Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

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 start date 16 February 2021
Experimental completion date 02 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

EC Number:

243-497-1

EC Name:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

Cas Number:

20073-13-6

Molecular formula:

C13H20O3

IUPAC Name:

methyl 2-[(1S,2S)-3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl]acetate

Specific details on test material used for the study:

Identification: Methyljasmonate
CAS Number: 39924-52-2
Batch: 0900175
Purity: 99.03%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 25 March 2023
Storage Conditions: Approximately 4 °C in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range finding test
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 analyzed on the day of sampling. Duplicate samples were taken and stored frozen for further analysis if required.

Definitive Test
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis.
Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary. In order to prove sufficient volume for duplicate samples at 72 hours, an additional aliquot of each test concentration was incubated alongside the test.

Test solutions

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 L of ASTM
media with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or
48 hours. After stirring samples were taken for chemical analysis after the following
pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 100 mL used to
pre-condition the filter was discarded)
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 500 mL used to
pre-condition the filter was discarded)
Discussion
The results obtained indicated that the test item was soluble in culture medium with the aid of
prolonged stirring. As a precautionary measure, following stirring any undissolved test item
which may remain is to be removed by filtration through a 0.2 μm Gelman Acrocap filter
(first approximate 500 mL discarded in order to precondition the filter).

Range finding test
A nominal amount of test item (1100 mg) was dispersed in 11 L of culture medium with the
aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring
was stopped and, as a precautionary measure, any undissolved test item was removed by
filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL used to
pre-condition the filter was discarded) to give a 100 mg/L stock solution. A series of
dilutions were made from this saturated solution to give further stock solutions of 10, 1.0 and
0.10 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated
with algal suspension (7.0 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solutions and each prepared concentration were inverted several times to ensure
adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (1100 mg) was dispersed in 11 L of culture medium with the
aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring
was stopped and, as a precautionary measure, any undissolved test item was removed by
filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL used to
pre-condition the filter was discarded) to give a 100 mg/L stock solution. A series of
dilutions were made from this stock solution to give stock solutions of 32, 10, 3.2 and
1.0 mg/L. An aliquot (1.0 L) of each of the stock solutions was separately inoculated with
3.7 mL of algal suspension to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solutions and each prepared concentration were inverted several times to ensure
adequate mixing and homogeneity.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures
of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and
Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll,
Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up
at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with
polyurethane foam stoppers and kept under constant agitation by orbital shaker
(approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density
was approximately 10^5 to 10^6 cells/mL.
A positive control test using potassium dichromate as the reference item was performed twice
in a 12 month period to demonstrate satisfactory conditions of the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24 ±1 ºC throughout the test

pH:

7.6 - 7.7 at 0 hours
7.8 - 9.2 at 72 hours

Nominal and measured concentrations:

Range finding test
Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L
Measure at 0 hours: 0.110, 1.05, 9.29, 98.4 mg/L

Definitive test
Nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
Measure at 0 hours: 1.11, 3.27, 10.7, 31.9, 111 mg/L

Details on test conditions:

Range-Finding Test
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 incubator) at
24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm
white lighting (380 to 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 hemocytometer and light
microscope.
It was not possible to monitor algal growth at 72 hours using a Coulter® Multisizer Particle Counter due to a technical issue.
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
analyzed on the day of sampling. Duplicate samples were taken and stored frozen for further
analysis if required.

Definitive Test
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 group 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 1.35 x 10^6 cells per mL. Inoculation of 1.0 L of test medium with 3.7 mL of this
algal suspension gave an initial nominal cell density of 5.00 x 10^3 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
incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux)
provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately
150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

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

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentration 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 test preparations at 0 hours showed measured concentrations to range from 0.11 to 98 mg/L (93% to 110% of nominal) and 72 hours showed measured concentrations to range from 0.069 to 97 mg/L (69% to 97% of nominal) indicating that the test item was stable at the concentrations selected for the definitive test under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 100% to 111% of nominal. There was no significant change in the measured concentrations at 72 hours (99% to 104% of nominal) and so the results are based on the nominal test concentrations only.

Inhibition of Growth Rate
Statistical analysis of the growth rate data was carried out for the control and all test
concentrations using a Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm
incorporating Cochran’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal
Distribution. There were no statistically significant differences between the control, 1.0 and
3.2 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 3.2 mg/L. Correspondingly the Lowest Observed Effect
Concentration (LOEC) based on growth rate was 10 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a
factor of 165 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.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 8.26 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control
cultures was 6% 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 to 72 hours) was 1% and hence satisfied the validation criterion given in the
OECD Guideline which states that this must not exceed 7%.

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 and 10 mg/L,
however some slightly misshapen cells were observed to be present in the test cultures at
32 mg/L and no intact cells were observed to be present in the test cultures at 100 mg/L.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless
solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L test cultures were
observed to be green dispersions, whilst the 32 mg/L test cultures were observed to be pale
green dispersions and the 100 mg/L test cultures were observed to remain clear colorless
solutions.

Results with reference substance (positive control):

A positive control (used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 21 December 2020 and 24 December 2020.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following
results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.37 to 0.52 mg/L
No Observed Effect Concentration based on growth rate: 0.125 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Inhibition of Yield

E_yC₁₀ (0 to 72 hour):   6.6 mg/L; 95% confidence limits 2.4 to 18 mg/L

E_yC₂₀ (0 to 72 hour):   11 mg/L; 95% confidence limits 5.2 to 22 mg/L

E_yC₅₀ (0 to 72 hour):   25 mg/L; 95% confidence limits 16 to 39 mg/L

Where E_yC_x is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out for the control and all test concentration using Williams Multiple Sequential t-test Procedure incorporating Trend Analysis by Contrasts, Levene’s test on Variance Homogeneity and Shapiro-Wilk’s test on Normal Distribution.  There were no statistically significant differences between the control and 1.0 mg/L test concentration (P³0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 1.0 mg/L.  Correspondingly the LOEC based on yield was 3.2 mg/L

Water Quality Criteria

Temperature was maintained at 24 ±1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 9.1 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 Guideline

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	6.42E+03	1.03E+06	-	-
	R2	6.01E+03	7.67E+05
	Mean	6.22E+03	8.99E+05
0.10	R1	5.49E+03	8.83E+05	[1]	7
	R2	5.28E+03	7.95E+05
	Mean	5.38E+03	8.39E+05
1.0	R1	5.98E+03	7.17E+05	4	22
	R2	6.28E+03	6.83E+05
	Mean	6.13E+03	7.00E+05
10	R1	5.75E+03	5.60E+05	10	42
	R2	6.19E+03	4.90E+05
	Mean	5.97E+03	5.25E+05
100	R1	5.43E+03	6.65E+03	104	100
	R2	5.19E+03	1.65E+03
	Mean	5.31E+03	4.15E+03

 

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

R       =  Replicate

-        =  Not applicable

[  ]     =  Increase in growth compared to controls

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	23 Hour	48 Hour	72 Hour	72 Hours
Control	R1	7.7	2.34E+04	1.50E+05	7.79E+05	9.1
	R2		2.64E+04	1.77E+05	8.61E+05
	R3		2.54E+04	1.64E+05	8.34E+05
	R4		2.68E+04	1.47E+05	7.04E+05
	R5		2.60E+04	1.67E+05	8.53E+05
	R6		2.54E+04	1.77E+05	9.28E+05
	Mean		2.56E+04	1.64E+05	8.26E+05
1.0	R1	7.7	2.26E+04	1.61E+05	7.59E+05	9.2
	R2		2.26E+04	1.79E+05	8.64E+05
	R3		2.31E+04	1.72E+05	7.81E+05
	Mean		2.27E+04	1.71E+05	8.01E+05
3.2	R1	7.6	2.17E+04	1.51E+05	7.64E+05	9.1
	R2		2.26E+04	1.59E+05	6.59E+05
	R3		2.46E+04	1.66E+05	7.55E+05
	Mean		2.30E+04	1.59E+05	7.26E+05
10	R1	7.6	2.10E+04	1.37E+05	7.36E+05	9.2
	R2		2.10E+04	1.44E+05	6.28E+05
	R3		2.25E+04	1.52E+05	6.77E+05
	Mean		2.15E+04	1.44E+05	6.80E+05
32	R1	7.6	1.53E+04	9.86E+04	3.88E+05	8.9
	R2		1.45E+04	8.76E+04	3.83E+05
	R3		1.40E+04	8.35E+04	2.87E+05
	Mean		1.46E+04	8.99E+04	3.53E+05
100	R1	7.6	9.65E+03	1.55E+04	2.53E+04	7.8
	R2		9.03E+03	1.30E+04	1.66E+04
	R3		7.67+E03	1.32E+04	1.65E+04
	Mean		8.79E+03	1.39E+04	1.95E+04

 

*        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

R       =  Replicate

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.067	0.074	0.069
	R2	0.072	0.076	0.066
	R3	0.071	0.075	0.068
	R4	0.073	0.068	0.065
	R5	0.072	0.074	0.068
	R6	0.071	0.078	0.069
	Mean	0.071	0.074	0.068

 

R       = Replicate

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.070	-	7.74E+05	-
	R2	0.072		8.56E+05
	R3	0.071		8.29E+05
	R4	0.069		6.99E+05
	R5	0.071		8.48E+05
	R6	0.073		9.23E+05
	Mean	0.071		8.21E+05
	SD	0.001		7.69E+04
1.0	R1	0.070	1	7.54E+05
	R2	0.072	[1]	8.59E+05
	R3	0.070	1	7.76E+05
	Mean	0.071	0	7.96E+05	3
	SD	0.001		5.57E+04
3.2	R1	0.070	1	7.59E+05
	R2	0.068	4	6.54E+05
	R3	0.070	1	7.50E+05
	Mean	0.069	2	7.21E+05	12
	SD	0.001		5.82E+04
10	R1	0.069	3	7.31E+05
	R2	0.067	6	6.23E+05
	R3	0.068	4	6.72E+05
	Mean	0.068	4	6.75E+05	18
	SD	0.001		5.43E+04
32	R1	0.060	15	3.83E+05
	R2	0.060	15	3.78E+05
	R3	0.056	21	2.82E+05
	Mean	0.059	17	3.48E+05	58
	SD	0.002		5.72E+04
100	R1	0.023	68	2.03E+04
	R2	0.017	76	1.16E+04
	R3	0.017	76	1.15E+04
	Mean	0.019	73	1.45E+04	98
	SD	0.003		5.05E+03

 

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

R       = Replicate

-        = Not applicable

SD    = Standard Deviation

[  ]     =  Increase in growth compared to controls

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated
over a 72 hour period and based on the nominal test concentrations gave the following results:
Growth Rate
EC50: 64 mg/L (59 - 68 mg/L 95 % confidence limits)
NOEC: 3.2 mg/L
LOEC: 10 mg/L

Yield
EC50: 25 mg/l (16 - 39 mg/L 95 % confidence limits)
NOEC: 1.0 mg/L
LOEC: 3.2 mg/L

Executive summary:

Introduction
A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as 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
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 the test item was soluble in test media with the aid of prolonged stirring. Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to solutions of the test item at nominal 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 atemperature of 24 ±1 °C. The test item  solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL used to pre-condition the filter was discarded) to produce a 100 mg/L
solution of the test item. This 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
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 100% to 111% of nominal. There was no significant change in the measured concentrations at 72 hours (99% to 104% of nominal) and so the results are based on the nominal test concentrations only.
Exposure of Raphidocelis subcapitata to the test item gave the following results based on the nominal test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	64	59	-	68	3.2	10
Yield	25	16	-	39	1.0	3.2