p-mentha-1,3-diene

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:

March to April 2018

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)

Specific details on test material used for the study:

Identity: Alpha-terpinene
Batch/Lot Number: 83100001
CAS Number: 99-86-5
EC Number: 202-795-1
Scymaris Reference Number: 1027.005
Purity: 92.7%
Water Solubility: Immiscible
Appearance: Clear colourless liquid
Expiry Date: 11 May 2018
Storage Conditions: Room temperature

Analytical monitoring:

yes

Remarks:

Measured using GCMS

Details on sampling:

The concentrations of Alpha-terpinene in the test solutions detailed below were measured at 0 and 48 hours for each test concentration using GC-MS. Samples for analysis at 0 hours were taken from the excess test solutions of the dilution water control, solvent control and each test concentration and at 48 hours from a single test replicate solution or pooled replicates of the dilution water control, solvent control and each test concentration.

Dissolved oxygen and pH were measured in the control, solvent control and in each test substance concentration at the start of the test using the excess test solutions after filling the test vessels, from the 24 hour ‘aged’ test solutions (from one replicate vessel from each test concentration), from the 24 hour ‘fresh’ test solutions (using excess test solutions after filling the test vessels) and at the end of the test from one replicate vessel from each test concentration.

Temperature values were measured daily by thermometer in an additional replicate vessel (without D. magna), maximum and minimum temperature values were also recorded daily.

Vehicle:

yes

Remarks:

DMF (dimethylformamide)

Details on test solutions:

Range finding study.
The nominal test solutions during the range finding study were a Control, Solvent Control, 0.625, 1.25, 2.5, 5.0 and 10 mg/l. The range finding test was a static test over 48 hours.

Definitive test
The dilution water used for testing (and maintenance of stock cultures) was Elendt M4 medium. Physical and chemical analyses were performed on the dilution water used for testing. pH of the medium was within the range 6-9. Hardness of the medium will be between 140 and 250 mg/L (as CaCO3). The dilution water was aerated for >2 hours before use.


This study was run with a dilution water control and solvent control together with nominal concentrations of 0.0625, 1.25, 2.5, 5.0 and 10 mg/L. The test was performed under semi-static conditions, test solutions were renewed after 24 hours.
The 4 replicate test vessels will each receive approximately 200 mL of test solution and the remaining solution will be retained for physical and chemical analysis.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organism was the freshwater crustacean, D. magna, <24 hours old at the start of the test, and not first brood progeny, derived from continuous laboratory cultures. They were derived from a healthy culture (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.). The D. magna used for testing was cultured in the dilution water and under the same photoperiod and temperature conditions used for the study. Test organisms were fed (with a dual algal diet of Pseudokirchneriella subcapitata and Chlorella vulgaris) prior to the start of the test in order to minimise potential starvation effects during the pre-test holding period.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Elendt M4 medium

Total exposure duration:

48 h

Remarks on exposure duration:

The test was a semi static test with daphnia transferred to a fresh test medium at 24 hours.

Hardness:

140-250 mg CaCO3/l

pH:

6-9

Details on test conditions:

Materials in contact with the dilution water, test solutions, test substance and D. magna were, where practical, restricted to glass and unplasticised plastics. The test vessels were glass beakers of 250 mL nominal capacity (to contain 200 mL of test solution), with four replicates per test concentration. The beakers were covered with loose fitting glass panes and positions of the treatments will be randomised within the test area.

The test was performed under semi-static conditions. The test was initiated by the addition of 5 randomly selected D. magna in <2.0 mL of dilution water to each test vessel. D. magna were transferred to each test vessel using a disposable plastic pipette. At 24 hours, D. magna were transferred to ‘fresh’ test solutions using the same method. Each treatment contained a total of 20 D. magna.

At least five test concentrations were used and these were arranged in a geometric series with a separation factor not exceeding 2.2.

The test solutions were maintained in the range 18 - 22°C kept constant to within ± 1°C, and a photoperiod of 16 hours light: 8 hours dark, with a 20 minute dawn:dusk transition period was provided. Test solutions were not aerated and D. magna were not fed during the course of this study.

For a semi-static test, dissolved oxygen and pH were measured in the control, solvent control and in each test substance concentration at the start of the test using the excess test solutions after filling the test vessels, from the 24 hour ‘aged’ test solutions (from one replicate vessel from each test concentration), from the 24 hour ‘fresh’ test solutions (using excess test solutions after filling the test vessels) and at the end of the test from one replicate vessel from each test concentration.

The test was performed without pH adjustment, the pH should not vary by more than 1.5 pH units in any one study. If the pH did not remain in the range 6 – 9, the pH of the stock solution (or individual test solutions) was adjusted to that of the dilution water control using hydrochloric acid (HCl) or sodium hydroxide (NaOH).

Temperature values were measured daily by thermometer in an additional replicate vessel (without D. magna), maximum and minimum temperature values were also recorded daily.

Test concentrations showing 100% effect at 24 and 48 hours were measured for pH and dissolved oxygen at that time.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

In this 48 -hour semi-static test, Daphnia were exposed to alpha-terpenene dissolved in dimethylformamide at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/l (mean measured concentrations equivalent to 0, 0.21, 0.39, 0.97, 1.7, 5.3 mg/l, respectively). No significant effects on mortlity or immobilisation were observed in the bottom three doses. A 50% effect was observed at a nominal concentration of 5.0 mg/l (mean measured concentration of 1.7 mg/l). There was uncertainty in the analytical measurement at the top dose at 24 hours and therefore the results at the top dose were omitted from statistical analysis. As such, an EC50 of 1.7 mg/l (geometric mean measurement) and a NOEC of 0.97 mg/l (geometric mean measurement) has been reported for this study.

Table 1. Range Finding Test Biological Data.

Nominal concentration (mg/L)	Number of dead/immobile at 48 hours	Total dead/immobile	Total tested	Percentage immobility
Control	0	0	10	0
Solvent Control	0	0	10	0
0.625	0	0	10	0
1.25	0	0	10	0
2.5	0	0	10	0
5	1	1	10	10
10	0	0	10	0

Table 2. Range Finding Test Analytical results

Nominal concentration (mg/L)	0 hour measured concentration (mg/L)	0 hour measured conc. (% of nominal)	48 hour measured concentration (mg/L)	48 hour measured conc. (% of nominal)	Mean measured concentration (Geometric mean) (mg/L)	Mean measured concentration (Geometric mean) (% of nominal)
Control	<LOQ	-	<LOQ	-	-	-
Solvent Control	<LOQ	-	<LOQ	-	-	-
0.625	0.44	70	0.069	11	0.17	27
1.25	0.94	75	0.16	13	0.39	31
2.5	2.1	82	0.15	6	0.56	22
5.0	5.1	101	0.33	7	1.3	26
10	8.9	89	0.73	7	2.6	26

Table 3. Definitive Test Biological Results

Nominal concentration (mg/L)	Number of dead/immobile at 24 hours	Number of dead/immobile at 48 hours	Total dead/immobile	Total tested	Percentage immobility (48 hours)
Control	0	0	0	20	0
Solvent Control	0	0	0	20	0
0.625	0	1	1	20	5
1.25	0	0	0	19*	0
2.5	0	0	0	20	0
5.0	1	10	10	20	50
10	15	19	19	20	95

*Only 4 daphnids found in one replicate of 1.25 mg/L at 48 hours – assumed to have been missed during 24 h transfer to new test solutions.

Table 4. Definitive Test Analytical Results

Nominal concentration (mg/L)	0 hour measured conc. (mg/L)	0 hour measured conc. (% of nominal)	24 hour measured conc. (aged) (mg/L)	24 hour measured conc. (aged) (% of nominal)	24 hour measured conc. (fresh) (mg/L)	24 hour measured conc. (fresh) (% of nominal)	48 hour measured conc. (mg/L)	48 hour measured conc. (% of nominal)	Time-weighted mean conc. (mg/L)
Control	<LOQ	-	<LOQ	-	<LOQ	-	<LOQ	-	-
Solvent Control	<LOQ	-	<LOQ	-	<LOQ	-	<LOQ	-	-
0.625	0.33	54	0.065	10	0.55	87	0.095	15	0.21
1.25	0.62	50	0.16	13	1.0	83	0.15	12	0.39
2.5	1.2	48	0.52	21	2.3	94	0.45	18	0.97
5.0	2.1	42	0.68	14	4.2	85	0.82	16	1.7
10	5.2	52	1.9	19	20*	197*	1.7	17	5.3

*Although stirred for the same length of time as other test solutions before sampling, it is possible that this test solution was not completely homogenous when the analytical sample was taken, hence the high concentration. Unfortunately no further sample was available to confirm this result. Due to the uncertainty this value, the nominal 10 mg/L concentration was omitted from the statistical analysis. This did not affect the endpoints (EC50, LOEC and NOEC) as the 50% effect was in the nominal 5.0 mg/L concentration.

Validity criteria fulfilled:

not specified

Conclusions:

An EC50 of 1.7 mg/l (geometric mean measurement) and a NOEC of 0.97 mg/l (geometric mean measurement) has been reported for this study.

Executive summary:

The effects of Alpha-terpinene on the mobilisation of Daphnia magna was determined over 48 hours, according to OECD guideline 202. A EC50 of 1.7 mg/l and a NOEC of 0.97 mg/l (based on geometric mean measured concentrations) have been reported for this study.

Description of key information

Key study: Test method according to OECD 202, GLP study. The effects of Alpha-terpinene on the mobilisation of Daphnia magna was determined over 48 hours. An EC50 of 1.7 mg/l and a NOEC of 0.97 mg/l (based on geometric mean measured concentrations) have been reported for this study.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.7 mg/L