Essential oil of Blue tansy obtained from the flowers and leaves of Tanacetum annuum (Asteraceae) by steam distillation

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 2019 - 25 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

June 6, 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"

Version / remarks:

February 08, 2019

Test guideline 4

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

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Tanacetum annuum

IUPAC Name:

Essential oil of Tanacetum annuum

Test material form:

liquid

Sampling and analysis

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 Blue Tansy 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).

Test solutions

Vehicle:

no

Details on test solutions:

Dosage of Test Item:
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 were tested.
Therefore, defined amounts of the test item were added directly to the test water for each test concentration and were carefully stirred for approximately 24 hours in the dark to dissolve as much of the test item as possible. The highest loading rate of 70 mg test item/L was prepared by mixing 142.0 mg test item into 2028.57 mL test water, for the loading rate of 31.8 mg test item/L, 65.0 mg test item were mixed into 2044.03 mL test water, for the loading rate of 14.5 mg test item/L, 29.3 mg were mixed into 2020.69 mL test water. The loading rate of 6.6 mg test item/L was prepared by mixing 13.4 mg into 2030.3 mL test water , the loading rate of 3 mg test item/L , 15.7 mg test item were mixed into 5233.33 mL test water and for 1.4 mg test item/L, 14.8 mg were mixed into 10571.43 mL test water. After mixing followed a period of 15 minutes of settling to allow phase separation. At the highest loading rate of 70 mg/L emulsion drops stayed in the water phase even after an one hour settling period. The highest loading rate of 70 mg/L was therefore filtered with a folded filter. The test media of all lower loading rates were withdrawn from a tap, which is located approx. 2 cm above the bottom after first flushing the tap. These accommodated fractions were used as test media for the test. The test media were prepared just before introduction of the daphnids (= start of the test).

Control: In the control, test water was used without addition of the test item.

Appearance of the Test Item in Test Medium: At the highest loading rate of 70 mg test item/L a colouration of the test medium was observed.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 0.5 to 19.5 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 (see 6.5). 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/L as CaCO3

Test temperature:

Test start: 19.8 to 20.5 °C
Test End: 19.6 to 19.8 °C

pH:

Test start: 7.8
Test End: 7.8

Dissolved oxygen:

Test start: 8.9 to 9.2 mg/L
Test End: 8.9 to 9.0 mg/L

Nominal and measured concentrations:

Nominal loading rates of 1.4, 3.0, 6.6, 14.5, 31.8 and 70 mg test item/L and a control.

Toc based on control,
TOC (t=0hr): TOC (t=48 hr):
n.a.: not applicable
* values below LOQ, indicative only

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 loading rate, divided into 4 groups of 5 animals, each group in 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 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 190 to 430 lux (measured once during the test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility: The mobility of the daphnids were determined by visual observation after 24 and 48 hours. 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
In a solubility pre-experiment WAFs of 100 mg/L with stirring periods of 24, 48, 72 and 96 h were analytically measured. The stirring period had no effect on the TOC content and therefore a stirring period of 24 h for the definitive test was determined avoiding any chemical processes during the stirring period and reduce volatility.

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

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

8.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 7.03 - 9.83

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

determined directly from the raw data

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

20.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Cl: 16.0 - 26.2

Details on results:

After 48 hours of exposure no immobilisation of the test daphids were observed in the control and up to and including the nominal loading rate of 3.0 mg test item/L. At the loading rate of 6.6 mg test item/L, five daphnids were immobile and 19 daphnids were immobile at the loading rate of 14.5 mg test item/L. At the loading rates of 31.8 and 70 mg test item/L all daphnids were immobile. WAFs were prepared correctly because of the dose dependent increase of TOC with increasing loading rate. Further the stability of the exposure based on TOC during the test (e.g. no volatilisation) can be concluded from the highest two 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 ≥ 8.5 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 February 2019, the EC50 after 24 hours was determined to be 0.932 mg test item/L. The results indicate 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 probit 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.

Any other information on results incl. tables

Influence of Blue Tansy Oil on the Mobility of Daphnia magna

Nominal loading rate [mg test item/L]	% of immobilised Daphnia
	24 h	48 h
Control	0	0
1.4	0	0
3.0	0	0
6.6	0	25
14.5	40	95
31.8	80	100
70	90	100

Applicant's summary and conclusion

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 Blue Tansy Oil for Daphnia magna was determined to be 8.31 mg test item/L based on nominal values.

Executive summary:

A study was performed to assess the acute toxicity of Blue Tansy Oil to Daphnia magna under static conditions in closed vessel design. The study was conducted in accordance with OECD Guideline 202 and GLP principles. Water Accommodated Fractions (WAFs) of Blue Tansy Oil were individually prepared at loading rates of 1.4, 3.0, 6.6, 14.5, 31.8 and 70 mg test item/L and used as test concentrations.Groups of twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to each WAF. The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses were taken at the start and at the end of the test. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. WAFs were considered properly prepared, based on a dose dependent increase of TOC concentrations with loading rate. Exposure concentrations were considered stable over the test period based on TOC analyses concluded from the two highest loading rates. Therefore all reported results refer to nominal values. The study met the acceptability criteria prescribed and the 48h-EL50 for Daphnia magna exposed to Blue Tansy Oil was 8.31 mg/L based on nominal values (95% confidence interval between 7.03 and 9.83 mg/L).