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EC number: 226-789-3
CAS number: 5468-75-7
mean of living offspring
coefficient of variation
n = number of daphnids exposed per concentration
The test item was tested in Daphnia magna (water flea) over 21 days in a
semistatic system (GLP, key). Since no effects were expected up to the
limit of solubility a limit test with a nominal concentration of 1 mg/L
A negative control (0 mg/L) and a nominal concentration of 1 mg/L were
tested. The tested nominal concentration is already above the limit of
solubility of the test substance in water ( <=
The test was conducted under semistatic conditions. The media and the
test solutions were renewed every Monday, Wednesday and Friday. For
every renewal new test solutions were prepared. This was done by
stirring the test substance for 48 hours in the test medium. The aqueous
phase of the concentration group was present as a slightly yellowish
clear solution during the entire exposure period. There were no visible
particulate matters. Water parameters were within the normal range.
Analytical monitoring of the concentration of the test substance in the
test water was not conducted since no relevant results could be
expected. The solubility limit of the test substance is below the
analytical detection limit. From the properties of the test substance it
could be concluded that the test substance is chemically stable in water
over the test intervals of 48 and 72 hours, respectively.
The concentration values given in this report are based on the nominal
In the control group no mortality occurred and the mean number of living
offspring produced per parent animal was 126.0, thus fulfilling the
validity criteria for the test.
No immobility occurred in the 1 mg/L (nominal concentration) and in the
control group. No difference in the onset of brood production was
observed in the concentration group in comparison to the control. The
reproduction rate in the concentration group showed no statistically
significant changes in comparison to the control.
In this prolonged toxicity study with C.I. Pigment Yellow 13 in Daphnia
magna over 21 days the following effect concentrations were obtained:
NOEC (immobility) = 1 mg/L (nominal concentration, solubility limit <=
NOEC (reproduction rate) = 1 mg/L (nominal concentration, solubility
limit <= 20 pg/L)
[%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure
Nominal Loadingof theSaturated Solution[mg/L]
of Juveniles in the Control and Test Group after 21 Days
Nominal Loading of the SaturatedSolution
Mean No. of Juveniles
Number of Juveniles in Replicate No.
MV ± SD
N= number of parents producing juveniles alive; Inc.= increase of
Appearance of Living Juveniles in the Individual Groups
Day of First Appearance of Living Juveniles
in Replicate No.
Length and Dry Weight of the Parental Daphnids
Total Length of the Parental Daphnids [mm]
Dry Weight [mg]
S= sum; N= number of parental daphnids alive;
Daphnia magna Reproduction Test (Semi-Static, 21 d) with the test
a limit test with the saturated solution* according
to OECD 211 (2008) from 2009-11-09 to 2009-12-03
1, D-31157 Sarstedt, Germany.
was Daphnia magna STRAUS (Clone 5). 10 test organisms,
individually held were used for the limit concentration and control. At
test start they were 2 to 24 hours old. The test method
was semi-static. The test solutions were renewed 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 on
parameters such as occurrence of aborted eggs and stillborn juveniles,
time of production of first brood, adult mortality, intrinsic rate of
natural increase, dry body weight and length of the parent animals.
A specific analytic of the test item was
not carried out, due to the low solubility of the test item in water.
All results given are based on the saturated solution* with the nominal
loading of 10 mg/L.
average number of juveniles per parental daphnid in the control
group was 116 and 117 at the saturated solution* after 21 days. The
reproductive output at the limit concentration is comparable to the
control group (Unpaired
t-test, p = 0.05).
coefficient of variation of the number of living offspring produced
per parent was 6 % at the control group and the saturated
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 IR of the saturated
solution* was comparable to
the control group (Unpaired
t-test, p = 0.05).
or immobilization of parental daphnids was observed
at the control group and at the saturated
to the total number of produced juveniles (dead + alive) the percentage
of dead juveniles and aborted eggs was lower than 1 % at the
saturated solution, which is seen to be not biologically significant. At
the control no dead juveniles or aborted eggs were observed.
broods were observed during the test period at all surviving parental
daphnids. The first brood was released till day 9 by all parent
animals of the control and the saturated solution*.
mean dry body weight and total
body lengths of
all parental daphnids of the test and control group determined at the
end of the study were comparable.
mean dry body weight of the parental daphnids was 0.87 mg per daphnid at
the control group and 0.86 mg per daphnid at
the saturated solution. The mean
body length of the parental daphnids at
the saturated solution was 5.35 mm per
daphnid and 5.38 mm per daphnid at the control group.
and ephippia (winter eggs) were
observed at control or test groups.
summary of all test item related effects is given in Table 1.
There was no biologically or statistically significant effect determined
at the saturated solution* of the test item.
Summary of all
Test Item Related Effects
on the saturated solution* of test item with
the nominal loading of 10 mg/L)
(Nominal Loading 10 mg/L)
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)
116 ± 7
117 ± 7
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent
Intrinsic Rate of Natural Increase
Appearance of First Brood [Mean Day]
Number of Broods
Stillborn Juveniles and Aborted Eggs
Adult Mortality after 21 days [%]
Parent Animals: Mean Dry Weight [mg]
Parent Animals: Mean Body Length [mm]
> Saturated Solution*
water quality parameters (pH-value, dissolved oxygen, water
hardness and temperature) were determined to be within the acceptable
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 dichromateas
reference item once per month.
The EC50 of the reference item at 1.67 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. *) Saturated solution: The
maximum dissolved concentration of the test item that can be achieved
under the test conditions in the test medium , acc. to OECD Series No.
No toxicity of Pigments Yellow 13 and 83, the structural analogues of
Pigment Yellow 14, was found in reliable (category 1) OECD 211-tests
(Daphnia magna Reproduction Test) with nominal concentrations well above
the solubility limit.
Therefore, the Diarylide Yellow Pigments are to be regarded as
chronically nontoxic to invertebrates.
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