Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

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 06, 2017 to July 06, 2017
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)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-hex-3-enyl isobutyrate
EC Number:
EC Name:
(Z)-hex-3-enyl isobutyrate
Cas Number:
Molecular formula:
(Z)-hex-3-enyl isobutyrate
Specific details on test material used for the study:
Name ( stated on the report) : Hexenyl-3-cis Isobutyrate
Appearance: Colourless to pale yellow liquid
Batch: VE00450298
Expiry date: July 15, 2017

Sampling and analysis

Analytical monitoring:
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 of 100 mg/L, samples were taken only from the first renewal period as all daphnids were immobile (i.e. dead) at Day 1. Additionally samples were taken from this test concentration at the start of the second renewal period as it was used as stock solution for the preparation of the test media with lower test concentrations.
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 preexperiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Test solutions

Details on test solutions:
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).
The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

Test organisms

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-Werke,
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.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

250 mg/L as CaCO3
Test temperature:
20 - 21 °C
7.7 - 7.9
Dissolved oxygen:
8.2 mg/L to 8.6 mg/L
Nominal and measured concentrations:
Nominal concentration: 4.6, 10, 22, 46 and 100 mg/L.
Mean Measured concentration: 3.8, 9.2, 22, 46 and 102 mg/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 in the range of 20-21 °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 approximately between 15 and 17 μmol m-2 s-1.

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
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 9.2 mg/L. At the mean measured concentrations of 22 and 46 mg/L, 35 and 75 % of the daphnids were found to be immobile, respectively. At the highest mean measured concentration of 102 mg/L, all test organisms were already immobile (i.e. dead) after 24 hours exposure time.
The 24-hour EC50 of the test item was calculated to be 32 mg/L with 95% confidence limits of 26 and 40 mg/L. The 24-hour EC0 and NOEC were at the mean measured concentration of 9.2 mg/L. The 24-hour EC100 was 102 mg/L (mean measured).
After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 9.2 mg/L. At the mean measured concentrations of 22 and 46 mg/L, 65 and 100% of the daphnids were found to be immobile, respectively.
The 48-hour EC50 was calculated to be 20 mg/L with 95% confidence limits of 18 and 23 mg/L.
The 48-hour EC0 and NOEC of HEXENYL-3-CIS ISOBUTYRATE were both at the mean measured concentration of 9.2 mg/L, since no toxic effect was observed up to and including this test concentration. The 48-hour EC100 was at the mean measured concentration of 46 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
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).
The test item HEXENYL-3-CIS ISOBUTYRATE had acute toxic effects on Daphnia magna in a 48-hour semi-static test.
The 48-hour EC50 was determined to be at the test concentration of 20 mg/L with 95%-confidence limits of 18 and 23 mg/L (based on mean measured concentrations).
Executive summary:

The acute toxicity of the test item HEXENYL-3-CIS ISOBUTYRATE 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.

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.

The nominal test item concentrations tested were 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The test media was prepared by completely dissolving the test item in test water using intense stirring for 24 hours in a closed vessel. The mixing vessel was nearly completely filled

(including a small headspace) and tightly sealed with glass stoppers. The measured concentrations of HEXENYL-3-CIS ISOBUTYRATE in the test media of all test concentrations were between 80 and 113% of the nominal values at the start of the two test medium renewal periods. Thus, the correct dosage of the test item could be verified.

At the end of the two renewal periods, 77 to 116% of the nominal values were found. Thus, the test item was sufficiently stable during the test medium renewal periods of 24 hours.

The biological results are based on the mean measured concentrations. These were calculated as follows: for each test medium renewal period (i.e. Day 0-Day 1, Day 1-Day 2), the mean concentration was calculated as the geometric mean of the test item concentrations measured at the start and the end of the test medium renewal period. 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 at the test concentration of 20 mg/L with 95%-confidence limits of 18 and 23 mg/L (based on mean measured concentrations).

A clear dose-response effect was observed after the exposure period of 48 hours: At the two lowest test concentrations no toxic effect was observed. At 22 mg/L (mean measured) 65% of

the daphnids were immobile. At the two highest test concentrations all daphnids were found to be immobile (i.e. dead) after 48 hours.