2-Propenoic acid, 2-methyl-, hydroxybutyl 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: 29 October 2012 Experimental Completion Date: 17 January 2013

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

Report date:

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

Test material form:

liquid

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:

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 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 4.

Test solutions

Vehicle:

no

Details on test solutions:

Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to
reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was
separately inoculated with algal suspension (3.6 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.
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 range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.


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

Amounts of test item (100 and 32 mg) were each separately dissolved in culture medium and the volumes adjusted to 1 liter to give 100 and 32 mg/L stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L.
An aliquot (900 mL) of each of the stock solutions was separately inoculated with 3.5 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.

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

The concentration and stability of the test item in the test samples were verified by chemical analysis at 0 and 72 hours (see attached Appendix 4).

Test organisms

Test organisms (species):

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

Details on test organisms:

Test Species
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master
cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and 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 Study Number: 41205049) used potassium dichromate as the reference item. Details of the positive control are given in
the attached Appendix 2.


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 3.

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 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 (see Table 2) was observed to increase from pH 7.7 at 0 hours to pH 8.1 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:

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: 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.27 x 106 cells per mL. Inoculation of
900 mL of test medium with 3.5 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, 26, 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.

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 3.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (see Appendix 2)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: 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 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and
100 mg/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours (see the attached Appendix 4) showed measured test concentrations to
range fromless than the limit of quantitation (LOQ) of the analytical method employed to 107% of nominal whilst measured concentrations in the
range of 48% to 104% of nominal were obtained at 72 hours. Examination of the data could show no cause for the low measured test concentrations obtained from the 1.0 and 10 mg/L test preparations. However, it was considered, based on the results from the 100 mg/L test preparations that the test item was stable over the test period.


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.


Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 96 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 : 6.62 x 103 cells per mL
Mean cell density of control at 72 hours : 6.34 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% 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 1% 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) : >100 mg/L
ErC20 (0 - 72 h) : >100 mg/L
ErC50 (0 - 72 h) : >100 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, 1.0, 3.2 and 10 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 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/L.


Inhibition of Yield

EyC10 (0 - 72 h) : >100 mg/L
EyC20 (0 - 72 h) : >100 mg/L
EyC50 (0 - 72 h) : >100 mg/L*

Where:

EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences between the control, 1.0, 3.2 and 10 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 10 mg/L.
Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 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 1.0, 3.2, 10 and 32 mg/L, however misshapen cells and cell debris 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 colorless solutions. After the 72-Hour test period all control and test
cultures were observed to be 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 7.7 at 0 hours to pH 8.1 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 the attached Appendix 4) showed measured test concentrations to range from 99% to 138%. Examination of the data could show no cause for the slightly higher than nominal measured concentrations obtained for the 3.2 and 32 mg/L test
preparations at 0 and 72 hours. Given that all other concentrations were within 80-120% of nominal it was considered appropriate to calculate the
results based on nominal test concentrations only.

Results with reference substance (positive control):

Appendix 2 Positive Control
A positive control (Harlan Study Number: 41205049) 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 1.0 – 1.3 mg/L
EyC50 (0 – 72 h) : 0.70 mg/L*

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 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:

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, 1.0, 3.2 and 10 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 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/L.

There were no statistically significant differences between the control, 1.0, 3.2 and 10 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 10 mg/L.
Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 mg/L.

Any other information on results incl. tables

Tables

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	6.93E+03	6.06E+05	-	-
	R2	7.55E+03	6.27E+05
	Mean	7.24E+03	6.17E+05
0.10	R1	5.63E+03	6.72E+05	[6]	[10]
	R2	5.99E+03	6.82E+05
	Mean	5.81E+03	6.77E+05
1.0	R1	7.17E+03	6.25E+05	5	13
	R2	7.73E+03	4.47E+05
	Mean	7.45E+03	5.36E+05
10	R1	6.67E+03	4.54E+05	8	30
	R2	7.63E+03	4.17E+05
	Mean	7.15E+03	4.35E+05
100	R1	8.14E+03	3.48E+05	15	39
	R2	9.11E+03	4.15E+05
	Mean	8.63E+03	3.81E+05

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

R₁ and R₂ = Replicates 1 and 2

[Increase in growth compared to controls]

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	26 h	48 h	72 h	72 h
Control	R1	7.7	6.98E+03	3.80E+04	1.74E+05	6.62E+05	8.1
	R2		6.50E+03	2.78E+04	1.32E+05	6.36E+05
	R3		8.02E+03	2.70E+04	1.43E+05	7.42E+05
	R4		6.50E+03	2.88E+04	1.20E+05	5.92E+05
	R5		6.96E+03	4.26E+04	9.25E+04	5.79E+05
	R6		4.80E+03	2.71E+04	1.29E+05	5.92E+05
	Mean		6.62E+03	3.19E+04	1.32E+05	6.34E+05
1.0	R1	7.6	7.04E+03	2.99E+04	1.12E+05	6.05E+05	8.2
	R2		5.41E+03	2.42E+04	1.17E+05	6.01E+05
	R3		6.21E+03	2.69E+04	9.99E+04	7.05E+05
	Mean		6.22E+03	2.70E+04	1.10E+05	6.37E+05
3.2	R1	7.6	7.87E+03	3.18E+04	1.07E+05	4.91E+05	8.2
	R2		7.63E+03	2.58E+04	8.99E+04	5.75E+05
	R3		6.64E+03	2.34E+04	1.03E+05	5.02E+05
	Mean		7.38E+03	2.70E+04	9.99E+04	5.23E+05
10	R1	7.5	8.24E+03	2.34E+04	1.24E+05	5.77E+05	8.1
	R2		8.43E+03	2.62E+04	9.78E+04	4.58E+05
	R3		6.85E+03	2.24E+04	1.08E+05	5.82E+05
	Mean		7.84E+03	2.40E+04	1.10E+05	5.39E+05
32	R1	7.5	5.36E+03	2.27E+04	9.87E+04	5.42E+05	8.1
	R2		6.90E+03	2.08E+04	9.12E+04	4.23E+05
	R3		5.66E+03	2.12E+04	1.02E+05	5.21E+05
	Mean		5.97E+03	2.16E+04	9.72E+04	4.95E+05
100	R1	7.5	6.66E+03	2.55E+04	9.51E+04	4.97E+05	8.1
	R2		6.46E+03	2.34E+04	9.89E+04	5.04E+05
	R3		7.65E+03	2.57E+04	7.38E+04	4.28E+05
	Mean		6.92E+03	2.49E+04	8.93E+04	4.76E+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

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	0.078	0.069	0.056
	R2	0.066	0.071	0.066
	R3	0.065	0.076	0.069
	R4	0.067	0.065	0.066
	R5	0.082	0.035	0.076
	R6	0.065	0.071	0.063
	Mean	0.070	0.065	0.066

 

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	0.068		6.55E+05
	R2	0.067		6.30E+05
	R3	0.069		7.34E+05
	R4	0.066	-	5.85E+05	-
	R5	0.066		5.72E+05
	R6	0.066		5.87E+05
	Mean	0.067		6.27E+05
	SD	0.001		6.12E+04
1.0	R1	0.067	0	5.98E+05
	R2	0.067	0	5.96E+05
	R3	0.069	[3]	6.99E+05
	Mean	0.068	[1]	6.31E+05	[1]
	SD	0.001		5.91E+04
3.2	R1	0.064	4	4.83E+05
	R2	0.066	1	5.67E+05
	R3	0.064	4	4.96E+05
	Mean	0.065	3	5.15E+05	18
	SD	0.001		4.53E+04
10	R1	0.066	1	5.69E+05
	R2	0.063	6	4.50E+05
	R3	0.066	1	5.75E+05
	Mean	0.065	3	5.31E+05	15
	SD	0.002		7.07E+04
32	R1	0.065	3	5.36E+05
	R2	0.062	7	4.16E+05
	R3	0.065	3	5.15E+05
	Mean	0.064	4	4.89E+05	22
	SD	0.002		6.43E+04
100	R1	0.064	4	4.90E+05
	R2	0.064	4	4.98E+05
	R3	0.062	7	4.20E+05
	Mean	0.063	5	4.69E+05	25
	SD	0.001		4.30E+04

 

*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁- R₆ = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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

Response Variable EC50 (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate >100* 10 32
Yield >100* 10 32

* It was not possible to calculate EC50 values as no concentration tested resulted in greater than 50% inhibition of growth.

Executive summary:

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 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 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

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

 

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

 

Response Variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>100*	10	32
Yield	>100*	10	32

 

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 99% to 138% of nominal and so the results are based on nominal test concentrations only.

 

General Information

*It was not possible to calculate EC₅₀ values as no concentration tested resulted in greater than 50% inhibition of growth.