Effects of Cd injection on osmoregulation and stress indicators in freshwater 3 Nile tilapia 4 5

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CBP ms.23614 Revised – part C 1

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Effects of Cd injection on osmoregulation and stress indicators in freshwater 3

Nile tilapia 4

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Sofia Garcia-Santos1*, Sandra Monteiro1, Salman Malakpour-6

Kolbadinezhad2, António Fontaínhas-Fernandes1, Jonathan Wilson2 7

8 1 Centre for the Research and Technology of Agro-Environmental and Biological Sciences, 9

CITAB, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, 5000-801 Vila 10

Real, Portugal 11 2 Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Rua dos Bragas 289, 12

4050-123 Porto, Portugal. 13

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*Corresponding author 15

Address: DeBA, UTAD, Apartado1013, 5001-801 Vila Real 16

Telephone: +351 259 350 245; 17

E-mail address: [email protected] 18

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Abstract 20

Freshwater tilapia (Oreochromis niloticus) were intraperitoneally injected with sublethal doses 21

of cadmium (1.25 or 2.5 mg Cd kg-1 body mass) and sampled after 1, 4 and 7 days in order to 22

evaluate the mechanisms of Cd toxicity at physiological and biochemical levels. Cd levels were 23

significantly elevated in the gill and kidney following injection however levels in kidney 24

continued to accumulate while in gills levels either did not change or decreased with time. Cd 25

caused a generalized stress condition as indicated by an increase in blood glucose, lactate and 26

cortisol levels as well as an oxidative stress indicated by increases in lipid peroxidation and 27

protein carbonyl content. Furthermore, tilapia exhibited impairment in their osmoregulatory 28

status based on the fall in plasma sodium levels. Concerning ion regulatory disruption, the 29

kidney was the most affected organ since there was a generalized increase in renal Na+/K+-30

ATPase activity after 1 day of exposure to Cd followed by a significant decrease by day 7. This 31

study provides some insights into the mechanisms of Cd toxicity at physiological and 32

biochemical levels and complements previously reported findings on O. niloticus. The 33

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disruption of ion homeostasis, alterations in Na+/K+-ATPase activity and oxidative damage are 34

effects of Cd exposure that can be integrated in a comprehensive model for Cd impacts. 35

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Keywords: Cadmium; Osmoregulation; Stress; Oreochromis niloticus 37

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1. Introduction 39

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Cadmium (Cd) is one of the most toxic metals in the environment. It is mainly produced as a by-41

product from mining, smelting and refining sulfide ores of zinc, coal combustion, electroplating 42

processes, and the production of iron, steel, pigments, fertilizers and pesticides (USEPA, 2001). 43

In freshwater, total dissolved Cd is usually less than 0.5 µg L-1 (Pan et al., 2010). In Europe, 44

levels of Cd in stream water range from < 0.002 µg L-1 to 1.25 µg L-1 for the most pristine sites 45

and contaminated sites, respectively (Pan et al., 2010). However, within the aquatic environment 46

Cd can reacts with other constituents and be removed from solution into sediments. In fact, 47

Lawrence et al. (1996) in a multi-year lake dosing experiment demonstrated that less than 1% of 48

the Cd input remained in the water column. Continuous Cd input into the aquatic environment 49

builds reservoirs of Cd in sediment that may be released back into the water column when 50

aquatic loadings are reduced (Stephenson et al., 1996). 51

In freshwater fishes, Cd accumulates maximally in the kidney, gills, liver and gut, to a lesser 52

extent in the blood, but not significantly in the brain or muscle, although the pattern of 53

accumulation differs depending on the exposure route (McGeer et al, 2011). This biologically 54

nonessential metal is regarded as a potential risk for fishes and other aquatic organisms. In 55

fishes, Cd can exert a wide range of pathological effects including oxidative damage within 56

tissues (Almeida et al., 2001, Cao et al., 2012, Roméo et al., 2000 and Shi et al., 2005). 57

Specifically, Cd may trigger redox reactions that generate free radicals and reactive oxygen 58

species (ROS) (Cao et al., 2012), resulting in lipid peroxidation (LPO) and protein carbonyl 59

(PCO) formation which are two important indicators of oxidative damage of macromolecules 60

induced by ROS (Shi et al., 2005). 61

In aquatic animals, Cd exposure has been shown to change plasma stress parameters (i.e 62

cortisol, glucose) (Garcia-Santos et al., 2011, Lin et al., 2011 and Pratap and Wendelaar Bonga, 63

1990), interfere in ion regulation (Firat and Kargin, 2010, McGeer et al., 2000 and Pratap et al., 64

1989), inhibit enzyme activities (Lionetto et al., 2000 and Sastry and Subhadra, 1985), and cause 65

skeletal deformities and calcium (Ca) balance disturbances (Kessabi et al., 2009 and Muramoto, 66

1981). In fact, Cd exposure has been associated with disruption of important ions like Ca2+ and 67

Na+. For example, waterborne Cd exposure of rainbow trout at 3 µg L-1 resulted in significant 68

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reductions in whole-body Na+ and Ca2+ levels over the first 4 days of exposure (McGeer et al., 69

2000). Similarly, a 10 µg L-1 Cd exposure of tilapia (O. mossambicus) resulted in reductions in 70

plasma Na+ and Ca2+ (Fu et al., 1990). Moreover, Cd has been shown to cause 71

morphopathological changes of varying severity in different fish organs (Garcia-Santos et al., 72

2007, 2006, Giari et al., 2007, Liu et al., 2011 and Thophon et al., 2003). 73

In their natural habitat, fish would be exposed to harmful substances in the water through 74

respiration, contact and feeding. Uptake via these routes is subject in variable biotic and abiotic 75

factors. In the present study, Cd was administered via intraperitoneal injection. Although 76

intraperitoneal metal injection does not mimick realistic environmental exposure, it has been 77

employed in fish studies to investigate the direct effects of cadmium (Sparus aurata, Garcia-78

Santos et al., 2011; Dicentrarchus labrax, Roméo et al., 2000; Lithognathus mormyrus, 79

Yudkovski et al., 2008; Oncorhynchus mykiss, Castaño et al., 1998; Pagrus major, Kuroshima, 80

1995). However, with this method fish receive a well defined Cd dose and the confounding 81

factors in metal uptake are avoided, making it possible to directly interpret changes in 82

physiological parameters. The sublethal Cd concentrations used in this work were selected based 83

on published studies in other fish species (Sparus aurata: 1.25 mg Cd kg-1, Garcia-Santos et al. 84

2011; 2.5 mg Cd kg-1; Vaglio and Landriscina, 1999, Oncorhynchus mykiss (0.5, 1, 2 mg Cd kg-85 1, Castaño et al., 1998) and Dicentrarchus labrax (LC50 3 mg kg-1; Roméo et al., 2000). 86

Nile tilapia (Oreochromis niloticus) belong to one of the most important groups of fishes, and 87

are recognized as good biological models, due to their ease in handling, culture, and 88

maintenance in the laboratory (e.g. Almeida et al., 2001 and Wu et al., 1999). The present work 89

was conducted to determine the impact of Cd on physiological stress, and osmoregulatory 90

performance of tilapia, O. niloticus. For this purpose, this study was conducted as a 91

multivariable approach examining osmoregulatory variables (gill and kidney Na+/K+-ATPase 92

expression and plasma ion levels), metabolic indicators (plasma lactate, triglycerides and 93

glucose values), stress response indicators (plasma cortisol, heat shock proteins - HSPs, lipid 94

peroxidation - LPO and protein carbonyl - PCO formation) and cell proliferation markers 95

(proliferating cell nuclear antigen - PCNA expression). 96

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2. Material and Methods 98

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2.1. Fish 100

Nile tilapia, O. niloticus, Linnaeus (1758) were raised in the Aquaculture Station of the 101

University of Trás-os-Montes and Alto Douro (UTAD, Vila Real, Portugal) in 600 L aerated 102

tanks supplied with a continuous flow (5 L min-1) of dechlorinated tap water (from the 103

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university) whose quality parameters (84/449/EEC Directives, Annex 5, method c1) were 104

maintained by mechanical and biological filtration (pH 6.5-7.5; dissolved oxygen, 89%; 105

alkalinity, 38.15 mg L-1 as HCO3; conductivity, 63 mS cm-1; hardness, 48.45 mg L-1 CaCO3; 106

Na+, 21.10 mg L-1; K+, 5.5 mg L-1; Ca2+, 7.2 mg L-1; Mg2+, 7.4 mg L-1; Cl-, 34.30 mg L-1; NO3-, 107

99.75 mg L-1; NO2-, n.d.; suspended solids, n.d.). Fish were maintained under a controlled 108

photoperiod of 12 h:12 h (dark:light) and were fed daily to satiation with commercial fish food 109

(Aquasoja-Sorgal, Ovar, Portugal: fiber 1.9%, lipid 4.3%, crude protein 37.2%, Ca2+ 2.2%, P 110

1.4% and vitamins A, C, D3 and E). 111

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2.2. Experimental design and sampling 113

Before the experiment, tilapia (n=120, 28.6 ± 1.5 g body mass) were randomly divided into one 114

of four groups in triplicate (Ctr0, Cd0, Cd1.25, Cd2.5), in 200 L recirculating tanks with the 115

same water and photoperiod conditions as used in the stock tanks. 116

After one week of acclimation, the fish were fasted for 24h, anaesthetized with 2-117

phenoxyethanol (0.5mL/L), weighed and injected intraperitoneally with cadmium solutions 118

(1.25 and 2.5 mg Cd kg-1 body mass in the form of CdCl2 in 0.9% NaCl) or vehicle alone (0.9% 119

NaCl) as a sham control (Cd0) and returned to the aquaria. The Ctr0 group was not subjected to 120

any treatment and was used to evaluate possible manipulation effects. 121

At days 1, 4 and 7, ten animals per treatment were anaesthetized with 2-phenoxyethanol (1 mL 122

L-1 water) and blood was collected by caudal puncture using heparinized needles and syringes. 123

Plasma was separated by centrifugation (3 min at 10,000 g) and aliquots were immediately 124

frozen in liquid nitrogen and stored at -80 ºC. Fish were euthanized by decapitation and from 125

each, small pieces from the posterior portion of the kidney and the gill arches were taken using 126

fine-point scissors. The tissues were placed in SEI buffer (300 mM sucrose/20 mM EDTA/50 127

mM imidazole, pH 7.5), and frozen at -80 ºC for later Na+/K+-ATPase activity measurement. 128

Livers were excised and their wet mass obtained to calculate the hepatosomatic index (HSI). 129

Larger samples of gill and kidney were taken, and frozen directly in liquid nitrogen and stored at 130

-80ºC for Cd concentration determination, immunoblotting, and for assessing oxidative stress 131

parameters. Additionally, a sample of white muscle was taken to analyze the percentage of 132

water in this tissue. 133

During the experiment no mortality was observed and the described experiments complied with 134

European Guidelines (86/609/EU) for the correct use of laboratorial animals. 135

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2.3. Analytical techniques 137

2.3.1. Muscle water content, Condition factor (K), Hepatosomatic index 138

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Muscle water content was determined by subtracting the dry mass of the sample (70ºC for 2 139

days) from the initial wet mass and dividing the product by the initial wet mass. 140

Condition was determined according to Abowei et al. (2009). K= 100M/L3, where K= condition 141

factor; M = fish mass (g); L= length of fish (cm). 142

The hepatosomatic index (HSI) was calculated according to Barton et al. (2002). HSI = (Liver 143

mass (g) / Body mass (g)) × 100. 144

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2.3.2. Metal analysis 146

To measure Cd accumulation, the tissues (gill and kidney) were first dried at 60 ºC for 24 h until 147

a constant mass was reached. Samples were then transferred into glass tubes and digested in a 148

1:2 perchloric acid and nitric acid (Merck) mixture at 120 ºC. After complete digestion (visual 149

inspection), samples were cooled and diluted with distilled water to be within the range of 150

standards that were prepared from stock standard solutions of Cd (Merck). All samples were 151

completely clear and not filtered. Cd concentrations, expressed as µg Cd g-1 of dry mass, were 152

determined by atomic absorption spectrophotometry (Unicam 939, Kassel, Germany). 153

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2.3.3. Plasma analysis 155

Glucose, lactate, triglyceride and calcium levels were measured with commercial kits from 156

Spinreact (Cat # 41010, 1001330, 1001310 and 1001060, respectively; Sant Esteve de Bas, 157

Girona, Spain) adapted to 96-well microplates. Plasma sodium (Na+) levels were determined in 158

diluted samples (1:500) by flame photometry (Jenway, UK). 159

Plasma cortisol levels were measured by Enzyme Linked ImmunoSorbent Assay (ELISA), using 160

a kit (Neogen ® Corporation) following extraction with diethylether. 161

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2.3.4. LPO and PCO content assays 163

Oxidative damage to lipids and proteins were evaluated according to Ferreira et al. (2008). 164

Briefly, gill samples were homogenized in ice-cold 100 mM potassium phosphate buffer, 1 mM 165

Na2EDTA, pH 7.5 and the supernatants were obtained by centrifugation. Lipid peroxidation was 166

measured as the amount of malondialdehyde (MDA) determined by the thiobarbituric acid-167

reactive substance (TBARS) method. Tissue homogenate was incubated with trichloroacetic 168

acid (TCA) 100%, and after centrifugation the supernatant was incubated at 100°C, for 30 min, 169

with thiobarbituric acid (TBA) 1%, NaOH 0.05 mol L−1 and butylated hydroxytoluene (BHT) 170

0.025%. The absorbance was measured at 532 nm. Lipid peroxidation (LPO) is expressed as 171

mmol MDA g-1 of tissue. For protein carbonyl determination, the homogenate was reacted with 172

1 2,4-dinitrophenylhydrazine (DNPH) in hydrochloric acid (HCl) for 1h at room temperature, in 173

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the dark, and precipitated with 20% TCA. After centrifugation, the pelleted protein was washed 174

twice in ethanol/ethylacetate (1:1), then resuspended in a guanidine hydrochloride solution and 175

incubated at 37 ºC until complete dissolution. The carbonyl content was measured 176

spectrophotometrically at 370 nm. Blank tubes were also incubated with 2N HCl without DNPH 177

for each sample. The results are expressed as nmoles of carbonyl mg-1 protein using the molar 178

extinction coefficient of 22000 M cm−1. All assays were performed in duplicate. 179

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2.3.5. ATPase assay 181

Gill and kidney Na+/K+-ATPase activities were measured using a microplate technique 182

developed by McCormick (1993) and described by Garcia-Santos et al. (2011). Briefly, gill and 183

kidney tissues were homogenized in SEID buffer (SEI buffer with 0.1% deoxycholic acid; 184

Sigma) and centrifuged at 5000 g for 30 s. The homogenates were pipetted into a 96-well plate 185

and each sample had two wells containing a solution with ouabain (1.0 mM; Sigma-Aldrich 186

Chemical Co., St. Louis, MO, USA) and two wells containing an assay mixture without 187

ouabain. The kinetic assay was read at 340 nm for 10 min with intermittent mixing. Ouabain 188

sensitive ATPase activity was detected by the enzymatic coupling of ATP dephosphorylation to 189

NADH oxidation. The assay was performed in a microplate reader (EL-340i, Bio-Tek 190

Instruments) using DeltaSoft3 software for Macintosh (BioMetallics Inc.). 191

Protein concentrations were measured using the method of Bradford (1976) adapted for 192

microplates, with a bovine serum albumin (BSA) standard, and activity expressed as µmol ADP 193

mg-1 protein h-1. 194

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2.3.6. Western blotting 196

For western blotting, proteins were prepared from gill and kidney tissue and analysis was done 197

as described by Garcia-Santos et al. (2011). Briefly, tissues were homogenized as described for 198

the ATPase assay and prepared in Laemmli’s buffer (Laemmli, 1970). Before loading onto gels, 199

the protein concentrations were adjusted to 1 mg mL-1. Samples were separated by SDS-PAGE 200

using a MiniProtean III system (Bio-Rad) and then equilibrated in transfer buffer and protein 201

bands transferred to polyvinylidenefluoride (PVDF) membranes, using a semidry transfer 202

apparatus (Bio-Rad). The membranes were rinsed in TTBS (0.05% Tween-20 in Tris buffered 203

saline, pH 7.4) and blocked with 5% powdered skim milk in TTBS. Following rinsing, 204

membranes were probed with the primary antibody diluted in 1% BSA in TTBS. After another 205

series of rinses, membranes were incubated with a horseradish peroxidase-conjugated secondary 206

antibody (goat anti-rabbit or anti-mouse) diluted in TTBS. Positive controls have been run on 207

separate membranes using the same antibodies. The blots were detected by chemiluminescence 208

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(Immobilon Milipore) using an imager (LAS4000mini Fuji-Film, Tokyo Japan). Images were 209

imported into an image analysis software program for band semi-quantification (SigmaScan Pro, 210

Image analysis, Version 5.0.0, SPSS Chicago, IL, USA). 211

Na+/K+-ATPase α-subunit was detected using the panspecific α5 mouse monoclonal antibody 212

(Takeyasu et al., 1988). The antibody was obtained as culture supernatant from the 213

Developmental Hybridoma Bank, University of Iowa, Iowa City, under contract N01-HD-7-214

3263 from the National Institute of Child Health and Human Development. We have already 215

used this antibody in different studies of teleost fishes (Garcia-Santos et al., 2011, 2006 and 216

Wilson et al., 2004). The proliferating cell nuclear antigen (PCNA) was detected using a mouse 217

monoclonal antibody (clone PC10; Abcam, Cambridge, UK) that has previously been shown to 218

react with teleost fish PCNA (e.g. Dang et al., 2000, Garcia-Santos et al., 2011 and Monteiro et 219

al., 2009;). Heat shock protein (HSP70) was detected with a mouse monoclonal antibody (clone 220

BRM-22; Sigma), which has been used before in teleost fish tissues (e.g. Burkhardt-Holm et al., 221

1998, Garcia-Santos et al., 2011). 222

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2.4. Statistical analysis 224

Data are expressed as means ± SEM. Prior to statistical analysis, normality and homogeneity of 225

variance were assessed. Differences among groups were tested by two-way ANOVA. When 226

significant differences were obtained, multiple comparisons were carried out using the post hoc 227

Student Neuman Keuls (SNK) test or the non-parametric equivalent (Kruskal-Wallis two-way 228

ANOVA on the ranks and Dunn’s test) (SigmaStat 3.0, SPSS). The fiducial limit was set at 229

0.05. 230

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2.5. Ethics statement 232

The animals used in the research described in this paper were treated in accordance with the 233

Portuguese Animals and Welfare Law (Decreto-Lei n◦ 197/96) approved by the Portuguese 234

Parliament in 1996 and with the European directive 2010/63/UE approved by the European 235

Parliament in 2010. Institutional animal approval by CIIMAR/UP and General Veterinary 236

Direction was granted for this study. 237

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3. Results 239

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For all measured parameters there were no differences between sham fish injected with vehicle 241

alone (0.9% NaCl, Cd0) and those not subjected to any manipulation (Ctr0) during the 242

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experiment (data not shown). Since sham injection had no effect we considered the Cd0 as the 243

control group in this experiment. 244

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3.1. Mortality, morphometric parameters and muscle water content 246

No mortality occurred in either control or Cd-treated groups after injection or during 247

experimental time course. The fish condition factor did not change significantly with Cd 248

treatment or time of exposure. In contrast, the hepatosomatic index increased significantly 7 249

days after injection in the 2.5 mg Cd kg-1 group (Table 1). 250

There were no significant differences in muscle water content, an indicator of osmoregulatory 251

status, between exposure groups over time or between any Cd-treatment groups (Table 1). The 252

mean muscle water content was 79.4 ± 0.17%. 253

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3.2. Tissue metal content 255

Cadmium concentrations measured in the gill and kidney of tilapia at 1, 4 and 7 days after i.p. 256

injection are presented in Table 2. The Cd concentrations in tissues from the control animals 257

were similar over the entire experiment period. Cd i.p. injection significantly increased Cd 258

levels in both tissues; however, over time gill and kidney showed differences in Cd level 259

patterns with levels in kidney being almost 10-fold higher than in gill. Kidney was the main 260

accumulation organ, with Cd levels reaching a maximum value of 418.64 ± 14.10 µg g-1 dry 261

mass 7 days after injection of 2.5 mg kg-1 Cd. In contrast, although not statistically different, 262

following i.p. injection gill Cd levels decreased by 37% over time at the higher dose (Cd 2.5) 263

from an initial Cd concentration of 57.04 ± 14.34 µg g-1 dry mass at day 1 or at the lower dose 264

(Cd 1.25) remained unchanged over time. 265

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3.3. Plasma parameters 267

The results presented in Table 3 show that there were statistically significant interactions 268

between the Cd dose and the time after injection for some plasma parameters. The effect of 269

different levels of Cd on glucose and triglycerides concentrations was dependent on time of 270

exposure. Cadmium treatment transiently enhanced plasma glucose concentration after 1 day of 271

exposure, after which it returned to basal levels. Regarding plasma triglycerides, there was no 272

significant effect of Cd treatment after 1 and 4 days of exposure. However at 2.5 mg Cd kg-1, 273

and a longer exposure (7days), triglycerides increased significantly. 274

Four days after injection, plasma lactate levels showed a tendency to increase. However, only 275

the dose of 1.25 mg Cd kg-1 resulted in a statistically significant increase relative to the control 276

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at day 4. After longer exposure (7 days), lactate levels returned to baseline values and no 277

significant treatment effects were noted. 278

Although within each sampling time no differences in plasma cortisol levels were detected 279

among Cd concentrations, the time of Cd exposition significantly affected plasma cortisol 280

levels. In fact, 7 days after the injection (at both concentrations) fish showed a significant 281

increase compared to the earlier exposure time. 282

No significant differences in plasma calcium levels with either Cd treatment or time of exposure 283

were observed. In contrast, plasma sodium levels showed a decreasing trend with Cd treatment 284

at days 1 and 7 after injection. The decrease was statistically significant on both days to 2.5 Cd 285

concentration and only on day 1 for the lower Cd concentration. On day 4, no differences 286

between the different Cd treatments were observed and, the control group showed a significant 287

decrease in plasma sodium levels when compared with those obtained at the other two sampling 288

times. 289

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3.4. Gill oxidative damages 291

Oxidative damage to lipids and proteins were evaluated in the gill of tilapia as a measure of 292

oxidative stress (Fig. 1). Lipid peroxidation products (Fig.1A) increased significantly in fish 293

exposed to the highest Cd concentration 1 and 4 days after injection. At day 7 no significant 294

differences were seen between the groups. 295

Protein oxidation (carbonyl content; Fig.1B) increased with both injected Cd concentrations at 296

days 1 and 4. However, with longer exposition, only the highest concentration maintained a 297

significant carbonyl content elevation compared to the control. Fish exposed to the 1.25 Cd 298

concentration showed a significant decrease in carbonyl levels at day 7, compared to the other 299

sampling times. 300

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3.5. Na+/K+-ATPase activity 302

The activity of gill Na+/K+-ATPase (Fig.2A) did not reveal significant differences among the 303

treatments at the different exposure times and no time-related effects were seen within each 304

treatment. On the other hand, in the kidney (Fig.2B), the effect of the Cd treatment on Na+/K+-305

ATPase activity depended on time of exposure. Tilapia injected with the highest Cd 306

concentration after 1 day showed a significant increase in renal Na+/K+-ATPase activity when 307

compared with the fish exposed to the lower Cd level or the sham control. However, this 308

increase did not remain over time, but rather decreased significantly after 7 days exposure. 309

310

3.6. Immunoblotting 311

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In gill and kidney western blots, the Na+/K+-ATPase α5 antibody immunoreacted with a single 312

band of about 100 kDa. Band intensity quantification indicated that Cd injection had no 313

significant effect on branchial Na+/K+-ATPase abundance (Fig.3A). In contrast, this enzyme's 314

catalytic α-subunit expression significantly increased in kidney after 1 day of exposure to Cd. 315

However, at day 7, the values decreased and no significant differences between the two 316

treatment groups and the control were noted (Fig.3B). 317

The PCNA antibody revealed a single cross-reactive band in the region of 30 kDa in both 318

tissues. The quantification of immunoreactive bands showed that in gill, Cd had a significant 319

effect on PCNA expression. In fact, the control group had a significantly lower expression 320

relative to those exposed to Cd and this was irrespective of exposure time (Fig.3C). However, in 321

kidney the time effect seemed to be most important since at day 7 there was significantly higher 322

expression when compared to the first day of exposure (Fig.3D) which reflects the lower 323

expression in the Cd groups at day 1. 324

In relation to HSP70 expression, the antibody reacted with a band of about 70kDa and the 325

analysis indicated that there were no significant differences among the treatments at the different 326

times of exposition in either kidney or gill (Fig.3E, F). 327

328

4. Discussion 329

330

The sensitivity as well as the nature of the responses to the impact of toxic metals is not uniform 331

among teleost fishes. That variability makes the establishment of safety limits for releasing 332

metals into the environment difficult. Our work provides relevant data that broadens the 333

knowledge about the toxic effects of metal contamination on aquatic animals and complement 334

previously reported findings on O. niloticus, a widely distributed and studied teleost fish. 335

Gross indices can offer information on potential pollutant impacts. Morphological parameters 336

that are often determined in research include the hepatosomatic index (HSI), to identify possible 337

liver diseases, and the condition factor (K), to assess the general condition of the fish (Van der 338

Oost et al., 2003). In the present work, the condition factor did not change significantly with Cd 339

treatment or time of exposure; however, the hepatosomatic index increased significantly 7 days 340

after injection in the 2.5 mg Cd kg-1 group. Although these parameters are not very sensitive and 341

may be affected by non-pollutant factors, they may serve as an initial screening biomarker, since 342

their response varies depending on the level of contaminant exposure. Demonstrated in a 343

previous study with a different species (Sparus aurata, Garcia-Santos et al., 2011), the 344

hepatosomatic index showed a significant increase in response to Cd that could be explained by 345

hyperplasia and/or hypertrophy and it may be associated with higher capacity to metabolize 346

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xenobiotics (Heath, 1995). In fact, in that previous work (Garcia-Santos et al., 2011), an 347

increase in liver cell proliferation in S. aurata exposed to Cd was reported. Further, Liu et al. 348

(2011) found that Cd exposure increased lipid deposits in the liver that may also contribute to 349

the higher HSI. 350

In freshwater fish, Cd accumulates predominantly in the kidney, gills, liver and gut, however, 351

the pattern of accumulation varies depending on the exposure route. During waterborne 352

exposure, the increase in Cd accumulation generally occurs in the order kidney > gills > liver > 353

intestine, whereas in dietary exposure the tissue Cd accumulation follows the order intestine > 354

kidney > liver > gills (McGeer et al., 2012; Szebedinszky et al., 2001; Chowdhury et al., 2005). 355

Our results corroborate these findings and it appears that whatever the route of contamination 356

(water, food, or i.p. injection) Cd accumulation in the kidney is always greater than in the gills. 357

Nevertheless, similar to what happens in dietary exposures (Szebedinszky et al., 2001; 358

Chowdhury et al., 2005), the gills accumulate considerable amounts of Cd after the i.p injection, 359

despite a lack of direct exposure. In fact, contrary to the kidney, the gills showed the highest Cd 360

concentration immediately 1 day after the exposure which then shows a decreasing trend over 361

time. According to this, we suggest that the gills could be a transient target organ of Cd 362

accumulation, and then Cd is transferred to other organs like kidney via the circulatory system 363

or excreted directly. 364

It has been suggested that metal concentrations in the organs of fish, rather than the metal 365

concentrations in the ambient water, could be used as a biomonitor for water pollution in natural 366

freshwaters (Pelgrom et al., 1995). The levels of Cd accumulation measured in the present work 367

are in agreement with others obtained from waterborne exposures (Wu et al., 2007; Firat et al., 368

2009). Firat et al. (2009) investigated the effects of 0.1 mg L-1 and 1.0 mg L-1 Cd on antioxidant 369

parameters and metal accumulation in O. niloticus. In that study, the values of Cd burdens in gill 370

after 7 days of exposure (25.77 ± 1.65 and 37.21 ± 0.49 µg Cd g-1dry mass) were similar to 371

those obtained in the present study using 1.25 and 2.5 mg Cd kg-1 i.p injections, respectively. 372

Furthermore, after 15 days of 0.5 mg L-1 Cd exposure, male hybrid tilapia (Oreochromis 373

niloticus×O. aureus) showed Cd levels in gill and kidney in the range of ours obtained at day 7 374

by i.p. injection (Wu et al., 2007). The waterborne Cd concentrations in the aforementioned 375

studies would be considered above the environmentally relevant values (<10µg L-1). In natural 376

freshwaters, Cd typically occurs at concentrations of less than 0.1 µg/L, however in impacted 377

environments, the concentrations can be several micrograms per liter or greater (USEPA, 2001), 378

particularly when a sudden and short-term discharge occurs into the aquatic environment. 379

Xenobiotic agents, including toxic metals, have the potential to produce reactive oxygen species 380

(ROS) such as hydrogen peroxide, and superoxide and hydroxyl radicals. When the rate of ROS 381

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generation exceeds the rate of its inactivation by antioxidant defenses, an oxidative stress 382

condition is established. Lipid peroxidation (LPO) and protein carbonyl (PCO) formation are 383

two consequences of oxidative stress and are used as biomarkers in monitoring aquatic 384

ecosystems (Abdel-Moneim et al., 2012, Almroth et al., 2005, Farombi et al., 2007 and 385

Valavanidis et al., 2006). Lipid peroxidation results in the production of lipid radicals and in the 386

subsequent formation of a complex mixture of lipid degradation products. These products react 387

with thiobarbituric acid (TBA) yielding a colored intermediate that is commonly used to 388

quantify damage to cellular lipids (Pretto et al., 2011). Protein carbonyl content is the most 389

general indicator and the most commonly used marker of protein oxidation (Dalle-Donne et al., 390

2003). It has been frequently used in research on oxidative stress in humans, however, there are 391

relatively fewer reports concerning its use in fishes exposed to environmental contaminants 392

(Almroth et al., 2005, Parvez and Raisuddin, 2005, Pretto et al., 2011 and Shi et al., 2005). 393

The present study revealed that Cd is responsible for oxidative stress in gill of tilapia as shown 394

by general increase in lipid peroxidation and protein carbonyl content. Our results are similar to 395

those obtained in other experiments with different species or even different contaminants (Dabas 396

et al., 2012, Parvez and Raisuddin, 2005 and Pretto et al., 2011). Interestingly, malondialdehyde 397

(MDA) production was significant only at the higher Cd dose and, at day 7, significant 398

differences between the groups were no longer seen in respect to this parameter. Protein 399

oxidation increased with both injected Cd concentrations at days 1 and 4, however, only the 400

highest concentration maintained a significant elevation with longer exposition. Shi et al. (2005) 401

showed that after 24h, Cd significantly increased lipid peroxidation (LPO) and protein carbonyl 402

(PCO) content at higher concentrations (0.5 and 5 mg L-1 for LPO; 0.05, 0.5 and 5 mg L-1 for 403

PCO) in liver of Carassius auratus. In the liver of Sparus aurata exposed to 0.5 mg L-1 Cd, 404

Souid et al. (2013) observed a significant increase in liver LPO values as well. Further, Dabas et 405

al. (2012) noted a significant increase in thiobarbituric acid reactive substances (TBARS) 406

formed in gill, liver and kidney of Channa punctatus after 96h of waterborne Cd exposure to a 407

range of concentrations (4.1, 8.2 and 12.3 mg L-1 Cd) compared to controls. Among the tissues, 408

the gills showed most LPO damage. In addition, induction of protein carbonyl was highest in the 409

gills amongst all the tissues studied by Parvez and Raisuddin (2005) in C. punctata exposed to 410

waterborne pesticides (deltamethrin, endosulfan and paraquat). 411

Our results demonstrate that the effect of Cd was more marked in PCO than in LPO levels. 412

Similar observations were made by Almroth et al. (2005) and Shi et al. (2005). In fact, 413

according to Stadtman and Oliver (1991), metals are known to cause the formation of protein 414

carbonyls, through metal catalyzed oxidation reactions, and are not as effective in damaging 415

13

lipids. Moreover oxidized proteins are more stable than malondialdehyde and the TBARS 416

method is less sensitive at detecting slight variations of LPO (Fan, 2002 and Shi et al., 2005). 417

Animals increase blood glucose levels and regulate energy metabolism in response to stress (Lin 418

et al., 2011 and Mommsen et al. 1999). Indeed, an increase in plasma glucose levels in fishes 419

under Cd stress was reported by Almeida et al. (2001), Chowdhury et al. (2004), Cicik and 420

Engin (2005) and Garcia-Santos et al. (2013). Corroborating these previous results, in the 421

present work, a significant elevation in plasma glucose was evident on day 1 in cadmium-422

exposed fish. The relationship between increased plasma glucose and cortisol levels is 423

frequently observed following exposure of fishes to water pollutants or other stressors, and the 424

relationship most likely is causal: usually, the primary response (elevated cortisol) leads to the 425

secondary (elevated glucose) via stimulation of gluconeogenesis (Wendelaar Bonga, 1997). Our 426

results contrast with earlier reports on the relationship between these two parameters, since the 427

increase in plasma glucose levels (day 1) apparently preceded the increase in plasma cortisol 428

(day 7) levels. This apparent discrepancy can be explained by the likelihood that there was a 429

peak of cortisol before 24 h which was not detected with the sampling protocol time course 430

used. In most fishes, cortisol reaches its highest concentration 1 h after being stressed and 431

returns to basal levels after 6 h (Iwama et al., 2006). The cortisol-induced gluconeogenesis 432

lagged behind and was detected at 24 h. 433

Another explanation could be the exposure method. Cd injected intraperitoneally is likely to be 434

quickly absorbed by nearby organs and thus made readily available to affect tissues and cells 435

(Costa and Costa, 2008). This would accelerate the timing of changes that occur and allow these 436

results to approach those that would be obtained in longer term exposure experiments where 437

metals accumulate more slowly. Monteiro et al. (2005) in a 21-day experiment analyzing the 438

effect of copper on biochemical parameters of O. niloticus, showed that plasma cortisol levels 439

increased significantly in the first days of exposure, dropped at day 7 (40 µg L-1) or 14 (400 µg 440

L-1) after which cortisol levels rose again. The authors concluded that an early adaptation to 441

stress occurred followed by a sustained decompensation in long-term exposures. The costs of 442

metal-induced stress are associated with three phases of stress: alarm, adaptation and exhaustion 443

(Lin et al., 2011). Considering the cortisol levels in Garcia-Santos et al. (2013) we notice that 444

the control levels after 1 day in the present study were concordant, showing that the injection 445

itself did not cause changes in cortisol levels that could mask a Cd specific effect. 446

Another parameter consistent and associated with a nonspecific stress response was plasma 447

lactate. In the present study plasma lactate was elevated after 4 days of Cd injection. According 448

to De Smet and Blust (2001), levels of both glucose and lactate often increase during the first 449

phase of the stress response due to an elevated breakdown of glycogen, particularly in liver and 450

14

muscle. Furthermore, increased lactate levels after toxic metal exposure are considered to result 451

from anaerobic metabolism caused by gill impairment (Zhang et al., 2013), suggesting possible 452

damage to gas exchange. 453

The exposure of O. niloticus to 2.5 mg Cd kg-1 induced a significant rise in blood triglycerides 454

after 7 days of the experiment corroborating other studies that demonstrated that the blood 455

triglycerides levels were significantly increased in fishes exposed to different metals (Atef and 456

Al-Attar, 2005 and Singh and Reddy, 1990). There are several reports indicating that 457

contaminants influence the thyroid function (Hontela et al., 1996, Levesque et al., 2003 and 458

Ricard et al., 1998). In fact, in a previous work (Garcia-Santos et al., 2013) we demonstrated 459

that waterborne Cd impacted thyroid functions in tilapia, O. niloticus. Since the decrease in 460

thyroid secretion greatly increases triglycerides level in the blood, the observed 461

hypertriglyceridaemia may be due to hypothyroidism induced by cadmium and/or liver 462

dysfunction because the liver is the main center of lipid metabolism (Atef and Al-Attar, 2005). 463

Gills and kidneys are the most important organs responsible for ionoregulation in freshwater 464

teleosts since the diffusional loss of ions and the osmotic influx of water are balanced by 465

absorption of ions in the gills and excretion of large amounts of urine by the kidneys (Marshall 466

and Grosell 2006). Morphology and physiology of the gill ionocytes have been studied 467

extensively, however the ion transport mechanisms in the kidney are less well understood. 468

Immunocytochemical studies demonstrated that Na+/K+-ATPase is located mainly in 469

mitochondrion-rich cells of gill epithelia (Wilson and Laurent, 2002) and epithelia of kidney 470

tubules (Ura et al., 1996) in euryhaline teleosts. 471

Data from the literature have shown that there were considerable variations (inhibition, 472

stimulation or even no changes) in the response of Na+/K+-ATPase activity in fishes exposed to 473

Cd (Garcia-Santos et al., 2013, 2006, Lionetto et al., 2000 and Saglam et al., 2013). In the 474

present work, the abundance of the gill Na+/K+-ATPase α-subunit, like its activity, was not 475

significantly affected by Cd. Most authors, when comparing the differences between tissues, 476

concluded that gill ATPases seem to be more sensitive to metal exposures than ATPases from 477

other tissues, because of structure and position of gill tissue which allows direct contact with the 478

external medium (Atli and Canli, 2013). However, in our study, Cd was injected 479

intraperitoneally, which means that the gills were not the main or the first target organ to the 480

toxic metal effects. Moreover, these results corroborate our previous work (Garcia-Santos et al., 481

2006), where no significant changes in gill Na+/K+-ATPase activity were seen in O. niloticus 482

exposed to waterborne Cd. In that study we concluded that the species was relatively insensitive 483

to the effects of Cd. 484

15

Instead, a generalized increase in renal Na+/K+-ATPase activity was observed after 1 day of 485

exposure to Cd which was positively correlated with its α-subunit protein expression suggesting 486

that alterations in activity were due to variations in the relative abundance of the enzyme. The 487

increase may reflect a compensatory response to the drop observed in plasma sodium levels 1 488

day after Cd injection. That sodium decline could result, in turn, from the slightly (although not 489

statistically significant) decrease in gill Na+/K+-ATPase. The reestablishment of plasma sodium 490

levels, at day 4, would reflect the transient success of the compensatory mechanism. Over the 491

longer term (7 d), the ionic imbalance returns, and is accompanied by a decrease in renal 492

Na+/K+-ATPase activity as plasma sodium values fall. 493

Based on the Na+/K+-ATPase response and plasma sodium level changes, kidney was the most 494

affected tissue concerning ionic disruption and that could be attributed to its important function 495

in osmoregulation. Indeed, although in fishes the gill plays a quantitatively larger role in ionic 496

regulation, the kidney is the primary organ for elimination of excess water. This is particularly 497

important for freshwater species in which efficiency of ion reabsorption mechanisms in this 498

organ minimize the loss of ions, which is a pivotal role (Atli and Canli, 2013). 499

Studies in fishes have demonstrated that several stressors, including pollutants and thermal 500

stress, can induce HSP expression (Iwama et al., 2004). The function of HSP during stress is 501

related to cytoprotection as these proteins can act to prevent and repair protein damage. 502

Although, the HSP response seems to vary with factors such as species, development stage, 503

tissue, stressor, concentration and duration of exposure (Hori et al., 2008), in the present work, 504

there was a slight trend to increase HSP70 after 1 day of exposure to Cd. Similar results have 505

been observed in our previous study in sea bream (Garcia-Santos et al., 2011), suggesting that 506

acutely exposed fish were experiencing a cellular stress and trying to cope with Cd toxicity. In 507

addition, the increased expression of PCNA observed in gill, also suggests a compensatory 508

response to Cd exposure (Dang et al., 2000) with an increase in cell turnover that helps maintain 509

tissue structure and function. 510

Our results provided an insight into the mechanisms of Cd toxicity at physiological and 511

biochemical levels. Studies involving environmentally relevant pollutants in water are usually 512

constrained by variables such as contaminant bioavailability and interactions, and the 513

characteristics of the entry pathway and detoxification (Costa and Costa, 2008). By using the 514

intraperitoneal injection, the present experiment, allows a more objective interpretation of the 515

toxicological effects of Cd, which is fundamental for understanding studies involving 516

environmental xenobiotics tested under laboratory or even field conditions. The disruption of 517

ion homeostasis, alterations in Na+/K+-ATPase activity and oxidative damage are effects of Cd 518

injection that could be integrated into a comprehensive model for understanding Cd impacts. 519

16

520

Acknowledgments. The present work was supported by the Portuguese Foundation for Science 521

and Technology (FCT) through a Ph.D. Grant to SGS (SFRH/BD/22750/2005). This work was 522

partially supported by European Regional Development Fund through the COMPETE - 523

Operational Competitiveness Program and national funds through FCT [PEst-524

C/MAR/LA0015/2011] to JMW. The authors thank Professor Ana Coimbra, colleague Ana 525

Luzio and the technicians Cesaltina Carvalho, and Donzília Costa for the invaluable support and 526

technical assistance in the experimental work. 527

528

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746

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Figure captions 747

748

Figure 1. Lipid peroxidation (A) and protein carbonylation (B) values in gill of tilapia exposed 749

by intraperitoneal injection to different concentrations of cadmium and sacrificed after 1, 4 and 750

7 days. The values are expressed as mean ± SEM (n=10). Comparisons between treatment 751

groups at the same exposure time with different lower cases are significantly different. Different 752

capital letters within each treatment group indicate significant differences between exposure 753

times. (P < 0.05) 754

755

Figure 2. Branchial (A) and renal (B) Na+/K+-ATPase activity of tilapia exposed by 756

intraperitoneal injection to different concentrations of cadmium and sacrificed after 1, 4 and 7 757

days. The values are expressed as mean ± SEM (n=10). All other details are identical to those in 758

Fig. 1 legend. 759

760

Figure 3. Effect of Cd intraperitoneal injection on Na+/K+-ATPase α-subunit, PCNA and 761

HSP70 expression determined by immunoblotting, in gill (A, C, E) and kidney (B, D, F) of O. 762

niloticus, after 1 and 7 days of exposition. The values are expressed as mean ± SEM (n=10) 763

relative to Cd0-1d. All other details are identical to those in Fig. 1 legend. 764 765

22

Figure 1

766

23

Figure 2

767

24

Figure 3

768

25

Table 1: Morphometric parameters and muscle water content of O. niloticus injected with different cadmium concentrations.

Data are presented as mean ± SEM (n=10). Values with different lowercase letters within the same time of exposure (in the same line) are significantly different. Within each Cd treatment, values with different capital letters (in the same column) are significantly different. (P < 0.05, two-way ANOVA, SNK test). Cd0 = fish injected with 0.9% NaCl (control); Cd 1.25 and Cd 2.5 = Fish injected with 1.25 and 2.5 mg Cd Kg-1, respectively. 769

Exposure days

Treatment

Cd 0 Cd 1.25 Cd 2.5

Condition factor (g cm-3)

1d 2.22 ± 0.10 2.11 ± 0.07 2.21 ± 0.16

4d 2.03 ± 0.11 2.21 ± 0.12 2.02 ± 0.09

7d 2.09 ± 0.06 2.35 ± 0.11 2.19 ± 0.11

Hepatosomatic index (%)

1d 0.89 ± 0.06 0.81 ± 0.06 0.86 ± 0.07 A

4d 1.03 ± 0.10 0.93 ± 0.06 1.03 ± 0.10 AB

7d 0.81 ± 0.06 (a) 0.82 ± 0.08 (a) 1.11 ± 0.07 B(b)

Muscle water content (%)

1d 79.37 ± 0.30 79.59 ± 0.32 79.92 ± 0.32

4d 79.83 ± 0.17 79.70 ± 0.19 79.80 ± 0.22

7d 79.68 ± 0.21 79.73 ± 0.19 79.29 ± 0.21

26

Table 2: Concentrations of Cd in µg g-1 dry mass in gills and kidney of tilapia intraperitoneally injected with either saline (Cd 0), 1.25 mg Kg-1 Cd (Cd 1.25), or 2.5 mg Kg Cd-1 (Cd 2.5).

Data are presented as mean ± SEM (n=3). Values with different lowercase letters within the same time of exposure (in the same row) are significantly different. Within each Cd treatment, values with different capital letters (in the same column) are significantly different. (P < 0.05, two-way ANOVA, SNK test). 770

Exposure days

Treatment

Cd 0 Cd 1.25 Cd 2.5

Gills

1d 1.07 ± 0.13 (a) 28.12 ± 1.14 (b) 57.04 ± 14.34 (b)

4d 1.00 ± 0.08 (a) 25.67 ± 8.68 (b) 38.32 ± 7.40 (b)

7d 1.14 ± 0.20 (a) 25.63 ± 6.23 (b) 35.98 ± 8.16 (b)

Kidney

1d 8.24 ± 2.20 (a)

1d

Ctrl 1,4829562 0,393

6

Cd1,25 33,85239013 5,643

9

Cd2,5 41,75478123 8,258

1

4d

Ctrl 1,582831878 0,331

9

Cd1,25 39,7258499 5,430

1 Cd2,5 71,01788141 13,18

7d

Ctrl 1,469178822 0,383

8

Cd1,25 74,17970173 19,22

6

Cd2,5 82,90123781 5,630

8

195.03 ± 37.99 (b) 198.28 ± 51.07 (b)A

4d 8.64 ± 1.43 (a) 167.06 ± 36.62 (b) 283.02 ± 57.69 (b)AB

7d 8.58 ± 2.12 (a)

84,19792498

349.11 ± 92.02 (b) 418.64 ± 14.10 (b)B

27

Table 3: Biochemical parameters measured in plasma of O. niloticus injected with different cadmium concentrations.

Data are presented as mean ± SEM (n=10). Values with different lowercase letters within the same time of exposure (in the same line) are significantly different. Within each Cd treatment, values with different capital letters (in the same column) are significantly different. (P < 0.05, two-way ANOVA, SNK test). Cd0 = fish injected with 0.9% NaCl (control); Cd 1.25 and Cd 2.5 = Fish injected with 1.25 and 2.5 mg Cd Kg-1, respectively.

Exposure days

Treatment

Cd 0 Cd 1.25 Cd 2.5

Glucose (mmol/L)

1d 1.40 ± 0.09 (a) 2.82 ± 0.51 A(b) 2.38 ± 0.17 A(b)

4d 1.80 ± 0.12 1.76 ± 0.07 B 1.83 ± 0.16 B

7d 1.54 ± 0.14 1.58 ± 0.23 B 1.60 ± 0.13 B

Triglycerides (mmol/L)

1d

4d

7d

0.63 ± 0.03 0.71 ± 0.12 0.60 ± 0.04 A

4d 0.89 ± 0.14 0.82 ± 0.10 0.69 ± 0.06 A

7d 0.71 ± 0.03 (a) 0.90 ± 0.08 (a) 1.35 ± 0.22 B(b)

Lactate (mmol/L)

1d 0.42 ± 0.10 0.60 ± 0.08 0.52 ± 0.08

4d 0.45± 0.06 (a) 0.92 ± 0.17 (b) 0.69 ± 0.14 (ab)

7d 0.41 ± 0.07 0.48 ± 0.07 0.40 ± 0.39

Cortisol (ng/L)

1d 34.6 ± 5.0 49.6 ± 14.1 A 38.4 ± 2.8 A

4d 51.6 ±15.9 50.6 ± 7.6 A 38.1 ± 5.5 A

7d 69.2 ± 9.6 136.3 ± 42.6 B 123.8 ± 40.6 B

Sodium (mmol/L))

1d 170.66 ± 11.29 A(a) 146.21 ± 4.52 (b) 138.63 ± 3.02 (b)

4d 135.81 ± 4.83 B 141.57 ± 5.72 150.68 ± 4.07

7d 162.34 ± 5.66 A(a) 149.94 ± 4.51(ab) 141.60 ± 2.45 (b)

Calcium (mmol/L)

1d 2.26 ± 0.30 2.29 ± 0.16 2.16 ± 0.26

4d 2.49 ± 0.39 2.57 ± 0.27 2.53 ± 0.25

7d 2.33 ± 0.24 1.92 ± 0.18 2.58 ± 0.43

Effects of Cd injection on osmoregulation and stress indicators in freshwater 3 Nile tilapia 4 5

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