Endocrine Disruptive Chemicals and Their Effects On Zebrafish (Danio

DEPARTMENT OF BIOLOGICAL AND
ENVIRONMENTAL SCIENCES
ENDOCRINE DISRUPTIVE CHEMICALS AND

THEIR EFFECTS ON ZEBRAFISH (DANIO
RERIO) BEHAVIOUR
A toxicologic study with an environmental aspect
Sanne Odenlund
Degree project for Master of Science (60 hec) with a major in Biology
BIO727 Physiology and Cell biology 60 hec
Second cycle
Semester/year: Autumn 2017- Spring 2018
Supervisor: Joachim Sturve and Lina Birgersson, Department of Biological and Environmental
Sciences
Examiner: Björn Thrandur Björnsson, Department of Biological and Environmental Sciences
Picture source; Sanne Odenlund
Table of content
Abstract ............................................................................................................................................ 3
Sammanfattning ............................................................................................................................... 4
Introduction ...................................................................................................................................... 5
Endocrine disruptive chemicals.................................................................................................... 5
Per- and polyfluorinated alkyl substances .................................................................................... 5
Adverse outcome pathways (AOPs) and EDC mixtures .............................................................. 6
The Hypothalamic-Pituitary-Thyroid axis ................................................................................... 7
What is behaviour? ....................................................................................................................... 8
How to study behaviour in fish and how it can be affected by EDCs .......................................... 9
Different ways to study the behaviour of fish........................................................................... 9
Zebrafish as a model species .................................................................................................... 9
Environmental effect............................................................................................................... 10
Aim and Hypothesis ................................................................................................................... 10
Aim ......................................................................................................................................... 10
Hypothesis .............................................................................................................................. 11
Materials and Method .................................................................................................................... 11
Animals and Exposure................................................................................................................ 11
Aquaria and Housing .................................................................................................................. 14
Beakers.................................................................................................................................... 14
Aquaria.................................................................................................................................... 15
Enrichment .............................................................................................................................. 16
Feeding.................................................................................................................................... 16
Behaviour tests ........................................................................................................................... 17
1 month after exposure- zebrafish fry locomotion (Zebrabox)............................................... 17
4 months after exposure- locomotion, schooling and braveness ............................................ 17
Locomotion, after 4 months .................................................................................................... 18
Schooling, after 4 months ....................................................................................................... 18
Braveness, after 4 months ....................................................................................................... 18
Calculations and data analyses ................................................................................................... 18
Survival rate ............................................................................................................................ 19
Results ............................................................................................................................................ 19
Observations ............................................................................................................................... 19
1 month after exposure- zebrafish fry locomotion (Zebrabox) .................................................. 20
1
Dark cycle ............................................................................................................................... 21
Light cycle .............................................................................................................................. 22
4 months after exposure- locomotion ......................................................................................... 23
4 months after exposure- braveness ........................................................................................... 24
4 months after exposure- schooling............................................................................................ 24
Survival rate ............................................................................................................................... 25
Discussion ...................................................................................................................................... 26
Exposure ..................................................................................................................................... 26
Environment ............................................................................................................................... 27
Survival rate ............................................................................................................................... 28
Morphology ................................................................................................................................ 28
Improvements ............................................................................................................................. 28
Experimental design ............................................................................................................... 28
Conclusion and Future studies ....................................................................................................... 29
Acknowledgements ........................................................................................................................ 29
References ...................................................................................................................................... 29
Appendix ........................................................................................................................................ 36
2
Abstract
One great challenge today is to understand the complex effects of mixtures of endocrine
disrupting chemicals (EDCs). Understanding the cocktail effects of these compounds is not as
simple as studying the toxicity of single chemicals. In this study, we wanted to investigate if
acute, early exposure to an EDC mixture had any delayed effects on behaviour (locomotion,
braveness and schooling) of zebrafish (Danio rerio) after 1 and 4 months. In addition, we wanted
to see if fish developed in different environments (poor and enriched) behaved differently in
combination with exposure to the mixture. Results 1 month after exposure showed a significant
difference in swimming activity, where the unexposed fish from the enriched environment had
significantly higher activity compared to the other groups. We could also see that exposed fish in
the poor environment had a lower swimming activity compared to the unexposed fish. After 4
months the zebrafish showed no significant difference in either behaviour after analysis with a
two- way ANOVA (p<0.05). However, due to a noticeable difference in locomotion activity
when only looking at the unexposed groups in the 2 environments we conducted a two-sample t-
test (p<0.05) which showed a significant difference where fish developed in enriched
environments had a higher swimming activity compared to fish reared in the poor. We also found
that fish in the exposed group without enrichment had a significantly lower survival rate
compared to exposed fish in the enriched environment, suggesting that an enriched environment
may reduce negative effects of EDCs. The results from the behaviour experiments in this study
did not resemble earlier findings where swimming activity of larval zebrafish was significantly
induced after exposure to a similar EDC mixture. This indicates that the effect of the EDC
mixture tested in this study may have been reduced or even disappeared over time.
3
Sammanfattning
En stor utmaning i dagens samhälle är att förstå de komplexa effekter som blandningar av
hormonstörande ämnen kan leda till. Förståelsen kring de cocktaileffekter som kan uppstå på
grund av dessa komponenter är betydlig mer komplicerad än att studera en kemikalie i taget. I
den här studien ville vi undersöka om en tidig akut exponering av en blandning av
hormonstörande ämnen påverkade zebrafiskars (Danio rerio) beteende (rörelseaktivitet, mod, och
stimtendens) efter 1 respektive 4 månader. Samtidigt ville vi också undersöka om fiskarnas
utveckling i 2 olika miljöer (fattig och berikad) påverkade deras beteende samt om interaktionen
mellan exponering och miljö hade betydande effekter på de beteenden som studerades. Resultaten
1 månad efter exponering visade en signifikant skillnad i rörelseaktivitet där zebrafiskar som inte
var exponerade för blandningen och hade utvecklats i en berikad miljö visade en högre aktivitet
jämför med de andra grupperna. Vi kunde också se att exponerade fiskar som utvecklats i en
fattig miljö hade en lägre rörelseaktivitet jämfört med de som inte var exponerade men uppväxta i
samma miljö. Resultat från en två faktors ANOVA efter 4 månader visade inga signifikanta
skillnader i fiskarnas beteende på grund av varken de hormonstörande ämnena eller de 2 olika
miljöerna, men på grund av en synbar skillnad i rörelseaktivitet mellan kontrollgruppen i de två
olika miljöerna undersöktes differensen i ett t- test som visade en signifikant skillnad mellan de 2
grupperna där fiskar i den berikade miljön hade en högre aktivitet. Då resultaten i den här studien
skiljer sig från tidigare studier där exponerade juvenila zebrafiskars aktivitet har varit högre
jämfört med kontrollfiskarna samt att resultaten efter 1 månad visade på signifikanta skillnader är
det möjligt att tidigare sedda effekter på grund av dessa hormonstörande ämnen har reducerats
eller även försvunnit.
4
Introduction
Endocrine disruptive chemicals
Anthropogenic chemicals are a huge part of our daily lives and some of these man-made
chemicals are endocrine disruptive chemicals (EDCs) which can affect the hormonal system of an
organism or its progeny. There are discussions of how to correctly define the definition of EDCs
but one definition that has been accepted is from Bergman et al. (2012) and reads as follow “An
endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine
system and consequently causes adverse health effects in an intact organism, or its progeny, or
(sub) populations.” EDCs are found in materials such as pesticides, personal care products and
metals (Bergman et al. 2012). They are transported in the body by the cardiovascular system and
may bind to hormones or multiple receptors (Bergman et al. 2012), and thereby affect
reproduction, growth, metamorphosis, development, digestion, immunity, learning and memory
(Bell 2004; Bergman et al. 2012; Blanton and Specker 2007; Harrison and Hester 1999; Segner
2009). It has been shown that exposure during early life stages may affect organisms later in life
and be permanent and irreversible (Colborn et al. 1993). However, it is critical to determine
different time- effects due to EDCs. This is because of exposure during development seems to
lead to irreversible effects whereas exposure during later life stages appears to in some cases
disappear when EDCs are removed. Another difficult interpretation of EDCs is that physiological
processes that occur due to EDCs do not often act dose- linear, which leads to difficulties in
extrapolating the consequences due to different concentrations of EDCs (Bergman et al. 2012).
Per- and polyfluorinated alkyl substances

Per- and polyfluorinated alkyl substances (PFASs) are chemicals that are suspected to be one of
the classes of EDCs. These are a group of anthropogenic environmental pollutants which are
expected to have endocrine disruptive properties (Joensen et al. 2009). PFASs are found in
firefighting foams, weatherproof clothes, detergents, pans, etc (Jensen and Leffers 2008) and are
stable against heat or chemical degradation due to the strong bond between carbon and fluorine
atoms. PFASs can more or less be hydrophobic or lipophobic (Buck et al. 2011; Stahl 2011) due
to one hydrophobic and one lipophobic tale. This results in mixtures of PFAS including both
characteristics which is one of the reasons for its wide usages in our daily lives. All molecular
structures of PFASs consist of carbon and fluorine atoms, some also include oxygen, hydrogen,
sulphur and/or nitrogen atoms, but the main characters that differ between the dissimilar
structures of PFASs are the chain lengths. The reason why most scientists study PFASs as one
group is because of its similar chemical reactions and toxicity (Environmental Protection Agency
[EPA] 2017). Aquatic systems are suspected to be a recipient and the main transport medium and
reservoir for PFASs and therefore it is important to understand how these chemicals affect the
aquatic wildlife (Kallenborn 2004; Prevedouros et al. 2006). Different PFASs are widely seen all
over the world in both aquatic and land-living wildlife (Lewis et al. 2014) and they are persistent
and bioaccumulative substances which has been found able to transfer in trophic levels and food
webs (Giesy and Kannan 2002; Haukås et al. 2007) resulting in liver samples taken from Alaskan
polar bears (Ursus maritimus) containing 180–680 ng/g perfluorooctane sulfonate (PFOS) (wet
wt) (Giesy and Kannan 2002). PFOS is a type of PFAS which end consists of a sulphonic acid
group. This molecule is often used in experimental studies due to its widely usage and effects on
the ecosystem and it has been found in eggs from birds and concentrations as high as 250 ng/g
5
(wet wt) has been detected in the eggs of brown trout (Salmo trutta) and lake whitefish
(Coregonus clupeaformis) (Giesy and Kannan 2002) suggesting maternal transfer during yolk
formation. The distribution of PFASs depends on whether they are long- or short- chained. Short-
chained PFASs are potentially more water soluble while the long-chained substances seem to
react with particles and accumulate in the food webs with the result of toxic effects in both
animals and humans (Ahrens et al. 2010; Ahrens et al. 2015). Since PFASs are widely used and
found in the environment it is of importance to investigate what kind of effects these substances
have on wildlife, not only physiological but also behavioural.  
Lipophilic persistent organic pollutants (POPs), such as DDT, polychlorinated biphenyls
(PCB), dioxins and brominated diphenyl ethers accumulates in lipids. PFASs differ from POPs
by being able to be lipophobic or hydrophobic (Joensen et al. 2009; Jensen and Leffers 2008).
They do not bind to lipids, instead, PFASs accumulate in blood, liver and kidneys where they
bind to proteins (Jensen and Leffers 2008; Kovarova et al. 2012). The primary target is the liver
where increased concentrations of PFASs may lead to weight loss and hepatocyte hypertrophy,
which means enlargement of the liver cells (Seacat et al. 2003; Kennedy et al. 2004). PFASs also
bind to membrane proteins which can change the fluidity of the membrane and disrupt the
signalling of the membrane receptors, and a study found that PFOS at the lowest concentration
effect 5-15mg/L changed the permeability in fish leucocytes (yue Hu et al. 2003). The strongest
effect in toxicity and change in behaviour due to PFASs depend on the carbon chain-lengths and
the attached functional group, where long-chained molecules with a sulfonic group show the
strongest effect on locomotor behaviour on zebrafish (Danio rerio) larvae (Ulhaq et al. 2013).
Other scientists examined the effect of different concentrations of 6:2 fluorotelomer alcohol (6:2
FOTH) on zebrafish and found endocrine disruptive effects, for example an increase in plasma
oestradiol and testosterone in both sexes (Liu et al. 2009) and Hoke et al. (2012) found that the
temperate freshwater fish fathead minnow (Pimephales promelas) had a 50% mortality after
exposure to a concentration of 32 mg/L of Perfluoro-n-pentanoic acid (PFPeA). Juvenile goldfish
(Carassius auratus) exposed to different concentrations (0, 0.5, 2, 8 and 32 mg/L) of PFOS
during 48 h showed effects on their swimming performance, for example, were their motion
distance reduced with an increased concentration (Xia et al. 2013).
PFASs are not the only group of chemicals that might cause physiological and behavioural
changes. Correlations between heavy metal, for example, cadmium (Cd), lead (Pb) and zinc (Zn),
and disorder in physiology and behaviour have previously been reported (Qi et al. 2017). A
change in physiological mechanisms can change the behaviour of an organism, for example, can
a heavy metal such as Cd induce the disorder of nerve, muscle and respiratory systems (Sthanadar
et al. 2013; Yılmaz et al. 2004) which can lead to changes in swimming, predator avoidance and
reproduction (Sullivan et al. 1978; Thomas 1989; Yılmaz et al. 2004). Other studies have shown
behavioural effects due to endocrine disrupters, one examples is the reaction of juvenile rainbow
trout (Oncorhynchus mykiss) chronically exposed to 2,3,7,8-Tetraklordibenso-p-dioxin (TCDD)
where the juveniles displayed lethargic swimming, feeding inhibition and no response to external
stimuli (Mehrle et al. 1988) and zebrafish exposed to the pesticide deltamethrin during
development has shown increased locomotor activity at the larval stage (Kung 2014).
Adverse outcome pathways (AOPs) and EDC mixtures

A cascade of effects starts when an organism is exposed to one chemical alone and this process
can be visualized and structured with adverse outcome pathways (AOPs) (figure 1). An AOP
6
starts with an initial molecular event and moves on through a cascade of key events which ends
with an adverse outcome for an organism or a population (Ankley et al. 2010), for example,
change in behaviour. Predict toxic effects is complex and cannot be done only by looking at one
of these steps but combining data from different researchers may provide information needed to
forecast these effects (National Toxicology Program 2017). Organisms are often exposed to many
kinds of chemicals at the same time (Jaeger et al. 1999) and substances such as EDCs can act
together with other molecules and thereby increase or decrease the effect of this chemical alone
(Olson and Christensen 1980). This leads to subsequent cascades of effects which are much more
complex and difficult to understand, for example, can one chemical start a reaction while another
one can stop it. Chemical safety levels are often based on lab studies with one chemical which
may be a problem because the toxicity of a chemical can be even worse due to the cocktail effect
(Celander 2011). Another reason for consideration of mixture effect is that an organism's
sensitivity for a chemical may increase due to exposure of PFASs or other EDCs, for example,
due to increased membrane permeability (yu Hu et al. 2003; Färber et al. 2007).
There are a lot of missing data when it comes to the toxicological effects of EDCs and
different mixtures, especially in aquatic animals (Ulhaq et al. 2013; Celander 2011), therefore it
is important to continue with research in this field to broaden our knowledge on the risks of these
different concentrations, structures and cocktail effects.
Figure 1. An illustrated description of AOPs, which start with a molecular initiating event that begins the toxicity process. Step
number 2 represents the key events of the reaction characterize the crusade of the toxicity. The last step is the adverse outcome
which can arise both on an individual or population level (National Toxicology Program 2017).
The Hypothalamic-Pituitary-Thyroid axis
7
The Hypothalamic-Pituitary-Thyroid (HPT) axis is a part of the neuroendocrine system which is
activated through actions from the brain (Zoeller et al. 2007). The action of the HPT axis can start
Figure 2. An explanation of the HTP axis in fish and how T4 and T3 are regulated and regulate it. The subtraction sign to the
left represents the negative feedback loop (Blanton and Specker 2007).
due to external stimuli such as temperature, olfactory cues and photoperiod but also due to
internal stimuli as growth and secretion of chemicals. Thyroid-stimulating hormone (TSH) is
produced by the pituitary and regulated by thyroid- releasing- hormone (TRH) which is secreted
by the hypothalamus (Blanton and Specker 2007). TSH further regulates secretion of thyroxine
(T4) and triiodothyronine (T3) which have a negative effect on the TSH release through a negative
feedback loop (figure 2) (Yoshiura et al. 1999). The thyroid hormone T3 is known to affect,
among other things growth, osmoregulation, metamorphosis and behaviours of fish (Power et al
2001). Some EDCs have been shown to affect the HPT axis (Blanton and Specker 2007) and a
novel study made within the EDC MixRisk project showed that the thyroid hormone system in
zebrafish was affected by a mixture of common EDCs correlated with disruption of growth in a
pregnancy cohort study (Birgersson et al. 2017), which affected zebrafish behaviour in turn of
swimming activity.
What is behaviour?
Behaviour is an organism’s visible response to internal and external factors, which arises from
both abiotic and biotic factors (Gerhardt 2007). An animal's behaviour has a plasticity which
means that the trait is not fixed. This phenotypic plasticity makes it possible for animals of the
same species to react differently to a stimulus even though the reaction is based on a single
genotype (West-Eberhard 1989).
According to Tinbergen (2005), there are 4 questions that must be answered to really
understand the reason for a specific behaviour. These questions assume to answer the reason for
immediate mechanism, ontogeny, phylogeny and function (Tinbergen 2005). These can further be
categorized into 2 different groups when it comes to behaviour studies; proximate and ultimate
explanations (Wilson 2009). Mechanism and ontogeny are found in the category of proximate
explanations. Mechanism explains the causation for an immediate behaviour, for example, what
type of hormone and disruption in this hormone production will cause an animal’s direct reaction
to a stimulus. Ontogeny refers to the development of a behaviour usually from the time of
fertilization to the organism's mature form. The ultimate explanation includes function, which
8
refers to how well an organism is adapted to its environment, and phylogeny, which includes
evolution and how an organism passes its genetic ability to survive and reproduce (Cohn and
MacPhail 1996).
How to study behaviour in fish and how it can be affected by EDCs

Different ways to study the behaviour of fish
Studying different behaviours of an organism to determine both physiological and ecological
processes and changes in the behaviour are a productive way of noticing toxic effects (Xia et al.
2010). There are several ways to study the behaviour of fish, one is by studying schooling
behaviour, which is when a group of fish are swimming together probably due to an anti-predator
and foraging strategy (Pavlov and Kasumyan 2000; Pitcher et al. 1982). Schooling seems to lead
to a wide variety of adaptive functions (Pavlov and Kasumyan 2000) and a study made by
Seghers (1974) he showed that fish in habitats where predation was common the guppies
(Poecilia reticulata) had more tendencies to school compared to environments where predation
on guppies was more unusual. In the non- present predator habitats, the most common social
groups were when a group of males followed a female also called courtship activity. This kind of
structure does not persist for more than a few seconds, and after the males moved on to court
other females. In the areas, with a high predator risk, the schooling behaviour was well developed
and the group including both sexes was still together even when not courting or foraging. The
biological phenomena of schooling behaviour can be extremely complex and have a huge
plasticity not only between species and physiological conditions but also between environmental
factors and condition and habitat type (Pavlov and Kasumyan 2000).
Another example on how to study the behaviour of fish is to introduce them to a novel
object, such as a Rubik’s cube and thereby test an organism fear for a novel object by measuring
it in a shy to a bold axis (Jutfelt et al. 2013). In a study made by Ballaz et al. (2007), they found
that the 5-HT7 receptor has a role in novel object discrimination and relation to novelty-seeking
behaviour in rats. The 5-HT7 receptor is a member of the G protein-coupled receptor (GPCRs)
family which act as molecule switchers inside cells and transmit and send signals from outside a
cell to its interior. The 5-HT7 receptor is activated by the neurotransmitter serotonin (5-
hydroxytryptamine, 5-HT) (Nikiforuk 2015) which plays a role in zebrafish fear/anxiety, stress
and aggression (Herculano and Maximino 2014) and it has been shown that serotonin plays an
inhibitory role in aggressive behaviour and a decreased locomotor activity in teleost fish
(Winberg and Nilsson 1993).
Changes in locomotion is another way to test fish reaction due to chemicals in
ecotoxicology and it is a sensitive way to test toxic stress (Kavitha and Rao 2007). A non- defect
locomotor activity is essential for an organism as it will affect both reproductive and non-
reproductive behaviour crucial to an organism's survival (Sárria et al. 2011). By using a video
recording device (Viewpoint Zebrabox®) it is possible to record the swimming activity in an in
vitro system and screen locomotion data from small fish without human errors (van Woudenberg
et al. 2014) which will lead to more trustworthy conclusions.
Zebrafish as a model species
Zebrafish are often used in scientific research experiments and 70% of protein-coding genes in
human are related to genes in zebrafish, which is one of the reasons why zebrafish is often used
as a model when studying human diseases (Howe et al. 2013). As a model species, they are easy
9
to handle, have a rapid development, low cost, high fecundity and a short reproductive cycle (Dai
et al. 2014). There are clearly documented impacts of EDCs in fish in the wild and model species
are often used in experiments when studying the effects of these chemicals (Ankley et al. 2009).
Fishes can easily absorb chemicals from the water through their gills and are therefore practical
to use when study exposure to chemicals (Goldsmith 2004). Zebrafish are also often used in
behaviour studies, and chronic exposure to PFOS has for instance been shown to cause
behavioural changes in both adult fish and larvae (Chen et al. 2013; Ulhaq et al 2013). Water is
the main transport medium and reservoir for PFASs (Prevedouros et al. 2006; Kallenborn 2004)
and it has been shown that PFASs concentrations in freshwater fish can be even higher than in
marine fish (Schuetze et al. 2010).
Environmental effect
Studies have been done comparing the behaviour of fish reared in environments with or without a
predator (Seghers 1974; Ives and Dobson 1987) and different habitat structures in vivo
(Angermeier and Karr 1984; Bassett 1994). These studies have found that different habitats may
cause variances in behaviour and a study made by Salvanes et al (2013) even described positive
effects on the neural plasticity and cognition in Atlantic salmon (Salmo salar) when introduced to
environmental enrichment. Comparing rich and poor environments in laboratory studies and its
effect on behaviour in toxicology studies are not common, which may lead to unanswered
questions whether differences in environments play a role when an organism is exposed to
chemicals. These discoveries and the fact that laboratory studies are often made in clean and
sterile environments make it even more interesting to see if there are differences in exposure
results when fish have been developed in a rich habitat with for example running water,
vegetation and gravel compared to a poor environment like a bare aquarium with only water.
Aim and Hypothesis
Aim
Ecotoxicology and behaviour are two well-studied fields in biology and there are a lot written and
published in these two separate fields. Ecology and toxicology are the two subjects combined
making the relatively new field ecotoxicology which is the science when contaminants impact on
the individual, population and ecosystem is analysed (Connell et al. 2009).  EDCs linking
developmental exposures to changed outcomes later in life is called developmental programming
(Nesan and Vijayan 2013) which means that exposure to EDCs may lead to inappropriate
hypothalamic programming during development resulting in reduced reproductive success in
adulthood (Gore 2008). Previous studies done by scientists from the EDC-MixRisk project
(EDC-MixRisk Project 2015) have demonstrated that a similar EDC mixture to the one used in
this experiment affected locomotion behaviour in exposed zebrafish embryos (Birgersson et al.
2017). These findings led to the first aim of this study which was to examine if short time
exposure to a similar mixture of EDCs under early development impacted behaviour later in life.
As previous studies have shown that environment can play a role in the behaviour of fish the
second aim of this study was to determine whether long-time development in enriched versus
poor environment had an impact on the behaviour of zebrafish.
The gap of environmental influence on behaviour in toxicological experiments led to the 3rd
aim of this study which was to investigate the interaction of environment and exposure and if it
would influence zebrafish behaviour.
10
Hypothesis
Experiments including exposure of zebrafish embryo to a similar EDC mixture used in this
experiment has shown results of increased locomotion behaviour in fish who were exposed which
led to the 1st hypothesis which was that fish exposed to the EDC mixture would behave
differently, and for example, have a higher swimming activity compared to the control group.
The hypothesis to the 2nd aim was that it would be a difference in behaviour between fish
developed in the enriched environment compared to the ones reared in the poor habitat as earlier
studies have shown differences in behaviour due to variances in habitats.
The hypothesis to the 3rd aim was that the interaction of environment and exposure would
influence zebrafish behaviour.
Materials and Method

Animals and Exposure
AB strain zebrafish eggs were purchased from Karolinska Institute (Stockholm, Sweden) and
transported to the Department of Biology and Environmental Sciences, Zoology building,
(Gothenburg, Sweden) at the end of October. The eggs were kept in petri dishes with zebrafish
embryo medium (ZEM; 245 mg of MgSO4·7H2O, 20.5 mg of KH2PO4, 6 mg of Na2HPO4, 145
mg of CaCl2·2H2O, 37.5 mg of KCl, 875 mg of NaCl dissolved in 1 litre Milli-Q water) in an
incubator in a temperature of 28°C with a 14:10h light/dark cycle for 2 days until 72 hours post
fertilization (hpf) when treatment started.
Due to a high mortality risk during early life stages, a total number of ≈400 zebrafish were
used (see table 1 for an exact number of fish). The EDC mixture was made by chemists from the
EDC-MixRisk project in Lund and table 2 shows its composition. The concentration of the EDC
mixture used in this project was a hundred times higher (100X) than the mean concentration
found in the serum of pregnant women in the EDC-MixRisk project (Bornehag et al. 2012;
Birgersson et al. 2017).
11
Table 1. The 20 beakers and the number of zebrafishes in it, including the treatment and day of exposure.
Beaker Exposure date Exposure Environment Start: End:

number Number of Number of
fish fish
1 17.10.21 Mix G1 100X Poor 20 1
2 17.10.21 Control Poor 19 8
3 17.10.21 Mix G1 100X Poor 19 4
4 17.10.21 Control Poor 20 6
5 17.10.21 Mix G1 100X Enriched 20 7
6 17.10.21 Control Enriched 20 9
7 17.10.21 Mix G1 100X Enriched 20 4
9 17.10.29 Mix G1 100X Poor 21 7
10 17.10.29 Control Poor 20 7
11 17.10.29 Mix G1 100X Poor 18 3
12 17.10.29 Control Poor 20 7
13 17.10.29 Mix G1 100X Poor 18 2
14 17.10.29 Control Poor 20 8
15 17.10.29 Mix G1 100X Enriched 19 9
17 17.10.29 Mix G1 100X Enriched 19 6
19 17.10.29 Mix G1 100X Enriched 19 7
Total 394 119
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Table 2. The composition of the EDC-Mixture (MIXG, Growth and Metabolism).
Compounds Concentration at
100X (mol/L)
DEP 3.204E-06
DBP 2.855E-06
BBZP 5.68E-07
DEHP 2.051E-06
DINCH 5.2E-08
Triclosan 3E-08
2-OH-PH 1.36E-07
DPP 4.9E-08
3-PBA 1.1E-08
PFOA 3.89E-07
PFOS 1.048E-06
PFHXS 3.28E-07
HCB 1.6E-08
p,p’-DDE 5.9E-08
MIXG1 exposure solutions were prepared from the MIXG1 stock solution (dissolved in
DMSO) by dilution to 100X in zebrafish embryo medium. The final concentration of DMSO was
0.01% in all solutions and the volume was 30 ml for each exposure. The exposure was divided
over 2 weeks, 40% of the total sample size was received from Karolinska Institute the first week
(n≈160) and 60% the second week (n≈240). ≈20 embryos randomly selected from at least 4
different breeding pairs were used for each exposure. The embryos were transferred by pipetting
to 100 ml glass Erlenmeyer flasks containing exposure solution (EDC mixture diluted to 100X in
ZEM or ZEM with 0.01% DMSO). When pipetted all the eggs hatched and the 72 hours post
fertilization (hpf) zebrafish embryos were exposed for 48 h and kept in an incubator with a
temperature of 28°C. After 48 h exposure, the embryos were moved with a pipette to 400 ml
beakers filled with 100 ml clean ZEM and kept in 28°C in the same incubator for 1 week (figure
3). To stimulate an enriched environment half of exposed and control group (table 1) were placed
over a stone mat. The same approach was repeated 1 week later (table 1) and the reason for this
division was time. Both the exposure and following behaviour studies took a lot of time and
thereby the total sample size had to be divided into 2 rounds.
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Figure 3. The beakers which were placed in the incubator holding a temperature of 28 degrees, with the poor environment to the
left and the enriched to the right.
Aquaria and Housing

Beakers
After 1 week in the incubator, the beakers were filled up with an extra 100 ml ZEM (total 200 ml)
and moved to aquaria with circulated water holding a temperature of 28 ± 1 °C, with the help of
immersion heaters (figure 4). After approximately 2 months the zebrafish were moved from the
beakers to the aquaria where they were kept for the rest of the experiment. To stimulate the
enriched environment the same stone mat as mentioned above was placed under the aquaria. The
first 2 months the zebrafish were contained in 400 ml beakers which were numbered 1-20. After
6 weeks the ZEM were diluted every day with water containing 0.28 g/l Instant Ocean Sea Salt
until fully exchanged. The beakers were cleaned every day and disposable transfer pipettes were
used to remove leftovers, faeces and dead fish. The water was fully exchanged once a week to
Figure 4. Here the beakers have been moved from the incubator to the self-circulating aquaria. This picture shows fish in the
enriched environment.
keep properly oxygenated and clean.
14
Aquaria
After 2 months the zebrafish were moved from the beakers to 3×7 aquaria with a 14:10h
light/dark cycle (each aquarium; 10×58×17cm) (total number of 21 where 20 were used)
containing 0.28 g/l Instant Ocean Sea Salt to a salinity between 500 and 600 μsiemens where
each aquarium contained 2.9 l water. Under the 21 small aquaria, 2 big ones were placed. The 2
big aquaria contained 3 water pumps (1 in 1 of them and 2 in the other) connected to silicone
hoses, T-crosses and taps to circulate water to the 20 small ones. Each small aquarium contained
a hole in the bottom which was blocked with a stopper with a hole in the middle where a sawn-
off serological pipette was placed. Due to this arrangement the water could circulate from the big
aquaria to the 20 small ones and back to the big ones creating a self-cleaning and oxygenating
circulation system. The top of the serological pipettes was covered with pieces of small nets
fastened with cable ties. This was necessary so the zebrafish in the aquaria were not sucked
through the pipettes. These nets were cleaned every day to reduce the risk of clogging and
thereby inundation. Figure number 5 shows the arrangement.
Figure 5. The poor aquaria and the water pumps which were connected to silicone hoses which were further connected to T-
crosses and flow regulators. The picture also shows how water was running through the pipettes back to the big aquaria under
creating a circulating system.
The temperature was measured every day and was 28 ±1° C (Avdesh et al. 2012). Water
salinity was also checked every day with a SENZ MSiemen, and due to evaporation, the big
aquaria had to be filled with deionized water (dH2O) when necessary. pH was checked once a
week and measured between 7 and 8 (Avdesh 2012). Other water quality measurements were also
performed once a week with a JBL EasyTest 6 in 1.
15
Enrichment
For 6 weeks the zebrafish only had a plastic stone mat under the enriched beakers (figure 4).
Stones and vegetation were added later due to high sensitivity among the zebrafish during early
life stages. After 6 weeks 1 stone (Bohusgranit) was added in the beakers simulated the enriched
environment. When fish were transferred to the small aquaria 6 stones and live vegetation were
added and the same stone mat as before was placed under the aquaria (figure 6). Before the stones
were added they were carefully cleaned with ethanol (70%) and after washed with H2O. The
Figure 6. 2 pictures of the enriched small aquaria, where the enrichment consisted of 6 stones, vegetation and a plastic stone
mat. The left picture is taken from the side of the aquaria and the right one is taken from above.
vegetation was washed under dH2O before placed in the aquaria.

Feeding
Zebrafish embryo was fed twice a day with dry food, ZM-000 (food size < 100 microns) the first
2.5 weeks and then dry food ZM-100 sizes between 80-200 microns for 3 days. After this
zebrafish were fed live food, brine shrimps (Artemia sp.) twice a day. Brine shrimps hatched after
2 days in continuous oxygenated saltwater (30 g/L) and were collected with a 25ml pipette. After
the brine shrimps were filtered and rinsed with ZEM before fed to the zebrafish.
16
Behaviour tests
The behaviours that were examined during this study were locomotion, schooling and braveness.
1 month after exposure- zebrafish fry locomotion (Zebrabox)

1 month after treatment zebrafish locomotion was tracked in a video recording device (Viewpoint
Zebrabox®). First, the fish were placed in a 24 well plate before being placed and acclimatized
for 1 h in a 28 °C incubator. Afterwards, each plate was tracked for 1 h and 55 minutes (including
15 minutes of acclimatization) at a constant temperature of 27 ±0.5° C. A protocol of alternating
light/dark cycles (5 minutes each) adapted from the protocol used by Ašmonaitė et al. (2016) was
used. The tracking software took measurements every 10th second and larval locomotion of
continuous movements longer than 2.4 mm was analysed.
4 months after exposure- locomotion, schooling and braveness
The fish that were going to be studied were removed from their housing aquaria ≈24 h before the
behaviour experiments and acclimatized in other aquaria.
Behaviour studies were done in custom designed experimental aquaria which were divided
into 3 parts, 2 smalls (4×5×10cm) and 1 big (19.5×5×10cm) (figures 7 and 8). The 2 small areas
of the aquaria where visually separated from the middle part of the aquaria with the help of 2
(5×10cm) non- transparent glass sheets placed on each side (figure 7) during the acclimation part
of the experiment. 2 aquaria were filmed at the same time and therefore the 2 glass sheets on each
aquarium were attached to laces which on the other side were attached to a rod. This was done so
the 4 glass sheets could be removed at the same time.
The left small part of the aquaria contained 5 zebrafish representing the schooling group
while the right small part of the aquaria was empty, just containing water (figure 8). During the
experiment, 1 fish in each experimental aquarium were filmed and examined. Before the
experiments started the fish was introduced to the experimental aquarium and acclimated for 10
minutes. After 10 minutes the 2 non- transparent glass sheets were removed, and the experiment
filmed with a GoPro Hero 5 lasted for 5 minutes.
Figure 7. The 2 experimental aquaria during the 10 minutes acclimation where 2 non- transparent glass sheets in each aquarium
separated the schooling group from the experimental fish. The golden laces were attached to a rod which was used to remove the
non- transparent glass sheets at the same time
17
Figure 8. The experimental aquaria with 18 drawn squares which were used during the locomotion test. The longitudinal line
separated schooling and non- schooling and the horizontal line separated shy from bold behaviour. 5 fish in the small left part of
the aquaria represented the schooling group.
Locomotion, after 4 months

18 squares were drawn on the experimental aquaria (figure 8) where the squares closest to the 2
small parts of the aquaria were smaller than the rest of the squares (small; 1.5×2.5cm big;
2.5×2.5cm). Locomotion data were collected from calculating the squares fish visited. Visited
squares where counted when the head of the fish crossed a line. This was later examined in the
tracking software Kinovea.
Schooling, after 4 months

For calculation of schooling behaviour, the experimental aquaria where divided into 2 squares.
This was done by drawing a longitudinal line in the middle of the aquaria (figure 8). The area on
the left (closest to the schooling group) was counted as schooling time and during the 5 minutes
long experiment the time spent on the schooling side was measured with the help of the tracking
software Kinovea.
Braveness, after 4 months

Braveness was calculated by measuring the time spent by the fish in the upper area of the
aquarium. The upper area was counted as the brave part, due to a higher predation risk in the
wild, and the upper and lower area of the aquaria were divided by a horizontal line drawn in the
middle of the aquaria (figure 8). Examination of the films was done in the tracking software
Kinovea.
Calculations and data analyses

Data from the Zebrabox where sorted in excel and in this study, the measurements from small
and large movements were used. These data symbolized the locomotion data, which are
measurements of how much and long fish travelled in the Zebrabox during the 1 hour and 40
minutes long experiment. Graphs over total distance travelled by the 4 groups were made in
Excel.
Light and dark cycles were also separated, and the average distance fish travelled in the dark
respective light cycle was calculated. As the locomotion data from fish both travelled in darkness
18
or light did not show normal distribution, neither after transformation (Lg10), but did show
homoscedasticity data was investigated with the non-parametric Kruskal- Wallis H test. To see
which group differed from which a Post hoc test was conducted at the same time as the Kruskal-
Wallis H test.

Due to a detected tank effect (one- way ANOVA (p> 0.05) and unequal sample size the mean of
the fish from each aquarium was calculated in Excel. This led to a total sample size of 20 (n=5
for each treatment). All statistical analyses were performed in IMB SPSS. Transformation (Lg10)
was performed which led to homoscedasticity and normal distribution of the behavioural data. A
two- way ANOVA (p<0.05) was conducted where environment and exposure were treated as
fixed factors and locomotion data as the dependent variable.
Survival rate
Survival rate was calculated in excel where the number of fish alive when the study started was
compared to the number of fish left when the experiment was over. Differences between the 4
groups were also investigated with a one- way ANOVA in IBM SPSS and presented in a bar
chart.
The study was conducted under the ethical permits 39-2014, held by Joachim Sturve.
Results
Observations
It was observed that fish in the enriched compared to the poor environment acted differently, for
example, did the fish in the enriched aquaria exhibit a "tighter schooling behaviour" meaning
they stayed in close proximity to each other.
From observations, we could also see a distinct difference in pigmentation between fish
developed in the enriched environment compared to the poor one (figure 9). The fish in the poor
environment had a pale scale tone (left) while the fish in the enriched environment had a much
Figure 9. The difference in scale tone between the 2 environmental treatments was observed during the experiment. Fish on
the left are from the poor environment and fish on the right are from the enriched environment. The size difference between
the groups has only occurred by chance at this photograph.
darker tone (right).
19
Locomotion activity of the zebrafish 1 month after exposure was measured in a Zebrabox where
the fish’s photo- motor response was measured. The total distance travelled/locomotion activity
was measured as counts of movements which was stimulated with cycles of darkness and light 5
minutes each. The graphs (figures 10 to 12) showed an unstable photo-motor response pattern
during the first 40 minutes of the recording and after this a consistently increase in the swimming
behaviour in the dark cycles until it dropped when switched to light cycles. Exposure to the EDC
mixture and the 2 different environments affected the zebrafish locomotion activity, which is seen
in figures 10 to 12 and which is statistically explained under the titles dark and light cycles.
Figure 10 shows a clear difference between the 2 treatments, where CTRL Enriched have a
higher locomotion activity during both dark and light cycles. The differences between the other
groups seen in figure 11 and 12 are not noticeable by just looking at the graphs. Whether or not
these data differed significantly are shown under subheads “Dark cycle” and “Light cycle”.
Figure 10. Photo- motor response of zebrafish in the CTRL and the CTRL Enriched group. The dark regions of the graph
illustrate the dark cycles in the Zebrabox where zebrafish activity was higher compared to the light cycles.
Figure 11. Photo- motor response in the CTRL and MIX group. The darker regions represent the dark cycles where zebrafish
activity was higher compared to the light cycles.
Figure 12. Photo- motor response in the CTRL and MIX Enriched group. The dark regions of the graph represent the dark cycles
where zebrafish activity was higher compared to the light cycles.
20
Dark cycle
Results show the difference in locomotion activity between the 4 treatments (N=114) during the
dark cycles in the Zebrabox. A Kruskal-Wallis H test (p<0.05) showed that there was a statistical
difference in locomotion between the 4 different groups (χ2(3) = 54.985, p = 0.000), with a mean
rank locomotion score of 92.32 for CTRL for CTRL Enriched, 144.44 for MIX, 59.74 and 105.50
for MIX Enriched. Table 3 shows the results from the Post hoc test where a significant difference
between every group except between CTRL and MIX Enriched (p= 1.000) was found (Appendix
A). The Boxplots (figure 13) clearly show how fish in the CRTL Enriched group had a higher
locomotion activity compared to the other treatments.
Table 3. P value between the 4 treatment groups during the dark cycles, where all were significant except between CTRL and
MIX100XEnriched. The red p- values are the significant ones (p<0.05).
Figure 13. Boxplots over the 4 groups where the lower quartile mark 25 % of the values, the upper 75% and the box 50%. The
line in the box represents the median and the dots outside the boxplots represent outliers. The significance is marked in the figure
as letters where equal letters mean no significance and different letters mean that there was a significant difference between the
groups.
21
Light cycle
The results show the difference in locomotion activity between the 4 treatments during the light
cycles. A Kruskal-Wallis H test (p<0.05) showed that there was a statistically significant
difference in locomotion between the 4 different treatments (χ2(3) = 70.711, p = 0.000) with a
mean rank locomotion score of 101.74 for CTRL for CTRL Enriched, 155.70 for MIX, 79.22 and
65.34 for MIX Enriched. A pairwise comparison between the 4 groups (N=114) and the p- values
are shown in table 4. There was a significant difference between each group except CTRL – MIX
(p= 0.310) and MIX – MIX Enriched (p= 1.000) (Appendix B). Figure 14 show boxplots over the
4 treatments, where CTRL Enriched had the highest locomotion activity.
Table 4. Results from the pairwise comparison during the light cycles in the Kruskal- Wallis H test. The red p-
values are the significant ones when p <0.05.
Figure 14. Boxplots over the 4 groups where the lower quartile mark 25 % of the values, the upper 75% and the box 50%
and the line in the box represents the median. The significance is marked in the figure as letters where equal letters mean no
significance and different letters mean that there was a significant difference between the groups.
22
4 months after exposure- locomotion
Studies on locomotion behaviour in a two- way ANOVA (p<0.05) showed, no significant effect
in either clean or enriched environment (F1.19=1.041, p=0.323), the exposed or unexposed
(F1.19=0.987, p=0.335) or the interaction between environment and exposure (F1.19=3.045,
p=0.100) were discovered.
Due to the noticeable trend of difference between CTRL and CTRL Enriched in a bar chart
(figure 15) a two-sample t-test (p< 0.05) was conducted without the interference of the mixture.
This test was done as data were normally distributed and had equal variance. The results showed
a significant difference between the 2 groups (t8= -2.649, p= 0.029), which is represented by the
asterisk in figure 15. When comparing the trend of difference between CTRL and MIX data had
not equal variance and a Mann- Whitney U test was conducted (p< 0.05). Here no significant
difference was found (U5.5 =6.000, p= 0.175).
Figure 15. Locomotion difference due to the environment with or without exposure. The asterisk represents the significant
difference between CTRL and CTRL Enriched from the two- sample t-test. Bars show means ± standard error for n=5.
23
4 months after exposure- braveness
Bar chart in figure 16 shows the difference in braveness between the 4 treatments. However, the
results of the braveness data from the two- way ANOVA (p<0.05) did not show any significant
difference when braveness was studied. The p- values were following p=0.983 for environment
(F1.19=2.231), p=0.155 for exposure (F1.19=0.000) and the p-value for the interaction between
environment and exposure was 0.682 (F1.19=0.174).
Figure 16. Bars represent the difference in the behaviour braveness due to the environment with or without exposure to the EDC
mix. Bars show the means ± standard error for samples n=5. Here no significance was detected.
4 months after exposure- schooling

Bar chart below (figure 17) shows the difference in schooling behaviour among the fish in each
treatment. Further on schooling results from the two- way ANOVA (p <0.05) showed no
significant difference in schooling behaviour when looking at environment (F1.19=0.234,
p=0.635). Neither did the results from exposure or the interaction of the 2 treatments show any
significant difference in schooling (F1.19=0.330, p=0.574 respectively F1.19=0.382, p=0.545).
Figure 17. The difference in schooling due to the environment with or without exposure to the EDC mix with no significant
difference found. Bars show the means ± standard error for n=5.
24
Survival rate
Zebrafish mortality during this experiment was very high at the beginning which was expected.
The survival rate was calculated as the number of fish that survived until the end of the
experiment compared to the number of fish at the beginning of the study (≈ 6 months). The
survival rate of the fish in the CTRL group was ≈ 36.4 %, ≈32.4 % for CTRL Enriched, ≈17.7%
for MIX and ≈34.0% for MIX Enriched (figure 18). The total survival rate was ≈30.2%.
Figure 18. Bar chart over the survival rate (%), where the number of fish in the beginning of the experiment was compared to the
fish in the end. The letters above the graphs show which treatments were significant or not, where equal letters meaning no
significance. The error bars represent ± standard error.
When comparing fish survival (survival rate) in the aquaria in each treatment with a one-
way ANOVA (p< 0.05) a significant difference was found between the groups (F3.19= 4.531, p=
0.018). The post hoc test showed significant differences between the MIX – CTRL (p =0.020)
and MIX – MIX Enriched (p= 0.045). Table 5 shows all p- values from the post hoc test.
Table 5. P values from the survival rate multiple comparisons between the groups. The red values are the significant ones
(p<0.05).
25
Discussion
Man-made waste products are a major issue which can lead to consequences for both us and all
the organisms living in our ecosystem. Some of these chemicals are EDCs and, in this study, we
examined zebrafish behaviour and their response to an acute exposure to an EDC mixture and
long-time development in 2 different environments.
In the present study, 3 different behaviours were filmed at the same time in the same
experimental aquarium, this could have led to different results compared if the 3 behaviours had
been studied separately. By observation, I noticed that many of the examined fish followed the
schooling group, in the smaller part of the aquarium. If the schooling group swam above the
horizontal line (the line which separated the brave versus shy part of the aquarium) the examined
fish often followed the schooling group. This may have led to an overestimation of the braveness
behaviour among some fish, especially the ones with a higher tendency to school. A similar
problem could have occurred when investigated locomotion versus schooling behaviour, where
fish with a high tendency to school would have a lower locomotion activity. The number of fish
in each aquarium also differed between 1 to 9 which could have influenced fish behaviour
without taking the effect of the 2 treatments into account. For example, a study by Ranta et al.
(1992) has shown tendencies towards that a fish of a different size compared to nearby fish have
a lower schooling activity. This could have led to a lower schooling activity in fish developed
alone or in small numbers which tended to have a larger body size due to more ingested food.
Exposure
The photo- motor response showed higher locomotion activity during the dark cycles compared
to the light at least after 40 minutes of recording. In comparison with other studies, this was
hypothesized as zebrafish tend to have a higher swimming activity in darkness (Ašmonaitė et al.
2016). The reason for the unstable pattern during the first 40 minutes may be due to differences in
behaviour between 5 days old fish, which showed a stable pattern during the screening session
(Birgersson et al. 2017), and the fish in this study which was 1 month old.
The mean locomotion activity was higher in the CTRL group compared to the MIX group in
both dark and light cycles, but the difference was only significant during the dark ones. These
findings are opposite to earlier findings on locomotion activity when exposed to a similar EDC
mixture where an increase in swimming activity has been found (Birgersson et al. 2017). It has
been shown that the similar EDC mixture effects the HPT axis, and more exactly the thyroid
system. Explained in the introduction thyroid hormones have been found to affect, among other
things, growth and behaviours in fish (Bell 2004; Power et al 2001) and there is a possibility that
the earlier findings on increased locomotion activity relate to the relation between these EDCs
and increased acceleration of growth (Bell 2004). When fish grow they requires an increased
amount of food which in turn may lead to an increased locomotion activity while searching. The
study performed by Birgersson et al. (2017) used 120hpf zebrafish after they had been exposed to
the similar EDC mixture (100X) for 48 h. In this study, the locomotion activity was measured 1
month after exposure (48 h) and did not generate similar results as the previous study. However,
results on the motion distance in juvenile goldfish from an earlier study by Xia et al. (2013)
showed a reduced activity due to PFOS exposure which is one of the PFASs active in the EDC
mixture used in this experiment.
26
The results from the two- way ANOVA showed no significant difference between the 4 groups
when studying exposure and environment separately. Neither was there a significant difference
between them when looking at the interaction of the 2 treatments. This led to a rejection of the
hypothesis that the EDC mixture would influence behaviour possible leading to an increase in
locomotion and a rejection of the hypothesis that the interaction would be significant. The effect
may have been reduced or disappeared through time, as earlier studies have shown significant
results (Birgersson et al. 2017; Ulhaq et al. 2013). However, these results can only say that this
mixture of EDCs in this concentration do not affect zebrafish when studied 4 months after
exposure, but they do not tell whether exposure during early life stages affect organisms later in
life when other behaviours are studied, which has been shown before (Colborn et al. 1993).
Another possibility to consider is the fact that fish who attributed to significant results 1 month
after exposure may have died when the study after 4 months was conducted. This theory can be
discussed due to the still relatively high mortality after 1 month. Therefore, further studies are
needed especially on possible physiological effects due to this exposure.
Environment
When comparing the locomotion activity between CTRL and CTRL Enriched there was a
significant difference between these groups during both dark and light cycles. The fish in the
enriched environment (CTRL Enriched) had a higher locomotion activity than the control group
(CTRL), which suggests that even poor enrichment (only a plastic stone mat) can affect the
results from behaviour studies. These findings fall in line with other results (Schroeder et al.
2014) but still, further studies are needed to find out more and draw any precise conclusions.
It is hard to explain the significant differences during the dark cycles between MIX and
MIX Enriched, due to the possible effect of the EDC mixture, but the explanation may be the
same as when comparing CTRL and CTRL Enriched.

Previous studies comparing poor and rich environment using this method and its effect on
behaviour are missing. However, several studies on rodents have been done comparing
environmental enrichment and its positive effect on the ability to learn (Falkenberg et al. 1992;
Harburger et al. 2007) and one study have found that environmental enrichment may also affect
regulation of neural plasticity leading to enhanced spatial learning abilities in Atlantic salmon.
Where Atlantic salmon bred in enriched environments made fewer mistakes in an experimental
maze compared to the fish in the control group (Salvanes et al. 2013) and it has also been
demonstrated that environmental variations can rapidly alter cell cycle dynamics in the zebrafish
brain (von Krogh et al. 2010). The results from the two- way ANOVA did not show any
significant results on the 3 behaviours but due to the noticeable trend of difference between
CTRL and CTRL Enriched in locomotion data this was studied separate (not including exposure).
Here a significant difference between these groups was found which correspond to previous
studies mentioned above that environment plays a role in fish behaviour. From observation of the
fish while still in the housing aquaria a difference in behaviour was detected. The fish in the
enriched environment appeared to swim more together and exhibit a “tighter schooling
behaviour” compared to fish on the poor environment. Even if this was not shown during the
27
statistical analyses it is important to mention and further investigate using other experimental
methods.
Survival rate
Zebrafish are known to have a high mortality rate at the beginning of their lives and one
especially critical time is when their egg yolk is fully consumed, and they must “catch” their own
food. This led to the low survival rate observed at the beginning of the experiment which was
expected. A difference in the survival rate was observed where the fish in the MIX group had a
much lower survival compared to the other groups. This negative survival effect has also been
shown in another study where mortality among fathead minnows exposed to PFPeA (32 mg/L)
was as high as 50 % (Hoke et al. 2012). From the one – way ANOVA significance in survival
between MIX – CTRL and MIX – MIX Enriched were found and a low but not significant p-
value between MIX and CTRL Enriched, compared to the multiple comparisons between the
other groups where the p values were higher. This indicates that exposure to this concentration of
EDC mixture lowers the survival rate of zebrafish. However, exposed fish in the enriched
environment did not have a significantly lower survival compared to the other groups (except
MIX) which can possibly be explained by a positive effect due to the enrichment. Maybe due to
an enhanced cell dynamic in the brain (von Krogh et al. 2010), and that the observed negative
survival effect of the EDC mixture can be reduced by an enriched environment.
Morphology
By observation, a distinct difference in scale tone between the fish in the enriched versus the poor
environment was detected. Fish in the enriched environment had a much darker tone compared to
the ones in the poor. This was expected since fish tend to change “colour” depending on the
environment. The floor in the poor aquaria was white which led to a high reflection and a much
brighter habitat. However, it would have been interesting to see if the colour shifted when fish
were moved from a treatment to the opposite (from poor to enriched and vice versa).
Improvements
Experimental design
At the beginning of the study, the total sample size (number of fish) was 394 compared to 119 at
the end of the study, which led to a survival rate of ≈ 30% throughout the experiment. Before
comparing the fish in a two- way ANOVA the mean behaviour of each aquarium (N=20) was
calculated, this was done to remove the detected tank effect and since a two- way ANOVA
requires an equal sample size. This led to a sample size of 5 in each treatment, which is quite low
and thereby led to a low power.
When redoing this experiment, a higher number of fish in each aquarium and a larger
number of aquaria in each treatment is highly recommended to increase the power of this
experiment.
Further on, in this study, I looked at locomotion, braveness and schooling behaviour and
there is a possibility that other behaviour test would be more favourable especially since
differences between the fish in the poor compared to the enriched were observed. I would also
suggest studying each behaviour separate due to the interaction of the schooling group and the
examined fish.
28
Conclusion and Future studies
The results of the study conducted after 1 month did support the 1st hypothesis; that fish exposed
to the EDC mixture would behave differently. However, the findings from the experiments
conducted after 4 months lead to a rejection of the 1st hypothesis. There are other possible effects
that may occur due to this mixture which cannot be ruled out, as some physiological effects may
not be able to detect by the behaviours studied in the present study. Maybe behaviour such as
reproduction would be preferable or that behaviours in this study should be examined via other
methods to yield clearer results. There is also a possibility that exposure to this mixture has a
delayed negative effect on different behaviours even later than 4 months or that the effect gets
reduced or even disappears with time, at least in the concentration used in this experiment.
In this project, we showed that even a little enrichment plays a role in zebrafish locomotion.
This and the fact that another study by Schroeder et al. (2014) has shown that pictures of gravel
cause a preference for enrichment in zebrafish it is important to consider environmental
differences when conducting behaviour experiments. However, results after 4 months showed
that environment had no influence on the behaviour when studying exposed and unexposed
groups together. This contradicts the observed differences between fish in poor and enriched
aquaria which may not be possible to detect using the methods in this experiment. Furthermore,
we did find that fish in the unexposed group developed in the poor environment had a lower
swimming activity compared to the unexposed fish reared in the enriched. Still, it is possible that
the enrichment was not enough to detect differences in the other 2 behaviours in this study
(schooling and braveness), or that the methods used in this experiment were not preferable to
detect possible differences.
After investigating survival rates in the different treatments, we could observe a reduction of
mortality in the exposed fish when reared in an enriched environment. This may be due to that
enrichment reduces the negative effect of the exposure which may be due to increased neural
plasticity. However, these assumptions need supplementary investigation.
In conclusion, enriched environments seemed to reduce possible negative effects due to
EDCs exposure. However, further studies are needed to investigate additional interactions
between these 2 factors.
Acknowledgements
I am grateful to my supervisors Joachim Sturve and Lina Birgersson at the Department of
Biological and Environmental Sciences, Gothenburg; Peter Tiselius at the Department of
Biological and Environmental Sciences, Gothenburg, for statistical discussions and help; Ferm
och Persson AB for providing the experimental aquaria; and at last to Jessica Lindborg for fish
husbandry.
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Appendix
Appendix A
Figure 19 explains the difference between the 4 treatments during dark cycles. The yellow lines represent a significant
difference.
Appendix B
Figure 20 explain the difference between the 4 treatments during light cycles. The yellow lines represent significance while the
black and missing line show which groups the difference was not significant.
36
A diverse living condition may lead to reduced effects of
EDCs.
We are living in a critical time where human waste is a major issue, which can lead to
consequences for both us and all the living organisms in various ecosystems. One group of such
waste products that we are commonly washing down our toilets are endocrine disruptive
chemicals (EDCs), which are chemicals that disrupt the hormonal system of an organism and
have shown associations to some of the most common chronic diseases and disorders of the
western world. These concerns are based on effects seen in the wild, findings in laboratories
experimenting with fish and other organisms combined with increased hormonal related diseases
in humans. A project called EDC MixRisk, which is founded by the European Commission, is
investigating the possible hazard of different EDCs and EDC mixtures such as their behavioural
and physiological effects on organisms. Exposure to EDCs can lead to problems with
development, growth and learning, which may lead to negative effects in several kinds of
organisms. Chemicals are not acting alone in the wild, however, and by studying the effect of
EDC mixtures and combining physiological and behavioural studies improved knowledge of the
adverse effects of these chemicals can be acquired.
Zebrafish are often used in laboratory toxicology studies and the main reason for this are the
similarities in their DNA towards human DNA. Around 70% of protein-coding genes in humans
are related to genes in zebrafish and by studying zebrafish response to chemicals assumptions
about the respective effect in humans may be drawn. This study was built on an ongoing study on
the presence and effects of EDCs in pregnant women and the concentration used was a hundred
times higher than the mean concentration found in the serum of the pregnant women. In this
study, two aspects were studied; long-time development in different environments and acute
exposure to an EDC mixture. Many laboratory experiments are conducted in sterile environments
and by studying the effects of two different environments, in this case, a poor and enriched
environment we wanted to investigate if different environments affected and interfered with our
results from the exposure. Exposed zebrafish studied one month after exposure showed a lower
swimming activity compared to the group that was not exposed and when we looked at the effect
of different environments we found that zebrafish developed in the enriched environment had a
higher swimming activity than fish reared in the poor. We also found differences in swimming
behaviour between the unexposed fish in the poor environment compared to the exposed fish in
the enriched, where the exposed fish showed a higher activity level than the unexposed. These
results indicate that EDCs influence swimming behaviour one month after exposure and that
development in different environments may play a role when it comes to resistance to EDCs. The
last assumption can also be underlined by the fact that exposed fish in the enriched environment
had a higher survival rate compared to exposed fish in the poor environment. Another study was
conducted four months after exposure where no differences in swimming behaviour between the
exposed and unexposed groups could be found. Neither did we find differences in two other
behaviours studied which suggests that some behavioural effects of EDCs can be reduced or even
disappear over time. Even though these findings are based on a small study with relatively few
fish, assumptions can be made that behavioural changes, due to this concentration of these EDCs,
investigated in this experiment may only be of concern shortly after exposure and that differences
in environment can influence resistance against EDCs.
37

Endocrine Disruptive Chemicals and Their Effects On Zebrafish (Danio

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DEPARTMENT OF BIOLOGICAL AND

ENDOCRINE DISRUPTIVE CHEMICALS AND

Per- and polyfluorinated alkyl substances

Adverse outcome pathways (AOPs) and EDC mixtures

The Hypothalamic-Pituitary-Thyroid axis

How to study behaviour in fish and how it can be affected by EDCs

Materials and Method

Beaker Exposure date Exposure Environment Start: End:

Aquaria and Housing

keep properly oxygenated and clean.

vegetation was washed under dH2O before placed in the aquaria.

1 month after exposure- zebrafish fry locomotion (Zebrabox)

Locomotion, after 4 months

Schooling, after 4 months

Braveness, after 4 months

Calculations and data analyses

4 months after exposure- locomotion, schooling and braveness

darker tone (right).

4 months after exposure- schooling

4 months after exposure- locomotion, schooling and braveness

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