Behavioural and transcriptional changes in the amphipod Echinogammarus marinus exposed to two antidepressants, fluoxetine and sertraline

Accepted Manuscript

Title: Behavioural and transcriptional changes in theamphipod Echinogammarus marinus exposed to twoantidepressants, Fluoxetine and Sertraline

Author: Maryline C. Bossus Yasmin Z. Guler Stephen J.Short Edward R. Morrison Alex T. Ford

PII: S0166-445X(13)00343-3DOI: http://dx.doi.org/doi:10.1016/j.aquatox.2013.11.025Reference: AQTOX 3697

To appear in: Aquatic Toxicology

Received date: 30-8-2013Revised date: 4-11-2013Accepted date: 27-11-2013

Please cite this article as: Bossus, M.C., Guler, Y.Z., Short, S.J., Morrison, E.R.,Ford, A.T.,Behavioural and transcriptional changes in the amphipod Echinogammarusmarinus exposed to two antidepressants, Fluoxetine and Sertraline, Aquatic Toxicology(2013), http://dx.doi.org/10.1016/j.aquatox.2013.11.025

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Behavioural and transcriptional changes in the amphipod 1

Echinogammarus marinus exposed to two antidepressants, 2

Fluoxetine and Sertraline 3

Maryline C. Bossus1, Yasmin Z. Guler1, Stephen J. Short1, Edward R. Morrison², Alex T. 4 Ford1* 5

1Institute of Marine Sciences, School of Biological Sciences, University of Portsmouth, Ferry 6 Road, Portsmouth, Hampshire, UK, PO4 9LY 7

²Higher Education Academy Psychology, Department of Psychology, University of 8 Portsmouth, Hampshire, UK, PO1 2DY 9

*Corresponding author: Email address: [email protected] 10 Tel.: +44-2392-845805 Fax: +44-2392-845800 11

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Abstract 14

In the past decade, there have been increasing concerns over the effects of pharmaceutical 15

compounds in the aquatic environment, however very little is known about the effects of 16

antidepressants such as the Selective Serotonin Re-uptake Inhibitors (SSRIs). Many 17

biological functions within invertebrates are under the control of serotonin, such as 18

reproduction, metabolism, moulting and behaviour. The effects of serotonin and fluoxetine 19

have recently been shown to alter the behaviour of the marine amphipod, Echinogammarus 20

marinus (Leach, 1815). The purpose of this study was to observe behavioural and 21

transcriptional modifications in this crustacean exposed to the two most prescribed SSRIs 22

(fluoxetine and sertraline) and to develop biomarkers of neurological endocrine disruption. 23

The animals were exposed to both drugs at environmentally relevant concentrations from 24

0.001 to 1 μg/L during short-term (1 hour and 1 day) and medium-term (8 days) experiments. 25

The movement of the amphipods was tracked using the behavioural analysis software during 26

12 min alternating dark/light conditions. The behavioural analysis revealed a significant 27

effect on velocity which was observed after 1 hour exposure to sertraline at 0.01 μg/L and 28

after 1 day exposure to fluoxetine as low as 0.001 μg/L. The most predominant effect of 29

drugs on velocity was recorded after 1 day exposure for the 0.1 and 0.01 μg/L concentrations 30

of fluoxetine and sertraline, respectively. Subsequently, the expression (in this article gene 31

expression is taken to represent only transcription, although it is acknowledged that gene 32

expression can also be regulated at translation, mRNA and protein stability levels etc.) of 33

several E. marinus neurological genes, potentially involved in the serotonin metabolic 34

pathway or behaviour regulation, were analysed in animals exposed to various SSRIs 35

concentrations using RT-qPCR. The expression of a tryptophan hydroxylase (Ph), a neurocan 36

core protein (Neuc), a Rhodopsin (Rhod1) and an Arrestin (Arr) were measured following 37

exposure to fluoxetine or sertraline for 8 days. The levels of Neuc, Rhod1 and Arr were 38

significantly down-regulated to approximately 0.5, 0.29 and 0.46 fold respectively for the 39

lower concentrations of fluoxetine suggesting potential changes in the phototransduction 40

pathway. The expression of Rhod1 tended to be up-regulated for the lower concentration of 41

sertraline but not significantly. In summary, fluoxetine and sertraline have a significant 42

impact on the behaviour and neurophysiology of this amphipod at environmentally relevant 43

concentrations with effects observed after relatively short periods of time. 44

Keywords: Antidepressants, SSRIs, Neuro-endocrine disruptor, Behaviour, Biomarker, 45

Crustacean 46

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1. Introduction 48

The issue of anthropogenic contaminants released in the aquatic environment acting as 49

endocrine disruptors has been well studied, but the research effort has mainly consisted of the 50

study of estrogenic substances and their effects on vertebrates (Hutchinson 2007; Weltje and 51

Schulte-Oehlmann 2007). The increasing use (over 60% in just the past decade) of 52

antidepressants, the improper disposal of unused pharmaceuticals, and their limited 53

biodegradability has raised concerns about their potential effects in the aquatic environment 54

(Demeestere et al. 2010; Santos et al. 2010). Antidepressants represent about 4% of the 55

therapeutic drugs found in the environment, and are present in coastal waters and estuaries 56

(Santos et al. 2010). Indeed, 30 to 90% of ingested drugs are excreted and released in the 57

environment in an active form (Kashiyama et al. 2010) which can potentially have an impact 58

on the organisms that inhabit these areas. 59

Much is still unknown about the ecotoxicological effects of pharmaceutical and personal care 60

products in aquatic organisms (Crane et al. 2006; Santos et al. 2010). However, recent 61

concerns regarding the impact of antidepressants, especially selective serotonin re-uptake 62

inhibitors (SSRIs), on aquatic organisms has been increasing (Johnson et al. 2007; Minagh et 63

al. 2009; Demeestere et al. 2010; Guler and Ford 2010; Styrishave et al. 2011). SSRIs inhibit 64

the serotonin re-uptake into the pre-synaptic nerve inducing an increased neuro-stimulation of 65

the post-synaptic nerve (Stahl 1998). These compounds act by modulating or mimicking the 66

effects of serotonin (Santos et al. 2010). Since its approval by the US Food and Drug 67

Administration in 1987, fluoxetine has become one of the most widely prescribed 68

antidepressants, being in the top five psychiatric drugs prescribed in 2011 after citalopram 69

and sertraline (Grohol 2012). Fluoxetine and sertraline are both SSRIs, primarily prescribed 70

for depression but also used to treat compulsive behaviour, social anxiety, panic and 71

personality disorders (AHFS 2013). 72

These drugs have been detected in the surface water and in wastewater effluent respectively 73

at levels up to 0.54 μg/L and 0.929 μg/L for fluoxetine and up to 0.08 μg/L and 0.087 μg/L 74

for sertraline (Brooks et al. 2003; Metcalfe et al. 2010; Styrishave et al. 2011; Silva et al. 75

2012). Fluoxetine has also been detected in groundwater at 0.056 μg/L (Silva et al. 2012). 76

The only record of fluoxetine in seawater is in the Chesapeake Bay (Maryland, Virginia, 77

USA) at 0.0026 μg/L (Pait et al. 2006). These findings make it clear that animals inhabiting 78

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aquatic ecosystems impacted by sewage effluent can be/are subjected to chronic exposure to 79

SSRIs. Concentrations of fluoxetine and its metabolite norfluoxetine has been found at 80

extremely high level (10 μg/kg) relative to the environmental background in the tissues of 81

fish collected near a municipal wastewater treatment plant, suggesting that these compounds 82

have the capacity to bioaccumulate (Orem and Dolph 2002). The chronic effects of SSRIs on 83

aquatic life are diverse (Brooks et al. 2003). For example, negatives impacts of fluoxetine 84

have been found on the reproduction and growth of invertebrates, vertebrates as well as algae 85

(Péry et al. 2008; Lister et al. 2009; Santos et al. 2010). The effects of sertraline on aquatic 86

organisms have been less studied. According to several studies comparing the effects of 87

SSRIs on diverse species, sertraline is the most toxic, seemingly more potent on daphnia 88

species than on fish (Christensen et al. 2007; Paterson and Metcalfe 2008; Minagh et al. 89

2009). 90

The majority of studies on the impact of antidepressants within invertebrates have focused on 91

reproduction and growth effects but few data sets are available on their behavioural effects 92

(Fong 1998; Péry et al. 2008; Gust et al. 2009; Minagh et al. 2009; Campos et al. 2012b). 93

Behavioural studies provide a link between physiological and ecological impacts, providing a 94

major endpoint to assess population health and fitness (Craddock and Sklar 2013). Light is 95

critical to a diverse range of behavioural and physiological processes such as diurnal rhythms, 96

reproduction and predator avoidance (Henry et al. 2004). Indeed, light exposure regulates 97

several neuro-modulatory systems; the activation of diverse photo-receptors modulates 98

neurological components which in turn adjust behaviour. Serotonin, also named 5-99

hydroxytryptamine (5-HT), acts as a neurotransmitter or a hormone depending on its location 100

and is a common modulator of animal behaviour in response to light. It is involved in many 101

biological endpoints in invertebrates, such as growth, maturation, reproduction, visual 102

perception and behaviour (Cezilly et al. 2000; Campos et al. 2012a). It has recently been 103

demonstrated that exogenous serotonin and fluoxetine in amphipods increase phototaxis 104

activity (Guler and Ford 2010). Acanthocephalan and trematode parasites can also act by 105

increasing the serotonergic activity leading to an increase in phototaxis activity (Tierney et al. 106

2004; De Lange et al. 2006; Guler and Ford 2010; Underwood et al. 2010) which can 107

increase susceptibility to predation (Cezilly et al. 2000; Lagrue et al. 2007; Perrot-Minnot et 108

al. 2007). Guler and Ford (2010) suggested that altered phototaxis behaviour in amphipods 109

following SSRI exposure could then conceivably make them more prone to predation. 110

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Gammarid amphipods are fundamental to many food chains and have an important role in 111

ecosystem dynamics (Donner et al. 1994). Therefore, they have often been used in 112

ecotoxicology studies, being considered as excellent bioindicators to monitor the health of 113

aquatic biotopes and the effects of anthropogenic contaminants (De Lange et al. 2006; Felten 114

et al. 2008; De Lange et al. 2009; Guler and Ford 2010; Issartel et al. 2010). 115

Echinogammarus marinus (Leach, 1815) is a ubiquitous intertidal marine amphipod which is 116

widely found throughout the coasts of northwest Europe. The aim of this study was to 117

develop behavioural biomarkers of SSRI antidepressants exposure and elucidate the 118

molecular mechanism of action through components of the serotonin pathway. 119

120

2. Materials and methods 121

2.1. Animals and exposure experiment 122

Echinogammarus marinus were collected on the intertidal zone beneath seaweed and stones 123

at low tide from Langstone Harbour, Portsmouth, UK (50º47’23.13N 1º02’37.25W). This 124

area is used for light recreational sailing and is a Special Protection Area (SPA), Site of 125

Special Scientific Interest (SSSI) and Special Area of Conservation (SAC) due to the use of 126

expansive mudflats by wading birds. Animals were sorted and adult males with no visual sign 127

of infection by trematodes (incorrectly reported as acanthocephalans by Guler and Ford, 128

2010) were isolated. These parasitised individuals were excluded for their known impacts on 129

host behaviour in response to light and modulation of serotonin in some species. The 130

amphipods were kept individually in plastic containers filled with 80 mL of mechanical-131

filtrated natural seawater (from Langstone Harbour) at 10 °C under a 12 hrs light/12 hrs dark 132

photoperiod and fed with fucoid seaweed. 133

After a week of acclimation, amphipods were exposed to the antidepressants fluoxetine and 134

sertraline. In addition to an unexposed control group of 30 animals, groups of 15 animals 135

were exposed to four nominal concentrations (0.001, 0.01, 0.1 and 1 μg/L) of each 136

compound. Mortality was recorded and water was renewed every 3 d. Both fluoxetine (CAS 137

no. 56296-78-7) and sertraline (CAS no. 79559-97-0) were obtained from Sigma-Aldrich® 138

(St. Louis, MO, USA). 139

2.2. Behavioural analysis 140

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Behavioural assays were performed after 1 hr, 1 d and 8 d of exposure to each condition 141

using DanionVisionTM (Noldus Information Technology, Wageningen, The Netherlands) and 142

its software EthoVision® XT. Animals were put in 6-wells plates and placed within the 143

DanioVision hardware for 2 min to allow settling prior to recording. The velocity (mm/s) 144

measurements of amphipods were recorded every 0.1 second (s) during 3 cycles of 2 minutes 145

(min) dark and 2 min light, thus for a total period of 12 min (Fig. 1). 146

Due to the complexity of the dataset, an average velocity of every 10 s of the raw data were 147

used to make heat maps for each condition by highlighting in green the 5th percentile, in 148

black the 50th percentile and in red the 95th percentile (percentile calculated on the entire 149

pool of data). This enabled a visual representation of periods when the amphipods were very 150

active (red) or inactive (green). Statistical analyses were conducted using SPSS® Statistics 151

v.20.0.0 software (IBM®) on the velocity of each amphipod during the 3 time periods (1 hr, 1 152

d and 8 d) of behavioural assays. The data was normalised with a cube-root transformation 153

and tested using a Kolmogorov-Smirnov test. Repeated Measure Analysis of Variance 154

(ANOVA) with Dunnett multiple comparison tests was used to determine whether significant 155

differences occurred over the 12 min recording period and between concentrations for both 156

drugs. This enabled us to determine whether the velocity of the amphipods changed over the 157

12 min dark/light regime, with SSRI concentrations or an interaction occurred between time 158

and concentration. Within subject factors (time over the dark-light cycles and interactions 159

between time and concentration) were tested using the Greenhouse-Geisser adjustments 160

whereby sphericity of data is not assumed. All statistical analysis used a significance level of 161

p < 0.05. 162

2.3. DNA/RNA isolation, purification and reverse transcription 163

After the 8 d behavioural assays, animals were anaesthetised using a mixture of clove oil and 164

seawater (0.2 μL/mL). The head of each amphipod was rapidly dissected, snap frozen in 165

liquid nitrogen and stored in Tri Reagent® (Ambion®, Life Technologies, Carlsbad, CA, 166

USA) at -80 °C before the extraction. DNA and RNA were extracted according to the 167

manufacturer protocol and used for the infection screening and real-time PCR, respectively. 168

After a DNAse step using DNAse I (RNAse free) (New England Biolabs, Ipswich, MA, 169

USA), RNA samples were cleaned on RNA clean and concentrator 5 columns (Zymo 170

Research, Orange, CA, USA) following the manufacturer instructions. Quantification of total 171

RNA and genomic DNA was performed with a NanoDrop® ND-100 Spectrophotometer 172

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(Nanodrop Technology Inc., Wilmington, DE, USA) and the integrity was checked using 173

1.5% agarose gel electrophoresis. For each sample, 250 ng of total RNA isolated was used to 174

obtain cDNA by reverse transcription using the GoScriptTM Reverse Transcription System 175

(Promega, Fitchburg, WI, USA) following the manufacturer protocol and using Oligo(dT)15 176

primers and recombinant RNasin® Ribonuclease Inhibitor. 177

2.4. Infection screening 178

The infection of E. marinus by parasites capable of inducing an increase in the serotonergic 179

activity (Guler and Ford 2010) might interfere with the response of this species to SSRIs or 180

create additional variation within controls. The E. marinus population used for this study has 181

been comprehensively screened and found to contain a single trematode species capable of 182

neurological modulation of its host (Yasmin Guler, unpublished data). Therefore, an infection 183

screen using PCR was performed to enable the removal of amphipods infected by this 184

trematode from the dataset used for the behavioural and transcriptomic analysis. PCR assays 185

were conducted on genomic DNA using primers designed to amplify the Internal Transcribed 186

Spacer (ITS) region of the ribosomal RNA gene for this trematode species (Yasmin Guler, 187

unpublished data). To check the quality of DNA sample, amplification of the glyceraldehyde 188

3-phosphate dehydrogenase (Gapdh) gene was used as a control (Table 1). PCR reactions 189

were performed in a final volume of 25 μL containing 1X GoTaq® Flexi Buffer, 2.3 mM of 190

MgCl2, 0.8 mM of each dNTPs, 0.4 μM of each primer, 1 U of GoTaq® DNA polymerase 191

(Kit GoTaq® Flexi DNA polymerase, Promega) and 30 ng of genomic DNA. The PCR 192

conditions were: initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 45 193

s, 59 °C for 45 s 72 °C for 2 min and 20 s and a final incubation at 72 °C for 5 min. The PCR 194

products were then analysed using agarose gel electrophoresis to check the presence of the 195

amplified trematode ribosomal sequence. 196

2.5. Primer design and real-time PCR 197

The transcriptome of E. marinus has recently been sequenced (unpublished data). The 198

generated expressed sequence tags were assembled to create a "transcriptome atlas" of 199

contiguous sequences (or contigs) and these contigs were annotated by comparison to non-200

redundant sequences in the UniProt and FlyBase database (BLASTX, E-value cut-off of 1e-5). 201

The contigs chosen for primer design were selected using the following criteria: (i) selection 202

of contigs that potentially represent genes involved in behaviour modulation, even if they 203

were not well annotated (E-value > e-5), on the basis that such contigs might represent poorly 204

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conserved genes involved in neurological pathways; or (ii) selection of genes with a 205

confident annotation (an E-value < e-5) potentially involved in serotonin or neurological 206

pathways; (iii) genes that appeared to show exclusively high expression in the head, on the 207

basis that these are more likely to represent genes with neurological functions. A pooled 208

sample of cDNA was used to test the suitability of each set of primers. All primers used in 209

this study, including those used as reference genes Gapdh and Calreticulin (Table 1) were 210

designed using Primer-3 software (Koressaar and Remm 2007; Untergrasser et al. 2012) and 211

synthesised by Eurofins MWG Operon (Ebersberg, Germany). 212

Quantitative real-time PCR (qPCR) analyses were performed using a real-time PCR cycler 213

(Eco Illumina®, San Diego, CA, USA) on 12 samples per condition (or 3 pools of 4 head 214

samples to test the primer pairs), using 7.5 μL of LabTAQTM Green (LabTech International 215

Ltd, Uckfield, UK), 1 μL of cDNA, 5.7 μL of ultra-pure water, 0.2 μL each of Rhod1 forward 216

and reverse primers and 0.4 μL each for all other primers (all primer volumes taken from a 10 217

μM stock). The PCR reactions were performed with an initial incubation at 95 °C for 2 min, 218

followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s with Rox normalisation. Following 219

the final cycle, the reactions underwent a 15 s, 95 °C denaturing step followed by a 15 s, 55 220

°C hybridisation step before PCR product melt curves were determined during a further 221

temperature increase to 95 °C. Standard curve analysis was used to determine the efficiency 222

of each primer pairs and melt curve analysis were performed for each gene to confirm the 223

specificity of the PCR product in each reaction. Ultra-pure water was used in the place of 224

template in the no template control reactions. Furthermore, minus RT reactions were 225

performed to control for the potential presence of residual genomic DNA. The control group 226

of animals (that were not exposed to antidepressants) were used as the reference sample. The 227

relative expression of each gene (in this article gene expression is taken to represent only 228

transcription, although it is acknowledged that gene expression can also be regulated at 229

translation, mRNA and protein stability levels etc.) was calculated using the ΔΔCt method 230

(Livak and Schmittgen 2001) and normalised with both Gapdh and Calreticulin as reference 231

genes. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) is a reference (housekeeping) 232

gene often used in several species (Barber et al. 2005) and particularly in crustaceans 233

(Underwood et al. 2010; Leelatanawit et al. 2012). Statistical analyses were conducted on the 234

square-rooted relative expression of each gene and results are expressed as the mean ± 235

standard deviation (s.d.). The normality was tested using a Kolmogorov-Smirnov test and 236

multiple comparisons and comparison of two mean values were performed following 237

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ANOVA using the Dunnett’s multiple comparison test using SPSS® Statistics and at a 238

significance level of p < 0.05. 239

240

3. Results 241

The mortality was very low during all exposure experiment with only 2 dead amphipods for 242

the sertraline exposure at 1 μg/L and 1 ng/L. No trematode-infected amphipods (as detected 243

by visual inspection and retrospective PCR) were used in the experiments. 244

3.1. Behavioural experiment 245

The average of each amphipods’ velocity during the 3 times 2 min dark/2 min light cycle for 246

each condition after 8 d exposure are shown in Fig. 1 as an example of the dataset generated. 247

Generally, when the light was switched on, the amphipods react and the velocity increases 248

almost instantly for each condition and time of exposure with the velocity gradually abating 249

after 30 s. 250

Multiple comparison tests (Tukey’s Multiple comparison; data not shown) revealed 251

significant differences (p < 0.001) between 30 s time bins occurred overwhelmingly between 252

the light and dark periods and were more pronounced for the 1st thirty seconds into the 2 min 253

light cycles. This pattern was consistent for all concentrations, drugs and exposure periods. 254

Interestingly, the 1st 30 s bin on the 1st of the 3 dark-light periods was also significantly 255

different (p < 0.001) from all other periods within the light apart from after 1 d for both drugs 256

(Fig. 2A.). For both drugs and at all exposure times, there was a significant effect of the 257

varying dark-light cycles over the 12 min on the amphipods velocity (p < 0.001; Table 2; Fig. 258

2 and 3). 259

For fluoxetine after 1 hr exposure, there was no significant effect of the different 260

concentrations (p > 0.05; Fig. 2 and Table 2) on velocity (mm/s) but there was a significant 261

interaction between dark-light cycles and concentration (p < 0.05). This interaction appears to 262

have occurred due to a divergence in velocity between concentrations over the three dark-263

light cycle which can be observed on the graph (Fig. 2A.). After 1 d exposure, a significant 264

difference in the velocity was observed between concentrations (p < 0.001). Dunnett’s 2-way 265

multiple comparison tests revealed that significant differences occurred between the controls 266

and all concentrations (0.001-1 µg/L: p < 0.01) with the highest velocities generally observed 267

in the concentration 0.1 µg/L (about 78% higher than the control) and the lowest increase at 268

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0.001 µg/L (about 43% higher than the control) (Fig. 2). Similarly, a significant interaction 269

between concentrations and the dark-light cycles was observed (p < 0.005). After the 8 d 270

exposure no significant difference was observed in the velocity of amphipods between 271

treatments (p > 0.05) and the interaction tests failed to reach the significant cut-off (p = 272

0.098). 273

For sertraline, after just 1 hr exposure, significant differences were observed in the velocity 274

between concentrations (p < 0.05; Fig. 3 and Table 2). Velocities were elevated in all 275

concentrations, apart from the lowest (0.001 µg/L), relative to the control. However, 276

Dunnett’s multiple comparison tests revealed that significant differences occurred only 277

between the controls and the 0.01 µg/L concentration (about 73% higher than control 278

velocity; p = 0.002; Fig. 3). A significant difference was also observed after 1 d of exposure 279

to sertraline with all (apart from 0.001 µg/L; velocity higher of about 69, 55 and 33% 280

respectively for 0.01, 0.1 and 1 µg/L) exposed groups recording higher average velocities 281

compared to the control (p < 0.001; Fig. 3). After 8 d exposure, no significant difference was 282

observed in the velocity between exposed and control groups, with the associated p-value just 283

failing to meet the significant criteria (p = 0.057; Fig. 3A.). For all sertraline exposure times, 284

no interaction was observed between concentration and time (p > 0.05; Table 2). 285

3.2. The expression levels of neurologically-related genes in amphipods 286

exposed to fluoxetine and sertraline 287

The expression level of reference genes (in this article gene expression is taken to represent 288

only transcription, although it is acknowledged that gene expression can also be regulated at 289

translation, mRNA and protein stability levels etc.), Gapdh and Calreticulin, did not change 290

for any concentrations of both fluoxetine and sertraline: for Gapdh: df = 8, F = 1.380 and p-291

value = 0.239; for Calreticulin: df = 8, F = 1.648 and p-value = 0.148. 292

RNA pooled from 12 individuals for each exposure group were used to test the suitability of 293

each primers pair associated to a set of 10 potential neurological biomarker genes (7 294

annotated with an E-value < e-5 and 3 unannotated). The serotonin receptor 1 (5HT1), the N-295

acetylserotonin O-methyltransferase-like protein (Acser), the inebriated neurotransmitter 296

(Ine1) genes and contig 11430 presented very low expression, making it hard to determine 297

expression from genomic contamination or the amplification of small amounts of artefact 298

(results not shown). Those primer sets were then subsequently abandoned. For the two 299

remaining unannotated genes (contig 9063 & 113810) the Ct value (the cycle number at 300

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which the fluorescent signal (ΔRn) crossed an arbitrary threshold set within the linear phase 301

of amplification) for both genes, was less than 22 cycles and no contamination by dimers or 302

hairpin hybridisation was evident. However, despite this high expression, no variation in their 303

expression with pooled cDNA was observed between each exposure (results not shown). 304

Four sets of primers, [Neurocan core protein (Neuc), Rhodopsin (Rhod1), Arrestin (Arr) and 305

tryptophan hydroxylase (Ph)] did present evidence of both high and altered expression using 306

the pooled cDNA and were therefore used to quantify the variation of gene expression among 307

each condition (drug and concentration after 8 d exposure). 308

The mRNA expression levels of these four genes in the head of E. marinus exposed for 8 d to 309

0, 0.001, 0.01, 0.1 and 1 μg/L of fluoxetine are illustrated in Fig. 4A. Significant differences 310

were observed between expression of Neurocan core protein (F = 6.632, df = 4, p = 0.007), 311

Rhodopsin (F = 4.367 df = 4, p = 0.027), and tryptophan hydroxylase (F = 3.917, df = 4, p = 312

0.036) but not for Arrestin (F = 1.313, df = 4, p = 0.330). Where significant differences were 313

observed, these were predominantly found to be down-regulated in treated samples when 314

compared to the control group for the lower fluoxetine concentrations (Dunnett’s Multiple 315

Comparison p < 0.05; Fig. 4A). 316

The mRNA expression levels of the four genes for 8 d exposure to sertraline are illustrated in 317

Fig. 4B. Significant differences in expression were observed for Rhodopsin (F = 7.868, df = 318

4, p = 0.004) and Arrestin (F = 3.527, df = 4, p = 0.048) but not for Neurocan core protein (F 319

= 2.860, df = 4, p = 0.081) and tryptophan hydroxylase (F = 2.137, df = 4, p = 0.151). 320

Multiple comparison tests found no significant differences from the control, although it is 321

worth noting that the expression of Neurocan core protein just failed to meet the significance 322

criteria for the lowest concentration (0.001 μg/L, p = 0.064) as well as Rhodopsin for 0.1 323

μg/L (p = 0.075). 324

325

4. Discussion 326

4.1. Effect of light on amphipod behaviour 327

Amphipods naturally avoid well lighted areas and favour shadowed or dark regions in the 328

intertidal zone where there is lower risk of predation (Cezilly et al. 2000). In this study, a 329

significant increase in the velocity was observed in the first 30 s of light periods with a higher 330

increase for the first of three light periods, at 1 hr and 8 d but not after 1 d of the beginning of 331

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the experiment. Sudden stimulation of the eyes could be interpreted by the amphipod as a 332

reduction in cover and results in an escape-related behaviour in order to avoid predation. The 333

decrease in the response to subsequent light periods indicates that the optic nerves may have 334

been overstimulated and that a time of recovery from the first stimulation is needed. One day 335

after the start of the experiment, the initial response to the light was reduced across all 336

exposures indicating that more time may be necessary to recover. 337

4.2. Effect of fluoxetine and sertraline on amphipod behaviour 338

The first purpose of this investigation was to assess the effect of two SSRIs on the swimming 339

behaviour of the amphipod E. marinus. In this study, amphipods were exposed to 340

concentrations from 0.001 to 1 μg/L of fluoxetine and sertraline, these concentrations fall 341

well within those currently being found in the aquatic environment (0.929 μg/L and 0.087 342

μg/L respectively) (Brooks et al. 2003; Metcalfe et al. 2010; Styrishave et al. 2011; Silva et 343

al. 2012). Interestingly, a significant interaction between the dark-light cycling and 344

concentration was observed for fluoxetine at short-term (1 hr and 1 d). This interaction was 345

due to a divergence in the response to light between the animals exposed to various 346

concentrations of fluoxetine and demonstrates that these antidepressants have an effect on 347

amphipod behaviour. There was a significant increase in the velocity over the 12 min time 348

period at 1 d exposure to 0.1 μg/L of fluoxetine (of about 78%) compared to the control, 349

which is consistent with the concentration used in the experiment to produce maximum 350

phototaxis behaviour of this species exposed to fluoxetine (Guler and Ford 2010). Guler and 351

Ford (2010) highlighted the non-monotonic concentration response curve, noting a peak of 352

phototaxis activity in the animals exposed at 0.1 μg/L of fluoxetine. The lack of significant or 353

reduced effects in higher concentrations of fluoxetine could be due to the inhibition of a finite 354

amount of endogenous serotonin or desensitisation, as also suggested by Guler and Ford 355

(2010). Amphipods exposed to 0.01 μg/L of sertraline showed a significant higher velocity 356

than the control after 1 hr exposure (about 69%), as well as from 0.01 to 1 μg/L after 1 d. 357

Sertraline’s mode of action is similar to fluoxetine, both being SSRIs. The effect of sertraline 358

was most prominent for the 0.01 μg/L concentration compared to the higher concentrations 359

for which the velocity was lower. This suggests that as well as for fluoxetine, higher 360

concentrations of sertraline might tend to more quickly reach a maximum level of serotonin 361

re-uptake inhibition or lead to a desensitisation. The larvae of the fish, P. promelas has a 362

suppression of predator avoidance after less than a week of exposure to 0.025 μg/L of 363

fluoxetine (Painter et al. 2009), although no alteration of this behaviour was found at higher 364

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concentrations. In adults, a decrease of the predator avoidance behaviour has also been 365

demonstrated when exposed at a concentration of 3 μg/L of sertraline for 28 d (Valenti et al. 366

2012). However, contrary to fluoxetine, the response to higher concentrations (10 and 30 367

μg/L) of sertraline was the same as for 3 μg/L in P. promelas. 368

The increased light-induced velocity of amphipods exposed to SSRIs is consistent with an 369

increase of the serotonin amount. This study did not test the preference of the amphipods to 370

lit areas [although this was observed by Guler and Ford (2010)], but rather the velocity of 371

shrimp within light or dark environments. The most consistent results from this experiment 372

indicated that amphipods are significantly more active both in light and dark phases of the 373

experiment (with some interactions between light and concentration observed) when exposed 374

to SSRIs as compared to untreated amphipods. Furthermore the recovery time (time to return 375

to the basal velocity level) to light stimulation is altered between exposures and control. It is 376

possible that the increased activity could also be due to the influence of serotonin on other 377

hormones [e.g. Crustacean Hyperglyceamic Hormone, CHH; (Fingerman 1997)] and/or 378

locomotor activity (McPhee and Wilkens 1989). However, changes in the transcription of 379

genes relating to phototransduction pathways measured during study add some weight for 380

linking the behavioural and gene responses. It will be beneficial in future studies to lengthen 381

the periods of light and dark to differentiate the behaviours further. 382

Studies investigating the effect of SSRIs on aquatic organisms have been mainly performed 383

using concentrations higher than those found in the environment and used in this study. 384

Impacts of fluoxetine on the reproduction of C. dubia were observed at 56 μg/L with a 385

decrease of fecundity (Brooks et al. 2003), and around 10 μg/L in D. magna (Péry et al. 386

2008). The acute toxicity of sertraline on animals has been demonstrated with a LC50 of 380 387

μg/L in fish following 96 hr of exposure (Minagh et al. 2009) and change in the behaviour of 388

fish was found from 3 μg/L (Valenti et al. 2012). Relatively few studies have been carried out 389

using environmentally relevant concentration of SSRIs (Painter et al. 2009; Guler and Ford 390

2010; Fong and Hoy 2012). However, the current study has found significant impacts as low 391

as 0.001 μg/L that fall well within concentrations considered environmental relevant in the 392

aquatic environment close to wastewater effluent and inhabited by this species (about 0.0026 393

μg/L in US estuaries, Paint et al. 2006). Furthermore, the degree of degradability of these 394

antidepressants in water is generally low and their half-lives is from 2 days to indefinite 395

(Johnson et al. 2005; Kwon and Armbrust 2006). The benthos is a reservoir for these 396

compounds as they tend to be absorbed by sediments or sludge (Kwon and Armbrust 2006). 397

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The amount of SSRIs in this compartment should also be investigated in order to better 398

evaluate the effects of antidepressants on amphipods. In this study, fluctuations in fluoxetine 399

and sertraline concentrations might have occurred due to the static renewal of water every 2 400

days and the potential binding to the exposure chamber. Furthermore, insignificant results 401

from the lower concentration range need to be carefully interpreted in light of the nominal 402

concentrations used and the potential for chemical breakdown. 403

The presence of antidepressants in the environment can be chronic due to a constant release 404

from the sewage water (Santos et al. 2010), thus a long-term analysis is essential to truly 405

understand the effect of prolonged exposure times on aquatic organisms. Our results 406

indicated that the most enhanced effects of fluoxetine and sertraline were observed following 407

short-term exposure, after 1 hr (sertraline only) and 1 d of exposure. Although, contrary to 408

this, Guler and Ford (2010) found a significant and continued preference of lit arenas still 409

after 3 weeks exposure to fluoxetine at 0.1 μg/L compared to controls. As suggested by our 410

higher concentrations of SSRIs in this study, a longer term exposure might lead to a 411

desensitisation effect or a lack of serotonin availability and explain why no significant effect 412

of both drugs was found after 8 d exposure. In mammals, it has been shown that the 413

responsiveness to fluoxetine decreases following chronic exposure due to a critical decrease 414

in the tryptophan levels, the precursor of serotonin (Delgado et al. 1999). Therefore, after 415

several days of exposure to SSRIs, the haemolymph tryptophan content might be nearly 416

depleted, reducing the drug effect on amphipods. Another hypothesis could be a negative 417

feedback loop in the serotonin pathways; amphipods might be compensating for the change 418

by producing less serotonin to flood the synapse or by increasing the expression of serotonin 419

re-uptake transporter (Pineyro et al. 1994). It would then be interesting to compare the impact 420

of these drugs on the serotonin pathway at short-term and long-term in further research. 421

4.3. Effect of fluoxetine and sertraline on amphipod gene transcription 422

The second aim of this study was to elucidate the molecular mechanism by which 423

behavioural changes may be taking place. The absence of variations in Calreticulin and 424

Gapdh expression supports their utilisation as reference genes. 425

Rhodopsin (Rhod1) is involved in behaviour regulation and is a light receptor and signal for 426

phototransduction in vertebrates and invertebrates (Orem and Dolph 2002). In invertebrates, 427

phototransduction cascade is mediated by rhodopsin, a light receptor which is transformed 428

into metarhodopsin by photo-isomerisation (Orem and Dolph 2002). The metarhodopsin 429

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activates a Gαq-type of G-protein, hydrolysing guanosine triphosphate (GTP) to guanosine 430

diphosphate (GDP), which then activates a phospholipase C (PLC). Finally, the PLC opens a 431

transient receptor potential (TRP) channels which induce a depolarisation of the cells. Rhod1 432

was significantly down-regulated in amphipods exposed to low concentrations of fluoxetine 433

(0.001 and 0.01 μg/L) and slightly up-regulated for those exposed to 0.001 μg/L of sertraline. 434

One explanation for the opposite gene expression patterns observed in E. marinus when 435

exposed to these two antidepressants may be the differences in their mode of action. 436

Therefore, one might speculate that the mis-regulation of Rhod1 could then modulate the 437

transduction of light stimulation and alter the behaviour of amphipods to light. However, 438

further studies will be necessary to better understand the role of rhodopsin in modulating 439

amphipod behaviour. The protein encoded by the arrestin (Arr) gene is also involved in the 440

phototransduction. In fact, this gene contributes to the arrest of the phototransduction cascade 441

(Kashiyama et al. 2010) by binding the active metarhodopsin and inhibits it by uncoupling 442

rhodopsin from the Gα-subunit protein (Orem and Dolph 2002). An example of their role in 443

crustacean is that arrestin and rhodopsin promote light-induced hatching in Triops granarius 444

(Kashiyama et al. 2010). In our study, Arr is down-regulated only in animals exposed to 445

0.001 and 0.01 μg/L of fluoxetine, which could be potentially linked to the down-regulation 446

of Rhod1 and components of the phototransduction pathway if followed by a protein down-447

regulation. 448

The neurocan core protein (Neuc) is a protein involved in cell adhesion and migration and is a 449

factor in bipolar disorder, manic-depressive disorder and schizophrenia (Cichon et al. 2011; 450

Mühleisen et al. 2012). In our study, Neuc mRNA expression significantly decreases for the 451

two lower concentrations of fluoxetine. Assuming a similar function of Neuc in amphipods 452

and mammals (Livak and Schmittgen 2001), a decrease in the expression of this gene if 453

followed by a decrease in amount of its protein might lead to behavioural changes. It might 454

then induce an increase of energy (Livak and Schmittgen 2001) which might tend to reduce 455

the predator avoidance behaviour. The role of this gene should be investigated in further 456

studies to define its function in amphipods. The enzyme tryptophan hydroxylase (Ph) 457

catalyses serotonin biosynthesis in the serotonergic nerves (Hasegawa and Nakamura 2010). 458

However, no significant variation in the expression of this gene has been found between each 459

condition, which suggests that this gene is not involved in the serotonin regulation inducing 460

the behavioural change observed when exposed to antidepressants. 461

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The expression variations of these four genes were relatively low in E. marinus and it is 462

unclear what impacts their down-regulation may have on amphipod behaviour. There is 463

paucity of research regarding the molecular processes that underlie serotonin pathways and 464

behavioural regulation in aquatic invertebrates. Further studies are essential in order to better 465

understand the role of these genes in crustaceans and their relationship to the behaviour 466

modification observed following antidepressant exposure. However, this study clearly 467

demonstrates that exposure to SSRIs can be associated with alteration in the expression of 468

genes with plausible links to amphipod behaviour and serotonergic activity. Recently, it has 469

been demonstrated in the crustacean Gammarus pulex that the histaminergic system is 470

involved in the reaction to light in association with the serotonergic system (Perrot-Minnot et 471

al. 2013). In the same study, looking at the influence of several 5-HT receptor antagonists and 472

agonists, it has been suggested that the serotonin receptor 5-HTR2 subtype might be involved 473

in the behaviour regulation of G. pulex. Furthermore, in D. magna, a transcriptomic analyses 474

using a custom microarray showed that more than 1200 genes have a mRNA expression 475

change when exposed to fluoxetine (Campos et al. 2013). Serotonin metabolism, neuronal 476

development processes, carbohydrate and lipid metabolism functions were found to be 477

differentially expressed when annotated by comparison to the functionally annotated 478

Drosophila genome. 479

480

4.4. Summary 481

This study has provided evidence that a crustacean’s behaviour and gene expression could be 482

abnormally altered in waters receiving antidepressants at concentrations as low as 0.001 483

μg/L. The use of behavioural analysis has been demonstrated as good biomarker of the 484

exposure of amphipods to antidepressants. The transcriptome of E. marinus is a rich resource 485

for neurological genes that are potentially involved in behavioural regulation and serotonin 486

related pathways. Therefore, future studies will be able to test an expanding number of 487

amphipod genes for transcriptional change following exposure to antidepressants. This study 488

has also provided further evidence for the non-monotonic concentration responses of some 489

antidepressants, which should be taken into account when designing and evaluating toxicity 490

tests. Whether other biological systems, for example: reproduction, moulting, metabolism and 491

the immune system are impacted following low SSRIs exposure remains an important 492

unanswered question. The effect of other SSRIs and their metabolites (Brooks et al. 2003; 493

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Stanley et al. 2007; Paterson and Metcalfe 2008; Metcalfe et al. 2010) on amphipods should 494

also be evaluated along with other types of antidepressant such as the serotonin-495

norepinephrine re-uptake inhibitors (SNRIs) and the serotonin antagonist and re-uptake 496

inhibitors (SARIs). The use of other types of antidepressants increases every year, with an 497

increase of about 60 % for the SNRI duloxetine the last two years (HSCIC and Prescribing 498

and Primary Care Services 2013). Considering that the mode of action for these other types of 499

antidepressants is different from the SSRIs, it is important to also determine their potential 500

impact on aquatic organisms. How multiple antidepressants, with multiple modes of action, 501

will act in mixtures is another challenge faced by ecotoxicologists. For example, it has been 502

demonstrated that mixtures of antidepressants have additive effects in aquatic organisms 503

(Christensen et al. 2007; Styrishave et al. 2011) and leads to a decrease in the predation 504

avoidance behaviour in the larvae of the fish P. promelas (Styrishave et al. 2011). The 505

organismal and ecological implications of these findings are difficult to deduce but coupled 506

with previous studies suggest that SSRIs present in the aquatic environment could 507

conceivably lead to population level effects through impacts on predation, feeding and 508

reproductive associated behaviour. 509

510

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Acknowledgments 510

The authors would like to acknowledge the following awarding bodies for supporting this 511

research: The EU INTERREG programme entitled Peptide Research Network of Excellence 512

(PeReNE) for supporting MB & ATF and the Natural Environmental Research Council 513

(NERC; NE/G004587/1) supporting YG, SS & ATF. We wish to thank J. Trevett for his help 514

with the exposure and behavioural experiment. We greatly appreciate the constructive 515

comments provided by two anonymous reviewers. 516

517

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Pait A.S., Warner R.A., Hartwell S.I., Nelson J.O., Pacheco P.A., Mason A.L., 2006. Human Use 667 Pharmaceuticals in the Estuarine Environment: A Survey of the Chesapeake Bay, Biscayne 668 Bay and Gulf of the Farallones. NOS NCCOS 7, Silver Spring, MD. 669 NOAA/NOS/NCCOS/Center for Coastal Monitoring and Assessment. 670

Paterson G., Metcalfe C.D., 2008. Uptake and depuration of the anti-depressant fluoxetine by the 671 Japanese medaka (Oryzias latipes). Chemosphere 74 (1):125-130. 672 http://dx.doi.org/10.1016/j.chemosphere.2008.08.022 673

Perrot-Minnot M.-J., Dion E., Cézilly F., 2013. Modulatory effects of the serotonergic and 674 histaminergic systems on reaction to light in the crustacean Gammarus pulex. 675 Neuropharmacology 75 (0):31-37. http://dx.doi.org/10.1016/j.neuropharm.2013.06.028 676

Perrot-Minnot M.J., Kaldonski N., Cézilly F., 2007. Increased susceptibility to predation and altered 677 anti-predator behaviour in an acanthocephalan-infected amphipod. Int J Parasitol 37 (6):645-678 651. http://dx.doi.org/10.1016/j.ijpara.2006.12.005 679

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Pineyro G., Blier P., Dennis T., de Montigny C., 1994. Desensitization of the neuronal 5-HT carrier 683 following its long-term blockade. J Neurosci 14 (5):3036-3047. 684

Santos L.H.M.L.M., Araújo A.N., Fachini A., Pena A., Delerue-Matos C., Montenegro M.C.B.S.M., 685 2010. Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic 686 environment. J Hazard Mater 175 (1–3):45-95. 687 http://dx.doi.org/10.1016/j.jhazmat.2009.10.100 688

Silva L.J.G., Lino C.M., Meisel L.M., Pena A., 2012. Selective serotonin re-uptake inhibitors (SSRIs) 689 in the aquatic environment: An ecopharmacovigilance approach. Sci Total Environ 437 690 (0):185-195. http://dx.doi.org/10.1016/j.scitotenv.2012.08.021 691

Stahl S.M., 1998. Mechnism of action of serotonin selective reuptake inhibitors: Serotonin receptors 692 and pathways mediate therapeutic effects and side effects. J Affect Disord 51 (3):215-235. 693 http://dx.doi.org/10.1016/S0165-0327(98)00221-3 694

Stanley J.K., Ramirez A.J., Chambliss C.K., Brooks B.W., 2007. Enantiospecific sublethal effects of 695 the antidepressant fluoxetine to a model aquatic vertebrate and invertebrate. Chemosphere 69 696 (1):9-16. http://dx.doi.org/10.1016/j.chemosphere.2007.04.080 697

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718

719

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Table and Figures captions 719

Table 1. Primer sequences used in this study and target genes associate. The primers couple 720

for serotonin receptor 1 have been design on alignment of several invertebrates’ sequences of 721

this gene and in very conserved area. Italic: the reference genes used to normalised the gene 722

expression; † Four set of primers found relevant for quantification; * Target gene unknown, 723

no annotation: E-value > e-5. 724

Table 2. Results of statistical analyses of velocity tracking during the 12 min of 2 min dark/2 725

min light periods in Echinogammarus marinus exposed to each concentrations of fluoxetine 726

and sertraline for each time of exposure. 727

Fig. 1. Mean velocity (mm/s) of 15 Echinogammarus marinus per treatment exposed to 728

fluoxetine and sertraline for 8 d recorded with DanioVision. 6-wells plates were used to track 729

the velocity of 6 amphipods at a time every 0.1 s over a 12 min period of alternate 2 min 730

dark/2 min light periods (A). Lines indicate mean values of replicates specimens. Black: 731

control, gradation of blue: fluoxetine (FLU) concentrations (B), gradation of orange: 732

sertraline (SER) concentrations (C). 733

Fig. 2. Estimated marginal means (A) and heat map (B) of the velocity (mm/s) average every 734

10 s during the 12 min of 2 min dark/2 min light periods for each fluoxetine concentrations 735

and time exposure. Heat map: green: the 5th percentile, black: the 50th percentile and red: the 736

95th percentile. Hr: hour, d: day(s). Asterisks indicate significant differences to the control (p 737

< 0.05). 738

Fig. 3. Estimated marginal means (A) and heat map (B) of the velocity (mm/s) average every 739

10 s during the 12 min of 2 min dark/2 min light periods for each sertraline concentrations 740

and time exposure. Heat map: green: the 5th percentile, black: the 50th percentile and red: the 741

95th percentile. Hr: hour, d: day(s). Asterisks indicate significant differences to the control (p 742

< 0.05). 743

Fig. 4. Relative mRNA expression of genes encoding Neurocan core protein (Neuc), 744

Rhodopsin (Rhod1), Arrestin (Arr) and tryptophan hydroxylase (Ph) in the head of 745

Echinogammarus marinus exposed to four fluoxetine (A) and sertraline (B) concentrations 746

for 8 d. The expression was normalised according to the expression of Gapdh and 747

Calreticulin. n = 3 pools of 4 amphipods. Data are expressed as the mean ± s.d. Asterisks 748

indicate significant differences to the control (p < 0.05). 749

750

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Primer Name

Nucleotide sequences

(from 5’ to 3’) Target Gene Uniprot or

GenBank ID Ref. Species E-value

5HT1-F

CAA CGC AGA GTA CGG GGT

TGG T

5HT1-R

GCA AAA CGG CGA AAT CGA

ACG GG

Serotonin receptor 1

Acser-F

AAA CCC ACA AAC GAC GAC

CA

Acser-R

AAG GTT ACT CTC TGC CAC

GC

N-acetylserotonin O-

methyltransferase-like protein

O95671 Homo sapiens 7E-25

Arr-F CTC CTT CGA CTC CAG GCT

TG

Arr-R GGC TAA CCT GGG CAT CAA

CA

Arrestin† P32122 Locusta migratoria

5.00E-50

Calret-F

AGA TCG GAG GCA TTG TTT

TG

Calret-R

AAC ACG TGG GCC GAG TAT

AG

Calreticuline Q7Z1E6 Bombyx mori 1.00E-155

Gapdh-F

ATA GTG TCC AAC GCC TCC

TG

Gapdh-R

CCA GTG GAG GAT GGA ATG

AT

GAPDH P56649 Panulirus versicolor 1E-164

Ine1-F CGT GGA

GGA GCC GTT GCC TG

Ine1-R CCT GTG CGG CAT CCC TCT

GC

Inebriated neurotransmitter NM057664.5 Culex

quinquefasciatus 4.00E-

05

Neuc-F CCC TAC CCT GTT TGC TCC

AG

Neuc-R

CCA TTT TGG TAG TTC GCG

GC

Neurocan core protein† P55066 Mus musculus 7.00E-

19

Ph-F GGT CAA GAC

CTG GAG CGC GG

Ph-R GGT GCT GTG GAA CAC GCG

GA

Tryptophan hydroxylase† AY099427.1 Aedes aegypti 6.00E-

142

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Rhod1-F

CCC GCC AAC ATG CTG CCT

GA

Rhod1-R

CGG GTG ACC GCA

GGC TCT TG

Rhodopsin† DQ85259 Neomysis americana

4.00E-74

9063-F TCA TCGACG AAC TTG GAG

CC

9063-R TCA TTG GCC TCT AGA AGC

GC

*

11381-F

TTC CGA ACT AAC GCC TGC

TC

11381-R

CCA ACA GTG CAG CAA CAT

CG

*

11430-F

GTG AGG AGG AGG TGT

GGG TA

11430-R

GGT ACA GGC GAG ACA ACA

GG

*

751

752

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Compound Exposure Period Concentration Time (Light-Dark

Cycles) Interaction: time*

concentration

F df p F df p F df p

1 Hour 0.585 4 0.675 27.335 12.35 <0.001 1.482 49.412 0.018

1 Day 7.199 4 <0.001 14.148 23 <0.001 1.694 53.017 0.002Fluoxetine

8 Days 1.087 4 0.368 13.787 23 <0.001 1.311 39.437 0.098

1 Hour 3.719 4 0.008 14.878 23 <0.001 1.061 53.725 0.358

1 Day 7.966 4 <0.001 14.341 23 <0.001 1.307 43.656 0.407Sertraline

8 Days 2.373 4 <0.057 15.451 23 <0.001 1.321 46.337 0.076

F: ratio of the between and within group variance estimates; df: degrees of freedom; p: p-753 value, in bold when significant. 754

755

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Figure 1 TIFF

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Figure 2 TIFF

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Figure 3 TIFF

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Figure 4 TIFF

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A.

0

5

10

15

20

25

30

35

1 601 1201 1801 2401 3001 3601 4201 4801 5401 6001 6601 7201

Velo

vity

(mm

/s)

Time (s)

SER-1 μg/L SER-0.1 μg/L SER-0.01 μg/L SER-0.001 μg/L control

B.

0

5

10

15

20

25

30

35

0 60 120 180 240 300 360 420 480 540 600 660 720

Velo

vity

(mm

/s)

Time (s)

FLU-1 μg/L FLU-0.1 μg/L FLU-0.01 μg/L FLU-0.001 μg/L control

C.

Figure 1 powerpoint

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0.8

1

1.2

1.4

1.6

1.8

2

2.2 1 hr

1 d

8 d

0.8

1

1.2

1.4

1.6

1.8

2

2.2

0.8 1

1.2 1.4 1.6 1.8

2 2.2

30

A.

B. 0 μg/L 2.886657 2.358449 2.193984 1.384431 1.373069 1.795282 2.093299 1.771583 1.609143 1.416285 1.223442 1.650953 12.55899 4.869178 3.689626 3.250424 2.425757 1.795195 1.457748 2.133145 1.688268 1.882117 2.132217 1.879808 2.081939 2.224629 2.688132 3.297604 2.446941 2.838797 3.576065 3.671236 2.931169 2.908485 2.632874 2.151974 5.078851 3.079855 2.63877 2.851213 2.560388 2.87643 1.528809 1.897737 1.675935 1.586168 1.918176 2.184642 1.94721 2.879723 2.499425 3.677339 4.079483 4.375287 3.779602 2.212264 1.766552 2.648355 2.628719 4.304175 5.568272 4.345471 3.735428 2.168544 1.240419 1.734982 1.843853 1.767176 2.478255 2.076904 2.000496 1.989453

0.001 μg/L 2.012875 1.902849 1.589011 1.783906 1.927633 1.645344 1.711528 2.061414 2.925264 2.694763 2.899505 2.125406 15.33474 9.41476 3.715187 2.093837 1.616851 1.143461 2.385731 3.309697 3.441371 1.756409 1.944495 2.093869 2.625489 1.329356 2.246214 2.659369 2.849843 2.123304 1.766107 1.362762 2.305882 2.368155 2.037358 2.562593 7.836312 5.115309 3.374531 2.50197 1.826538 3.070203 3.677164 1.231633 1.225747 1.259598 2.609064 3.24794 2.269369 2.08517 2.744145 2.817599 3.181165 2.264294 1.648303 1.930062 2.085841 2.483533 2.89322 2.79785 8.384385 6.663695 4.250508 3.109464 3.808681 3.703814 3.171017 1.509995 2.571695 4.095201 2.310445 2.15191

0.01 μg/L 1.607891 1.316814 1.38717 1.462111 1.294223 1.515081 1.477941 1.916817 2.461364 2.003026 2.579568 2.087923 16.9164 7.986433 5.654762 3.48069 3.021987 2.566768 2.194215 2.701271 3.519313 3.231556 1.853729 1.270221 2.89959 5.18098 4.495765 4.121522 3.481181 4.31612 4.074042 1.686821 1.438589 1.480896 1.701034 2.472893 6.766298 5.011882 3.462872 1.244366 2.243375 1.85581 1.473716 3.117004 2.591328 3.415926 3.477566 2.499302 2.293159 2.448306 3.938336 1.760276 2.027383 2.05137 3.207641 4.023968 2.75751 1.552547 1.956631 1.842242 6.17216 6.314945 2.777874 2.562492 2.862029 1.264071 1.40422 1.377203 1.167407 1.329792 1.449619 1.644069

0.1 μg/L 1.163981 1.211017 1.248804 1.116518 1.22632 1.163161 1.890077 1.82563 1.860918 1.228416 1.344331 1.19196 13.13503 9.964685 6.185629 2.868488 1.629127 2.418917 1.494427 2.115949 1.525468 1.976804 2.014325 2.261659 2.527621 1.678442 1.667167 1.884557 1.325823 0.93032 1.246716 1.665886 2.073871 1.754397 1.894288 1.593825 6.896136 5.58935 5.593806 3.906367 2.361752 2.487217 1.450245 1.812549 1.754006 1.542004 2.047061 2.231692 1.915931 2.269775 3.005123 2.755374 3.410277 3.23758 4.19899 4.841976 3.631472 3.05958 2.933734 3.293089 8.829376 7.24433 2.20276 1.379675 1.977821 2.994152 3.714772 3.065819 3.896625 3.063105 2.734708 2.631381

1 μg/L 1.696323 1.485309 1.596229 1.499152 1.317652 1.08621 0.683675 0.908694 1.209669 0.898533 1.156006 1.01762 6.59086 8.505008 6.36296 3.646206 2.973108 1.946085 1.688361 2.244517 1.764174 1.648043 1.345816 1.474754 2.113893 1.232183 1.491774 1.423843 1.44989 1.70314 1.478579 1.368271 0.779129 1.120992 1.383077 1.943324 4.330941 3.97036 2.507885 2.548389 1.799348 1.740631 1.585191 2.322212 1.451276 2.158959 2.460084 2.320828 1.845307 1.728793 1.964066 1.507541 2.086348 2.195902 2.58716 2.109329 2.720564 3.517953 2.811286 2.786335 5.526756 4.520035 3.383706 2.843597 2.86072 2.912131 3.62436 3.54457 4.264386 4.257333 4.048479 3.62459

0 μg/L 3.514557 3.632781 3.877227 3.476375 3.044919 2.707888 2.296975 2.125692 1.614394 1.578315 1.454061 1.415881 5.454525 3.752891 3.713311 3.00528 2.38366 2.278852 1.74122 1.732597 1.551355 1.887836 1.745164 1.975603 1.881382 1.769342 2.043023 2.640703 1.837218 1.595498 1.498372 1.590235 1.378095 1.901908 1.453086 1.240868 4.533109 5.349041 3.906038 3.035437 1.925691 1.240432 1.14081 1.172099 1.507934 1.728954 1.385289 1.635584 1.51375 1.309399 2.413539 1.856539 1.400436 1.37283 1.643491 2.326351 1.76491 1.988805 1.71552 1.726757 3.555741 4.93944 2.849457 2.159952 2.117591 1.660586 1.533136 2.167742 1.600402 1.555058 1.381231 1.490898

0.001 μg/L 3.164938 2.714742 3.294577 3.240361 2.463557 2.287519 2.526587 2.455624 2.421 2.752403 3.167411 4.015235 6.768376 6.333811 5.310733 3.163016 2.189944 3.111157 1.721817 2.344142 2.882181 2.717846 3.515683 2.074018 1.820349 1.848886 1.700668 1.670366 2.404766 2.028193 2.292509 2.392111 1.75335 1.469762 1.864321 2.874313 7.543125 6.663749 4.813952 3.852054 2.000244 2.506383 1.967803 2.407437 3.25273 3.147595 2.02086 2.066359 2.005908 1.599254 2.350552 1.826696 3.06013 3.140632 2.975638 3.080768 3.161503 3.439001 2.579527 3.71903 7.659688 7.221884 6.105713 3.712349 1.373856 1.978453 3.506305 5.207103 4.883668 4.416704 3.087693 2.729928

0.01 μg/L 5.175446 7.209059 6.443372 5.968459 5.673421 4.448028 5.360077 5.268993 4.009393 3.543983 2.693718 2.617349 7.537607 6.485698 3.527314 3.309934 1.71154 1.88912 2.395688 2.066695 3.314149 1.978056 2.443861 2.260609 2.369798 1.997159 2.748961 3.112373 2.954325 2.546757 2.860613 3.607759 3.74295 4.458081 4.374249 4.613573 6.850912 5.109487 5.308387 4.694228 2.548205 1.798651 2.752943 2.450158 3.023406 3.242042 3.986086 2.342312 1.746068 1.319696 1.776887 2.363137 2.680879 3.247985 3.444645 3.090392 3.746845 3.96174 3.249944 3.113541 6.556742 5.017605 4.415679 3.105179 2.869065 2.908559 3.99673 3.496381 2.760556 3.757037 3.732801 3.097069

0.1 μg/L 4.24983 5.189183 4.573537 5.136766 3.654517 3.27269 3.968739 3.4221 3.245062 2.928692 2.859294 3.746667 5.943556 5.797639 6.106602 5.156193 5.141954 4.602888 2.126255 2.7855 2.037737 2.490523 4.257646 3.530521 2.876112 2.93624 3.106117 3.271318 3.100501 3.404687 3.28253 3.397091 4.180788 4.286647 3.888153 4.258852 6.806663 7.862207 6.249636 5.185389 3.467139 2.768256 1.564263 2.33944 4.538705 4.051234 3.358895 3.75988 3.759552 3.578738 2.405508 3.250348 3.3951 3.247832 3.15634 3.959999 4.794896 4.27022 2.782158 3.781661 6.625407 6.956713 4.29689 3.275258 4.797637 4.770016 4.961928 4.362264 2.914105 1.672857 3.424063 3.650029

1 μg/L 2.408204 2.828638 2.651512 2.826635 2.91867 3.184196 3.190482 2.716147 2.259493 2.396664 2.455147 2.667888 6.276137 6.009556 6.603431 4.467021 4.706925 4.77071 3.293948 3.500996 3.228839 2.941153 2.511488 2.635822 3.140335 1.946184 2.251761 2.530752 2.903338 1.971465 3.35057 3.343793 2.745113 2.40067 2.353838 3.16767 6.236799 6.455753 4.492202 3.206118 3.237899 3.648793 3.043198 3.127638 2.550501 2.860525 3.486192 3.893447 2.347805 2.052186 2.110883 2.6058 2.352405 2.837855 3.827308 3.863243 3.331814 3.416191 3.748544 3.081141 7.084799 7.737957 5.531342 3.992043 3.87017 3.908512 4.178419 6.446718 3.82876 2.947908 4.576864 2.975654

0 μg/L 2.584915 2.32796 2.838807 2.466969 2.462582 2.371471 2.63734 2.802569 2.861699 3.220043 2.793194 2.786032 13.76131 9.093318 5.305224 4.562098 2.536187 2.378621 1.888508 2.839923 2.472647 2.211354 2.280984 2.655565 3.947683 3.28061 3.521064 3.662951 3.371955 3.618848 4.201468 2.834652 2.205982 2.357598 2.254375 2.70227 5.859397 5.775058 3.646024 2.272913 1.865606 1.81077 1.477657 1.411335 2.692705 2.14929 2.075657 2.409499 2.359551 2.032697 2.358476 2.401654 2.55126 2.764051 4.152976 4.032177 3.840351 4.032856 3.473193 2.870565 5.213127 3.614397 3.173645 2.714591 2.659865 2.455439 2.387591 2.366769 1.987541 1.781198 2.100802 1.989209

0.001 μg/L 3.150574 3.192127 3.604994 2.762287 2.591862 2.498 2.776113 1.925753 2.087491 2.252297 2.226738 1.677658 8.924057 6.725748 4.033625 3.190782 2.91986 2.904826 2.094234 2.036798 2.270977 1.589464 2.594306 2.812672 1.937909 1.924245 1.953115 1.40306 1.276231 1.269645 1.540776 2.097948 1.896517 2.370283 2.470635 2.797483 6.981863 7.345307 6.180092 3.143291 2.953059 2.753735 2.224119 2.109478 2.446503 2.288066 1.892299 2.39661 1.383113 1.411378 1.534538 1.668434 2.889939 3.061419 1.848251 1.918358 1.368841 1.759302 2.122023 1.662853 6.153097 7.255189 6.198776 4.921721 3.048536 2.809527 2.623843 2.684692 2.929293 3.776862 2.62431 2.852315

0.01 μg/L 2.800993 2.453808 2.662871 3.526438 3.289736 3.002099 3.042604 4.129004 4.284856 5.163199 3.849265 3.103779 11.19956 6.840644 5.166063 4.239276 4.359025 3.346805 2.855352 3.910006 3.595461 3.377416 2.829421 2.138899 2.425374 2.512953 4.008503 5.370398 6.348397 4.425651 5.264611 3.082248 3.587497 4.129888 4.751355 4.413395 6.212159 5.438069 6.29919 5.858018 3.153123 2.752352 2.633572 2.420886 1.663734 2.02946 2.324886 2.223048 1.991936 2.518342 3.019233 3.529364 3.836569 4.589134 3.389376 3.937075 4.294444 4.053681 4.425743 5.371785 7.049185 6.24673 3.391423 3.165472 2.307978 3.315577 3.817163 3.054716 3.113206 3.028894 2.820493 3.234267

0.1 μg/L 1.928295 1.313572 2.173019 1.960337 2.367371 2.546525 2.513739 3.085884 3.623866 4.415006 3.116994 2.997211 8.149974 5.476557 3.73676 2.618837 2.442704 2.215037 3.037882 2.826197 2.926874 1.566577 1.800552 2.390109 2.833145 3.123408 4.880066 7.60775 6.623226 6.057516 5.430427 4.943371 4.432866 4.207955 3.964188 4.2482 7.020815 5.533973 3.14188 2.945597 1.849449 1.504582 1.915789 1.263113 1.556638 1.379259 1.889562 2.102816 2.237225 1.913197 3.807523 5.277633 6.32261 6.001977 6.287969 6.450888 5.49739 4.316557 3.879556 3.878014 5.963263 5.180732 3.600419 2.592311 1.812388 1.710128 2.721281 4.488445 3.421329 1.744082 1.615121 1.664723

1 μg/L 4.730265 4.799377 5.347657 5.86388 4.768618 4.304595 4.736199 4.723052 5.100531 5.63107 4.78584 3.734695 9.481107 8.236449 7.209599 5.180716 6.7271 3.704364 4.225134 3.407757 2.67544 2.798066 3.354545 3.698493 3.684454 2.600764 3.113597 3.374401 3.737436 3.783 2.852187 2.779095 3.67117 3.152084 3.289499 3.028375 7.18024 6.975643 6.72083 4.594631 4.942543 4.135136 2.73484 4.124032 2.691979 2.140513 2.260575 1.903474 2.69205 2.244516 4.110515 6.389972 5.786323 5.31201 4.658005 5.19781 3.639353 5.008493 3.966693 3.75913 5.360827 5.760351 5.139524 4.190967 3.3812 2.138317 2.750221 3.70329 2.913976 3.77849 4.336977 2.904287

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0 μg/L 2.886657 2.358449 2.193984 1.384431 1.373069 1.795282 2.093299 1.771583 1.609143 1.416285 1.223442 1.650953 12.55899 4.869178 3.689626 3.250424 2.425757 1.795195 1.457748 2.133145 1.688268 1.882117 2.132217 1.879808 2.081939 2.224629 2.688132 3.297604 2.446941 2.838797 3.576065 3.671236 2.931169 2.908485 2.632874 2.151974 5.078851 3.079855 2.63877 2.851213 2.560388 2.87643 1.528809 1.897737 1.675935 1.586168 1.918176 2.184642 1.94721 2.879723 2.499425 3.677339 4.079483 4.375287 3.779602 2.212264 1.766552 2.648355 2.628719 4.304175 5.568272 4.345471 3.735428 2.168544 1.240419 1.734982 1.843853 1.767176 2.478255 2.076904 2.000496 1.989453

0.001 μg/L 3.085822 2.25135 1.495718 1.655078 3.212018 2.774719 1.873414 2.335589 2.283066 1.883535 2.878263 3.301908 8.724943 5.496047 4.842238 2.103072 2.38607 1.960007 1.967517 1.759793 1.416428 2.031909 1.474035 1.012368 3.680735 2.663791 2.898179 2.530853 2.791989 1.706782 2.216087 3.405421 2.813997 1.958929 3.967649 3.673892 7.733969 4.321527 1.938964 1.439257 2.092047 2.450581 1.921628 2.169314 1.335805 2.338196 1.774107 2.042014 1.474519 1.595839 1.991654 2.539865 2.580467 3.09231 3.395875 2.545992 1.806542 3.193921 5.001843 2.797221 6.67312 3.265164 1.992586 1.848093 1.573203 2.039018 2.393527 1.902302 1.506419 2.065001 2.985677 1.72071

0.01 μg/L 2.770576 3.188998 2.965858 3.415502 4.6581 3.778295 4.358406 5.125689 3.93534 3.834663 3.170135 3.624395 8.237855 8.207407 7.91303 6.692224 3.67803 2.788148 2.183829 2.746553 3.129178 3.409016 4.047015 4.841298 2.829596 3.272401 5.420213 6.629197 7.07989 7.524112 6.141389 5.959031 5.772897 4.000588 2.974797 3.85316 7.762615 8.452451 7.295587 3.518592 5.083796 5.464049 3.137536 3.226172 2.668915 3.210593 2.260005 5.042862 2.454572 3.6681 4.553903 5.628228 4.066765 2.483543 2.532173 3.503809 4.242887 6.916318 6.4618 4.217025 10.59578 9.236606 7.079545 4.149091 2.534304 4.473084 3.635436 2.726126 4.954456 5.156504 6.080809 4.604154

0.1 μg/L 3.18434 3.036165 3.611949 3.085975 2.489988 2.86422 2.453468 2.531094 2.290955 2.485991 1.55244 1.447203 8.704394 2.410098 3.652196 3.813271 4.196211 2.620694 2.406177 1.691312 1.855186 1.629999 1.446986 2.18383 1.961695 1.650249 2.228569 2.005049 3.467006 2.876925 1.876904 2.196016 1.807566 2.452589 1.687111 1.472309 3.530625 3.881584 4.385098 4.756113 3.460338 2.872784 2.363598 1.933029 1.791841 2.039749 2.81334 3.040649 2.414027 1.495621 2.546417 2.66898 1.604626 2.012334 2.601705 2.899575 3.068357 3.26045 2.365069 3.240309 5.153588 4.230783 4.28625 2.018323 1.6023 2.291754 1.502702 2.261053 2.355026 2.808384 3.628456 2.648244

1 μg/L 2.203903 1.909665 1.648143 1.6542 1.874863 1.717632 1.428613 1.410177 1.28606 1.416122 1.694152 1.281997 5.864718 6.403883 6.691598 4.251814 3.967044 2.77921 1.901394 1.689597 1.719883 2.189367 2.468502 1.850564 2.616347 1.594877 1.574278 2.37594 2.164961 1.980006 2.233211 2.054211 1.404661 2.679324 3.576632 3.33089 6.515571 4.598858 4.514015 4.575517 4.81409 2.38072 1.333906 1.598219 1.948636 3.231919 3.135887 3.041639 3.156041 2.142596 2.140021 3.994612 3.239404 3.384056 2.466134 2.889822 2.87832 3.811277 2.447471 1.951583 5.829374 5.486755 4.563913 3.715612 2.577017 2.838617 4.015828 3.847726 3.946685 3.921945 2.050595 2.290551

0 μg/L 3.514557 3.632781 3.877227 3.476375 3.044919 2.707888 2.296975 2.125692 1.614394 1.578315 1.454061 1.415881 5.454525 3.752891 3.713311 3.00528 2.38366 2.278852 1.74122 1.732597 1.551355 1.887836 1.745164 1.975603 1.881382 1.769342 2.043023 2.640703 1.837218 1.595498 1.498372 1.590235 1.378095 1.901908 1.453086 1.240868 4.533109 5.349041 3.906038 3.035437 1.925691 1.240432 1.14081 1.172099 1.507934 1.728954 1.385289 1.635584 1.51375 1.309399 2.413539 1.856539 1.400436 1.37283 1.643491 2.326351 1.76491 1.988805 1.71552 1.726757 3.555741 4.93944 2.849457 2.159952 2.117591 1.660586 1.533136 2.167742 1.600402 1.555058 1.381231 1.490898

0.001 μg/L 2.802766 2.132673 1.786392 2.441239 1.851054 1.876852 2.012799 1.542062 1.572664 1.767084 1.956638 1.461864 5.158261 4.318319 4.89932 2.680819 1.672357 1.840527 1.302268 1.562264 1.537292 1.272431 2.212563 1.683741 2.176561 1.608925 1.295619 1.337111 1.495495 1.183312 1.412626 1.559885 1.245325 1.308645 1.370885 1.409058 4.582001 4.731584 2.394686 3.407227 2.486188 1.603606 2.097627 1.307405 1.174648 0.88664 0.789912 0.994524 1.699173 1.157817 1.261018 1.773737 1.906856 1.160774 1.118074 1.340215 1.336274 1.87206 1.357325 1.084769 2.756899 3.349284 3.580445 3.651704 2.22574 1.905697 2.066896 1.889786 1.452143 1.108975 1.381865 1.383401

0.01 μg/L 5.923654 5.902591 5.206103 5.205155 4.475828 4.64305 4.158957 4.325603 3.317397 2.931112 1.826624 2.266145 4.196769 4.156652 4.229341 5.387233 5.166132 3.654544 3.466245 3.189554 3.606925 3.245761 3.195076 3.413253 2.646782 3.243229 3.845643 4.135935 3.868857 4.462014 4.812579 4.17294 3.356503 2.546377 2.386864 3.082013 5.067458 4.798817 5.657226 5.097352 6.129039 5.085567 2.406183 2.107252 3.682451 2.741298 3.459312 4.044427 2.037365 1.850508 2.673664 2.951049 2.467496 1.584916 2.628012 2.850149 3.01852 3.269957 3.448614 2.125893 6.089345 5.981928 6.151008 4.822267 4.189969 3.453632 2.262097 2.927017 3.559118 2.778034 3.45251 2.751442

0.1 μg/L 3.981167 4.587594 4.601412 4.916419 5.407628 4.79702 4.426127 3.564415 3.523781 3.617072 3.190598 3.19505 6.597182 6.835163 5.28773 3.663031 3.093658 3.198204 2.29913 2.453947 3.353883 3.303749 3.657744 2.392885 2.265052 2.188872 3.028002 3.7827 4.112115 4.098368 4.040465 3.951667 3.05058 2.572222 2.765619 2.751531 4.065198 3.601445 4.67696 3.215589 4.883015 4.158348 3.635069 3.048674 2.933318 3.294792 2.562516 1.64046 2.016034 1.874914 2.220704 2.946308 2.897185 2.807033 3.261533 2.90031 3.35155 2.678242 2.707023 2.463322 4.767946 4.99878 4.065822 3.170888 2.78118 3.179342 2.579637 2.17606 2.340678 2.539458 2.911596 3.056336

1 μg/L 4.111845 4.325152 4.283489 4.335209 3.557773 3.605 3.312629 3.639018 3.43844 2.757141 3.19092 2.85745 4.257591 3.376116 3.800947 4.862215 3.584478 3.58538 3.032001 1.450583 1.121761 1.586999 1.915054 1.714542 2.478485 1.913833 2.323272 3.406272 3.727116 4.266779 3.243123 2.672344 2.500197 3.308854 1.840684 1.446664 3.948576 2.804944 2.965785 3.404058 3.279791 3.974805 2.510556 2.38065 2.242053 1.821276 1.819673 2.10179 2.277446 1.887879 2.506023 2.445874 2.843503 2.579745 2.979973 2.893653 3.094203 3.246648 3.447303 2.341118 5.143799 4.757556 3.585086 3.237113 3.017128 1.894729 1.774488 1.361428 1.733433 3.469443 3.224382 1.921899

0 μg/L 2.584915 2.32796 2.838807 2.466969 2.462582 2.371471 2.63734 2.802569 2.861699 3.220043 2.793194 2.786032 13.76131 9.093318 5.305224 4.562098 2.536187 2.378621 1.888508 2.839923 2.472647 2.211354 2.280984 2.655565 3.947683 3.28061 3.521064 3.662951 3.371955 3.618848 4.201468 2.834652 2.205982 2.357598 2.254375 2.70227 5.859397 5.775058 3.646024 2.272913 1.865606 1.81077 1.477657 1.411335 2.692705 2.14929 2.075657 2.409499 2.359551 2.032697 2.358476 2.401654 2.55126 2.764051 4.152976 4.032177 3.840351 4.032856 3.473193 2.870565 5.213127 3.614397 3.173645 2.714591 2.659865 2.455439 2.387591 2.366769 1.987541 1.781198 2.100802 1.989209

0.001 μg/L 1.680334 1.642772 1.489292 1.077563 1.220264 1.631924 1.5909 2.096195 2.050051 2.420758 1.824954 1.952959 6.127786 2.795202 3.640484 2.320575 1.7581 1.961366 1.224366 2.13346 2.305499 2.305021 1.886294 1.091381 2.978366 3.117317 2.667113 2.604139 2.559452 2.937127 3.256728 2.02343 1.520255 1.803241 1.693033 1.173013 5.052869 3.746728 2.456487 1.560378 1.615854 1.819865 3.13407 1.635647 1.43259 0.846428 1.552842 1.398575 2.62265 2.189201 1.833319 1.713933 2.400865 2.314861 2.648458 1.032509 1.248275 1.177263 1.322544 1.034096 3.677971 3.269142 3.484437 2.974069 2.617986 1.092031 1.201389 1.393378 1.364285 1.494941 1.749638 1.43432

0.01 μg/L 2.984699 3.050923 2.448697 2.745155 2.612186 2.976808 2.108726 2.009796 2.289205 3.069539 2.893203 2.766159 8.04794 6.494278 7.133858 4.172294 4.635927 3.941811 3.562428 4.155106 3.603227 3.370213 3.715649 3.53984 3.966571 2.769848 2.96423 2.7626 3.303661 2.639672 4.0966 3.929952 5.462356 6.842263 4.819398 4.144749 7.521066 6.698725 8.39647 5.519163 4.862463 3.183162 3.30264 3.590443 2.582386 3.861909 3.972409 4.322023 2.489702 1.8685 2.710002 2.08862 3.219717 3.910256 3.596953 3.986203 4.702229 2.661794 1.989062 3.729968 7.294498 6.613517 6.051192 3.474575 3.953412 4.659658 3.884201 4.164467 2.723494 2.849251 1.550796 1.824608

0.1 μg/L 3.50284 3.740867 3.093441 3.230594 3.382573 3.355146 3.174247 3.297041 3.839125 3.075467 3.172477 3.945605 7.327709 6.145721 4.80255 5.065776 4.627337 3.486022 3.199979 2.245308 4.031852 2.016779 1.683955 2.346263 2.099792 1.957833 2.446205 2.492965 3.011272 3.378822 3.940406 4.345518 5.102282 4.606535 4.796679 3.103111 5.423916 5.938974 4.709045 3.244145 3.335591 2.948864 1.719541 2.540845 2.481268 2.894635 2.239782 1.795465 2.502642 2.121276 2.163802 1.763275 1.777506 2.295873 2.61513 3.120597 2.533864 2.027785 1.844837 2.012794 4.903151 5.784673 5.528052 5.240993 4.056736 3.181908 2.559281 1.629876 1.581327 3.057748 2.778537 2.750717

1 μg/L 3.109494 3.248224 2.328123 1.820493 1.741201 2.00748 2.354445 1.639682 2.367582 1.503149 1.854425 1.676052 10.34218 8.936402 8.576127 6.291801 4.973865 4.527531 3.226555 2.697197 2.639029 2.803045 2.313552 1.7446 2.710339 1.710004 1.817857 1.871784 2.209339 2.197105 2.112358 2.017805 1.909762 2.244892 2.393267 2.7424 4.339177 3.800965 3.643107 4.87574 3.00458 3.496113 2.943351 2.798777 2.621644 1.647077 2.620491 1.741969 2.782338 1.447062 2.029792 2.20495 2.618721 2.034971 2.672353 2.278564 2.207352 2.303021 1.722964 1.90122 3.028189 2.827517 3.275928 3.68283 2.749129 3.392163 2.421227 1.813181 1.740813 2.386876 1.747098 2.049238

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Figure 4 powerpoint