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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:
2019
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEFM177269
- Expiration date of the lot/batch: 19.09.2022
- Purity test date: 15.10.2018

Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 40.9, 51.2, 64.0, 80.0, 100.0 mg/L test item
- Sample storage conditions before analysis: Specimens were diluted with methanol (1:1) and were stored deep frozen (≤ -18°C) until they were analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution was prepared by adding the test item Isobornyl acetate to test medium.
The stock solution was treated with ultrasonic for 30 minutes and stirred for 110 minutes on a magnetic stirrer. The stock solution was visible cloudy.
Stock solution and test vessels were prepared in the following way:

- A stock solution was prepared by weighing of 103.0 mg test item into a graduated flask and filling up to 1000 mL using test medium
(= stock solution A, corresponding to a concentration of 103.0 mg/L test item).

The test solutions of the chosen concentrations were prepared directly in the test vessels by the dilution of the stock solution A with the test medium immediately before the transfer of the Daphnia into the test vessels.

- Controls: dilution water. The control vessels contained test medium (dilution water) only. 5 days before the start of the test, the test medium was prepared and aerated overnight.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: RWTH Aachen University, Institute for Environmental Research (Biology V)
Worringerweg 1
52074 Aachen
Germany

- Age at study initiation (mean and range, SD): 24 h
- Method of breeding: breeding was performed in glass beakers (50 mL) under the same conditions as in the test; test animals were separated from the adults by means of a pipette

- Source:
RWTH Aachen University, Institute for Environmental Research (Biology V)
Worringerweg 1
52074 Aachen
Germany

- Feeding during test : none


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
test animals were separated from the adults by means of a pipette
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
231 mg/L CaCO3
Test temperature:
20.1 – 20.6 °C (recorded continuously)
pH:
7.66 to 8.02
Dissolved oxygen:
7.72 to 9.32
Nominal and measured concentrations:
Control (test medium only), 40.9, 51.2, 64.0, 80.0, 100.0 mg test item /L
Control (test medium only) 9.54, 14.3, 17.9, 22.5, 26.1 mg isobornyl acetate/L

Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker (25 mL)
- Type (delete if not applicable): open
- Volume of solution: 20 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): renewal after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 + 3 additional vessels (1 for measuring of water parameters, 1 for analysis, 1 retain specimen)
- No. of vessels per control (replicates): 4 + 3 additional vessels (1 for measuring of water parameters, 1 for analysis, 1 retain specimen)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water: reconstituted water according to ISO 6341, prepared 5 days in advance; conductivity of deionised water: ≤ 10 µS/cm; measured: 0.9 µS/cm)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8
- Light intensity: 16/8 light/dark phases (on average 20 µE *m-2*s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
19.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: immobilization
Details on results:
- Behavioural abnormalities: none
- Mortality of control: none
- Other adverse effects control: none
- Immobilisation of control: none
- Abnormal responses: No abnormal behaviour or appearance was observed (e.g. trapping at surface of water or discoloration).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: unclear under test conditions, but results were based on geometric mean measuired concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50
- Dose-response test: yes
- ECx: EC50 1.89 mg/L Potassium dichromate
Reported statistics and error estimates:
Fisher`s Exact Binomial Test for 24 hours; Step-down Cochran-Armitage Test for 48 hours, p <= 0.05, one-sided greater

Number of immobilisedDaphnia magnaand percentage immobility

 

Treatment group

mg/L test item, nominal

(mg/L IBA, mean measured)

ImmobilisedDaphnia(number)

Immobility ofDaphnia(%)

 

3 h

24 h

48 h

3 h

24 h

48 h

Control (test medium only)

0

0

0

0.0

0.0

0.0

40.9

(9.54)

0

0

0

0.0

0.0

0.0

51.2

(14.3)

0

0

2

0.0

0.0

10.0 +

64.0

(17.9)

0

0

8

0.0

0.0

40.0 +

80.0

(22.5)

0

1

14

0.0

5.0

70.0 +

100.0

(26.1)

0

4

18

0.0

20.0

90.0 +

+significantly different from the control

(Fisher`s Exact Binomial Testfor 24 hours; Step-down Cochran-Armitage Test for 48 hours, p<=0.05, one-sided greater)

Validity criteria fulfilled:
yes
Conclusions:
Daphnia magna 48 h-EC50: 19.3 mg isobornyl acetate/L
Executive summary:

 Juckeland (2019) performed an acute invertebrate immobilisation test according to OECD 202 under GLP to assess the effects of the test item Isobornyl acetate (IBA) to Daphnia magna during 48 hours of semi-static exposure.

 Concentrations of the active ingredient in the test media were analytically verified. As a result, the measured concentrations of Isobornyl acetate remained within a range of 74 – 86% of nominal concentrations in the freshly prepared test solutions at the start of the test and at the renewal in the freshly prepared test solutions at 24 hours after test start. The Isobornyl acetate concentrations in the spent test solutions were determined at 5 – 11% of nominal concentrations at the renewal and at the end of the test (48 hours).

 The calculated endpoints are based on the nominal concentrations for the test item and on the geometric mean measured concentration for the test item as active substance.

 Significant effects on Mobility were found using Step-down Cochran-Armitage Test (alpha = 0.050, one-sided greater) at the nominal test concentrations ≥51.2mg/L test item at 48 hours. As a result, a LOEC of 51.2 mg/L test item (equivalent to 14.3 mg/L IBA, mean measured) was determined. The corresponding NOEC was 40.9 mg/L test item (equivalent to 9.54 mg/L IBA, mean measured).

 The EC10,EC20 and EC50 values for immobilisation based on nominal concentrations were calculated to be 51.1, 57.0 and 70.3 mg/L test item at 48 hours (equivalent to 13.9, 16.6 and 19.3 mg/L IBA, mean measured).

All validity criteria were achieved.

 

 

Description of key information

Isobornyl acetate EC50 (48h): 19.3 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
19.3 mg/L

Additional information

The Klimisch 1 study from Juckeland (2019) is the key study. Further Klimisch 2 QSAR information is available, but not considered as relevant as the experimental result and therefore not reported here.

Juckeland (2019) performed an acute invertebrate immobilisation test according to OECD 202 under GLP to assess the effects of the test item Isobornyl acetate (IBA) to Daphnia magna during 48 hours of semi-static exposure.

 Concentrations of the active ingredient in the test media were analytically verified. As a result, the measured concentrations of Isobornyl acetate remained within a range of 74 – 86% of nominal concentrations in the freshly prepared test solutions at the start of the test and at the renewal in the freshly prepared test solutions at 24 hours after test start. The Isobornyl acetate concentrations in the spent test solutions were determined at 5 – 11% of nominal concentrations at the renewal and at the end of the test (48 hours).

 The calculated endpoints are based on the nominal concentrations for the test item and on the geometric mean measured concentration for the test item as active substance.

 Significant effects on Mobility were found using Step-down Cochran-Armitage Test (alpha = 0.050, one-sided greater) at the nominal test concentrations ≥51.2mg/L test item at 48 hours. As a result, a LOEC of 51.2 mg/L test item (equivalent to 14.3 mg/L IBA, mean measured) was determined. The corresponding NOEC was 40.9 mg/L test item (equivalent to 9.54 mg/L IBA, mean measured).

 The EC10,EC20 and EC50 values for immobilisation based on nominal concentrations were calculated to be 51.1, 57.0 and 70.3 mg/L test item at 48 hours (equivalent to 13.9, 16.6 and 19.3 mg/L IBA, mean measured).

The EC50 of 19.3 mg isobornyl acetate/L will be used for the risk assessment.