Schinus molle, ext.

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:

21 February 2018 - 24 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)

Version / remarks:

April 13, 2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15, 2000

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Schinus molle oil
Batch No.: 0008691103
Purity: 100% UVCB
Certificate of Analysis Date: December 19, 2017
Aggregate State at Room Temperature: Liquid
Colour: Colorless to pale yellow
Relative Density D20/4: 0.8475
Expiry Date: 28-Jun-2018
Storage Conditions at Test Facility: At 20 +/- 5 °C, in the dark

Analytical monitoring:

yes

Remarks:

TOC analysis

Details on sampling:

Sampling:
The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment. The samples remained undiluted until analysis.

Storage:
All samples were stored in a fridge (4°C ± 4°C), protected from light until analysis was performed. Afterwards the samples were again stored cooled (4°C ± 4°C) and will be kept stored up to the date of the final report.

Analyses:
The dissolved fraction of the test item Schinus molle oil was analysed in the duplicate test media samples from all test concentrations and the control samples from both sampling times (0 and 48 hours).

Vehicle:

no

Details on test solutions:

The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. A defined amount of the test item was added directly to the test water for each loading rate and was carefully stirred for 72 hours in the dark to dissolve as much of the test item as possible. The highest test item loading rate of 100 mg test item/L was prepared by mixing 124.8 μL test item into 1060 mL test water, for the test item loading rate of 50 mg test item/L, 62.4 μL test item were mixed into 1060 mL test water, for the loading rate of 25 mg test item/L, 31.2 μL were mixed into 1060 mL test water. The loading rate of 12.5 mg test item/L was prepared by mixing 15.6 μL into 1060 mL test water and for 6.25 mg test item/L, 18.4 μL were mixed into 2500 mL test water. After cessation of mixing and a following period (one hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal loading rate. The test media were prepared just before introduction of the daphnids (= start of the test).

Appearance of the Test Item in Test Medium: There were no remarkable observations

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 3 to 22 hours old
Sex: Female
Origin: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

Test start: 19.4 to 20.1 °C
Test End: 18.6 to 19.2°C

pH:

Test start: 7.7 to 8.0
Test End: 7.8 to 7.9

Dissolved oxygen:

Test start: 8.4 to 8.9 mg/L
Test End: 8.8 to 8.9 mg/L

Nominal and measured concentrations:

Nominal loading rates: control, 6.25, 12.5, 25, 50 and 100 mg test item/L

TOC based on mean value of all measured samples per treatment group(n=2). The results were corrected by the mean value of the control.

TOC (t=0 hr) n.a, 0.589*, 2.13, 5.40, 8.16 and 16.1 mg/L
TOC (t=48 hr) n.a, 0.568*, 1.81, 5.30, 8.17 and 15.7 mg/L

n.a.: not applicable
* TOC content of this loading

Details on test conditions:

TEST SYSTEM
- Type and Size: Glass beakers of approximately 110 mL volume containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some mL), kept closed during the whole period of the study with conical glass stoppers to avoid loss of the test item due to volatilisation.
- Introduction of Daphnids: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in approximately 110 mL test medium
- Replicates: The test was performed with four replicates per treatment group.
- Exposure Time: 48 hours
- Test Procedure: A static test was performed.

TEST MEDIUM:
- Reconstituted Water (Elendt "M4")

TEST CONDITIONS:
- Measurement of pH, Dissolved Oxygen and Water Temperature: The water temperature, pH-values and dissolved oxygen concentrations were determined at - test start and test end in each treatment group.
- Light Regime: 16 h light : 8 h dark
- Light Intensity: The light intensity was 160 to 600 Lux (measured once during the test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility: The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

RANGE-FINDING STUDY
- Non-GLP pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

9.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: <6.25 - 17.4 mg/L

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

9.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Cl: <6.25-17.4 mg/L

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and including the loading rate of 6.25 mg test item/L. At the higher loading rates of 12.5, 25, 50 and 100 mg test item/L, all 20 daphnids were immobile. WAFs were considered properly prepared, based on a dose dependent increase of TOC concentrations with loading. Exposure concentrations were considered stable over the test period based on TOC analyses (≥ 85% of initial TOC at 48h, based on quantified loadings). Therefore all reported results refer to nominal loading rates.

Control Immobilisation Rate: Was 0 % and furthermore no daphnid showed signs of disease or stress; thus the validity criterion was met.
Dissolved Oxygen Concentration: Was ≥ 8.8 mg O2/L in the control and test vessels at the end of the test; thus validity criterion was met.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate performed in June 2017 the EC50 after 24 hours was determined to be 1.58 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EL50, EL20 and EL10 and the 95 % confidence limits were calculated by Weibull analysis. The NOEL and LOEL after 24 and 48 hours were determined directly from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Influence of Schinus molle oil on the Mobility of Daphnia magna

Nominal loading rate [mg test item/L]	% of immobilised Daphnia
	24 h	48 h
Control	0	0
6.25	0	0
12.5	100	100
25	100	100
50	100	100
100	100	100

Validity criteria fulfilled:

yes

Remarks:

<10% immobilization in control group, O2 conc. at end is >=3 mg/L in control and test vessels

Conclusions:

The acute 48-hour EL50 value of Schinus molle oil for Daphnia magna was 9.73 mg test item/L based on nominal values.

Executive summary:

A study was performed to assess the acute toxicity of Schinus molle oil in Daphnia magna under static conditions in closed vessels. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP principles. Groups of twenty daphnids (less than 24 hours old) were exposed for 48 hours to five concentrations of the substance dispersed in test water via WaterAccomodated Fraction procedure and an untreated control. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours. WAFs were considered properly prepared, based on a dose dependent increase of TOC concentrations with loading. Exposure concentrations were considered stable over the test period based on TOC analyses (≥ 85% of initial TOC at 48h, based on quantified loadings). Therefore all reported results refer to nominal loading rates.The 48-hour EL50 value was determined as 9.73 mg test item/L.

Description of key information

A study was performed to assess the acute toxicity of Schinus molle oil in Daphnia magna under static conditions in closed vessels. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP principles. Groups of twenty daphnids (less than 24 hours old) were exposed for 48 hours to five concentrations of the substance dispersed in test water via WaterAccomodated Fraction procedure and an untreated control. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours. WAFs were considered properly prepared, based on a dose dependent increase of TOC concentrations with loading. Exposure concentrations were considered stable over the test period based on TOC analyses (≥ 85% of initial TOC at 48h, based on quantified loadings). Therefore all reported results refer to nominal loading rates.The 48-hour EL50 value was determined as 9.73 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9.73 mg/L

Additional information

Result relates to nominal loading rate, hence is expressed as EL50.