Cyclopropanemethanol, 1-methyl-2-[(1,2,2-trimethylbicyclo[3.1.0]hex-3-yl)methyl]-

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February, 1999 - 13 October, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

17 July, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

April, 1996

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

JAVANOL :
Batch No. : 8825
Purity : 88.4% (sum of 2 isomers)
Expiry Date : 27 November, 2000

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

Singular samples were taken from each vessel at each concentration for Days 0, 7, 14, 21 and 28 of the exposure phase and on Day 2 from the lowest, middle and highest exposure concentrations.

A volume of 1 L was taken for all expsoure levels, except for the 0.20 mg/L (nominal) exposure level where 500 mL were sampled.

Samples were analysed on the day of sampling except on Day 14, where the samples were pre-extracted with dichloromethane and stored until analysis.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

A stock solution was prepared of JAVANOL at a concentration of 100 mg/mL in acetone. A volume of 0.1 mL of this stock solution was added per Litre of test medium and this solution stirred overnight. Two days prior to use in the test, this solution was filtered through a 0.2 micron filter to obtain the fraction actually dissolved. This solution was prepared weekly and a total of 5 L was required for 7 days of dosing. A sample for analysis was taken at each weekly renewal of the stock, i.e. from both the "old" and the "fresh" solution. This solution was stored in the dark at 7 - 10°C under continuous stirring. On a daily basis, the required amounts to be dosed were taken from this solution. A pre-test measurement demonstrated that the concentration of this solution was relatively stable during the 7-day period.

Dosing System :
The solutions with test substance were dosed via a computer controlled dosing system consisting of 6 dispensers. Via this system the dosed volume entered a mixing flask separately from the dilution water supply. The dilution water was also supplied via a dispenser connected with a six-way valve. The flow-rate was 600 mL per hour. The dosed volume was mixed with the dilution water in a mixing flask under continuous stirring. The whole system was verified daily.

The system comprised of two stainless-steel vessels of 1.5 L per concentration, each with 2 mesh containers of stainless-steel.

The test solutions in the test vessels were fortified on day 2 prior to introduction of the test organisms.

The target Nominal concentrations to be studied were :
0.20 mg/L
0.10 mg/L
0.05 mg/L
0.02 mg/L
0.01 mg/L
Solvent Control (with a maximum concentrtion of acetone of 0.1 mL/L)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Holding of Brood Stock :
Medium : UV sterilised tap water, filtered by bio-filtration
Water Conditions : NO3 / NO2 < 1 ppm; NH3 / NH4 < 1 ppm; O2 > 5 ppm; Temperature 27 °C
Age of Brood Stock : 5 - 9 months
Ratio Male / Female : 3 : 1
Spawning Tank : Spawning tank equipped with a false mesh bottom to enable collection of fertilised eggs
Feeding : Crumble feed own formulation (50% protein; 6% fat; 9% ash; 4% fiber)
Time of Fertilisation : Males and females put together in spawning tanks in the late afternoon prior to spawning; Spawning starts the following day after lights have been switched on. One hour after lights have been switched on, the spawning tanks are cleaned and all eggs are removed and discarded. Final collection takes place two hours after cleaning, to ensure a maximum age difference of two hours. The Spawning Tanks are fitted with a mesh through which the spawned eggs drop.

Test type:

flow-through

Water media type:

freshwater

Remarks:

ISO Medium

Limit test:

no

Total exposure duration:

30 d

Hardness:

180 mg CaCO3/L

Test temperature:

23 - 26 °C

pH:

pH 7.3 to 8.0

Dissolved oxygen:

> 6 mg O2/L

Nominal and measured concentrations:

The Nominal and Geometric mean meaured exposure concentrations were, as follows :

Nominal (mg/L) Mean Measured (mg/L)
Solvent Control Not Detected
0.01 0.012
0.02 0.022
0.05 0.055
0.1 0.104
0.2 0.225

Details on test conditions:

Test Type : Flow-Through
Test Duration : 30 days
Introduction of Eggs : 2 - 4 hours after fertilisation
Test Chambers : 1.5 L stainless-steel in duplicate per concentration, each with 1 mesh container of stainless-steel
Experimental design : on Day 0, 60 fresh and healthy fertilised eggs, between 2 - 4 hours old, were introduced and exposed per test concentration. The fertilised eggs were randomly distributed and divided equally over two petri-dishes. Each dish contained 30 eggs while the Control group counted 120 eggs distributed over 4 petri-dishes. On the second day the eggs (already hatched larvae) were transferred to the stainless-steel meshes.
Ligh Period : 16 hours light / 8 hours dark (700 - 800 Lux)
Loading Rate : < 0.5 g/L/24h
Feeding : At Embryo-stage - No feeding; Newly hatched larvae : Protein (0.1g/50 mL); Juvenile Stage . Artemia Nauplii 24h old. Surplus food and faeces were removed daily.

Reference substance (positive control):

no

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.055 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

The most sensitive end-points observed were for Body Weight and Body Length, with an NOEC of 0.055 mg/L.

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

0.104 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

The most sensitive end-points observed were for Body Weight and Body Length, with an OEC of 0.104 mg/L.

Basis for effect:

weight

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

JAVANOL did not induce any signficant, visible effects on the development of Zebrafish embryos at or below 0.225 mg/L, the highest exposure concentration tested. JAVANOL did not affect time of hatching or the hatching success at any of the concentrations tested. At or below the highest tested concentratio of 0.225 mg/L, JAVANOL did not affect survival and development of larvae during the yolk-sac period (first 8 Days).

Exposure to 0.225 mg/L induced > 80% mortality in larval Zebrafish. No lethal effects were observed in larvae at or below 0.104 mg/L. The 30-day LC50 was determined to be 0.14 mg/L.

JAVANOL affected larval growth (weight and length) of Zebrafish to a statistically significant level at 0.104 mg/L. No effects were observed at 0.055 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

JAVANOL affected larval growth (weight and length) of Zebrafish to a statistically significant level at 0.104 mg/L. No effects were observed at 0.055 mg/L.

The overall NOEC was determined to be 0.055 mg/L.

The 30-day LC50 was determined to be 0.14 mg/L.

Description of key information

JAVANOL affected larval growth (weight and length) of Zebrafish to a statistically significant level at 0.104 mg/L. No effects were observed at 0.055 mg/L.

The overall NOEC was determined to be 0.055 mg/L.

The 30-day LC50 was determined to be 0.14 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.055 mg/L

Additional information

JAVANOL affected larval growth (weight and length) of Zebrafish to a statistically significant level at 0.104 mg/L. No effects were observed at 0.055 mg/L.

The overall NOEC was determined to be 0.055 mg/L.

The 30-day LC50 was determined to be 0.14 mg/L.