Accepted Manuscript

Analytical Methods

Feasibility of microwave-induced combustion for trace element determination

in Engraulis anchoita by ICP-MS

Juliana V. Maciel, Camila L. Knorr, Erico M.M. Flores, Edson I. Müller, Marcia

F. Mesko, Ednei G. Primel, Fabio A. Duarte

PII: S0308-8146(13)01221-1

DOI: http://dx.doi.org/10.1016/j.foodchem.2013.08.119

Reference: FOCH 14607

To appear in: Food Chemistry

Received Date: 29 August 2012

Revised Date: 21 November 2012

Accepted Date: 28 August 2013

Please cite this article as: Maciel, J.V., Knorr, C.L., Flores, E.M.M., Müller, E.I., Mesko, M.F., Primel, E.G., Duarte,

F.A., Feasibility of microwave-induced combustion for trace element determination in Engraulis anchoita by ICP-

MS, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.08.119

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1

Feasibility of Microwave-induced Combustion for Trace Element 1

Determination in Engraulis anchoita by ICP-MS 2

3

Juliana V. Maciel,a Camila L. Knorr,b Erico M. M. Flores,b Edson I. Müller,b Marcia F. 4

Mesko,c Ednei G. Primel,a Fabio A. Duartea* 5

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a Escola de Química e Alimentos, Universidade Federal do Rio Grande, 96203-900 Rio 7

Grande, RS, Brazil 8

b Departamento de Química, Universidade Federal de Santa Maria, 97105-900 Santa 9

Maria, RS, Brazil 10

c Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de 11

Pelotas, 96010-610, Pelotas, RS, Brazil 12

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*Corresponding author: +55 53 3233 6967 24

E-mail address: fabioand@gmail.com 25

2

Abstract 26

A procedure based on microwave-induced combustion (MIC) was developed for fish 27

(Engraulis anchoita) digestion and subsequent determination of As, Cd, Co, Cr, Cu, Fe, 28

Mn, Mo, Ni, Se, and Zn by inductively coupled plasma mass spectrometry (ICP-MS). A 29

reflux step (5 min) was applied to improve absorption and recovery of analytes. Nitric 30

acid was investigated as absorbing solution and suitable results were achieved using 5 31

mol L-1 HNO3. Microwave-assisted digestion in closed vessels using concentrated 32

HNO3 was also evaluated for comparison of results. Both sample preparation methods 33

were considered suitable for sample digestion but MIC was preferable not only because 34

diluted HNO3 can be used as absorbing solution but also because it provides higher 35

efficiency of digestion and also better limits of detection. Accuracy was evaluated by 36

the analysis of certified reference materials (DORM-2 and TORT-2) after MIC 37

digestion with subsequent determination by ICP-MS. Agreement with certified values 38

was better than 94%. 39

40

Keywords: Microwave-induced combustion; Engraulis anchoita; Trace element 41

determination; Inductively coupled plasma mass spectrometry. 42

3

1. INTRODUCTION 43

Seafood is an important source of components with significant nutritional value, 44

such as proteins, vitamins and minerals. It is considered the largest reserve of 45

polyunsaturated fatty acids, especially omega-3 compounds. These compounds have 46

brought numerous benefits to human health, such as reduced risk of cardiovascular 47

disease and stroke (Cundiff, Lanou, & Nigg, 2007), anti-inflammatory action (Pilon et 48

al., 2011), low triglyceride and total cholesterol (Mozaffarian & Wu, 2011). Despite 49

their recognized benefits, fish and other seafood may represent a risk for human health 50

since they can accumulate contaminants from aquatic environment and suffer 51

biomagnification along the food chain (Maceda-Veiga, Monroy, & Sostoa, 2012). 52

Contamination of aquatic ecosystems with trace elements has been of interest 53

worldwide and a lot of studies in the environment have been published lately (Türkmen, 54

Türkmen, Tepe, Töre, & Ates, 2009; Djedjibegovic, Larssen, Skrbo, Marjanovic’, & 55

Sober, 2012; Pereira et al., 2012). 56

Fish consumption in Brazil is about two times lower than the world average 57

(Ministry of Fisheries and Aquaculture, 2012). It can be attributed to the lack of 58

diversification of fish processing industry along with economic and cultural factors. The 59

southwestern area of Atlantic ocean is a large region characterized by its marine 60

diversity and different oceanographic conditions (Haimovici, 2007). One of the fish 61

species found in the South of Brazil, Engraulis anchoita, is a common anchovy species 62

(total length 100-104 mm) in Brazilian coast. Although it has been poorly explored, 63

Engraulis anchoita has potential for sustainable use (Carbonera & Espírito Santo, 2010) 64

with short life cycle and rapid growth with a major role in the trophic chain on the 65

continental shelf in southeastern and southern Brazil. Its distribution ranges from 66

Vitória city (20º S, Brazil) to the center of Patagonia (47º S, Argentine) (Haimovici, 67

4

2007). Recently, Engraulis anchoita has been used in Brazil as an alternative to sardine 68

in the preparation of fishmeal and school snacks. However, the determination of trace 69

elements in this fish species has not been still reported. 70

Analytical techniques, such as inductively coupled plasma optical emission 71

spectrometry (ICP OES) (Medeiros et al., 2012), inductively coupled plasma mass 72

spectrometry (ICP-MS) (Tuzen, 2009), graphite furnace atomic absorption spectrometry 73

(GF AAS) (Fallah, Zeynali, Saei-Dehkordi, Rahnama, & Jafari, 2011) and flame atomic 74

absorption spectrometry (F AAS) (Ghanomi, Nikpour, Omidvar, & Maryamabadi, 75

2011) have been used for element determination in fish samples. Flame AAS has been 76

widely used in analytical determinations because of its relatively low cost, easy 77

operation and good selectivity. However, the determination of trace elements is difficult 78

due to the unsuitable limit of detection (LOD) for many elements. On the other hand, 79

plasma-based techniques (ICP OES and ICP-MS) have been widely employed for trace 80

element determination due to their multielemental capacity, large linear range and 81

suitable LODs (Dressler, Antes, Moreira, Pozebon, & Duarte, 2011; Antes et al., 2010). 82

Sample preparation has received much attention lately, since this step may 83

represent a high potential source of errors in the analytical sequence. Wet digestion with 84

oxidizing acids is the most common sample preparation procedure due to its availability 85

for many laboratories. In this sense, microwave assisted digestion (MAD) in closed 86

vessels has been widely applied for the digestion of many matrices due to its high 87

efficiency and reduced risks of losses and contamination in comparison with 88

conventional digestion procedures using open vessels (Matusiewicz, 2003). It is 89

important to mention that MAD has also been successfully applied for the digestion of 90

biological samples using diluted HNO3 under oxygen atmosphere (Bizzi, Barin, Garcia, 91

Nobrega, Dressler, & Flores, 2011; Bizzi, Flores, Barin, Garcia, & Nobrega, 2011). 92

5

Combustion techniques have also been considered alternatives for sample digestion of 93

complex matrices due to its suitability for metal and non-metal determination by using 94

diluted acids. Recently, the feasibility of microwave-induced combustion (MIC) and its 95

respective advantages over conventional combustion methods for sample preparation 96

have been reported for many samples (Mesko et al., 2010; Mello et al., 2009, Pereira et 97

al., 2010). Some of the advantages are related to the possibility of digesting relatively 98

large amounts of samples (up to 500 mg), consuming low volumes of acids and 99

minimizing the generation of laboratory effluents. It is important to point out that 500 100

mg of sample mass could also be digested using MAD, but the residual carbon content 101

(RCC) is not reduced in the same low level when compared to MIC (typically below 102

1%) (Mesko, Moraes, Barin, Dressler, Knapp, & Flores, 2006; Antes et al., 2010; 103

Duarte et al., 2009). 104

The main goal of this study was to develop a MIC method for the determination 105

of As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, and Zn by ICP-MS in Engraulis anchoita in 106

order to reduce the volume of concentrated reagents and decrease the amount of 107

effluents in combination to a suitable digestion efficiency of sample matrix. The 108

operational parameters of MIC, such as the type of absorbing solution and an additional 109

reflux step were investigated. Accuracy was evaluated by using certified reference 110

materials (CRM) of dogfish muscle (DORM-2) and lobster hepatopancreas (TORT-2). 111

112

2. MATERIALS AND METHODS 113

114

2.1. Instrumentation 115

A microwave digestion system (model Multiwave 3000, Anton-Paar, Graz, 116

Austria), equipped with a rotor for eight high-pressure quartz vessels (capacity of 80 117

6

mL, maximum pressure and operation temperature of 80 bar and 280 ºC, respectively), 118

was used for the MIC and MAD procedures. Commercial quartz holders were used to 119

place the sample into the quartz vessel for the MIC method. 120

An inductively coupled plasma mass spectrometer (PerkinElmer-SCIEX, model 121

Elan DRC II, Thornhill, Canada) equipped with a concentric nebulizer (Meinhard 122

Associates, Golden, USA), a cyclonic spray chamber (Glass Expansion, Inc., West 123

Melbourne, Australia) and a quartz torch with a quartz injector tube (2 mm i.d.) were 124

used for element determination. Iron measurements by ICP-MS were performed with 125

dynamic reaction cell (DRC) using ammonia (minimum purity of 99.999%) as reaction 126

gas. The DRC parameters were adjusted in order to minimize the interferences on 56Fe+ 127

isotope. Adjustment of the reaction gas flow rate and the rejection parameter q (RPq) 128

were optimized in order to obtain the lowest LOD. NH3 and RPq values were 0.5 mL 129

min-1 and 0.5, respectively. 130

Residual carbon content determination in digests was performed by using an 131

inductively coupled plasma optical emission spectrometer with axial view configuration 132

(model Spectro Ciros CCD, Spectro Analytical Instruments, Kleve, Germany) equipped 133

with a cross flow nebulizer coupled to a double pass-Scott type spray chamber. 134

Measurements were performed in agreement with previously described conditions 135

(Gouveia, Silva, Costa, Nogueira & Nóbrega, 2001). Plasma, auxiliary and nebulizer 136

gas flow rate, RF power and other operational conditions used for the determinations by 137

ICP OES and ICP-MS are described in Table 1. For the determinations using ICP OES 138

and ICP-MS, argon (99.996%, White Martins - Praxair, São Paulo, Brazil) was used for 139

plasma generation, auxiliary and nebulization gas. 140

141

2.2. Reagents and standards 142

7

All solutions were prepared with analytical grade reagents in ultrapure water 143

(18.2 MΩ cm) generated by a purification system (model Milli-QTM Plus, Millipore 144

Corp., Bedford, USA). Concentrated nitric acid (65%, Merck, Darmstadt, Germany) 145

was purified by a sub-boiling system (Model Duopur, Milestone, Bergamo, Italy). A 146

solution of 6 mol L-1 ammonium nitrate was prepared by dissolving the correspondent 147

salt (Merck) in water. This solution was used as aid for ignition for the combustion 148

process. A multielement stock solution (SCP 33 MS, SCP Science, Quebec, Canada) 149

containing 10 mg L-1 of each element was used to prepare the calibration curves by 150

sequential dilution in 5% (v/v) HNO3 in the range from 0.025 to 10 g L-1. 151

152

2.3. Sample collection 153

Eight samples of Engraulis anchoita (named A, B, C, D, E, F, G, and H) were 154

collected in October 2010, at different locations along the southwestern Atlantic ocean. 155

Sample “A” was used for method optimization. Sampling points are described in Table 156

2. Approximately 10 kg fish were collected in each sampling point. Afterwards, samples 157

were transported to the laboratory in ice boxes. For sample preparation, a subsample 158

(about 1.0 kg) was manually filleted, homogenized and frozen at -20 ºC. Finally, it was 159

freeze-dried (Terroni, Model Lh-2000/3, São Carlos, Brazil). Samples were then ground 160

in a cryogenic mill (SpexCertiPrep, Model 6750, Metuchen, USA) by 2 runs for 2 min 161

(Duarte et al., 2009). 162

Accuracy was evaluated by CRMs, i.e., dogfish muscle (DORM-2) and lobster 163

hepatopancreas (TORT-2) purchased from National Research Council Canada (NRCC, 164

Ontario, Canada). Spiked samples were also performed by adding suitable amounts of 165

multielement stock solution (SCP 33 MS) containing all analytes above the sample 166

pellet before the combustion step. 167

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168

2.4. Sample preparation procedures 169

For the proposed MIC method, pellets of samples were prepared by a hydraulic 170

press (Specac, Orpington, UK) set at 5 ton. Pellets were weighed (up to 500 mg) and 171

transferred with the filter paper to the quartz holder of the MIC system. The holder 172

containing the sample was placed into a quartz vessel which was previously charged 173

with 6 mL of the absorbing solution (1.4, 3.5, 5, 7 or 14 mol L-1 HNO3). A small disc of 174

filter paper (10 mm diameter, 10 mg) with low ash content (Black Ribbon Ashless, 175

Schleicher and Schuell GmbH, Dassel, Germany) was used as auxiliary for the 176

combustion process. A solution of NH4NO3 (6 mol L-1, 50 µL) was added and after 177

closing the vessels and capping the rotor, vessels were pressurized with oxygen at 20 178

bar. The microwave irradiation program used for the MIC procedure was 1400 W for 5 179

min (reflux step) and 0 W for 20 min (cooling). Digests were diluted with ultrapure 180

water up to 30 mL in polypropylene vessels. As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se 181

and Zn were then determined by ICP-MS. 182

Sample digestion was also performed by MAD in high-pressure closed vessels. 183

The operational conditions for MAD were in agreement with previous studies (Soares et 184

al. 2012; Pereira et al., 2012). In this case, about 500 mg fish were weighted inside 185

quartz vessels and 6 mL of 14 mol L-1 HNO3 were added to it. The heating program was 186

carried out as follows: 1000 W for 10 min (ramp of 10 min), 1400 W for 10 min and 0 187

W for 20 min (cooling). After cooling, digests were diluted with ultrapure water up to 188

30 mL in polypropylene vessels for further element determination. After each run, 189

vessels were soaked in concentrated HNO3 for 10 min and rinsed with ultrapure water. 190

9

All statistical calculations were performed using GraphPad InStat software 191

(GraphPad InStat Software Inc, Version 3.06, 2007). A 95% significance level was 192

adopted for all comparisons. 193

194

3. RESULTS AND DISCUSSION 195

196

3.1. Operational conditions for microwave-induced combustion 197

The MIC conditions used in this study for Engraulis anchoita digestion were 198

adapted to those previously used for biological samples. With MIC system up to 500 mg 199

fish can be digested and the reached temperature (up to 1400 ºC) assures a complete 200

oxidation of the organic matrix (Mesko, Moraes, Barin, Dressler, Knapp, & Flores, 201

2006; Duarte et al., 2009). After MIC procedure, the absorbing solution was completely 202

clear without any visible particle or solution turbidity. Moreover, despite the high 203

temperature, no damage was observed in holders and vessels, even after about 200 204

combustion cycles. 205

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3.2. Influence of the absorbing solution on the MIC procedure 207

Choosing the right absorbing solution is important in order to achieve suitable 208

recoveries (Müller et al., 2011). Therefore, it has been widely discussed in the literature 209

that the best recoveries are obtained by a reflux step, which ensures quantitative analyte 210

recoveries by comparison with combustion without a reflux step (Antes et al., 2010; 211

Pereira et al., 2009). As a result, in this study the reflux step was performed for 5 min 212

without opening the vessels after combustion. 213

The HNO3 concentration (1.4, 3.5, 5, 7 and 14 mol L-1) was evaluated as 214

absorbing solution for the determination of As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, and 215

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Zn. Spike recoveries in sample “A” were evaluated for each absorbing solution. Results 216

obtained with different absorbing solutions are shown in Table 3. Recoveries below 217

55% were obtained for Cr and Cu when 1.4 mol L-1 HNO3 was used as the absorbing 218

solution. Using the same solution, recoveries for As, Cd, Co, Fe, Mn, Mo, Ni, and Zn 219

ranged from 67 to 94%. Despite the better results for these analytes, the relative 220

standard deviation (RSD) with 1.4 mol L-1 HNO3 were considered relatively high (up to 221

21%). Recoveries for Se ranged from 96 to 104% with RSD below 13% for all 222

absorbing solutions under evaluation. 223

Recoveries above 96% were obtained for As, Cd, Cu, and Ni when 3.5 mol L-1 224

HNO3 was used as absorbing solution with a RSD below 12%. On the other hand, 225

results obtained in the same conditions for Co, Cr, Fe, Mn, Mo, and Zn showed lower 226

recoveries, ranging from 62 to 90%, and RSD values up to 16%. 227

However, using 5 mol L-1 HNO3, recoveries for all elements were above 94% 228

and RSD values were below 7%. Similar results were obtained when solutions of 7 and 229

14 mol L-1 HNO3 were used. Results indicated that, with these solutions, recoveries 230

were above 94% for all elements and RSD ranged from 2 to 7%. Although 7 or 14 mol 231

L-1 HNO3 solutions could also have been employed, their use was not necessary, since 232

satisfactory recoveries were obtained for most elements with 5 mol L-1 HNO3, which 233

corresponds to 35% of concentrated HNO3. It can be considered as an advantage, taking 234

into account the consumption of reagents, suitable level of blanks and, consequently, the 235

lower generation of laboratory residues. Therefore, 5 mol L-1 HNO3 was selected as the 236

absorbing solution for subsequent studies. 237

238

3.3. Element determination in Engraulis anchoita 239

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The proposed procedure was applied to the determination of As, Cd, Co, Cr, Cu, 240

Fe, Mn, Mo, Ni, Se and Zn in eight samples (A to H) of Engraulis anchoita using 5 mol 241

L-1 HNO3 as absorbing solution. Results for element determination by ICP-MS are 242

shown in Table 4. In general, results using MIC for all analytes in sample “A” were in 243

agreement with values obtained by the MAD method (P > 0.05). It is noteworthy that 244

the RCC in MIC digests was always below 0.6%, while the RCC in digest after MAD 245

was about 6%. Since only solutions relatively diluted (5 mol L-1 HNO3) are required for 246

the proposed MIC method, after a 5-times dilution digests were suitable to be analyzed 247

by ICP-MS (resultant HNO3 concentration was 1 mol L-1 or lower). 248

Among the elements determined in this study, only As, Cd and Cr presented 249

values higher than the maximum limits (1.0, 0.1 and 0.1 µg g-1, respectively) established 250

in Brazil for fish and fish products (ANVISA, 1998). Results found for As, Cd and Cr 251

ranged from 5.49 to 6.53, 0.221 to 0.271 and 48.5 to 58.4 µg g-1, respectively. In 252

addition, the result for As was about 6 times higher than the obtained for S. brasilienses 253

species (Medeiros et al., 2012). Other studies reported Cd and Cr contents of 0.08 ± 254

0.01 and 1.2 ± 0.1 µg g-1, respectively, for Sardine pilchardus from Greece (Zotos & 255

Vouzanidou, 2012). 256

Some elements, such as Co, Cu, Fe, Mn, Se and Zn, are considered essential for 257

the human body and diet. Lack of these elements can cause several problems in human 258

health, such as anemia, disturbance of the nervous system and acute adverse effects 259

(Cornelis, Caruso, Crews, & Heumann, 2005). The values found for Co, Cu, Fe, Mn, Se 260

and Zn in the samples under study ranged from 0.101 to 0.150, 6.08 to 6.98, 201 to 251, 261

5.62 to 6.08, 2.22 to 2.76 and 64.7 to 88.9 µg g-1, respectively. These concentrations 262

were higher than the ones reported for S. brasilienses (Medeiros et al., 2012). 263

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The levels found for Mo and Ni ranged from 1.64 to 1.98 and 0.912 to 1.20 µg g-264

1, respectively. By comparison with other fish species, such as S. undosquamis, 265

M.barbatus and S. aurata, Ni concentration ranged from 4.9 to 8.2, 0.8 to 2.1 and 1.7 to 266

3.2 µg g-1, respectively (Türkmen, Türkmen, Tepe, & Akyurt, 2005). 267

The variability of the inorganic contaminants levels in different fish species 268

depends on several factors, such as aquatic ecosystems (Medil, Demirci, Tuzen, & 269

Soylak, 2010), geological weathering (Bienfang, De Carlo, Christopher, DeFelice, & 270

Moeeller, 2009) and discharge of agricultural, residential or industrial waste products 271

(Islam & Tanaka, 2004). 272

273

3.4. Analytical performance 274

The linear working range was between 0.025 and 10 µg L-1 for all the analytes. 275

The limits of detection and quantification were calculated in agreement with IUPAC 276

recommendations: i) limits of detection = 3 σ, where σ = standard deviation of ten 277

measurements of blank solution, n = 10; ii) limits of quantification = 10 σ, where σ = 278

standard deviation of ten measurements of blank solution, n = 10. The better efficiency 279

of MIC procedure was important to assure a complete digestion of relatively large 280

amount of sample (up to 500 mg) and to obtain LODs 2 to 4 times lower when MAD 281

method was used. The linear correlation coefficients of the calibration curves were 282

above 0.999 indicating a suitable linearity. 283

Accuracy was evaluated by using CRMs of dogfish muscle (DORM-2) and 284

lobster hepatopancreas (TORT-2). The CRMs were diluted in the same way as the 285

sample. Results are shown in Table 5. All results were in agreement with certified, 286

ranging from 94 to 108%. Likewise, precision was estimated by the RSD; it was below 287

13

10%. Taking into account the multielemental analysis, it was considered reasonable 288

precision. 289

290

4. CONCLUSIONS 291

The proposed MIC procedure for Engraulis anchoita was considered suitable as 292

a sample preparation method for the determination of As, Cd, Co, Cr, Cu, Fe, Mn, Mo, 293

Ni, Se, and Zn by ICP-MS. MIC made it possible to obtain low RSDs, low LODs and 294

sample throughput at least 2 times higher by comparison with MAD procedure. The 295

digestion efficiency was also higher then MAD (RCC below 0.6%). The proposed MIC 296

procedure enables complete oxidation of the organic matrix along with a safe operation. 297

In addition, the use of diluted HNO3 (5 mol L-1) leads to lower effluent generation by 298

comparison with conventional procedures of digestion. In addition, taking into account 299

that concentrated acids were not necessary, the proposed procedure is in agreement with 300

green chemistry recommendations. Since the digestion time was relatively low, 301

simultaneous digestion of eight samples was carried out in 25 min. The concentration of 302

As, Cd and Cr in Engraulis anchoita were above the maximum levels established by the 303

Brazilian legislation that implies in a control of these elements for fish consumption. 304

305

ACKNOWLEDGEMENTS 306

The authors are grateful to CNPq, CAPES and FAPERGS for supporting this study. 307

308

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Table 1. Operational parameters for element determination by ICP OES and ICP-MS. 436

Parameters ICP OES ICP-MS

RF power (W) 1500 1400

Plasma gas flow rate (L min-1) 14 15

Auxiliary gas flow rate (L min-1) 1.0 1.2

Nebulizer gas flow rate (L min-1) 0.70 1.09

Dwell time (ms) - 50

Sweeps/reading - 5

Readings/replicate - 3

Replicates - 3

Data collection mode - Peak hopping

Sampler and skimmer cones - Pt

Isotopes (m/z) - 75As, 111Cd, 59Co, 53Cr, 65Cu, 56Feb, 55Mn, 96Mo, 60Ni, 82Se,

and66Zn

Wavelengths (nm) Ca 193.091 - a RCC measurements were performed according to Gouveia, Silva, Costa, Nogueira & Nóbrega, 437 2001. 438 b Use of DRC-ICP-MS. 439

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Table 2. Sampling points of Engraulis anchoita. 440

441 442 Samples Latitude Longitude

A 32° 1'44.00" S 52° 5'53.99" W

B 32° 29'18.99" S 51° 54'51.00" W

C 33° 0'7.00" S 51° 54'55.01" W

D 33° 15'34.00" S 52° 8'59.00" W

E 33° 27'21.00" S 52° 13'18.99" W

F 33° 41'27.00" S 52° 43'38.00" W

G 32° 34'46.00" S 52° 18'58.99" W

H 32° 19'36.00" S 52° 11'54.00" W

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Table 3. Influence of the absorbing solution on sample “A” after digestion by MIC and 443

determination by ICP-MS (values represent spike recovery and standard deviation, 444

n=3). 445

Absorbing solution HNO3 (mol L-1)

Elements 1.4 3.5 5.0 7.0 14.0

As 94 ± 8 100 ± 7 101 ± 4 102 ± 5 100 ± 4

Cd 86 ± 11 96 ± 7 97 ± 3 97 ± 3 96 ± 4

Co 77 ± 16 79 ± 11 98 ± 7 99 ± 7 97 ± 6

Cr 55 ± 6 60 ± 5 95 ± 2 93 ± 3 96 ± 2

Cu 47 ± 6 97 ± 4 101 ± 3 100 ± 2 103 ± 3

Fe 72 ± 9 62 ± 10 97 ± 4 98 ± 6 97 ± 3

Mn 73 ± 8 64 ± 6 94 ± 5 94 ± 5 103 ± 4

Mo 67 ± 2 68 ± 3 97 ± 3 100 ± 4 99 ± 2

Ni 69 ± 14 97 ± 11 96 ± 7 97 ± 8 98 ± 7

Se 96 ± 12 104 ± 8 102 ± 8 100 ± 9 96 ± 7

Zn 92 ± 4 90 ± 5 96 ± 3 95 ± 2 101 ± 2 446

Table 4. Results for the determination of As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se and Zn in Engraulis anchoita by ICP-MS after digestion by

MAD (sample A) and MIC (samples A to H). Values represent the mean in µg g-1

and standard deviation, n=3.

MAD MIC

Elements Sample A Sample A Sample B Sample C Sample D Sample E Sample F Sample G Sample H

As 5.74 ± 0.42 6.13 ± 0.38 6.44 ± 0.21 6.38 ± 0.15 5.93 ± 0.33 6.17 ± 0.42 5.49 ± 0.28 6.53 ± 0.11 6.22 ± 0.32

Cd 0.233 ± 0.041 0.257 ± 0.022 0.221 ± 0.019 0.260 ± 0.027 0.247 ± 0.015 0.271 ± 0.024 0.259 ± 0.026 0.241 ± 0.020 0.233 ± 0.012

Co 0.111 ± 0.011 0.134 ± 0.015 0.150 ± 0.009 0.116 ± 0.010 0.101 ± 0.006 0.147 ± 0.008 0.133 ± 0.014 0.139 ± 0.009 0.123 ± 0.011

Cr 52.3 ± 4.2 55.1 ± 1.9 57.3 ± 2.5 53.6 ± 3.4 48.5 ± 3.8 50.9 ± 1.8 49.6 ± 3.9 58.4 ± 4.0 54.0 ± 2.8

Cu 6.22 ± 0.43 6.29 ± 0.21 6.45 ± 0.53 6.71 ± 0.32 6.19 ± 0.39 6.94 ± 0.55 6.08 ± 0.28 6.98 ± 0.33 6.37 ± 0.22

Fe 214 ± 6 224 ± 4 246 ± 7 249 ± 6 208 ± 15 251 ± 12 201 ± 7 236 ± 10 223 ± 9

Mn 6.05 ± 0.18 5.86 ± 0.26 5.90 ± 0.43 6.08 ± 0.19 5.62 ± 0.31 6.00 ± 0.52 5.75 ± 0.36 5.82 ± 0.21 5.71 ± 0.40

Mo 1.73 ± 0.19 1.86 ± 0.16 1.98 ± 0.11 1.73 ± 0.08 1.66 ± 0.10 1.87 ± 0.12 1.64 ± 0.10 1.96 ± 0.07 1.80 ± 0.13

Ni 1.13 ± 0.10 1.11 ± 0.12 1.20 ± 0.06 1.16 ± 0.04 0.912 ± 0.072 0.992 ± 0.080 1.01 ± 0.05 1.18 ± 0.09 1.06 ± 0.08

Se 2.23 ± 0.30 2.42 ± 0.34 2.76 ± 0.23 2.55 ± 0.18 2.35 ± 0.21 2.22 ± 0.20 2.37 ± 0.17 2.67 ± 0.16 2.41 ± 0.31

Zn 72.3 ± 2.9 71.7 ± 1.7 76.0 ± 3.6 88.9 ± 4.2 69.8 ± 4.1 80.8 ± 5.4 64.7 ± 5.1 73.5 ± 2.9 82.2 ± 5.0

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Table 5. Results for the determination of As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se and 451

Zn in DORM-2 and TORT-2 by ICP-MS after the optimization of the MIC method. 452

Values represent the mean in µg g-1 and standard deviation (uncertainty for CRMs), 453

n=3. 454

455

DORM-2 TORT-2

Elements LOD MIC Certified value MIC Certified value

As 0.003 17.8 ± 0.8 18.0 ± 1.1 20.9 ± 0.6 21.6 ± 1.8

Cd 0.001 0.043 ± 0.004 0.043 ± 0.008 26.1 ± 0.3 26.7 ± 0.6

Co 0.002 0.189 ± 0.008 0.182 ± 0.031 0.551 ± 0.043 0.51 ± 0.09

Cr 0.005 34.1 ± 1.8 34.7 ± 5.5 0.814 ± 0.073 0.77 ± 0.15

Cu 0.004 2.25 ± 0.20 2.34 ± 0.16 101 ± 8 106 ± 10

Fe 0.015 134 ± 9 142 ± 10 109 ± 10 105 ± 13

Mn 0.002 3.57 ± 0.19 3.66 ± 0.34 12.9 ± 0.7 13.6 ± 1.2

Mo 0.005 0.320 ± 0.023 - 0.904 ± 0.051 0.95 ± 0.10

Ni 0.004 19.7 ± 1.3 19.4 ± 3.1 2.64 ± 0.13 2.50 ± 0.19

Se 0.016 1.44 ± 0.13 1.40 ± 0.09 5.42 ± 0.50 5.63 ± 0.67

Zn 0.006 24.4 ± 1.6 25.6 ± 2.3 181 ± 4 180 ± 6

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Highlights 456 - Trace metals were determined for the first time in Engraulis anchoita species. 457 - MIC enabled an effective digestion of up to 500 mg of sample with low RCC and low 458 acid consumption. 459 - Proposed MIC procedure can be considered as in agreement with green chemistry 460 recommendations. 461 462 463 464