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Coumarin 47 and permethrin effects on zebrafish embryos:
FET tests and behavioural challenges
Stefania Rabasco
Independent project Örebro University 2018 Supervisors: Mélanie Blanc, Steffen Keiter
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1. INTRODUCTION ... 3
1.1 General Background ... 3
1.2 Coumarin 47 ... 4
1.3 Permethrin ... 5
1.4 The Zebrafish Model ... 7
1.5 Fish embryo Acute Toxicity (FET) Test ... 8
1.6 Locomotor Activity ... 8
2. MATERIALS AND METHODS ... 9
2.1 Well-plate pre-exposure ... 9
2.2 Adult fish Maintenance ... 10
2.3 FET Tests ... 10
2.4 Measurements of larva photomotor response ... 12
2.5 Statistics ... 13
3. RESULTS AND DISCUSSION ... 13
3.1 FET ... 13 3.1.1 Coumarin 47 ... 13 3.1.2 Permethrin ... 15 3.2 Behavioural Challenges ... 15 3.2.1 Coumarin 47 ... 15 3.2.2 Permethrin ... 16 4. CONCLUSION ... 19 ACKNOWLEDGMENTS ... 19 REFERENCES ... 20 SUPPLEMENTARY DATA ... 23
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1. Introduction
1.1 General Background
An environment is defined as polluted when contaminants are introduced within it and cause adverse effects to its ecosystem. Chemical pollutants can migrate and often affect several environmental compartments: for instance, chemicals can leach from polluted soil into groundwater, or they can evaporate and disperse into the atmosphere.
The recent advent of industrialisation and urbanisation has brought on the daily discharge of harmful chemicals, such as heavy metals, organic pollutants and radioactive materials, into the environment via a high number of contamination sources, both commercial and industrial. More and more chemicals are identified as harmful to humans and the environment every year; in regard to the seemingly unsurmountable number of new ones being constantly introduced to the market, it is important that authorities keep improving the efficiency of regulatory procedures and safety testing. This includes supporting scientific research which is fundamental for improving our understanding of the mechanisms of action of these chemicals in order to handle them properly.
In the present study, FET (Fish Embryo acute Toxicity) tests and behavioural challenges were performed on zebrafish (Danio rerio) embryos and larvae to investigate coumarin 47 and permethrin in terms of toxicity and effect on larval photomotor response. A change in locomotor activity in response to a light-to-dark transition stimulus is often the ultimate cause of exposure to specific neurotoxic agents, making it a useful endpoint in assessing central nervous system toxicity, while FET tests are a guideline tool employed in the determination of developmental toxicity and teratogenicity.
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Overall, the results obtained provided a significant understanding on the toxicity endpoints of the selected test compounds to be employed as references for future investigations.
1.2 Coumarin 47
Coumarins (1-benzopyran-2-one) are organic compounds of the benzopyrone class which are naturally found in many plants, such as tonka bean, woodruff and bison grass (Poumale, Hamm et al. 2013). Coumarin congeners have different biological and chemical activities. Coumarin 47 (C14H17NO2, IUPAC name 7-(diethylamino)-4-methyl-2H-chromen-2-one; CAS No 91-44-1) is used as a dye in laser systems (Axner and Magnusson 1985), as well as a fluorescent whitening agent and as such it can be added during the manufacturing process of fabrics and papers to improve colour temperature (Park, Lee et al. 2007, Jung, Seok et al. 2012).
Coumarin 47 is produced and/or imported in the European Economic Area in large quantities (up to 10 tonnes per year). Its production is
achieved by the reaction of Figure 1. Reaction scheme for the production of coumarin 47
3-diethylaminophenol with ethyl acetoacetate, as shown in Figure 1) (PubChem Identifier, CID 7050). Limited information is found on its physicochemical properties; some of are presented in Table 1.
Table 1. Some physicochemical properties of coumarin 47 (does not
include predicted values) (PubChem Identifier, CID 7050)
Endpoint Description
Appearance Dry powder
Colour Light tan
Melting point 89 °C
Solubility in water Slightly soluble
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The European Chemical Agency (ECHA) lists coumarin 47 as toxic to aquatic life with long lasting effects (H400/410) and as causing eye irritation (H319), skin irritation (H315), respiratory irritation (H335), acute dermal toxicity (H312), and to be harmful if inhaled (H332), yet scientific literature on this compound is extremely limited and not much research, especially in vivo studies, has been conducted to investigate its ecotoxicological properties. No reliable data on the environmental fate of coumarin 47 can be accessed nor is there any OECD-complying study on its toxicity on human or the environment. Most of the sections of its safety data sheet lack data, and it is thus not possible to fully characterise this compound.
1.3 Permethrin
Permethrin (C21H20Cl2O3, IUPAC name (3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate; CAS No 52645-53-1) is a synthetic pyrethroid (insecticide) mainly present as an active substance in creams and lotions which are sold commercially for topical administration in the treatment of lice, mites, ticks and arachnids. Permethrin is also used in the treatment of textile fibres (especially carpets and rugs) to control keratin-feeding textile pests such as moths and beetles (Biocidal Products Committee 2014). Environmental exposure scenarios for permethrin include emission to wastewater via washing of clothes, treated carpets, rugs and wet cleaning of treated surfaces; human exposure is limited by advertisement of proper handling procedures for commercially available products containing permethrin (Biocidal Products Committee 2014).
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The principal technique for the industrial production of permethrin is condensation of 3-phenoxybenzyl alcohol (C13H12O2) with (1RS)-cis/trans-3-(2,2 - dichlorovinyl) - 2,2 - dimethylcyclo propane carboxylic acid (C8H10Cl2O2). This is illustrated in Figure 2. Chemical and physical properties are listed in Table 2 (PubChem Identifier, CID 40326).
Figure 2. Reaction scheme for the production of
permethrin
Table 2. Some physicochemical properties of permethrin (PubChem
Identifier, CID 40326).
Endpoint Description
Appearance Liquid or crystals
Colour Pale yellow
Boiling point 220 °C at 5.00E-02 mm Hg
Melting point 34 °C
Solubility in water Soluble
Solubility in organic solvents Soluble in most organic solvents except ethylene glycol
Density 1.19 - 1.27 at 20 °C
Vapour pressure 5.18x10-8 mm Hg at 25 °C
Log Kow 6.50
Koc 10,471 to 86,000 (estimated)
Henry’s law constant 2.4x10-6 atm-cu m/mole (estimated)
The Global Harmonised System (GHS) categorises permethrin as harmful if swallowed (H302) or inhaled (H332), a potential source of allergic skin reaction (H317) and very toxic to aquatic life with long lasting effects (H400/410). At present, ECHA has classified permethrin as not having endocrine disrupting or Carcinogenic, Mutagenic, Reprotoxic (CMR) properties. Thus, it does not meet the criteria to be laid down in Article 5 nor 10 of Regulation (EU) No 528/2012, and as such it is not presently considered as a candidate for substitution (i.e. manufacturers are not obliged to substitute with similar alternative products). It is nevertheless considered by ECHA as a potential environmentally persistent compound and its human toxicity has not been classified yet, so it is in need of further assessment (Biocidal Products Committee, 2014).
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Synthetic neurotoxins, including insecticides, can affect the central nervous system of insects causing a range of adverse effects such as hyperactivity, tremors and eventually death (WHO, 1990, Alzogaray, Fontán et al. 1997). Permethrin blocks the movement of sodium ions across the neuronal cell membrane which leads to neural system paralysis. As other insecticides, exposure to permethrin can also be detrimental to non-target species, such as fish and mammals (Rice, Drewes et al. 1997, Farag, Goda et al. 2006). The onset and degree of hyperactivity reported are wildly dependent on animal species, permethrin cis/trans isomeric ratio, exposure pathway and other variable experimental settings.
1.4 The Zebrafish Model
Zebrafish (Danio rerio) has been used for many years as one of the main vertebrate model system alternative to mammal models in a variety of scientific disciplines, including toxicology, medicine and pharmacology (Blechinger, Warren Jr et al. 2002, Topczewska, Postovit et al. 2006, Bencan, Sledge et al. 2009).
The advantages of using zebrafish are numerous: the optical clarity of zebrafish embryos and their ex utero development allows for cell tracing and real-time, high-resolution imaging, while their small size, short reproductive cycle and frequent spawning make them ideal for the laboratory research environment (Parng, Roy et al. 2007, Sullivan and Kim 2008, Fent, Weisbrod et al. 2010).
After being identified in the 1980s as a genetically tractable organism, zebrafish entered the Wellcome Sanger Institute genome sequencing project in 2001; since then, its assembly has been completed and is periodically reviewed by the Genome Reference Consortium (GRC) (Howe, Clark et al. 2013). The latest update of the sequence (GRCz11) was released in 2017, and the close relationships that the zebrafish genome sequence shares with the human genome
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keeps being successfully exploited for the study of human genetic disease (Golzio, Willer et al. 2012, Carter, Cortés-Campos et al. 2017).
1.5 Fish embryo Acute Toxicity (FET) Test
The FET test was first drafted in 2006 and subsequently adopted in 2013 as introduced in the OECD guideline 236 (OECD 2006, OECD 2013). It is based on the testing of chemicals on zebrafish embryos up to 96 hours post fertilization (hpf), a time when they still do not need to feed actively. This is of particular importance as the European Directive 2010/63/EU on the protection of animals used for scientific purposes does not categorise non-feeding developmental stages as protected, this test has effectively been used as an alternative to animal testing (Braunbeck, Kais et al. 2015).
The OECD guideline 236 describes the required test criteria when testing chemicals using FET tests. These criteria include: tests must be run until 96 hpf; overall mortality of negative and solvent control specimens must not exceed 10%; overall mortality of positive control specimens must be above 30%; water temperature and oxygen concentrations must be kept within the accepted limits (i.e. 26 ± 1 °C and >80% saturation, respectively) (OECD 2013).
1.6 Locomotor Activity
Behavioural challenges have long been used for the study of drug neurotoxicity on animals (Berlin, Grant et al. 1975, Eriksson and Fredriksson 1996, Wallace, Gudelsky et al. 1999). Traditionally, these tests employed human observation and manual scoring of results. It is only in the past few years that automated tracking methods have made it possible to efficiently and consistently track animal behaviour without the bias of human error; these methods also allow for multiple variables (e.g. distance moved, velocity, localization) to be tracked simultaneously and for long periods of time (Noldus, Spink et al. 2001).
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Although mice and rats are still nowadays the most commonly employed model for behavioural challenges, zebrafish is gaining popularity as a useful tool to complement studies on rodents, as a bridge between in vitro and mammalian models (Ahmad, Noldus et al. 2012).
Several behavioural studies have demonstrated how light conditions have an influence over zebrafish embryo development and larva locomotor behaviour; this influence may appear in the form of changes in swimming behaviour. Light/dark preference tests have previously been conducted on both juvenile and adult specimen (Champagne, Hoefnagels et al. 2010, Steenbergen, Richardson et al. 2011) and the findings have been that zebrafish strongly prefer light environments and display anxious behaviour when exposed to dark environments. This can be explained by the fact that zebrafish is a diurnal species and is thought to perceive sudden darkness as the threat of an incoming predator which would shield sunlight while swimming or flying above its preys (Dill 1974, Csányi 1986, Hall and Suboski 1995, Hall and Suboski 1995, Gerlai, Lahav et al. 2000).
Within toxicological science, this characteristic is used to monitor how exposure to chemicals can affect the photomotor activity of zebrafish larvae in response to a transition between light and dark periods. This aims at bringing evidence on the neurobehavioral toxicity of a compound (Porsolt et al. 2002).
2. Materials and Methods
2.1 Well-plate pre-exposure
96-well test plates were pre-exposed with exposure solutions, negative control or solvent controls 24 hours prior to the start of each experiment. This was done to saturate the well walls to limit sorption during exposure which would reduce chemical availability.
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All solutions were prepared in ISO water, i.e. 99% millipore water and 1% of each salt: CaCl2·2H2O (24.4 g/L), MgSO4·7H2O (12.3 g/L), NaHCO3 (6.3 g/L) and KCl (0.55 g/L) according to the guideline ISO 7346-1. The ISO water was kept oxygenated using an air pump, and temperature maintained at 27 °C.
Five nominal concentrations were selected for testing of each of the two compounds (Table 3). Serial dilutions were performed to reach the desired concentrations; solvent content (Dimethylsulfoxide (DMSO)) was adjusted throughout the process in order to be kept consistent after the dilutions, i.e. 0.01% for coumarin 47 and 0.015% for permethrin. All reagents and chemicals were purchased from Sigma Aldrich (St Louis, MO).
Table 3. Nominal FET test concentrations of coumarin 47 and permethrin Coumarin 47 (mg/L) Permethrin (µg/L) Concentration 1 (C1) 5 600 Concentration 2 (C2) 2.5 300 Concentration 3 (C3) 1.25 150 Concentration 4 (C4) 0.625 75 Concentration 5 (C5) 0.3125 37.5
2.2 Adult fish Maintenance
Adult zebrafish were kept in tanks on a 14 h light:10 h dark cycle (light on at 8:00 am), fed twice a day with Tetra flakes (TetraRubinÒ, Tetra) or artemias (Ocean NutritionÒ) and their environmental conditions (water temperature, pH, conductivity) were kept consistent; overall maintenance followed standard protocols (Westerfield, C. 2007).
2.3 FET Tests
The evening before spawning, adult zebrafish (Figure 3a) were randomly selected to spawn in groups (ratio 3 males:2 females) and placed into spawning tanks (Figure 3b). Several hundred eggs were then collected in the morning for testing. After collection, eggs were promptly placed
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in the test beakers to allow for immediate exposure (i.e. within 2 hpf). They were subsequently transferred in 250 µl of exposure solution into test plates, one egg per well. The healthiest eggs, i.e. those that were fertilised, intact and most suitable for testing, were chosen via visual observation; coagulated embryos were easily spotted (and discarded) as they distinctly appeared, to the naked eye, white instead of transparent as can be observed in Figure 3c.
Figure 3a. Zebrafish room and
aquarium
Figure 3b. Fish spawning tank
filled with water
Figure 3c. A coagulated fish
egg appears white at naked eye
Two plates were prepared for each compound and 12 wells per plate were assigned for each concentration and control solution. A graphic representation of the plate is shown in Figure 4. Randomization was optimized for the behavioural challenge tests. During incubation, all plates were covered with a plastic film (VWR International, United States) to avoid cross-contamination between wells and limit evaporation.
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With the start of exposure, the FET test was initiated and the embryos started developing under the influence of the test compounds. Plates were kept incubated at 27 °C on the usual 14 h light:10 h dark cycle.
Endpoints for embryo and larva development were checked under microscope at 24, 48, 72 and 96 hpf. Table I in Supplementary Data lists all significant parameters and their time relevance. In order to keep concentrations constant and avoid loss of chemicals due to evaporation, degradation or sorption, 50% of the medium from each well was renewed gently every 24 hours during the course of each FET test.
2.4 Measurements of larva photomotor response
Locomotor activity was measured at the end of the FET test, i.e. 96 hpf, and followed a predefined test method, as described below.
The photomotor response of the larvae was monitored using the DanioVision™ observation system (Noldus, Wageningen, Netherlands) and accompanying video tracking software package EthoVisionÒ XT. The DanioVision™ comprised a chamber to hold well plates while recording, and a temperature control unit to circulate water at 27 °C around the plate to ensure constant temperature conditions.
A 20-minute light-dark challenge template was created (Figure 5), which comprised the following: 10 minutes with the light switched on (Light ON 1), of which the first 5 minutes (non-analysed) were to acclimate the larvae to the chamber and 5 minutes were for data recording; 5 minutes of darkness (Light OFF); 5 additional minutes with the light switched on (Light ON 2).
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Figure 5. Light/dark challenge template with light periods
Positive control larvae were not included in the data recordings, as well as dead or malformed (edema, scoliosis) larvae; this was done so that the statistical analysis would take into consideration only the individuals showing no physical toxic effect that could affect their behaviour.
2.5 Statistics
All analyses were performed with GraphPad Prism® 7 software. Behavioural data were analysed with two-way ANOVA (light and exposure) when data distribution followed normality; otherwise the non-parametric Kruskall-Wallis test was applied at each light condition separately. A non-linear regression analysis with variable slope was conducted on FET data to calculate LC50 and LC10 values.
3. Results and Discussion
3.1 FET
3.1.1 Coumarin 47
Overall, three out of five independent FET tests were valid according to OECD 236 and were considered for statistical analysis. The effect that was observed was coagulation of embryos at 24 hpf. Figure 6 illustrates overall mortality at 96 hpf and 24 hpf; it is noticeable that percentage of dead embryos does not differ significantly between the two time points. The average mortality at 24 hpf was 93% in C1 embryos. Exposure longer than 24 h to lower concentrations (C3-5) did not cause higher mortality rates.
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Figure 6. Mortality percentage vs log concentration mg/L, coumarin 47, 24 vs
96 hpf
The LC50 and LC10 were calculated at each time point; these are presented in Table 4; the LC50 at 96 hpf was determined to be 1.3 mg/L.
Table 4. LC50 and LC10 of coumarin 47 at each FET test
time point
24 hpf 48 hpf 72 hpf 96 hpf
LC50 (mg/L) 1.4 1.3 1.3 1.3
LC10 (mg/L) 0.7 0.8 0.8 0.7
Little is known about the toxicity of coumarins. Few studies can be found on coumarin 47 congeners, such as warfarin, 2H-Chromen-2-one and dicoumarol (Wardrop and Keeling 2008, Pinho, Santos et al. 2013, Weigt, Huebler et al. Reproductive Toxicology). A single study by Jung et al (2011) looked at the effect of coumarin 47 on zebrafish embryos until 72 hpf, and their reported LC50 was 0.9 mg/L (Jung, Seok et al. 2012), which the present study corroborates. In 2017, coumarin 47 was detected in the water of the German river Holtemme at a concentration of 13.7 µg/L (0.0137 mg/L) by Muschket et al (Muschket, Di Paolo et al. 2017). Its antiandrogenic activity was also studied and confirmed in the laboratory on Japanese killfish (medaka) at the same concentration which occurred in the river; this suggested high environmental risks for the aquatic organisms of Holtemme and similarly contaminated water
-0.5 0.0 0.5 1.0 1.5 -50 0 50 100 150 Log concentration mg/l Mortality % Coumarin 47, mortality 24 vs 96 hpf 96 h PC NC SC 24 h
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bodies, as androgen receptor antagonists interfere with hormone signalling and can cause physiological adverse outcomes (such as endocrine disruption, i.e. impair the fertility and reproduction abilities of an organism) (Singh, Gauthier et al. 2000). In addition, according to the present study coumarin 47 causes acute toxicity in zebrafish at levels that are 100 times higher than what was reported in the river Holtemme. Altogether, this points toward the need for additional investigations both on its environmental occurrence and on the associated ecotoxicological risks.
3.1.2 Permethrin
Overall, 8 FET tests for permethrin were performed, and all of them resulted valid according to OECD 236. None of the official FET endpoints were reported. The only effect observed was hyperactivity in 96 hpf larvae exposed to C1 and C2. Here, hyperactivity as an endpoint is defined as quick random movements, pointless swimming, and violent and frequent spasms. No embryos or larvae in the lowest permethrin concentrations (C3, C4, C5) showed any visible signs of hyperactivity at any time point, and mostly appeared healthy. An LC50 could not be calculated, and higher concentrations might have been needed for mortality to be a significant effect.
3.2 Behavioural Challenges
3.2.1 Coumarin 47
The average distance moved by the larvae was calculated for each exposure condition, at each period of light separately. As illustrated by Figure 7, all larvae followed the expected pattern of moving more during the dark period, and less during the light periods. In addition, no difference was reported between exposed and control larvae; therefore, this indicates that no apparent neurotoxicity was caused by exposure to coumarin 47.
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Figure 7. Light period/exposure concentration vs distance
moved for coumarin 47
3.2.2 Permethrin
Permethrin-exposed larvae exhibited revealing behaviour patterns. The swimming activity in the light periods was different upon exposure concentrations, and so was the swimming activity in the dark period. Table II in Supplementary data shows the average of the distance travelled (mm) by larvae for every concentration and light period. Results were analysed and interpreted as follows: basal activity level was extracted from the distance travelled in Light ON periods while anxiety level was reflected in the distance travelled in the Light OFF period.
Basal activity level (Light ON)
In both Light ON 1 and Light ON 2 (Figure 7a and 7b), a clear dose-dependent hyperactivity effect over the course of the five concentrations was reported. A significant increase between SC and C1 (67% in Light ON 1, 62% in Light ON 2) and between SC and C2 (27% in Light
Ligh t ON (1) C 3 Ligh t OFF C3 Ligh t ON (2) C 3 Ligh t ON (1) C 4 Ligh t OFF C4 Ligh t ON (2) C 4 Ligh t ON (1) C 5 Ligh t OFF C5 Ligh t ON (2) C 5 Ligh t ON (1) SC Ligh t OFF SC Ligh t ON (2) SC 0 50 100 150 200
Coumarin 47, distance moved vs light period C3, C4, C5 and SC
Light Period
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ON 1, 18% in Light ON 2) was observed. The increase in activity from SC to C3, C4 and C5 was lower and did not result to be statistically significant.
Figure 7a. Light ON 1 permethrin C1-5 and SC Figure 7b. Light ON 2 permethrin C1-5 and SC
Anxiety level (Light OFF)
In Light OFF (Figure 8), C2, C3 and C4 larvae exhibited a significant increase in activity (15, 8 and 22%, respectively). In contrast, no difference in the activity level was reported in C1 and C5 larvae compared to SC. While C5 does not seem to affect embryos at all, the absence of increase in activity of C1 larvae is likely due to the reported effects on the basal activity level (Figure 9) as described further on.
Interestingly, the highest increase in activity was observed in C4 larvae. Overall, this suggests that exposure to permethrin induces higher anxiety levels at lower concentrations, while higher concentrations primarily affect locomotor activity which either suppresses or masks the anxiety response.
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Figure 8. Light OFF permethrin C1-5 and SC
Loss of the light-to-dark response in C1 larvae
Figure 9 illustrates the light-to-dark response of larvae exposed to C1, C2 and SC. While C2 to C5 larvae do not seem to be affected and follow a predictable light-to-dark response, C1 larvae manifest no specific increase in the swimming activity during the Light OFF period compared to Light ON periods.
Figure 9. Light period/exposure concentration vs
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Acute exposure to permethrin can induce tremors, involuntary muscles spams and as such can result in a lack of specific anxiety-like response. Such tremors have been observed during the FET test. In addition, C1 larvae show strong basal hyperactivity, with a level in Light ON as nearly as high as the level of SC larvae in Light OFF. Therefore, physical and energetic limitations may be an additional explanation to the lack of increase in response to a dark stimulus.
Overall, the behavioural data on permethrin exposed fish underline that the specificity of neurotoxic effects of this compound is highly dependent on the concentration. While hyperactivity was expected, anxiety-like behaviour observed at lower concentrations is a specific effect that is distinct from its primary mechanism of action.
4. Conclusion
The results obtained during this project demonstrate that coumarin 47 and permethrin induce toxic responses in zebrafish embryos and larvae. Coumarin 47 generates early embryo coagulation and death in 24 hpf-old embryos. Permethrin is neurotoxic and stimulates hyperactivity at higher concentrations and anxiety at lower concentrations in 96 hpf-old larvae. Finally, it can be concluded that further studies on the effects of these compounds, such as lower concentration ranges for coumarin 47 and long-term exposure tests for permethrin, need to be performed as to allow them to be adequately assessed.
Acknowledgments
The author wishes to acknowledge Mélanie Blanc and Greta Nilén for their continuous guidance and devoted contribution; thanks to Steffen Keiter and the MTM department at the
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University of Örebro for allowing the realisation of this project. Additional thanks to Stan for his support in our shared time and workplace.
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Supplementary Data
Table I. Endpoints for assessing embryo toxicity. If any of the first four endpoints are met,
an embryo is defined as dead according to the OECD guideline 236.
Endpoint 24 hpf 48 hpf 72 hpf 96 hpf
Coagulated embryo x x x x
Lack of somite formation x x x x
Non-detachment of the tail x x x x
Lack of heartbeat x x x
Underdevelopment of the eyes x x x x
Lack of blood circulation x x x
Unfertilised egg x x x x
Abnormal heartbeat /blood circulation x x x
Hatched too early x x x
Epiboli stage x x x x
Edema (pericard/yolk) x x x
Lack of pigmentation x x x
Deformation of the embryo x x x
Deformation of the tail x x x
Underdeveloped embryo x x x x
Table II. Means of distance moved (mm) versus light period for
C1-5 and SC, permethrin behaviour challenges
C1 C2 C3 C4 C5 SC
Light ON 1 130.9 99.6 88.9 83.8 83.5 78.5
Light OFF 133.7 152.0 143.0 161.7 135.8 132.9
