1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo-1-piperidinyl)phenyl]-, ethyl ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2012 to 12 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Definitive Test

Based on the results of the pre-study media preparation trial and range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Experimental Preparation

An amount of test item (550 mg) was dispersed in 11 liters 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 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 10 mg/L. A series of dilutions was made from this saturated solution to give stock solutions of 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with 11.6 mL of algal suspension to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 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 solutions were verified by chemical analysis at 0 and 72 hours.


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 1.94 x 105 cells per mL. Inoculation of 450 mL of test medium with 11.6 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, 23, 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 20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

An amount of test item (550 mg) was dispersed in 11 liters 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 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 10 mg/L. A series of
dilutions was made from this saturated solution to give stock solutions of 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (450 mL) of each of the stock
solutions was separately inoculated with 11.6 mL of algal suspension to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 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 solutions were verified by chemical analysis at 0 and 72 hours.

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 (Section 3.3). The master cultures were maintained in
the laboratory under constant aeration and constant illumination at 21 ± 1 deg 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 deg C until the algal cell density was approximately 104 - 105 cells/mL.

A positive control (Harlan Laboratories Ltd Project Number: 41104038) used potassium dichromate as the reference item. Details of the positive
control are given in Appendix 2. The positive control was conducted between 10 October 2011 and 13 October 2011.


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.

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

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Range-Finding Test
The results obtained from the pre-study media preparation trial conducted indicated that a dissolved test item concentration of approximately 10 mg/L could be obtained using a saturated solution method of preparation.

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 0.010, 0.10, 1.0 and 10 mg/L for a period of 72 hours.

An amount of test item (550 mg) was dispersed in 11 liters 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 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 10 mg/L. A series of dilutions was made from this saturated solution to give further stock solutions of 1.0, 0.10 and 0.010 mg/L. An aliquot (450 mL) of each of the
stock solutions was separately inoculated with algal suspension (7 mL) to give the required test concentrations of 0.010, 0.10, 1.0 and 10 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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 ± 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.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

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 pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 8.3 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.

e aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an
increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the EEC Guidelines (1.5 pH units after 72
hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control
cultures exceeded the validation criterion given in the Test Guidelines.

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.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

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 0.010, 0.10, 1.0 and 10 mg/L for a period of 72 hours.

Details on test conditions:

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 constant illumination at 21 ± 1 deg 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 deg C until the algal cell density was approximately 104 - 105 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.

Procedure
Pre-Study Media Preparation Trial
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 categorized 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.


Range-Finding Test
The results obtained from the pre-study media preparation trial conducted indicated that a dissolved test item concentration of approximately
10 mg/L could be obtained using a saturated solution method of preparation.

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 0.010, 0.10, 1.0 and 10 mg/L for a period of 72 hours.

An amount of test item (550 mg) was dispersed in 11 liters 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 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 10 mg/L. A series of dilutions was made from this saturated solution to give further stock solutions of 1.0, 0.10 and 0.010 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (7 mL) to give the required test concentrations of 0.010, 0.10, 1.0 and 10 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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 ± 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.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

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 pre-study media preparation trial and range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Experimental Preparation
An amount of test item (550 mg) was dispersed in 11 liters 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 1 liter discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 10 mg/L. A series of dilutions was made from this saturated solution to give stock solutions of 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with 11.6 mL of algal suspension to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and
10 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 solutions were verified by chemical analysis at 0 and 72 hours.


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 1.94 x 105 cells per mL. Inoculation of
450 mL of test medium with 11.6 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, 23, 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 20ºC for further analysis if necessary.

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

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RESULTS
Range-finding Test
The results showed no effect on growth at the test concentrations of 0.010 and 0.10 mg/L. However, growth was observed to be reduced at 1.0 and 10 mg/L

Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal thereby 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. 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 155 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 : 4.89 x 103 cells per mL
Mean cell density of control at 72 hours : 7.58 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% 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 6% 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) : 8.0 mg/L
ErC20 (0 - 72 h) : 8.9 mg/L
ErC50 (0 - 72 h) : 11 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, 0.10, 0.32, 1.0 and 3.2 mg/L test
concentrations (P0.05), however the 10 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect
Concentration" (NOEC) based on growth rate was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth
rate was 10 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 2.9 mg/L
EyC20 (0 - 72 h) : 3.7 mg/L
EyC50 (0 - 72 h) : 6.5 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 decreases in yield between the control, 0.10, 0.32, 1.0 and 3.2 mg/L test concentrations (P0.05), however the 10 mg/L test concentration was significantly different (P<0.05) and,
therefore the "No Observed Effect Concentration" (NOEC) based on yield was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 10 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 0.10, 0.32, 1.0 and 3.2 mg/L, however cell debris was observed to be present in the test cultures at 10 mg/L.


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, 0.10, 0.32, 1.0 and 3.2 mg/L test cultures were observed to be pale green dispersions whilst the 10 mg/L test cultures were observed to be extremely pale green dispersions.


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 8.1 at 0 hours to pH 8.3 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) showed measured test concentrations to range from 81% to 99% of nominal and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

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) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate 11 3.2 10
Yield 6.5 3.2 10

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 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.

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).

Table1     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	5.10E+03	9.09E+05	-	-
	R2	5.07E+03	1.28E+06
	Mean	5.09E+03	1.10E+06
0.010	R1	5.10E+03	1.20E+06	0	[3]
	R2	5.01E+03	1.06E+06
	Mean	5.05E+03	1.13E+06
0.10	R1	5.10E+03	9.63E+05	0	[3]
	R2	5.12E+03	1.29E+06
	Mean	5.11E+03	1.13E+06
1.0	R1	5.05E+03	9.84E+05	8	34
	R2	5.06E+03	4.54E+05
	Mean	5.05E+03	7.19E+05
10	R1	4.74E+03	3.44E+05	25	78
	R2	3.82E+03	1.49E+05
	Mean	4.28E+03	2.46E+05

Table2     Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	23 h	48 h	72 h	72 h
Control	R1	8.1	4.55E+03	2.07E+04	7.39E+04	4.44E+05	8.3
	R2		5.12E+03	2.93E+04	1.78E+05	8.74E+05
	R3		4.98E+03	2.91E+04	1.11E+05	6.52E+05
	R4		4.65E+03	2.70E+04	1.35E+05	7.40E+05
	R5		5.11E+03	3.35E+04	1.78E+05	9.28E+05
	R6		4.96E+03	2.90E+04	1.73E+05	9.13E+05
	Mean		4.89E+03	2.81E+04	1.41E+05	7.58E+05
0.10	R1	8.0	4.98E+03	3.28E+04	1.75E+05	1.07E+06	8.2
	R2		5.04E+03	3.65E+04	2.07E+05	1.14E+06
	R3		5.14E+03	3.70E+04	2.06E+05	1.14E+06
	Mean		5.05E+03	3.54E+04	1.96E+05	1.12E+06
0.32	R1	7.9	5.08E+03	3.43E+04	1.72E+05	1.02E+06	8.1
	R2		5.18E+03	2.62E+04	1.27E+05	7.47E+05
	R3		4.76E+03	3.36E+04	1.87E+05	1.08E+06
	Mean		5.01E+03	3.14E+04	1.62E+05	9.51E+05
1.0	R1	7.8	4.88E+03	2.18E+04	1.07E+05	6.30E+05	8.0
	R2		4.80E+03	3.04E+04	1.60E+05	8.91E+05
	R3		4.94E+03	2.56E+04	1.50E+05	7.80E+05
	Mean		4.87E+03	2.59E+04	1.39E+05	7.67E+05
3.2	R1	7.7	3.78E+03	2.60E+04	1.56E+05	8.73E+05	8.0
	R2		5.14E+03	2.54E+04	1.36E+05	7.46E+05
	R3		5.21E+03	2.18E+04	1.15E+05	6.44E+05
	Mean		4.71E+03	2.44E+04	1.36E+05	7.55E+05
10	R1	7.7	4.75E+03	2.24E+03	2.65E+04	1.78E+05	7.9
	R2		2.95E+03	2.92E+03	1.92E+04	1.34E+05
	R3		5.35E+03	3.41E+03	8.11E+03	4.28E+04
	Mean		4.35E+03	2.86E+03	1.79E+04	1.18E+05

Table3     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	0.062	0.051	0.075
	R2	0.077	0.072	0.066
	R3	0.077	0.054	0.074
	R4	0.073	0.064	0.071
	R5	0.083	0.067	0.069
	R6	0.076	0.072	0.069
	Mean	0.075	0.063	0.071

 

Table4     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	0.062	-	4.39E+05	-
	R2	0.072		8.69E+05
	R3	0.068		6.47E+05
	R4	0.069		7.36E+05
	R5	0.073		9.22E+05
	R6	0.072		9.08E+05
	Mean	0.069		7.54E+05
	SD	0.004		1.88E+05
0.10	R1	0.075	[9]	1.07E+06
	R2	0.075	[9]	1.13E+06
	R3	0.075	[9]	1.14E+06
	Mean	0.075	[9]	1.11E+06	[48]
	SD	0.000		3.93E+04
0.32	R1	0.074	[7]	1.02E+06
	R2	0.070	[1]	7.42E+05
	R3	0.075	[9]	1.08E+06
	Mean	0.073	[6]	9.46E+05	[26]
	SD	0.003		1.79E+05
1.0	R1	0.067	3	6.25E+05
	R2	0.072	[4]	8.86E+05
	R3	0.070	[1]	7.75E+05
	Mean	0.070	[1]	7.62E+05	[1]
	SD	0.003		1.31E+05
3.2	R1	0.072	[4]	8.70E+05
	R2	0.070	[1]	7.41E+05
	R3	0.067	3	6.39E+05
	Mean	0.070	[1]	7.50E+05	0
	SD	0.003		1.16E+05
10	R1	0.050	28	1.73E+05
	R2	0.046	33	1.31E+05
	R3	0.030	57	3.75E+04
	Mean	0.042	39	1.14E+05	85
	SD	0.011		6.93E+04

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitatahas been investigated over a 72-Hour period and gave the following
results:
 
Growth Rate: Response Variable EC50 11 mg/L, No Observed Effect Concentration (NOEC) 3.2 mg/L, Lowest Observed Effect Concentration (LOEC) 10 mg/L

Yield: Response Variable EC50 6.5 mg/L, No Observed Effect Concentration (NOEC) 3.2 mg/L, Lowest Observed Effect Concentration (LOEC)
10 mg/L

Executive summary:

SUMMARY

Introduction

A study was perford to assess the effect of the test item on the growth of the green algaPseudokirchneriella 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

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional method of preparation e.g. ultrasonication and high shear mixing.

 

A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 10 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

 

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at measured concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (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 1 liter discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a nominal concentration of 10 mg/L. 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 ofPseudokirchneriella subcapitatato the test item gave the following results:

 

Response Variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	11	3.2	10
Yield	6.5	3.2	10

 

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 81% to 99% of nominal and so the results are based on nominal test concentrations only.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitatahas been investigated over a 72-Hour period and gave the following

results:

 

Growth Rate: Response Variable  EC50 11 mg/L,  No Observed Effect Concentration (NOEC) 3.2 mg/L,  Lowest Observed Effect Concentration (LOEC) 10 mg/L

Yield: Response Variable  EC50 6.5 mg/L,  No Observed Effect Concentration (NOEC) 3.2 mg/L,  Lowest Observed Effect Concentration (LOEC)  10 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L