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CAS number: -
of Juveniles in the Control and Test Groups after 21 Days
Mean No. of Juveniles
Number of Juveniles in Replicate No.
MV ± SD
Number of parents producing juveniles alive
± SD =
Mean value ± standard deviation
Coefficient of variation
Increase on reproductive output in comparison to the control group
Statistical significance (One
Way Analysis of Variance,
p = 0.05)
Statistically not significant
Not applicable, due to mortality of the parental animal
Not applicable, due to the mortality of all daphnids
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
Adult Mortality [%]
Body Length and Dry Weight of the Parent Animals
Total Length of the Parent Animals
magna Reproduction Test with Natural River Water (Semi-Static, 21 d)
of the test item Tallowtripropylenetetramine (CAS
batch number S001255)
was conducted according to OECD 211 (2008) from 2009-07-22
to 2010-01-18, with the definitive
exposure phase between 2009-07-22 to 2009-08-12 at
1, D-31157 , Germany .
system was Daphnia magna STRAUS (Clone 5). 10 test organisms,
individually held, were used per concentration level and control. At the
beginning of the test 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 adult
mortality, intrinsic rate of natural increase, time of production of
first brood, occurrence of aborted eggs and stillborn juveniles, dry
body weight and length of the parent animals.
Tallowtripropylenetetramine (CAS No.
68911-79-5) is insoluble in water and also has a strong tendency to
adsorb to negatively charged surfaces such as suspended matter, algae
and test vessels or organic material (including dissolved organic matter
such as humic acids). Many cationic substances in general but long chain
alkyl polyamines in particular rank among the most difficult substances
to test in environmental toxicology. Standard guideline studies are
inappropriate to test substances with such properties and the current
REACH Guidance Documents do not provide sufficient guidance concerning
bioavailability and exposure assessment for cationic surface-active
substances like the polyamines as these were written with normal
hydrophobic chemicals in mind, failing to take into account the lack of
bioavailability that occurs in the environment with these substances.
The aquatic ecotoxicity tests with
polyamines were therefore performed in river water to allow a PECaquatic,bulk/PNECaquatic,bulkapproach
and is considered to be conservative but more environmentally realistic
than the standard method. This approach is based on PEC estimations
representing ‘total aquatic concentrations’. To characterize the risk to
the aquatic compartment the PECaquatic,bulk is compared with
the PNECaquatic,bulk derived from river water ecotoxicity
studies (ECETOC, 2001).
In order to class standard
laboratory toxicity study valid, it is of particular importance that -
besides information on test substance, test method / conditions and test
organism used - suitable precautions are taken to prevent the loss of
test substance by adsorption and that exposure concentrations are based
upon measured levels.
For ecotoxicity tests performed using
the bulkapproach, however, adsorption to suspended matter and DOC
is acceptable and only adsorption to glassware should be accounted for.
For a valid bulk approach test the concentration-effect relationship
should be based on the sum of adsorbed and dissolved substance in the
volume of the medium tested. One of the advantages of the bulk approach
tests with these difficult substances is that in the presence of
suspended matter, humic acids and/or algae, the residual sorption to
glassware will be negligible. The results of these bulk approach tests
are therefore much easier to interpret, more environmental realistic,
and if compared to PECbulk clearly provide a more appropriate
assessment of risks for the environment.
Nominal concentrations of the test
item Tallowtripropylenetetramine were selected after a preliminary acute
immobilization test (48 h, static) as follows: 0.128 - 0.320 - 0.800 -
2.00 - 5.00 mg/L. The test item Tallowtripropylenetetramine was
analytically verified in the concentration levels 0.320, 0.800 and 2.00
mg/L and in the control via LC-MS/MS of samples taken on days 0, 7, 9,
14 (fresh media) and on days 2, 9, 16 (old media, 48 h) and 12 (old
media, 72 h). Due to the mortality of all parent animals in
concentration level 2.00 mg/L on day 3, the concentration level 0.800
mg/L was analytically verified instead at the remaining sampling. The
freshly prepared stock solutions of 10 mg test item/L were analytically
verified too and gave recoveries in the range of 86 to 133 % of the
Recoveries between 85 to 115 % were obtained in the fresh media for the
test groups exposed to 0.800 and 2.00 mg/L. In the fresh media recovery
rates between 49 to 121 % were obtained in the test group exposed to
0.320 mg test item/L.
In the old media recoveries decreased
to values in the range of 25 to 86 % of the nominal values. The old
media samples were analysed including the sorbed fraction to glassware.
In general lower recovery rates were observed for the samples at the end
of every batch independent of the exposure concentration. Biodegradation
as possible reason for the observed decrease of the test concentration
during the exposure period is very unlikely considering the short time
frame between the refreshments of the test solutions. The sorption of
the test item to glassware was determined exemplarily at the
concentration level 0.800 mg/L. The concentration observed was
0.189 mg/L, which corresponds to 24 % of the nominal test concentration.
The observed concentration decrease between fresh and old media is thus
only for a small fraction caused by sorption to glassware. The remaining
reduction is most likely caused by thermodynamically more favourable
redistribution of the sorbed fraction resulting in an additional
sorption to suspended matter and DOC. The results of the chemical
analyses show that the test organisms were fully exposed to the test
substance during the test. Therefore, all effect values are given based
on the nominal test item concentrations.
· The average number of juveniles
per parent in the control group was 144 after 21 days. The reproductive
output was not statistically significant increased or reduced in any
of the tested concentration levels when compared to the control (One Way
Analysis of Variance, p = 0.05). At the concentration levels 2.00 and
5.00 mg/L no offspring was produced due to mortality of all parental
daphnids before day 3 of the exposure phase. The estimation of an EC10-
or EC50-value for the reduction or increase of the
reproductive output could not be performed due to the absence of effects
coefficient of variation of the mean number of living offspring produced
per parent alive in the control group was 5 %.
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 One Way
Analysis of Variance (p = 0.05).
There was no statistical significance determined at any of the tested
juveniles and aborted eggs were produced by the control group.
Related to the total number of produced juveniles (dead + alive) the
percentage of dead juveniles was below 1 % at all test groups, which is
not biologically significant.
mean day of release of the first brood was day 8.0 in the control
group. At the tested concentration levels 0.128 to 0.800 mg/L the mean
day of release of the first brood was in the range of 7.7 to 8.1. At the
concentration levels 2.00 and 5.00 mg/L no daphnid survived until the
end of the test. The first brood was released until day 9 by all
surviving daphnids of the control group and the tested concentration
levels. Five broods were released by all surviving animals of the
control and the test groups.
study the test item did not induce statistically significant effects on
reproduction. A summary of all test item related effects on reproduction
is given in Table 1.
The estimation of an EC10- or EC50-value for the
reduction or increase of the reproductive output could not be performed
due to the absence of effects on reproduction.
Related Effects on Reproduction (NOEC and EC50)
Nominal Concentration [mg/L]
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)
144 ± 7
145 ± 15
160 ± 9
144 ± 20
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent
Mean Intrinsic Rates of Natural Increase
Appearance of First Brood (Mean Day)
Number of Broods
with Confidence Interval (CI) p = 95 %
(CI: Not applicable)
applicable due to the mortality of all parent animals
of test item related effects concerning the adult mortality and growth
(weight, length) is given in Table 2
(see next page).
test item induced significant adult
mortality of 100 % at the tested concentration levels 2.00 and 5.00 mg/L
after 21 days. At the other
concentration levels and the control no significant mortality (≤ 20 %)
of parent animals was observed. The EC10 for adult mortality
after 21 days was calculated to be 0.712 mg/L. The EC50for
adult mortality after 21 days was
calculated to be 0.974 mg/L
(CI: 0.819 - 1.16 mg/L).
mean dry body weight of
the parent daphnids was biologically significantly reduced to 0.94 mg
per daphnid at the tested concentration level 0.800 mg/L when compared
to the control (reduction: 32 %). The mean dry body weight at the
concentration levels 0.128 and 0.320 mg/L were determined to be 1.26 and
1.28 mg per daphnid, which is comparable to the control group with a
mean dry body weight of 1.39 mg per daphnid.
The mean value of the total body lengthin the concentration
levels 0.128 and 0.320 mg/L were determined to be 5.67 and 5.81 mm per
daphnid and 5.86 mm per daphnid in the control group. At the
concentration level 0.800 mg/L the mean total body length was
statistically significantly reduced to 5.28 mm per daphnid. The total
body length of the concentration levels 0.128 to 0.800 mg/L were
compared to the control using Kruskal-Wallis
One Way Analysis of Variance on Ranks (p = 0.05), because normality test
sensitive effect in this study was the reduction of the mean
dry body weight and mean total body length of the parent
daphnids at the concentration level 0.800 mg/L. At the concentration
levels ≤ 0.320 mg/L no effects were observed.
Related Effects on Adult Mortality (EC10 and EC50)
and Growth (weight, length)
Adult Mortality after 21 Days [%]
Parent Animals: Mean Dry Weight [mg]
Parent Animals: Mean Body Length [mm]
EC10, Adult Mortality
EC50, Adult Mortality
(CI: 0.819 - 1.16 mg/L)
20 % biologically significant
Statistically significant (Kruskal-WallisOne
Way Analysis of Variance on Ranks, p
and ephippia (winter eggs) were
observed at the control or test groups.
quality parameters as pH-value, dissolved oxygen, water hardness and
temperature were determined to be within the acceptable limits.
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