(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-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

Study period:

27 January – 01 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and all test concentrations.
- Sampling method: taken at the start (t = 0 h) and every 24 h thereafter in old and new solutions and the control, ca. 0.5 mL were taken for each sample, which was diluted in half with acetonitrile into vials and analysed by HPLC-DAD.
- Sample storage conditions before analysis: samples were analysed directly after sampling.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Given the volatility of the test item (vapour Pressure: 530 Pa at 25 °C), the stock and test solutions were prepared under closed conditions and gently stirred to avoid production of a dispersion. Four stock solutions (for the fresh mediums at t = 0, 24, 48 and 72 h) were prepared by slow-stirring. The mixing vessel (10 L) was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. A magnetic stirring bar was placed in the vessel and 10 L of the test water was added. Then an excess of the test item (approximately 7-10 g) was carefully added directly to the surface of the test water. Mixing was initiated with the vortex in the centre extending maximally around 10 % vessel depth from the top to the bottom of the vessel. The stirring speed was kept as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 ± 2 h of gentle stirring, the saturated aqueous phase was taken out of the drain port. The first 100 mL were discarded and samples were taken from the following stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary into 5-L volumetric flasks to obtain the required test concentrations. Each prepared concentration was inverted several times before filling of aquaria to ensure adequate mixing and homogeneity. After filling the vessels were closed immediately after introduction of fish. The test sample formed a clear colourless solution and appeared to be completely soluble when mixed with test water at the concentrations prepared.
- Controls: Test water without test substance but treated in the same way as the test substance solutions

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio
- Source: Elevage de la Grande Rivière, Saint Forgeux, France
- Length at study initiation (mean and range): Range-finding test: 2.71 cm (2.54-2.83 cm); final test: 2.33 cm (2.10-2.67 cm)
- Feeding during test: No

ACCLIMATION
- Acclimation period: At least 12 days
- Type of food: Pelleted fish food
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): 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:

no

Total exposure duration:

96 h

Post exposure observation period:

No data

Hardness:

250 mg/L as CaCO3

Test temperature:

21.1-23.2 °C

pH:

7.58-8.01 (all groups, whole test period)

Dissolved oxygen:

7.81-9.07 mg O2/L (all groups, whole test period)

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: 0.10, 0.20, 0.30, 0.60 and 1.00 mg/L
- Measured (geometric mean) concentrations: 0.08, 0.14, 0.19, 0.26 and 0.58 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Type: closed by placing a glass plate on the aquarium and sealed with vaseline
- Volume: 3.5 L
- Headspace: ca. 1 cm
- Aeration: no
- Renewal rate of test solution: daily
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: < 1.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Test water: reconstituted water, as prescribed by the OECD TG 203
- Conductivity: < 10 µS/cm
- Culture medium same as test medium: yes
- Intervals of water quality measurement: pH, nitrate and nitrite concentration, ammonia concentration, total water hardness and dissolved oxygen were measured once a week. Temperature was recorded every day.

OTHER TEST CONDITIONS
- Photoperiod: 16h light : 8 h dark

EFFECT PARAMETERS MEASURED
- Mortality at 3, 6, 24, 48, 72 and 96 h following the start of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Range finding study: yes
- Test concentrations: 0.5, 1.0 and ca. 2.0 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Mortalities were 0, 66.7, 100 and 100 % at 0, 0.5, 1.0 and ca. 2 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non-concurrent)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.303 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL = 0.187-0.578

Details on results:

- Mortalities (96 h) at 0 (blank), 0.08, 0.14, 0.19, 0.26 and 0.58 mg/L mg/L (measured) were 0, 0, 0, 0, 14.3 and 100 %, respectively
- LC50 values at 24, 48 and 72-96 h were calculated to be 0.632, 0.392 (0.291-0.591) and 0.303 (0.187-0.578) mg/L, respectively
- LC0 = 0.187 mg/L
- LC100 = 0.578 mg/L

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- LC50 (24 h): 179.39 mg/L (historical data, July 2012)

Reported statistics and error estimates:

The software ToxRat® Professional was used for the determination of the effective concentrations. The LC50 value was determined by Probit analysis using linear max. likelihood regression.

Sublethal observations / clinical signs:

ANALYTICAL RESULTS

- Concentrations measured in freshly prepared solutions and in 24-h old solutions revealed that the concentration of the test item was not satisfactorily maintained within ± 20 % of the initial concentration throughout the test (except some concentrations the second day). Since the deviation of the exposure concentrations of the test substance was greater than ± 20 % of the initial concentrations, the results were expressed in terms of geometric means of the exposure concentrations.

- See table 6.1.1/1 for more details.

Table 6.1.1/1. Mean measured test item concentrations (mg/L) in test water - final test

Nominal concentration (mg/L)	Geometric mean 0 - 24 h (Day 0 – Day 1) (mg/L)	Geometric mean 24 - 48 h (Day 1 – Day 2) (mg/L)	Geometric mean 48 - 72 h (Day 2 – Day 3) (mg/L)	Geometric mean 72 - 96 h (Day 3 – Day 4) (mg/L)	Arithmetic Mean 0 - 96 h (Day 0 – Day 4) (mg/L)
Control	N.A.*	N.A.*	N.A.*	N.A.*	N.A.*
0.1	0.05	0.105	0.117	0.05	0.081
0.2	0.092	0.254	0.123	0.073	0.136
0.3	0.202	0.318	0.134	0.094	0.187
0.6	0.388	0.329	0.166	0.135	0.255
1.0	0.636	0.785	0.313	N.A.*	0.578

*N.A.: not applicable.

Validity criteria fulfilled:

yes

Remarks:

mortality in control was 0 %; semi-static test conditions; oxygen content was ≥ 60 % ASV in the control and test vessels throughout the test; results were based on measured concentrations

Conclusions:

The 96h-LC50 for (-)-alpha-pinene was 0.303 (0.187-0.578) mg/L in zebrafish (Danio rerio).

Executive summary:

In an acute aquatic toxicity study performed according to OECD guideline 203 and in compliance with GLP, groups (7/concentrations) of zebrafish (Danio rerio) were exposed to (-)-alpha-pinene at concentrations of 0 (blank), 0.08, 0.14, 0.19, 0.26 and 0.58 mg/L [measured (geometric mean)] for 96 h under semi-static conditions. Mortalities were recorded at 3, 6, 24, 48, 72 and 96 h. A preliminary range-finding test was conducted with 3 fishes/concentration at 0.5, 1.0 and ca. 2.0 mg/L (nominal) and cumulative mortalities after 96 h were recorded as 66.7, 100 and 100 %, respectively.

In the main test, mortalities (96 h) at 0 (blank), 0.08, 0.14, 0.19, 0.26 and 0.58 mg/L mg/L (measured) were 0, 0, 0, 0, 14.3 and 100 %, respectively. LC50 values at 24, 48 and 72-96 h were calculated to be 0.632, 0.392 (0.291-0.591) and 0.303 (0.187-0.578) mg/L, respectively.

Therefore, the 96h-LC50 for (-)-alpha-pinene was 0.303 (0.187-0.578) mg/L in zebrafish (Danio rerio).

Description of key information

96h-LC50 for freshwater fish for (-)-alpha-pinene is 0.303 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.303 mg/L

Additional information

One reliable study is available for the substance itself and is selected as key study. Fish (Danio rerio) were exposed to (-)-alpha-pinene at nominal test concentrations of 0.10, 0.20, 0.30, 0.60 and 1.00 mg/L over a test period of 96 hours (semi-static test, daily renewal, closed conditions). Concentrations were analytically verified at the start and daily until the end of the test. The concentration of the test item was not satisfactorily maintained within ± 20% of the initial concentration throughout the test. Thus, the endpoint were based on the geometric means of the measured concentrations. The 96h-LC50 was determined to be 0.303 mg/L, the LC0 and LC100 were 0.187 mg/L and 0.578 mg/L, respectively.

Thus the 96h-LC50 of 0.27 mg/L is valid for key value for chemical safety assessment.