Introduction

Spirulina is a photosynthetic, filamentous non-differentiated, spiral-shaped, multicellular, and blue-green microalgae (cyanobacteria) that grows naturally in warm climates (Sánchez et al., 2003). It is a ubiquitous organism that was used as food in Mexico 400 years ago during the Aztec civilization. They are found in variety of environments: soil, sand, marshes, brackish water, sea water, and fresh water. In recent year, the most commonly used Spirulina are S. platensis and S. maxima (Khan et al., 2005). Today, there are several companies producing Spirulina as a food supplement, which is sold in many health food stores around the world (Belay, 1993). On the other hand, about 30% of the current world production of 2000 ton is sols for animal food applications (Belay et al., 1996). Spirulina is being grown in the United States, Hawaii, Thailand, Taiwan, Chile, Vietnam, India, Japan, Cuba, Spain, Argentina, Mexcio and other countries (Fox, 1996). Worldwide medical research has discovered that Spirulina with its unique blend of nutrients (good quality proteins, balanced fatty acids profile, antioxidant vitamins, and minerals) has helped to combat many health problems like diabetes, arthritis, anemia, cancer and so forth. Spirulina capsules have also proved effective in lowering blood lipid level, and in decreasing white blood corpuscles (Ruan et al., 1988; 1990), as well as improving immunological function. In addition, Spirulina also is used for health food, feed and for the biochemical products since 1980s (Becker, 1988; Borowitzka, 1988; Richmond, 1988).

Metals like lead, mercury, cadmium, and arsenic are the most likely to adulterate Spirulina products. Each is found as a trace contaminant in certain pesticides and fertilizers, so they are common in agricultural areas. Nickel, copper, and zinc are common fertilizer components or contaminants, but they are substantially less toxic and have a narrow range of optimum concentrations for algae, at least in the case of Chlorella and Spirulina (Kallqvist and Meadows, 1978; Gribovskaya et al., 1980; Pande et al., 1981; Kotangale et al., 1984). They are likely to terminate algae growth before being accumulated at levels toxic to humans. Tin, chromium, selenium, and aluminum are not such a universal threat, but local conditions must be appraised before they can be completely eliminated as a possible hazard.

Cyanobacteria may be especially effective accumulators: Certain types excrete hydroxamate chelating agents (Murphy et al., 1976) that can act as carrier molecules or increase the trace metal pool available near the cell surface. Experiments demonstrate that Spirulina platensis accumulates trace metals more effectively than Chlorella vulgaris (Gribovskaya et al., 1980), an advantage with regard to trace elements essential to humans but a liability if toxic metals are present.  Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been validated for analysis of trace metals in plants (Leiterer et al., 1997). However, ICP-MS is currently being used to detect metals in seaweeds (Netten et al., 2000), and algae food products (Dawczynski et al., 2007). In order to quantify the metal concentration/contamination, the present investigation was carried out in 25 commercially available Spirulina products from different countries of origin for quantify the available metals concentration using ICP-MS. The results of these data would be benefit future nutritional and quality studies.

Materials and Methods

Spirulina sample collection

Totally 25 Spirulina samples in the form of tablets and capsules were obtained from specialist shops from seven different country of origin. The product code, form and country of origin are summarized in table 1.

Table 1: List of Spirulina products and their country of origin.

Elemental determination

The sealed samples of Spirulina were purchased by the author from worldwide producers. Triplicate samples of each product were digested for heavy metal analyses. 100 mg of each triplicate was put into a Teflon PFA type digestion vessel and dissolved in 65% HNO₃ (Merck, Germany; Suprapur grade) using a microwave sample preparation system (CEM Co. Model MDS-200). The temperature in the interior of the vessels can be monitored with the 300 Automatic Temperature Control Probe. Maximum operating temperature and pressure were 300 ^oC and 100 bar, respectively. Repeated concentration-dilution procedures were performed and the final volume was 10-20 ml which was used for heavy metal determination.

Heavy metal content was determined by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) using a Perkin Elmer apparatus model Elan – 6000 (Waltham, Massachusetts, USA) following the manufacturer’s recommended standard operating procedure. The instrumental parameters of ICP-MS were as follows: forward power (1400 W), sample cone (Ni) – 1.0 mm and skimmer cone (Ni) – 0.75 mm, spray chamber temperature 10^oC and diffusion chamber temperature 18^oC, gas controls nebuliser (0.7 ml/min), coolant (13 ml/min, auxiliary 0.5 ml/min), sample flow rate (1.0 ml/min) and sampling distance (10 mm) from load coil and vacuum controls expansion stage (1.8 mbar), intermediate (<104 mbar), analyser (5×106 mbar) and data acquisition for surveying, scanning mode (200 sweep, 4-245 amu), dwell time (160 μs, 2048 channels), and run time 65 s.  Standard concentrations for each element were prepared and used according to the recommendations by the manufacturer to calibrate the instrument before using it on real samples.

Statistical analysis

The statistical analysis of mean ± SD and correlation co-efficient were carried out using PASW statistics 18 software package.

Results

The results of metals analysis of 25 Spirulina products in dry weight are summarized in Table 2. The order of metals concentrations determined in this study for Spirulina samples was Cr>Cd>As>V>Pb>Cu>Fe, while the concentrations (mg/kg d.w.) ranging from 0.003 to 0.018 (Cr) followed by 0.003 – 0.069 (Cd), 0.006 – 0.578 (As), 0.005 – 1.199 (V), 0.100 – 1.206 (Pb), 0.017 – 3.155 (Cu) and 0.336 – 54.68 (Fe).

Lead

The table 2 shows that Pb concentrations for different Spirulina samples are highly variable. The Pb content ranged between 0.100 to 1.206 mg/kg dw and the highest value of Pb (1.206 mg/kg dw) was detected in sample S1 from Australia. Among the 25 samples tested, the maximum level of Pb content  at 1.206 and 0.835 mg/kg d.w. were found in only in two samples namely S1 and S15  respectively. The samples namely S1 and S15 were purchased from Australia and United Kingdom respectively. Whereas the remaining 23 samples, less amount of Pb content was detected ranging from 0.100 to 0.470 mg/kg dw. The order of lead concentration determined from the samples was S1>S15>S6>S7>S8>S16>S25>S5>S21>S25>S22>S18>S17>S4>S3>S14>S10>S20>S13>S11>S9>S23>S12>S9>S2 (Table 2 and Fig. 1a).

Table 2: Heavy metal concentration in Spirulina samples.

Arsenic

The As contents ranged (mg/kg d.w.) between 0.006 detected in sample S1 and 0.578 found in samples S8. Altogether Spirulina products tested, only two samples namely S8 and S9 found maximum As contents were 0.578 and 0.515 mg/kg dw respectively. The both S8 and S9 Spirulina products procured from USA. Nevertheless, the three samples S7, S4 and S5 had the As contents were 0.111, 0.206 and 0.229 respectively. The rest of 23 samples, the order of As concentration was detected as S1>S10>S13>S12>S11>S23>S6>S25> S24>S3>S17>S22>S2>S21>S15>S14>S18>S19>S20>S16 (Table 2 and Fig. 1b).

Figure 1 a-g: Graphical effect of heavy metal concentration (mean ± SD) in commercially available Spirulina products.   Click here to View figure

Cadmium

The differences in Cd content were not very pronounced in all the Spirulina samples tested. The Cd content ranged between 0.003 to 0.069 mg/kg d.w. and the highest value of Cd (0.069 mg/kg d.w.) was detected in sample S2. Among the 25 samples tested, in all the samples had less amount of Cd content was detected (Table 2 and Fig. 1c). A significant positive correlation (P<0.001) was observed between Cd and Pb (r = 0.923) (Table 3).

Chromium                                          

In all the samples of Spirulina (Table 2) had concentrations below the method limit of detection. The Cr contents ranged between 0.003 mg/kg dw detected in sample S1 and 0.018 mg/kg dw in sample S14. The differences in Cd contents were also not very pronounced in all the tested Spirulina samples (Table 2 and Fig. 1d)

Vanadium

We tested 25 samples including 16 tablets and nine capsule forms of Spirulina products (Table 1). The V contents ranged between 0.005 mg/kg dw, detected in sample S1, and 1.199 mg/kg dw found in samples S9. Among the Spirulina products tested, only the two samples S8 (1.199 mg/kg dw) and S9 (1.199 mg/kg dw) were found maximum V contents. On the other hand, another two samples S4 and S5 had the V contents were 0.510 and 0.518 respectively. The remaining of 21 samples, the V concentration was detected between 0.005 and 0.365 (Table 2 and Fig. 1e). A significant positive correlation (P<0.001) was observed between V and As (r = 0.947) (Table 3).

Copper

Table 2 shows that Cu concentrations for different Spirulina samples are highly variable. The highest value (3.155 mg/kg dw) corresponded to the sample of S2 from Australia. The samples such as S17, S11, S15, S7, S3, S19 and S13, the Cu concentration was 1.028±0.068, 1.048±0.077, 1.176±0.061, 1.276±0.031, 1.375±0.793, 1.379±0.127 and 1.627±0.141 mg/kg dw, respectively. While the remaining 17 samples, the Cu concentration were detected between 0.015 and 0.969, the order of concentration was S21>S5>S22>S1>S10>S9>S18>S16>S8>S4>S25>S20>S14>S24>S6>S23>S12 (Table 2 and Fig. 1f). A significant positive correlation (P<0.005) was observed between between Cu and Pb (r = 0.772), and also Cu and Cd (r = 0.730) (Table 3).

Table 3: Correlation co-efficient matrix for heavy metals in Spirulina samples.

Critical r =0.725 at P < .005, or r =0.900 at P < .001 (n = 24).

Iron

The Fe contents ranged between 0.279 mg/kg dw, detected in sample S12, and 69.24 mg/kg dw found in samples S9. In total 25 Spirulina products tested, only four samples namely S6, S11, S12 and S23 were found < 1mg/kg dw. Nevertheless, the five samples such as S10 (51.51±3.519 mg/kg dw), S17 (53.36±0.523), S16 (54.68±3.864 mg/kg dw), S19 (59.92±2.346 mg/g dw) and S9 (69.24±5.66 mg/kg dw) were detected highest Fe content, the level was >50 mg/kg dw. While the 16 samples, the Fe concentrations was detected between 3.440 mg/kg dw and 48.230 mg/kg dw (Table 2 and Fig. 1g).

Discussion

The monitoring system of continuous surveillance of contaminant content in food and pharmaceutical products is crucial for consumer protection and facilitates international trade. Risk assessment is a continually evolving process since information on contaminants, the health effects involved and their occurrence in food are all factors that should be continuously studied and monitored (Kuhnlein and Chan, 2000). The heavy metals are the metals which belong to the transition elements with higher specific gravity. However, only seven of them, Pb, As, Cd, Cr, V, Cu and Fe, have received more attention, due to their detrimental effect on health and possibility of food contamination. The recommend daily intake heavy metal elements are summarized in table 4.

Table 4: Recommend daily intake heavy metal elements (Iyengar, 1985).

This study reveals that the most abundant metals in the Spirulina samples analyzed are Fe, Cu, and the least abundant are usually Cd and As (Table 2). The Pb content of twenty-five Spirulina samples was analysed, all the samples did not exceed the recommend daily intake (300mg/daily of Pb). In all the samples, the lead content is <1.3 mg/kg dw. Taiwanese and Japanese manufacturers allow up to 20 mg/ kg in Pb for Spirulina products, a value not attained by the samples analysed (Ortega-Calvo et al., 1993; Hsu et al., 2001). Tolerable Daily Intakes (TDI) of lead recommended by the WHO for an adult weighing 70 kg (0.250 mg/day) (WHO, 1993). The Pb contents in Spirulina products (Table 2) showed in general good agreement with previous reports (Ortega-Calvo et al., 1993 and Almela et al., 2006).

Accordingly, the total amount of As ingested by humans depends greatly on the amount of Spirulina included in their diet. It is well-known that organic and inorganic species of As differ widely in their toxicity (Oygard et al., 1999), inorganic forms being in general more toxic than organic ones (López et al., 1994). The total As contents found varied between 0.100 and 1.206 mg/kg dry weight. Most of the samples had total As concentrations less than 1 mg/kg d.w. In the present experimental results revealed that total As in all the Spirulina samples were comparable to other reports, although values of the samples were lower (Netten et al., 2000). Furthermore, our results strongly agreed with those of Almela et al. (2002) and Besada et al. (2009).

Ingestion of the daily dose of Spirulina (10 g) recommended for human consumption by the commercial source.  The Natural health products directorate of Canada recommended daily tolerable level of Cd < 0.09 mg/kg b.w. /day (Gershwin and Belay, 2008). The previously reported Cd and Cr level in Spirulina samples were 0.2 and 7.1 mg/kg d.w. respectively (Ortega-Calvo et al., 1993).  It was interesting to note that cadmium and chromium were all below the detection limit in most of Spirulina samples. The present investigation the highest Cd content (0.069 mg/kg d.w.) was determined in Spirulina samples S2 produced by General Nutrition Corporation, Pittsburgh, USA. In all the Spirulina samples analysed the concentrations of Cd not exceed the maximum values permitted by France (0.5 mg/kg dw) (Mabeau and Fleurence, 1993). In general, value for total Cr level (0.003 – 0.018 mg/kg d.w.) was highly similar to Cd value.

Significant differences in V levels between the Spirulina samples from different region were found (Table 2). In this study, much higher content (1.199 mg/kg d.w.) of this element were observed from S8 and S9 Spirulina samples produced by Jarrow Formulas, Los Angeles, USA and Earthrise Nutritionals LLC, USA respectively. The V content in Spirulina samples are yet not studied and reported. Cu concentrations were similar to those reported elsewhere (Netten et al., 2000; Topcluogu et al., 2003, Besada et al., 2009). Cu level ranged between 0.015 mg/g d.w. and 3.155 mg/kg d.w. recorded from Spirulina samples S21 and S2 respectively. On the other hand, in 60% of the Spirulina samples Cu level is <0.5 mg/kg d.w. The range of Cu content found in present study is very less compare than those previously reported (Ortega-Calvo et al., 1993).  Fe content of Spirulina samples also differed depending on sample origin; sample S9 had much higher value (69.24 mg/kg d.w.) than that of other samples. The average Fe content in the present study was very less to the levels previously reported by Ortega-Calvo et al. (1993) and Hsu et al. (2001).

Conclusions

The heavy metals in 25 marketed Spirulina food samples were identified and quantified in this study. Results showed that the contents of Pb, As, Cd, Cr, V, Cu and Fe in the entire test Spirulina food samples were all within the daily intake levels. Therefore, all the tested Spirulina food samples were considered to be safe food.

Acknowledgments

This work is supported fully by Addiriyah Chair for Environmental Studies, Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.

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