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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:
April 01 to November 12, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
GC-FID
Details on sampling:
Analysis of the Test Item Concentrations
For the determination of the actual test item concentration, duplicate samples were taken from
the treatment and control group at the start and end of each test medium renewal period.
For sampling from the aged test medium, the contents of the respective replicates were
combined prior to sampling.
The samples were analyzed immediately after sampling.
The concentrations of SCENTAURUS JUICY were analytically measured in one of the
duplicate samples of the single concentration from all sampling dates and the control.

Preparation of the Internal Standard Solution:
The internal standard (9.81 mg of dodecane) was dissolved in 10 mL cyclohexane. This solution
was further diluted in cyclohexane giving a final internal standard concentration of 5.05 mg/L.
Details on test solutions:
Dosage:
At the start of the test and before each test medium renewal, the undiluted equilibrated test
medium with a loading rate of 100 mg/L was prepared following the slow-stirring method (see
Section 5.5.1) with a stirring period of 72 hours at room temperature in the dark. For this,
256.7 μL of test item were carefully applied (pipetted) onto the surface of 2300 mL test water.
This volume is equivalent to a loading rate of 100 mg/L, considering the density of the test item
of 0.8958 g/cm3. No auxiliary solvent or emulsifier was used.

This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item
only, at the water saturation concentration, was used as the single test concentration
The test medium was clear with no evidence of undissolved test item.
The test medium was prepared just before the introduction of the daphnids (i.e., start of the test
and prior to the test medium renewal).
The preparation of the test medium was based on the OECD Guidance Document No. 23
(Second Edition) on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals,
2019.
The measured water saturation concentrations obtained for the 100 mg/L loading rate undiluted
filtrate in the dose range finding experiment were very similar to that obtained in the final Limit
Test (Table 5 and Table 6 of the Analytical Appendix to Report attached) and demonstrate the
reproducibility of the slow-stir preparation approach for this test item, despite the very low
water solubility.

Since the test item may be volatile, the test was performed in glass vessels completely filled
(without headspace) with 60 mL test medium and tightly sealed with glass stoppers to avoid
losses of volatile components of the test item by evaporation (closed system).
The test vessels were labeled with the study number and all necessary additional information
to ensure unique identification.
Test organisms (species):
Daphnia magna
Details on test organisms:
The study was performed with young daphnids of the species Daphnia magna Straus. A clone
of this species (originally from the Daphnia Collection of the University of Basel/Switzerland
in 2015) is successfully bred in IES Ltd Laboratories. The cultivation of the parental daphnids
is performed in reconstituted water of the quality identical to the water quality used in the tests
(with respect to pH, main ions, and total hardness) and under temperature and light conditions
identical to those of the tests (see below).
During breeding, daphnids were generally fed three times a week with an algal suspension of
the green algae Desmodesmus subspicatus, supplied by the Collection of Algal Cultures (SAG,
Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and
cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal
suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH,
49324 Melle/Germany). At the start of the test, the organisms used in the test were 6-24 hours
old and were not first brood progeny.
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium
dichromate is tested as a positive control twice a year. The result of the latest positive control
test in April 2021 (24-hour EC50: 2.0 mg/L, IES Study 20210091) showed that the sensitivity
of the test organisms was within the internal historical range (24-hour EC50 from 2015 to 2021:
0.78-2.0 mg/L).
After the end of the test the organisms in the treatment including the control were disposed.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Based on the analytical results of the range-finding test, a semi-static main test with test medium
renewal after 24h was performed to keep the concentration of the test item in the test
medium as constant as possible during the test period of 48h.
Post exposure observation period:
The daphnids were observed for immobility after 24 and 48 hours of exposure (daphnids not
being able to swim within 15 seconds after gentle agitation of the test beaker were considered
to be immobile).
Hardness:
Water hardness: 250 mg/L as CaCO3
Test temperature:
21°C
pH:
in the range of 7.7 to 7.9
Dissolved oxygen:
The dissolved oxygen concentrations in the test medium and the control were at least 8.2 mg/L
Salinity:
5.8 mg/L NaHCO3
Nominal and measured concentrations:
In the analyzed test medium samples (loading rate 100 mg/L) from the start of the renewal
periods, 32.5 and 9.41 μg/L were found, respectively. At the end of the test medium renewal
periods the measured test item concentrations were 5.03 and 2.09 μg/L, respectively
corresponding to 16 and 22 % of the initially measured concentrations.
The mean measured concentration was calculated as the geometric mean of the test item
concentrations measured at the start and the end of the test medium renewal periods (i.e. Day 0-
Day 1, Day 1-Day 2). From the two geometric mean values obtained, the mean measured test
item concentration during the test period of 48 hours was calculated as an arithmetic mean and
was 8.6 μg/L.
Details on test conditions:
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21 °C.
The daphnids were not fed during the test.
A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity
during the light period was 16-17 μmol m-2 s-1.

Study Design for Main Test:
Based on these results and in agreement with the Sponsor, a limit test was performed in
accordance with the test guidelines to demonstrate that the test item has no toxic effect on the
test organisms up to its limit of solubility in the test water. Thus, a single loading rate of the test
item of 100 mg/L was tested. Additionally, a control (test water without test item) was tested
in parallel.
The main test was performed in a closed system to avoid losses of the test item by evaporation.
Based on the analytical results of the range-finding test, a semi-static main test with test medium
renewal after 24 hours was performed to keep the concentration of the test item in the test
medium as constant as possible during the test period of 48 hours. After 24 hours, the test
organisms were transferred by wide-bore pipette in clean test vessels with freshly prepared test
medium of the corresponding concentration.
For each treatment, 20 daphnids were used distributed into four replicates of five daphnids each.
The volume of test medium provided for each daphnid was 12 mL. Thus, the requirement of
the test guidelines for a minimum volume of 2 mL test medium per daphnid was fulfilled. The
daphnids were randomly distributed to the test vessels at the initiation of the test.
The test duration was 48 hours.

Determination of Immobility and EC Values:
The daphnids were observed for immobility after 24 and 48 hours of exposure (daphnids not
being able to swim within 15 seconds after gentle agitation of the test beaker were considered
to be immobile).
The NOEC, EC0, EC50 and EC100 were directly determined from the raw data.

Calculation of Mean Measured Test Item Concentration
The biological results were based on mean measured test item concentration. For each test
medium renewal period, the mean concentration was calculated as geometric mean of the test
item concentrations measured at the start and the end of the test medium renewal period. From
the values obtained, the mean measured test item concentration during the test period was
calculated as an arithmetic mean provided in the study report attached.

Monitoring of Experimental Conditions
At the start and end of each test medium renewal, the pH values, dissolved oxygen
concentrations and water temperature were determined in each treatment. In addition, the
appearance of the test medium was visually controlled and documented at the same time.
Reference substance (positive control):
yes
Remarks:
Potassium dischromate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 8.6 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Physical effects, behaviour and appearance
Remarks on result:
other: No effects observed up to and including water saturation (equivalent to a loading rate of 100 mg/L test item)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 8.6 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: No effects observed up to and including water saturation (equivalent to a loading rate of 100 mg/L test item)
Details on results:
In the analyzed test medium samples (loading rate 100 mg/L) from the start of the renewal
periods, 32.5 and 9.41 μg/L were found, respectively. Over the test medium renewal periods of
24 hours, the test item concentration in the test medium decreased. At the end of the test
medium renewal periods the measured test item concentrations were 5.03 and 2.09 μg/L,
respectively corresponding to 16 and 22 % of the initially measured concentrations.

The mean measured concentration was calculated as the geometric mean of the test item
concentrations measured at the start and the end of the test medium renewal periods (i.e. Day 0-
Day 1, Day 1-Day 2). From the two geometric mean values obtained, the mean measured test
item concentration during the test period of 48 hours was calculated as an arithmetic mean and
was 8.6 μg/L.

Therefore, the 48-hour NOEC and EC0 of SCENTAURUS JUICY to Daphnia magna were
determined to be at least 8.6 μg/L. The 48-hour NOEC and EC0 might even be higher, but
concentrations of the test item exceeding the loading rate 100 mg/L, above the solubility limit
in the test water were not tested, in accordance with the test guidelines. The 48-hour EC50 and
EC100 were clearly higher than 8.6 μg/L, 100 mg/L loading rate.
The test item had no acute toxic effects on Daphnia magna up to and including the mean
measured concentration of 8.6 μg/L, 100 mg/L loading rate, its solubility limit in test water
under the conditions of the test.

The test medium was a clear solution throughout the test period.

The test is considered to be valid, as in the control no daphnids showed immobilization or other
signs of disease or stress (e.g., discolouration or unusual behaviour such as trapping at the
surface of water). Furthermore, the dissolved oxygen concentration at the end of the test was
>=3 mg/L in all test vessels.
Results with reference substance (positive control):
In the control and at the mean measured test item concentration of 8.6 μg/L, 100 mg/L loading
rate and the equivalent of the water saturation concentration, no immobilized test organisms
were observed during the test period of 48 hours.

The biological test results (based on mean measured test item concentration) were as follows:












































Parameter (0-48h) 


Immobility
Mean measured concentration
[μg/L]
Loading
Rate
(mg/L)
24-hour EC50> 8.6> 100
24-hour EC0 / NOEC>= 8.6 >= 100
24-hour EC100> 8.6 > 100
48-hour EC50 > 8.6 > 100
48-hour EC0 and 48-hour NOEC >= 8.6 >= 100
48-hour EC100 > 8.6 > 100
Validity criteria fulfilled:
yes
Conclusions:
The test item had no acute toxic effects on Daphnia magna up to and including the mean
measured concentration of 8.6 μg/L, 100 mg/L loading rate, its solubility limit in test water
under the conditions of the test.
Executive summary:

The acute toxicity of the test item SCENTAURUS JUICY to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.



A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the Daphnia magna up to the loading rate of 100 mg/L and equivalent to the water saturation concentration. Thus, a single loading rate of the test item of 100 mg/L was tested. A control was run in parallel (test water without addition of the test item).
Loading rates of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines.
As the test item may be a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). A semi-static test design was used.
As the test item is a liquid with low water solubility, the slow-stirring method (to avoid formation of micro-droplets) was applied for preparation of a saturated test item solution. For preparation of the single concentration of test medium, the test item was carefully applied (pipetted) onto the surface of the test water at a loading rate of 100 mg/L. Thereafter slow-stirring was applied for 72 hours in a closed vessel.
After this treatment the lower part of the equilibrated test medium was carefully harvested from the stirring vessel through a tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item only, was used as the test medium.
The preparation of the test medium was based on the OECD Guidance Document No. 23 (Second Edition) on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.
In the analyzed test medium samples (loading rate 100 mg/L) from the start of the renewal periods, 32.5 and 9.41 μg/L were found, respectively. At the end of the test medium renewal periods the measured test item concentrations were 5.03 and 2.09 μg/L, respectively corresponding to 16 and 22 % of the initially measured concentrations.
The mean measured concentration was calculated as the geometric mean of the test item concentrations measured at the start and the end of the test medium renewal periods (i.e. Day 0-Day 1, Day 1-Day 2). From the two geometric mean values obtained, the mean measured test item concentration during the test period of 48 hours was calculated as an arithmetic mean and was 8.6 μg/L.
The biological results are based on the mean measured test item concentration.
In the control and at the mean measured test item concentration of 8.6 μg/L, 100 mg/L loading rate and the equivalent of the water saturation concentration, no immobilized test organisms were observed during the test period of 48 hours.


The biological test results (based on mean measured test item concentration) were as follows:












































Parameter (0-48h) 


Immobility
Mean measured concentration
[μg/L]
Loading
Rate
(mg/L)
24-hour EC50> 8.6> 100
24-hour EC0 / NOEC>= 8.6 >= 100
24-hour EC100> 8.6 > 100
48-hour EC50 > 8.6 > 100
48-hour EC0 and 48-hour NOEC >= 8.6 >= 100
48-hour EC100 > 8.6 > 100


In conclusion, the test item SCENTAURUS JUICY had no acute toxic effects on
Daphnia magna up to its solubility limit in test water under the conditions of the test.



Validity criteria:
The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration, surface trapping, reduced swimming etc.).
Furthermore, the dissolved oxygen concentration at the end of the test was >= 3 mg/L in all test vessels.

Description of key information

The acute toxicity of the test item SCENTAURUS JUICY to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.


The mean measured concentration was calculated as the geometric mean of the test item concentrations measured at the start and the end of the test medium renewal periods (i.e. Day 0-Day 1, Day 1-Day 2). From the two geometric mean values obtained, the mean measured test item concentration during the test period of 48 hours was calculated as an arithmetic mean and was 8.6 μg/L.
The biological results are based on the mean measured test item concentration.
In the control and at the mean measured test item concentration of 8.6 μg/L, 100 mg/L loading rate and the equivalent of the water saturation concentration, no immobilized test organisms were observed during the test period of 48 hours.


Therefore: 48h EC50 > 8.6 microg/L based on mobility


In conclusion, the test item SCENTAURUS JUICY had no acute toxic effects on
Daphnia magna up to its solubility limit in test water under the conditions of the test.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
> 8.6 µg/L

Additional information

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), Scentaurus Juicy doesn't need to be classified as Hazardous to the Aquatic Environment Acute 1 classification (Acute results greater than the aqueous saturation concentration of the test item).