Direct analysis of marine macroalgae for determination of macro minerals by energy dispersive X-ray fluorescence

Accepted Manuscript Direct analysis of marine macroalgae for determination of macro minerals by energy dispersive X-ray fluorescence

Geysa B. Brito, Leonardo S.G. Teixeira, Maria Das Graças A. Korn PII: DOI: Reference:

S0026-265X(17)30409-5 doi: 10.1016/j.microc.2017.05.001 MICROC 2822

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

14 March 2016 23 February 2017 2 May 2017

Please cite this article as: Geysa B. Brito, Leonardo S.G. Teixeira, Maria Das Graças A. Korn , Direct analysis of marine macroalgae for determination of macro minerals by energy dispersive X-ray fluorescence, Microchemical Journal (2017), doi: 10.1016/ j.microc.2017.05.001

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ACCEPTED MANUSCRIPT Direct analysis of marine macroalgae for determination of macro minerals by energy dispersive X-ray fluorescence

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Geysa B. Brito1, Leonardo S. G. Teixeira1, Maria das Graças A. Korn1*

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Instituto de Química, Universidade Federal da Bahia, Campus de Ondina,

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CEP: 40170-115, Salvador, Bahia, Brazil

*Corresponding author – Tel.: +55 7132836830; Fax: +55 7132355166 E-mail address: [email protected] (MGA Korn)

ACCEPTED MANUSCRIPT Abstract Determination of the macro mineral composition of macroalgae is important considering

the

relevant

applications

and

potential

consequences

of

macroalgae. The application of direct solid analysis is attractive, as it decreases costs, consumes less reagents, has higher sample throughput, generates less

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residue, and avoids analyte loss and contamination. The aim of this study is to

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propose a method for the direct determination of Ca, K and Mg in marine

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macroalgae using energy dispersive X-ray fluorescence (EDXRF). This method was calibrated using samples previously analyzed by inductively coupled

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plasma optical emission spectrometry (ICP OES) as calibration standards. In a parallel procedure applied for comparison purposes, macroalgae samples were

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also analyzed by ICP OES after an acid digestion procedure. Analysis of three Certified Reference Materials (CRM) of different plant materials were performed

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to evaluate the accuracy of the method. The coefficients of correlation (R) from

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the calibration curves, the precision (%), the limit of quantification (mg kg-1) and the recovery (%) were: Ca (0.961, 2.07, 109.5 and 85.0 to 89.3), K (0.998, 3.82,

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207.0 and 126.6 to 129.6) and Mg (0.978, 4.07, 195.6 and 92.7 to 115.4). The proposed method was then applied in the determination of Ca, K and Mg in

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macroalgae samples collected in Todos os Santos Bay, Bahia, Brazil.

Keywords: macroalgae, macrominerals, EDXRF, direct analysis.

ACCEPTED MANUSCRIPT 1. INTRODUCTION

There are approximately 1.5 million species of algae on Earth, including planktonic algae that are unicellular and usually live suspended in water bodies and macroalgae that are multicellular and generally live fixed on rocks, coral,

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sediments and other surfaces as part of the benthos [1,2]. Factors essential for

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macroalgae to be present in a given environment include light, nutrients,

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temperature, salinity and substrate. They are photosynthetic organisms and contribute to coastal ecosystems. In addition, macroalgae are large oxygen

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repositories in the environment that absorb and transform inorganic nutrients as part of the primary link of the food chain. In the marine environment,

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macroalgae also serve as shelter, spawning grounds and food for many species of animals [2,3]. There are three divisions of algae based mainly on its

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coloration according to the abundance of the major pigments in their stems:

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brown, red and green. Botanists refer to these broad groups as Phaeophyceae, Rhodophyceae and Chlorophyceae, respectively [4].

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Seaweeds are used in environmental studies and can be used in the energy and food industry. Due to the concentration of metals and other

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elements in their stems [3,5] at levels above the sediment and bodies of water they inhabit, algae have been used as an environmental biomonitor [6-10]. Other works have studied the efficiency of elemental biosorption, mainly in the treatment of effluent water [11-13]. Many industries use macroalgae as a source of raw material, employing hydrocolloids extracted from algae (agar, alginate and carrageenan) in the manufacture of thickeners, stabilizers and emulsifiers, especially in the food, pharmaceutical and textile industry. Several other

ACCEPTED MANUSCRIPT applications are developed using seaweed as fertilizer, in cosmetic products, as energy in biodiesel manufacturing and in medicinal uses in the treatment of rheumatism, osteoporosis and others [14]. Algae are of great importance not only due their broad applicability but also because of their nutritional value [14]. A very important function of algae is

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their use as a direct food source (fresh or flour) for animals [15] and especially

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to humans [16,17]. Studies have verified its importance as a source of fatty

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acids, fiber, protein, carotenoids and amino acids [18,19]. In addition to these compounds, the macroalgae are important sources of minerals [20]. The main

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minerals present in algae that are important for human development are the macronutrients Na, K, Ca, Mg, and P and the micronutrients I, Fe, Zn, Cu, Se

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and Mn [21-23].

Many methods to assess the elemental concentrations in marine

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macroalgae have been developed [24-27]. The most common technique used

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for elemental determination in algae is atomic absorption spectrometry, requiring sample pre-treatment by complete destruction of the organic matrix by

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digestion [28-31]. Normally, the digestion is performed in the presence of large amounts of oxidants and concentrated mineral acids under aggressive

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conditions in a time consuming procedure. In addition, the sample is diluted in these protocols, causing a loss in sensitivity such that the analyte often becomes undetectable, depending on the measurement technique used. Systematic errors can also be observed due to contamination or loss by volatilization, which affects the accuracy and precision of the results [32]. Direct analysis with minimal manipulation of the sample is an alternative that would overcome these limitations. Some advantages are incorporated into

ACCEPTED MANUSCRIPT the method, and certain disadvantages are minimized or even eliminated. These improvements include: 1) simplified sample pretreatment, which reduces the time spent in this stage, thereby enhancing the analytical frequency; 2) minimized contamination due to using less reagents, decreasing sample manipulation and/or lowering the exposure to the environment; 3) minimized

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analyte loss; 4) decreased risk by avoiding the use of toxic or corrosive

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reagents; 5) minimized waste generation; and 6) increased detection, as the

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samples are not diluted [33].

A multi-element technique that allows direct analysis in a solid sample is

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X-ray fluorescence spectrometry (XRF). The XRF technique has characteristics that overcome the limitations of many spectrometric techniques, including: 1)

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XRF is able to conduct simultaneous multi-element measurements (typically sodium to uranium); 2) XRF is compatible with solid and liquid samples; and 3)

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XRF is non-destructive [34]. XRF is considered a selective technique with

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simple spectra when compared to ultraviolet emission. It has high strength, and the wavelengths of characteristic X-ray lines are independent of the physical

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and chemical state of the element since because the electronic transitions that occur do not involve electrons participating in chemical bonding.

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On the other hand, XRF does exhibit some interference. In addition to effects that occur due to the physical structure of the matrix (for whole, powder and pressed solid), there are also known spectral interferences. This type of interference is caused primarily by the phenomena of absorption or intensification of the emission signal by the other elements in the sample matrix [34]. Nevertheless, in analysis of solids, the use of standards that simulate the matrix, mathematical corrections and chemometric tools can minimize these

ACCEPTED MANUSCRIPT effects, allowing qualitative and quantitative analyses on various matrices [3541]. In this work, EDXRF was employed for direct determination of the elemental composition of untreated seaweed samples. Considering the environmental, industrial and nutritional importance of marine macroalgae, the objective of this work was to develop a method for the

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direct and rapid analysis of macroalgae for determination of Ca, K and Mg by

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EDXRF, which would minimize the use of reagents, sample manipulation, time

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and cost.

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2.1 Reagents and standards

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2. EXPERIMENTAL

All closed vessels, polyethylene flasks and plastic containers were cleaned with

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HNO3 (65% w/v, diluted 1/10 with high-purity water) for 24 h and rinsed with

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high purity water. Subsequently, all material was dried and stored under cleanair conditions. All plastic containers, polyethylene flasks, pipette tips, PFA

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Teflon digestion vessels (Milestone SRL, Sorisole, Italy) and reagents that came into contact with the samples or standards were checked for

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contamination. All solvents and reagents were of the highest commercially available purity grade. Deionized water with a resistivity of ≥18 MΩ cm was obtained by means of a Milli-Q Plus pure water generating system (Millipore Molsheim, France) and was used to prepare all standard and sample solutions. Analytical grade nitric acid (Merck, Darmstadt, Germany) was doubly distilled in a model duoPUR 2.01E sub-boiling system (Milestone, Bergamo, Italy). Monoelemental, high-

ACCEPTED MANUSCRIPT purity grade stock solutions (1 g L-1) of Ca, K and Mg were purchased from Merck (Darmstadt, Germany). Plasma torch argon purity was higher than 99.99%.

2.2 Sample collection and preparation

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Forty-six seaweed samples (from fifteen different species) were collected from

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rock sand stones in the sub littoral zone (0.5–3 m). The collections were made

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four times in March, April, May and November 2014 in the Penha beach, area of Itaparica Island, Bahia, Brazil. The taxa were Acanthophora spicifera (Vahl)

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Boergesen, Agardhiella sp. F. Schmitz, Bryothamnion triquetrum (S. G. Gmelin) M. A. Howe, Caulerpa cupressoides (Vahl) C. Agardh, Caulerpa racemosa var.

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occidentalis (J.Agardh) Boergesen, Caulerpa scalpelliformis (R. Brown ex Turner) C. Agardh, Codium isthmocladum Vickers, Cryptonemia crenulata (J.

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Agardh) J. Agardh, Dictyopteris jamaicensis (W. R. Taylor), Dictyota spp.,

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Gracilaria domingensis (Kützing) Sonder ex Dickie, Hypnea musciformis (Wulfen) J. V. Lamouroux, Padina spp., Sargassum spp. and Ulva lactuca

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(Linnaeus). Algae collected at different days, designated by A, B, C and D, are shown in Table 1.

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The samples were washed at the sampling site in seawater and then transferred to the laboratory in polyethylene bags containing seawater. After delivery to the laboratory, the plant material was cleaned of epiphytes and organic and mineral particles. The sample was washed with tap water and then rinsed in deionized water before being frozen until pretreatment. Seaweed samples were dried using freeze dryers between 24 and 72 h under vacuum and then ground in a ball mill (Spex SamplePrep, 8000M Mixer /

ACCEPTED MANUSCRIPT Mill) equipped with tungsten carbide balls and bottles from 60 to 150 s. The samples were then stored in plastic containers and maintained in a desiccator. After this process, the samples were mashed to below 100 μm. Sample powder (150 mg) were pressed (in triplicate) into uniform pellets of a 13 mm diameter using a hydraulic press machine (Shimadzu, SSP-10A, P/N 200-64175) under

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80-90 kN with a standing time of 5 min.

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2.3. EDXRF measurements

An energy dispersive X-ray spectrometry (Bruker, S2 Ranger) with a palladium

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target X-ray tube was used for the measurements. After testing several different atmospheres, the measurements were performed in a vacuum, which resulted

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in higher analytical signals with 100 s of irradiation. The voltage, maximum current and power were 50 kV DC, 2 mA DC and 50 W, respectively. The

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equipment was calibrated with a copper disk placed inside the 40 mm diameter

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ring (steel sampler) supplied with the system before each application was made. After this procedure, the samples were placed into the center of the

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spectrometer cell, a polyethylene cup, using Mylar film as a sample support. The mask between the sample and the collimator was set to view a 27 mm

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diameter area (the polyethylene cup’s circular area). The pellets had a 13 mm diameter, so a filter paper mask (Boeco - Germany, grade 388.84 g m-2) was created to cover the area of the polymer film that was unfilled to both center the pellets on the sampler cup and avoid scattering loss by radiation.

2.4 Comparative procedure

ACCEPTED MANUSCRIPT Acid digestion of the macroalgae samples was performed, using a validated method [10], with a commercial high-pressure laboratory microwave oven (Milestone Ethos 1600 Microwave Labstation, Sorisole, Italy), operating at a frequency of 2450 Hz with an energy output of 900 W. This microwave digestion system was equipped with ten vessels made with perfluoroalcoxi polymer (PFA)

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with a volume of 100 mL. Approximately 250 mg of freeze-dried samples were

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digested with 7 mL of doubly distilled nitric acid and 1 mL of hydrogen peroxide.

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The maximum temperature was 210ºC. Three replicates of each sample were analyzed. Blank assays were also carried out. The extracts were stored under

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refrigeration in PET-HD flasks that were previously cleaned with nitric acid. An inductively coupled plasma optical emission spectrometer (ICP OES)

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with an axially viewed configuration (VISTA PRO, Varian, Mulgrave, Australia) equipped with a charge coupled device (CCD) detector was used for the

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determination of the analytes as a comparative method. The V-groove nebulizer

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coupled with a Sturman-Masters chamber was used as the sample introduction system. The operating conditions of the ICP OES for elemental determination

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were carried out using the manufacturer recommended conditions for power (1.3 kW), RF generation (40 MHz), plasma gas flow (15.0 L min-1), auxiliary gas

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flow (1.5 L min-1) and nebulizer gas flow (0.7 L min-1) that were previously tested as described in a previous study [10]. The spectral lines were selected according to interference, sensitivity, calibration, and the highest analytical signal/background ratio (SBR). The analytical wavelengths (nm) chosen were as follows: Ca(II) 396.847, K(I) 759.897 and Mg(II) 280.270.

3. RESULTS AND DISCUSSION

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3.1 Evaluation of EDXRF method Analysis of the pellets samples was carried out to determine the levels of three elements (Ca, K and Mg) in each sample. Fifteen samples of different species previously analyzed by ICP OES were chosen for calibration. The calibration

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curves obtained for each analyte are shown in Table 2. As seen, elements

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showed curves with good coefficients of determination, with correlation

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coefficients (R) of 0.961, 0.998 and 0.978 for Ca, K and Mg, respectively. Different algae species were used as standards for calibration curves, resulting

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in minimization of deviations provided by matrix effects [42, 43]. The limits of detection, regarded as being the minimal analyte signal that

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can be differentiated from the background signal, was calculated by the blank signal, YB (or a from equation calibration curve), plus three standard deviations

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of the blanks, SB (or error Sy/x) [44], and the results are shown in Table 2.

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Because this is a direct analysis technique that does not use sample decomposition, which would result in a dilution of analytes, these limits are

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sufficient for determination of these elements in macroalgae samples. The pellet structure and the efficiency of the measurements depend on

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the particle size distribution. To carry out the analysis, the incident X-rays must irradiate the larger area that contains all of the particles in the sample [45]. A sample was randomly selected, and ten pellets were prepared to evaluate the possible interference on the physical structure of the sample from the preparation of the pellets (surface, grain size, compression, etc.) and to check the precision of the method by means of the relative standard deviation (RSD). The precision obtained for all elements, evaluated as relative standard deviation

ACCEPTED MANUSCRIPT (RSD), was between 2.1 and 4.1% (n= 10), indicating that the preparation of the tablet was efficient to perform analysis by EDXRF. Certified reference materials (CRM) of plant origin from NIST were used to evaluate the accuracy of the proposed procedure: NIST 1515 (Apple Leaves), NIST 1547 (Peach Leaves) and NIST 1570a (Trace Elements in

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Spinach Leaves). The CRMs were prepared using the same procedure

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employed for the macroalgae. The results given by EDXRF for Ca, K and Mg

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were compared with the certificates for the analysis of CRMs. The results are listed in Table 3. A t-Test was performed for each certified reference material,

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and there was no significant difference between the certified values and the values obtained at a confidence level of 95%. Thus, the method showed good

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accuracy for the studied application.

The accuracy of the proposed method was also verified comparing the

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results obtained by EDXRF of the macroalgae samples not involved in the

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calibration with those obtained by decomposition of samples and quantified by ICP OES. The results were compared graphically to evaluate the relationship

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between the both methods. Comparative plots are shown in Figure 1 and, as seen, the correlations obtained for Ca, K and Mg were between 93 to more than

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99%. The plots for Ca, K and Mg show inclination angles near 45° (around the angular coefficients to 1 unity). These results demonstrate that these elements are well determined by EDXRF compared with ICP OES, showing good applicability of these elements by the proposed method.

3.2 Elements levels in macroalgae

ACCEPTED MANUSCRIPT The proposed procedure was applied in the determination of Ca, K and Mg in the analysis of macroalgae from Itaparica Island, Bahia, Brazil and the results are shown in Table 4. In general, samples from the green group had lower concentration of Ca and K, showing similar levels of Mg in relation to the brown and red groups. For Ca, the red algae showed highest concentrations; in

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particular, the Briotamnion triquetum species had the highest Ca levels. In the

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group of brown macroalgae, samples of Padina spp. had high levels of Ca with

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levels close to those obtained by B. triquetum. For K, samples from red and brown groups presented similar concentrations. However, the taxon Sargassum

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spp. showed the highest levels for this element. For Mg, concentrations obtained from samples of all groups were similar, but the species of Ulva

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lactuca, Codium isthmocladum and Bryotamnion triquetum present the highest levels.

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A compilation of data for Ca, K and Mg in marine macroalgae from

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different geographical regions can be seen in Table 5. Comparing the data obtained in this study to the results from different research conducted in recent

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years, it is clear that the elemental values of the Itaparica Island samples have average values within the previously reported range. However, the maximum

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value for Ca in this study was the largest found compared with the data in Table 5 and was obtained for a sample of the red alga B. triquetrum, which is not listed in other research. With regard to the data from different classifications of algae, according to Table 5, the lowest values for Ca, K and Mg were for samples of red, green and red groups, respectively. For larger amounts of these analytes, the results highlight the levels in red, red and brown algae groups, respectively. Similarly,

ACCEPTED MANUSCRIPT the lower values obtained in this work for K and Mg were found in the red and green algae groups as well as higher values were found for red algae for all analytes. These results demonstrate the general characteristic of red algae compositions present higher Ca and K, while the green algae have lower values

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for these elements.

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4. CONCLUSIONS

The direct analysis of marine macroalgae samples by EDXRF provided a simple

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approach for determination Ca, K and Mg by avoiding drastic or time-consuming sample pretreatment. The method is rapid and involves little sample

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preparation. The use of samples of the same matrix for calibration made determination of these elements possible, and good limits of detection and

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quantification were obtained, especially considering the low sensitivity of this

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analytical technique. With regard to the algae, the average concentrations of Ca, K and Mg obtained for the macroalgae from Itaparica Island are in

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agreement with previous reported range. In addition, the red algae compositions presented, in general, higher Ca and K, while the green algae had lower values

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for these elements.

Acknowledgments The authors are grateful to the Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB, Brazil), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), and the Coordenação de Aperfeiçoamento de

ACCEPTED MANUSCRIPT Pessoal de Nível Superior (CAPES, Brazil) for providing grants, fellowships and financial support.

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Lankosz, X-ray fluorescence study of the concentration of selected trace and

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[37] A. Mir-Marqués, M. Martínez-García, S. Garrigues, M.L. Cervera, M. de la Guardia, Green direct determination of mineral elements in artichokes by

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infrared spectroscopy and X-ray fluorescence, Food Chem. 196 (2016) 1023-

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ACCEPTED MANUSCRIPT [39] I. Reinholds, V. Bartkevics, I.C.J. Silvis, S.M. Ruth, S. Esslinger, Analytical techniques combined with chemometrics for authentication and determination of contaminants in condiments: A review, J. Food Compos. Anal. 44 (2015) 56-72. [40] L. Bonizzoni, A. Galli, M. Gondola, M. Martini, Comparison between XRF, TXRF, and PXRF analyses for provenance classification of archaeological

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bricks, X-Ray Spectrom. 42 (2013) 262–267.

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[41] A.C. Neiva, M.A. Marcondes, H.P.F Pinto, P.A.D. Almeida, Analysis of

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photographs and photo-paintings by energy-dispersive X-ray fluorescence spectroscopy, Radiat. Phys. Chem. 95 (2014) 378-380.

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[42] M. Mantler, M. Schreiner, X-ray fluorescence spectrometry in art and archaeology. X-ray spectrum. 29 (2000) 3-17.

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[43] E.P. Bertin, Principles and Practice of X-Ray Spectrometric Analysis, Plenum Press, Nova York, 1970.

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[44] J.N. Miller, J.C. Miller, Statistics and Chemometrics for Analytical

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Chemistry, sixth ed., Pearson Education Limited, England, 2010. [45] R. Jenkins, X-Ray Spectrometry, v 99, chapter 07 and 09, John Wiley&

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Sons, Canadá, 1988.

[46] A.N. Syad, K.P. Shunmugiah, P.D. Kasi, Seaweeds as nutritional

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supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii, Biomed Prev Nutr 3 (2013) 139-144. [47] R. Domínguez-Gonzalez, A. Moreda-Piñeiro, A. Bermejo-Barrera, P. Bermejo-Barrera, Application of ultrasound-assisted acid leaching procedures for major and trace elements determination in edible seaweed by inductively coupled plasma-optical emission spectrometry, Talanta 66 (2005) 937-942.

ACCEPTED MANUSCRIPT [48] L.S. Ferreira, R.P. Lopes, M.N.C. Ulbrich, T. Guaratini, P. Colepicolo, N.P. Lopes, R.C. Garla, E.C. Oliveira Filho, A.M. Pohlit, O.L.A.D. Zucchi, Concentration of inorganic elements content in benthic seaweeds of Fernando de Noronha Archipelago by synchrotron radiation total reflection X-ray fluorescence analysis (SRTXRF), Int. J. Anal. Chem. 2012 (2012) 1-8.

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Vasconcelos, M. Roriz, L.M. Rodríguez-Alcalá, A.M.P. Gomes, A.C. Duarte,

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Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal, Food Chem. 183 (2015) 197-207.

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[50] M.H. Norziah, C.Y. Ching, Nutritional composition of edible seaweed

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D

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Gracilaria changgi, Food Chem. 68 (2000) 69-76.

ACCEPTED MANUSCRIPT Figures captions

Figure 1. Regression curves obtained in this work, correlating results of macroalgae analysis by ICP OES versus EDXRF for (a) Ca, (b) K and (c) Mg, in

AC

CE

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D

MA

NU

SC

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PT

mg kg-1.

ACCEPTED MANUSCRIPT

Figure 1.

y = 1.1637x - 10876 R² = 0.93

100000 80000 60000

PT

mg kg-1 by EDXRF

120000

40000 20000 0

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0

20000 40000 60000 80000 100000 120000

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mg kg-1 by ICP OES

(a)

60000 40000 20000 0 0

20000

(b)

40000

60000

80000

100000

8000

10000

D

mg kg-1 by ICP OES

8000

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10000

y = 1.0765x - 734.65 R² = 0.9314

6000 4000

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mg kg-1 by EDXRF

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y = 0.9473x + 487.8 R² = 0.9886

80000

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mg kg-1 by EDXRF

100000

2000 0

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0

(c)

2000

4000

6000

kg-1

by ICP OES

mg

ACCEPTED MANUSCRIPT Table 1. Taxa of macroalgae collected in different sampling periods.

Sample

A

B

C

D

-

1

Ulva lactuca

x

x

x

x

Green

2

Caulerpa scalpelliformis

x

x

x

x

Green

3

Padina spp.

x

x

x

x

Brown

4

Bryothamnion triquetrum

x

x

x

5

Codium isthmocladum

x

x

x

Green

6

Caulerpa racemosa

x

x

x

x

Green

7

Sargassum spp.

x

x

x

Brown

8

Hypnea musciformis

x

x

9

Dictyopteris jamaicensis

x

10

Acanthophora spicifera

x

x

x

x

Red

11

Dictyota spp.

x

x

x

x

Brown

12

Cryptonemia crenulata

13

Agardhiella sp.

14 15

D

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n°

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Group

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Collections

SC

RI

x

x x

Red

Red Brown

x

Red x

Red

Gracilaria domingensis

x

x

Red

Caulerpa cupressoides

x

x

Green

CE

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x

AC

The letters A, B, C and D correspond to different sampling days.

ACCEPTED MANUSCRIPT Table 2. Limit of detection (LOD), in mg kg-1, equations for calibration curves and coefficients of correlation (R) of the studied elements determined by EDXRF. LOD

Calibration curve

R

Ca

401.4

y = 0.0427x - 322.65

0.961

K

84.75

y = 0.0136x + 13.191

0.998

Mg

27.88

y = 0.0139x + 11.141

AC

CE

PT E

D

MA

NU

SC

RI

y: intensity x: concentration

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Analyte

0.978

ACCEPTED MANUSCRIPT

Table 3. Analysis of Certified Reference Materials (CRM), in mg kg-1, by EDXRF.

K

CRM

Mg

Ca

Certified

Obtained

Certified

Obtained

Certified

NIST 1547

24300

31492 ± 873

4320

4003 ± 128

15600

NIST 1515

16100

20377 ± 913

2710

3126 ± 104

15260

NIST 1570a

29000

37323 ± 972

-

5448 ± 149

C S U

N A

D E

T P E

A

C C

M

I R

T P

15260 ± 660

Obtained

13401 ± 896 13621 ± 979

12964 ±1 007

ACCEPTED MANUSCRIPT Table 4. Determination of Ca, K and Mg (mg g-1) in macroalgae samples by EDXRF. Mg

B. triquet rum H. muscif ormis A. spicife ra C. crenul ata Agard hiella sp. G. domin gensis

C

D

A

B

C

D

A

B

C

D

NC

15.5 8±0. 66 12.6 3±1. 30

15.5 9*

NC

1.08 6±0. 058

3.72 8±0. 185

9.10 0±0. 941 3.54 3±0. 462

9.76 4±0. 112 3.18 2±0. 236

5.79 0*

1.43 1±0. 216

1.58 1±0. 328 1.11 3±0. 117

NC

26.2 1±0. 570

1.96 2±0. 094 1.56 3±0. 155

1.59 0*

16.9 9±1. 05

17.4 5±0. 38 28.0 5±2. 72

38.2 1±1. 90 11.3 5±0. 76

63.7 8±1. 14 3.76 4±0. 321

13.1 6±1. 49 2.82 8±0. 289

30.8 6±0. 96 NC

2.68 0±0. 165 1.55 5±0. 145

1.13 9±0. 404 3.21 5±0. 297

2.80 3±0. 319 2.62 3±0. 250

2.39 8±0. 082 NC

3.60 6±0. 022 3.83 9±0. 210

3.95 1±0. 371 7.83 9±0. 674

2.94 5±0. 118 7.85 7±0. 747

3.68 9±0. 047 NC

NC

NC

NC

NC

NC

84.2 5±4. 69 18.2 1±2. 66 NC

27.1 7±9. 54 99.2 7±2. 40 43.6 6±0. 69 NC

NC

84.1 8±7. 89 71.6 1±8. 02 61.4 4±5. 43 41.4 0±2. 64 107. 6±1. 3 24.1 1±1. 13 49.0 9±2. 58 NC

9.02 8±0. 291 62.2 3±4. 45 23.1 4±2. 20 NC

5.74 8±0. 467 4.03 1±0. 552 NC

3.75 2±0. 132 4.77 4±0. 085 6.08 0±0. 027 NC

47.4 3±3. 64 93.9 1±4. 67 16.1 1±1. 62 28.5 7±3. 09 NC

42.5 0±0. 35 61.5 1±0. 46 NC

4.77 8±0. 258 8.48 1±0. 264 4.74 2±0. 378 5.98 4±0. 730 NC

5.17 6±0. 155 6.08 7±0. 136 NC

NC

43.9 8±4. 29 107. 0±1 0.8 21.3 2±2. 50 33.6 8±2. 16 46.6 6±2. 51 NC

5.79 1±0. 254 7.69 8±0. 432 4.24 7±0. 163 5.07 7±0. 040 8.90 7±0. 793 4.94 0±0. 303 6.57 2±0. 125 NC

3.47 3±0. 071 5.69 7±0. 402 4.92 9±0. 284 NC

7.58 2

13.0 1

4.66 8

4.68 0

NC

NC

5.08 7±0. 394

20.9 0±0. 12

2.53 5±0. 283

4.36 1±0. 037

56.1 7±1. 16 NC

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RI

PT

B

1.65 6±0. 031 2.20 9±0. 036 6.62 1±0. 156 NC

2.03 7±0. 059 3.79 0±0. 763 4.04 8±0. 430 3.42 3±0. 071 NC

3.47 7±0. 087 6.44 5±0. 376 NC

NC

2.30 7±0. 140 3.53 9±0. 172 3.50 4±0. 064 5.03 7±0. 399 5.40 0±0. 618 NC

11.5 2

5.18 2

NC

4.03 4±0. 280 8.60 6±0. 829 5.10 1±0. 310 6.69 2±0. 291 6.14 8±0. 077 NC

NC

NC

85.5 9±3. 43

5.16 5±0. 244

NC

NC

NU

3.84 4±0. 180 2.04 3±0. 206 46.3 8±4. 01 NC

3.64 0±0. 131 5.80 4±0. 307 3.81 5±0. 233 6.89 6±1. 004 3.67 8±0. 092 4.96 4±0. 626 4.41 3±0. 552 NC

2.40 6±3 18 53.6 8±5. 56 NC

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Sarga ssums pp. D. jamaic ensis Dictyo ta spp.

A

D

Red

Sampl es U. lactuc a C. scalpe lliform is C. racem osa C. isthmo cladu m C. cupres soides Padina spp.

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Bro wn

K Period sampling

CE

Gre en

Ca

AC

Mac roal gae gro up

3.73 6±0. 220 NC

4.38 0±0. 061

6.31 8±0. 159 NC

The letters A, B, C and D correspond to different sampling days. NC: not collected. *Sample without replica

26

ACCEPTED MANUSCRIPT

Table 5. Concentration for Ca, K and Mg in mg g-1, in macroalgae samples from different geographical areas. Gr ou p

Taxa Fucus vesiculosus Laminaria digitata (kombu)

Hizikia fusiformis (Hijiki) Ascophylum nodosum (rock weed, egg wrack)

Undaria pinnatifida (Wakame), Himanthaliae longata (sea spaguetti), and Laminaria ochroleuca (kombu)

Dictyopteris justii

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Dictyopteris plagiogramma Padina gymnospora

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Gelidiella acerosa

D

Sargassum sp.

Porphyra columbina

Palmaria palmata (Dulse)

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Gracilaria salicornia (Gorillaogo) Gracilaria parvispora (Green ogo) Porphyra spp. (Nori, Laver) Gracilaria coronolopifolia (Red ogo) Jania rubens Pterocladia capillacea Porphyra (nori) and Palmaria (Dulse) Gracilaria changgi Asparagopsis taxiformis Dictyurus occidentalis

43.22±0. 46 115.79± 1.28 86.99±1. 44 2.8304± 0.1415 63.30±0. 66 20.80±0. 45 4.31±0.0 5 37.075.0 40.5275 ±0.0012 7.3701± 0.0044 10.5935 ±0.0024 9.9101± 0.0005 27.1963 ±0.0008 31.84±0. 00 35.00±0. 71 1.763.19

9.94±0 .13 6.59±0 .06 11.81± 0.34 1.03±0 .05* 5.86±0 .04 7.26±0 .26 6.99±0 .06 33.067.0

8.0-11.0

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Dictyota cervicornis

Porphyra vietnamensis

9.38±0.0 7 10.05±0. 05 9.31±0.3 8 0.7875± 0.0472 11.96±0. 17 29.04±0. 89 10.03±0. 05

SC

bro wn

Undaria pinnatifida (Wakame)

Porphyra tenera (Nori)

Mg

RI

Sargassum wightii

Chondrus crispus (Irishmoss)

K

PT

Undaria pinnatifida (Wakame)

Ca

red

44.3133 ±0.0010 18.3949 ±0.0043 56.3977 ±0.0022 3.0284± 0.0006 14.0350 ±0.0007 4.20±0.2 2 3.90±0.1 7 1.406.12 4.245.87 0.4500± 0.0245 15.18±0. 93 3.74±0.4 4 2.25±0.0 2 2.92±0.0 3 1.94±0.0 9 0.4140.626 0.2650.744 2.0-6.0

136±14

30 30 46 28 28 28 47 48

Nd

48

Nd

48

Nd

48

Nd

48

0.1290.423 0.2950.509 14.059.0

0.5224± 0.0222 81.19±0. 41 201.0±2. 0 155.30± 0.29 16.69±0. 17

30

Nd

7.32±0 .06 5.65±0 11 4.005.90 5.196.11 0.203± 0.012* 4.91±0 .07 2.59±0 .08 4.24±0 .04 4.29±0 .02 2.68±0 .22 0.3000.520 0.1570.221 7.018.0

Nd

Refer ence

30 30 31 7 46 28 28 28 28 28 29 29 47

6.510±0. 052 88.91

Nd

Nd

49

1.988

Nd

48

15.3666 ±0.0003

49.5233 ±0.0013

Nd

48

27

ACCEPTED MANUSCRIPT Galaxaura rugosa Galaxaura obtusata Galaxaura marginata Three Enteromorpha Ulva sp. Enteromorpha spp. (Aonori, Green laver) gre en

1.0928± 0.0040 2.6113± 0.0070 15.6554 ±0.0006 2.30013.80 Nd 26.96±0. 14 7.02±0.2 2 33.42±0. 18 0.0460.075 0.5041± 0.0051

PT

Codium edule (Limu, wawae’iole)

22.9496 ±0.0016 41.5006 ±0.0068 82.6063 ±0.0009 47.6051.20 7.0613.06 4.89±0.0 7 9.83±0.4 9 4.58±0.1 3 0.4730.978 20.6398 ±0.0093

Ulva lactuca (sea lettuce)

RI

Ulva lactuca Caulerpa verticillata -1

48

Nd

48

Nd

48

8.90015.60 27.732.9 9.57±0 .13 15.61± 0.02 22.52± 0.16 0.0900.146 Nd

50 7 28 28 28 29 48

SC

*mg kg

Nd

AC

CE

PT E

D

MA

NU

Nd – not determined

28

ACCEPTED MANUSCRIPT Highlights



EDXRF technique was suitable for direct analysis of marine macroalgae samples Different algae species were used as standards for calibration curves



The proposed method is fast, requires little sample preparation,

PT



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D

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Red algae compositions present higher Ca and K

AC



RI

minimizing sample contamination

29