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EC number: 224-809-5
CAS number: 4500-29-2
of Juveniles in the Control and Test Groups after 21 Days
Mean No. of Juveniles
Number of Juveniles in Replicate No.
MV ± SD
Juveniles and Aborted Eggs after 21 Days
Nominal Test Item
Mean Stillborn Juveniles and Aborted Eggs per Parent alive
0.67 ± 1.15
3.10 ± 1.91
2.90 ± 2.13
0.70 ± 1.06
0 ± 0
Appearance of Living Juveniles in the Individual Groups
Day of First Appearance of Living Juveniles
in Replicate No.
[%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure
Nominal Test Item Concentration
Adult Mortality [%]
Body Length and Dry weight of the Surviving Parental Daphnids
Total Length of the Parent Animals
magna Reproduction Test (semi-static, 21 d) of the test item 2,2’-(Cyclohexylimino)diethanol(batch
number ESD0011452) was
conducted according to OECD 211 (2008) from 2011-08-31 to
2011-09-22, with the definitive
exposure phase from 2011-08-31 to 2011-09-21,
1, 31157Sarstedt, Germany.
species was Daphnia magna STRAUS (Clone 5). Ten daphnids,
individually held, were used per concentration level, pH-control and
control. At test start the daphnids were 2 to 24 hours old. The
study was carried out under
semi-static conditions with renewal of the test solutions 3 times per
week. Aim of the Daphnia Reproduction Test over 21 days was to assess
effects on the reproduction capacity and other test item-related effects
or parameters such as time of production of first brood, adult
mortality, intrinsic rate of natural increase, occurrence of aborted
eggs and stillborn juveniles and body length of the parental daphnids.
Nominal concentrations of
2,2’-(Cyclohexylimino)diethanolwere selected as follows: 6.40 - 16.0 -
40.0 - 100 - 250 mg/L.
The concentrations of the test item
2,2’-(Cyclohexylimino)diethanol were analytically verified by
LC-MS/MS of samples taken on days 0, 7, 14 (fresh media, 0 hours) and on
days 2, 9, 16 (old media, 48 hours) of all concentration levels, the
pH-control and the control.
The recoveries in the fresh media (0
hours) and in the old media (48 hours) were in the range of 92 to 106 %,
demonstrating that concentrations of 2,2’-(Cyclohexylimino)diethanolwere
successfully maintained for the duration of the test.
Therefore, all effect values given are based on the nominal
concentrations of the test item 2,2’-(Cyclohexylimino)diethanol.
· The average number of juveniles
per parent in the control group was 131 after 21 days. The reproductive
output was statistically significant reduced in comparison to the
control at the nominal test item concentrations of 40.0 and 100 mg/L.
The other concentration levels of 6.40 and 16.0 mg/L were not
statistically significant reduced when compared to the control (Kruskal-Wallis
One Way Analysis of Variance on Ranks, Dunn’smethod,
p 0.05). No reproductive output was observed in the concentration level
250 mg/L and in the pH-control (containing 250 mg test item/L) due to
100 % mortality of the daphnids.
The EC10 for the reduction of the reproductive output was
calculated by sigmoidal dose-response regression to be 24.3 mg/L (95%
confidence limits: 20.3 – 29.1 mg/L). The EC50 was
calculated to be 54.0 mg/L (95 % confidence limits: 49.1 – 59.7 mg/L),
coefficient of variation of the number of living offspring produced
per parent was 3 % in the control.
Lowest Observed Effect Concentration (LOECReproduction)
and the No Observed Effect Concentration (NOECReproduction)
after 21 days based on the reproductive output as the most sensitive
effect are summarized in Table 1.
intrinsic rates of natural increase (IR)of the surviving parent
animals accounting for generation time and number of offspring were used
for calculation of population growth and maintenance. The mean IR of the
surviving daphnids of the treatment groups were compared to
the control by Kruskal-WallisOne
Way Analysis of Variance on Ranks, Dunn’smethod
(p 0.05). There was a
statistically significant difference at the concentration levels of 40.0
and 100 mg/L. No intrinsic
rate could be calculated for the concentration level 250 mg/L and the
pH-control (containing 250 mg test item/L) due to 100 % mortality of the
daphnids. The other concentration
levels of 6.40 and 16.0 mg/L were not statistically significant when
compared to the control.
to the total number of produced juveniles (dead + alive) the percentage
of dead juveniles and aborted eggs was ≤
3 % at the tested concentration levels 6.40 to 100 mg/L (Table
In the control no dead juveniles or aborted eggs were observed. In
the concentration level 250 mg/L and in the pH-control (containing 250
mg test item/L) no juveniles were observed due to 100 % mortality of the
broods were observed during the test period at all surviving parental
daphnids in the concentration levels of 6.40 to 40.0 mg/L. In the
concentration level 100 mg/L the number of broods was significantly
reduced to ≤ 3 broods per surviving daphnid during the exposure phase (Kruskal-WallisOne
Way Analysis of Variance on Ranks, Dunn’smethod,
p 0.05). In
the concentration level 250 mg/L and in the pH-control no broods were
observed due to 100 % mortality of the daphnids. The first brood
was released till day 9 by all surviving parental daphnids of the
control and the nominal test item concentrations of 6.40 to 40.0 mg/L.
In the concentration level of 100 mg/L the first brood was released
after day 15, which is regarded to be biologically significantly
Effects on Reproduction (EC10, EC50, NOEC and LOEC)
Nominal Test Item Concentration [mg/L]
Mean Number of Juveniles
per Producing Parental Daphnid
(Reproduction Rate ± SD)
131 ± 4
128 ± 4
126 ± 7
92 ± 102)
20 ± 182)
Mean Intrinsic Rates of Natural Increase
Appearance of First Brood [Mean Day]
EC10, Reproduction(with 95 % confidence limits)
24.3 mg(L (Cl: 20.3 – 29.1 mg/L)
EC50, Reproduction(with 95 % confidence limits)
54.0 mg/L (Cl: 49.1 – 59.7 mg/L)
of test item related effects concerning the adult mortality (EC10, Adult
Mortality and LOECAdult
Mortality) and growth
(dry weight, body length) is given in Table 2.
test item induced biologically substantial adult
mortality of 70 % in the
nominal test item concentration of 100 mg/L
and 100 % in the concentration level of 250 mg/L and in the pH-control
after 21 days. According to the
guideline adult mortality of > 20 % is seen to be biologically
concentration levels of 6.40 to 40.0 mg/L and in the control all
parental daphnids survived until the end of the study.
The EC10 for the adult mortality was calculated by sigmoidal
dose-response regression to be 73.8 mg/L (95% confidence limits: 73.7 –
73.8 mg/L. The EC50 was calculated to be 91.9 mg/L (95 %
confidence limits: 40.0 – 250 mg/L), respectively.
mean body lengths of the surviving parental daphnids in the
nominal test item concentrations 6.40 to 100 mg/L were determined to be
in the range of 4.33 to 5.50 mm per daphnid and 5.25 mm per daphnid in
the control group, respectively (Table 2
and Table 10).
The body length of the surviving daphnids was not statistically
significantly reduced at any of the tested concentration levels in
comparison to the control (Kruskal-Wallis
One Way Analysis of Variance on Ranks, Dunn’smethod,
The mean dry
body weight was determined to be in the range of 0.03 to 0.71 mg per
daphnid in the test groups and 0.66 mg per daphnid in the control,
Related Effects on Adult Mortality (EC10,
EC50, NOEC and LOEC)
Adult mortality after 21 days [%]
Mean body length of parental daphnids [mm]
Dry weight of parental daphnids [mg]
EC10, Adult Mortality(with 95 % confidence limits)
73.8 mg(L (Cl: 73.7 – 73.8 mg/L)
EC50, Adult Mortality(with 95 % confidence limits)
91.9 mg/L (Cl: 40.0 – 250 mg/L)
EC100, Adult Mortality
and ephippia (winter eggs)
occurred in control or test groups.
quality parameters as
pH-value, dissolved oxygen, water hardness and temperature were
determined to be within the acceptable limits.
order to prove the validity of the test system and test
conditions at the test facility, an acute immobilization test according
to DIN 38412 L 11 was carried out with potassium dichromate as
reference item once per month.
The EC50 of the reference item at 1.70 mg/L after 24 hours
was within the prescribed concentration range of 1.0 - 2.5 mg/L of
quality criteria according to AQS P 9/2 (05/1996)
for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value
of the reference item is also within the recommended range of 0.6 - 2.1
mg/L according to OECD-Guideline 202.
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