Tricyclo[3.3.1.13,7]decane-1-acetic acid, .alpha.-[[(1,1-dimethylethoxy)carbonyl]amino]-3-hydroxy-, (.alpha.S)-

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:

Experimental Starting Date: 3 August 2012 Experimental Completion Date: 31 August 2012

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:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

BMS-528233-01

IUPAC Name:

BMS-528233-01

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

The integrity of the supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Identification: BMS-528233-01
Description: white powder
Batch: 2C80150N
Purity: 100%
Expiry / retest date: not provided
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

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. 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 -20 ºC for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached Appendix 3.

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 with the aid of high shear mixing at approximately 7500 rpm for 30 minutes and the
volume adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution 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 (3.5 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 ± 1 ºC 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.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test
conditions. 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: 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 high shear mixing at approximately 7500 rpm for 30 minutes and the
volume adjusted to 1 liter to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution 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 (4.8 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 3).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

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 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 103 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 104 - 105 cells/mL.

A positive control (Harlan Laboratories Ltd Study Number: 41104038) used potassium dichromate as the reference item. Details of the positive control are given in the attached Appendix 1.


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 the attached Appendix 2.

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 were observed to increase from pH 7.5 at 0 hours to pH 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.

The test item vessels showed a concentration dependant decline in pH at 0 hours in the range of pH 7.4 at 6.25 mg/L through to pH 4.2 at 100 mg/L. Given
that this was considered to be due to an intrinsic property of the test item no adjustments were made to the pH prior to the addition of the algal cells.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

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: 6.25, 12.5, 25, 50 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 characterized by a cell density of 5.20 x 105 cells per mL. Inoculation of
500 mL of test medium with 4.8 mL of this algal suspension gave an initial nominal cell density of 5 x 103 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.


Physico-Chemical Measurements
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.


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. 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 -20 ºC for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached 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.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits (mg/L) 54 - 67

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% Confidence Limits (mg/L) 54 - 67

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits (mg/L) 54-67

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% Confidence Limits (mg/L) 29 - 36

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% Confidence Limits (mg/L) 29 - 36

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% Confidence Limits (mg/L) 29 - 36

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.

The test item vessels showed a concentration dependant decline in pH at 0 hours in the range of pH 7.4 at 6.25 mg/L through to pH 4.2 at 100 mg/L. Given that this was considered to be due to an intrinsic property of the test item no adjustments were made to the pH prior to the addition of the
algal cells.

Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 72 hours (see attached Appendix 3) showed measured test concentrations to
range from 95% to 102% of nominal indicating that the test item was stable over the test duration.


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 the attached Figure 1. Percentage inhibition values are plotted against test concentration in the attached Figure 2 and Figure 3.


Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 144 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 : 4.06 x 10E+3 cells per mL
Mean cell density of control at 72 hours : 5.85 x 10E+5 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 0% 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 Table 2 and Table 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) : 36 mg/L
ErC20 (0 - 72 h) : 43 mg/L
ErC50 (0 - 72 h) : 60 mg/L; 95% confidence limits 54 - 67 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) : 16 mg/L
EyC20 (0 - 72 h) : 21 mg/L
EyC50 (0 - 72 h) : 33 mg/L; 95% confidence limits 29 - 36 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 4.2.2.1. 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. 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 colorless solutions. After the 72-Hour test period all control, 6.25, 12.5 and 25 mg/L test cultures were observed to be pale green dispersions. The 50 mg/L test cultures were observed to be very pale green dispersions whilst the 100 mg/L test cultures were observed to be clear colorless solutions.


Physico-Chemical Measurements
The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC 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 7.8 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.

The test item vessels showed a concentration dependant decline in pH at 0 hours in the range of pH 7.4 at 6.25 mg/L through to pH 4.2 at 100 mg/L. Given that this was considered to be due to an intrinsic property of the test item no adjustments were made to the pH prior to the addition of the
algal cells.


Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours (see attached Appendix 3) showed measured test concentrations to range from 90% to 107% of nominal and so the results are based on nominal test concentrations only.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Study Number: 41104038) 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) : 1.4 mg/L; 95% confidence limits 1.2 – 1.7 mg/L
EyC50 (0 – 72 h) : 0.59 mg/L, 95% confidence limits 0.53 – 0.65 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.

See Appendix 1 for full results.

Reported statistics and error estimates:

Determination of ECx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a
line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments (Litchfield
and Wilcoxon, 1949).


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

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	6.38E+03	1.02E+06	-	-
	R2	6.80E+03	1.01E+06
	Mean	6.59E+03	1.02E+06
0.10	R1	6.69E+03	6.18E+05	7	35
	R2	6.10E+03	7.16E+05
	Mean	6.39E+03	6.66E+05
1.0	R1	5.78E+03	6.24E+05	9	43
	R2	6.02E+03	5.41E+05
	Mean	5.90E+03	5.83E+05
10	R1	5.68E+03	4.75E+05	9	42
	R2	6.32E+03	6.99E+05
	Mean	6.00E+03	5.87E+05
100	R1	6.34E+03	8.78E+03	101	100
	R2	5.74E+03	2.37E+03
	Mean	6.04E+03	5.58E+03

 

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

Table 2     Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	4.19E+03	2.28E+04	9.29E+04	5.77E+05	7.8
	R2		3.41E+03	1.97E+04	9.28E+04	5.81E+05
	R3		3.95E+03	1.68E+04	9.84E+04	5.99E+05
	R4		4.10E+03	1.67E+04	8.85E+04	5.75E+05
	R5		4.69E+03	1.71E+04	6.31E+04	5.82E+05
	R6		4.05E+03	1.65E+04	8.06E+04	5.94E+05
	Mean		4.06E+03	1.83E+04	8.61E+04	5.85E+05
6.25	R1	7.4	4.24E+03	1.88E+04	8.40E+04	5.63E+05	7.8
	R2		4.27E+03	1.88E+04	9.03E+04	5.70E+05
	R3		4.22E+03	1.59E+04	7.98E+04	5.55E+05
	Mean		4.25E+03	1.78E+04	8.47E+04	5.63E+05
12.5	R1	7.3	4.40E+03	1.66E+04	8.54E+04	6.15E+05	7.7
	R2		3.63E+03	1.45E+04	7.90E+04	6.16E+05
	R3		3.46E+03	1.95E+04	9.88E+04	5.97E+05
	Mean		3.83E+03	1.69E+04	8.77E+04	6.09E+05
25	R1	7.1	3.57E+03	1.84E+04	9.48E+04	4.23E+05	7.6
	R2		4.24E+03	2.42E+04	8.74E+04	3.53E+05
	R3		3.76E+03	1.71E+04	9.15E+04	3.77E+05
	Mean		3.86E+03	1.99E+04	9.12E+04	3.84E+05
50	R1	6.5	3.31E+03	2.21E+04	3.39E+04	1.47E+05	7.4
	R2		3.36E+03	1.24E+04	3.44E+04	1.28E+05
	R3		3.84E+03	1.43E+04	3.96E+04	1.48E+05
	Mean		3.50E+03	1.63E+04	3.60E+04	1.41E+05
100	R1	4.2	4.53E+03	9.30E+03	6.72E+03	1.07E+04	4.6
	R2		4.46E+03	3.52E+03	8.26E+03	6.19E+03
	R3		4.02E+03	8.48E+03	6.26E+03	6.00E+03
	Mean		4.34E+03	7.10E+03	7.08E+03	7.63E+03

*    Cell densities represent the mean number of cells per mL calculated from the mean 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	0.063	0.059	0.076
	R2	0.057	0.065	0.076
	R3	0.050	0.074	0.075
	R4	0.050	0.070	0.078
	R5	0.051	0.054	0.093
	R6	0.050	0.066	0.083
	Mean	0.054	0.065	0.080

 

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	0.066		5.73E+05
	R2	0.066		5.78E+05
	R3	0.066		5.95E+05
	R4	0.066	-	5.71E+05	-
	R5	0.066		5.78E+05
	R6	0.066		5.90E+05
	Mean	0.066		5.81E+05
	SD	0.000		9.44E+03
6.25	R1	0.066	0	5.58E+05
	R2	0.066	0	5.66E+05
	R3	0.065	2	5.51E+05
	Mean	0.066	1	5.58E+05	4
	SD	0.001		7.34E+03
12.5	R1	0.067	[2]	6.11E+05
	R2	0.067	[2]	6.12E+05
	R3	0.066	0	5.94E+05
	Mean	0.067	[1]	6.06E+05	[4]
	SD	0.001		1.05E+04
25	R1	0.062	6	4.19E+05
	R2	0.059	11	3.49E+05
	R3	0.060	9	3.73E+05
	Mean	0.060	9	3.80E+05	34
	SD	0.002		3.59E+04
50	R1	0.047	29	1.44E+05
	R2	0.045	32	1.25E+05
	R3	0.047	29	1.44E+05
	Mean	0.046	30	1.37E+05	76
	SD	0.001		1.11E+04
100	R1	0.011	83	6.18E+03
	R2	0.003	95	1.73E+03
	R3	0.003	95	1.98E+03
	Mean	0.006	91	3.30E+03	99
	SD	0.005		2.50E+03

 

*In accordance with the OECD test guideline only the mean 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:

CONCLUSION

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:

Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Lowest Observed Effect
Concentration (NOEC) (mg/L) Concentration (LOEC) (mg/L)
Growth Rate 60 54 - 67 12.5 25
Yield 33 29 - 36 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 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

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

 

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

 

 

Results

Exposure 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	60	54	-	67	12.5	25
Yield	33	29	-	36	12.5	25

 

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 90% to 107% of nominal and so the results are based on nominal test concentrations only.