Savory, Satureja hortensis, ext.

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:

3-6 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:

OECD Guideline for Testing of Chemicals No. 202, adopted 13. Apr. 2004: ”Daphnia sp., Acute Immobilisation Test“

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 20.0 – 23.8 °C, and therefore not in the desired range (18.0 – 22.0°C) and fluctuated by more than ± 1 °C. As no immobility was observed in the blank control this was stated as uncritical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No. 440/2008, Method C.2. “Daphnia sp. Acute Immobilisation Test”, adopted 30. May 2008

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 20.0 – 23.8 °C, and therefore not in the desired range (18.0 – 22.0°C) and fluctuated by more than ± 1 °C. As no immobility was observed in the blank control this was stated as uncritical.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD guidance document no. 23, second edition, GUIDANCE DOCUMENT ON AQUEOUS-PHASE AQUATIC TOXICITY TESTING OF DIFFICULT TEST CHEMI-CALS, adopted 06. Jul. 2018

Version / remarks:

06 July 2018

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 20.0 – 23.8 °C, and therefore not in the desired range (18.0 – 22.0°C) and fluctuated by more than ± 1 °C. As no immobility was observed in the blank control this was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Remarks:

The organic carbon content of the test item (82.21 %) was determined by elemental analysis under non-GLP conditions (average of 2 measured values). The concentrations to be tested are based on the result of a non-GLP pre-test.

Test material

Test material information

1

Reference substance name:

Savory, Satureja hortensis, ext.

EC Number:

283-922-8

EC Name:

Savory, Satureja hortensis, ext.

Cas Number:

84775-98-4

IUPAC Name:

Essential oil of Savory, Satureja hortensis, obtained from whole, dried herb by steam distillation

Test material form:

liquid

Specific details on test material used for the study:

Designation in Test Facility: 19032806G
Date of Receipt: 28. Mar. 2019
Condition at Receipt: Room temperature, in proper conditions
The following information concerning identity and composition of the test item was provided by the sponsor.
Name: Summer savory oil
Batch no.: SSV1.ES.sample
Appearance: clear, light-yellow liquid
Composition: see the substance identification profile (SIP)
CAS No.: 8016-68-0
EC-No.: 283-922-8
Molecular formula: unknown
Molecular weight: unknown
Purity: Not applicable, UVCB sub-type 3 (natural complex substance (NCS)
Homogeneity: homogeneous
Vapour pressure: 115.04 Pa (calculated based on partial vapour pressure of 11 main constituents vapour pressure)
Stability in solvents: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility in solvents: H2O: 0.001 – 1.25 g/L (calculated on the basis of the range of calculated or measured values of components identified) ; EtOH: > 1 g/L; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date: 08. Jan. 2019
Expiry date: 08. Jan. 2021
Storage: The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5 °C).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Selection of Daphnia: 18 hours and 10 minutes before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settlingin period, animals which showed no apparent damage were used for the test.
Deviation: no acclimation period of 48 h was performed, because no any detrimental effects on Daphnia were obtained switching animals from M4-medium (husbandry) to Dilution water (test). This deviation from OECD Guideline No. 202 and EU-Method C.2, can be considered uncritical.
Study performance: Using a glass tube, the Daphnia were caught and lifted from the beaker. They were put on a small sieve, and the medium surrounding the animals was sucked off using absorbent paper. Immediately after that, the animals were put into the respective test solution.
The test vessels were left to stand for 48 hours. After 24 and 48 hours, the immobilised daphnia were counted. The daphnia are considered as being immobilised when they do not move for 15 seconds after the test vessel was gently shaken. During observation, no anomaly in behaviour or appearance besides immobilisation was recorded. Daphnia which are trapped at the surface of the test solution are also considered immobilised.
The pH, the concentration of dissolved oxygen and the content of TOC in the test vessels were measured at the beginning and at the end of the test.

Test solutions

Vehicle:

yes

Details on test solutions:

The water-accommodated fractions (WAF) were prepared for the test. The test item was pipetted under the hood. This was done by mixing the nominal loads of 4.6 / 10.0 / 22.0 / 46.0 / 100.0 mg/L resp. 5.0 / 10.9 / 23.9 / 49.9 / 108.6 µL/L test item (based on a density of 0.921 g/mL stated in the SDS provided by the sponsor) with the corresponding amount of dilution water and stirring moderate for 24 hours. The lower phase was used unfiltered as test solutions.The water-accommodated fractions (WAF) were prepared for the test. The test item was pipetted under the hood. This was done by mixing the nominal loads of 4.6 / 10.0 / 22.0 / 46.0 / 100.0 mg/L resp. 5.0 / 10.9 / 23.9 / 49.9 / 108.6 µL/L test item (based on a density of 0.921 g/mL stated in the SDS provided by the sponsor) with the corresponding amount of dilution water and stirring moderate for 24 hours. The lower phase was used unfiltered as test solutions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna
Authority: STRAUS
Strain: Berlin
Sex: female
Age: between 0 and 24 hours
Origin: Umweltbundesamt Berlin
In-house breeding since: 27. September 2007
Selection of the test system was made following the proposal of the guidelines.
Animal Husbandry
Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), version 12 from 02. Feb. 2015.
Vessels: preserving glasses, nominal volume 2 L
Medium: M4-Medium (recipe of ELENDT)
Food: green algae (Desmodesmus subspicatus)
Medium renewal: twice a week
Photo period: 16/8 hours, using neon tubes
Temperature: 20 ±2°C
18 hours and 10 minutes before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediate-ly placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test.
Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia, therefore no acclimation period of 48 h was performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which can be considered uncritical.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

For each test concentration and the blank control, 20 Daphnia were exposed to the test item for 48 hours in a static test system and after 24 and 48 hours, the immobilised Daphnia were counted.

Post exposure observation period:

Not available

Test conditions

Hardness:

Data not available

Test temperature:

20.0 – 23.8 °C

pH:

Nominal Concentration in mg/L pH
0 h 48 h
Blank control 7.7 7.7
4.6 7.7 7.7
10 7.7 7.7
22 7.7 7.7
46 7.8 7.7
100 7.8 7.7

Dissolved oxygen:

Nominal Concentration in mg/L O2-Concentration in mg/L
0h 48h
Blank control 9.1 8.5
4.6 8.7 8.5
10 8.7 8.5
22 8.7 8.5
46 8.7 8.5
100 8.6 8.3

Salinity:

Data no available

Conductivity:

Data no available

Nominal and measured concentrations:

At the start and at the end of the test, the content of the test item in the test solutions was determined using measurement of total organic carbon (TOC). The measured concentrations lay between 25 % and 57 % of the nominal concentrations at the beginning of the test and between 16 % and 38 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see § 23 OECD 202).
Nominal Concentration Test Item mg/L Geometric Mean of measured concentrations mg/L
Blank control -
4.6 1.4
10 2.0
22 8.9
46 20.2
100 46.2

Details on test conditions:

Date of performance: 04. – 06. June 2019
Treatments 4.6 / 10 / 22 / 46 / 100 mg/L resp. 5.0 / 10.9 / 23.9 / 49.9 / 108.6 µL/L based on a density of 0.921 g/mL (nominal concentration)
The concentrations to be tested are based on the result of a non-GLP pre-test.
Temperature: 20.0 – 23.8 °C
Duration: 48 hours
Observation times: 24 and 48 hours
Medium renewal: none
Test vessels: glass beakers, nominal volume 50 mL, tall shape
Replicates (Treatments) 4 vessels, each containing 20 ± 5 mL test solution and 5
daphnia
Replicates (Blank control) 4 vessels, each containing 20 ± 5 mL dilution water and 5 daphnia
Feeding: no feeding during the test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control. The 24h-EC50 value was determined as 1.48 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of K2Cr2O7 as specified in the OECD guideline.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

>= 8.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

>= 8.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

>= 5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

All concentrations showed toxicity between 5 and 100% immobilisation. None of the animals was immobilised in the blank control.
- Behavioural abnormalities: immobilisation
- Observations on body length and weight:no observed
- Other biological observations:discoloration
- Mortality of control: no obtained
- Other adverse effects control:no obtained
- Immobilisation of control:no observed
- Abnormal responses: unusual behaviour such as trapping in the surface of water
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no obtained
- Effect concentrations exceeding solubility of substance in test medium:no observed

Results with reference substance (positive control):

Results with reference substance (positive control) K2Cr2O7 (CAS No. 7778-50-9)
- Results with reference substance valid? yes
- Relevant effect levels:yes
- Limit test:
- Dose-response test: yes
- ECx: EC50 at 24h =1.48mg/L between 0.6 - 2.1 mg/L as specified in the OECD guideline.

Reported statistics and error estimates:

The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in chapter 17 Annex 4: Statistical calculation using ToxRat® Professional 3.3.0.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment with 5 concentrations (nominal concentration) ranging from 4.6 to 100 mg/L was performed. For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.
All concentrations showed toxicity between 5 and 100% immobilisation. None of the animals was immobilised in the blank control.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.
At the start and at the end of the test, the content of the test item in the test solutions was determined using measurement of total organic carbon (TOC). The measured concentrations lay between 25 % and 57 % of the nominal concentrations at the beginning of the test and between 16 % and 38 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see § 23 OECD 202).

The following results were determined for the test item Summer savory oil (species: Daphnia magna).
48h-NOEC = 2.00 mg/L
48h-LOEC = 8.90 mg/L
24h-EC50 = 8.25 mg/L
48h-EC50 = 5.20 mg/L

N.B. According to the guideline, LOEC is determined by comparing of the respective treatment with the blank control. Statistically significant variation is considered as “lowest observed effect”, although the EC50 which is read from the graph toxicity vs. concentration

Executive summary:

In order to evaluate the toxicity of the test item Summer savory oil in an acute immobilisation test under static conditions towards Daphnia magna was performed.

Daphnia magna Straus was chosen in the guideline as a typical species of zoo-plankton.

Young daphnia, aged less than 24 hours at the start of the test, were exposed to the test item for a period of 48 hours. Immobilisation was recorded at 24 hours and 48 hours and compared with control values. Not more than 10% of the control Daphnia show immobilisation or other signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water that was in consistence with OECD 202.The results were analysed in order to calculate the EC50at 24 h and at 48 h. 

A positive control with K2Cr2O7 was tested in a separate study to assure that the test conditions are reliable.

The following deviations from the Study Planand Guidline were observed:

 - The temperature in the experiment was in the range 20.0 – 23.8 °C, and therefore not in the desired range (18.0 – 22.0 °C). As no immobility was observed in the blank control this was stated as uncritical.

 -  The temperature in the experiment was in the range of 20.0 – 23.8 °C and therefore fluctuated by more than ± 1 °C. As no immobility was observed in the blank control this was stated as uncritical.

The result of the test is considered valid because all validity criteria were met: Immobilisation in the controls was 0 % and the concentration of dissolved oxygen at the end of the test must be at least 3 mg/L (the lowest concentration of dissolved oxygen at the end of the test was 8.3 mg/L).