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EC number: 227-813-5
CAS number: 5989-27-5
6.1.4/1: Endpoints observed in the 21-day reproduction test with Daphnia
Actual concentrations (TWM) μg/L
Average number of offspring
Mortality among parent animals (%)
Average time to which the first offspring were present (days)
Average number of dead offspring
Average number of aborted broods per replicate
raises in all test concentrations because the test is performed in
closed flasks. Only in the highest test concentrations, pH are above 9,
which is due to the low number of offspring available to eat the algae,
which is causing the pH rise. The oxygen saturations recorded
simultaneously is >95 % during the whole test in all test
validation criteria of the test guideline for the control series are
The total male development and mortality of parent animals in the
control did not exceed 20 % at the end of the test, as no females died
and there were no males observed.
The mean number of live offspring produced per female animal alive at
the end of the test was ≥60. The result was 63.9 offspring per female.
study was conducted to
test the inhibitory effects of d-limonene on the reproduction of
freshwater crustacean Daphnia magna according to Guideline OECD
211 and in compliance with GLP.
was tested at the following nominal concentrations: 0 (control), 2.5, 4,
6.5, 10 and 16% of a saturated solution of the test item in the test
medium. As the test item is volatile, a closed test system with a minor
headspace was used. The test was carried out as a semi-static test in
50-mL glass flasks sealed with PTFE-coated screw caps. All test
solutions were renewed every Monday, Wednesday and Friday. The
concentrations of the test item were determined by chemical analyses
during the exposure period. The
actual test concentrations of d-limonene was used in the calculation of
the Time Weighted Mean (TWM), which was applied in the data analysis of
the effect concentrations.
of the measured concentrations were 23, 50, 80, 173 and 363 μg/L.
first offspring was observed after 8-10 days in the controls. Few
aborted broods were observed whereas dead offspring was observed in all
test concentrations and in the controls, indicating that the effect was
not treatment-related. Very few offspring was observed at the highest
tested concentration. Mean number of live offspring produced per female
animal alive at the end of the test were 63.9, 80.1, 88.4, 76.4, 42.8
and 1.5 offspring per female at 0, 23, 50, 80, 173 and 363 μg/L.
taking into account the number of living offspring per surviving female
as effect parameter, the EC10 and EC50 were 153 µg/L (95% CL: 83 -222
µg/L) and 188 µg/L (95%CL: 118 -257 µg/L), respectively. The NOEC was 80
µg/L and the LOEC was 173 μg/L. The effect parameters are based on the
TWM of the measured concentration. The
validity criteria were fulfilled.
The chronic toxicity of the daphnids was
determined using a validated QSAR for the Mode of Action in question
(MOA 1, non-polar narcosis). The QSAR is based on validated data for a
training set of 30 chemicals derived from 21-day test on daphnids, for
which the concentrations of the test item had been determined by
chemical analyses over the test period.
The chronic toxicity of d-limonene to
daphnids has been investigated using a QSAR model that predicts
reduction of daphnid reproduction in an OECD 211 study. d-Limonene,
falls within the applicability domain of the model as demonstrated in
The 21d-NOEC was predicted as 0.050 mg/L
with 95%-Confidence Limit between 0.035 and 0.070 mg /L. The NOEC was
based on reproduction.
This toxicity study is acceptable and can be
used for that endpoint.
The substance exhibits a 21-day EC10 of 0.153 mg/L for freshwater invertebrates.
One experimental data and a valid QSAR
prediction are available and are provided as endpoint study records.
The experimental study investigated the
effects of substance d-limonene on the reproduction of freshwater Daphnia
magna during 21 days. The study was performed according to OECD test
guideline 211 in a semi-static exposure system. The endpoint assessed is
the number of living offspring per surviving female. A
concentration-effect relationship was clearly observed. The endpoint
values (NOEC, LOEC, EC10, EC50) were based on measured concentrations in
abiotic groups that were run in parallel to the test as it was not
possible to acquire meaningful results by sampling directly from the
beakers containing daphnids. Thus, the results acquired of measured
concentrations may be slightly higher than the “true” concentrations to
which the daphnids were exposed during 21 days. The EC10 and EC50 were
153 µg/L (95% CL 83 -222 µg/L) and 188 µg/L (95% CL 118 -257 µg/L),
respectively. The NOEC was 80 µg/L and the LOEC was 173 µg/L.
Besides, an endpoint value from High
Accuracy QSARs is also available. This provides a 21-day NOEC of 50 µg/L
The EC10 value from experiment is obtained
by an appropriate statistical method for modelling the
concentration/response relationship. Besides, the NOEC is a test
concentration for which the substance is observed to have no statistical
difference effect by comparison with the control, and thus is being
subjective to choice of nominal test concentrations. The NOECs and the
LOECs straddle the classification threshold, with NOEC below 0.1 mg/L,
but the geometric mean (MATC) of the NOECs and LOECs of the three
studies is greater than 0.1 mg/L suggesting that the “true” effect
concentration is higher than this classification threshold.
For the purposes of setting a self-classification
and defining a key value for chemical safety assessment, the EC10 for
long-term toxicity to aquatic invertebrates is used preferentially to
NOEC for the same endpoint and the same duration.
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