Savory, Satureja hortensis, ext.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 – 7 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

Deviations from the guidline were related to the time for autoclaving the stock solutions, the temperature during pre-culture incubation and in the experiment. All deviations can be considered uncritical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

Deviations from the guidline were related to the time for autoclaving the stock solutions, the temperature during pre-culture incubation and in the experiment. All deviations can be considered 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 CHEMICALS, adopted 06 Jul. 2018

Version / remarks:

06 July 2018

Deviations:

yes

Remarks:

Deviations from the guidline were related to the time for autoclaving the stock solutions, the temperature during pre-culture incubation and in the experiment. All deviations can be considered uncritical.

GLP compliance:

yes (incl. QA statement)

Remarks:

The carbon content of the test item (82.21%) was determined by elemental analysis under non-GLP conditions. The concentrations to be tested are based on non-GLP pre-tests.

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:

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: Room Temperature (20 ± 5 °C)
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:

For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.470 mL) to achieve a cell concentration of 2.5*103 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.822 mL). In this mixture, the pH-value was measured.
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of the test. Samples for the analytical determination were taken from the replicates without algae.

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 1.0 / 3.3 / 10.0 / 32.0 / 100.0 mg/L resp. 1.1 / 3.6 / 10.9 / 34.7 / 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 algal medium (demineralised water enriched with minerals but without algae) and stirring moderately for 24 hours. The lower phase was used unfiltered as test solutions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 23.8 – 25.5°C). The resulting culture grew exponentially. The light intensity is checked monthly and recorded in a control chart. The lighting corresponded to the specified range of 4440 - 8880 lux. The light intensity is around 5000 lux.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter.

Post exposure observation period:

Not available

Test conditions

Hardness:

Data not available

Test temperature:

19.3 – 22.8 °C

pH:

Nominal Concentration in mg/L 0h 72h
Blank control 7.9 8.8
1 7.8 8.7
3.2* 5.9 5.0
10 7.5 8.2
32 8.0 7.6
100 8.1 7.5

Salinity:

No data available

Conductivity:

No data available

Nominal and measured concentrations:

Nominal Calculated Calculated % of Nominal % of Nominal
Concentration Concentration Concentration concentration concentration
Test Item Test Item Test Item
t = 0 h t = 72 h t = 0 h t = 72 h
mg/L mg/L mg/L % %
Blank control -- -- -- --
1 0.3 0.8 31 81
3.2 1.5 1.8 46 55
10 4.7 4.2 47 42
32 16.3 11.0 51 34
100 52.7 38.9 53 39

Details on test conditions:

Details on test conditions

Date: 04. – 07. June 2019
Treatments tested:1 / 3.2 / 10 / 32 / 100 mg/L
resp. 1.1 / 3.5 / 10.9 / 34.7 / 108.6 µL/L based on a density
of 0.921 g/mL (nominal concentration)
The concentrations to be tested are based on non GLP
pre-tests
Number of replicates:6 replicates for the blank control
3 replicates for each treatment
1 replicate for each treatment and blank control without al-
gae (for analytical determination)
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: within the specified range (4440 – 8880 Lux)
The light intensity is checked monthly and recorded in a
control chart. The lighting corresponded to the specified
range of 4440 - 8880 lux. The light intensity is around
5000 lux.
Temperature: 19.3 – 22.8 °C (see chapter 13 Deviations)
Replicates (Blank control): 45 ±1 mL algal medium and algae
Replicates (Treatments): 45 ±1 mL test solution and algae

Reference substance (positive control):

yes

Remarks:

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 13.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

>= 5.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

>= 3.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 12.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 6.3 <= mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Details on results:

Significant inhibition of algal growth was observed at the following concentrations:
10 – 100 mg/L (for yield)
32 – 100 mg/L (for growth rate)
Treatment 3.2 mg/L showed a turbidity of all replicates after 24 h incubation. Therefore, determination of the cell number was biased, which result to wrong, much too high inhibition values. In addition, a much lower pH value was measurable than in the other treatments and the blank control. This could be due to contamination. For this reason, this treatment was not used for evaluation. With the other four concentrations a clear dose-effect relationship was observed.
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Unusual cell shape: normal cell shape
- Colour differences: no available
- Flocculation: no available
- Adherence to test vessels: no available
- Aggregation of algal cells: no available
- Other:
- Any stimulation of growth found in any treatment: no observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:contamination
- Effect concentrations exceeding solubility of substance in test medium:no found

Results with reference substance (positive control):

Growth rate of K2Cr2O7 EC50 0.65-1.10 mg/L ; yield EC50- 0.21-0.66 mg/L

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in chapter 20 Annex 6: Statistical calculation using ToxRat® Professional 3.3.0.

Any other information on results incl. tables

Parameter			Value		95 % confidence interval
NOEC (Growth Rate) 72 h			4.40 mg/L		--
NOEC (Yield) 72 h			0.50 mg/L		--
LOEC (Growth Rate) 72 h			13.40 mg/L		--
LOEC (Yield) 72 h			4.40 mg/L		--
72h	ErC10		5.48 mg/L		4.31 – 6.97 mg/L
72h	EyC10		3.56 mg/L		2.58 – 4.91 mg/L
72h	ErC50		12.12 mg/L		9.13 – 15.94 mg/L
72h	EyC50		6.30 mg/L		4.05 – 9.58 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One 72 h valid experiment was performed with Desmodesmus subspicatus.
1. The growth rate µ and the yield2 determined from the cell number at the respective observation times showed significant inhibition of algal growth at the following concentrations:
10 – 100 mg/L (for yield)
32 – 100 mg/L (for growth rate).

Endpoint NOEC LOEC EC10 EC50
Growth Rate 4.40 mg/L 13.40 mg/L 5.48 mg/L 12.12 mg/L
Yield 0.50 mg/L 4.40 mg/L 3.56 mg/L 6.30 mg/L

2. Due to contamination and a much lower pH value measured than in the other treatments and the blank control, the treatment 3.2 mg/L was not used for evaluation. With the other four concentrations a clear dose-effect relationship was observed.
3. At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the total organic carbon (TOC) content in the test solutions using a carbon analyser. The measured concentrations lay between 31 % and 53 % of the nominal concentrations at the beginning of the test and between 34 % and 81 % of the nominal concentrations at the end of the test. Because the measured concentrations were not in the range ± 20 % of the nominal values, the determination of the results was based on the geometric mean of the measured concentrations (see § 39 OECD 201).
4. The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.
5. The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.9 units in the blank control.
6. All validity criteria were met.
7. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Executive summary:

The freshwater green algae Desmodesmus subspicatus was chosen as a typical species of phytoplankton in order to evaluate the toxicity of Summer savory oil.

The system response was the reduction of growth in a series of algal cultures (test units) exposed to the test item. The response was evaluated as a function of the exposure concentration in comparison with the average growth of replicate, unexposed blank control cultures. For full expression of the system response to toxic effects (optimal sensitivity), the cultures were allowed unrestricted exponential growth under nutrient sufficient conditions and continuous light for a sufficient period of time to measure reduction of the specific growth rate.

A positive control potassium dichromate K2Cr2O7(CAS No. 7778-50-9) was used as positive control in a separate reference test to assure that the test conditions are reliable.

The following results for the test item Summer savory oil were determined:

 

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	4.40 mg/L	13.40 mg/L	5.48 mg/L	12.12 mg/L
Yield	0.50 mg/L	4.40 mg/L	3.56 mg/L	6.30 mg/L

Therefore, this toxicity study is considered acceptable and meets validity criteria according to OECD 201.