Pin-2(3)-ene

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

September 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to recommended test guideline. The validity criteria were met and results were based on the measured concentration since deviation from the nominal concentration was greater than 20%. At a mean measured concentration of 0.60 mg/L, 100% of mortality was reported while no mortality was observed at a mean measured concentration of 0.12 mg/L. Therefore, the 96h-LC50 was approximated at 0.27 mg/L by calculating the geometric mean between the LC0 (0.12 mg/L) and the LC100 (0.60 mg/L).

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

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment number 23, 2000.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples for possible analysis were taken from all test concentrations and the control. The test samples were analysed freshly on the day of sampling. If required, the samples of 1, 2 or 3 ml were diluted with ISO-medium to obtain an end volume of 10 ml. To all samples, 100 µl acetonitrile was added. Acetonitrile was added to obtain a similar end solution as the end solution of the calibration solutions.
- Frequency: at the start of the test and after 72 hours from the freshly prepared solutions / at the first renawal (t=24 h) and the end of the test from the 24-hour old solutions
- Volume: varying from 1 to 10 mL from the approximate centre of the test vessels depending on concentration

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Soluble Fraction
- Differential loading: main test: 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): the final test solutions were all clear and colourless.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Zodiac, proefacc, 'De Haar Viessen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation (length definition, mean, range and SD): range-finding test: 2.3 ± 0.1 cm/ main test: 2.5 ± 0.2 cm
- Weight at study initiation (mean and range, SD): range-fnding test: 0.30 ± 0.04 g / main test: 0.39 ± 0.1 g
- Method of breeding: F1 from a single parent-pair bred in UV-treated water
- Feeding during test: no feeding from 24 hours prior to the test and during the total test period


ACCLIMATION
- Acclimation period: at least 12 days after delivery
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: pelleted fish food, no data on amount
- 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 post-exposure observation period

Hardness:

180 mg CaCO3/L

Test temperature:

at the start of the test: 21.9 -22.3°C
During the test: 20.9- 21.7 °C

pH:

at the stast of the test: 7.7-7.8
during the test: 7.4-8.0

Dissolved oxygen:

at the start of the test: 8.5-8.6 mg/L
during the test: 5.9 - 9.0 mg/L

Salinity:

Not relevant

Nominal and measured concentrations:

- Nominal concentrations: 4.6, 10, 22, 46, 100 % of a WSF prepared at a loading rate of 100 mg/L
- Average exposure concentrations: 0.015, 0.031, 0.047, 0.12 and 0.60 mg/L. See table 1 in "Any other information on materials and methods incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass vessel
- Type (delete if not applicable): closed (air-tight vessels )
- Material, size, headspace, fill volume: Control, 4.6 en 10 % of WSF 6.5 litres and the remaining concentrations 3.5 litres, all-glass, containing 6.2 and 3.2 litres of test solution, respectively.
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: Control, 4.6 en 10% of WSF 0.44 g fish/litre, i.e. 7 fish per 6.2 litres of test medium. 22, 46 and 100% of WSF 0.85 g fish/litre, i.e. 7 fish per 3.2 litres of test medium g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment
- Photoperiod: 16 hours photoperiod daily
- Light intensity: 5 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and other effects

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100% of a WSF prepared at a loading rate of 100 mg/L

Reference substance (positive control):

yes

Remarks:

pentachlorophenol; tested at concentration of 0.10, 0.22 and 0.46 mg/L in ISO-medium.T

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.27

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Geometric mean between LC0 (0% mortality at 0.12 mg/L) and LC100 (100% mortality at 0.60 mg/L)

Duration:

96 h

Dose descriptor:

other: The highest concentration without observed effect

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No clinical effects were observed but mortality was reported at 48h.
See the following table in the "Any other information results incl. tables" section:
Table 2: Incidence of mortality and total mortality during the main test

Results with reference substance (positive control):

The 96h-LC50 for carp exposed to PCP was estimated to be 0.15 mg/l (95 % confidence interval between 0.10 and 0.22 mg/l). This effect was already reached within 24 hours The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests.

Reported statistics and error estimates:

No reported statistics and error estimates

Sublethal observations / clinical signs:

Table 2: Incidence of mortality and total mortality during the main test

ALPHA PINENE % WSF prepared at 100 mg/l	Initial number of fish	Cumulativemortality					Total Mortality (%)
		4h	24h	48h	72h	96h
control	7	0	0	0	0	0	0
4.6	7	0	0	0	0	0	0
10	7	0	0	0	0	0	0
22	7	0	0	0	0	0	0
46	7	0	0	0	0	0	0
100	7	0	0	7	7	7	100

Validity criteria fulfilled:

yes

Remarks:

mortality of control not exceed 10%, constant conditions of test were maintained, the dissolved oxygen concentration was > 60% of the air saturation

Conclusions:

At the loading rate of 100 mg/L (mean measured concentration of 0.60 mg/L), 100% of mortality was reported while no mortality was observed at the dilution of 46% WSF (mean measured concentration of 0.12 mg/L). Therefore, the 96h-LC50 was calculated at 0.27 mg/L, corresponding to the geometric mean between the LC0 (0.12 mg/L) and the LC100 (0.60 mg/L).

Executive summary:

In a 96-h acute toxicity study conducted according to the European recommended guidelines, carp (Cyprinus carpio) were exposed to alpha pinene at 0, 4.6, 10, 22, 46 and 100 % of a WSF prepared at a loading rate of 100 mg/L under semi-static conditions.

Because alpha-pinene was sensitive to light and volatile, all handlings were performed under yellow or dimmed light using air-tight closed test systems.

Analysis of the samples taken at the start of the test showed group mean measured concentrations of 0.03, 0.06, 0.13, 0.27 and 0.52 mg/l in the solutions containing 4.6, 10, 22, 46 and 100% WSF, respectively. After 24 hours of exposure the test substance concentrations measured decreased to 13 - 30% of initial (group average). The group mean measured concentrations in the fresh samples taken at t=72h were 0.02, 0.04, 0.10, and 0.18 mg/l at 4.6, 10, 22 and 46% of WSF, respectively. The measured concentrations decreased to 25 – 47% of the initially measured in fresh solution (group mean). The mean measured concentration taken from the 100% WSF directly after all fish died in this group (i.e. 6 hours after renewal of test solutions) was 0.91 mg/l.

The average exposure concentrations were determined to be 0.015, 0.031, 0.047, 0.12 and 0.60 mg/L, corresponding to 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L

At the loading rate of 100 mg/L (mean measured concentration of 0.60 mg/L), 100% of mortality was reported while no mortality was observed at the dilution of 46% WSF (mean measured concentration of 0.12 mg/L). Therefore, the 96h-LC50 was calculated at 0.27 mg/L, corresponding to the geometric mean between the LC0 (0.12 mg/L) and the LC100 (0.60 mg/L).

 

Results Synopsis

Test organism : carp (Cyprinus carpio)

Test Type): static renewal

LC₅₀: 0.27 mg/L (geometric mean between LC0 (0% mortality at 0.12 mg/L) and LC100 (100% mortality at 0.60 mg/L))

Endpoint(s) Effected: mortality

Description of key information

96h-LC50 for freshwater fish for alpha-pinene is 0.27 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.27 mg/L

Additional information

One reliable study is available for the substance itself and is selected as key study. The toxicity of alpha-pinene to freshwater fish was studied according to OECD TG No. 203 and GLP. Fish (Cyprinus carpio) were exposed to substance at 0, 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L. Analysis of the samples taken at the start of the test showed group mean measured concentrations of 0.03, 0.06, 0.13, 0.27 and 0.52 mg/L in the solutions containing 4.6, 10, 22, 46 and 100% WSF, respectively. The 96h-LC50 was determined to be 0.27 mg/L, corresponding to the geometric mean between LC0 (0.12 mg/L) and LC100 (0.60 mg/L).

In addition to this key study, a reliable study is available on (-)-alpha pinene. Fish (Danio rerio) were exposed to substance 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.