2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 March 2009 to 15 May 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No dose-response curve; solvent addition.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 2: Semi-static method)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

- Storage conditions: At room temperature in the dark
- Stability under storage conditions: Stable
- Solubility in water: 14 mg/L

Batch No. : LDP0800001
Purity : 97.0%
Date of Expiry : 01 May, 2010

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

- Concentrations: all test concentrations
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control according to the following: at the start of the test and after 72 hours from the freshly prepared solutions; at the first renewal (24h) and the end of the test from the 24h old solutions. In addition, after 48h from the solutions with no surviving fish. Volume = 15 mL from the approximate centre of the test vessels. Additionally, reserve samples of 15 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

yes

Remarks:

DMSO at a concentration of 0.1 mL/L

Details on test solutions:

- Preparation of the test solution: Daily preparation of the test solutions started with stock solutions in DMSO ranging from 14 to 140 mg/mL. Subsequently, 0.1 mL of the stock solution was added per litre of test medium in order to reach the test concentrations. After a 15-minute stirring period, test solutions up to and including 4.5 mg/L were clear and colourless while test solutions from 8.2 mg/L and higher contained a floating layer.
Control: Test medium without test substance but with DMSO in the same amount as used in the treatment of the stock solutions.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
- Source: Zodiac, proefaccn "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Mean length (10 fish of the batch used for the test were weighed and measured prior to the start of the test): Range-finding test = 2.5 +/- 0.3 cm; Final test = 2.7 +/- 0.3 cm
- Mean weight (10 fish of the batch used for the test were weighed and measured prior to the start of the test): Range-finding test = 0.48 +/- 0.17 g; Final test = 0.54 +/- 0.21 g
- Characteristics: F1 from a single parent-pair bred in UV-treated water
- Reason for selection: This system had been selected as an internationally accepted species
- Total fish used: 51

HOLDING
- Quarantine / Acclimatisation: At least 12 days after delivery
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: CaCl2.2H2O = 211.5 mg/L; MgSO4.7H2O = 88.8 mg/L; NaHCO3 = 46.7 mg/L; KCl = 4.2 mg/L.
- Measurements: pH, nitrate and nitrite concentration and ammonia concentration, once a week. Temperature: every day. In addition, pH and temperature were measured before transferring the fish to the test system.
- Water quality parameters: Were kept within the optimum limits for the respective fish species.
- Feeding: Daily with pelleted fish food (Cyprico Cumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)
- Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

TEST PROCEDURE
- Feeding: No feeding from 24 hours prior to the test and during the total test period
- Introduction of fish: Within 25 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 0.5°C.
- Euthanasia: At the end of the test, the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg CaCO3/L

Test temperature:

Measurement daily in all vessels with surviving fish, beginning at the start of the test (day 0): From 20.8 to 21.6°C

pH:

Measurement daily in all vessels with surviving fish, beginning at the start of the test (day 0): From 7.7 to 8.1

Dissolved oxygen:

Measurement daily in all vessels with surviving fish, beginning at the start of the test (day 0): From 6.9 to 9.3 mg/L.

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal concentrations: 1.4, 2.5, 4.5, 8.2 and 14 mg/L.
- Mean measured concentrations: 1.2, 2.1, 4.0, 6.7 and 8.5 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
10 litres, all glass, containing 9.8 litres of test solution.
- Type (delete if not applicable): closed
. Since the test substance was volatile the study was performed in air-tight vessels, using Perspex plates sealed with silicon glue.
- Aeration:
The test media were not aerated during the test
- Type of flow-through (e.g. peristaltic or proportional diluter):
semi-static
- Renewal rate of test solution (frequency/flow rate):
Daily renewal of test solutions
- No. of organisms per vessel:
7 fish per concentration and control
- No. of vessels per concentration (replicates):
1
- No. of vessels per vehicle control (replicates):
1
- Biomass loading rate:
0.39 g fish/litre, i.e. 7 fish per 9.8 litres of test medium

TEST MEDIUM / WATER PARAMETERS
Adjusted ISO medium with a hardness of 180 mg CaCO3/L.
The pH of culture medium was 7.8.
The temperature of culture medium was 21.9°C.

OTHER TEST CONDITIONS
- Adjustment of pH:
no
- Photoperiod:
16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality and other effects: At 2 1/2, 24, 48, 72 and 96 hours following the start of the exposure. In addition, every afternoon from day 0 and every morning from day 1 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Preliminary data: A non-GLP pretest was performed by Givaudan. This experiment shows that a solution of the test substance in ISO medium does not remain stable (65.8-70.3% of initial) without agitation at least for 96 hours in closed glass containers (full or not). When stored in an open vessel, the substance had evaporated out of the solution almost totally after 96 hours (1.4% initial).
- Range finding study
: A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions: Three fish per concentration were exposed to 0.14, 1.4 and 14 mg test substance/L; Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration with surviving fish; Test vessels were closed with Perspex plates sealed with laboratory film (Parafilm); The first recording of mortality and other effects was made after 6 hours of exposure; Samples for possible analysis were taken from all concentrations.
After 48h of exposure, all fish exposed to a nominal concentration of 14 mg/L had died. This concentration contained a floating layer, indicating that 14 mg/L was above the solubility limit of the test substance in test medium. No mortality, clinical effects and floating layers were observed at any of the lower test concentrations (0.14 and 1.4 mg/L) during the test period. Therefore, the expected LC50 was between nominal concentrations of 1.4 and 14 mg/L. Based on these results, samples taken from nominal 1.4 and 14 mg/L used during the first refreshment period were analysed. The initial concentrations were 1.3 and 8.2 mg/L. These concentrations decreased to 0.71 and 6.3 mg/L during the test period (55-76% of initial). In order to keep the actual test concentrations more stable during the refreshment periods, it was decided to seal the lids on the test vessels with silicon glue during the final test.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (PCP)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI: 2.1 - 4.0 mg/L

Details on results:

- Measured concentrations: See table 6.1.1/1 in "Any other information on results incl. tables". Analysis of the samples taken at the start of the first and the last refreshment period were in agreement with nominal (93 -105%) for concentrations ranging from 1.4 to 8.2 mg/L (1st period) or 1.4 to 4.5 mg/L (last period). At the nominal concentration of 14 mg/L, the actual test concentration was slightly lower (73% nominal). During the 24 -hour refreshment periods all concentrations slightly decreased (60 -99% of initial). Based on these results, the average exposure concentrations were calculated to be 1.2, 2.1, 4.0, 6.7 and 8.5 mg/L.
- Mortality and other effects: See tables 6.1.1/2 and 3 in "Any other information on results incl. tables". At the end of the test all fish exposed to average concentrations of 4.0 mg/L and higher had died, while all fish exposed to lower concentrations (1.2 and 2.1 mg/L) survived. Concentrations of 6.7 mg/L and higher contained a floating layer.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- LC50: The 96h-LC50 for carp exposed to PCP was 0.15 mg/L (9% CI between 0.10 and 0.22 mg/L).

Sublethal observations / clinical signs:

Table 6.1.1/1: Concentrations of the test substance in test medium (final test)

Time of sampling (hours	Date of sampling (dd-mm-yy)	Date of analysis* (dd-mm-yy)	Concentrations
			Nominal (mg/L)	Analysed (mg/L)	Relative to nominal (%)	Relative to initial (%)
0 Fresh	30-03-09	06-04-09	0 1.4 2.5 4.5 8.2 14	n.d. 1.45 2.50 4.37 7.66 10.2	n.a. 104 100 97 93 73
24 Old	31-03-09	06-04-09	0 1.4 2.5 4.5 8.2 14	n.d. 0.874 1.67 3.16 5.83 7.06	n.a. 62 67 70 71 50	n.a. 60 67 72 76 69
48 Old	01-04-09	06-04-09	8.2 14	6.83 7.57	83 54
72 Fresh	02-04-09	06-04-09	0 1.4 2.5 4.5	n.d. 1.43 2.62 4.31	n.a. 102 105 96
96 Old	03-04-09	06-04-09	0 1.4 2.5 4.5	n.d. 1.02 1.91 4.27	n.a. 73 76 95	n.a. 72 73 99

* Samples were stored in the freezer until the day of analysis.

n.d.: not detected

n.a.: not applicable

Table 6.1.1/2: Incidence of mortality and total mortality during the final test

Average concentrations of test substance (mg/L)	Initial number of fish	Cumulative mortality					Total mortality (%)
		2 1/2h	24h	48h	72h	96h
Control 1.2 2.1 4.0 6.7 8.5	7 7 7 7 7 7	0 0 0 0 0 0	0 0 0 0 0 5	0 0 0 0 7 7	0 0 0 2 7 7	0 0 0 7 7 7	0 0 0 100 100 100

Table 6.1.1/3: Clinial effects observed during the final test

Average concentrations of test substance (mg/L)	Time of recording (hours)	Specification of effects	Relative number
Control, 1.2	2 ½ - 96	No abnormalities	7/7
2.1	2 ½ - 48	No abnormalities	7/7
	72	Immobile No abnormalities	2/7 5/7
	96	Immobile Loss of equilibrium No abnormalities	2/7 1/7 4/7
4.0	2 ½ - 24	No abnormalities	7/7
	48	Immobile No abnormalities	1/7 6/7
	72	Immobile	5/5
6.7	2 ½	No abnormalities	7/7
	24	Discoloured and immobile Discoloured and loss of equilibrium	3/7 4/7
8.5	2 ½	No abnormalities	7/7
	24	Discoloured and immobile Discoloured and loss of equilibrium	1/2 1/2

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 value was determined at 2.9 mg/L (95% CI between 2.1 and 4.0 mg/L).

Executive summary:

This study was performed to assess the acute toxicity of the test substance in Carp according to OECD Guideline 203 with GLP compliance.

Based on the results of a range-finding test, a final test was performed. Daily preparation of the test solutions started with stock solutions in DMSO ranging from 14 to 140 mg/mL. Subsequently, 0.1 mL of the stock solution was added per litre of test medium in order to reach the test concentrations. After a 15-minute stirring period, test solutions up to and including 4.5 mg/L were clear and colourless while test solutions from 8.2 mg/L and higher contained a floating layer. Seven fish per concentration were exposed to a control group and nominal test substance concentrations of 1.4, 2.5, 4.5, 8.2 and 14 mg/L. Test vessels were closed and the total test period was 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the first and the last refreshment period.

Analysis of the samples taken at the start of the first and the last refreshment period were in agreement with nominal (93 -105%) for concentrations ranging from 1.4 to 8.2 mg/L (1st period) or 1.4 to 4.5 mg/L (last period). At the nominal concentration of 14 mg/L, the actual test concentration was slightly lower (73% nominal). During the 24 -hour refreshment periods all concentrations slightly decreased (60 -99% of initial). Based on these results, the average exposure concentrations were calculated to be 1.2, 2.1, 4.0, 6.7 and 8.5 mg/L.

The study met the acceptability criteria prescribed by the protocol and was considered valid.
The test substance induced no visible effects in carp at 1.2 mg/L and the 96h-LC50 value was determined at 2.9 mg/L (95% CI between 2.1 and 4.0 mg/L).

Description of key information

OECD Guideline 203, GLP, key study, validity 2:

96h-LC50 (Cyprinus carpio) = 2.9 mg/L (95% CI: 2.1 - 4.0 mg/L) based on measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

2.9 mg/L

Additional information

To assess the short-term toxicity of the registered substance to fish, one valid experimental study is available.

 

This study was performed according to OECD Guideline No. 203 with GLP compliance to assess the acute toxicity of the registered substance to the carp Cyprinus carpio.

Following a preliminary range-finding test, seven fish per concentration were exposed to a control group and nominal test substance concentrations of 1.4, 2.5, 4.5, 8.2 and 14 mg/L (prepared with a solvent DMSO), for a period of 96 hours under semi-static and closed test conditions. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the first and the last refreshment period and the number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined throughout the study.

Analysis of the samples taken at the start of the first and the last refreshment period were in agreement with nominal (93 -105%) for concentrations ranging from 1.4 to 8.2 mg/L (1st period) or 1.4 to 4.5 mg/L (last period). At the nominal concentration of 14 mg/L, the actual test concentration was slightly lower (73% nominal). During the 24 -hour refreshment periods all concentrations slightly decreased (60 -99% of initial). Based on these results, the average exposure concentrations were calculated to be 1.2, 2.1, 4.0, 6.7 and 8.5 mg/L.

At the end of the test all fish exposed to average concentrations of 4.0 mg/L and higher had died, while all fish exposed to lower concentrations (1.2 and 2.1 mg/L) survived. Concentrations of 6.7 mg/L and higher contained a floating layer. In conclusion, the 96h-LC50 value was determined at 2.9 mg/L (95% CI between 2.1 and 4.0 mg/L).