Terpenes and Terpenoids, sinpine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Remarks:

Daphnia magna (Crustacea, Cladocera)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January - 25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Terpenes and Terpenoids, sinpine

EC Number:

272-842-9

EC Name:

Terpenes and Terpenoids, sinpine

Cas Number:

68917-63-5

IUPAC Name:

Reaction mass of 1-methyl-4-(propan-2-ylidene)cyclohex-1-ene and 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene and 2,6,6-trimethylbicyclo[7.1.1]hept-2-ene and 1-isopropyl-4-methylcyclohexa-1,4-diene

Test material form:

not specified

Specific details on test material used for the study:

Identification
Sinpine P
IUPAC name
Reaction mass of 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 1-methyl-4-(propan-2-ylidene)cyclohex-1-ene and 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene and 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene
EC name
Terpenes and Terpenoids, sinpine
Empirical formula:
C10H16 and C10H18O *
Molecular weight:
136 g/mol and 154 g/mol *
CAS Number
68917-63-5
EC Number
272-842-9
Description
Pale yellow to yellow liquid
Batch
VE00319578

* the test item consists of unsaturated monoterpene hydrocarbons (C10H16, MW 136 g/mol) and oxygen-containing monoterpenes (C10H18O, MW 154 g/mol) in a typical ratio of 3.5:1.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. A single peak, which derives from an oxygen-containing monoterpene component of the test substance, was used for quantification.
Frequency at t=0 h and t=48 h
Volume 12 ml from the approximate centre of the test vessels
Storage Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead, samples were taken from one replicate of each group.
Additionally, reserve samples of 12 ml were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Sinpine P tested was a pale yellow to yellow liquid not completely soluble in test medium at the loading rates initially prepared. Since the test item consists of multiple components with different water solubility, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).
Preparation of test solutions was based on the results obtained in a pre-test performed under the same project number. Water Accommodated Fractions (WAFs) were prepared at loading rates ranging from 0.46 to 100 mg/l. All test solutions were prepared separately. A 24-hour period of magnetic stirring was applied to accelerate the dissolving of the test substance in the test medium. Thereafter, the obtained solutions were allowed to settle for a period of 1 hour. The aqueous WAFs were collected by siphoning and used as test solutions. The final test solutions were all clear and colourless.
During preparation the solutions were protected from light.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

ISO Medium

Total exposure duration:

48 h

Test conditions

Hardness:

The hardness: 180 mg/l expressed as CaCO3

Test temperature:

Temperature of medium 18-22°C

pH:

pH: 7.7 ± 0.3.

Nominal and measured concentrations:

WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6 and 10 mg/l

Details on test conditions:

Test duration 48 hours
Test type Static
Test vessels 50 ml, all-glass, closed airtight with minimum headspace
Medium Adjusted ISO medium
Number of daphnids 20 per concentration
Loading 5 per vessel containing 50 ml of test solution
Light Study was performed in dark
Feeding No feeding
Aeration No aeration of the test solutions.
Introduction of daphnids Within 20 minutes after preparation of the test solutions

Reference substance (positive control):

yes

Remarks:

Reference test using potassium dichromate to check sensitivity of test system performed 9-11 March 2015.

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: results based on nominal WAF loading rates

Details on results:

Since the test item consists of multiple components with different water solubility (i.e. unsaturated monoterpene hydrocarbons and oxygen-containing monoterpenes), the test was performed using Water Accomodated Fractions (WAF) and the effect parameters based on nominal loading rates.

TOC analysis and chemical specific analysis (based on an oxygen-containing component) demonstrated that exposure to the WAF was maintained throughout the 48h exposure period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the 48h-EL50 was 2.8 mg/l based on nominal WAF loading rates.

Executive summary:

Acute Toxicity Study in Daphnia magna with Sinpine P. The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000. The batch of Sinpine P tested was a pale yellow to yellow liquid not completely soluble in test medium at the loading rates initially prepared. Since the test item consists of multiple components with different water solubility (i.e. unsaturated monoterpene hydrocarbons and oxygen-containing monoterpenes), for the purposes of the test, the test item was prepared as a Water Accomodated Fraction (WAF). A final test was performed based on the results of a combined limit/range-finding test. Water Accommodated Fractions (WAFs) were prepared at loading rates ranging from 0.46 to 10 mg/l and used as test concentrations. The final test solutions were all clear and colourless. During preparation the solutions were protected from light. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/l. The total exposure period was 48 hours and samples for analytical confirmation of Sinpine P (based on an oxygen-containing component) and TOC concentrations were taken at the start and at the end of the test to demonstrate that exposure to the WAF was maintained. The exposure was performed in airtight closed vessels and in dark due to volatility and light sensitivity of Sinpine P. The actual concentrations measured at the start of the test increased with the applied dose from 0.30 mg/l at the lowest to 9.4 mg/l at the highest WAF. The measured concentrations remained stable during the test (95-101% of initial). Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the effect parameters were based on nominal loading rates. The study met the acceptability criteria prescribed by the protocol and was considered valid. Under the conditions of the present study, the 48h-EL50 was 2.8 mg/l based on loading rates initially prepared.