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EC number: 227-813-5
CAS number: 5989-27-5
6.1.2/1: Results from chemical analyses of d-Limonene
T = 0 days (mg/L)
T = 3 days - old (mg/L)
T = 3 days - new (mg/L)
T = 6 days - old (mg/L)
T = 6 days - new (mg/L)
T= 8 days - old (mg/L)
Time weighted mean value (mg/L)
Sampled at T = 5 days, because all eggs/larvae were observed dead
2.5 % solutions were not analysed as they were not relevant for the
determination of the EC or NOEC values
Table 6.1.2/2: Mean measured
concentration, survival and mean length
Time-weighted mean concentration (mg/L)
Survival (N = 30)
Mean length (mm)
Overall hatching success and post-hatch success in the control was
greater than or equal to 60 and 70 %, respectively.
The dissolved oxygen concentration was between 60 and 100 % of the air
saturation value (ASV) throughout the test.
At one occasion, however, (one out of 3 replicates in the highest test
concentrations at day 5) the dissolved oxygen concentration was 50 %.
Due to the need for sealed test containers during the test it was not
possible to remove dead eggs and larvae. It is expected that the low
oxygen percentage observed was due to decomposition of dead eggs and
larvae. As all organisms died in the highest test concentration in all 3
replicates the low oxygen concentration observed in the single replicate
is not expected to have any influence of the overall results obtained.
The water temperature did not differ by more than ± 1.5 °C between test
chambers or between successive days at any time during the test (within
the range 25 ± 2.0 °C).
short-term toxicity test on embryo and sac-fry stages with Pimephales
performed according to OECD Guideline 212 and in compliance with GLP.
was tested at the following nominal concentrations: 0 (control), 2.5,
5.3, 11.0, 23.2 and 48.6% of a saturated solution of the test item in
test medium. The test was carried out as a semi-static test in 100 mL
Pyrex glass flasks sealed with PTFE-coated screw caps. Approx. 95% of
the test solutions were renewed at day 3 and day 6. At termination of
the test the growth of the hatched larvae was determined and during the
test, hatching, survival, abnormal appearance and behaviour was observed
and recorded daily. The duration of the test was 8 days. The
concentrations of the test item were determined by chemical analyses
during the exposure period.
the highest test concentration (0.67 mg/L), the hatching rate is
increased. Significant effect on the survival rate (100% mortality at
0.67 mg/L). Slight to moderate effects were observed on the appearance
and behaviour at 0.37 mg/L. The effect on growth rate, measured as
length of the surviving larvae at the end of the test was significant at
0.19 and 0.37 mg/L (both 4% reduction).
the NOEC for hatching, abnormal appearance & behaviour and growth were
0.37, 0.19 and 0.059 mg/L; LOEC for hatching, abnormal appearance &
behaviour and growth were 0.67, 0.37 and 0.19 mg/L; LC50 was determined
to be 0.41 mg/L (95% Cl: 0.31-0.53 mg/L) and the LC10 was determined to
be 0.32 mg/L (95%CI: 0.21 -0.41 mg/L. The effect on the growth rate
observed at 0.19 and 0.37 mg/L was less than 10% and data did not allow
the calculation of EC10 and EC50. Bot the EC10 and EC50 for growth are
between 0.37 and 0.67 mg/L.
The chronic toxicity to fish 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 26 chemicals derived from 14 to 32-day test on fish, for which
the concentrations of the test item had been determined by chemical
analyses over the test period.
The chronic toxicity to fish of d-limonene
has been investigated using a QSAR model that predicts fish lethality in
an OECD 210 study. d-Limonene falls within the applicability domain of
the model as demonstrated in the QPRF.
The 28d-NOEC for d-limonene was 0.080 mg
test item/L with 95%-Confidence Limit of 0.056-0.11 mg test item/L. The
NOEC was based on sublethal effects, like growth and hatchability.
This toxicity study is acceptable and can be
used for that endpoint.
The substance exhibits an estimated EC10 for freshwater fish higher than 0.37 mg/L.
One experimental data and a valid QSAR
prediction are available and are provided as endpoint study records.
The experimental study investigated the
short-term toxicity on embryo and sac-fry stages of substance d-limonene
on freshwater fish Pimephales promelas. The study was performed
according to OECD Guideline 212 under semi-static conditions for 8 days.
Hatching, survival, abnormal appearance and behaviour were observed
daily. Growth length was measured at 8 days (4 days post hatch). The
concentrations of d-limonene were determined by chemical analyses during
the exposure period. The analysis of concentrations were performed by
sampling analysis from additional test flasks without fish eggs (abiotic
groups). At the highest measured concentration of 0.67 mg/L (time
weighted mean), the hatching rate is increased. However, 100% mortality
was observed at 0.67 mg/L. The effect on growth rate, measured as length
of the surviving larvae at the end of the test was significant at 0.19
and 0.37 mg/L (both 4% reduction) but no clear concentration / response
was observed for growth endpoint. The endpoint values are based on the
measured concentration of d-limonene in abiotic replicates. Thus, the acquired
results of measured concentrations may be slightly higher than the
“true” concentrations to which the fish were exposed during 8 days. The 8-day
EC10 are estimated between 0.37 and 0.67 mg/L.
Besides, an endpoint value from High
Accuracy QSARs is also available. This provides a 28-day NOEC of 0.08
(0.056-0.11) mg/L for sublethal effects on fish. The value of NOEC is
the test concentration immediately below the LOEC. Where EC10-values are
available, these are preferred over NOEC values for the same endpoint.
Justification on the choice of OECD TG
In the ECHA Guidance R.7B, 184.108.40.206, Guideline
OECD 212 is listed under the long-term fish tests and mentioned as one
of the tests to be used for refinement of the PNEC value. The OECD 212
test includes the same sensitive life stages as OECD 210. It misses one
stage: where the alevins start feeding by themselves. The current OECD
212 study with d-limonene concludes a NOEC with a statistically
significant effect on growth of only 4%. The 4% growth reduction is
observed at two consecutive test concentrations (0.19 and 0.37 mg/L),
thus there is no dose-effect relation in that section of the curve. The
level with 10% effect is actually above 0.37 mg/L, and although the EC10
could not be derived statistically, 0.37 mg/L forms the appropriate
basis for the classification. In view of the difficulties in testing
this substance (poorly soluble in water, biodegradable, highly volatile,
potentially photodegradable) a longer test duration than 7 days would be
technically extremely difficult to perform as was already encountered in
the daphnid reprotoxicity study due to the problems of solution renewal,
the volumes necessary, biodegradation due to bacterial growth etc. This
study would be even more complicated to perform than the daphnid test..
Moreover, the test would then include free living vertebrate test
species for which prior approval would be needed.
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