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.

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 each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

The pH value of the control cultureswas observed to increase from pH 7.5 at 0 hours to pH 7.7 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.

See results section for physico-chemical measurement results.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.

Details on test conditions:

EXPOSURE CONDITIONS
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation 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 6.61 x 10E5 cells per ml. Inoculation of 500 ml of test medium with 3.8 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.


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.


Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Remarks on result:

other: 95% confidence limits 4.7 -8.0 mg/l loading rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield

Remarks on result:

other: 95% CL not stated

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield

Remarks on result:

other: 95% CL not stated

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 ( any other information on results including tables section.)
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 reduced at 100 mg/l.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were selected for the definitive test.
Chemical analysis of the 1.0, 10 and 100 mg/l test preparations at 0 and 72 hours (see Appendix 4 attached in background material section) showed near nominal test concentrations were obtained indicating that the test item was stable under test conditions.

DEFINITIVE TEST:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (any other information on results including tables section). Daily specific growth rates for the control cultures are given in Table 3 (any other information on results including 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(any other information on results including tables section ).

The mean cell densities versus time for the definitive test are presented in Figure 1 attached in background material section. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (see attached in background material section).

VALIDATION CRITERIA:
The following data show that the cell concentration of the control cultures increased by a factor of 44 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 : 4.99E+03 cells per ml
Mean cell density of control at 72 hours : 2.19 E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% 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 Pseudokirchneriella subcapitata (CCAP 278/4) were 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) : 1.3 mg/l
ErC20 (0 - 72 h) : 6.0 mg/l
ErC50 (0 - 72 h) : 86 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 between the control and 1.0 mg/l test concentration (P equal to or greater than 0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.0 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.2 mg/l.
* 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.

INHIBITION OF YIELD
EyC10 (0 - 72 h) : 0.48 mg/l
EyC20 (0 - 72 h) : 1.2 mg/l
EyC50 (0 - 72 h) : 6.1 mg/l; 95% confidence limits 4.7 – 8.0 mg/l
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 between the control and 1.0 mg/l test concentration (P equal to or greater than 0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.0 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.2 mg/l.

PHYSICO-CHEMICAL MEASUREMENTS
The pH values of the control and test preparations are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.5 at 0 hours to pH 7.7 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 4 attached in background material section) showed measured test concentrations to range from 88% to 119% of nominal indicating that the test item was stable over the test duration and so the results are based on nominal test concentrations only.

Results with reference substance (positive control):

A positive control 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 to 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

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

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.02E+03	4.96E+05
	R2	5.02E+03	5.05E+05	-	-
	Mean	5.02E+03	5.01E+05
0.10	R1	5.08E+03	8.44E+05
	R2	5.16E+03	5.46E+05	[6]	[39]
	Mean	5.12E+03	6.95E+05
1.0	R1	5.01E+03	7.80E+05
	R2	5.12E+03	5.11E+05	[5]	[29]
	Mean	5.06E+03	6.45E+05
10	R1	5.01E+03	4.41E+05
	R2	5.04E+03	4.25E+05	3	14
	Mean	5.02E+03	4.33E+05
100	R1	5.06E+03	8.61E+04
	R2	5.09E+03	1.04E+05	36	82
	Mean	5.07E+03	9.50E+04

*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 (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	4.70E+03	1.88E+04	5.04E+04	2.28E+05	7.7
	R2		5.48E+03	1.34E+04	4.46E+04	2.23E+05
	R3		4.99E+03	1.36E+04	4.67E+04	2.09E+05
	R4		5.64E+03	1.32E+04	4.71E+04	2.28E+05
	R5		4.53E+03	1.37E+04	4.60E+04	2.12E+05
	R6		4.62E+03	1.30E+04	4.64E+04	2.11E+05
	Mean		4.99E+03	1.43E+04	4.69E+04	2.19E+05
1.0	R1	7.5	5.30E+03	1.25E+04	4.76E+04	1.99E+05	7.5
	R2		4.63E+03	1.28E+04	4.88E+04	1.97E+05
	R3		4.93E+03	1.22E+04	4.78E+04	2.00E+05
	Mean		4.95E+03	1.25E+04	4.81E+04	1.99E+05
3.2	R1	7.4	5.08E+03	1.06E+04	3.75E+04	1.62E+05	7.4
	R2		4.43E+03	6.75E+03	1.73E+04	1.36E+05
	R3		5.02E+03	7.76E+03	4.12E+04	1.02E+05
	Mean		4.85E+03	8.38E+03	3.20E+04	1.33E+05
10	R1	7.4	5.02E+03	6.69E+03	1.88E+04	6.11E+04	7.4
	R2		5.34E+03	9.27E+03	2.77E+04	8.81E+04
	R3		5.08E+03	7.78E+03	1.54E+04	8.11E+04
	Mean		5.15E+03	7.91E+03	2.06E+04	7.67E+04
32	R1	7.3	4.47E+03	5.18E+03	1.99E+04	5.85E+04	7.4
	R2		4.70E+03	7.89E+03	1.59E+04	4.98E+04
	R3		5.32E+03	6.42E+03	1.38E+04	4.46E+04
	Mean		4.83E+03	6.50E+03	1.66E+04	5.10E+04
100	R1	7.3	5.38E+03	4.26E+03	1.98E+04	5.46E+04	7.4
	R2		4.94E+03	3.16E+03	1.29E+04	3.87E+04
	R3		4.25E+03	3.36E+03	9.92E+03	2.17E+04
	Mean		4.86E+03	3.59E+03	1.42E+04	3.83E+04

*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	0.055	0.041	0.063
	R2	0.041	0.050	0.067
	R3	0.042	0.051	0.062
	R4	0.041	0.053	0.066
	R5	0.042	0.051	0.064
	R6	0.040	0.053	0.063
	Mean	0.044	0.050	0.064

 

R₁- R₆= Replicates 1 to 6

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

Nominal Concentration (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.053		2.23E+05
	R2	0.053		2.18E+05
	R3	0.052		2.04E+05
	R4	0.053	-	2.23E+05	-
	R5	0.052		2.07E+05
	R6	0.052		2.06E+05
	Mean	0.053		2.14E+05
	SD	0.001		8.78E+03
1.0	R1	0.051	4	1.94E+05
	R2	0.051	4	1.93E+05
	R3	0.051	4	1.95E+05
	Mean	0.051	4	1.94E+05	9
	SD	0.000		1.14E+03
3.2	R1	0.048	9	1.57E+05
	R2	0.046	13	1.32E+05
	R3	0.042	21	9.71E+04
	Mean	0.045	14	1.29E+05	40
	SD	0.003		2.99E+04
10	R1	0.035	34	5.61E+04
	R2	0.040	25	8.27E+04
	R3	0.039	26	7.60E+04
	Mean	0.038	28	7.16E+04	66
	SD	0.003		1.39E+04
32	R1	0.034	36	5.41E+04
	R2	0.032	40	4.51E+04
	R3	0.030	43	3.92E+04
	Mean	0.032	40	4.61E+04	78
	SD	0.002		7.47E+03
100	R1	0.033	38	4.92E+04
	R2	0.028	47	3.38E+04
	R3	0.020	62	1.75E+04
	Mean	0.027	49	3.35E+04	84
	SD	0.007		1.59E+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

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 over a 72-Hour period and gave the following results:

Growth Rate:
EC50: 86 mg/l

Yield:
EC50: 6.1 mg/l

NOEC (growth rate and yield): 1.0 mg/l
LOEC (growth rate and yield): 3.2 mg/l

Executive summary:

Introduction. 

A study was perford 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 440/2008.

Methods.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 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 of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC50(mg/l)	95% Confidence Limits (mg/l)			No Observed Effect Concentration (NOEC) (mg/l)	Lowest Observed Effect Concentration (LOEC) (mg/l)
Growth Rate	86		*		1.0	3.2
Yield	6.1	4.7	-	8.0	1.0	3.2

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 88% to 119% of nominal indicating that the test item was stable over the test duration and so the results are based on nominal test concentrations only.

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.