Bicyclo[3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl-, 2-acetate, (1R,5S)-

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:

26-29 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline No 202.

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:

yes

Remarks:

hardness of the water was ≥ 250 mg/L as CaCO3

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations: single samples for analysis were taken from the control and all test concentrations from replicates without daphnids (except at the end of the test : replicates with daphnids).

Sampling method: samples were taken at the start (t = 0 h) and every 24 h thereafter in old and new solutions and the control.

Storage: samples were analysed directly after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Method: 2 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 was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L of the test water was added. This was done to use a maximum volume and to minimize head space during preparation. Then an excess of the test item (approximately 2 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 and this was visually observed to be the case. After 24 ± 2 h of gentle stirring with no standing time (stirring was stopped before drawing off the aqueous phase), 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 analyzed. Then the stock solution was diluted with test water as necessary into 200-mL volumetric flasks (filled up to the meniscus) to obtain the required test concentrations based on the measured concentration of the stock solution (22.5 mg/L for the fresh medium at t = 0 h and 22.45 mg/L for the fresh medium at t = 24 h). 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 screwcaps after introduction of daphnids.
The test sample formed a clear colourless solution and appeared to be completely soluble and stable 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 (Daphnia)
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: at least 48 h
Acclimation conditions: same as test
Type and amount of food: suspension of freshwater algae (mix of 3 algae strains)
Feeding frequency: daily
Only daphnids not older than 24 hours were used for the test

VALIDITY OF BATCH OF DAPHNIDS
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

Post exposure observation period:

no

Hardness:

≥ 250 mg/L as CaCO3

Test temperature:

19.5-20.3 °C

pH:

7.63-7.94

Dissolved oxygen:

8.22-9.19 mg O2/L

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 5, 6.8, 9.1, 12.3, 16.7 and 22.5 mg/L
Mean measured concentrations: 4.381, 6.259, 8.879, 12.012, 17.327 and 21.667 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel: glass test tubes (approximately 20 mL) sealed with screwcaps
Aeration: no
Renewal rate of test solution (frequency): after 24 h
No. of organisms per vessel: 5
No. of vessels per concentration (replicates): 4
No. of vessels per blank control (replicates): 4
Moreover, 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 h and t = 24 fresh) and another for sampling from the 24-h old solutions (t = 24 old).

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 at the start of the test (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h) in all vessels.

OTHER TEST CONDITIONS
Photoperiod: 16 h light and 8 h dark
Light intensity: ca. 400 lux

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: 1.35

Range finding study
Nominal test concentrations: 1, 5, 10, 15 and 24 mg/L (maximum solubility)
Results used to determine the conditions for the definitive study: in the range-finding test, immobilisations after 48 h of exposure were 0, 0, 0, 20 , 70 and 100 % at 0, 1, 5, 10, 15 and 24 mg/L (nominal), respectively. The chemical analyses revealed that test item concentrations were not stable during the test period. Based on these results, test concentrations of 5, 6.8, 9.1, 12.3, 16.7 and 22.5 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.:

11.946 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL = 5.864–23.990

Details on results:

Mortality of control: no

After 24 h of exposure, immobilizations were 0 % at 4.381, 8.879 and 12.012 mg/L, 5 % at 6.259 mg/L, 60 % at 17.327 mg/L and 90 % at 21.667 mg/L.

After 48 h of exposure, immobilizations were 0 % at 4.381 mg/L, 5 % at 6.259 and 8.879 mg/L, 40 % at 12.012 mg/L and 100 % at 17.327 and 21.667 mg/L.

EC50 values at 24 and 48 h were 16.324 and 11.946 (5.864–23.990) mg/L, respectively.

Results with reference substance (positive control):

Results with reference substance valid: yes
Historical data (dated: March 2013): EC50 (24 h) = 1.02 mg/L

Reported statistics and error estimates:

EC50 values including the 95 % confidence interval were calculated using Probit-analysis via the computer program ToxRat® Professional.

none

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

Under the test conditions, the 48h-EC50 of nopyl acetate for Daphnia magna was estimated to be 11.946 (5.864–23.990) mg/L, based on mean measured concentrations.

Executive summary:

An acute aquatic toxicity study was performed on Daphnia magna in accordance with GLP and OECD Guideline No 202. Following a preliminary range-finding test and previous tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of nopyl acetate at the required nominal test concentrations of 5, 6.8, 9.1, 12.3, 16.7 and 22.5 mg/L. Immobilisation of 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 measured at the start (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h). Concentrations measured in freshly prepared solutions and in 24-h old solutions revealed that the concentrations of the test item were not satisfactorily maintained within ± 20 % of the initial concentrations, except the first day at 6.8, 9.1, 12.3, 16.7 and 22.5 mg/L (nominal). Thus, the evaluation of the effects on Daphnia magna was based on the geometric mean of the measured concentrations: 4.381, 6.259, 8.879, 12.012, 17.327 and 21.667 mg/L.

After 24 h of exposure, immobilizations were 0 % at 4.381, 8.879 and 12.012 mg/L, 5 % at 6.259 mg/L, 60 % at 17.327 mg/L and 90 % at 21.667 mg/L. After 48 h of exposure, immobilizations were 0 % at 4.381 mg/L, 5 % at 6.259 and 8.879 mg/L, 40 % at 12.012 mg/L and 100 % at 17.327 and 21.667 mg/L. The EC₅₀ values at 24 and 48 h were 16.324 and 11.946 (5.864–23.990) mg/L, respectively.

Therefore, the 48h-EC₅₀ of nopyl acetate for Daphnia magna was estimated to be 11.946 (5.864–23.990) mg/L, based on mean measured concentrations.

Description of key information

An acute aquatic toxicity study was performed on Daphnia magna in accordance with OECD Guideline No 202.
The 48h-EC50 of nopyl acetate for freshwater invertebrates is 11.95 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

11.95 mg/L

Additional information

Key study conducted according to OECD 202 and EU C2 Guidelines under GLP without deviation.

Daphnids were exposed to nopyl acetate at nominal concentrations of 5, 6.8, 9.1, 12.3, 16.7 and 22.5 mg/L. Immobilisation of daphnids was recorded in a semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test substance were measured at the start of the study, at 24 h (new and old solutions) and at the end of the test. The concentrations were not satisfactorily maintained within ± 20% of the initial concentrations throughout the test, thus the EC₅₀ were based on the geometric mean of the measured concentrations. The 48h-EC₅₀ determined in this study can be used as key value for chemical safety assessment.