Cyclopentene

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Remarks:

Methods to prevent losses of test item due to volatilisation have been employed.

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:

Sampling for Analysis of Test Concentrations

Frequency: At the start (t=0 h) and the end of the test (t=48 h). If analytical results show that a concentration has decreased below the LOD /LOQ before the end of the test period, no further sampling was needed at that concentration.

Concentrations: Samples for analysis were be taken from all concentrations and the control immediately before transfer to the individual replicates. Care was be taken not to include any floating layer, test item film or undissolved material in separate vessels. At the end of the test samples will be taken from the approximate center of the pooled solutions (provided that solutions are still homogeneous) of the vessels at each concentration. In case of volatile items solutions will not be pooled before sampling. Instead, samples will be taken from at least one replicate.

Number of samples: Sampling will consist of single samples per treatment. Should the analytical validation require duplicate or multiple samples per treatment, this will be followed without prior notification. In case undissolved particles were removed from the test solutions before the start of the test, this residue will be retained for possible analysis.

Volume: Typically, volumes of 2 mL will be taken, but depending on the limit of detection of the analytical method used in relation to the test concentrations the volume may differ.

Storage: If stability of test concentrations under freeze conditions is ensured, the samples will be stored in a freezer (≤-15°C) until analysis. Optionally, samples can be stored under different conditions (e.g. room temp. or in refrigerator (2-8°C)) if stability under these conditions is ensured.

Extra samples: In case single samples are taken, which are known to be stable under the storage conditions, extra samples will be taken from all concentrations and stored for possible analysis until delivery of the final report with a maximum of three months.

Analyses: The entire volume of each sample used for analysis was taken for further dilution or pre-treatment.

Measurements and Recordings
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Vehicle:

no

Details on 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 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. Any residual volumes were discarded.

The following salts and vitamins were added to freshly prepared test medium to reach the following concentrations:
Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L



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

Full test 1
Nominal test item concentrations: 0, 10, 18, 32, 56 and 100 mg/l

Full test 2
Nominal test item concentrations: 0, 10, 18, 32, 56 and 100 mg/l

Medium M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).


Numbers of organisms
Range finding test: 10 daphnia per concentration (5 per vessel)
Full test 1: Nominal test item concentrations: 20 daphnia per concentration (5 per vessel)
Full test 2: Nominal test item concentrations: 20 daphnia per concentration (5 per vessel)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L

Test temperature:

18-22 °C

pH:

6-9

Dissolved oxygen:

≥3 mg/L

Nominal and measured concentrations:

Combined limit/range finding test: nominal 0.10, 1.0, 10 and 100 mg/L (equivalent to 1.9, 2.4, 5.1 and 57 mg/L measured, repsectively)
Main test: nominal 10, 18, 32, 56 and 100 mg/L (equivalent 5.3, 1.9, 1.2, 2.0 and 5.7 mg/L measured, respectively)

Details on test conditions:

Test Procedure and Conditions
Test duration: 48 hours
Test type: Static
Test vessels: All-glass, with sufficient volume to accommodate the organisms. In case of volatile items test vessels will be closed air-tight and the headspace will be reduced to a minimum.
Test medium The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON

Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.

Number of daphnia: 20 per concentration
Loading: 5 per vessel (with at least 2 mL of test solution per daphnia)
Light: 16 hours photoperiod daily; should the test item be light sensitive, the test will be performed in the dark.
Temperature: 18-22°C, constant within ±1°C.
Oxygen concentration ≥ 3 mg/L at the end of the test.
pH: Between 6 and 9. Should normally not vary by more than 1.5 unit.
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnia: Daphnia are introduced into the test medium as soon as possible after preparation of the test solutions.

Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a nominal concentration of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to nominally 0.10, 1.0 and 10 mg/L in the combined range-finding test.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Test Concentrations
Cyclopentene 10, 18, 32, 56 and 100 mg/L.
Control Test medium without test item or other additives.

Reference test
The objective of the study was to evaluate potassium dichromate (K2Cr2O7) for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 48 hours of exposure (Charles River Den Bosch Study Number 20161586).
The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.
Start of first exposure: 02 Jul 2018
Completion last exposure: 04 Jul 2018
The reference test was carried out to check the sensitivity of the test system as used by Charles River Den Bosch. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
The reference item, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

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-finder
No biologically relevant immobility was observed in the control and the three lowest test concentrations throughout the test. At the highest test concentration, all daphnids were observed to be immobile at 24 and 48 hours of exposure.
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 1.9, 2.4, 5.1 and 57 mg/L in nominally 0.10, 1.0, 10 and 100 mg/L, respectively. During the exposure period, the concentrations decreased and were at 3.4-86% of the initial concentration at the end of the test.

It should be noted that a concentration of 6.2 mg/L was detected in the control at the start of the test. Considering that no immobility or other signs of disease or stress were observed during the test and that no test item could be detected in the sample taken at the end of the test, it was assumed that the contamination occurred during sampling or sample preparation.

All test conditions were maintained within the limits prescribed by the study plan.

This range-finder was deemed suitable to decide on test concentrations for the main study which were 0, 10, 18, 32 and 56 mg/l.

First full test
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 5.3, 1.9, 1.2, 2.0 and 5.7 mg/L in nominally 10, 18, 32, 56 and 100 mg/L, respectively, and thus clearly lower than the concentrations measured during the combined limit/range-finding test. It was assumed that the lower concentrations were related to the volatile properties of the test item and it was decided to repeat the test. Consequently, no samples were analysed at the end of the test.

No immobility was observed in the control and any of the test concentrations throughout the exposure period.

Considering the results of the chemical analysis, it was assumed that the lack of effects was related to the lower exposure concentrations supporting the decision to repeat the test.

pH and oxygen concentrations remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: >= 3 mg/L at the end of the test).

Second full test
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 7.7, 2.2, 1.5, 3.5 and 13 mg/L in nominally 10, 18, 32, 56 and 100 mg/L, respectively. These concentrations decreased to 35-71% of the initial concentration at the end of the test.

The measured concentrations were still below of what was measured during the combined limit/range-finding test indicating that the test solutions could not be prepared reproducibly with the employed protocol for preparing the test solutions.

No biologically relevant immobility was observed in any of the test concentrations throughout the exposure period. In the control, 5 and 15% of daphnids were observed to be immobile at 24 and 48 hours of exposure, respectively. However, this was accepted as no other daphnids in the control showed other signs of disease or stress such as discoloration, or unusual behaviour such trapping at the surface of the medium. Furthermore, not more than 5% of daphnids per test concentration group were immobilised indicating that the immobility in the control was an isolated occurrence.

Due to the discrepancies between the results of the combined limit/range-finding test and the full tests, an exact EC50 value could not be determined. Instead only an estimate can be provided.

These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: >=3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18 22°C, constant within ±1°C).

Conclusions

None of the daphnia studies produced 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.

No conclusions can be drawn from these test results because even though there were detected effects at a “measured” concentration of 56.6 mg/l this analytical detection is not reliable.

In the combined limit/range-finding test, all daphnids exposed to an average concentration of 43 mg/L were observed to be immobile from 24 hours of exposure onwards. In the second final test, one daphnid exposed to the highest average concentration of 10 mg/L was observed to be immobile after 24 and 48 hours of exposure, respectively. Consequently, the 24h- and 48h-EC50 values are expected to be between 10 and 43 mg/L, although this analytical result is not reliable enough to draw conclusions and actual concentrations may have been much higher before dilution during analysis.

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L.
The raw data from this study are kept in the Charles River Den Bosch archives. The test described above was performed under GLP with a QA-check.

Analytical validation

Accuracy and Repeatability

Water QC sample

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

Combined limit/range-finding test

Immobilization recorded during the test

Time (h)	Replicate	Test item; Nominal conc. (mg/L)
		Control	0.10	1.0	10	100
0	A	5	5	5	5	5
	B	5	5	5	5	5
	C	5				5
	D	5				5
	Total introduced	20	10	10	10	20
24	A	0	0	0	0	5
	B	0	0	0	0	5
	C	0				5
	D	0				5
	Total immobilised	0	0	0	0	20
	Effect %	0	0	0	0	100
48	A	0	0	0	0	5
	B	0	0	0	1	5
	C	0				5
	D	0				5
	Total immobilised	0	0	0	1	20
	Effect %	0	0	0	10	100

Results of the chemical analysis (analysed on day of sampling)

Time of	Concentration		Relative	Relative
sampling √	[mg/L]		to nominal	to initial
hours	Nominal √	Analysed √	[%] √	[%] √
0	0	6.16	n.a.
	0.10	1.87	1865
	1.0	2.43	243
	10	5.12	51
	100	56.6	57
48	0	n.d.	n.a.	n.a.
	0.10	0.0633	63	3.4
	1.0	0.679	68	28
	10	4.41	44	86
	100	32.0	32	57

First full test

Immobilization recorded during the test

Time (h)	Replicate	Test item; Nominal conc. (mg/L)
		Control	10	18	32	56	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0	0	0	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0

Results of the chemical analysis (analysed on day of sampling)

Time of	Concentration		Relative
sampling √	[mg/L]		to nominal
hours	Nominal √	Analysed √	[%] √
0	0	n.d.	n.a.
	10	5.28	53
	18	1.93	11
	32	1.22	4
	56	2.03	4

 

Because we thought, that a mistake had been made with preparing the test solutions, it was decided to not analysethe samples at the end of the first full test and to repeat the test.

  

Second full test

Immobilization recorded during the test

Time (h)	Replicate	Test item; Nominal conc. (mg/L)
		Control	10	18	32	56	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	0
	B	0	0	0	0	0	0
	C	1	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	1	0	0	0	0	0
	Effect %	5	0	0	0	0	0
48	A	0	0	0	0	0	0
	B	0	0	0	1	0	0
	C	2	0	0	0	0	1
	D	1	0	0	0	0	0
	Total immobilised	3	0	0	1	0	1
	Effect %	15	0	0	5	0	5

Results of the chemical analysis (analysed on day of sampling)

Time of	Concentration		Relative	Relative
sampling √	[mg/L]		to nominal	to initial
hours	Nominal √	Analysed √	[%] √	[%] √
0	0	n.d.	n.a.
	10	7.71	77
	18	2.19	12
	32	1.53	5
	56	3.51	6
48	0
	10	n.d.	n.a.
	18	2.72	27	35
	32	1.33	7	61
	56	0.928	3	61

Validity criteria fulfilled:

yes

Conclusions:

A full OECD 202 short-term toxicity of cyclopentene to Daphnia Magna 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 202 short-term toxicity of cyclopentene to Daphnia Magna 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.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 short-term toxicity to Daphnia magna 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 drawn and adequate conclusion.