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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April 2019 – 16 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 10%, 15%, 25%, 40%, 62.5% and 100% (nominal concentrations expressed in % saturated solution)
- Sampling method: Samples were taken 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). Approximately 1 mL was taken for each sample, then put in a vial and analysed by HPLC-UV.
- Sample storage conditions before analysis: All the solutions were kept in refrigerator.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was found poorly soluble and volatile, then a special solubilisation protocol in the test medium (M4 Elendt) was carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
The mixing protocol consisted in three independent stock solutions at 100 mg/L (nominal concentration) which were stirred slowly 24 hours at 20 ± 2°C, at darkness and without headspace. After this step, a settling period of 30 minutes was performed and then, an aliquot was sampled at the bottom of the flask after removing the first 50 mL.
Chemical analysis was performed and the stock solution with the highest measured concentration was chosen for the preparation of the test solutions by serial dilution of this stock solution. Considering the measured value of the selected stock solution, the proposed concentration range was in a geometric series: 100% - 62.5% - 40.0% - 25% - 15% and 10 % (expressed as % v/v saturated solution).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, clone number 5, from at least the third generation (i.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
- Source: no data
- Feeding during test: No

ACCLIMATION
- Acclimation period: not specified.
- Acclimation conditions (same as test or not): The daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4. Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 μm and 800 μm (corresponding to daphnids less than 24 hours old).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

240 mg/L as CaCO3

Test temperature:

19.8 - 20.0ºC

pH:

7.8-8.0 (fresh solutions) and 7.7-7.8 (old solutions)

Dissolved oxygen:

9.0-9.1 mg O2/L (fresh solutions) and 8.6-9.0 mg O2/L (old solutions)

Nominal and measured concentrations:

Nominal concentrations (%v/v saturated solution): 0 (control), 10%, 15%, 25%, 40%, 62.5% and 100%
Geometric mean measured concentrations: 0 (control), 0.19, 0.25, 0.41, 0.65, 1.08 and 1.78 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: closed tube
- Material, size, headspace, fill volume: glass, without headspace, 10 mL fill volume.
- Aeration: not specified.
- Renewal rate of test solution (frequency/flow rate): 24 h (semi-static test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 2 mL of test media per daphnia.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium M4 ELENDT was prepared from concentrated stock solutions in ultrapure water as described in the OECD 202 guideline.
- Intervals of water quality measurement: pH and dissolved oxygen were measured before and after renewal of solutions (including the controls) and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: complete darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of the daphnid’s immobility and behaviour were done after 24 and 48 hours in control and exposed groups. Any sign of stress observed on the daphnids after 48 hours of exposure to the test item was reported.

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1 and 10 mg test item/L along with negative control.
- Results used to determine the conditions for the definitive study: In the range finding test the per cent immobilisation of daphnia at 48h was 0, 0, 0, and 100% at the tested concentrations of 0, 0.1, 1 and 10 mg/L, respectively. The chemical analyses revealed that test item concentrations were not stable during the test period. Consequently, the main test was carried out under semi-static conditions which means that all solutions were renewed at 24h. Based on these results, the main test was conducted on six concentrations levels obtained from serial dilutions of a saturated stock solution (100 mg/L nominal concentration): 100% - 62.5% - 40.0% - 25% - 15% and 10% (expressed as % v/v saturated solution).

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Confidence interval = [0.34 – 0.48]

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Confidence interval = [0.49– 0.69]

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Confidence interval = [0.14– 0.26]

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Confidence interval = [0.21– 0.35]

Details on results:

- Behavioural abnormalities:
- Mortality of control: No mortality was observed over a period of 48 h in the control group.
- Abnormal responses: no signs of stress or other abnormal behaviour was reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the last toxic response obtained on the reference item K2Cr2O7 (April 15, 2019) indicates that the daphnids sensitivity is correct (within the accepted interval of 0.6–2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.
- EC50-24 h: 1.57 mg/L (1.41 – 1.78).

Reported statistics and error estimates:

EC10 and EC50 were calculated with the software Minitab 17 with Probits analysis (based on geometric mean of the measured test item concentrations).

Table 1: pH and dissolved oxygen values during the test

Nominal test item concentration  (% v/v)	pH
	T0 Fresh solution	T24h Old solution	T24h Fresh solution	T48 h Old solution
Control	8.0	7.7	8.0	7.8
10	8.0	7.7	8.0	7.7
15	8.0	7.7	8.0	7.7
25	8.0	7.7	7.9	7.7
40	8.0	7.8	7.9	7.7
62.5	8.0	7.8	7.8	7.7
100	8.0	7.8	/	/

Nominal test item concentration (mg/L)	O2(mg/L)
	T0 Fresh solution (Air: 9.4 mg/L)	T24h Old solution (Air: 9.5 mg/L)	T24h Fresh solution (Air: 9.5 mg/L)	T48 h Old solution (Air: 9.4 mg/L)
Control	9.0	8.9	9.0	8.8
10	9.0	8.9	9.0	8.8
15	9.0	9.0	9.0	8.8
25	9.1	8.8	9.0	8.6
40	9.1	8.9	9.0	8.7
62.5	9.1	8.9	9.1	8.7
100	9.1	8.9	/	/

Table 2: Results on daphnia immobilization

Nominal test item concentration  ( % v/v)	Geometric mean measured test item concentration (mg/L)	Number of daphnids exposed at the start	Immobilised daphnids (%)
			at 24h	at 48h
Control	0	20	0 (0%)	0 (0%)
10	0.19	20	0 (0%)	0 (0%)
15	0.25	20	1 (5%)	6 (30%)
25	0.41	20	8 (40%)	11 (55%)
40	0.65	20	10 (50%)	14 (70%)
62.5	1.08	20	17 (85%)	20 (100%)
100	1.78	20	20 (100%)	20 (100%)

Table 3: Conclusion on the chemical analysis

Nominal test item concentration (% v/v)	Results of the determination of test item analysis (mg/L)
	0 hour Start of the exposure interval (Fresh solutions)	24 hours End of the exposure interval (Old solutions)	Deviation (%)	24 hours Start of the exposure interval (Fresh solutions)	48 hours End of the exposure interval (Old solutions)	Deviation (%)
Control	< 0.1	< 0.1	NA	< 0.1	< 0.1	NA
10	0.16	0.17	+6.3	0.24	0.20	-16.7
15	0.24	0.20	-16.7	0.33	0.23	-30.3
25	0.44	0.33	-25.0	0.51	0.38	-25.5
40	0.64	0.45	-29.7	0.88	0.70	-20.5
62.5	1.18	0.77	-34.7	1.36	1.12	-17.6
100	2.32	1.37	-40.9	NA	NA	NA

Table 4: Calculation of geometric mean of the measured concentrations

Nominal test item concentration (% v/v)	Results of the determination of test item analysis (mg/L)
	0 hour Start of the exposure interval (Fresh solutions)	24 hours End of the exposure interval (Old solutions)	24 hours Start of the exposure interval (Fresh solutions)	48 hours End of the exposure interval (Old solutions)	Geometric mean measured test item concentration (mg/L)
10	0.16	0.17	0.24	0.20	0.19
15	0.24	0.20	0.33	0.23	0.25
25	0.44	0.33	0.51	0.38	0.41
40	0.64	0.45	0.88	0.70	0.65
62.5	1.18	0.77	1.36	1.12	1.08
100	2.32	1.37	NA	NA	1.78

Taking into account that the test item is poorly soluble and the main test was performed under semi-static conditions, the test results were determined and expressed relative to the geometric mean of the measured concentrations. This geometric mean was calculated with the formula in Annex 2 of the Guidance document on aquatic toxicity testing of difficult substances and mixtures OECD n°23, 15/12/00.

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L)

Conclusions:

In a short-term toxicity test to Daphnia magna performed in semi-static conditions, the 48h-EC50 of the test substance was found to be 0.4 mg/L.

Executive summary:

An acute aquatic toxicity study with Daphnia magna was conducted on the test item according to OECD guideline 202, following GLP. The test item was found poorly soluble and volatile, then a special solubilisation protocol in the test medium (M4 Elendt) was carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures. No vehicle was needed to be used. In a preliminary range finding study the per cent immobilisation of daphnia was 0, 0, 0 and 100% at the tested concentrations of 0, 0.11, 1 and 10 mg/L, respectively. Also, the chemical analyses revealed that test item concentrations were not stable during the test period. Consequently, the main test was carried out under semi-static conditions for a total period of 48 h (solutions renewed at 24h). Based on these preliminary results, the main test was conducted on six concentrations levels obtained from serial dilutions of a saturated stock solution (100 mg/L nominal concentration): 100% - 62.5% - 40.0% - 25% - 15% and 10% (expressed as % v/v saturated solution). The results were determined and expressed relative to the geometric mean of the measured concentrations in fresh and old solutions during the test period which were calculated to be 0 (control), 0.19, 0.25, 0.41, 0.65, 1.08 and 1.78 mg/L (according to OECD nº 23, 15/12/00). The test was conducted in darkness in closed tubes filled with 10 mL daphnia medium and without headspace. 4 replicates with 5 daphnids per replicate were performed. Test medium alone was used as negative control and a solution of potassium dichromate was used as reference substance. A validated analytical method based on HPLC/UV was used to monitor the concentration of the active ingredient in the test solutions. There was no immobilization of daphnia in the negative control and dissolved oxygen measured before and after renewal of solutions and at the end of the test was higher than 3 mg/L in control and test vessels. The immobilization of daphnia was 0, 0, 5, 40, 50, 85 and 100 % at 24 hours exposure at 0 (control), 0.19, 0.25, 0.41, 0.65, 1.08 and 1.78 mg/L. The immobilization of daphnia was 0, 0, 30, 55, 70, 100 and 100 % at 48 hours exposure at 0 (control), 0.19, 0.25, 0.41, 0.65, 1.08 and 1.78 mg/L. Based on these results, the 24h-EC10 was 0.29 mg/L (95% CL: 0.21 – 0.35), the 24h-EC50 was 0.58 mg/L (95% CL: 0.49 – 0.69), the 48h-EC10 was 0.21 mg/L (95% CL: 0.14 – 0.26) and the 48h-EC50 was 0.40 mg/L (95% CL: 0.34 – 0.48).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted similarly to OECD guideline 202 with minor deviations (no data on hardness of dilution water, acclimation period or reference substance. EC50 value could not be obtained). The report is sufficiently well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(no data on hardness of dilution water, acclimation period and reference substance. The EC50 value could not be obtained. Only the LC50 value was reported).

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 124, 248, 372, 496 and 620 μg/L
- Sampling time: 0, 24 and 48 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water, stirring in closed vessel
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (620 µg/L liq-liq equil.)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: ERL-Duluth, USA
- Age at study initiation: < 24 hours

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

45.4 mg CaCO3/L (SD=0.2)

Test temperature:

20.2ºC (SD=0.2)

pH:

7.8 (SD=0.09)

Dissolved oxygen:

7.8 mg O2/L (SD=0.4)

Salinity:

42.9 mg CaCO3/L (SD=0.1)

Nominal and measured concentrations:

Nominal concentrations: 0, 124, 248, 372, 496 and 620 μg/L
Average measured concentrations: <32, 75.7, 223, 301, 407 and 542 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed; stainless steel screen enclosures (forty mesh) were placed in each chamber for the daphnia exposures
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 75.7-542 μg/L
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 20 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (°C), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hours/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Final mortality counts were taken at termination. Daphnia were removed from test screens and observed under a dissecting scope to confirm responses.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 542 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Main test

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 542 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Main test

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 1 440 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Based on two additional tests

Details on results:

- Mortalities (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively.
- Effects (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively.
-Additional tests: LC50 and EC50 could not be determined from the results obtained in the main test. Thus, two additional tests were performed. The test concentrations were 920 and 1965 ug/l, respectively. An approximate LC50 value of 1440 ug/l was calculated based on one mortality in the first test and total mortality in the second test.

Results with reference substance (positive control):

Not applicable
 

Reported statistics and error estimates:

- LC50 or EC50 was calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour	Control (0 µg/L)	A (124 µg/L)	B (248 µg/L)	C (372 µg/L)	D (496 µg/L)	E (620 µg/L)
Initial	15	15	15	15	15	15
48	0	0	0	0	0	2

Table 2: Effects

Hour	Control (0 µg/L)	A (124 µg/L)	B (248 µg/L)	C (372 µg/L)	D (496 µg/L)	E (620 µg/L)
Initial	15	15	15	15	15	15
48	0	0	0	0	0	2

 

Validity criteria fulfilled:

yes

Remarks:

(immobilisation in control < 10%; dissolved oxygen concentration > 3 mg O2/L)

Conclusions:

The 48h-LC50 of d-alpha pinene to Daphnia magna was calculated to be 1440 μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of Daphnia magna were exposed to d-alpha pinene at concentrations of <32, 75.7, 223, 301, 407 and 542 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively. Treatment-related effects (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively. Based on these results, LC50 and EC50 could not be determined. Thus, two additional tests were performed. The test concentrations were 920 and 1965 µg/l, respectively. An approximate LC50 value of 1440 µg/l was calculated based on one mortality in the first test and total mortality in the second test.

Description of key information

Key study. Test method according to OECD TG 202, GLP study. The 48h-EC50 of the test substance to Daphnia magna was found to be 0.40 mg/L.

Supporting study: Test method similar to OECD TG 202. The 48h-LC50 of d-alpha pinene to Daphnia magna was calculated to be 1440 μg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.4 mg/L

Additional information

Key study: An acute aquatic toxicity study with Daphnia magna was conducted on the test item according to OECD guideline 202, following GLP. The main test was carried out under semi-static conditions for a total period of 48 h (solutions renewed at 24h) with six concentrations levels obtained from serial diltutions of a saturated stock solution (100 mg/L nominal concentration): 100% - 62.5% - 40.0% - 25% - 15% and 10% (expressed as % v/v saturated solution). The results were determined and expressed relative to the geometric mean of the measured concentrations in fresh and old solutions during the test period which were calculated to be 0 (control), 0.19, 0.25, 0.41, 0.65, 1.08 and 1.78 mg/L (according to OECD nº 23, 15/12/00). The test was conducted in darkness in closed tubes filled with 10 mL daphnia medium and without headspace. 4 replicates with 5 daphnids per replicate were performed. A validated analytical method based on HPLC/UV was used to monitor the concentration of the active ingredient in the test solutions. There was no immobilization of daphnia in the negative control and dissolved oxygen measured before and after renewal of solutions and at the end of the test was higher than 3 mg/L in control and test vessels. The immobilization of daphnia was 0, 0, 30, 55, 70, 100 and 100 % at 48 hours exposure at 0 (control), 0.19, 0.25, 0.41, 0.65, 1.08 and 1.78 mg/L. Based on these results, the 48h-EC50 was 0.40 mg/L (95% CL: 0.34 – 0.48).

Supporting study: In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of Daphnia magna were exposed to d-alpha pinene at concentrations of <32, 75.7, 223, 301, 407 and 542 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively. Treatment-related effects (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively. Based on these results, LC50 and EC50 could not be determined. Thus, two additional tests were performed. The test concentrations were 920 and 1965 µg/l, respectively. An approximate LC50 value of 1440 µg/l was calculated based on one mortality in the first test and total mortality in the second test.