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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2018 - May 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 30, 2008
according to 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
according to 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

Constituent 1
Reference substance name:
Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by supercritical CO2 extraction
Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by supercritical CO2 extraction
Test material form:
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
Known Constituents Content (according to certificate of analysis):
- Gingerol: 44.7 %
- Shogaol: 3.4 %,
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

Sampling and analysis

Analytical monitoring:
TOC analysis
Details on 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 loading rates 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 loading rates during the test period, duplicate samples from the test media of all test loading rates 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.

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.

The dissolved fraction of the test item Ginger extract (non-volatile) was analysed in the duplicate test media samples from all test loading rates and the control samples from both sampling times (0 and 48 hours).

Test solutions

Water accomodated fraction was used
Details on test solutions:
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 was tested. Therefore, 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 30 mg test item/L was prepared by mixing 31.8 mg test item into 1060 mL test water, for the test item loading rate of 13.6 mg test item/L, 14.4 mg test item were mixed into 1060 mL test water, for the loading rate of 6.2 mg test item/L, 14.9 mg were mixed into 2400 mL test water. The loading rate of 2.8 mg test item/L was prepared by mixing 16.2 mg into 5800 mL test water and for 1.3 mg test item/L, 14.8 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 daphnids (= start of the test).

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

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 4 to 20.75 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. 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:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

250 mg/L as CaCO3
Test temperature:
Test start: 19.8 to 20.1 °C
Test End: 19.6 to 19.8 °C
Test start: 7.9 to 8.3
Test End: 7.9 to 8.0
Dissolved oxygen:
Test start: 8.5 to 8.8 mg/L
Test End: 8.7 to 8.9 mg/L
Nominal and measured concentrations:
Nominal loading rates of 1.3, 2.8, 6.2, 13.6 and 30 mg test item/L and a control.

TOC based on mean value of all measured samples per treatment group (n=2). The results were corrected by the mean value of the control .

TOC (t=0hr): n.a, 0.484*, 0.64*, 2.54, 5.40 and 11.6 mg/L
TOC (t=48 hr) n.a, 0.445*, 0.40*, 2.31, 5.29 and 11.3 mg/L

n.a.: not applicable
* TOC content of this loading
Details on test conditions:
- 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 a conical glass stopper to avoid loss of the test item due to volatilisation.
- Introduction of Daphnids: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in approximately 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 450 to 730 Lux (measured once during the test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility: The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those 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).

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):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
48 h
Dose descriptor:
Effect conc.:
9.01 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL: 7.98 - 10.17
24 h
Dose descriptor:
Effect conc.:
9.42 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% Cl: 8.28 - 10.17 mg/L
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item at the loading rate of 6.2 mg test item/L. At the loading rate of 13.6 and 30.0 mg test item/L, all 20 animals were immobile. 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 30 mg test item/L was found to contain approximately 11.4 mg carbon/L over the course of the test. The carbon content of the lowest WAF loading rate of 1.3 mg test item/L was found to be below the Limit of Quantification of 1 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.

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 ≥ 8.7 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 January 2018, the EC50 after 24 hours was determined to be 1.06 mg test item/L, indicating 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 Ginger extract (non-volatile) on the Mobility of Daphnia magna

Nominal loading rate [mg test item/L]

% of immobilised Daphnia

24 h

48 h



















Applicant's summary and conclusion

Validity criteria fulfilled:
<10% immobilization in control group, O2 conc. at end is >=3 mg/L in control and test vessels
The acute 48-hour EL50 value of Ginger hot flavor extract (non-volatile) for Daphnia magna was determined to be 9.01 mg test item/L based on nominal values.
Executive summary:

A study was performed to assess the acute toxicity of Ginger hot flavor extract (non-volatile) in Daphnia magna under static conditions in closed vessels. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP principles. Groups of twenty daphnids (less than 24 hours old) were exposed for 48 hours to five concentrations of the substance, dispersed in test water via Water Accomodated Fraction procedure, and an untreated control. The incidence of immobilisation was recorded for each test and control group at 24 and 48 hours.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 analyses (≥ 91% of initial TOC at 48h, based on quantified loadings). Therefore all reported results refer to nominal loading rates. Validity criteria was met and the 48h EL50 was determined to be 9.01 mg test item/L.