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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 October 2017 - 05 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Deviations:
yes
Remarks:
Age of juveniles was within a range of 20.5 – 24.5 h instead of <24 hrs old. No presumed effect on the study.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 30, 2008
Qualifier:
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
Qualifier:
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. certificate)
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 of all test concentrations 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 concentrations during the test period, duplicate samples from the test media of all test concentrations 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.

Storage:
The samples of test start were stored in a refrigerator (4 ± 4 °C), protected from light until analysis was performed. The samples of test end were analysed immediately after sampling. Afterwards the samples were again stored in a refrigerator (4 ± 4 °C) and will be kept stored up to the date of the final report.

Analyses:
The concentrations of the test item Geranium oil were analysed in the duplicate test media samples from all test concentrations, and in the duplicate
Vehicle:
no
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 concentration and was carefully stirred for 72 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 281.7 μL test item into 2500 mL test water, for the test item loading rate of 45.5 mg test item/L, 128.2 μL test item were mixed into 2500 mL test water, for the loading rate of 20.7 mg test item/L, 58.31 μL were mixed 2500 mL test water. The loading rate of 9.4 mg test item/L was prepared by mixing 26.48 μL into 2500 mL test water and for 4.3 mg test item/L, 12.11 μL were mixed into 2500 mL test water. After cessation of mixing and a following period (1 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 concentration. 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 (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 20.5 to 24.5 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
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
Test start: 20.5 to 21.9 °C
Test End: 20.3 to 20.4 °C
pH:
Test start: 7.9 to 8.3
Test End: 7.9 to 8.0
Dissolved oxygen:
Test start: 8.2 to 9.2 mg/L
Test End: 8.9 to 9.0 mg/L
Nominal and measured concentrations:
Nominal loading rates of 4.3, 9.4, 20.7, 45.5 and 100 mg test item/L and a control.

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

TOC (t=0hr): n.a, 4.24, 5.92, 11.70, 26.47 and 51.25 mg/L
TOC (t=48 hr) n.a, 3.89, 5.86, 11.26, 26.32 and 50.53 mg/L

n.a.: not applicable
Details on test conditions:
TEST SYSTEM
- 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 conical glass stoppers 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 100 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 260 to 420 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).

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
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
14.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: not determinable
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
28.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Cl: 22.9- 35.4 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 loading rate of 4.3 mg test item/L. At the loading rate of 9.4 mg test item/L, one animal was immobile. At a loading rate of 20.7 mg test item/L 20 daphnids were immobile. At the two highest loading rates of 45.5 and 100 mg test item/L 19 and 20 daphnids were immobile, respectively. 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 (≥ 92% of initial TOC at 48h). Therefore all reported results refer to nominal values.

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 ≥ 8.9 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 June 2017 the EC50 after 24 hours was determined to be 1.58 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 calculated by Step-down Cochran-Armitage test procedure. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Influence of Geranium oil on the Mobility of Daphnia magna

Nominal loading rate [mg test item/L]

% of immobilised Daphnia

24 h

48 h

Control

0

0

4.3

0

0

9.4

0

0

20.7

30

100

45.5

80

95

100

100

100

Validity criteria fulfilled:
yes
Remarks:
<10% immobilization in control group, O2 conc. at end is >=3 mg/L in control and test vessels
Conclusions:
The acute 48-hour EL50 value of Geranium oil for Daphnia magna was determined to be 14.5 mg test item/L based on nominal values.
Executive summary:

A study was performed to assess the acute toxicity of Geranium oil to Daphnia magna under static conditions in closed test vessels. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP principles. Groups of twenty daphnids (≤ 24.5 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 hours and at 48 hours and the 48h EL50 was 14.5 mg test item/L. 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 (≥ 92% of initial TOC at 48h). Therefore all reported results refer to nominal values.

Description of key information

A study was performed to assess the acute toxicity of Geranium oil to Daphnia magna under static conditions in closed test vessels. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP principles. Groups of twenty daphnids (≤ 24.5 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 hours and at 48 hours and the 48h EL50 was 14.5 mg test item/L. 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 (≥ 92% of initial TOC at 48h). Therefore all reported results refer to nominal values.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
14.5 mg/L

Additional information

Result relates to nominal loading rate, hence is expressed as EL50.