Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by 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:

Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by supercritical CO2 extraction

IUPAC Name:

Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by supercritical CO2 extraction

Test material form:

liquid

Details on test material:

Batch number: 861213

Specific details on test material used for the study:

Name: Ginger extract (non-volatile)
Synonym: Ginger Hot Flavor CO2-to extract, type no. 014.008
Batch No.: 861213
Analysis Date: July 20, 2016
Type: UVCB
Aggregate State at Room Temperature: Liquid
Colour: Brown
Retest Date: July 2019
Storage Conditions at Test Facility: At 20 +/- 5 °C, in the dark

Known Constituents Content:
Gingerol: 44.7 %
Shogaol: 3.4 %,
according to certificate of analysis

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 (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 and of the maintenance of the test item concentrations during the test period, adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated during test period under the same conditions as in the actual test and from these test media duplicate samples were taken at the end of the test period. The collecting of samples after 72 hours from the actual test itself was not possible, since the test media volumes in the test were too small for the analytical requirements. 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 Ginger extract (non-volatile) was analysed in the duplicate test media samples from all test concentrations and the control samples from both sampling times (0 and 72 hours).

Test solutions

Vehicle:

no

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 105.9 mg test item into 1059 mL test water, for the test item loading rate of 32 mg test item/L, 33.5 mg test item were mixed into 1047 mL test water, for the loading rate of 10 mg test item/L, 24.4 mg were mixed into 2440 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 of half an 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: The visual appearance of the test item in test water was determined daily in all test item loading rates. At test start coloration was observed at the loading rates of 100, 32 and 10 mg test item/L. The coloration was not visible after 24 h at the loading rate of 10 mg test item/L and after 48 h not at the loading rate of 32 mg/L anymore. A slight coloration remained at the highest loading rate after 72 h

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:

22.4 to 23.1 °C

pH:

Test start: 8.1 to 8.3
Test end: 8.3 to 9.8

Nominal and measured concentrations:

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

Measured TOC corrected by the mean value of the control
t=0 hr : n.a, 0.521*, 0.975*, 3.48, 11.0 and 29.3 mg Carbon/L
t=72 hr: n.a, 0.618*, 1.15*, 2.85, 9.56, 28.1 mg Carbon/L

Mean value of all measured samples per treatment group. The results were corrected by the mean value of the control
* TOC content of this loading

Details on test conditions:

TEST CONDITIONS:
- Type and Size: Erlenmeyer flasks of 50 mL volume of test medium 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 a conical glass stopper to avoid loss of the test item due to volatilisation.
- Control end cells density: 95.122 [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: 7555 lux (range: 7100 to 8000 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.:

65.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 61.3 - 69.7 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 18.7 - 26.4 mg/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 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 the highest tested loading rate.

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 EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis (yield) and Weibull analysis (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 TOC content of the test item was determined in the test media samples from all loadings at test start and test end. The highest WAF loading rate 100 mg test item/L was found to contain approximately 28.7 mg carbon/L over the course of the test. The carbon content of the lowest WAF loading rate of 1 mg test item/L was found to be below the Limit of Quantification of 2.5 mg carbon/L. The analytical results show that the 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) was shown.

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	2.196	-	18.93	-	94.622	-
1.0	2.069	5.8	17.025	9.4	89.750	5.1
3.2	1.498	31.8	15.062	19.9	86.663	8.4
10	1.688	23.1*	11.462	39.0*	66.150	30.1*
32	1.308	40.4*	7.600	59.6*	39.119	58.7*
100	0.800	63.6*	1.120	94.0*	1.180	98.8*

 

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)

 

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	1.682	-	1.819	-	1.748	-
1.0	1.635	2.8	1.778	2.3	1.732	0.9
3.2	1.373	18.4*	1.718	5.6*	1.720	1.6
10	1.474	12.4*	1.587	12.8*	1.631	6.7*
32	1.285	23.6*	1.392	23.5*	1.457	16.7*
100	0.953	43.3*	0.587	67.7*	0.402	77.0*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control(tested with Williams t-test (24h and 48h) Bonferroni-Welch t-test (72h),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 65.4, 22.2 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 group were exposed to WAFs prepared at loading rates of 100, 32, 10, 3.2 and 1.0 mg mg Ginger hot flavor extract (non-volatile) per litre in closed vessels. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. 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 analysis (≥ 82% of initial TOC at 72h, based on quantified loadings). Therefore all reported results refer to nominal loading rates.The ErL50, ErL10 and NOELR for Pseudokirchneriella subcapitata exposed to Ginger hot flavor extract (non-volatile) were 65.4, 22.2 and 3.2 mg test item /L respectively.