Biological Trace Element Research https://doi.org/10.1007/s12011-018-1397-8
Mineral and Trace Metal Concentrations in Seaweeds by Microwave-Assisted Digestion Method Followed by Quadrupole Inductively Coupled Plasma Mass Spectrometry Suman Thodhal Yoganandham 1 & Vasantharaja Raguraman 1 & GobalaKrishnan Muniswamy 1 & Gayathri Sathyamoorthy 1 & Remya Rajan Renuka 1 & Jayaseelan Chidambaram 1 & Thirugnanasambandam Rajendran 1 & Kumar Chandrasekaran 1 & Radhika Rajasree Santha Ravindranath 1 Received: 7 April 2018 / Accepted: 24 May 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract This study reports the total concentrations of mineral and trace metals sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc, and manganese in the seaweeds Padina tetrastromatica, Turbinaria ornate, Sargassum wightii, Sargassum swartzii, Gracilaria edulis, Ulva lactuca, Chaetomorpha antennina, and Halimeda opuntia collected from mandapam coastal regions, Southeast coast of India. Microwave-assisted digestion was used for sample preparation prior to mineral and trace metal analysis. Mineral and trace metal analyses were determined by inductively coupled plasma mass spectrometry. The ranges of concentrations of mineral and trace metals in algae were 27.04 ± 2.54–194.08 ± 2.36 mg/kg for manganese, 1.88 ± 0.10–121.5 ± 0.70 mg/ kg for sodium, 6.5 ± 0.56–90.5 ± 2.12 mg/kg for magnesium, 59.07 ± 0.34–672 ± 2.82 mg/kg for potassium, 13.15 ± 2.08– 135.13 ± 1.59 for sulfur, 0.003 ± 0.001–3.44 ± 0.13 mg/kg for cobalt, 0.39 ± 0.19–8.95 ± 0.38 mg/kg for copper, 0.72 ± 0.28– 25.72 ± 0.39 mg/kg for zinc, and 6.01 ± 0.27–188.47 ± 1.92 mg/kg for iron.The results were evaluated statistically, and the significant difference was observed in the mean concentrations of all mineral and trace elements, except Co, Cu, and Zn, among the type of seaweeds. Keywords Inductively coupled plasma mass spectrometry . Microwave-assisted digestion . Seaweeds . Mineral . Trace metals . Multielement
Introduction Seaweeds are macro algae that grow in rocky landscape or on coral reefs or can grow at great depths if sunlight can penetrate through the water [1]. Most of the seaweeds can be seen thriving in underwater beds floating along the sea surface attached to rocks [2]. According to previous report, 221 seaweed species (125 red (Rhodophyta), 32 green (Chlorophyta), and 64 brown (Phaeophyta) seaweeds) are being used as fertilizer, food fodder, medicine, and now used in industry for the productions like carrageenan, algin, and agar [3]. Seaweeds are easily cultivated and achieve rapid growth for several species
* Suman Thodhal Yoganandham
[email protected] 1
Ecotoxicology Division, Centre for Ocean Research, Col. Dr. Jeppiar Research Park, Sathyabama Institute of Science and Technology, Chennai, India
[4]. Seaweeds are traditionally consumed by humans as part of daily diet in Asian countries mainly Japan, Korea, and China. Seaweed contains rich sources of minerals; sometimes, their content is as high as 40% [5, 6]. This is because seaweeds accumulate metal ions from salt water and concentrate those substances as carbonate salts in their fronds [7]. Generally, seaweeds constitute in polysaccharides, minerals, vitamins, and other bioactive substances like proteins, polysaccharides, polyphenols, and lipids with antifungal, antibacterial, and antiviral properties [8, 9]. The most common minerals found in seaweeds are phosphorus, potassium, iodine, sodium, magnesium, calcium, iron, copper, and fluorine [10]. The seaweeds show great variation in the mineral content based on environmental factors as water temperature, salinity, light, and nutrients [11]. To fully exploit the nutritional value of seaweeds, several studies conducted on nutritional values and biochemical composition of various seaweeds collected from different parts of the world have been conducted [12–16]. Compared to land plants, the chemical composition of seaweeds has been poorly investigated.
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An interesting strategy to sample preparation of seaweed is a microwave-assisted digestion. In the most recent years, the utilization of closed vessels combined with microwave radiation has been described as a possible sample preparation technique, which presents high efficiency of digestion, the loss of volatile elements is avoided, and the risk of contamination may be reduced [17]. Historically, atomic absorption spectrometry was widely used to determine metal elements, but in recent years, it has been replaced by inductively coupled plasma mass spectrometry [18–20]. Inductively coupled plasma mass spectrometry technique is useful to analyze the sensitive range of elements from 7 to 250 (Li to U) [20]. Inductively coupled plasma mass spectrometry is widely used for determining low concentration (ppb) and ultra-low concentration (ppt) in environmental and biological samples owing to its unique analytical capabilities. It offers several advantages that include extremely low detection limit, a large linear range, and possibilities to detect isotope composition of elements. However, the inductively coupled plasma mass spectrometry analysis is subject to spectral and non-spectral interferences, which are mostly associated with the quadruple inductively coupled plasma mass spectrometry instrument [21]. Spectral interferences occur mainly due to the fact that polyatomic interferences are usually due to the sample matrix, sample diluent, and/or the inert gas used during ionization. Non-spectral interferences named matrix effects are caused by sample matrix; as a result, it allows the formation of species that may determine with analyte determination [22]. In this paper, a microwave assisted digestion procedure of sea weed and determine the elemental profile Padina tetrastromatica, Turbinaria ornate, Sargassum wightii, Sargassum swartzii, Gracilaria edulis, Ulva lactuca, Halimeda opuntia, and Chaetomorpha antennina. The studied seaweeds were sleeted according to their natural presence on rameshwaram. To the best of our knowledge, this is the common seaweeds from rameshwaram. Because of expanding industrialization alongside with environment pollution, the determination of trace element concentration in sea weed is crucial. Hence, in this study, minerals and trace elements such as sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc, and manganese were analyzed by inductively coupled plasma mass spectrometry.
Materials and Methods Sample Collection and Sample Preparation Seaweed Sampling A total of nine seaweeds such as Padina tetrastromatica, Turbinaria ornate, Sargassum wightii, Sargassum swartzii, Gracilaria edulis, Ulva lactuca, Kappaphycus alvarezii,
Chaetomorpha antennina, and Halimeda opuntia were collected from mandapam coastal regions, Southeast coast of India. After collection, seaweed samples were rinsed with freshwater to remove sand, epiphytes, and detritus, transported to the laboratory and dried in oven at 65 °C.
Reagents and Apparatus Ultra-pure water (18 MΩ cm−1) from a Milli-Q water purification system (Milli-Q SP ICP-MS, Millipore, USA) and nitric acid of Suprapur grade (purchased from Merck) were used in all the digestion experiments. Ultra-pure argon (99.9999% purity). Volumetric flasks, tubes, and polypropylene labware were immersed with 10% nitric acid solution for at least 24 h and subsequent washing through ultra-pure water.
Microwave-Assisted Acid Digestion The seaweed samples were acid digested using Mars 6 One Touch Technology Microwave lab station (CEM Microwave Technology Ltd., North Carolina, USA). Briefly, 0.500 g of seaweed sample was digested with nitric acid supra pure metal. Approximately 5 mL of nitric acid supra pure metal 65% was added in Teflon vessel and the sample could predigest for 20 min and the pre-digested seaweed samples were capped. The Teflon vessels were placed in mars 6 microwave digester, which was operated at the temperature that was set 200 °C for 20 min and hold temperature was 200 °C for 15 min. At the end of the digestion, all seaweed samples were cooled at room temperature. Then, the digests inside the vessels were quantitatively transferred into 50-mL polyethylene centrifuge vials.
Analysis of Digest Digested samples were analyzed using Agilent 7700x with an octopole-based collision/reaction cell that was used for all measurements. The instrument was equipped with Agilent Integrated Autosampler and Mass Hunter version A.01.02 software. Sample introduction system for the Agilent 7700X ICP-MS instrument consisted of Tygon peristaltic pump tubing (1.02 mm ID), a glass concentric pneumatic nebulizer, a double-pass Scott type chamber, a standard quartz torch (2.5 mm ID), a nickel (Ni)-plated sampling cone, and a platinum (Pt) skimmer cone. Argon (99.99%) and helium (99.99%) from Atlas (CA, USA) were used as plasma and collision gases, respectively. Instrument parameters plasma gas (l/min) 15, helium flow (mL/min) 1.5, carrier gas (L/ min) 0.45, make up gas (L/min) 0.69, plasma RF power (W) 1550, plasma RF matching (V) 1.70, sampling depth (mm) 7.5, S/C temp (°C) 2, extract 1 lens (V) 1.1, extract 2 lens (V) − 200, omega bias (V) − 75, omega lens (V) 9.8, cell entrance (V) − 36, cell exit (V) − 60, deflect (V) 14, plate bias (V) − 35, energy discrimination (V) 4.5, OctP Bias (V) − 8.0, Oct RF
Mineral and Trace Metal Concentrations in Seaweeds by Microwave-Assisted Digestion Method Followed by...
(V) 180, acquisition mode spectrum: peak pattern 1, replicates 3, sweeps/replicate 1000, integration time/mass (s) 0.7 were used for all the validation studies and sample analyses. The following elements/isotopes were detected sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc, and manganese. Calibration standards were prepared from 100-mg L−1 multielement standard solutions.
Statistical Analysis Statistical analysis was carried out with graph pad prism 7.0 software package. The experimental results are expressed as mean ± SD of triplicates. A significant difference among the means of samples was obtained by Tukey’s test, and analysis of variance was performed by one-way ANOVA. Results with p < 0.05 were considered statistically significant.
Result and Discussion Considerable differences in the concentrations of the analyzed minerals and trace metals were observed when the seaweed samples were grouped as green, brown, and red. Figure 1 shows the mean concentrations of the mineral and trace metals that showed significant differences (p < 0.05) among the groups. Thus, Co, Cu, and Zn were not included in the figure because there were no significant differences observed. The K concentration in red seaweed had the highest mean and showed statistically significant (p < 0.05), when compared to green and brown seaweed group, whereas it was stated that the element content in seaweeds may be dependent on various environmental factors, including
Fig. 1 Mean concentrations including the standard deviation of Mn, Na, Mg, K, S, Fe according to the type of seaweed. Different letters indicate that the differences between mean values are significant (p < 0.05)
concentrations of elements in water [23], interactions between elements, salinity, pH, light intensity, and metabolic factors such as dilution of element contents due to seaweed growth [24]. The green seaweed had the highest mean in Mn concentration and showed statistically significant (p < 0.05) within the group. Magnesium content in both the green and brown seaweeds was not statistically significant. Interestingly, magnesium content was found to be higher in red seaweed when compared to the other seaweed groups [25]. The sulfur content in red seaweed had the highest mean and showed statistically significant (p < 0.05) when compared to the other groups. On the other hand, the green seaweed had the highest mean in Fe concentration when compared to red and brown seaweeds that showed statistically significant (p < 0.05). Red seaweeds contain significantly higher levels of iron compared to that of green and brown seaweeds [25]. Trace element content in marine algae species was found to be in the range of 27.04 ± 2.54–194.08 ± 2.36 mg/kg for manganese, 1.88 ± 0.10–121.5 ± 0.70 mg/kg for sodium, 6.5 ± 0.56–20.14 ± 0.24 mg/kg for magnesium, 59.07 ± 0.34–404.66 ± 2.34 mg/kg for potassium, 13.15 ± 2.08–135.13 ± 1.59 for sulfur, 0.003 ± 0.001–3.44 ± 0.13 mg/kg for cobalt, 0.39 ± 0.19–8.95 ± 0.38 mg/kg for copper, 0.72 ± 0.28–25.72 ± 0.39 mg/kg for zinc, and 6.01 ± 0.27–188.47 ± 1.92 mg/kg for iron (Table 1). The difference in ion levels in different algal species depended on their physicochemical parameters, chemical composition, growth and metabolic factors such as dilution of element contents during the growth of seaweeds [26, 27]. The lowest and highest levels of copper were found as 0.39 ± 0.19 mg/kg in Gracilaria edulis and 8.95 ± 0.38 mg/kg in Halimeda opuntia (Fig. 2). The lowest and highest levels of manganese
Red
Green
Brown
Table 1
8.25 ± 1.76
194.08 ± 2.36 137.43 ± 3.33
Halimeda opuntia Gracilaria edulis
Kappaphycus alvarezii
37.78 ± 0.85 27.04 ± 2.54 50.70 ± 1.22 28.36 ± 1.30
Sargassum wightii Sargassum swartzii Ulva lactuca Chaetomorpha antennina
41 ± 1.41 29.10 ± 1.70
Mn
Turbinaria ornate
Padina tetrastromatica
Seaweed list
25.66 ± 2.34
34.76 ± 2.49 49.91 ± 0.72
1.88 ± 1.04 4.84 ± 0.58 17.34 ± 1.54 23.32 ± 1.51
2.12 ± 0.43
121.5 ± 0.70
Na
90.5 ± 2.12
20.14 ± 0.24 6.5 ± 0.56
20.8 ± 0.28 13.91 ± 0.57 7.75 ± 0.51 12.99 ± 0.14
8.53 ± 0.55
8.80 ± 0.13
Mg
672 ± 2.82
91.74 ± 1.37 404.66 ± 2.34
59.07 ± 0.34 162.8 ± 1.23 266.82 ± 2.12 176.38 ± 0.72
121.9 ± 1.83
201.5 ± 2.12
K
48.44 ± 2.04
41.33 ± 1.99 135.13 ± 1.59
13.95 ± 2.58 48.92 ± 1.97 18.41 ± 2.17 48.78 ± 2.05
13.15 ± 2.08
28.02 ± 1.41
S
Cu 4.09 ± 0.62 7.05 ± 0.35 1.44 ± 0.07 2.78 ± 0.21 0.52 ± 0.13 6.21 ± 0.59 8.95 ± 0.38 0.39 ± 0.19 5.25 ± 0.35
Co 1.22 ± 0.29 1.09 ± 0.20 0.05 ± 0.03 0.03 ± 0.01 0.003 ± 0.001 3.44 ± 0.13 1.31 ± 0.19 0.05 ± 0.02 3.2 ± 0.28
7.75 ± 1.06
0.72 ± 0.28 11.99 ± 0.62
2.15 ± 0.21 18.33 ± 0.84 25.72 ± 0.39 12.98 ± 0.82
4.72 ± 0.31
14.12 ± 0.43
Zn
22 ± 1.41
8.15 ± 0.21
19.88 ± 0.91 188.47 ± 1.92
6.01 ± 0.27 13.31 ± 0.75 40.44 ± 1.24 22.79 ± 1.66
8.68 ± 0.89
Fe
Concentrations (mean ± SD; minimum–maximum) of mineral and trace metal for different genera of seaweeds collected from mandapam coastal regions, Southeast coast of India
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Fig. 2 Copper concentration of different seaweeds
were found as 8.25 ± 1.76 mg/kg in Kappaphycus alvarezii and 194.08 ± 2.36 mg/kg in Halimeda opuntia (Fig. 3). The levels of metal concentration ranged from 28.26 mg/100 g for iron, 5.28 mg/100 g for zinc, 3.84 mg/100 g for manganese, 0.58 mg/100 g for copper in the Sargassum swartzii collected from saurashtra coast of India [6]. The lowest and highest levels of sodium were found as 1.88 ± 0.10 mg/kg in Sargassum wightii and 121.5 ± 0.70 mg/kg in Padina tetrastromatica (Fig. 4). The levels of metal concentration ranged from 2261.46–2288.57 mg/100 g for calcium, 1517.90–1519.76 mg/100 g for potassium, 1309.04– 1320.67 mg/100 g for sodium, 830.24–855.67 mg/100 g
Fig. 3 Manganese concentration of different seaweeds
Mineral and Trace Metal Concentrations in Seaweeds by Microwave-Assisted Digestion Method Followed by...
Fig. 4 Sodium concentration of different seaweeds
Fig. 6 Potassium concentration of different seaweeds
for magnesium, 0.04–0.11 mg/100 g for chromium, 00.23– 0.27 mg/100 g for Cu, 17.02–17.56 mg/100 g for iron in the Sargassum wightii collected from south east coast of India [28]. The lowest and highest levels of magnesium were found in Gracilaria edulis 6.5 ± 0.56 mg/kg and 90.5 ± 2.12 mg/kg in Kappaphycus alvarezii (Fig. 5). The levels of metal concentration ranged from 1.21–7.13 mg/g for magnesium, 0.4–6.4 μg/g for Fe, 0.11–0.8 μg/g for manganese, 0.011 μg/g for zinc, 0.012–0.17 μg/g for chromium, 0.024– 0.158 μg/g for lead, 0.0001–0.004 μg/g for mercury in the Padina tetrastromatica at different beaches of Karachi coast [29]. The lowest and highest levels of potassium were found
as 59.07 ± 0.34 mg/kg in Sargassum wightii and 672 ± 2.82 mg/kg in Kappaphycus alvarezii (Fig. 6). The lowest and highest levels of sulfur were found as 13.15 ± 2.08 mg/ kg in Turbinaria ornate and 135.13 ± 1.59 mg/kg in Gracilaria edulis (Fig. 7). The lowest and highest levels of cobalt were found as 0.003 ± 0.001 mg/kg in Ulva lactuca and 3.44 ± 0.13 mg/kg in Chaetomorpha antennina (Fig. 8). The levels of metal concentration ranged from 1.13% for sodium, 8.05% for potassium, 2.05% for calcium, and 1.64% for manganese in the Chaetomorpha antennina at Visakhapatnam, east coast of India [30]. The lowest and highest levels of zinc were found as 0.72 ± 0.28 mg/kg in
Fig. 5 Magnesium concentration of different seaweed
Fig. 7 Sulfur concentration of different seaweeds
Suman et al.
Fig. 8 Cobalt concentration of different seaweeds
Fig. 10 Iron concentration of different seaweeds
Halimeda oputia and 25.72 ± 0.39 mg/kg in Ulva lactuca (Fig. 9). The levels of metal concentration ranged from 574.6–558.1 mg/100 g for calcium, 77.32–77.67 mg/100 g for potassium, 92.20–93.63 mg/100 g for sodium, 825.8– 842.4 mg/100 g for manganese, 0.32–0.34 mg/100 g for chromium, 2.05–2.08 mg/100 g for copper, 163.80– 167.80 mg/100 g for iron in the Ulva lactuca collected from south east coast of India [28]. The lowest and highest levels of iron were found in 6.01 ± 0.27 in Sargassum wightii and 188.47 ± 1.92 mg/kg in Gracilaria edulis (Fig. 10). The levels of metal concentration ranged from 3.81% for sodium, 5.42% for calcium, 17.35% for potassium, 8.36% for
chlorine, 7.82% for sulfur in the Gracilaria edulis collected from south east coast of India [31]. The potassium, sodium, phosphorus, calcium, magnesium, iron, copper, zinc, and manganese were determined in seaweeds belonging to different genera of brown, red, and green seaweeds. There were clear differences in the concentrations between the considered genera. Green seaweed had the highest mean iron (1260 mg/kg), copper (7.46 mg/kg), and manganese (15.0 g/kg) concentrations and brown seaweed had the highest mean calcium (10.3 g/kg) and potassium (31.4 g/ kg) concentrations compared with red (3.11 g/kg for calcium and 14.1 g/kg for potassium) and green (7.58 g/kg for calcium and 13.9 g/kg for potassium) seaweed [32]. Mineral composition of Codium adhaerens, Codium vermilara, Bifurcaria bifurcate, Cystoseira usneoides, Fucus guiryi, Fucus serratus, Fucus spiralis, Laminaria ochroleuca, Pelvetia canaliculata, Saccharina latissima, Sargassum muticum, Sargassum vulgare, Gigartina sp, Gracilaria vermiculophylla. Total measured mineral content ranged from 71.0 g kg − 1 DM in Cadmium adhaerens to 10.9 g kg−1 dry matter (DM) in Gracilaria vermiculophylla, calcium being the mineral generally found in higher amounts [33].
Conclusion
Fig. 9 Zinc concentration of different seaweeds
The microwave-assisted digestion of sea weed samples in nitric acid allows the determination of trace elements in quadrupole inductively coupled plasma mass spectrometry that presents considerable advantages, which include good precision, reduced contamination, safety, accuracy, and speed.
Mineral and Trace Metal Concentrations in Seaweeds by Microwave-Assisted Digestion Method Followed by... Acknowledgments Authors are thankful to Sathyabama Institute of Science and Technology, Jeppiaar Nagar, Chennai for providing necessary facilities.
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