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

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: 2 June 2014 Experimental Completion Date: 17 June 2014

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:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. The test item was initially dissolved in dimethylformamide at a concentration of 100 mg/10 mL. An aliquot of this solvent stock solution was then dispersed in an appropriate volume of test media with the aid of magnetic stirring for approximately 10 minutes prior to removing any undissolved test item present by filtration (0.2 µm Gelman Acrocap, first approximate 500 mL discarded in order to pre-condition the filter) to give a nominal concentration of 1.0 mg/L.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

BMS-589152-01

IUPAC Name:

BMS-589152-01

Test material form:

solid: particulate/powder

Details on test material:

Identification: BMS-589152-01
Physical state/Appearance: Tan-colored powder
Batch: 2G72631N
Purity: 99.5%
Expiry date: 19 June 2014
Storage conditions: Room temperature in the dark
Intended use/Application: Chemical intermediate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentrations:
1 mg/l defintiive test group

Sampling method:
Samples were taken from the solvent control and the 1.0 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Given the unstable nature of the test item under test conditions, additional test samples were prepared at the start of the test for analysis after 24 and 48 hours. All 0, 24 and 48-Hour test samples were stored frozen prior to analysis alongside the 72-Hour test samples.

Sample storage conditions before analysis:
The 0, 24 and 48-Hour test samples were stored frozen prior to analysis alongside the 72-Hour test samples. All work was carried out under non-actinic light and amber vials used. Duplicate samples were taken.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
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. A test concentration of 1.0 mg/L (by visual inspection) was obtained using a preliminary solution in dimethylformamide.

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. For more information regarding the media preparation trial, please see the section details on analytical methods (which can be found above).

EXPERIMENTAL PREPARATION:
An amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (200 µL) of this solvent stock solution was dispersed in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 1.0 mg/L stock solution. An aliquot (1 liter) of this stock solution was inoculated with 17.9 mL of algal suspension to give the required nominal test concentration of 1.0 mg/L.

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

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting whilst all sample bottles were foil wrapped.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours (for more information on this please see the section above, details on analytical methods).

Test organisms

Test organisms (species):

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

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.

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 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E4 - 10E5 cells/mL.

ACCLIMATION
- Acclimation period: Not recorded.

- Any deformed or abnormal cells observed: None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test, control and solvent control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded

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 value of the control cultures (please see table 2, which can be found in the any other information on results section) was observed to increase from pH 7.6 at 0 hours to pH 8.3 at 72 hours whilst the pH of the solvent control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not recorded.

Salinity:

Not applicable as this is a fresh water study

Nominal and measured concentrations:

Range finding study - Nominal concentrations used: These were 0.010, 0.10 and 1.0 mg/L

Definitive study - Nominal concentration used: 1mg/L

Definitive study - Time weighted mean measured concentration: 0.33 mg/L

Details on test conditions:

TEST SYSTEM
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control, solvent control and 1.0 mg/L treatment group.

The control group was maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.80 x 105 cells per mL. Inoculation of 1 liter of test medium with 17.9 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.

CULTURE MEDIUM
- Standard medium used: yes
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 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 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 with 0.1N NaOH or HCl.


OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Yes
- Photoperiod: under constant illumination
- Light intensity and quality: warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples were taken at 0, 24, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test, control and solvent control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

For more information, please see the evaluation of data section within the additional information on methods section

- Chlorophyll measurement:
Not recorded

RANGE FINDING STUDY:
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.75 mg/L could be obtained using a solvent spike solution method of preparation.

The test concentration to be used in the definitive test was 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 and 1.0 mg/L for a period of 72 hours.

An amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (100 µL) of this solvent stock solution was dispersed in 1 liter of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 475 mL discarded in order to pre-condition the filter) to give a 1.0 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 0.10 and 0.010 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (6.4 mL) to give the required nominal test concentrations of 0.010, 0.10 and 1.0 mg/L.

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting whilst all sample bottles were foil wrapped.

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, solvent control and test concentration.

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 frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Based on the results of the range-finding test, a "limit test" was conducted at a nominal concentration of 1.0 mg/L to confirm that at the highest attainable test concentration, no effect on algal growth was observed.

POSITIVE CONTROL:
A positive control (Harlan Study Number 41303826) used potassium dichromate as the reference item. The positive control was conducted between 21 October 2013 and 24 October 2013. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

VALIDATION:
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%.

VERIFICATION OF TEST CONCENTRATIONS:
Samples were taken from the solvent control and the 1.0 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Given the unstable nature of the test item under test conditions, additional test samples were prepared at the start of the test for analysis after 24 and 48 hours. All 0, 24 and 48-Hour test samples were stored frozen prior to analysis alongside the 72-Hour test samples.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

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 results showed no effect on growth at the test concentrations of 0.010, 0.10 and 1.0 mg/L.

Based on this information a single test concentration of six replicates, of 1.0 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Chemical analysis of the 1.0 mg/L test preparations at 0 and 72 hours showed measured test concentrations of 0.74 and 0.47 mg/L respectively were obtained indicating that the test item was unstable over the test duration.

Table 1 can be found in the any other information on results section.

Definitive test:
Analysis of the 1.0 mg/L test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.60, 0.36, 0.20 and 0.32 mg/L respectively were obtained. Given the decline in measured test concentration over the test duration it was considered appropriate to estimate the results based on the time-weighted mean measured test concentration in order to a “worst case” analysis of the data.

The time-weighted mean measured test concentration was determined to be:

Nominal Concentration (mg/L) (TWMM) Concentration (mg/L) Expressed as a % of the 0-Hour Measured Test Concentration
1.0 0.33 55

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.

Tables 1-4 can be found in the any other information on results section (including tables).

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 not affected by the presence of the test item at a time-weighted mean measured test concentration of 0.33 mg/L over the 72-hour exposure period.

The test concentration of 0.33 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in test media and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline. Accordingly the following results were determined from the data:

Inhibition of Growth Rate:
ErC50 (0 - 72 h) : >0.33 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 solvent control and 0.33 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P≥0.05), between the solvent control and 0.33 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.33 mg/L.

Inhibition of Yield:
EyC50 (0 - 72 h) : >0.33 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P≥0.05), between the solvent control and 0.33 mg/L test group and therefore the NOEC based on yield was 0.33 mg/L.

Observations on Cultures:
All test, control and solvent control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control, solvent control or test cultures.

Observations on Test Item Solubility:
At the start of the test all control, solvent control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, solvent control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41303826) 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) : 1.1 mg/L, 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L, 95% confidence limits 0.45 – 0.59 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:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the solvent control and the 1.0 mg/L test concentration to determine any statistically significant differences between the test and solvent control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

Table1     Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells permL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.47E+03	7.86E+05	-	-
	R2	5.14E+03	1.08E+06
	Mean	5.30E+03	9.33E+05
Solvent Control	R1	6.54E+03	1.02E+06	-	-
	R2	5.15E+03	9.19E+05
	Mean	5.85E+03	9.69E+05
0.010	R1	5.58E+03	1.11E+06	[3]	[16]
	R2	5.94E+03	1.13E+06
	Mean	5.76E+03	1.12E+06
0.10	R1	4.74E+03	1.14E+06	[6]	[14]
	R2	5.14E+03	1.06E+06
	Mean	4.94E+03	1.10E+06
1.0	R1	5.15E+03	1.10E+06	[3]	[6]
	R2	5.86E+03	9.44E+05
	Mean	5.50E+03	1.02E+06

 

*    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.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table2     Cell Densities and pH Values in the Definitive Test

Time-Weighted Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells permL)				pH
		0 h	0 h	24 h	49 h	72 h	72 h
Control	R1	7.6	6.30E+03	2.54E+04	1.17E+05	5.10E+05	8.3
	R2		5.63E+03	2.47E+04	1.06E+05	5.42E+05
	R3		5.78E+03	2.43E+04	1.21E+05	5.75E+05
	R4		5.54E+03	2.32E+04	1.03E+05	5.58E+05
	R5		6.02E+03	2.42E+04	1.08E+05	5.56E+05
	R6		6.58E+03	2.56E+04	8.70E+04	5.23E+05
	Mean		5.97E+03	2.46E+04	1.07E+05	5.44E+05
Solvent Control	R1	7.6	6.30E+03	2.32E+04	1.16E+05	5.24E+05	8.2
	R2		5.87E+03	2.33E+04	1.25E+05	5.79E+05
	R3		5.39E+03	2.75E+04	1.25E+05	5.90E+05
	R4		5.54E+03	2.42E+04	1.06E+05	4.77E+05
	R5		6.05E+03	2.33E+04	1.03E+05	5.80E+05
	R6		5.57E+03	2.59E+04	1.22E+05	5.76E+05
	Mean		5.79E+03	2.46E+04	1.16E+05	5.54E+05
0.33	R1	7.6	5.50E+03	2.61E+04	9.24E+04	5.84E+05	8.0
	R2		6.02E+03	2.39E+04	9.84E+04	5.22E+05
	R3		5.60E+03	2.26E+04	8.92E+04	6.06E+05
	R4		5.94E+03	2.40E+04	1.03E+05	5.68E+05
	R5		5.78E+03	2.45E+04	7.01E+04	5.77E+05
	R6		5.52E+03	2.12E+04	9.93E+04	5.82E+05
	Mean		5.73E+03	2.37E+04	9.21E+04	5.73E+05

 

*    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

Table3     Daily Specific Growth Rates for the Control and Solvent Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.077	0.061	0.064
	R2	0.076	0.058	0.071
	R3	0.075	0.064	0.068
	R4	0.073	0.060	0.073
	R5	0.075	0.060	0.071
	R6	0.077	0.049	0.078
	Mean	0.076	0.059	0.071
Solvent Control	R1	0.073	0.064	0.066
	R2	0.073	0.067	0.067
	R3	0.080	0.061	0.067
	R4	0.075	0.059	0.066
	R5	0.073	0.060	0.075
	R6	0.078	0.062	0.067
	Mean	0.075	0.062	0.068

R₁- R₆= Replicates 1 to 6

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

Time-Weighted Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition
Control	R1	0.067		5.04E+05
	R2	0.068		5.36E+05
	R3	0.069		5.69E+05
	R4	0.069	-	5.53E+05	-
	R5	0.069		5.50E+05
	R6	0.068		5.16E+05
	Mean	0.068		5.38E+05
	SD	0.001		2.44E+04
Solvent Control	R1	0.068		5.18E+05
	R2	0.069		5.73E+05
	R3	0.069		5.85E+05
	R4	0.066	-	4.71E+05	-
	R5	0.069		5.74E+05
	R6	0.069		5.71E+05
	Mean	0.068		5.49E+05
	SD	0.001		4.46E+04
0.33	R1	0.069	[1]	5.78E+05	[5]
	R2	0.068	0	5.16E+05	6
	R3	0.070	[3]	6.00E+05	[9]
	R4	0.069	[1]	5.62E+05	[2]
	R5	0.069	[1]	5.71E+05	[4]
	R6	0.069	[1]	5.76E+05	[5]
	Mean	0.069	[1]	5.67E+05	[3]
	SD	0.001		2.79E+04

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to the solvent control]

Analytical investigations: Results:

Results for Spiked Recovery Samples

Nominal Concentra­tion of Test Item cnom	Fortified Concentra­tion of Test Item in the Spiked Sample cfort	Measured Concentra­tion of Test Item in the Sample Vial x	Sample Preparation Factor F	Determined Concentra­tion of Test Item in the Spiked Sample c	Mean Analytical Recovery Rate R	Precision (Relative Standard Deviation of Recovery)
[mg/L]	[mg/L]	[mg/L]		[mg/L]	[%]	[%]
1.0	0.992	0.459	2	0.917	93	0.58
	0.992	0.462	2	0.924
	0.992	0.464	2	0.928
	0.992	0.465	2	0.930
	0.992	0.464	2	0.927
Acceptance Target					80-120	<10

For reporting purposes, x has been calculated from c retrospectively.

Results for Test Samples

Time point	Nominal Concentration of Test Item in Test Sample cnom	Measured Concentration  of Test Item in Sample Vial x	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample   c	% of Nominal Concentration
[hours]	[mg/L]	[mg/L]		[mg/L]	[%]
0	Control	<LOQ	2	<LOQ	-
	1.0	0.299	2	0.598	60
24	1.0	0.178	2	0.356	36
48	1.0	0.0985	2	0.197	20
72	Control	<LOQ	2	<LOQ	-
	1.0	0.162	2	0.323	32

 

LOQ                      =             Limit of Quantification

-                              =             not applicable

For reporting purposes, x has been calculated from c retrospectively.

Discussion:

The detection system was found to have acceptable linearity. The analytical procedure was found to have acceptable recoveries of test item in test medium. The method of analysis was validated and proven to be suitable for use.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 0.33 mg/L based on the time-weighted mean measured test concentrations. The NOEC was 0.33 mg/L.

This study showed that there were no toxic effects at the limit of solubility.

Executive summary:

Introduction:

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

  

Methods:

Information provided by the Sponsor indicated the test item was insoluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.75 mg/L was obtained from a solvent spike 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 a solution of the test item at a nominal concentration of 1.0 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solution was prepared by dispersing 200µLof a 100 mg/10 mL solvent stock solution in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes. After stirring any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter).

 

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting.

 

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:

Analysis of the 1.0 mg/L test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.60, 0.36, 0.20 and 0.32 mg/L respectively. Given the decline in measured test concentration over the test duration it was considered appropriate to estimate the results based on the time-weighted mean measured test concentration in order to assume a “worst case” analysis of the data.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values based on the time-weighted mean measured test concentration of greater than 0.33 mg/L. The No Observed Effect Concentration was determined to be 0.33 mg/L.

This study showed that there were no toxic effects at the limit of water solubility.