Cyclohexane-1,3-dione

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:

11 December 2009 and 4 February 2010

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:

2010

Report date:

2010

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15-09-2009 Date of Signature: 26-11-2009

Test material

Test material information

Constituent 1

Reference substance name:

1,3-Cyclohexanedione

IUPAC Name:

1,3-Cyclohexanedione

Details on test material:

Sponsor's identification :1,3-Cyclohexanedione
Description :pale beige solid
Batch number :1091224
Date received :24 November 2009
Expiry date :18 May 2010
Storage conditions :approximately 4°C in the dark
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor. A Certificate of Analysis for the test item supplied by the Sponsor is given in Appendix 1 (see in attached section).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Verification of test 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. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in attached Appendix 4.

Test solutions

Vehicle:

no

Details on test solutions:

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 in attached Appendix 3.

Procedure
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 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 item was dissolved directly in culture medium.
An amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes 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 (200 ml) of each of the stock solutions was separately inoculated with algal suspension (1.8 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.

Definitive test
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 mg/l.

Experimental Preparation
For the purpose of the definitive test, the test item was dissolved directly in culture medium.
An amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 50, 25, 12.5 and 6.25 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (8.3 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 item in the test preparations were verified by chemical analysis at 0 and 72 hours (see attached Appendix 4).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, 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 andconstant illumination at 21 ± 1ºC.


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 in Appendix 3 (see in any other information on materials and methods section).



- 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.

- Age of inoculum :
Not recorded

- Method of cultivation:

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

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 10000 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 100000 - 1000000 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 with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

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.


- 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 values of the control cultures (see Table 2 in any other information on materials and methods section) were observed to increase from pH 7.2 at 0 hours to pH 7.5 – 7.6 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.
At the start of the test a concentration dependant decline in pH in the range of pH 6.9 at 6.25 mg/l through to pH 4.5 at 100 mg/l was observed. Given that this was considered to be due to an intrinsic property of the test item no attempt was made to alter the pH prior to exposure.


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 temperature within the incubator was recorded daily.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

Verification of test concentrations
Analysis of the test preparations at 0 hours (see Appendix 4) showed measured test concentrations to range from 96% to 98% of nominal. Analysis of the test preparations at 72 hours showed that whilst near nominal concentrations were obtained for the 6.25 and 12.5 mg/l test samples, measured concentrations of 18%, 59% and 78% of nominal were obtained for the 25, 50 and 100 mg/l test samples respectively.
Stability analyses showed the test item to be stable at test concentrations of 6.25 and 100 mg/l, however on a number of occasions analysis of samples at a nominal concentration of 25 mg/l showed a decline in the measured concentration. In all instances where this decline was observed, chromatography showed the presence of multiple unidentified peaks. Examination of the chemical structure of the test item and the stability data generated could not provide an explanation for this analytical artefact.
Given that it was not possible to accurately determine EC50 and NOEC values based on the geometric mean measured test concentrations it was considered appropriate to base the results on the nominal test concentrations alone.

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 240000 cells per ml. Inoculation of 500 ml of test medium with 8.3 ml of this algal suspension gave an initial nominal cell density of 4000 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.

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 in Appendix 3.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 33 - 38

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 33 - 38

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 33 - 38

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % CL = 23 - 25

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % CL = 23 - 25

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % CL = 23 - 25

Details on results:

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 1.
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 6.25, 12.5, 25, 50 and 100 mg/l 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.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 69 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 : 4.18 x 10-3 cells per ml
Mean cell density of control at 72 hours : 2.89 x 10-5 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 35% 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 3% 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 item over the 72-Hour exposure period.
Accordingly the following results were determined from the data based on the nominal test concentrations:

Inhibition of growth rate
ErC10 (0 - 72 h) : 21 mg/l
ErC20 (0 - 72 h) : 26 mg/l
ErC50 (0 - 72 h) : 35 mg/l; 95% confidence limits 33 - 38 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, 6.25 and 12.5 mg/l test concentrations (P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 12.5 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 25 mg/l.

Inhibition of yield
EyC10 (0 - 72 h) : 20 mg/l
EyC20 (0 - 72 h) : 21 mg/l
EyC50 (0 - 72 h) : 24 mg/l; 95% confidence limits 23 - 25 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 between the control, 6.25 and 12.5 mg/l test concentrations (P≥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 12.5 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 25 mg/l.


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 6.25, 12.5, 25 and 50 mg/l, however no intact cells were 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, 6.25 and 12.5 mg/l test cultures were observed to be pale green dispersions. The 25 mg/l test cultures were observed to be very pale green dispersions whilst the 50 and 100 mg/l 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 – 7.6 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.
At the start of the test a concentration dependant decline in pH in the range of pH 6.9 at 6.25 mg/l through to pH 4.5 at 100 mg/l was observed. Given that this was considered to be due to an intrinsic property of the test item no attempt was made to alter the pH prior to exposure.

Verification of test concentrations
Analysis of the test preparations at 0 hours (see Appendix 4) showed measured test concentrations to range from 96% to 98% of nominal. Analysis of the test preparations at 72 hours showed that whilst near nominal concentrations were obtained for the 6.25 and 12.5 mg/l test samples, measured concentrations of 18%, 59% and 78% of nominal were obtained for the 25, 50 and 100 mg/l test samples respectively.
Stability analyses showed the test item to be stable at test concentrations of 6.25 and 100 mg/l, however on a number of occasions analysis of samples at a nominal concentration of 25 mg/l showed a decline in the measured concentration. In all instances where this decline was observed, chromatography showed the presence of multiple unidentified peaks. Examination of the chemical structure of the test item and the stability data generated could not provide an explanation for this analytical artefact.
Given that it was not possible to accurately determine EC50 and NOEC values based on the geometric mean measured test concentrations it was considered appropriate to base the results on the nominal test concentrations alone.

Results with reference substance (positive control):

Appendix 2 Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) 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:

Statistical analysis
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	4.66E+03	4.79E+05
	R2	4.01E+03	5.99E+05	-	-
	Mean	4.33E+03	5.39E+05
0.10	R1	4.11E+03	5.22E+05
	R2	4.68E+03	5.74E+05	0	[2]
	Mean	4.40E+03	5.48E+05
1.0	R1	4.43E+03	5.58E+05
	R2	3.92E+03	6.12E+05	[3]	[9]
	Mean	4.18E+03	5.85E+05
10	R1	4.19E+03	3.81E+05
	R2	4.44E+03	5.95E+05	1	9
	Mean	4.32E+03	4.88E+05
100	R1	4.64E+03	2.16E+04
	R2	4.70E+03	6.79E+03	78	98
	Mean	4.67E+03	1.42E+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]

Table 2              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	7.2	4.36E+03	1.25E+04	3.52E+04	3.27E+05	7.6
	R2	7.2	4.06E+03	1.29E+04	5.19E+04	3.30E+05	7.6
	R3	7.2	4.17E+03	1.30E+04	3.96E+04	3.01E+05	7.6
	R4	7.2	4.25E+03	1.34E+04	2.88E+04	2.47E+05	7.5
	R5	7.2	4.10E+03	1.33E+04	4.56E+04	2.72E+05	7.5
	R6	7.2	4.16E+03	1.28E+04	3.85E+04	2.55E+05	7.5
	Mean		4.18E+03	1.30E+04	3.99E+04	2.89E+05
6.25	R1	6.9	4.25E+03	8.77E+03	4.58E+04	3.37E+05	7.4
	R2	6.9	4.61E+03	7.66E+03	2.92E+04	3.40E+05	7.4
	R3	6.9	4.10E+03	8.39E+03	3.91E+04	3.35E+05	7.4
	Mean		4.32E+03	8.27E+03	3.80E+04	3.38E+05
12.5	R1	6.4	4.12E+03	7.28E+03	3.68E+04	3.22E+05	7.3
	R2	6.4	4.11E+03	1.14E+04	3.90E+04	3.76E+05	7.3
	R3	6.4	4.22E+03	8.38E+03	3.66E+04	3.11E+05	7.3
	Mean		4.15E+03	9.01E+03	3.75E+04	3.36E+05
25	R1	5.5	3.94E+03	8.97E+03	2.45E+04	1.31E+05	6.9
	R2	5.5	4.22E+03	9.51E+03	3.48E+04	1.37E+05	6.9
	R3	5.5	4.34E+03	6.41E+03	3.39E+04	1.03E+05	6.9
	Mean		4.17E+03	8.30E+03	3.11E+04	1.23E+05
50	R1	4.9	3.92E+03	2.43E+03	4.78E+03	1.15E+04	4.9
	R2	4.9	4.57E+03	3.38E+03	6.78E+03	8.29E+03	4.9
	R3	4.9	4.23E+03	3.53E+03	3.44E+03	8.71E+03	4.9
	Mean		4.24E+03	3.11E+03	5.00E+03	9.50E+03
100	R1	4.5	4.62E+03	1.83E+03	1.64E+03	2.87E+03	4.5
	R2	4.5	4.54E+03	1.54E+03	3.38E+03	2.25E+03	4.5
	R3	4.5	4.06E+03	2.45E+03	2.24E+03	1.31E+03	4.5
	Mean		4.41E+03	1.94E+03	2.42E+03	2.14E+03

*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		0.048		0.043		0.093
		R2		0.049		0.058		0.077
		R3		0.049		0.047		0.084
		R4		0.051		0.032		0.090
		R5		0.050		0.051		0.074
		R6		0.049		0.046		0.079
		Mean		0.049		0.046		0.083

R₁- R₆= Replicates 1 to 6 

Table 4              Inhibition of Growth Rate and Yieldin 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	0.061		3.23E+05
	R2	0.061		3.26E+05
	R3	0.060		2.97E+05
	R4	0.057	-	2.43E+05	-
	R5	0.059		2.68E+05
	R6	0.058		2.50E+05
	Mean	0.059		2.85E+05
	SD	0.002		3.60E+04
6.25	R1	0.062	[5]	3.33E+05
	R2	0.062	[5]	3.35E+05
	R3	0.062	[5]	3.31E+05
	Mean	0.062	[5]	3.33E+05	[17]
	SD	0.000		2.15E+03
12.5	R1	0.061	[3]	3.18E+05
	R2	0.063	[7]	3.72E+05
	R3	0.060	[2]	3.07E+05
	Mean	0.061	[4]	3.32E+05	[17]
	SD	0.002		3.46E+04
25	R1	0.048	19	1.27E+05
	R2	0.049	17	1.32E+05
	R3	0.045	24	9.84E+04
	Mean	0.047	20	1.19E+05	58
	SD	0.002		1.82E+04
50	R1	0.015	75	7.58E+03
	R2	0.010	83	3.72E+03
	R3	0.011	81	4.48E+03
	Mean	0.012	80	5.26E+03	98
	SD	0.003		2.04E+03
100	R1	-0.005	108	-1.75E+03
	R2	-0.008	114	-2.29E+03
	R3	-0.015	125	-2.75E+03
	Mean	-0.009	116	-2.26E+03	101
	SD	0.005		5.00E+02

*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]

Please see attached Figures 1 to 3       Figure1 Mean Cell Densities v Timefor the Definitive Test, Figure 2 Inhibition of Growth Rate Based on Nominal Test Concentrations & Figure 3 Inhibition of Yield Based on Nominal Test Concentrations

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results based on nominal test concentrations:
Response Variable EC50 (mg/l) 95% Confidence Limits (mg/l) (NOEC) (mg/l) (LOEC) (mg/l)
Growth Rate 35 33 - 38 12.5 25
Yield 24 23 - 25 12.5 25

Executive summary:

Introduction. 

A study was perford to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus. The method 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 a preliminary range-finding test,Desmodesmus subspicatuswas exposed to an aqueous solution of the test item at nominal 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 of Desmodesmus subspicatus 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	35	33	-	38	12.5	25
Yield	24	23	-	25	12.5	25

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 96% to 98% of nominal. Analysis of the test preparations at 72 hours showed that whilst near nominal concentrations were obtained for the 6.25 and 12.5 mg/l test samples, measured concentrations of 18%, 59% and 78% of nominal were obtained for the 25, 50 and 100 mg/l test samples respectively. 

Stability analyses showed the test item to be stable at test concentrations of 6.25 and 100 mg/l, however on a number of occasions analysis of samples at a nominal concentration of 25 mg/l showed a decline in the measured concentration. In all instances where this decline was observed, chromatography showed the presence of multiple unidentified peaks. Examination of the chemical structure of the test item and the stability data generated could not provide an explanation for this analytical artefact.

Given that it was not possible to accurately determine EC₅₀and NOEC values based on the geometric mean measured test concentrations it was considered appropriate to base the results on the nominal test concentrations alone.

CONCLUSION

The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results based on nominal test concentrations:

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	35	33	-	38	12.5	25
Yield	24	23	-	25	12.5	25