The Effects of Carbamazepine on the Growth and Reproduction of Daphnia magna
An Honors Thesis (HONR 499)
by
Courtney Schroeder
Thesis Advisor Dr. Melody Bernot Signed
Ball State University Muncie, Indiana May 2015 Expected Date of Graduation May 2015 Abstract
Pharmaceuticals and personal care products (PPCPs) have been found in freshwater
ecosystems and have the potential to affect macroinvertebrate organisms. At this
time, the effects of PPCPs on macroinvertebrates are not completely understood.
Carbamazepine (CBZ) is an anticonvulsant drug that has been found in freshwater
ecosystems. This drug is of potential concern to ecosystems due to its toxicity and
recalcitrance. To better understand the effects of CBZ on freshwater
macroinvertebrates, Daphnia magna, a macroinvertebrate species found in
freshwater systems, were exposed to a range of environmentally relevant
concentrations of CBZ. The lifespan, age at which sexual maturity was reached, and
offspring/birth were analyzed over a 3 week period. D. magna exposed to higher
concentration of CBZ did not differ from D. magna exposed to no or low
concentrations of CBZ. These results could be due to a lack of toxicity level of CBZ or
short exposure time. Though results did not coincide with the hypotheses made at
the beginning of the experiment, they indicate that more research should be done
concerning CBZ toxicity in freshwater ecosystems with a longer exposure time and
higher CBZ concentrations.
Introduction
The potential influence of pharmaceuticals on the environment has become
of increasing concern. Prescription drug sales are anticipated to exceed one trillion
dollars, reaching 1,017 billion dollars by 2020 (EvaluatePharma 2014). As the
human population has continued to rise, the use of pharmaceuticals and personal
care products (PPCPs) has increased as well. This has resulted in the detection of
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pharmaceutical chemicals within the environment (Buser et al. 1999). These PPCPs
are dangerous because they are meant to elicit a response in certain target species;
however, they have the potential to do so in non-target species as well (Dussault et
al. 2009). PPCPs include prescription and OTC drugs, veterinary drugs, and an
assortment of other personal care products including fragrances and insect
repellent (Graber & Gerstl 2011). PPCPs enter the ecosystem through water vectors
within the environment. PPCPs are not entirely absorbed or metabolized by the
body; therefore, they are excreted and passed into wastewater in homes (Graber &
Gerstl 2011) and also through hospital wastewater streams (Debska et al. 2004).
Expired drugs that have not been used also get discharged into the environment
(Debska et al. 2004) through water vectors.
Although PPCP presence in freshwaters is ubiquitous across studies, the
potential and unintended environmental effects of these PPCPs are largely unknown
(Lawrence et aJ. 2005). Certain PPCPs are of particular concern due to their
recalcitrance, abundance, and potential toxicity (Murray et al. 2010). One such
pharmaceutical that made its way into the ecosystem through water vectors is
Carbamazepine (CBZ). CBZ is one of the most common pharmaceuticals detected in
freshwater ecosystems on a global scale (Hughes et al. 2013).
CBZ is an anticonvulsant drug that treats epilepsy and bipolar disorder and
relieves pain due to trigeminal neuralgia. It also aids in attention deficit disorder
and schizophrenia. It is available in suspension, tablet, and capsule forms
(Carbamazepine 2015). Previous research has documented acute toxicity of CBZ on
organisms. For example, CBZ was found to be lethal to a freshwater teleost, C.
2
carpio, at 59.70 mgjL, though other researchers have quantified acute toxicity of
CBZ <100 mgjL (Malarvizhi 2012).
Macroinvertebrates plan an important role in freshwater ecosystems by
facilitating nutrient cycling through translocation and decomposition (Wallace &
Webster 1996). However, macroinvertebrates can be affected by the watershed
conditions as well as anthropogenic stressors (Gueker et al. 2009). These stressors
can reduce biodiversity within the ecosystem and alter the function of
macroinvertebrates. Because macroinvertebrates are important components of food
webs and sensitive to anthropogenic contaminants, they are ideal bioindicators for
ecosystem health (Magbanua 2013) and little is known about macroninvertabrate
response to PPCPs.
One important macro invertebrate in pelagic systems is Daphnia magna,
which is a species of daphnia that resides in freshwater ecosystems and are filter
feeders. D. magna are asexual organisms. The female produces a clutch of
parthenogenic eggs ever 3-4 days after she molts. The development of an egg is
rapid; these eggs hatch usually within one day, but stay in the brooding chamber for
approximately 3 days until maternal death. The number of eggs produced in a clutch
ranges among Daphnia species. Egg sizes range from 1-2 eggs to even 100 eggs
(Ebert 2005). D. magna are useful in laboratory experiments because reproduction
is quick and analysis of these organisms is easily observed under the microscope.
The objective of my study was to analyze the effects of CBZ on the growth
rate and reproduction of D. magna to better understand what levels of CBZ are toxic
to these organisms. By performing this study, I hoped to provide more research to
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this field to spread awareness to individuals about the use of ppeps and their
unintentional effects on non-target species. I hypothesized that exposure to higher
concentrations of eBZ would result in D. magna reaching sexual maturity at a later
stage. I also hypothesized that exposure to higher concentrations of eBZ would
result in less offspring produced/birth and a shorter life span. Those exposed to no
eBZ and the lowest concentration of eBZ would reach sexual maturity earlier,
produce more offspring/birth, and live longer.
Methods
Pregnant adult D. magna were randomly collected and observed to obtain 64
newborns that were born within one day of each other. Three levels of treatment
were chosen along with a negative control. Each treatment group and negative
control had 16 D. magna split into 150 mL beakers with 4 in each beaker. Aged tap
water (140 mL) was added to each beaker. Each day, between 17:00 and 20:00, the
Daphnia were given Chlorella, a green algae, for food and water was replenished to
account for water evaporation. The negative control contained no carbamazepine.
The first treatment group was given 30 !AI of carbamazepine stock solution (100
ng/L). The second treatment group was given 300 !AI of carbamazepine stock
solution. The third treatment group was given 3000 !AI of carbamazepine stock
solution. The samples were kept in a research laboratory at room temperature. The
research laboratory was locked at all hours of the day. This limited the possible
contamination or disruption ofthe experiment from outside factors. Daphnia were
incubated over a 24-day period. The growth and reproduction rate ofthe D. magna
were recorded each day between 17:00 and 20:00 before the food and water were
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replenished. This was done by analyzing the individual D. magna under the
microscope. Age of sexual maturity, number of offspring produced, and life span
were all recorded. The controlled variables included the temperature, amount of
food given, amount of water in environment, time of observation, exterior
environment, the age of each D. magna, the number of D. magna in each beaker, the
method of administering the carbamazepine to each treatment group, and the
amount of carbamazepine for each individual treatment group. The dependent
variables included the growth rate of each D. magna, and the numbers of babies
born to each were recorded by visual observation of the D. magna under the
microscope.
Results
The cumulative mortality of D. magna among the control and treatment
groups was not significantly different (Table 1, p = 0.34). The treatment group that
was exposed to 3000 ul of eBZ stock solution had the highest cumulative mortality
of 15 deaths. The control group that was exposed to no eBZ had the second highest
cumulative mortality of 14 deaths. The treatment group that was exposed to 30 ul of
eBZ stock solution had the third highest cumulative mortality of 13 deaths. The
treatment group that was exposed to 300 ul of eBZ stock solution had the lowest
cumulative mortality of 12 deaths.
5
16
14
I
0,0 ,0 , 0Q-e,0
P 12
rn
t
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+=' rn
:J 10
8
6
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4
0
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,,,,,
trtrnt1
- - -T­ - - " -6 , -
trtmt2
trtrnt3
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o
5
10
15
20
25
30
Days
Figure 1. Comparison of cumulative mortality of the control and treatment groups
over a 24-day period
There was no significant difference between the numbers of D. magna that
reached sexual maturity among those that were living in the control and treatment
groups (Table 1). The control group had 66.67% ofthe living D. magna reach sexual
maturity in the second week of the experiment and 33.33% in the third week of the
experiment In each treatment group, 100% of the living D. magna reached sexual
maturity in the third week. Though 66.67% of the living D. magna in the control
group reached sexual maturity earlier than all ofthe treatment groups, there was no
significant difference found.
6
Group
Week 2
Week 1
Week3
Control
0
66.67%
Trtmt 1
Trtmt 2
0
0
0
0
Trtmt 3
0
0
33.33%
100%
100%
100%
Table 1. Comparison of the % living D. magna that reached sexual maturity in each
week in control and treatment groups
There was no significant difference found when comparing the offspring
produced from the first birth of the control and treatment groups (Figure 2, p =
0.66). The control group produced the most offspring from the first birth and the
first treatment group that was exposed to 30 ul of CBZ stock solution produced the
least offspring from the first birth.
18
16
14
12
Cl
c:
-;::
10
~
0
8
c..
T
T
6
4
2
I
I
0
control
trtmt1
trtmt2
trtmt3
Figure 2. Comparison of offspring produced from first birth between the control
and treatment groups
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Discussion
CBZ had no effect on the growth, sexual maturity, or offspring/birth of D.
magna during this experiment. These results did not support my hypotheses that D.
magna exposed to higher concentrations of CBZ would reach sexual maturity later,
reproduce less offspring/birth, and live a shorter amount of time than those D.
magna who were unexposed or exposed to only a low concentration of CBZ. Our
results conflict with previous research that suggested macroinvertebrate richness
would decline when exposed to higher concentrations ofCBZ (Munoz et al. 2009;
Beketov et al. 2013). The inconsistency found between our research and previous
findings may be contributed to a lack of toxicity to the samples in this experiment at
environmentally relevant concentrations of CBZ (Oetken et al. 2005; Dussault et al.
2008). This inconsistency could also be due to the duration of macroinvertebrate
exposure to CBZ. Rarely are macroinvertebrates exposed to a drug or chemical for
only a short period of time; rather, they are continually exposed to a range of
substances in freshwater, with only slight temporal and spatial variations in the
concentration of the chemical Oones et al. 2003). Thus, the inconsistencies found in
our research may be contributed to the aforementioned experimental conditions.
Multiple studies have found CBZ to be prevalent at specific concentrations in
certain bodies of water. Munoz et al. (2009) found that high concentrations (>10000
ng/L) of pharmaceuticals led to a reduction in macro invertebrate richness. CBZ has
also been detected in sewage treatment plants and rivers in Germany at
concentrations up to 6.3 ug/L-l (Ternes 1998). Ho Wing Leung et al. (2013) detected
CBZ in tap water at concentrations between 1.3-6.7 ng/L in 23-33% of their
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samples. These detections of CBZ support that it can be found in bodies of water at
high concentrations, and these concentrations will continue to rise as the population
rises exponentially.
Due to an anticipated rise in both human population and pharmaceutical
drug use, PPCPs and their metabolites will be more and more likely to enter into the
ecosystem. The ultimate fate of these PPCPs is unknown in terms of biodiversity
impact and overall impact on freshwater ecosystems (Jones et al. 2003). Because
this field of research is relatively new and there is limited knowledge on the
concentrations of PPCPs within the environment (Chunyan et al. 2006), it is
necessary for more research to be conducted so that potential changes could be
made in the regulation of these PPCPs to avoid creating toxic concentrations within
freshwater ecosystems. It is also necessary that efforts be made in order to promote
awareness of the possible effects that certain PPCPs may have on the environment.
There have been minor efforts made in order to limit the effects of PPCPs on
the environment. Pharmaceutical return programs, the development of "green"
pharmaceuticals, and the control of the source by segregation of sources have all
been proposed in order to limit the presence of pharmaceuticals in the environment
(Fatta-Kassinos et al. 2010). National regulation of exposed or unwanted PPCPs as
well as the implementation of an "extended producer responsibility" have been
desired by the u.s. EPA in order to facilitate and prevent the introduction of more
PPCPs into freshwater and wastewater systems (Daughton 2004). These efforts,
though steps in the right direction for promoting awareness about the possible
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harmful effects of ppeps, are small in comparison to the efforts that still need to be
made.
Because the potential harmful effects of ppeps on non-target species are
largely unknown, it is necessary to continue to do more research in this field. Little
effort has actually been done in order to stop ppeps from making their way into the
environment in the first place. Traces of pharmaceutical compounds are difficult to
analyze as well and pose a great challenge for those working in this field (Fatta­
Kassinos et al. 2010). Understanding the toxicity of certain ppeps within the
environment could provide crucial information on the mode of action and toxicity of
these compounds (Malarvizhi et al. 2011). This knowledge could help understand
how macroinvertebrates are affected as a result. Though it is uncertain how toxic
ppeps are to macroinvertebrates living in freshwater ecosystems, researching the
unintended affects of ppeps on non-target species may be beneficial to saving
macroinvertebrate life within the ecosystem.
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