Cyclopentene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study could not produce reliable results due to test item phys-chem properties.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Remarks:

MEthods adapted to account for high volatility of test item.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Cyclopentene
Appearance: Clear colourless liquid
Batch: 0000015386
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions until: Unknown
Test Facility test item number: 209393/A
Purity/Composition correction factor: No correction factor required
Stability at higher temperatures: Stable at temperatures << 44°C
Not stable at higher temperatures, potential to polymerize and form peroxides
Chemical name (IUPAC, synonym or trade name: Cyclopentene
CAS number: 142-29-0
EC number: 205-532-9

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=72 h.
Volume: 1.0 mL from the approximate centre of the test vessels.
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

At the end of the exposure period, the replicates with algae were not pooled at each test concentration before sampling. Instead, samples of the control and the limit concentration were taken from separate replicates. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at nominally 100 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Measurements and Recordings
pH: At the beginning and at the end of the test, for the limit concentration and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the limit test, microscopic observations were performed on the 100 mg/L test concentration and the control to observe for any abnormal appearance of the algae.

Vehicle:

no

Details on test solutions:

Preparation of test solutions

The batch of Cyclopentene tested was a clear colourless liquid with a purity of 98.9% and visually completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest concentration of 100 mg/L applying a period of 14-15 minutes of magnetic stirring to accelerate dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. Due to the volatile nature of the test item, the preparation of test solutions was performed in closed vessels with reduced headspace.

After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.


Range finding test
Nominal test item concentrations: 0, 0.10, 1.0, 10 and 100 mg/l.

Limit test
Nominal test item concentrations: 0 and 100 mg/l

Test organisms (species):

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

Details on test organisms:

Species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture: Algae stock cultures are started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505, formulated using Milli-RO water and with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture are inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture is maintained under the same conditions as used in the test. The cell density is measured immediately before use.
Pre-culture medium: Adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 300 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 7.1 ± 0.3
HEPES buffer 6 mmol/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21-24°C

pH:

7.1 ± 0.3

Details on test conditions:

Range-Finding Test
A range-finding test was performed to provide information about the range of concentrations to be used in the limit test. Test procedure and conditions were similar to those applied in the limit test with the following exceptions:
• Three replicates of exponentially growing algal cultures were exposed to a range of nominally 0.10 to 100 mg/L increasing by a factor of 10 and to a control.
• Cell densities were recorded only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration.
• At the end of the test, algae were not observed under a microscope to verify a normal and healthy appearance.

Limit test
Cyclopentene: 100 mg/L.
Control: Test medium without test item or other additives.
Replicates: 6 replicates each of the control and the limit concentration,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
2 replicates of the limit concentration without algae.

Test Procedure and Conditions
Test duration: 72 hours
Test type: Static
Test vessels: 40 mL, airtightly closed with minimal headspace to prevent loss of the test item due to volatilization.
Test Medium: Adjusted M2 (see paragraph 4.7.)
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 86 to 87 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

not determinable

Details on results:

Analytical method validation

Specificity: Several small peaks and one large peak were observed in the chromatogram of the QC sample. It was assumed that the large peak derives from the major component of the test item. The peak area of the major component was used as response in the calculations. A small response at the retention time of the test item was detected in the chromatogram of the blank QC sample. The contribution of the response to the LOQ level (see ‘8.5.4 Limit of quantification’) was 15%. Since this value was < 30%, the specificity requirements were met and the analytical method was found to be specific for the test item.

Calibration Curve: There was a linear relationship between response and test item concentration in the range of 0.0500 – 10.0 mg/L (in end solution). Since the coefficient of correlation (r) was > 0.99 and the back calculated accuracies of the data points were in the range 85-115% the calibration line was accepted. An additional calibration curve in the concentration range of 0.0500 – 12.0 mg/L was prepared and analyzed to perform the research on the storage stability of samples and on additional QC at 10 mg/L concentration level. The curve was similar to the validation curve and fell within the criteria ranges.

Accuracy and Repeatability: Since the mean accuracy at concentration levels between 0.1 and 10 mg/L fell in the criterion 70-110% and the coefficient of variation was ≤ 20% the analytical method was accepted for the analysis of the test item in water in the target concentration range of 0.1 – 10 mg/L. Additional QC samples in ISO-medium and M2-medium were analyzed. The mean accuracies for QC samples spiked in the range of 0.1-10 mg/l were in the range of 70-110%. The analytical method was accepted for the support of ecotoxicological studies.

Limit of Quantification: The limit of quantification (LOQ) was assessed at 0.1 mg/L in water.

Stability of the Analytical System and End Solutions: For 0.1 mg/L QC samples the analyzed concentrations were between 0.05 and 0.15 mg/L concentration level, which were stable, (the coefficient of variation of the 0.05 mg/L concentration level was slightly above 20%). For the 10 mg/L QC samples the analyzed concentration were between 0.15 and 12 mg/L and the coefficient of variation of the 12 mg/L was slightly above 20%. Therefore the results are accepted and the analytical system and end solutions were considered stable over at least a 7.45 hour time interval.

Stability of Stock Solutions: The coefficient of variation on the response factors of the calibration solutions prepared with fresh and stored stock solutions was 11%. Since the value was ≥ 10% the stock solutions were not stable when stored at room temperature for at least 1 days.

Storage Stability of Samples: Since the mean accuracy of the frozen QC samples fell outside the criterion 70-110% the samples were not stable when stored in the freezer (≤ -15°C) due to volatility and should be analyzed fresh.

Range-Finding Test
No inhibition of algal growth rate or yield was found at the three lowest test concentrations while slight inhibition of algal growth was observed at nominally 100 mg/L.
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were at 4.8-8.3% of the nominal concentrations. At the end of the exposure period, the concentrations had decreased to 31-80% of the initial concentrations. All test conditions were maintained within the limits prescribed by the study plan.

Limit test
Samples taken from the limit concentration and the control were analysed. The measured concentrations at the start of the test were 0.42 and 1.1 mg/L, respectively, in the samples taken from vessels with and without algae. The concentrations measured in the samples incubated with algae were 0.036 and 0.33 mg/L after 24 and 72 hours of exposure, respectively. As the concentration measured at t=24h was about 10 times lower than at t= 0 and 72 h (with algae), this concentration was excluded from the calculations of an average exposure concentration. Note that concentrations at 24 hours were measured in a separate vessel. It is most probable that volatilisation of the test item occurred from this test vessel and an unidentified stage of the test. The concentrations measured in the samples without algae were 0.44 and 0.16 mg/L after 24 and 72 hours of exposure, respectively.

The concentrations measured at the start of the limit test were noticeably lower than at the start of the range-finding test.

Conclusions

Both of these tests failed to produce consistent measured v nominal concentration values, therefore the test solution preparation is not appropriate for this test substance and is insufficient to get reproducible and reliable results.

As shown in the analytical validation analysis, analytical confirmation of actual concentrations in the test is unreliable at the higher doses. It is unclear from the results if the concentrations measured were influenced by the need to dilute the samples to within the calibration range of the method or, if this is a product of losses during the preparation or sampling stages of the test.

Under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of Cyclopentene tested.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded an Average concentration of 0.38 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was beyond the range tested, i.e. exceeded an average concentration of 0.38 mg/L.
The 72h-NOEC for growth rate inhibition was 0.38 mg/L (average concentration).
The 72h-NOEC for yield inhibition was 0.38 mg/L (average concentration).

However, this analytical result is not reliable enough to draw full conclusions and actual concentrations may have been much higher before dilution during analysis.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate and yield of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.

Analytical method validation

Accuracy and Repeatability

QC water accuracy and repeatability

Concentration [mg/L]			Accuracy [%]		Coefficient of variation [%]
Target	Nominal	Analyzed	Individual	Mean
0.1	0.100	0.0961	96	92	7.9
	0.100	0.0832	83
	0.100	0.0923	92
	0.100	0.102	102
	0.100	0.0881	88
10	10	8.13	81	80	7.6
	10	7.83	78
	10	7.73	77
	10	7.42	74
	10	9.01	90
100	100	11.7	12	7.0	39
	100	6.98	7.0
	100	5.92	5.9
	100	5.76	5.8
	100	4.79	4.8
100	100	6.15	6.2	6.5	15
	100	7.68	7.7
	100	6.93	6.9
	100	6.60	6.6
	100	5.06	5.1

QC Samples Alternative Matrices

Matrix	Concentration [mg/L]			Accuracy [%]
	Target	Nominal	Analyzed	Individual	Mean
ISO-medium	0.1	0.100	0.100	100	92
		0.100	0.0846	85
	10	10.0	9.54	95	88
		10.0	8.10	81
	100	100	6.38	6.4	6.9
		100	7.35	7.3
	100	100	4.12	4.1	5.3
		100	6.42	6.4
M2-medium	0.1	0.100	0.0810	81	85
		0.100	0.0888	89
	10	10.0	7.17	72	75
		10.0	7.76	78
	100	100	8.92	8.9	7.2
		100	5.55	5.6
	100	100	4.98	5.0	5.0
		100	5.01	5.0

Range finding test

Mean Cell Densities (x10⁴Cells/mL) during the Range-Finding Test

Time (h)	Cyclopentene; Nominal conc. (mg/L)
	Control	0.10	1.0	10	100
0	1.0	1.0	1.0	1.0	1.0
72	210.5	205.5	207.1	209.0	186.8

Percentage Inhibition of Growth Rate during the Range-Finding Test

Cyclopentene Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.783	0.0097	3
0.10	1.775	0.0015	3	0.45
1.0	1.778	0.0107	3	0.31
10	1.781	0.0058	3	0.13
100	1.743	0.0154	3	2.2

Percentage Inhibition of Yield during the Range-Finding Test

Cyclopentene Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	209.5	6.15	3
0.10	204.5	0.95	3	2.4
1.0	206.1	6.71	3	1.6
10	208.0	3.64	3	0.72
100	185.8	8.53	3	11

Limit test

Average Versus Nominal Concentrations

Cyclopentene Average conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.773	0.0266	6
0.38	1.765	0.0204	6	0.45

¹without algae; * not used in further calculations of average exposure concentrations.

Growth Rate and Percentage Inhibition for the Total Test Period

Cyclopentene Average conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.773	0.0266	6
0.38	1.765	0.0204	6	0.45

Growth Rate and Percentage Inhibition at Different Time Intervals 

Cyclopentene Average conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.653		1.792		1.875
0.38	3	1.622	1.8	1.772	1.1	1.902	-1.4

Yield and Percentage Inhibition for the Total Test Period

Cyclopentene Average conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	203.792	15.8051	6
0.38	198.762	12.4037	6	2.5

Effect Parameters

Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	0.38	>0.38	>0.38	>0.38
Yield	0.38	>0.38	>0.38	>0.38

Validity criteria fulfilled:

yes

Conclusions:

A full OECD 201 toxicity of cyclopentene to freshwater algae has been conducted with adaptations to account for the volatility of the substance. The results were inconclusive as they could not achieve consistent measured concentrations and the biological data was inconclusive. It is therefore concluded that these tests are technically not feasible and any data produced contains too much uncertainties to draw an adequate conclusion.

Executive summary:

A full OECD 201 toxicity of cyclopentene to freshwater algae has been conducted with adaptations to account for the volatility of the substance. The results were inconclusive as they could not achieve consistent measured concentrations and the biological data was inconclusive. It is therefore concluded that these tests are technically not feasible and any data produced contains too much uncertainties to draw and conclude toxicity of the substance.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

A full justification pertaining to this waiver is reported in section 13.

Description of key information

A full guideline OECD 201  toxicity to algae study has been conducted on cyclopentene.This study was adapted to account for the volatility of the test item, to prevent losses during testing.
The results of this study were inconclusive because of significant uncertainties in the reproducibility and reliability of the test media preparation and in the chemical analysis.
As such, it has been concluded that ecotoxicity studies of this nature are technically not feasible and hazard to the aquatic environment is unlikely. Further details are provided in Section 13 of this Dossier.

 

Additional information

The method of chemical analysis is not deemed suitable for concentrations in the media or water at dosed concentrations of >10 mg/l.
Any measured concentrations above 10 mg/l are concluded to be unreliable and any samples dosed at concentrations >10 mg/l cannot be reliably reproduced.
Full tests have been performed on algae and daphnia according to the OECD guidelines with adaptations to account for the volatility of the substance.
The results were inconclusive as they could not achieve consistent measured concentrations and the biological data was inconclusive.
It is therefore concluded that these tests are technically not feasible and any data produced contains too much uncertainties to draw an adequate conclusion.