5(or 6)-methyl-7(or 8)-(1-methylethyl)bicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

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)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item Name: MACEAL
Givaudan Product Code: 6576003
Batch Number: SC00018703
Appearance: Colorless to pale yellow liquid
Storage Conditions: (as provided by the Sponsor): Dry, well ventilated, preferably full, hermetically sealed.
Ambient temperature / 10-30 °C. Protect against light.
IES Code: 10843

Analytical monitoring:

yes

Details on sampling:

For the determination of the actual test item concentrations, duplicate samples were taken from each test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods.

From the highest test concentration (dilution 1:2.2), samples were taken only from the first renewal period as all daphnids were immobile (i.e. dead) at Day 1. For confirmation of the dosage, additionally samples were taken at the start of both renewal periods from the stock solution (i.e. the undiluted equilibrated test medium with a loading rate of 100 mg/L) and at the start of the second renewal period from the highest test concentration (dilution 1:2.2).

For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.

All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on analytical pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

The concentrations of MACEAL were analyzed in one of the duplicate test medium samples taken from the dilutions 1:22 to 1:2.2 at all sampling dates and in one of the duplicate test medium samples from the stock solution. The samples of the dilutions 1:100 and 1:46 were not analyzed since these concentrations were below the 48-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results.

Vehicle:

no

Details on test solutions:

Range-Finding Test for Selection of the Test Concentration of the Main Test
For determination of the appropriate test concentrations for the main test, a static range-finding test in a closed system was performed. Two replicates with 5 daphnids each per treatment were tested in parallel with a control.
The test concentrations were the undiluted equilibrated test medium with a loading rate of 100 mg/L and the dilutions 1:10 and 1:100. For preparation of the undiluted equilibrated test medium, the slow-stirring method was applied. The stirring time was 96 hours based on the results of the stirring pre-experiment. The undiluted equilibrated test medium was used in a series of dilutions with test water for preparation of the lower concentrated test media (dilutions 1:10 and 1:100).

Main Test
Based on the results of the stirring experiment and the range-finding test, the following concentrations of MACEAL were selected for the main test: the dilutions 1:2.2, 1:4.6, 1:10, 1:22, 1:46 and 1:100 of the equilibrated test medium with a loading rate of 100 mg/L. 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 test design with test medium renewal after 24 hours was chosen to further improve the maintenance of the test item concentrations during the test period of 48 hours. After 24 hours, the test organisms were transferred by wide-bore pipette in to clean test vessels with freshly prepared test medium of the corresponding concentration.
For each treatment, 20 daphnids were randomly distributed into four replicates of five daphnids each. The volume of test medium provided for each daphnid was 12 mL (60 mL per replicate). Thus, the requirement of the test guidelines for a minimum volume of 2 mL test medium per
daphnid was fulfilled

Dosage
At the start of the test and at the start of the renewal period after 24 hours (on Day -4 and Day -3), a stock solution (i.e. the undiluted equilibrated test medium with a loading rate of 100 mg/L), was prepared following the slow-stirring method with a stirring period of 96 hours at room temperature in the dark.
On both preparation dates, 239.5 μL of test item were carefully applied (pipetted) onto the surface of 2310 mL test water. This volume is equivalent to a loading rate of 100 mg/L, considering the density of the test item of 0.9639 g/cm3 (at 20 °C). This undiluted equilibrated test medium was used in a series of dilutions with test water for preparation of the test media. All solutions were clear with no evidence of undissolved test item. No auxiliary solvent or emulsifier was used.
The test media were prepared just before the start of the test (i.e., introduction of the daphnids to the test media) and prior to the test media renewal after 24 hours.
The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000

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 October 2017 (24-hour EC50: 1.3 mg/L, IES Laboratories Study 20170366) showed that the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60-2.1 mg/L).

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

The test was performed in a temperature-controlled room with continuous monitoring of the
room temperature. The water temperature was maintained at 20-21 °C.

Nominal and measured concentrations:

Range finding: dilutions 1:10 and 1:100 with a loading rate of 100 mg/L

Definitive test: dilutions 1:2.2, 1:4.6, 1:10, 1:22, 1:46 and 1:100 of the equilibrated test medium with a loading rate of 100 mg/L
Mean Measured Concentration of the Test Item during the 48-Hour Test Period: 8.2, 4.4, 2.1, 0.98 mg/L (The samples of the dilutions 1:46 and 1:100 were not analyzed since these concentrations were below the 48-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results)

Details on test conditions:

Material
Since the test item was determined to 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 the volatile substance by evaporation (closed system).

Experimental Conditions
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 between 15 and 17 μmol m-2 s-1.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 0.98 mg/L. At the next higher test concentration, 2.1 mg/L, all test organisms were found to be mobile but discolored. At the two highest mean measured concentrations of 4.4 and 8.2 mg/L, 40 and 100 % of the daphnids were found to be immobile (i.e. dead), respectively. In addition at 4.4 mg/L, the surviving daphnids showed reduced swimming activity.

After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 0.98 mg/L. At the next higher test concentration, 2.1 mg/L, 3 test organisms were found to be immobile, corresponding to an immobilization rate of 15%. In addition at 2.1mg/L, the surviving daphnids were found to be discoloured. At the test concentration of 4.4 mg/L, all daphnids were found to be immobile (i.e. dead). At the highest test concentration of 8.2 mg/L, all daphnids were found to be immobile (i.e. dead) still after 24 hours.

The 48-hour EC50 of the test item were calculated to be 2.7 mg/L with a lower 95% confidence limit of 2.2 mg/L and the upper one could not be determined. The 48-hour EC0 and NOEC were at the mean measured concentration of 0.98 mg/L.

No remarkable observations were made concerning the appearance of the test media. All test media remained clear solutions throughout the renewal periods.

At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.6 and 7.9. The dissolved oxygen concentrations in the test media and control were at least 8.2 mg/L and the water temperature was maintained at 20-21°C during the test.

Validity criteria fulfilled:

yes

Conclusions:

The test item MACEAL had acute toxic effects on Daphnia magna in a 48-hour semi-static test in a closed system. The biological test results (based on mean measured concentrations) were as follows:
The 48-hour EC50 of the test item were calculated to be 2.7 mg/L with a lower 95% confidence limit of 2.2 mg/L and the upper one could not be determined. The 48-hour EC0 and NOEC were at the mean measured concentration of 0.98 mg/L.

Description of key information

In the original REACH submission in 2016, no experimental data was available for short-term toxicity to aquatic invertebrates. Thus use of predicted values in a weight of evidence approach was used. Two predictions were performed using the OECD QSAR Toolbox 3.4.0.17. One using a category formed from the aquatic toxicity databases available in the QSAR Toolbox (2016a) and one using a category formed from Givaudans' proprietary database (2016b). The predicted 48 hour daphnid EC50 values were 1.82 and 1.77 mg/L respectively. The prediction results are considered reliable according to OECD principles. The prediction models are scientifically valid and the target chemical falls within the applicability domain of the prediction. The models are independent from each other (e.g. based on different training sets). Thus, the agreement among the predictions increases the confidence in the reliability of the predictions. Furthermore, the predicted values are within the same acute aquatic toxicity classification band of >1 and <=10mg/L and as such are considered adequate for regulatory purposes.

An experimental study became available in 2018 which was performed for regulatory requirements in Korea. The acute toxicity of the test item MACEAL to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals, No. 202. As the test item is 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 with a test medium renewal after 24 hours was used. Due to the decrease of test item concentrations during the test medium renewal periods, the mean concentration was calculated as the geometric mean of the test item concentrations measured at the start and the end of each test medium renewal period (i.e. Day 0 to Day 1, Day 1 to 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. The 48 hour EC50 was determined to be 2.7 mg/L based on mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.7 mg/L