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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Sample name: SANTAL ELLE CAL, Lot# 2582250, production date 4.11.2016
Analytical monitoring:
yes
Details on sampling:
Chemical analyses were taken from additional replicates in order to avoid disturbing the daphnids at t = 24h and to prevent potential test item losses on opening test tubes. Single samples for analysis were taken from the control and all test solutions at the start (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h). Concentration of dissolved organic material was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.
Vehicle:
no
Details on test solutions:
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t = 0 h and t = 24 h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion. The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L, except for the preparation of the lowest loading rates where a 10 L glass bottle were used. A magnetic stirring bar was placed in each mixing vessel and 5.0 to 5.2 L of test water (depending on the brim capacity of the bottles; 11.0 L for the lowest loading rates) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vertex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ±2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use.
The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna (Straus), clone 5, females, originating from LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine, Campus Bridoux - Bât. IBISE. 8, rue du Général Delestraint – 57070 METZ, bred in the Laboratoires des Pyrenees et des Landes. Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals. Age at test start: < 24 hours old.
Daphnids were cultured in the Laboratoires des Pyrenees et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1 - 0.2 mg / C.Daphnia and day. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
A semi-static test was performed with renewal of test solutions after 24 hours (because of the properties of the test item). 20 daphnids per treatment group (including controls) were exposed, divided into 4 groups of 5 animals.
Hardness:
~250 mg/L (as caCO3)
Test temperature:
Controlled environment cabinet (20 °C ±2 °C)
pH:
7.8 - 8.2
Dissolved oxygen:
7.8 - 8.4 mg/L throughout test
Nominal and measured concentrations:
Based on the results of a previous GLP test, where a 48-hour EL50 of 0.998 mg/L was determined, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 1.78): 0.32, 0.57, 1.01, 1.80 and 3.21 mg/L.
Controls: Test water without test substance but treated in the same way as the test substance solutions.
Details on test conditions:
Light regime: 16h light : 8h dark; No aeration of the test solutions occurred throughout the test. No feeding! Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
0.92 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.835 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
After 24 hours of exposure, immobilisation rates were 0% at 0.32 mg/L (loading), 5% at 0.57 mg/L (loading), 75% at 1.01 mg/L (loading), 90% at 1.00 mg/L (loading) and 100% at 3.21 mg/L (loading). After 48 hours of exposure, immobilisation rates were 0% at 0.32 mg/L (loading), 15% at 0.57 mg/L (loading), 75% at 1.01 mg/L (loading), 95% at 1.00 mg/L (loading) and 100% at 3.21 mg/L (loading).
The 24 and 48-hour EL50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat (8).
Results with reference substance (positive control):
The sensitivity of the test clone was checked by using potassium dichromate (K2Cr2O7) as reference substance.
Identification: Potassium dichromate
Manufacturer: VWR
Batch No.: 14F110009
Purity: 99.8%
Test substance storage: Between +5 °C to +30 °C
Expiry date: 30 March 2017
On January 10, 2017, the 24h-EC50 was 1.22 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg/L to 2.1 mg/L) at this time. For information, on April 4, 2017 (most recent test performed on another batch of reference substance), the 24h-EC50 was 0.97 mg/L.
Reported statistics and error estimates:
The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional (ToxRat® Professional - ToxRat® Solutions GmbH, Naheweg 15. D-52477 Alsdorf (www.toxrat—solutions.de)) was used for the determination of the effective concentrations.

Water quality parameters and environmental conditions throughout the test: The results of measurement of pH and oxygen concentrations (mg/L) remained within the limits prescribed by the study plan (pH: 6.0 - 9.0) not varying by more than 1.5 units; oxygen:≥60% of the air-saturation value at the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.8 and 20.9 °C throughout the test (average value: 20.9 °C), and complied with the requirements as laid down in the study plan (20 °C±2 °C, constant within 1 °C).

Analytical results: The detailed results of analysis of the samples taken during the final test are described in the Table of the Analytical Report. Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h).

The analytical results of this test showed that WAFs were overall stable throughout the test period. TOC content in WAFs was maintained around±20 - 30% during the first part of the test (t = 0 h – t = 24 h old), and was satisfactorily preserved within±20% of the initial concentration during the second exposure period (t = 24 h fresh -t = 48 h), except at 0.32 mg/L with a gain of 35%. It would be difficult to determine the specific cause of this last observation, as well as gains observed in controls.

However, this was considered not to affect the results and the integrity of the study regarding properties of the test item and the non-specific analytical method.

It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation...). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture. Therefore, results are provided as Effective Loading Rate 50, according to the OECD No. 23.

Validity criteria fulfilled:
yes
Remarks:
In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. Dissolved oxygen concentration at the end of the test was ≥60% of the air-saturation value in controls and test vessels.
Conclusions:
The toxic effect of test item SANDALWOOD AUSTROCALEDONIEN OIL to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value estimated was 0.835 mg/L (loading).
Executive summary:

A study was performed to assess the acute toxicity of test item SANDALWOOD AUSTROCALEDONIEN OIL to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202. "Daphnia sp., Acute lmmobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266) and with the "Guidance document on aquatic toxicity testing of difficult substances and mixtures" (OECD No. 23). The criterion measured was the EL50 (Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a previous test, where a 48-hour ELED of 0.998 mg/L was determined, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 0.32, 0.57, 1.01, 1.80 and 3.21 mg/L and to a control. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t = 0 h), at t = 24 h (new and old solutions) and at the end of the test (t = 48 h).

The analytical results of this test showed that WAFs concentrations were overall stable between the start and the end of each exposure period. After 48 hours of exposure, immobilisation rates were 0% at 0.32 mg/L (loading), 15% at 0.57 mg/L (loading), 75% at 1.01 mg/L (loading), 95% at 1.30 m/L (loading) and 100% at 3.21 mg/L (loading).

Thus, EL50 at 24 hours was found being 0.920 mg/L (95% confidence limits 0.781 — 1.085 mg/L) and at 48 hours 0.835 mg/L (95% confidence limits 0.708 — 0.986 mg/L).

Description of key information

The toxic effect of test item SANDALWOOD AUSTROCALEDONIEN OIL to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal concentrations, the 24 hour EL50 value estimated was 0.92 mg/L and the 48-hour EL50 value estimated was 0.835 mg/L (loading).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.835 mg/L

Additional information