Volatile oil of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by selective supercritical CO2 extraction

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:

March 2018 - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

adopted March 23, 2006, corrected July 28, 2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

August 24, 2009

Test guideline 3

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

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(3R)-3-(6-methylhept-5-en-2-yl)-6-methylidenecyclohex-1-ene; (5R)-2-methyl-5-[(2R)-6-methylhept-5-en-2-yl]cyclohexa-1,3-diene

IUPAC Name:

(3R)-3-(6-methylhept-5-en-2-yl)-6-methylidenecyclohex-1-ene; (5R)-2-methyl-5-[(2R)-6-methylhept-5-en-2-yl]cyclohexa-1,3-diene

Test material form:

liquid

Specific details on test material used for the study:

Name: Ginger extract (volatile)
Synonym: Ginger CO2-se extract, type no. 014.001
Batch No.: 461108
Known Constituents Content:
Volatile oils: 88.9 %
Gingerol: 2.2 %
Shogaol: 0.42 %

Analysis Date: August 02, 2016
Type: UVCB
Aggregate State at Room Temperature: Liquid
Colour: Yellow
Density: 0.8838 g/cm³ (20°C)
Retest Date: July 2021
Storage Conditions at Test Facility:
At 20 +/- 5 °C, in the dark

Sampling and analysis

Analytical monitoring:

yes

Remarks:

GC-FID peak area

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 (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.

Storage:
All samples were extracted with an organic solvent stand-by immediately after sampling. The extracts were stored in a refrigerator (4 ± 4°C), protected from light until analysis was performed. Afterwards the samples were again stored refrigerated and will be kept stored up to the date of the final report.

Analyses:
The dosage of the test item Ginger extract (volatile) was analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples, from both sampling times (0 and 72 hours).

Test solutions

Vehicle:

no

Remarks:

Water accomodated fractions were used

Details on test solutions:

Dosage of Test Item:
A defined amount of the test item was added directly to the test water for each test loading rate and was carefully stirred for 24 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 105.2 mg test item into 1052 mL test water, for the test item loading rate of 32 mg test item/L, 33.6 mg test item were mixed into 1050 mL test water, for the loading rate of 10 mg test item/L, 10.5 mg were mixed into 1050 mL test water. The loading rate of 3.2 mg test item/L was prepared by mixing 18.6 mg into 5800 mL test water and for 1.0 mg test item/L, 11.4 mg were mixed into 11400 mL test water. After cessation of mixing and a following period (0.5 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 test loading rate. The test media were prepared just before introduction of the algae (= start of the test).

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

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata (KORSHIKOV)
- Strain: No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Origin: The algae were supplied by the "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: Algae cells were taken from an exponentially growing pre-culture 4 days prior
- Culturing media and conditions (same as test or not): pre-culture medium and test medium are the same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

22.1 to 23.2 °C

pH:

Test start: 8.2 to 8.3
Test end: 8.6 to 9.9

The pH in the control increased by more than 1.5 units. However, this does not invalidate the tests since the validity criterion was met.

Nominal and measured concentrations:

Water accommodated Fractions (WAF) nominal loading rates: Control, 1.0, 3.2, 10, 32 and 100 mg/L

Dose dependent presence of test item based on peak area was shown on t=0h and t=72h (<= 54% of initial).

Details on test conditions:

TEST CONDITIONS:
- Type and Size: Erlenmeyer flasks of 50 mL volume containing as much test medium as possible, i.e. at least 50 mL in order to reduce the remaining head space to a technical possible minimum of some mL, kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
- Control end cells density: 85.202 [10000/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Light Regime: Continuous illumination
- Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 6953 lux (range: 6100 to 7660 lux)

GROWTH MEDIUM
- Standard medium used: yes - OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
- pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
- Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- 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

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

40.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 37.0 - 44.0 mg test item/L)

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

13.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 10.8 - 16.0 mg test item/L)

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

- Observation of abnormalities: no -The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test loading rate of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test loading rate, the highest loading rate.

No remarkable observations, clear test medium
The pH in the control increased by more than 1.5 units. However, this does not invalidate the tests since the validity criterion was met.

Results with reference substance (positive control):

Results with reference substance are valid. Historical ranges not included
72h-ErC50: 1.02 mg/L (95% C.I. 0.984-1.05 mg/L)

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErL50 and the 72 hours EyL50, the corresponding EL20 and EL10 values and where possible their 95 %-confidence limits were calculated by Probit analysis for yield and by Weibull analysis for growth rate.
For the determination of the 72 hours LOEL and the 72 hours NOEL, the calculated growth rates and yields at each test loading rate were tested for significant differences compared to the control values by Bonferroni-Welch t-test. 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

Analytical Results:

The qualitative analysis of the test item Ginger extract (volatile) in the test samples was performed using gas chromatography with flame ionization detection. From the analytical data it was shown that WAFs were prepared correctly based on increase in peak areas with loading rate. At test end the blank corrected total peak areas could not be adequately quantified for the two lowest loading rates. However, peak patterns could still be differentiated compared to the control, therefore confirming the presence of test item components at test end. For the three higher loading rates up to 54% of initial peak areas was observed at test end.

Yield y and Percentage Inhibition of y during the Test Period

 

Nominal loading rates [mg test item/L]	Yields y [10000 cells/mL] and % inhibition of y
	0-24 hrs		0-48 hrs		0-72 hrs
	y	%	y	%	y	%
control	1.933	-	14.950	-	84.702	-
1.0	1.560	19.3	14.121	5.5	83.476	1.4
3.2	0.316	83.7*	13.328	10.9*	84.053	0.8
10	1.187	38.6*	4.893	67.3*	38.994	54.0*
32	0.000	100.0*	0.330	97.8*	14.626	82.7*
100	0.067	96.5*	0.000	100.0*	0.000	100.0*

 

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control(tested with Bonferroni-Welch t-test (24h and 72h) and Williams t-test (48h),a = 0.05, one-sided)

 

Growth Rates μ and Percentage Inhibition of μ during the Test Period

Nominal loading rates [mg test item/L]	Growth rates μ [1/day] and % inhibition of μ
	0-24 hrs		0-48 hrs		0-72 hrs
	μ	%	μ	%	μ	%
control	1.552	-	1.714	-	1.713	-
1.0	1.415	8.8	1.688	1.6	1.707	0.3
3.2	0.468	69.8*	1.659	3.2	1.709	0.2
10	1.210	22.0*	1.188	30.7*	1.454	15.1*
32	0.000	100.0*	0.194	88.7*	1.135	33.7*
100	0.115	92.6*	0.000	100.0*	0.000	100.0*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control(tested with Bonferroni-Welch t-test,a = 0.05, one-sided)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%

Conclusions:

The ErL50, ErL10 and NOELR were 40.4, 13.2 and 3.2 mg test item /L respectively.

Executive summary:

Algae toxicity was assessed in an OECDTG 201 static concentration-response GLP study with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per treatment group were exposed to WAFs prepared at loading rates of 100, 32, 10, 3.2 and 1.0 mg Ginger extract (volatile) per litre in closed vessels.The qualitative analysis of the test item Ginger extract (volatile) in the test samples was performed using gas chromatography with flame ionization detection. From the analytical data it was shown that WAFs were prepared correctly based on increase in peak areas with loading rate. Presence of test item (up to 54% of initial) at test end was also shown for the three highest loading rates. All reported results refer to nominal loading rates.The 72-hour ErL50, ErL10 and NOELR were 40.4, 13.2 and 3.2 mg test item /L respectively.