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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 to 08 March 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-01-11

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Single sample for analysis was taken from any control and test concentration.
- Sampling method: Samples were taken at the start of the test (t = 0 h), at t = 24 h (new and old solutions) and the end of the test (t = 48 h). Approximately 500 µL were taken for each sample and analysed by HPLC-DAD.
- Sample storage conditions before analysis: Samples were analysed directly after sampling.

Vehicle:

no

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. Two stock solutions (one for the fresh medium at t = 0 h and another for the fresh medium at 24 h) were prepared by slow-stirring. The mixing vessel (1 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 1 L of the test water was added. Then an excess of the test item (approximately 4 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 200 mL volumetric flasks to obtain the required test concentrations. Each prepared concentration was inverted several times before filling of test tube (without headspace) to ensure adequate mixing and homogeneity. After filling the vessels were sealed immediately with screw-caps after introduction of daphnids. No small bubble was observed in the test tubes. 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain: Daphnia magna (Straus), clone 5
- Source: In-house laboratory cultures
- Age at study initiation: < 24 h
- Method of breeding: Parthenogenesis
- Feeding during test: No

ACCLIMATION
- Acclimation period: 48 h
- Acclimation conditions: Same as test
- Type and amount of food: Suspension of freshwater algae (mix of 3 algae strains)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

No

Hardness:

250 mg/L as CaCO3

Test temperature:

19.5-20.3 °C

pH:

7.56-7.88

Dissolved oxygen:

8.21-8.97 mg O2/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: 0.2, 0.3, 0.4, 0.7, 1.0 and 1.5 mg/L
- Mean measured concentrations: 0.156, 0.242, 0.361, 0.511, 0.685 and 1.190 mg/L. See table 6.1.3/1 for more details.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass test tubes (approximately 20 mL) sealed with screwcaps
- Type: Closed
- Aeration: No
- Renewal rate of test solution (frequency): daily
- No. of organisms per vessel: Five
- No. of vessels per concentration (replicates): Four
- No. of vessels per blank control (replicates): Four
- Three abiotic replicates for the control and per test concentration were prepared: two for sampling for analysis of test concentrations from the freshly prepared solutions (t = 0 and 24 h) and another for sampling from the 24-h old solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, as prescribed by the OECD Guideline 202
- Conductivity: < 10 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded continuously throughout the test. Dissolved oxygen concentrations and pH were recorded daily in all vessels.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility and abnormal behaviour were determined by visual observation after 24 and 48 h. The daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study: yes (test concentrations: 0, 0.3, 1.0, 1.5 and 2.0 mg/L (nominal))
- Results used to determine the conditions for the definitive study: In the range-finding test, immobilisations after 48 h of exposure were 0, 0, 60, 100 and 100 % at 0, 0.3, 1.0, 1.5 and 2.0 mg/L, respectively. The chemical analyses revealed that test item concentrations were not stable during the test period. Based on these results, test concentrations of 0.2, 0.3, 0.4, 0.7, 1.0 and 1.5 mg/L (nominal) were selected for the main test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non-concurrent)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.475 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mobility and behaviour

Remarks on result:

other: 95 % CL = 0.248-1.132

Details on results:

See table 6.1.3/2.
- Mortality of control: No
- After 24 h of exposure, immobilizations were 0, 0, 0, 0, 0, 25 and 50 % at 0, 0.156, 0.242, 0.361, 0.511, 0.685 and 1.190 mg/L, respectively. After 48 h of exposure, immobilizations were 0, 0, 35, 40, 35, 60 and 100 % at the respective concentrations.
- EC50 values at 24 and 48 h were 1.124 (0.934-1.598) and 0.475 (0.248-1.132) mg/L respectively, based on the average of measured concentrations.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- Historical data (dated: 09 January 2013): EC50 (24 h): 0.97 mg/L

Reported statistics and error estimates:

- Effective concentrations including the 95 % confidence intervals were determined using Probit analysis by the software ToxRat® Professional.

Table 6.1.3/2: Acute immobilization of daphnids after 24 and 48 hours in the final test

Nominal concentration (mg/L)	Replicate	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.2	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.3	1 2 3 4	5 5 5 5	0 0 0 0	0	1 3 1 2	35
0.4	1 2 3 4	5 5 5 5	0 0 0 0	0	2 4 1 1	40
0.7	1 2 3 4	5 5 5 5	0 0 0 0	0	3 1 1 2	35
1.0	1 2 3 4	5 5 5 5	1 2 2 0	25	2 4 4 2	60
1.5	1 2 3 4	5 5 5 5	3 2 2 3	50	5 5 5 5	100

Validity criteria fulfilled:

yes

Remarks:

mortality in control < 10 % (no immobilisation observed); dissolved oxygen concentration at the end of test ≥ 60 % of the air-saturation value in all vessels

Conclusions:

The 48 h EC50 for (-)-alpha-pinene to Daphnia magna was estimated to be 0.475 (0.248-1.132) mg/L, based on the average of measured concentrations.

Executive summary:

An acute aquatic toxicity study was performed on Daphnia magna with (-)-alpha-pinene in accordance with GLP and OECD Guideline 202. Following a preliminary range-finding test and a previous test, twenty daphnids (four replicates, five daphnids/replicate) were exposed to an aqueous solution of the test item, (-)-alpha pinene, at the required nominal test concentrations of 0.2, 0.3, 0.4, 0.7, 1.0 and 1.5 mg/L. The immobility of the daphnids was determined in a semi-static 48-h test by visual observation after 24 and 48 h.

The concentrations of the test item were determined by chemical analyses at the start (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h). The test item levels were found to be not stable throughout the test. Indeed, 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. Thus, the evaluation of the effects on Daphnia magna was based on the geometric means of the measured concentrations: 0.156, 0.242, 0.361, 0.511, 0.685 and 1.190 mg/L.

After 24 h of exposure, immobilizations were 0, 0, 0, 0, 0, 25 and 50 % at 0, 0.156, 0.242, 0.361, 0.511, 0.685 and 1.190 mg/L, respectively. After 48 h of exposure, immobilizations were 0, 0, 35, 40, 35, 60 and 100 % at the respective concentrations. The EC50 values at 24 and 48 h were 1.124 (0.934-1.598) and 0.475 (0.248-1.132) mg/L respectively, based on the average of measured concentrations.

Description of key information

48h-EC50 for freshwater invertebrates of (-)-alpha-pinene was 0.475 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.475 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 Daphnia magna was studied according to OECD 202 and GLP. Daphnids were exposed to substance at nominal test concentrations of 0.2, 0.3, 0.4, 0.7, 1.0 and 1.5 mg/L over a test period of 48 hours (semi-static test, daily renewal, closed conditions). Concentrations were analytically verified at the start and daily until the end of the test (old and new test media). The concentration of the test item was not satisfactorily maintained within ± 20% of the initial concentration throughout the test. Thus, the endpoint was based on the geometric means of the measured concentrations. The 48h-EC50 was determined to be 0.475 mg/L, the EC0 and EC100 were 0.156 mg/L and 1.190 mg/L, respectively.

Thus the 48h-EC50 is valid for key value for chemical safety assessment.