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- Abstract …………………………………………………………….…….….. 2
- Introduction ……………………………………………………………….…… 3
- Aquatic toxicology …………………………………………….….….….. 3
- Model organism ………………………………………………………….. 5
- Research study …………………………………………………………… 8
- Pharmaceutical drugs exposure …………………………………… 10
- Potential suggestion to problem ………………………………..….…… 16
- The aim ………………………………………………………..….…….. 17
- Methods and material ………………………………………………………. 18
- Chemicals ……………………………………………………..………… 18
- Experiment …………………………………………………..…………. 18
- Test organisms …………………………………………..…….…. 18
- Sample preparation ……………………………..…….………….. 19
- Behavioural and metabolism analysis …………………….……… 20
- Data analysis ………………………………………………….…….….. 21
- Results …………………………………………………………………….… 22
- Ibuprofen exposure …………………………………………….…….… 22
- Caffeine exposure ……………………………………….………….….. 27
- Discussion ………………………………………………………….……….. 31
- References ………………………………………………………………….. 35
Pharmaceutical drugs are widely used around the world to relief pain, increase or decrease activity and cure diseases. Recently, the disposal of these active pharmaceuticals has come into light and has become a major issue for our ecosystem, especially the aquatic environment. As the drugs are easily transferred into the waterways as improper disposal and accidental spillage, it is slowly creating an everlasting impact on the organisms that reside in the waterways. Hence, the aim of this study is to recognise the impacts of neurotoxins on the behaviour and metabolic activity of zebrafish larvae, to find a correlation between the changes at sub-lethal levels, before ecological death occurs. Two famous pharmaceutical drugs, caffeine and ibuprofen, were used to analyse 3-dpf larvae behaviour under light-dark periods, while detecting the fluorescence to study the metabolism. The results obtained demonstrate a correlation between behaviour change and metabolic activity of the larvae, and a range where ibuprofen acts as a toxicant is also discovered. Provided the limitations in prior studies, the outcomes of this study will provide sub-lethal end points to these drugs, which will assist scientist setting better measures to protect the aquatic environment.
Keywords: Zebrafish larvae, exposure, ibuprofen, caffeine, behaviour change, metabolism.
The influence of toxicants on organisms and the whole of ecosystem has been of a great concern in recent years . Several studies have found chemicals that are detrimental to organism health, have been found in freshwaters, drinking waters and other water ways . The field of ecotoxicology desires to study the influences of toxicants on a wider population in our ecosystem to measure its health by using biomarkers, in order to highlight the risk associated with environmental toxicants . Biomarker are known as a parameter to measure the presence of diseases. Although, there is a strong link between ecotoxicology and environmental public health, the field of ecotoxicology aims to limit the risk of chemicals on organisms, their population and the whole ecosystem, whereas environmental public health is concerned about the stressor and toxic chemicals, which poses a risk to individuals or the wider human population . Due to the increasing stress in the environment, organisms are experiencing ecological death. Ecological death is defined as the inability of an organism to function in its ecological context, where they usually experience changes in behaviour, predation, reproduction and swimming speed, which ultimately leads to death . Behaviour change is one of the earliest responses to ecological death. Presence of pollutants in the environment has caused several species to migrate from their normal ecological setting or in worst cases go extinct. Aquatic pollution is particularly an area of concern as aquatic organisms are captive to continual life-cycle, multigenerational exposure. The possibility for continual but undetectable or unnoticed effects on aquatic organisms could accumulate so slowly that major changes go undetected until the cumulative level of these effects finally cascades to irreversible change – change that would otherwise be attributed to ecologic succession .
The release of pharmaceutically active compound in the aquatic environment has certainly raised concerns for the wellbeing of our ecosystem. Pharmaceutically active compounds are produced very large volumes and disposed, and their use and diversity increase every year . These compounds are usually discovered in our waterways, causing harm to the species that live in them. According to a review by Jones et al. , it was discovered that in the 2000, UK was found to have exceeded the usage of pharmaceuticals by 10 tonnes per year for the top 25 compounds and the figure for the top three compounds (paracetamol, metformin hydrochloride and ibuprofen) surpassed 100 tonnes per year. These substances have only become worse in the ecosystem, which now cause detrimental impacts to the organism health. In the past, studies in toxicology have generally focused on assessing the acute lethal concentrations (e.g.: median lethal concentrations, LC50) and chronic sub-lethal impacts on the developmental and reproductive endpoints as such techniques allow access to results that can potentially be related to organismal health and fitness . Knowing the effects of pharmaceuticals on behaviour are importance as they link directly to the ecological significance, as behaviours are closely linked to individual fitness and population persistence .
Although pharmaceutical drugs are meant to cure and prevent diseases and improve medical health of an organism. However, an over-exposure of the drugs can also cause adverse effects . Recent studies have also explored the influence and dangers of the toxicants on the environment. A review on zebrafish larvae to assess their neurotoxicity by J. Legradi et al. , highlights that the zebrafish larvae form malformations when exposure to toxicants, which also leads to changes in their behaviour. Some studies about pharmaceutical in aquatic environments like Brodin T  review, highlights that ‘antidepressants, psychiatric drugs and antihistamines can induce behaviour changes in fish at concentrations ranging from low µg/L-1 to low mg/L-1.’ As studies have practiced experiments with a range of concentrations, most concentrations have only demonstrated a minor to no deformities. Pharmaceuticals used in the industry are entering the aquatic environment primarily because of excretion and their therapeutic uses. These pharmaceuticals drugs that enter the environment are always diluting, and potentially metabolizing or degrading, to levels well below that are likely to result in any physiological effects from residues in drinking water  but are definitely known to cause gradual impact on the well-being of aquatic organisms.
2.2 The model organism
Exposure to noxious substances can change the usual action of the nervous system and lead to adverse reactions on all kinds of nervous tissues from central and peripheral nervous system to sensory organs. This refers to the term of neurotoxicity, where the exposure to neurotoxic substances can lead to headaches, dizziness and other biological changes . In recent years, due to an increase in the number of people with neurological disorder such as autism and Parkinson’s, testing for neurotoxicity has increased remarkably . The recent upsurge in environmental disturbance and its impact organisms has caused experts to examine toxicity . Aquatic vertebrates have numerous factors that must be considered during experiments such as different personalities/individuals . Zebrafish embryo is evolving as an essential tool for examining toxicity test as well as behaviour analysis . Zebrafish is a small aquatic vertebrate that is rapidly growing as the model organism in the medical field to study a vast amount of disorders, due to its resemblance to human genetics and physiology . Zebrafish have a more complex genome than the human genome, because they have two more pairs of chromosomes than the twenty-three pairs of human chromosomes . In fact, zebrafish possess the major neurotransmitter systems described for mammals, which allows for a detailed pharmacological investigation regarding the influence of different compounds on the central nervous system . A lot is known about the normality of the zebrafish, including their morphological, biochemical, and physiological information at all stages of early development in juveniles and adults of both sexes. This makes zebrafish an ideal model for toxicology research where the ultimate objective is to identify adverse effects of chemical exposure .
Zebrafish is not only a popular model organism for behavioural studies due to its similarities and complexities of genetics and physiology, but they possess several advantages, such as their rapid development; they are comparatively low-cost, transparency of larvae, they are easily maintained, and have an abundant offspring (up to 200 eggs in one mating) [4,11]. All those features can result in shorter experimental examination periods in addition to that decrease the number of toxicants use to express affects. Prior studies have used adult zebrafish for conducting behavioural and toxicity studies. But in recent years, zebrafish embryos and larvae have been a popular model for studying functions of vertebrate nervous system and behavioural phenotypes . Compared to larger fishes, the minute size of the larvae minimizes the quantities of dosing solutions required such as the experimental chemicals, drugs, pollutants, which thereby creates low volumes of waste for disposal and reduces quantities of labware and chemicals for both treating and maintaining live fish, and for performing numerous assays (low quantities of reagents) and histological assessments (small amount of embedding materials and microscope slides) .
Despite numerous zebrafish advantages, there are several limitations of this model organism that have been recognised. In the past, zebrafish underwent a whole-genome duplication events during which, some duplicated genes were destroyed and did not contain in the same tissue as the orthologs, while some others possessed new functions. Consequently, zebrafish paralogs may under-represent the severe phenotype . There is also a limited amount of anti-bodies that have been specified for the zebrafish. Although zebrafishes are cost effective when it comes to using them in an experiment, but the cost of the zebrafish facility can be expensive. As known, fish embryos have a protective envelope, called a chorion, where diffusion of specific chemicals such as ethanol is limited [22-23].
In contrast to the disadvantages, the study conducted using zebrafish have had valid and reliable outcomes. The study commenced by Giancarlo Brunisome , explored the details on how unusual action of behaviour can lead to the discovery of most neuroactive drugs. It is highlighted through this study that the basic knowledge of drug molecules, that impact our central nervous system, can assist us in understanding their functionality in-depth. Differentiation and classification of neuroactive drugs can be difficult according to uncertainty of phenotype and compound structures. So far, invention of new technologies has helped researchers in discovering new drugs with the use of phenotypic assay, which has the ability of high throughput. High throughput assays are greatly dependent on huge amount of data and mathematical modelling.
Behavioural phenotype has been broadly believed to have a critical role in the discovery of new drugs. Most neuroactive drugs were discovered throughout two periods of time, pre –history and the mild -1900s. Compound like nicotine, alcohol and morphine and their identification were based on unusual behaviour phenotype. Drugs like antipsychotics and antidepressants were also discovered in the mid-1900, where the second wave of drugs were recognized based on the same aspect of unexpected behavioural phenotypes.
The study also used zebrafish as the model organism due to its advantages in providing reliable results. Zebrafish behaviour can be discrete in different ways. Their behaviours can have close connection with human behaviour and at other instances, some zebrafish behaviour can lack these connections. Nevertheless, chemical drugs can influence zebrafish behaviours but these changing on behaviour can vary between adult zebrafish and in larvae. At the larval stage, the impression of the chemical was translated as a spontaneous swimming or photometer reaction, whereas in the adult zebrafish, the same chemical resulted in changing the swimming pattern. Despite great similarities between other vertebrates and zebrafish, researchers have found that many compounds have the ability to work well with human, but the same compound can have an opposite effect in the zebrafish and vice versa. On the other hand, some compounds appear to have the same influence in both human and zebrafish. Phenotypic and molecular level between human and zebrafish are different. This stands as one possibility where the response among all those reactions can differ. The problem is rooted out from none to a little knowledge about central nervous system (CNS) disorders that is related to neuropharmacology. According to this article, the field of science can hope to see several neuroactive drugs discovered by chance.
2.3 Our research study
In this study, we are using 3-days-post-fetilized (dpf) zebrafish larvae to study the impacts of two very widely used pharmaceutical drugs, Caffeine and Ibuprofen on the larval behaviour and their metabolic activity. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxico-dynamics of the chemical . In mammalian studies, applying neuroactive drugs such as caffeine, ethanol and cocaine in different model species can lead to a better understanding of how the nervous system works as well as allow us to assess its function . Therefore, lethal dose 10 (LC10) of caffeine is used to establish sub-lethal endpoints for toxicity and a range finding behaviour analysis for ibuprofen was carried out, as not many past studies had verified a low lethal endpoint for ibuprofen. Since both pharmaceutical drugs cause diverse effects on the behaviour and metabolic activity of the zebrafish larvae. The behavioural analysis test is performed after treatment with caffeine and Ibuprofen. As an anticipated result, each drug would show a distinct pattern of behaviour. In fact, the type of drug and dose used would be the main factor responsible for fluctuating behaviour patterns. Both of these pharmaceutical drugs are known to have different effects on the mind, Caffeine is known to increase the activity in organisms whereas ibuprofen is known to decrease the activity and responsible for calming the organism. We are using a variety of concentrations by performing serial dilutions to study their response at 24 hours and 48 hours for both behaviour and metabolism.
As a part of our behaviour study, the zebrafish larvae were also tested for the relative change in their metabolic activity. Alamar blue dye is a redox indicator that was used as an indicator to the metabolic activity. It read the fluorescence present in each concentration well where zebrafish larvae were present. Alamar blue is a non-fluorescent blue colour dye that undergoes colorimetric change in response to cellular metabolic reduction, which reduces from the non-fluorescent blue dye to a highly fluorescent pink colour. A high reduction of the dye causes the medium to change its colour from blue to pink and finally becomes colourless. Alamar blue dye has many advantages. As this dye is innocuous, it does not have any harmful side-effects on the cell; in other words, to obtain a measurement in the experiments, it does not kill the cell. Therefore, cells can be re-used for further examination. Considering the economical part, this dye necessitates fewer reagents to get result, meaning limited waste production. It is a non-toxic dye which is safe for the cell, handler and to the environment. It is a highly recommended for cytotoxicity and viability assay that is a fast and simple assay, designed to distribute precise measurement over time .
The behaviour analysis was performed with the Viewpoint Zebra box. A zebra box is a high-throughput analysis machine, which is especially designed to analyse the behaviour of zebrafish. The fluorescence analysis was carried out using a ClairoSTAR by BMGtech. It is a machine that transmits light and uses high-speed absorbance to measure any fluorescence present in each well-plate to provide a statistical analysis of metabolic activity.
2.3.1 Pharmaceutical drugs exposure (caffeine and ibuprofen)
Pharmaceutical products are used in medicine are of great importance in the treatment of disease. However, because they are designed to be biologically active, concerns have been raised about trace levels (ng/L) of some of these compounds that have been measured in drinking water. Caffeine is broadly consumed as a psychoactive agent and it is used as central nervous system stimulant . It is from tea leaves, coffee beans, seed and from variety of plant species around the world. Like any other substance, the excessive use of caffeine can have harmful effect on any organism. The study undertaken by Luana C. Santos et al , confirmed positive properties of caffeine, while other studies reflect a variety of side-effects. In fact, high concentration of caffeine can cause dizziness, nausea and sleeplessness. Even more, since caffeine is a psycho stimulant substance and due to that, this drug has been known to cause addictive properties in consumer. Extravagant usage of caffeine can cause intoxication and ultimately lead to demise of an individual. Focusing on the positive side of this drug, researchers have shown, caffeine can potentially increase awareness and reduces exhaustion.
In Luana C. santos et al  reflects that studies on exploring the effects of neurotoxicants drug on zebrafish larvae behaviour showed that the higher dose of caffeine (50 mg/L) declined distance travelled by the larvae, whereas a lower dose of caffeine (25 mg/L) appeared to have increased the distance travelled by the zebrafish larvae. Move over same studies shows the opposite result illustrating freezing behaviour, where the highest dosage of caffeine, in this case 50 mg/L, increased freezing behaviour in zebra fish than compared to less dosage of caffeine, that ranged from 0-25 mg/L.
According to most evidence shown by researchers, it can be confirmed that caffeine as a stimulant, has a direct impact on the central nervous system and for it to perform in a distinct manner, the amount of dose and time of exposure plays a vital role. To conclude, although caffeine consumption does not have any legal consequences, but as shown by the results, a high dose of caffeine can lead to harmful consequences, whereas a moderate usage of the drug can improve performance. Conclusively, this study proves the use of zebrafish as a reliable and precise model organism for testing the impacts of neurotoxicants.
Similarly, a study carried out by Luiz Vinícius Rosa et al.  about caffeine and how it impacts our central nervous system activity highlights that as the CNS is a main part that gets affected by high caffeine consumption, it can lead to the blockage of adenosine receptors, which can further lead to various neurophysiological reactions. The main type of behaviours phenotypes resulting from adenosine blockage is anxiety and antagonism. It has been reported that one-third of the population has experienced anxiety – related conditions.
This literature focused on the effect of caffeine on behaviour of two zebrafish populations, short-fin wild-type (wt) and leopard (leo) zebrafish. The study discovered that the locomotor activity in WT population under the influence of caffeine did not show any distinct changes, whilst the same concentration of caffeine (200mg/L) substantially decreased the distance travelled by the leo population. A similar study has conducted another test on these two fish populations for caffeine concentrations ranging from 50 and 100mg/L. The wild-type population again did not show any change in erratic movement, while the freezing bouts increased. Unlike the wt population, the higher dosage of caffeine increased the erratic movement in leo population with no changes on freezing bouts. Furthermore, about cortisol level this study has found, under the influent of caffeine (50,100 and 200 mg/L) the cortisol level for both of the fish population experienced an increase.
The study concludes by exploring several details about the drug. It highlighted the effect of the caffeine dose is highly dependent on the population selected and that different organisms can react to certain endpoints. Further investigation is required to discover if adenosine plays a role on changing the threshold for caffeine on both fish population.
Figure 1. Effects of different caffeine concentrations (25–200 mg/L) on locomotor activity of WT (A) and leo (B) zebrafish populations. Data are expressed as means ± S.E.M and analysed by one-way ANOVA.
Ibuprofen is also used as one of the neurotoxicant in this study. Although not a lot of studies have been conducted on this drug, hence a range finding test was carried out as a part of this project. Ibuprofen is a famous drug that is an anti-inflammatory and is widely used to treat and relief pain. Possible adverse effects and persistent properties of this drug on the aquatic environment have been of a great concern in recent years . Liang Xia et al.  conducted a study on the effects of ibuprofen, diclofenac and paracetamol on motor behaviour of zebrafish embryos/ larvae. Their study discovered the spontaneous movement of zebrafish significantly decreasing during high concentration exposure of Ibuprofen. Furthermore, the same study discovered a similar behaviour pattern during exposure to high concentration of diclofenac and found that paracetamol exposure does not detect any activity. The higher dosage of ibuprofen (50-500 µg/L) significantly decreased the swimming distance by 34% and 41% compared to the control.
Figure 1. Hatch rate of zebrafish exposed to ibuprofen, diclofenac and paracetamol (*P < 0.05, **P < 0.01) permission from RightsLink®).
Figure 2. Spontaneous movement at 28 hpf affected by ibuprofen, diclofenac and paracetamol (**P < 0.01) (permission from RightsLink®).
Liang xia et al.  revealed from the locomotion test revealed that, under the dark cycle, ibuprofen had substantial effects on the larval activity, whereas diclofenac was found to cause similar effects under the light cycle. Based on the outcome, this study suggested that, both Ibuprofen and diclofenac had similar destructive consequences on the zebrafish development in the contaminated solution. On the other hand, paracetamol is found to have less harmful effects on the environment in comparison to the two drugs tested. Ibuprofen and diclofenac were noted to cause delays in the hatch period, but not in paracetamol. In a similar previous study by David and Pancharatna , ibuprofen was reported to have dose-dependent increase of anomalies caused at similar exposure level (0 mg/L to 500 mg/L) and the presence of ibuprofen was detected to increase hatch time and cause the zebrafish embryo hatch to fail. Due to such results, further investigation is required in order to gain better knowledge about the molecular mechanism of mentioned drugs on behaviour activity of the aquatic organism. Thou, since this study was conducted under short term effects, further caution is need during practicing paracetamol treatments to testify additional impacts.
Similar studies about the effects of toxins on the aquatic ecosystem have discovered that the annual production and the consumption levels of ibuprofen have increased significantly. The major risk associated with this drug is that, it gets to the environment in easy and rapid way. In fact, due to incomplete metabolization and through the urination of human as well as animal, this drug finds its way to get to surroundings environment . An increase in studies have reported the global detection rate of ibuprofen is much higher than ever before. Researchers have reported that the detection of ibuprofen has increased compared to previous years as in this drug has been detected on water surface of some waterways. Surface water and sediments of the Turia River Basin and in the sewage and seawater of Spain and Norway, were detected with ibuprofen contamination levels of (0.18e7.20 mg/L-1), (N.D.-3.90 mg/L-1) and (0.10e20.00 mg/L-1), respectively . Former studies have reported the detriment of hatching rate, growth developmental retardation and other behavioural changes in zebrafish populations after exposure to ibuprofen. Major changes that ibuprofen exposure causes relate to embryo loco-motivity, swimming distance, duration and speed .
This study by Yue Song et al.  mainly focused on how stereoselective consequence of ibuprofen can alter the adult zebrafish metabolism and finding a better way to limit the harmful effect of this drug on our environment, particularly in the marine environment. To accomplish this goal, 22 amino acids with 3 antioxidant enzymes were chosen to conduct enzyme assay.
This research obtained results that clearly show the detrimental impacts of ibuprofen on the aquatic environment. Ibuprofen was found to have an influence on the metabolization of nucleotide and can cause stress. It was also revealed that the role of co-enzymes can fluctuate with varying effects of enantiomers of ibuprofen. Additionally, the 15 amino acids are reported to change, when exposed to ibuprofen, with no changes observed in the remaining amino acids. This study concluded that the enantiomers of Ibuprofen plays a critical role and have significant effects in aquatic organism, since it induces different toxicities in the ecosystem .
While limited studies have been conducted using ibuprofen as a drug of threat to the aquatic environment, the results presented by the two different studies highlight the possible harm that ibuprofen in high doses can cause.
The effect produced by these two chemicals drugs (Caffeine and Ibuprofen) in developing embryo, might provide us with an insight on its influence on other vertebrates and assist in building measures to sustain the ecosystem .
2.4 Suggestion to the growing problem
There are several challenges faced by researchers and the major one involves the validation of the outcome to ensure the data derived is useful to be applied for further research. Ecological validity of results, which describes the work carried in vivo conditions, that is necessary to obtain a reliable result. In behavioural aquatic toxicology, components of a good experimental design is the necessity of a high external validity to ensure that the experiment is biologically relevant to the observations made in the laboratory . As the increment of toxicants in the environment has become a major problem, it is extremely crucial to use safety measures in order to limit this problem from mounting out of control.
Although the environment has been exposed to a variety of drugs/chemicals and have experienced a combined effect from them, most studies about neurotoxicants have been performed on a learning about the effects of a single drug. Hence, for a better investigation in the future, Further examination into the impacts of exposure of combined drugs would be a valuable study, especially if it is used in conjunction with monitoring programmes, which would assist in demonstrating where specific types of mixtures occur and whether an additional level of risk assessment is required . Very limited is also known about the impact of pollutants on the food-web of aquatic organism. Aquatic systems have been exposed to pharmaceutical drugs that may have already caused damage that is, in some cases, inevitable. As pharmaceuticals have been inflowing to our natural freshwater systems for over several decades, the extend of the damage to the ecosystem is still limited to our knowledge and it is about time that we learn more about their movement in the environment to protect the organisms from their ecological death.
2.5 The aim
The main objective of this research is to examine the impacts of neurotoxins and anti-depressants on the behavioural and metabolic activity of zebrafish larvae at sub-lethal endpoints to establish a correlation between behaviour change and metabolism. It is hypothesised that there is a direct relationship between the fluctuations in behaviour and the metabolic activity of the zebrafish larvae when exposed to the two pharmaceutical drugs, where caffeine would increase the metabolic activity and ibuprofen would have a decline in the metabolic activity.
- Methods and Materials
Caffeine (C8H10N4O2)powder (from 6-sigma.co.ltd) was dissolved in E3 medium in a 100 mL beaker, followed by serial dilutions to prepare the solution
Ibuprofen (C13H18O2) was purchased from 6-sigma as a powder and dissolved in 1% of Dimethyl sulfoxide (DMSO) in a 100 mL beaker, followed by serial dilutions.
Resazurin (Alamar Blue™, ThermoFisher, USA) is a redox indicator which is widely used to evaluate metabolic activity in cells.
E3 media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 5 % Methylene Blue was used for zebrafish embryos culture. Methylene blue used to act as a mild fungicide.
3.2.1 Test organisms
Zebrafish (Danio rerio) was used to conduct the testing. Adult Zebrafish were kept in recirculating tanks on a 14:10 h light: dark cycle, and maintained at 27±0.5 °C, and pH at 7.0–7.5 at Monash University FishCore Aquatic Facility. Zebrafish embryos were acquired from the pair-wise mating and natural spawning of the male and female adult Zebrafish. Embryos were collected within the first hour of spawning and transferred on to Petri dishes containing zebrafish embryo culture medium (E3) for optimal growth. Any unfertilised embryos or debris were identified using a light microscope and removed from the petri dishes using a plastic pipette. (Figure1) The embryos were incubated at 28 ℃ (± 0.5 ℃) in the laboratory until hatched. 3 days-post-fertilization (dpf) larvae were used to perform the testing. The experiment was conducted under the RMIT University Animal Ethics Committee and Monash University Animal Ethics Committee.
Fig. 1. Scheme of the FET test procedure (from left to right): collection of the eggs, pre-exposure of the eggs immediately after fertilization in crystallization dishes, selection of fertilized eggs with an inverted microscope or binocular and distribution of the fertilized eggs into prepared 24-well microtiter plates. (Reproduced with permission from RightsLink®)
3.2.2 Sample preparation
To prepare the 48 well-plate, two control groups were set with E3 and E3 with 1% alamar blue. E3+1% AB was made by directly diluting E3 medium with 1% of the volume of Alamar blue. Caffeine value for LC10 of 200 mg/L and to find the range for ibuprofen, 40 mg/L was used to prepare the solution and perform serial dilutions. Solid caffeine powder was measured to 200 mg/L and dissolved in E3 medium to make the master solution. Stock solution in tubes was prepared by adding 1 mL of the master solution into tube 1 containing 29 mL of E3+ 1% Alamar blue along with caffeine to make up to 30mL. Serial dilutions were performed by eliminating 3mL of solution from tube 1 and adding it to the 27mL tube 2 with E3+ 1% AB. This was performed to all the tubes to make 5 folds of dilutions (Table 1).
Table 1. Range of concentrations (serial dilutions) for caffeine.
|Concentration||Caffeine 1||Caffeine 2||Caffeine 3||Caffeine 4||Caffeine 5|
Similarly, the master solution was prepared with weighing solid ibuprofen powder to 40 mg/L and dissolving in 800µL of DMSO. The stock solution was made with 500 mg/L of ibuprofen with 1% DMSO, from which 100 µL of ibuprofen was added into E3+1% AB. E3+1% AB was made by mixing 200mL of E3 with 2 mL of Alamar blue. 50 mL of the stock solution was added to Tube 1 as concentration 1. Serial dilutions were performed by taking 25 mL of solution from tube 1 and transferring into tube 2 with 25 mL of E3+1% AB to make up to 50 mL, and so forth (Table 2).
Table 2. Range of concentrations (serial dilutions) for caffeine.
|Concentration||Ibuprofen 1||Ibuprofen 2||Ibuprofen 3||Ibuprofen 4|
Briefly, 48 fertilised larvae at 3 dpf were transferred to a 48-well plate using 1ml plastic pipette, with one larva per well. The larvae were transferred by separating the larvae required onto a clean petri dish and removing any existing E3 medium from the petri dish with a plastic pipette. The required concentration was pipetted onto the petri dish and the larvae was transferred into the well using a plastic pipette. Each well contained 1mL of the specific toxicant concentration and for each concentration, there were 3 replicates in addition to 2 columns of control wells per plate. The plates were incubated at 28 ℃ (± 0.5 ℃) in the incubator.
3.2.3 Behaviour test and fluorescence test
Zebrabox (ViewPoint Life Sciences, Lyon, France) was used to monitor the photo-response behaviour of the larvae. The 64 minutes light-dark cycle test was performed to study behaviour patterns, where the photo period (light and dark) changed every 4 minutes. Movement of each larvae was monitored and recorded by the software. The total moving distance (mm) was used to quantify the photo – motor response of the larvae.
The fluorescence test to study the metabolism activity of the zebrafish embryo was conducted using the Clariostar plate reader (BMG LABTECH, Germany). The excitation wavelength was 530nm, and the emission wavelength was 590nm, and fluorescence intensity readings in each well were recorded. Both 24hr readings and 48 hr readings were taken. All data obtained was exported as excel format for analysis.
- Data analysis
The data collected was analysed separately for the behaviour test and the fluorescence test. A customised macro function was used to sort the behavioural data. The sorted data from each replicate was combined and averaged for every well containing different concentrations of toxicants. The moving trajectories of larvae in each well were automatically generated by the software. Sorted data were presented in graphs, i.e. Total distance travelled plotted against light and dark cycle, movement patterns in the light-dark period plotted as a line graph, etc (please refer to the results section for graphs). Similarly, the fluorescence results were averaged, relative change was found by dividing the absolute change with the E3+1% AB average. Outliers were also identified in the data points and the ANOVA test was performed for statistical analysis.
4.1 Ibuprofen exposure
4.1.1 Ibuprofen exposure impact on behaviour activity of zebrafish larvae.
Figure 2: A & B) Behavioural response of zebrafish larvae on the effects of different ibuprofen concentrations (31.25–250 mg/L) under light – dark period (8-minute cycle) for 64 minutes after 24 hours of exposure. b) Data are expressed as means of total distance travelled at light and dark period.
Figure 3: A & B) Behavioural response of zebrafish larvae on the effects of different ibuprofen concentrations (31.25–250 mg/L) under light – dark period (8-minute cycle) for 64 minutes after 48 hours of exposure. b) Data are expressed as means of total distance travelled at light and dark period.
Fig 2A represents the behaviour response curve of the highest concentration (250 mg/L) and the lowest concentration (31.25 mg/L) against the control at 24 hours after exposure of ibuprofen. In the control, the distance travelled by the zebrafish larvae is recorded the highest in the dark period. The larvae travel relatively high at the second interval of the dark period. The control curve gradually decreasing after the 4th light-dark cycle, with greater response time during the dark period. The highest concentration increases and has a greater response in the dark period. However, the lowest concentration shows a high photo-motor response at the 6th and 8th cycle for the light period with a higher photo-motor. (Fig 3A) Likewise, at low concentration levels, higher activity by the larvae is observed during the light period than at dark periods. At high concentration, the larvae display greater activity in the dark period, peaking at the 6th cycle.
Additionally, for ibuprofen at 24 hours displays a significant trend (figure 2B), where the blank and control show an almost similar value for light and dark average, with the distance travelled in dark period peaking at 1319.2 and 1210.0, respectively. Figure 3B, 48 hours after exposure, shows an identical trend for the blank and control, 2893.6 and 2900.1, respectively; with a high photo-motor response in the dark period. As the concentrations shift from low to high, the two lowest concentrations, 31.25 mg/L and 62.5 mg/L, where the light period average has a greater distance travelled compared to the dark period, which is similar to the results for 31.25 mg/L and 62.5 mg/L in Figure 3B, 2589.6 and 2522.3, respectively. In contrast both Figure 2B and 3B, at concentration levels of 125 – 250 mg/L, show an increment in the behavioural activity of zebrafish in the darker period, which translates to a higher distance travelled by the larvae in the dark period. This trend is particularly significant in figure 3B as the highest concentration has the most distance travelled.
Figure 4: Total distance (sum of all means) travelled at different concentrations of ibuprofen (31.25 – 250 mg/L) after 24 hour and 48 hours of exposure.
Figure 4 represents that the distance travelled at 48 hours after exposure is much greater than the distance travelled at 24 hours after exposure. The control at 24 hours was recorded as 2539.1 mm, whereas the total distance travelled by zebrafish larvae at 48 hours after exposure was 5219.9 mm. Similarly, this trend was seen across all the concentrations. The highest concentration of ibuprofen had a total distance travelled for 24 hours and 48 hours as 2508.8 mm and 5619.6 mm, respectively. With the lowest concentration of 31.25 mg/L, 24 hours distance moved was 2439.0 mm and 48 hours was recorded at 4645.8 mm.
4.1.2. Ibuprofen exposure effects on metabolic activity of zebrafish larvae.
Figure 5: Relative change in the metabolic activity (plotted means) of zebrafish larvae at 24 hours and 48 hours of exposure of different concentrations of ibuprofen (with ± standard error bars).
Table 3. P-values obtained using ANOVA testing for ibuprofen exposure
|p-values||31.25 mg/L||62.5 mg/L||125 mg/L||250 mg/L|
Figure 5 displays the negative relative change of the metabolic activity in zebrafish larvae. The metabolism rate at 24 and 48 hours does not have a major significance in the readings for low concentration (31.25 mg/L) and the highest concentration (250 mg/L). The readings at 24 hours after exposure are lower in each concentration compared to the activity in 48 hours. The p-values at 24 hours after exposure were recorded lower than 0.05 (table 3). This is also similar in lower concentration at 31.25 mg/L and the highest concentration at 250 mg/L, where the p-values were below 0.05, 0.002 and 0.005, respectively. This signifies that high concentration levels impact the metabolic activity during changes in behavioural patterns. When compared to the respective control for each period, every concentration had a greater value than the control, meaning the zebrafish larvae felt the impact of ibuprofen.
- Caffeine exposure
- Caffeine exposure impact on behaviour activity on zebrafish larvae.
Figure 6: A & B) Effects of a variety of caffeine concentrations (0.214 – 2136 µg/L) on behavioural activity of zebrafish larvae under light – dark period (8-minute cycle) for 64 minutes after 24 hours of exposure. b) Data are expressed as means of total distance travelled at light and dark period.
Figure 7: a & b) Effects of a variety of caffeine concentrations (0.214 – 2136 µg/L) on behavioural activity of zebrafish larvae under light – dark period (8-minute cycle) for 64 minutes after 48 hours of exposure. b) Data are expressed as means of total distance travelled at light and dark period.
Fig 6A highlights the photo-motor response trends by zebrafish larvae at 24 hours after exposure to caffeine, where the control, highest and lowest concentrations have a similar photo-moto response recorded. At the highest concentration of caffeine (2136 µg/L), the larvae display a high activity during the 2nd light-dark cycle, whereas the residual concentration levels remain constant thought out the 64 minutes. Likewise, the graph in figure 7A represents the high and low concentration levels after 48 hours exposure of caffeine. The distance travelled at the control and lowest concentration by the zebrafish larvae is recorded the highest in the dark period compared to the light period. The travel distance is maintained at the low concentration whereas the at the control, the curve slowly decreases. The highest concentration has the least travelled distance compared to the control and low concentration (Fig 7A) and is significantly lower than the readings obtained at 24 hours for the highest concentration. (fig 6a). At the beginning of the 2nd light-dark cycle, activity during the light period was observed to be than at dark periods, this can be seen reducing overtime.
Figure 8: Average of total distance travelled at different concentrations of caffeine (0.214 – 2136 µg/L) after 24 hour and 48 hours of exposure.
Figure 8 signifies the distance travelled by the larvae under caffeine influence is much higher at 24-hour post-exposure compared to the distance travelled at 48 hours. The control had a high reading for total distance travelled at 24 hours at 9634.4 mm, whereas the distance moved at 48 hours after exposure was recorded at 6164.6 mm. This trend was present across all the concentrations. The most significant distance recorded was at the highest dose (2136 µg/L) of 11073.4 mm for 24 hours and 2137.5 mm at 48 hours. Such a significant trend was also noticed for concertation levels of 2.14 µg/L (12559.9 mm and 6858.8 mm), 21.36 µg/L (8971.9 mm and 6469.1 mm) and 213.6 µg/L (9974.9 mm and 2137.5 mm) for 24 hours and 48 hours, respectively.
4.2.2. Caffeine exposure effect on the metabolic activity of zebrafish larvae
Figure 9: Relative change in the metabolic activity (plotted means) of zebrafish larvae at 24 hours and 48 hours of exposure for different concentrations of caffeine (with ± standard error bars).
Table 4. P-values obtained using ANOVA testing for caffeine exposure
|p-values||0.214 µg/L||2.136 µg/L||21.36 µg/L||213.6 µg/L||2136 µg/L|
Figure 9 represents the relative change in metabolism of zebrafish larvae. The metabolic activity recorded at 24 hours was higher at all concentration levels when compared to the respective control. The most significant increase was observed at 213.6 µg/L with a p – value of 0.00002, followed by the highest concentration of 2136 µg/L with p – value of 0.000121, with both p-values being lower than 0.05 (table 4). Whereas, the activity at 48 hours for these concentrations was lower compared to the concentrations at 24 hours. Concentration 21.36 µg/L and 213.6 µg/L were found to have p values under 0.05, 0.04 and 0.0000003, respectively. This confirms the impacts of high concentration on the metabolic activity of the zebrafish larvae. The activity of behaviour and the corresponding metabolic rate drop significantly at the highest concentration, demonstrating a direct connection amongst both activities.
The aim of this research was to explore the effects of neurotoxins and anti-depressants on the behavioural and metabolic activity of zebrafish larvae to establish a correlation between behaviour change and metabolism. The hypothesis of our study was to confirm the direct link between the changes in behaviour and the metabolic activity of the zebrafish larvae when exposed to the pharmaceutical drugs. It was anticipated that exposure to caffeine will increase the metabolic activity and ibuprofen would decrease the activity.
The results demonstrate a high activity of zebrafish larvae for caffeine under dark conditions for both 24 and 48 hours after exposure (fig 6 & 7). Fig 6A, shows that high dose of caffeine increased the behaviour patterns whereas at 48 hours (fig 7A), the same dose had an adverse effect and the behaviour activity had slowed down. At 24 hours, the larvae felt the impact of caffeine on their body and begin to act excited (fig 6A, 9 & table 3). As ANOVA test was used, the p-values obtained confirmed that the concentration levels had an impact on the metabolism rate of the larvae when there was a change in the behaviour patter. The correlation between behaviour and metabolism for doses 213.6 µg/L and 2136 µg/L of caffeine at 24 hours post-exposure, had p-values under 0.05. At 48 hours after exposure, the p-values for 213.6 µg/L and 21.36 µg/L are under 0.05 and experience more fluorescence, which translates to an increase in the metabolism. Lower concentrations remain unchanged. The metabolic rate of change and the behavioural changes are directly dependent on each other and results implies that caffeine increases the metabolic activity, during the early hours of consumption and decrease activity and attentivity overtime (fig.7A & 9).
Prior studies have confirmed similar outcomes, where a high dose of caffeine decays the photo-motor response in the zebrafish larvae. The study by Luana C. santos et al , shows a decrease in the travel rate of larvae at higher dose (50 mg/L), which was 2136 µg/L in our study. A lower dose of 25 mg/L was found to increase the activity in the larvae, which was similar to the results obtained in our study at 24 and 48 hours at concentration 0.214 µg/L. An analysis conducted by Luiz Vinícius Rosa et al.  on two fish populations for caffeine concentrations ranging from 50 and 100mg/L, showed no distinct changes to the locomotor activity in the wild-type population of zebrafish larvae, under caffeine influence, whereas the same 200mg/L dose of caffeine caused a noticeable decline in the distance travelled in the leo population. The time of exposure and the amount of dose play a significant role in caffeine impacting the central nervous system, directly.
Additionally, the range finding test for ibuprofen was able to provide with an insight on the amount of dose required for the drug to act as a toxicant. As 250 mg/L of ibuprofen was the highest concentration for the experiment, more locomotion was observed during the 48 hours after exposure. Total distance travelled at 24 hours after exposure was lower than 48 hours (fig 2A &3A). Fig. 5 reflects the changes in the metabolic activity to the distance travelled by the larvae, which confirms the impact of behaviour change through the statistical significance. The p-values for each does at 24 hours of exposure was recorded under 0.05, confirming an increase in metabolism. It is suggested that the increase in behavioural activity, decreases the metabolism rate in the larvae, therefore decreasing the overall activity in the zebrafish larvae.
The behaviour activity did not decay as much as anticipated, which may due to the high dose of ibuprofen acting as a toxicant. Previously, when tested under the light-dark cycle, ibuprofen exposure had a significant decline in the swim activity of the larvae in the first 10 minutes of exposure to dark conditions. Overtime, the response during dark conditions was very slow, which suggested that the effect of ibuprofen under dark condition needed a longer time to take place . David and Pancharatna , also found ibuprofen to cause delays in the hatching of zebrafish embryos. The adverse effects observed in this study were suggested to be influenced by the potential neurotoxicity of ibuprofen that controlled the overall motion decrease, leading to a significant delay in hatching of the embryos. These studies confirm the potential risk of ibuprofen that can be caused to the aquatic environment, if used in high dose concentrations.
The results obtained in this research project have provided the field of science another topic for exploration. Understanding the impacts of certain chemicals that are released in the environment is necessary in order to maintain a healthy and balanced aquatic environment. Caffeine is the most popular drug that is consumed on a daily basis and ibuprofen is also a very widely used medication. Although, not many studies had explored the lethal endpoints for ibuprofen, by conducting the range determining experiment, this study has contributed to provide a starting point for further research. The exploration on caffeine has also assisted the field of science for further research, to understand the long-term impacts of caffeine on organisms. Organisms are vulnerable and are indirectly harmed in numerous ways through the disposal of toxins. It not only impacts the well-being of the aquatic animals, but also humans. Therefore, developing safety measures to limit disposal of toxicants in waterways will ultimately avoid ecological death of organisms.
This project was carried out under the supervision of PhD students, Yutao Bai and Milanga Walpitagama in Prof. Donald Wlokowic’s laboratory at RMIT university Bundoora. I would like to appreciate their effort and for their assistance throughout the completion of this report.
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