Oligomerisation products of alpha-pinene and beta-pinene

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 2018-02-05 to 2018-02-15

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

Remarks:

signed on 2017-01-10

Specific details on test material used for the study:

Not applicable.

Analytical monitoring:

yes

Details on sampling:

Duplicate samples for analysis were taken from the control and the limit test loading rate at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). The quantification method was targeted on the terpenic dimers representing 79.2% of the test item and very probably the most soluble constituent.
A liquid-liquid extraction of the water samples was carried out in order to pre-concentrate by a factor of ten to lower the limit of quantification.

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each mixing vessel and 5.2 to 5.3 L of test water (depending on the brim capacity of the bottle) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessel were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 +/- 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless. The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin:LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202.

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3).

Test temperature:

The temperature of the test medium was situated between 20.7 and 20.9°C throughout the test (average value: 20.8°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

pH:

pH: 7.80 - 8.53
pH requirements: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

8.36 - 9.07
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Limit test : 1.0 mg test item/L loading rate nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (control and loading rate of 1.0 mg.L-1), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202.

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

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all treatment group.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Range finding study: 5 daphnids per vessel, in duplicate were exposed to the nominal loading rates 1, 100 and 1000 µg.L-1 and to a control.
- Results used to determine the conditions for the definitive study:, immobilisation rates were 0% at at 1000 µg.L-1 after 24 hours and 48 hours of exposure.

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loadig rate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rate

Details on results:

The evaluation was based on the nominal WAFs concentrations.
After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 1.0 mg.L-1.
Based on these results, the 24 and 48-hour EL50 values were therefore > 1.0 mg.L-1.

Results with reference substance (positive control):

On August 17, 2017 (KD17-003; most recent test), the 24h-EC50 was 0.92 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

Not applicable.

Biological results

Acute immobilisation of daphnids after 24 and 48 hours in the final test.

Nominal concentration*(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.0	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0

Analytical results

The results of analysis of the samples taken during the final test are described in the Table I of the appended Analytical Report (Appendix IV). Samples taken fromthe control and the loading rate of 1.0 mg.L^-1were analysed at the start ofthe test (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Due to the very low solubility of the test item in test water, it was not possible to quantify and detect the presence of test item in the limit test loading rate of 1 mg.L^-1.However, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on the nominal test loading rates.

Water quality parameters and environmental conditions throughout the test

pH-values during the final test

		Nominal concentration (mg test item.L^-1)*
		Control	1
Start t=0h		8.53	8.35
t=24h	Old	8.07	7.8
	Fresh	8.35	8.14
End t=48h		8.03	8.04

Dissolved oxygen concentrations (mg.L^-1) during the final test

		Nominal concentration (mg test item.L^-1)*
		Control	1
Start t=0h		8.77	8.64
t=24h	Old	8.97	8.6
	Fresh	8.99	9.07
End t=48h		8.87	8.36

Validity criteria of the study:

Controls In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [0₂] Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels. 

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item TERPENIC OLIGOMERS to freshwater invertebrate Daphnia magna was investigated in a closed semi-static limit test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 was estimated to be higher than 1.0 mg.L-1. Moreover, the highest nominal loading rate without observed effects value was estimated to be at least 1.0 mg.L-1.

Executive summary:

A study was performed to assess the acute toxicity of test item TERPENIC OLIGOMERS to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 1.0 mg test item.L^-1(well above the water solubility of the test item) and to a control.

The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item were determined by chemical analysesat the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Due to the very low solubility of the test item in test water, it was not possible to quantify and detect the presence of test item in the limit test loading rate of 1.0 mg.L^-1. However, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on the nominal test loading rates.

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 1.0 mg.L^-1.

Therefore, the 48-hour EL50 was estimated to be higher than 1.0 mg.L^-1 and the highest nominal loading rate without observed effects value was estimated to be at least 1.0 mg.L^-1.

Description of key information

A study was performed to assess the acute toxicity of test itemTERPENIC OLIGOMERStoDaphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphniasp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Twenty daphnids (four replicates, five daphnids per replicate) were exposed toWater Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 1.0 mg test item.L^-1(well above the water solubility of the test item) and to a control.

The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item were determined by chemical analysesat the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).Due to the very low solubility of the test item in test water, it was not possible to quantify and detect the presence of test item in the limit test loading rate of 1.0 mg.L^-1.However, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based onthe nominaltest loading rates.

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 1.0 mg.L^-1.

Additional information

The 48-hour EL50 was estimated to be higher than 1.0 mg.L^-1and the highest nominal loading rate without observed effects value was estimated to be at least 1.0 mg.L^-1.