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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
according to guideline
EU Method C.1 (Acute Toxicity for Fish)
according to guideline
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Principles of method if other than guideline:
List of study plan deviations
1. Range-finding test: Mortality and other effects were recorded after 75 hours of exposure.
Evaluation: The results could still be used for the determination of a range of concentrations for the final test.
The study integrity was not adversely affected by this deviation.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
Identification: Precyclemone B
Appearance: Clear colourless liquid (determined by Charles River Den Bosch)
Batch: SC00016633
Purity/Composition: 99.5% (sum of two isomers)
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 31 October 2016 (expiry date)

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: At the start and the end of the first and the last 24-hour renewal periods
Volume: 15 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

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 after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Details on test solutions:
Range-finding test (static)
All test solutions were prepared separately and started with loading rates of 1.0, 10 and 100 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for one day where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations.

Final test (semi-static)
Considering the fact that the main components of Precyclemone B are isomers it was decided upon request of the sponsor that the test concentrations did not have to be prepared separately. Preparation of test solutions started with a loading rate of 10 mg/L applying two days of magnetic stirring in a closed vessel to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one day where after the clear and colourless Saturated Solution (SS) was siphoned out. All test concentrations were prepared by subsequent dilutions of the SS in test medium.

Test organisms

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: 3.2 ± 0.2 cm, Final test: 2.4 ± 0.2 cm
Mean weight: Range-finding test: 0.52 ± 0.12 g, Final test: 0.16 ± 0.04 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

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)

Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. 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 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%.

Study design

Test type:
Water media type:
Total exposure duration:
96 h

Test conditions

Test temperature:
Measured at the start and the end of each renewal period in all vessels with surviving fish. All test conditions remained within the ranges prescribed by the study plan 20-24°C, constant within 2°C
pH: 6.0-8.5
Dissolved oxygen:
Measured at the start and the end of each renewal period in all vessels with surviving fish. All test conditions remained within the ranges prescribed by the study plan oxygen > 60% of air saturation)
Nominal and measured concentrations:
Range finding (static): Three fish per concentration were exposed to WAFs at 1.0, 10 and 100 mg/L;
Final test (semi-static): Nominal: 2.2, 4.6, 10, 22 and 46% of a SS at 10 mg/L
Measured (average exposure concentration): 0.027, 0.17, 0.40, 0.90 and 1.9 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 treated as volatile the study 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: 0.35 g fish/Litre, i.e. 7 fish per 3.2 litres of test medium
Illumination: 16 hours photoperiod daily, under dimmed light.
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 20 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°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.
Reference substance (positive control):
pentachlorophenol (PCP)

Results and discussion

Effect concentrations
72 h
Dose descriptor:
Effect conc.:
> 1.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
After 96 hours of exposure no mortality was observed at any of the test concentrations and the control. However, fish exposed to the average concentration of 1.9 mg/L showed clinical effects.

Applicant's summary and conclusion

Validity criteria fulfilled:
The 96h-LC50 was >1.9 mg/L based on average exposure concentrations.