6,6-dimethoxy-2,5,5-trimethylhex-2-ene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.1. "Acute Toxicity for Fish".

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: 205601/B
Identification: Methyl Pamplemousse
Appearance: Colourless to pale yellow liquid
Batch: AS00239830
Test item storage: At room temperature protected from light
Stable under storage conditions until: 11 March 2017 (expiry date)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations with surviving fish and from the control according to the schedule below.

Frequency: At the start and the end of the first and the last 24h-renewal period
Volume: 4.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤ -15°C) until analysis.

Additionally, reserve samples of 4.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Details on test solutions:

The test item was completely soluble in test medium at loading rates up to and including 46 mg/L. No correction was made for the purity/composition of the test item. Weighing of test item and preparation of test solutions was performed under dimmed light.

Range-finding test (static)
Preparation of test solutions started with two loading rates of 10 and 100 mg/L applying one day of magnetic stirring in closed and dark vessels to reach the maximum dissolution of the test item in the test medium. The resulting aqueous mixture obtained with the 100 mg/L loading rate was left to stabilize for one hour where after the clear and colourless Saturated Solution (SS) was siphoned out and used as the highest test concentration. The 10 mg/L loading rate was observed to be dissolved after the stirring period and therefore used as such (nominal concentration). One lower test concentration of 1.0 mg/L was prepared by subsequent dilution of the 10 mg/L concentration in test medium.

Final test (semi-static)
Preparation of test solutions started with three loading rates of 10 (in duplicate), 22 (singular) and 46 (singular) mg/L applying one day of magnetic stirring in closed and dark vessels to reach the maximum dissolution of the test item in the test medium. All three loading rates were observed to be dissolved after the stirring period and therefore used as such (nominal concentrations). Lower test concentrations, namely 2.2 and 4.6 mg/L, were prepared by subsequent dilutions of one of the 10 mg/L concentrations in test medium.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length: Range-finding test: 2.6 ± 0.3 cm, Final test: 2.6 ± 0.2 cm
Mean weight: Range-finding test: 0.27 ± 0.10 g, Final test: 0.31 ± 0.07 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has 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: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH andtemperature 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 Crumble 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 type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

Measured daily in all vessels with surviving fish, beginning at the start of the test (day 0). Temperature 20-24°C, constant within 2°C

pH:

Measured daily in all vessels with surviving fish, beginning at the start of the test (day 0). pH: 6.0-8.5, constant within 1 unit

Dissolved oxygen:

Measured daily in all vessels with surviving fish, beginning at the start of the test (day 0). Oxygen > 60% of air saturation

Nominal and measured concentrations:

Final Test Nominal Concentrations: 2.2, 4.6, 10, 22 and 46 mg/L
Final Test Average Exposure Concentrations (geometric mean measured concentrations) : 1.3, 2.0, 6.2, 15 and 32 mg/L

Details on test conditions:

Test duration: 96 hours
Test type: Semi-static, with daily renewal of test solutions
Test vessels: 3.5 litres, all-glass, containing 3.2 litres of test solution. Since the test item was volatile the test was performed in air-tight vessels.
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Number of fish: 7 fish per concentration and control
Loading: 0.68 g fish/Litre, i.e. 7 fish per 3.2 litres of test medium
Illumination: 16 hours photoperiod daily using dimmed light (15 lux)
Aeration: The test media were not aerated during the test
Feeding: No feeding from 24 hours prior to the test and during the total test period.
Introduction of fish: Within 45 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 1.0°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.

Key result

Duration:

96

Dose descriptor:

LC50

Effect conc.:

22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% Confidence interval between 15 and 32 mg/l

Details on results:

Analysis of the samples taken at the start of the first and the last renewal period showed measured concentrations which were < 80% of nominal. Concentrations decreased to 43-87% of initial during the 24-hour renewal periods. Therefore the average exposure concentrations were calculated. These were based on the geometric means of the concentrations measured in samples taken at the start and end of the 24h renewal periods - see full study report for details.

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 was 22 mg/L (95% confidence interval between 15 and 32 mg/L) based on average exposure concentrations (geometric mean measured concentrations).

Description of key information

The acute toxicity of the test item to Carp has been investigated. The 96h-LC50 was determined to be 22 mg/L (95% confidence interval between 15 and 32 mg/L) based on average exposure concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

22 mg/L

Additional information

A semi-static test was performed based on the results of a preceding static range-finding test. Preparation of test solutions started with three loading rates of 10 (in duplicate), 22

(singular) and 46 (singular) mg/L applying one day of magnetic stirring in closed and dark vessels to reach the maximum dissolution of the test item in the test medium. All three

loading rates were observed to be dissolved after the stirring period and therefore used as such (nominal concentrations). Lower test concentrations, namely 2.2 and 4.6 mg/L, were

prepared by subsequent dilutions of one of the 10 mg/L concentrations in test medium.

Seven fish per concentration were exposed to a control and to nominal concentrations of 2.2, 4.6, 10, 22 and 46 mg/L. The total test period was 96 hours and test solutions were renewed daily. Samples for analytical confirmation of exposure concentrations were taken at the start and the end of the first and the last renewal period.

Analysis of the samples taken at the start of the first and the last renewal period showed measured concentrations which were < 80% of nominal. Furthermore, these concentrations

decreased to 43-87% of initial during the 24-hour renewal periods. Therefore, the LC50 was determined from the average exposure concentrations, which were calculated to be 1.3, 2.0, 6.2, 15 and 32 mg/L.