UNIVERSITY OF CINCINNATI

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1 U UNIVERSITY OF CINCINNATI Date: I,, hereby submit this original work as part of the requirements for the degree of: in It is entitled: Student Signature: This work and its defense approved by: Committee Chair: Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature:

2 The Toxicity Assessment of Heavy Metals and Their Species in Rice A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE in the Department of Chemistry of the College of Art and Science 2009 by Julie Zhiling Zhang B.S., Chemistry, Jiling University, China, 1989 Committee Chair: Joseph A. Caruso, Ph.D.

3 Abstract There is an accumulation of minerals and other elements in rice. Related to health, and particularly nutritional concerns, twenty (20) rice samples of different varieties and origins obtained from the US market were investigated for twenty-four (24) elements by using inductively coupled plasma mass spectrometry (ICP-MS). Arsenic species in Jasmine brown and white rice (Royal Thailand), Basmati brown and white rice (Royal India), and Nishiki brown and white rice (JFC California, US) were also studied by coupling high performance liquid chromatography (HPLC) with ICP-MS. Selenium species in Basmati brown and white rice (Royal India) were also studied by HPLC-ICP- MS. The study indicates essential elements are accumulated more in brown rice. There are heavy metals of cadmium, lead, antimony, and chromium in the rice samples tested, but the content is not enough for concern. 34 to 333 ppb of arsenic is found in the rice. The arsenic content in the most of white rice samples is in the range of 150 to 225 ppb. The brown rice shows more arsenic. As (III), As (V), and dimethylarsinic acid (DMA) are the species found in the three type of rice and these varied among different rice types. The Basmati rice, regardless to the origins, trends to contain more aluminum and selenium. Little selenium (IV) is presented in Basmati white rice (Royal India). ii

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5 Acknowledgements I would like to thank my advisor Dr. Joseph Caruso for his guidance and instruction during the research process. In addition to my advisor, I would also like to recognize my committee member Dr. Thomas Ridgway for his invaluable advices. Heather Trenary and Yaofang Zhang as members of the Caruso group provided a support to me. I would like to put my appreciation on them. The research was completed in the International Paper Cincinnati Technology Center. Providing financial, technological, and knowledge supports, International Paper allowed me to achieve my dream. Mr. Dennis Crawshaw (the International Paper Analytical Science Group Manager), Dr. Ewa Bucher, and Dr. Chuck Lohrke helped and supported me throughout the thesis. I would like to put my gratitude on them. Finally, my acknowledgement is to my family. My husband, George He, and my daughter, Grace He, gave me encouragement that cannot be forgotten. The influx of their euphonious words made this experience more inspiring and joyful. iii

6 Table of Contents Abstract Acknowledgement List of Figures List of Tables ii iii vii ix Chapter I Introduction Introduction of rice Heavy metal accumulation in rice General toxicological profile of heavy metals Antagonistic affects between selenium and arsenic Objective of the research 18 Chapter II Methodologies Inductively coupled plasma mass spectrometry (ICP-MS) Instrument description and theory Interference removal with collision and reaction cell High performance liquid chromatography (HPLC) 28 Chapter III Experimental Instrumentation and apparatus Reagents and standards 34 iv

7 3.3 Samples Experimental procedure Instrument tuning Determination of detection limit (DL) 46 Chapter IV Results and discussion The elemental profile in different rice varieties Detection limits (DL) and limits of quantification (LOQ) of the ICP-MS simultaneous multi-element analysis Quality assurance and quality control (QA/QC) The total element profile in different rice varieties from origins Trifluoroacetic acid (TFA) extraction Optimization of Collision or reaction gas flow QA/QC issues in TFA extraction Efficiency of TFA extraction Arsenic speciation analysis in TFA extracts Inorganic selenium speciation analysis in Basmati white and brown rice (Royal, India) Methanol/ water extraction Efficiency of methanol/water (50/50) extraction Arsenic speciation analysis in the methanol/water (50/50) extracts 73 v

8 Chapter V Conclusions 75 References 78 vi

9 List of Figures Figure 1.1 Diagrams of Rice Plant and Rice Grain Figure 1.2 Images of Rough rice, Brown Rice and White Rice Figure 1.3 Images of long Grain, Medium Grain and Short Grain Rice Figure 2.1 A General schematic of an inductively coupled plasma mass spectrometer Figure 2.2 A schematic of an ICP plasma torch adapted from Agilent ICP-MS Primer Figure 2.3 A schematic of a quadrupole mass analyzer Figure 2.4 Tow possible mechanisms for 40 Ar 35 Cl interference removal in determination of 75 As Figure 2.5 The kinetic energy discrimination mechanism Figure 2.6 Coupling HPLC to ICP-MS Figure 2.7 Analyte competitions between stationary and mobile phase Figure 3.1 Photograph of HPLC-ICP-MS used in the study Figure 4.1 The distribution of elemental recoveries in digestion and analytical procedures Figure 4.2 The distribution of P, S, K, and Mg in different rice varieties and origins Figure 4.3 The distribution of Ca, Zn, Mn, Fe, and Na in different rice varieties Figure 4.4 The distribution of Al, Ni, Ba, Ti, and Mo in different rice varieties Figure 4.5 The distribution of As, Cd, Cr, and Pb in different rice varieties Figure 4.6 The profile of arsenic in different rice varieties and origins Figure 4.7 The distributions of Selenium and arsenic vii

10 Figure 4.8 Optimization of the reaction/collision gas flow for arsenic and selenium determination Figure 4.9 Comparison of chromatograms obtained from HPLC-ICP-MS with a Hamilton PRP-100 anion exchange column and Luna SCX 100A column Figure 4.10 The profile of the sum of arsenic species in the Jasmine, Basmati, and Nishiki brown and white rice Figure 4.11 The profile of arsenic species in Basmati brown and white rice, Royal, India Figure 4.12 The profile of arsenic species in Nishiki brown and white rice, JFC California US Figure 4.13 The profile of arsenic species in Jasmine brown and white rice, Royal, Thailand Figure 4.14 Comparison of arsenic species among Jasmine white rice, Nishiki white rice and Basmati white and brown rice Figure 4.15 The chromatograms of inorganic selenium speciation analysis Figure 4.16 The yields of six sequential extractions Figure 4.17 The ph meter responses for several methanol/water mixtures Figure 4.18 The chromatogram of arsenic speciation analysis in methanol/water (50/50) extracts viii

11 List of Tables Table 1.1 Composition of White Rice and Brown Rice adapted from Thai food composition table (1999), Institute of Nutrition, Mahidol University, Thailand Table 1.2 The chemical forms of selected arsenic species and their toxicity Table 2.1 Gases for collision and/or reaction cell Table 3.1 Sample list Table 3.2 Operational parameters for ICP-MS Table 3.3 The HPLC conditions Table 3.4 Target tune values for 1ppb of Agilent tuning solution containing 0.5 % of HCl and 0.1 second integration time Table 4.1 The detection limits (DL) and limits of quantification (LOQ) of ICP-MS simultaneous multi-element analysis Table 4.2 The recovery of calibration verification standards in the analysis of total elements concentration Table 4.3 The total arsenic in TFA extracts and the efficiency of arsenic TFA extraction Table 4.4 The total selenium in TFA extracts and the efficiency of selenium TFA extraction Table 4.5 Quantitative figures of merit Table 4.6 The summary of arsenic speciation analysis Table 4.7 The extraction efficiency of arsenic species in methanol/water (50/50) ix

12 Chapter I - Introduction Rice as the main staple food for half of the world s population provides more than one fifth of calories consumed by humans worldwide [1], [2]. In developing countries, rice is a major nutrient source for lower income households. In these families, 30-55% of their total iron comes from rice [3]. Rice is a good micronutrient source of manganese, selenium, magnesium and other essential minerals when grown where these elements are present. White rice, also called milled rice, is the most accepted dietary form. The seeds of the rice plant are first milled to remove the hull becoming brown rice, and the process may be continued to polish the brown rice, producing white rice. The rice spoilage and storage time also can be improved, as the high oil bran tends to make the rice seeds rancid. However, the rice seeds lose the most of their nutrients in this process, because rice bran contains a high level of dietary fiber, lipids, amino acids, various antioxidants, vitamins, cofactors, and dietary minerals. Today, brown rice and wild rice as whole grain rice are suggested to provide more nutrients and health benefits than the more common white rice. These can make a significant difference in human health in offering some protection against cardiovascular disease, diabetes, insulin resistance, and obesity. [4], [5], [6], [7], [8], [9]. 1

13 However, research has revealed that rice plants accumulate heavy metals from soil and irrigation water. Affected by the application of fertilizers, pesticides, wastewater irrigation and other human activities, the issue gets worse. Certain heavy metals and excessive minerals may have a significant negative impact on humans, because many trace minerals are toxicity at high in-taking doses [10]. In order to assess the potential toxicity accurately, it is necessary to investigate the metals and metal species in different rice varieties and origin. The bioavailability of metal species depends their mobility, and also may be affected by the presence of other metals, because the species may bond with the present other metals to lower their solubility [11], [12]. 1.1 Introduction of rice Rice plants are grass type annual plants, which like to grow in wet and warm regions. There are thousands of varieties around the world. Rice plants can grow to 0.6 to 1.8 meters tall, depending on the variety and soil fertility. The rice grass has long, slender leaves and multiple inflorescences on a tiller. The inflorescence is a group of flowers arranged on a stem. The tiller is a branch of rice plant which consists of roots, stem, and leaves. Figure 1.1 illustrates the rice plants. Normally, one rice plant has 5 to 15 tillers and 200 flowers in one tiller. Thus, thousands of grains can be produced by one plant. The edible rice grains consist of hull, rice bran, white rice, and germ which are also 2

14 showed in Figure 1.1 Figure 1.1 Diagrams of Rice Plant and Rice Grain There are two types of rice cultivation: lowland and upland. Lowland rice is grown in a flooded rice paddy. About 85% of rice worldwide is produced by the lowland method. In the low rainfall, high humidity, or high soil fertile regions, upland rice can be cultivated. The upland rice, which grows without surface water, is less productive than lowland rice, because the lower soil moisture, and the less nutrient uptake. The lowland rice, also called paddy rice, usually is sown in a seedbed and then the young plants are transplanted into a flooded field. It is labor intensive but limits weeds because young plants shade them from needed sun. In America, the rice seeds first are socked in water and then distributed onto a flooded field using a low-flying aircraft. Herbicides are 3

15 usually used for weed control. In order to have higher yields, Pesticide and fertilizer are used in most rice farms. The life span of rice plant is 3 to 6 months depending on variety and fertility. After rice plant harvesting and drying, the grains are ready for the milling process. The rough rice passes through a sheller to remove its inedible hull. The brown rice with bran layer and germ results from this process. Brown rice can be used as is or further milled by rubbing grains together to produce white rice without bran and germ. The images of rough rice (with hull), brown rice (with bran), and white rice are illustrated in Figure 1.2 Rough Rice Brown Rice White Rice Figure 1.2 Images of Rough rice, Brown Rice and White Rice Rice is a carbohydrate complex, which means starch and fiber. The white rice contains mostly starch and 6 to 10 % of protein. Rice bran has diverse composition which includes protein, fiber, lipids, vitamins, antioxidants, and minerals. Brown rice with bran has more nutrients. The composition of white and brown rice is listed in table

16 Table 1.1 Composition of White Rice and Brown Rice adapted from Thai food composition table (1999), Institute of Nutrition, Mahidol University, Thailand Nutrition Facts Serving: 100g White Rice Brown Rice Calories, kcal Moisture (water), g Total Fat, g Dietary Fiber, g Calcium, mg 8 12 Phosphorus, mg Potassium, mg Sodium, mg Vitamin B1, mg Vitamin B2, mg Niacin, g Protein, g Carbohydrates, g The composition of rice shows little difference with rice varieties and origins. There are more than 40,000 varieties, but only over 100 of them grow worldwide. Today, more than 100 countries cultivate rice. Rice has ranked the number one human consumption food and number two in production among cultivated grains. Rice is generally classified into long grain, medium grain and short grain rice with their length. The images of long grain, medium grain, and short grain rice are illustrated in Figure 1.3 5

17 Figure 1.3 The Images of long Grain, Medium Grain and Short Grain Rice Some rice varieties are consumed only in where they are grown, and others are well known, such as Basmati, Jasmine, KoKuho (Shushi sweetheart), and Nishiki (Sushi rice). Additionally, wild, black and red rice, as small consumed varieties, are also favorite of some people. The wild, black and red rice are unpolished and have black and red skin. 1.2 Heavy Metal Accumulation in rice With development of stabilizing lipid degradation, brown rice becomes highly recommended by the nutritionist due to its high nutritional value and health benefits. Rice bran, the out layer of brown rice, has high level of several compounds which are potentially mitigate against some chronic diseases such as high cholesterol, osteoporosis, cardiovascular disease and cancers. Brown rice also is a good source of essential minerals. Kaneda et al. (2007) revealed that manganese mainly contributes to the anti-oxidative property of rice bran [13]. Selenium is another important essential element for the human 6

18 body, which involves several major metabolic pathways. Klein E. A. (2004) and Vogt T. M. et al. (2003) suggested selenium has the potential reduce the risk of certain cancers [14], [15]. Additionally, research has revealed that rice contains more heavy metals than other grains, and there are a much higher concentration of heavy metals in the rice bran that in white rice. Inappropriate levels of heavy metals may caused by genetic constitution, contaminated environment or different processes. Research studies are many for metals analyses of rice, which can be summarized by in the following four areas. 1. Investigation of heavy metals in rice and rice plants. The first report of human cadmium toxicity was in Japan in 1950s. Rice paddy fields were contaminated with cadmium by a zinc mining operation [16]. Cadmium exposure causes higher risk for renal impairment and bone diseases. Kaneta, M. et al. (1986) had investigated the distribution and chemical form of cadmium in rice and wheat plants. Their results suggested that there were three major organic compounds and a small amount of inorganic salt in the stem of rice plants. In addition, copper, lead, and nickel in rice and wheat were also studied. The results showed that these compounds were primarily organometals [17]. From a nutritional view, accurate determination of the trace level of metals is the first step to assess the nutritional benefits and toxic risk. Manjusha, R. et al. (2008) mineralized the organic matrix of rice by treating rice samples with ultraviolet (UV) radiation and concentrated HNO 3 and 30% of H 2 O 2. They measured trace metals by ETAAS and ICP-AES. The trace metal profile was established on Indian rice samples, 7

19 and included K, Mn, Fe, Cu, Zn, Cr, Cd, and Pb. The data showed that the white rice contained lower micronutrients (Fe, K, and Zn) than the brown rice. The iron content in the rice varieties was in the range of 0.32 to 4.2 mg/100g. The red and black rice had higher concentration of iron at 3 to 4 mg/100g. The basmati rice and black rice had higher amount of zinc. Cadmium, lead, and chromium were present in rice samples, but their concentrations were below maximum permissible levels [18]. 2. Establishment the mechanism for heavy metal uptake. The reason why rice accumulates heavy metals may be explained through genetics. Cheng W. D. et al. (2006) investigated Cd, Cr, As, and Pb on nine rice species grown in six locations in China for two successive years. Their study showed there was a significant genotype and environmental interaction on the concentration of five heavy metals in grains. Cultivar choices showed rice has low heavy metals for a certain location [19]. Under heavy metal stress, rice plants produce metal-binding peptides by phytochelatins which are small cystein-rich peptides synthesized from glutathione via metal dependent enzymatic pathways. Yan, S.L. et al. (2000) purified phytochelatins synthesized from rice seeds by treating with copper ions. Their study also indicated cadmium was the most phytochelatin producing, following by lead, copper, silver, cobalt and other divalent cations. Calcium and magnesium had no effect. Therefore, the composition of soil also influenced rice metabolizing heavy metals [20]. 8

20 The distribution of heavy metals in rice plants raises questions about the metabolism of heavy metal uptake. Mehdi, S. M. et al. (2003) carried out a study to see the effect of industrial effluent on mineral nutrition of rice and soil. In this study, they found there was a sufficient accumulation of all the heavy metals in both the straw and paddy samples. The straw samples accumulated much more heavy metals than paddy samples [21]. Zoiopoulosand, P. E. et al. (2008) assessed two rice mill byproducts in Western Greece, named broken rice and rice bran for their characterization as ingredients of feed. The trace elements and heavy metals analysis indicated there was a higher level accumulation in rice bran [22]. 3. Influence of anthropogenic activities in heavy metal accumulation. Human activities have often negatively impacted the environment. The research on quantification and distribution of heavy metals in soil has been expanded around urban and industrial point sources. The effect of industrial effluent, the practice of wastewater irrigation, and application of different fertilizers and pesticides on rice field soil and rice plants are normally involved, although not always at the same time. Methdi, S. M. et al. (2003) carried out a research to see the effect of industrial effluents on mineral nutrition of rice and for soil health. The metals (Zn, Cu, Fe, Mn, Pb, Ni, Cd, and Sr) were analyzed in rice paddy samples of, straw, and soil before transplanting the rice and after the rice harvest. Their study indicated that there was a sufficient accumulation of all the heavy metals in both the straw and paddy samples. A 9

21 slight decrease in ph, electrical conductivity, and soil sodium adsorption ratio were also found, after harvesting the rice. The mineral metals in soil were further increased [21]. Pakistan is a county with shortage of freshwater and also lack of infrastructure and facilities for sewage treatment. The farmers in Pakistan use polluted water to irrigate rice fields. The practice may introduce unwanted metal ions into the rice plants. Nawaz, A. et al (2006) conducted a study to assess the effects of industrial effluents, contaminated with heavy metals (Cu and Cd). The heavy metal content in soil increased, but still remained within safe limits for the rice. The heavy metals were higher in upper layers of the soil. The chemical analysis in paddy and straw showed that most of Cu absorbed by rice plant was retained in straw and minute quantity was translocated to the grain. The concentration of Cd, both in paddy and straw was within safe limits, and there was no significant difference between paddy and straw [23]. The application of fertilizers and pesticides can increase heavy metals in soil. Rahaman, A.K.M.M. et al. (2007) conducted an experiment in pots in the green house of Department of Soil Science, Bangladesh Agricultural University. The concentration of cadmium in experimental soil and with different fertilizer sources was determined by using atomic absorption spectrometry. The results showed that the application of inorganic and organic fertilizer significantly increased the cadmium concentration in rice plants. Inorganic fertilizer, which is composed of simple chemicals and minerals such as NP and NPKS Zn, showed a higher influence than organic fertilizer, which composed decayed plant/animal matter. The experiments also verified the soil ph had a major 10

22 influence on rice plant uptake of heavy metals. The soil with ph tended to accumulate more cadmium [24]. 4. Arsenic study Arsenic is a natural carcinogen, present in soil and drinking water around the world. World Health Organization (WHO) permissible limit for drinking water is 0.01 mg/l [37]. Recently, research studies have revealed that certain rice contained more arsenic than other grains, and the arsenic concentration reported was problematic. Arsenic may be present as a number of chemical species in soil. How these species were accumulated by rice was investigated by Abedin, M. J. et al. (2002) [25]. Arsenite, arsenate, dimethylarsinic acid (DMAA) acid, and monomethylarsonic acid (MMAA) were included in their study. These arsenic species were found in soil solutions from a greenhouse experiment where rice was irrigated with arsenate contaminated water. The short-term uptake kinetics for these four species was determined in 7 day old excised rice roots. The results showed that there were two mechanisms for roots of rice plant uptaking arsenate and arsenite. One was called high-affinity uptake system which was at lower arsenate and arsenite concentrations, and another mechanism was named low-affinity uptake system which was at high arsenite and arsenate concentrations. At lower substrate concentration, arsenite was taken up at nearly the same amount as arsenate. Greater uptake of arsenite than arsenate was found at higher substrate concentration. Competitive inhibition of uptake with phosphate showed phosphate inhibited the arsenate uptake, but did not affect arsenite. DMAA and MMAA had much lower rates of uptake than 11

23 inorganic arsenic species. Abedin M. J., et al. were also concerned the uptake rate might be related to physiological or morphological attributes of root systems. Greater length and smaller diameter of roots would result in higher surface area per unit mass roots, and result in higher uptake. Rice straw retained the most arsenite. 1.3 General Toxicological Profile of Heavy Metals The term heavy metals has been used for over 60 years, but there is no definition made by any authoritative organization. Some authors classify heavy metals on the basis of a specific gravity of greater than 4.0 or 5.0. Over the last two decades, heavy metals has been used as a general term for the metals and metalloids with potential toxicity or ecotoxicity [26]. The human toxicity of heavy metals is limited by many of factors such as the total dose absorbed, the chemical form taken, the age of the person, the route of exposure and whether the exposure was acute or chronic. Many of elements can be considered heavy metals such as lead, mercury, and cadmium, because they do not have any benefit for human biochemical processes. Other elements, for example zinc, copper, iron, manganese, selenium, chromium, and molybdenum are all essential elements. Another set of metals used therapeutically include even arsenic which is toxic, but used in a group of medicines called antineoplastics. Aluminum, bismuth, gold, gallium, lithium, and silver are all part of the 12

24 medical armamentarium. Any of these elements has pernicious effects if taken in quantity or used unproper. Some elements may have different toxic profiles due to the presence of differing chemical forms. Redox state, organometallic, and biomolecules are all need be considered. Chromium is an essential element, but is considered a heavy metal and its principle inorganic forms are trivalent and hexavalent chromium. Trivalent chromium is nontoxic and human body system need trace level for sugar and lipid motablism. Hexavalent chromium is toxic and carcinogen. Arsenic is a very toxic element. The world arsenic has become the synonymous of poison. Numerous asenicals have been found in enviromental or biological systems, including both inorganic and organic compounds. Inorganic forms are considered the most toxic, especially the trivalent arsenic compounds. Organic compounds trend to less toxic or non-toxic. For example, large concentrations of asenobetaine are found in marine organisms and may be found in mushrooms, but there is little danger in eating them because it has very low toxicity. The chemical forms and their LD50 (rat) of selected arsenicals compounds are listed in the Table 1. 2 [12]. LD50 is a toxicity index which is the dose required to kill 50% of the mumbers in a tested population. Selenium is an essential element trace levels, but it is also consided to be toxic in larger amounts. USDA has recommended 70 µg selemium daily for human health. Selenium is generally considered and antioxidant when it is incorporated into proteins. Selenoproteins help prevent cellular damage from free radicals, which contribute to the 13

25 development of cancer and heart disease [27], [28], and also play an important role on regulating the thyroid function and immune system [29], [30], [31], [32].[33]. However, selenium also can lead to problems if the selenium intake is elevated. The discoloration of the skin and nails, gastrointestinal disturbances, fatigue, hair loss, nervous system abnormalities, and garlic breath are all symptoms of selenium toxicity, also called selenosis. The toxicity and mobility of selenium is species dependent, so it is necessary to determine the chemical form(s). Selenite (IV) and selenate (VI) are the two forms primarily involve in inorganic selenium speciation analysis. 14

26 Table 1.2 The chemical forms of selected arsenic species and their toxicity 15

27 1.4 Antagonistic affect between selenium and arsenic Antagonistic affect is another issue which should be given attention on the assessment of the toxic heavy metals and their species in rice. In 1938, Moxon, A. L. discovered that arsenic had remarkable ability to protect against the toxicity of selenium [34]. A lot of studies have been generated in attempt to explain this metabolic antagonism between arsenic and selenium. Levander, O. A. (1977) conducted a study on metabolic interrelationships between arsenic and selenium. His study revealed sodium arsenite gave full protection against the liver damage and depression caused by a diet containing high selenium from seleniferous wheat. Sodium arsenite and sodium arsenate were equally effective in preventing the toxicity of selenium, but insoluble arsenic sulfide, AsS 2, and AsS 3 were essentially inactive. Several organic arsenicals have shown partial protective action against selenosis. Sodium methyl arsenate and calcium methyl arsenate had little or no beneficial effect in minimizing selenium poison. The protective effect of arsenic has been observed through enhancing the biliary excretion of selenium. Arsenite stimulated the excretion of selenium into the bile, and selenite stimulated the excretion of arsenic into the bile. Even though the chemical mechanism by which arsenic detoxifies selenium is still unknown, it is a fact that arsenic and selenium increase the biliary excretion of the other [35]. Inorganic arsenic in drinking water has been recognized as a public health hazard in many countries. Epidemiological studies have documented associations between arsenic 16

28 exposure from drinking water and elevated risks of premalignant skin lesions, skin and internal cancer, and cardiovascular diseases. IARC also has classified arsenic as a group 1 human carcinogen [36], [37], [38]. Animal studies have suggested that there is a potential antagonism between arsenic and selenium in the body by urinary and/or biliary routes [39], [40]. Therefore, it has been hypothesized that arsenic toxicity can be decreased by dietary selenium intake. In experimental studies, arsenic exposure has been also associated with greater production of free radicals and increased oxidative stress that may be reduced by taking selenoprotein [41]. In order to determine the protective effect of selenium on risks of arsenic-related disease, Chen, Y., et al. (2007) conducted a case-cohort study to evaluate the association between arsenic-related premalignant skin lesions and prediagnostic blood selenium levels in 303 cases of skin lesions diagnosed between November 2002 and April 2004 and 849 randomly selected from 8092 participants with available baseline blood and urine samples collected in The study found the risk of pr gnant skin lesions was consistently greater among participants with blood selenium lower than the average level. Their research also suggested that a higher dietary selenium intake may reduce the risk of arsenic related skin lesions, and selenium that the recommended daily intake may be inadequate in the presence of stressors, such as chronic arsenic exposure from drinking water [42]. 17

29 1.5 Objective of the research The principal objective of this study is to investigate trace metals and heavy metals in a variety of rice samples, and establish speciation analysis on certain significant rice samples. Assessment of the potential toxicity of heavy metals and their species on different rice varieties and origins is also included in the study. The rice samples in the study were collected from US market available products. The samples include a wide range of varieties and origins, which is hoped to help establish the comparison in terms of varieties and origins. White rice and brown rice samples can give the information about rice bran. Some whole grain rice varieties also may be involved. The investigated trace metals and heavy metals will cover twenty-four (24) elements: sodium, potassium, calcium, zinc, aluminum, cobalt, copper, nickel, iron, beryllium, molybdenum, vanadium, magnesium, manganese, cadmium, chromium, arsenic, selenium, antimony, lead, barium and titanium. Phosphorus and sulfur are also included. The wide range of elemental monitoring can help estimate the interaction among metals. The potential toxicity assessment will be studied from the prospective of bioavailability of heavy metals species. The bioavailability of chemical forms is limited by their solubility. To determine concentrations of species in different solvent extracts was used to study the matter In this study, inductively coupled plasma mass spectrometry (ICP-MS) will be used for trace metal and heavy metal analysis. High performance liquid chromatography 18

30 (HPLC) with ion exchange will be combined with ICP-MS for heavy metal speciation analysis. 19

31 Chapter II- Methodologies 2.1 Inductively coupled plasma mass spectrometry (ICP-MS) Inductively coupled plasma mass spectrometry is an elemental analysis technique which was developed in the 1980 s. This technique combines the effective ionization source of an inductively coupled plasma with a high sensitive and a low detection limit mass spectrometer together to achieve multi-element analysis at the part per trillion levels. Its capabilities of qualitative, semi-quantitative, quantitative analysis and ability to utilize isotope ratios have been utilized in many of different fields, such as environmental contaminant monitoring, geology and soil science, food science, mining, metallurgy, and medicine. Moreover, ICP-MS has been often coupled with separation techniques such as gas chromatography, liquid chromatography, and capillary electrophoresis as an element selective detector. This combination expands the application of ICP-MS to more than just trace element total analyses Instrument description and theory An ICP-MS mainly consists of four parts: a sample introduction system, ICP ionization source, mass filter and electron multiplier (EM) detector. In a conventional ICP-MS, a quadrupole mass analyzer is chosen for mass filter due to its ease of use, 20

32 robustness, mass range, high scanning speed and relatively low cost. Also a collision or reaction cell is often used for reducing polyatomic interferences. A schematic of ICP-MS is illustrated in Figure 2.1. Figure 2.1 A general schematic of an inductively coupled plasma mass spectrometer There are several methods to introduce sample to ICP ionization source such as laser ablation, continuous nebulization, flow injection and electrothermal vaporization. The flow injection and aerosol generation is the most common. Aqueous samples are introduced into ICP-MS system by a nebulizer which aspirates samples by a high velocity argon gas stream, forming a fine aerosol. The mist passes through a spray chamber to the ICP torch. The large droplets in aerosol will be removed by the spray chamber, and only about 2 % of original aerosol is transported to the ICP. This process is necessary to produce small enough droplets for plasma to evaporate. A Scott-type double pass spray chamber maintained with thermoelectric cooling is commonly used in ICP-MS to prevent 21

33 signal drift and reduce solvent loading. The reduced solvent loading maintains plasma s high temperature, reduces oxide interference, and assists the matrix decomposition. Once the sample aerosol enters the plasma torch, the aerosol droplets are rapidly dried, vaporized, decomposed, atomized, and ionized. The ICP torch is made of three concentric quartz tubes surrounded by a cooled copper coil which is connected to a radio-frequency (RF) generator. The RF can establish an oscillating electric and magnetic fields with maximal energy focused on the flowing gas stream. Argon ions formed by a spark will be affected in the magnetic field and collide with other argon atoms to form a argon discharge or plasma, which is an excellent ionization source due to its temperature of ºK. The ionization process in ICP discharge is ideally achieved by removing one electron from each atom. A schematic of ICP torch is presented in Figure 2.2 Figure 2.2 A schematic of ICP plasma torch adapted from Agilent ICP-MS Primer The positive ions will be extracted and focused into an ion beam by passing through a sampling cone, a skimmer cone, ion lenses and a collision/reaction cell before entering 22

34 the quadrupole mass analyzer. The neutral species and photons in the ion beam are removed by ion lenses to minimize false signal and lower background. For example, Agilent ICP-MS uses an off-axis ion lens which minimizes photon transmission which would also trigger the detector. Figure 2.1 shows an off-axis lens ICP-MS. A quadrupole mass analyzer separates ions by their mass to charge ratio (m/z). The resolution of quadrupole mass analyzer (M/ M) is approximately 400, so it typically offers unit mass resolution or a bit better for elemental analysis. Only ions having a certain m/z can pass through the mass analyzer, others having unstable trajectories will collide with the rods and be annihilated. The mass analyzer can select an ion with particular m/z by scanning the rf and dc voltages. Figure 2.3 illustrates a quadrupole mass filter. Figure 2.3 A schematic of quadrupole mass analyzer An electron multiplier (EM) device is applied for a detector in ICP-MS. The EM detector has the characteristics of high sensitivity, wide dynamic range, and low random background. Normally, the detector can be operated in two modes, pulse and analog. The analog mode allows the ICP-MS to measure higher count rates, which would result in pulse pile-up in the pulse or digital mode. 23

35 Mass spectrometers require high vacuum conditions. ICP-MS uses a rotary pump and two turbo-molecular pumps to maintain sufficiently low pressure. The three different stages are at progressively lower pressures, which are also illustrated in Figure Interference removal with collision/reaction cell The main spectral interferences in ICP-MS are isobaric interference, polyatomic ions, oxides and doubly charged species. The isobaric interference can be avoided by choosing different isotopes. Oxides and doubly charged species can be significantly reduced by proper plasma tuning. The polyatomic spectral interferences, derived from the plasma gas (argon), solvent, matrix and atmospheric gases, limit the sensitivity and detection limit of ICP-MS. Most recently, a collision and/or reaction cell (CRC) has become an important technique in atomic mass spectrometry to remove or eliminate polyatomic ion interferences. [43] Usually, a CRC is positioned before quadrupole mass analyzer such as the illustration presented in Figure 2.1. The CRC consists of a multipole (quadrupole or hexapole or octopole) with an applied radio frequency (RF) voltage, which serves to separate the interferences and as a focusing device. Gerichand D. (1992) found that the octopole provides good ion transmission efficiency for a maximum to minimum ion mass ratio of about 100 [44]. Controlled gas flow is applied into the CRC to interact with ions. There are four types of mechanism for interference removal depending on the gas utilized. Table 2.1 shows a brief summary of cell gases and their mechanisms of interaction. 24

36 Table 2.1 Gases for collision and/or reaction cell adapted from Koppenaal et al [43] Collision induced dissociation is using cell gases dissociate the polyatomic ions to make the polyatomic ions and remove interferences at a particular m/z. However, this is useful for only a few polyatomic ions. Kinetic energy discrimination (KED) refers to the application of a potential barrier between collision cell and mass analyzer to restrict the polyatomic ions from entering mass analyzer via a voltage barrier. [43]. In this process, polyatomic ions collide with cell gases such as helium, which reduces their kinetic energy more than that of the analyte ion because of the larger collision cross section for the polyatomic. Figure 2.4 is an example of the two possible mechanisms of 40 Ar 35 Cl interference removal in determination of 75 As. 25

37 Figure 2.4 Two possible mechanisms for 40 Ar 35 Cl + interference removal in determination of 75 As. Barrier energy is added between CRC and mass analyzer by building a positive electric field. The analytes or polyatomic ions can not pass through the energy barrier if their kinetic energy is not high enough. Figure 2.5 illustrates the function. 26

38 Figure 2.5 The kinetic energy discrimination mechanism. The chemical reaction mechanism is simply to use reaction gases such as hydrogen to react with polyatomic ions; thereby moving the reaction product to a different m/z value and limit the interference as is illustrated with Ar dimer. 40 Ar 40 Ar + +H 2 40 ArH + 40 ArH + 27

39 Electron transfer is a process to remove the charge of polyatomic ions to reaction gases by collision. Once the charge of the polyatomic ions is removed, the mass analyzer will no long detect them. 40 Ar 40 Ar + +H 2 40 Ar 40 Ar + H 2 + The sensitivity and detection limit are not always increased, because analytes are also lost through the various polyatomic ion interference removal processes. Therefore, the following is required for cell gas selection. 1. high efficiency for interfering ion, low efficiency for analytes 2. minimal side reactions 3. minimize any new interferences Hydrogen meets all these criteria, so it is a good choice for reaction gas to eliminate interferences. [43]. Helium is the favorite collision gas due to its small size and inert characteristics. 2.2 High performance liquid chromatography (HPLC) Even though ICP-MS provides highly sensitive, low detection limits for multielement quantitative analysis at ultra-trace level, it cannot give any detail about the parent molecules of these elements. Moreover, the information about element s oxidation states and molecular species in samples is very valuable in studying their toxicity, bioavailability, and transportability. In order to satisfy this requirement, ICP-MS is 28

40 coupled with HPLC. The combination is accomplished by simply connecting the outlet of chromatography column to the entrance of ICP-MS nebulizer. The instrumental set up is illustrated in the Figure 2.6. Figure 2.6 Coupling HPLC to ICP-MS HPLC is a form of column chromatography used to separate compounds. It utilizes a column packed with materials called stationary phase to separate compounds, and a pump drives a solvent, called mobile phase, through the column to elute the separated compounds from the stationary phase. The retention time of each compound depends on the interactions among the compound, stationary phase and mobile phase. The compounds can come out off column in a certain order by choosing a proper column (stationary phase) and mobile phase. If the compounds contain ICP-MS detectable elements, the ICP-MS can identify and quantify them by using time resolve mode as long as retention times are known. 29

41 There are several chromatographic separation techniques depending on the columns used. Commonly used chromatographic techniques include normal phase, reversed phase, ion-pairing reversed phase, ion-exchange and size exclusion chromatography. Selection of the separation technique is based on the physical and chemical property of analytes of interest. In this study, an ion-exchange column was used for the separations. Ion-exchange chromatography (IEC) separates compounds according to charge and size. Cation exchange columns carry a negative charge, so they can retain a compound with positive charge. They can be classified to strong and weak cation exchange columns depending on the functional groups covalently bonded to the stationary phase. The functional groups of strong cation exchange columns are sulfonic acid groups, -SO - 3, which remain their charged state over all ph ranges. The weak cation exchange columns contain the carboxylic acid group, -COO -. The charge on carboxylic acid groups can be removed by a change of ph. Anion exchange columns contain positive functional groups + to retain compounds with negative charge. The quaternary amino group, -NR 3 is used in strong anion exchange column. The functional groups of weak anion columns are primary, secondary, or tertiary amines [45]. Figure 2.7 shows how they work. 30

42 Figure 2.7 Analyte competitions between stationary and mobile phase 31

43 Chapter III Experimental 3.1 Instrumentation and apparatus Inductively coupled plasma mass spectrometer (ICP-MS): The ICP-MS used for total element analysis and speciation analysis in this study was an Agilent 7500cx (Agilent Technologies, Santa Clara, CA). The instrument includes a concentric nebulizer, a Peltier cooled Scott-type double pass spray chamber, a high matrix introduction system, a shielded torch, off-axis ion lens, an octopole collision and/or reaction system with helium and hydrogen gas, a quadrupole mass analyzer, and an electron multiplier detector. High performance liquid chromatography (HPLC): An Agilent 1200 series liquid chromatograph (Agilent Technologies Santa Clara, CA) was employed for liquid chromatography. The instrument consists of a quaternary pump, a vacuum de-gasser, a manual injector, and a UV detector. The ion exchange chromatography in the study was carried out with a Luna SCX 100A column (5 µm x 4.6 mm id x 150 mm) from Phenomenex (Torrance, CA) and a Hamilton PRP-X100 anion exchange column (10 µm x 4.1 mm id x 250 mm) from Hamilton Company, (Reno, NV). Figure 3.1 shows the HPLC-ICP-MS set up 32

44 18780 Pierce evaporating unit: The unit (Pierce Reacti-Therm, Rockford, IL) was used for evaporating methanol and water in this study. It includes a heating and stirring module. Spex 6700 freezer/mill: The apparatus (Spex CertiPrep, Metuchen, NJ) was applied for grinding rice samples which were used during methanol/water extraction process. Liquid Nitrogen was needed for freezing samples. IEC Centra CL2 centrifuge: the centrifuge (Thermo Scientific, Waltham, MA) was used during extraction process. Fisher Vortex: A vortex (Fisher Scientific) was used during extraction process. Figure 3.1 Photograph of HPLC-ICP-MS used in the study 33

45 3.2 Reagents and standards MilliQ water was used in preparing all the solutions for ICP-MS. Water, Chromasolv Plus, for HPLC (Sigma-Aldrich, Bellefonte, PA) was used in the HPLC mobile phase. Concentrated nitric acid (HNO 3 ), trace metal grade (Fisher Scientific) was applied for digesting samples and preparing 2 % nitric acid used in diluting samples and preparing standards. Hydrogen peroxide (H 2 O 2 ), Certified ACS 30% (Fisher Scientific) was used for digesting samples. Trifluoroacetic acid (TFA), 99.5% (Acros Organics, Morris Plains, NJ) was diluted to 2 M with MilliQ water. TFA 2 M was used in arsenic and selenium extraction process. Methanol, Certified ACS (Fisher Chemical) was used in sample extraction process. Methanol, Chromasolv Plus, for HPLC 99.9% (Sigma-Aldrich, Bellefonte, PA) was used in HPLC mobile phase. Ammonium phosphate monobasic (NH 4 H 2 PO 4 ), HPLC Grade Crystalline ACS (Fisher Scientific) was used for preparing HPLC mobile phase. Pyridine, Certified ACS (Fisher scientific) was used for making the HPLC mobile phase. Ammonium hydroxide (NH 3 H 2 O), 20% solution in water (Acros Orgainic, Morris Plains, NJ) was used for adjusting ph of HPLC mobile phase. 34

46 Formic Acid 20% (w/w) (Reagents Incorporated, Charlotte, NC) was used for adjusting ph of HPLC mobile phase. Certified element standards were purchased from two sources (Spex CertiPrep, Metuchen, NJ and High-Purity Standards, Charleston, SC). These standards include single element standards of Na, K, P, and S with initial concentration of 1000 parts per million (ppm), and custom blend made standards with 200 ppm of Ca, 100 ppm of Al and Mg, 40 ppm of Fe, and 10 ppm of Se, As, Cr, Cd, Pb, Sb, Be, Co, Ba, Ti, V, Zn, Ni, Mo, and Cu. The standards from one source were used for calibration standards, and those from another source were applied for quality assurance and quality control. These standards were also used for spikes during the study ppm of As (III), As (V), Se (VI), and Se (IV) was purchased from Spex CetiPrep (Metuchen, NJ) for speciation analysis. Dimethylarsinic acid (DMA) was obtained from Strem Chemicals, Inc. (Newburyport, MA) for speciation analysis. An internal standard was obtained from VHG Labs (Manchester, NH). It was diluted 100 times from initial concentration of 100 µg/ml of Bi, Ga, In, Li (6), Sc, Tb, and Y. The diluted internal standard was used in ICP-MS measurement process. Tuning solution was purchased from Agilent (Santa Clara, CA) for daily instrument tuning. P/A factor solution from VHG labs (Manchester, NH) was diluted 100 times from initial concentration of 2.5 ppm of Tb and Y, 5 ppm of Al, Ba, Bi, Co, Cr, Cu, In, Ir, 35

47 Li(6), Ln, Mn, Na, Sc, Sr, Th, Ti, Tl, and V, 10 ppm of Ge, Mg, Mo, Ni. Pb, Pd, Ru, Sb, and Sn, and 20 ppm of As, Be, Cd, and Zn for daily instrument tuning. This solution was used for complementing the detector transition from pulse mode to analog mode. 3.3 Samples Twenty (20) rice samples were investigated in this study. The rice samples were collected from local supermarkets (Meijier, Cincinnati Asian Market and Jungle Jims), which covered as many varieties and origins as possible. Brown rice and white rice from India, Thailand and the US were included, as well as wild rice, red rice and black rice. Table 3.1 shows a sample list. Table 3.1 Sample List No. Sample ID (Varieties) Origins Images comments 1 Black Sweet Rice US 2 Red Sweet Rice, US US 3 Wild Rice US 36

48 No. Sample ID (Varieties) Origins Images comments 4 Black Sweet Rice China 5 Brown Rice US 6 KoKuHo Rice US 7 CalRose BoTan Rice US 8 Sweet Rice US 9 Basmati Rice US 10 Nishiki Rice US 11 Jasmine Rice, US 37

49 No Sample ID (Varieties) Origins Images Comments 12 Long Grain Rice US Enriched with iron, Niacin, and thiamin 13 Jasmine Rice Thailand, Thailand 14 Basmati Rice, Pakistan Pakistan 15 Jasmine Rice, Royal Thailand 16 Jasmine Brown Rice, Royal Thailand 17 Basmati Rice, Royal Indian 18 Basmati Brown Rice, Royal Indian 19 Nishiki Rice, JFC California US 20 Nishiki Brown Rice, JFC California US 38

50 3.4 Experimental procedure Determination of the solid content of rice. In order to make results more reliable, the final reporting data were all calculated on the basis of solid sample weight. The percentage of solid is a factor used for the correcting results on the solid basis. To measure the solid content, a weighing bottle was dried in a forced air oven controlled at 105±2ºC and cooled in a desiccator to a constant weight. A weighed rice sample in the dried bottle, also dried at 105±2ºC was cooled in a desiccator to a constant weight. The % solid content was calculated as follows: Weight of dried bottle with rice Weight of dried bottle % Solid = X 100 Weigh of sample Determination of the elemental concentrations in rice. Approximately 1 g of asreceived rice samples was digested with 8 ml of concentrated HNO 3 and 4 ml of 30% H 2 O 2 in a heating block controlled at 115ºC. The digestion was carried out in a graduated polypropylene vial (Capital Vials) covered with a polypropylene watch-glass. The process could last up to 6 hr until the solution became clear and the volume was reduced to 1 ml. The digested sample was removed from the heating block to cool and diluted to 50 g with MilliQ Water. The diluted sample was filtered with a 0.45 µm filters and analyzed by using ICP-MS. External calibration standards were applied with certified element standards (High-Purity Standard). 1 ppm of internal standard (VHG Labs) was 39

51 introduced in all the ICP-MS measurements to compensate for matrix interferences. For quality assurance and quality control, a blank and a certified standard mixture (Spex CertiPrep) were involved in the digestion and sample analysis procedure. Calibration verification standards, prepared from Spex CertiPrep, also were utilized in each calibration, after every tenth sample, and at the end of the sample run. The solid content was also determined at the same time as the digestion. The final results are reported on a solid weigh basis. The measurement parameters of the ICP-MS are listed in the Table 3.2. Table 3.2 Operational parameters for ICP-MS No CRC gas mode RF power 1550 W Plasma gas flow 15.0 L/min Auxiliary gas flow 0.9 L/min Carrier gas flow 1.0 L/min Makeup gas flow 0 L/min Optional gas 0 % Spray chamber condition temperature 2 ºC Cell entrance -30 V Quadrupole focus 2 V Cell exit -30 V Octopole bias -6 V Quadrupole bias -1.6 V Monitored isotopes 95 Mo, 121 Sb, 137 BA, 206 pb Dwell time 45 ms per isotope Hydrogen CRC mode RF power 1550 W 40

52 Plasma gas flow 15.0 L/min Auxiliary gas flow 0.9 L/min Carrier gas flow 1.0 L/min Makeup gas flow 0 L/min Optional gas 0 % Spray chamber condition temperature 2 ºC Cell entrance -30 V Quadrupole focus -9 V Cell exit -40 V Hydrogen gas 3.5 L/min Octopole bias V Quadrupole bias V Monitored isotopes Dwell time Helium CRC mode RF power Plasma gas flow Auxiliary gas flow Carrier gas flow Makeup gas flow 78 Se 103 ms per isotope 1550 W 15.0 L/min 0.9 L/min 1.0 L/min 0 L/min Optional gas 0 % Spray chamber condition temperature 2 ºC Cell entrance -30 V Quadrupole focus -11 V Cell exit -40 V Hydrogen gas 4.5 L/min Octopole bias V Quadrupole bias V 41

53 Monitored isotopes 23 Na, 24 Mg, 27 Al, 31 P, 34 S, 39 K, 44 Ca, 47 Ti, 51 V, 5 3Cr, 55 Mn, 56 Fe, 59 Co, 60 Ni, 63 Cu, 66 Zn, 75 As, 111 Cd Dwell time 128 ms per isotope Speciation analysis of arsenic and inorganic selenium in rice by using the trifluoroacetic acid (TFA) extraction technique. [46], [47] Approximately 1 g of asreceived rice samples in a glass digestion tube. 4 ml of 2 M of TFA was added to the tube, covered with a watch-glass, and allowed to stand over night at room temperature. The following day, the tube was placed in a heating block controlled at 100ºC for at least 7 hr. The watch-glass was removed, the extraction mix was dried, and the residue was transferred to a 50 ml of polypropylene centrifuge tube, dissolved with MilliQ water to about 50 g. Once whirled well with a vortex, the residue would fall apart in the solution. The resulting solution was filtered with 0.2 µm filters and analyzed for total arsenic and selenium concentrations with ICP-MS. The filtered solution was also used for speciation analysis of arsenic by using HPLC- ICP-MS with a Luna SCX 100A column (5 µm x 4.6 mm id x 150 mm) and Hamilton PRP-X100 anion exchange column (10 µm x 4.1 mm id x 250 mm). A fixed 20 µl sample loop was utilized in both of the chromatographies. The mobile phase of 0.5 mm pyridine buffer in 3% methanol, adjusted to ph = 2.7 with formic acid, was employed for the Luna SCX 100A column, and the pump flow rate was 0.5 ml/min. In Hamilton PRP- 42

54 X100 anion exchange column chromatography, the mobile phase was 20 mm of NH 4 H 2 PO 4 buffer in 2% methanol, adjusted to ph =5.9 with ammonium hydroxide and the flow rate was 1 ml/min. The external calibration with standards of As (III), As (V) and DMA was utilized for quantitative analysis. In quantitative analysis sample amount and 2M of TFA extraction solvent were increased to make the signal higher than detection limit. The solid contents were determined at the same time as the extraction. The final results were calculated on the solid weigh basis. In inorganic selenium speciation analysis, Se (IV) and Se (VI) were identified by comparing retention times using Spex CertiPrep standards. The HPLC-ICP-MS with Hamilton PRP-X100 anion exchange column and a mobile phase of 20 mm NH 4 H 2 PO 4 buffer in 2% methanol adjusted ph to 7.9, was applied. The sample loop was 20 µl, and the flow rate was 1 ml/min. Determination of the total amount of arsenic and selenium in a methanol /water (50/50) extraction, as well as the analysis of arsenic species [48], [49]. For methanol/water extraction, the rice sample was first milled very fine by using a Spex 6700 freezer/mill. Approximately 2.5 g of the milled rice sample added to a polypropylene centrifuge tube, followed by 10 ml of methanol/water (50/50) (Certified ACS methanol from Fisher Chemical), whirled it 5 minutes by using a vortex and centrifuged it at 3000 rpm for 15 minutes. The clear supernatants were transferred to glass digestion tubes. The residue was extracted five times with the methanol/water mixture. The combined supernatants were 43

55 evaporated to dryness by an evaporating unit (18780 Pierce) at about 35 ºC. The residue was dissolved with MillQ water and brought up to about 50 g. The extraction solution was filtered by 0.2 µm filter, and the total arsenic and selenium measured by using ICP- MS. The solid contents were determined at the same time as the extraction. The total arsenic and selenium results were calculated on the solid weigh basis. The arsenic species were analyzed by HPLC-ICP-MS with the Hamilton PRP-X100 anion exchange column. The procedures of HPLC-ICP-MS measurement were the same for the TFA extraction. The HPLC condition are shown in Table 3.3 Table 3.3 The condition of HPLC Ion exchange chromatography for arsenic species with the Luna SCX 100A column Mobile phase 0.5 mm pyridine buffer in 3% methanol ph = 2.7 with formic acid. Flow rate 0.5 ml/min Injector volume 20 µl Run Isocratic Ion exchange chromatography for arsenic species with Hamilton PRP-100 anion exchange column Mobile phase 20 mm NH4H2PO4 buffer in 2% methanol ph =5.9 with ammonium hydroxide. Flow rate 1 ml/min Injector volume 20 µl 44

56 Run Isocratic Ion exchange chromatography for inorganic selenium species with Hamilton PRP-100 anion exchange column Mobile phase 20 mm NH4H2PO4 buffer in 2% methanol ph =7.9 with ammonium hydroxide. Flow rate 1 ml/min Injector volume 20 µl Run Isocratic 3.5 Instrument tuning Before analysis, the ICP-MS was tuned with Agilent tuning solution in three modes. The tuning for no gas mode to make sure there is sufficient sensitivity for complete mass range, and also to assure low oxide ions and doubly charge ions. The hydrogen and helium mode should reduce interferences to a low enough level while maintaining enough sensitivity. The pulse and analog modes were also updated with P/A factor solution (VHG Labs) to make sure the system smoothly transitions between pulse and analog modes. Table 3.4 lists the target tune values for this study. 45

57 Table 3.4 Target tune values for 1ppb of Agilent tuning solution containing 0.5 % of HCl and 0.1 second integration time 3.6 Determination of detection limit (DL) Detection limit (DL) is the lowest quantity of analyte that can be distinguished from the absence of analyte (blank) at a known confidence level. DL depends on the ratio of the magnitude of the analyte signal to the size of blank signal fluctuation. It can be obtained from three times (95% confidence level) of standard deviation of the noise which can be calculated from blank measurements. There are a numbers of different DLs. Limit of qualification (LOQ) is one used commonly, which is defined ten times of standard deviation of the blank. [50], [51]. 46

58 In this study, the DL and LOQ of ICP-MS multi-elements analysis were determined by three and ten times of standard deviation for 7 replicates of 2 % nitric acid blank respectively. The DL and LOQ of arsenic speciation analysis, using HPLC-ICP-MS with Hamilton PRP-100 anion exchange column, was calculated as three and ten times the standard deviation obtained from seven replicated measurements of a TFA extraction blank respectively. 47

59 Chapter IV- Results and Discussion 4.1 Elemental profiles in different rice varieties Detection limits (DL) and Limits of quantification (LOQ) of the ICP-MS simultaneous multi-element analysis The DL and LOQ were determined before multi-elemental analysis. The results are listed in the Table 4.2. There is a relatively higher DL and LOQ for isotopes of 34 S, 31 P, 39 K, 44 Ca, and 23 Na, but they are still within the ppb level. The DL and LOQ for others are in ppt level. In this study, the beryllium, vanadium, and antimony concentrations are lower than their DL and LOQ. The concentration of others is higher than the DL and LOQ Quality assurance and quality control (QA/QC) In the ICP-MS analysis procedure, a calibration verification standard was done following each calibration, after every tenth sample, and at the end of the sample run for QC/QA. There are three measurements of the calibration verification standard during the total elemental analysis. Recoveries of calibration verification standard are in the range of 92 ± 2 to 111 ± 3 %, excluding 44 Ca at 133±1 %. The interference between 40 Ar + and 40 Ca + precludes its measurement at this isotope. Therefore, the less abundant 44 Ca + was chosen to monitor. The 44 Ca + gave a higher LOQ (7.18 ppb) because 48

60 Table 4.1 The detection limits (DL) and limits of quantification (LOQ) of ICP-MS simultaneous multi-element analysis. 49

61 of more deviation provides during the measurement. The deviation is reduced with higher calcium content. In this study, a few ppm of calcium present in the sample solution is much higher than LOQ, and the accurate determination of calcium is not possible or a priority. The details of the recoveries were listed in the Table 4.2. A certified standard mixture from Spex CertiPrep was included in the digest and ICP- MS measurement process. The recoveries range from 83 % to 129 %, and the most of them are in the range of 100±15 %. The recovery distribution confirms the reliability of the digestion process. Figure 4.1 shows the distribution scatter. Figure 4.1 The distribution of elemental recoveries from the digestion and analytical procedures. 1-Na, 2-Mg, 3-Al, 4-P, 5-S, 6-K, 7-Ca, 8-Ti, 9-V, 10-Cr, 11-Mn, 12-Fe, 13- Co, 14-Ni, 15-Cu, 16-Zn, 17-As, 18-Se, 19-Mo, 20-Cr, 21-Sb, 22-Ba, 23-Pb. 50

62 Table 4.2 The recovery of calibration verification standards in the analysis of total elements. 51

63 4.1.3 The total element profile in different rice varieties from origins The rice samples listed in the table 3.3 were digested with concentrated nitric acid and 30% hydrogen peroxide on a heating block, and twenty-four (24) elements were analyzed with ICP-MS. The results indicate the following. 1. P, S, K, and Mg are the abundant elements compared with other elements in rice. The whole grain rice (with rice bran) contains much more P, S, K, and Mg than the polished rice (white rice). These elements are accumulated more in rice bran from according to the data for brown and white rice (Jasmine, Basmati, and Nishiki). Figure 4.2 shows the results. Figure 4.2 The distribution of P, S, K, and Mg in different rice varieties. The black arrows indicate the whole grain rice. The x-axis represents 1- Black Sweet Rice, US, 2- Red Sweet Rice, US, 3- Wild Rice, US, 4- Black Sweet Rice, china, 5- Brown rice, US, 6- KoKuHo Rice, US, 7- CalRose BoTan Rice, US, 8- Sweet Rice, US, 9- Basmati rice,us, 10- Nishiki Rice, US, 11- Jasmine Rice, US, 12- Long Grain Rice, US, 13- Jasmine Rice Thailand, 14- Basmati, Pakistan, 15- Jasmine Rice, Royal Thailand, 16-Jasmine Brown Rice, Royal Thailand, 17- Basmati rice, Royal Indian, 18-Basmati 52

64 Brown Rice, Royal Indian, 19- Nishiki Rice, Califonia US JFC, 20-Nishiki Brown Rice, California US JFC 2. Rice is a major source of mineral nutrients for food consumption. The distribution of essential elements of Ca, Zn, Mn, Fe, and Na in these rice varieties are presented in Figure 4.2. The Figure illustrates that brown rice contains more these elements, especially calcium. The US long grain rice has more iron, because it was enriched rice. Figure 4.3 The distribution of Ca, Zn, Mn, Fe, and Na in different rice varieties The x-axis represented 1- Black Sweet Rice, US, 2- Red Sweet Rice, US, 3- Wild Rice, US, 4- Black Sweet Rice, china, 5- Brown rice, US, 6- KoKuHo Rice, US, 7- CalRose BoTan Rice, US, 8- Sweet Rice, US, 9- Basmati rice,us, 10- Nishiki Rice, US, 11- Jasmine Rice, US, 12- Long Grain Rice, US, 13- Jasmine Rice Thailand, 14- Basmati, Pakistan, 15- Jasmine Rice, Royal Thailand, 16-Jasmine Brown Rice, Royal Thailand, 17- Basmati rice, Royal Indian, 18-Basmati Brown Rice, Royal Indian, 19- Nishiki Rice, California US JFC, 20-Nishiki Brown Rice, California US JFC 53

65 3. Figure 4.4 illustrates the Al, Ni, Ba, Ti, and Mo profiles in these rice varieties. Generally, the whole grain rice contains more of these elements than the white rice. Chinese black sweet rice and Indian Basmati brown rice have much higher aluminum than others. The Basmati white rice, regardless of origin, contains higher levels of aluminum than other white rice. Aluminum is classified as Generally Regarded As Safe (GRAS) by FDA. However, there are studies recognizing aluminum toxicity, such as aluminum related osteodystrophy and neuron degenerative disease [52]. JECFA established a provisional tolerable weekly intake (PTWI) of 7 mg/kg of body weight in According the PTWI and a body weight of 60 kg consuming 500 g/day of rice, the aluminum content in rice does not provide a health risk. Figure 4.4 illustrates the distribution. Figure 4.4 The distribution of Al, Ni, Ba, Ti, and Mo in different rice varieties. The arrows show higher aluminum content. The x-axis represented 1- Black Sweet Rice, US, 2- Red Sweet Rice, US, 3- Wild Rice, US, 4- Black Sweet Rice, china, 5- Brown rice, US, 6- KoKuHo Rice, US, 7- CalRose BoTan Rice, US, 8- Sweet Rice, US, 9- Basmati rice,us, 10- Nishiki Rice, US, 11- Jasmine Rice, US, 12- Long 54

66 Grain Rice, US, 13- Jasmine Rice Thailand, 14- Basmati, Pakistan, 15- Jasmine Rice, Royal Thailand, 16-Jasmine Brown Rice, Royal Thailand, 17- Basmati rice, Royal Indian, 18-Basmati Brown Rice, Royal Indian, 19- Nishiki Rice, California US JFC, 20-Nishiki Brown Rice, California US JFC 4. Cadmium, lead, arsenic, and chromium are the heavy metals of highest interest. Figure 4.5 indicates the distribution of these heavy metals in the rice samples investigated. Arsenic is the most abundant heavy metal (actually metalloid or semi-metal) in the rice samples except for US black sweet rice which showed appreciable lead. Figure 4.5 The distribution of As, Cd, Cr, and Pb in different rice varieties The axis represented 1- Black Sweet Rice, US, 2- Red Sweet Rice, US, 3- Wild Rice, US, 4- Black Sweet Rice, china, 5- Brown rice, US, 6- KoKuHo Rice, US, 7- CalRose BoTan Rice, US, 8- Sweet Rice, US, 9- Basmati rice,us, 10- Nishiki Rice, US, 11- Jasmine Rice, US, 12- Long Grain Rice, US, 13- Jasmine Rice Thailand, 14- Basmati, Pakistan, 15- Jasmine Rice, Royal Thailand, 16-Jasmine Brown Rice, Royal Thailand, 17- Basmati rice, Royal Indian, 18-Basmati Brown Rice, Royal Indian, 19- Nishiki Rice, California US JFC, 20-Nishiki Brown Rice, California US JFC 55

67 The Cr and Cd content in the studied rice samples range from 19 to 61 ppb and 6 to 48 ppb, respectively. The Pb content is in the 5 to 19 ppb range except for US sweet black rice, which has 126 ppb of Pb. FDA/WHO has established the Provisional Tolerable Weekly Intake (PTWI) for Cd, Cr and Pb, which is 7 µg/kg of body weight per week for Cd, 23.3 µg/kg of body weight per week for Cr, and 25 µg/kg body weigh per week for Pb. According the PTWI and a body weight of 60 kg where 500 g/day of rice are consumed, there should be no safety concerns for Cd, Cr and Pb for the samples used in this study. The exclusion is US sweet black rice. However, the US sweet black rice is not the commonly consumed rice variety. Arsenic is accumulated in rice from the soil and irrigation water. There is 34 ppb to 333 ppb of arsenic in the investigated rice samples. The range of 150 ppb to 225 ppb is for the most part the usual expected arsenic content in white rice, based on the arsenic scatter plot presented in Figure 4.6. The Figure also illustrates that the US NIshiki brown rice, US brown rice, Thailand brown Jasmine rice, and US long grain enriched rice contains more arsenic. Basmati rice, no matter what the origins, has lower arsenic concentration. US wild rice and US black sweet rice contain the least arsenic, which is 34 ppb and 63 ppb, respectively. There is not a clear consensus from US EPA scientists about the arsenic exposure limits. The more conservative exposure level from US EPA (1998) is 56

68 0.3 ug As/kg body weight per day. According to these exposure limits, there may be concern about the arsenic content in these rice samples. Figure 4.6 The profile of arsenic in different rice varieties and origins The x-axis represents 1- Black Sweet Rice, US, 2- Red Sweet Rice, US, 3- Wild Rice, US, 4- Black Sweet Rice, china, 5- Brown rice, US, 6- KoKuHo Rice, US, 7- CalRose BoTan Rice, US, 8- Sweet Rice, US, 9- Basmati rice,us, 10- Nishiki Rice, US, 11- Jasmine Rice, US, 12- Long Grain Rice, US, 13- Jasmine Rice Thailand, 14- Basmati, Pakistan, 15- Jasmine Rice, Royal Thailand, 16-Jasmine Brown Rice, Royal Thailand, 17- Basmati rice, Royal Indian, 18-Basmati Brown Rice, Royal Indian, 19- Nishiki Rice, California US JFC, 20-Nishiki Brown Rice, California US JFC 5. Selenium is an essential element in low content, but is toxic at higher levels. It is demonstrated below that there also is a Se/As antagonism. There is 35 ppb to 282 ppb of selenium in the rice samples. The US Nishiki brown rice contains more selenium than its white rice, but the Thailand Jasmine brown rice and Basmati brown rice have similar or even less amounts of selenium relative to their white 57

69 rice counterparts. In addition, the US long rice and Basmati rice trend to have more selenium, which is indicated in Figure 4.7. Figure 4.7 The distributions of Selenium and arsenic. The arrow represented the higher content of selenium. The x-axis represents 1- Black Sweet Rice, US, 2- Red Sweet Rice, US, 3- Wild Rice, US, 4- Black Sweet Rice, china, 5- Brown rice, US, 6- KoKuHo Rice, US, 7- CalRose BoTan Rice, US, 8- Sweet Rice, US, 9- Basmati rice,us, 10- Nishiki Rice, US, 11- Jasmine Rice, US, 12- Long Grain Rice, US, 13- Jasmine Rice Thailand, 14- Basmati, Pakistan, 15- Jasmine Rice, Royal Thailand, 16-Jasmine Brown Rice, Royal Thailand, 17- Basmati rice, Royal Indian, 18-Basmati Brown Rice, Royal Indian, 19- Nishiki Rice, California US JFC, 20-Nishiki Brown Rice, California US JFC 4.2 Trifluoroacetic acid (TFA) extraction Optimization of collision or reaction gas flow 58

70 The isotopes of 75 As and 78 Se were chosen for analysis in this study. Polyatomic ion 40 Ar 35 Cl interferes with 75 As, as does 38 Ar 40 Ar with 78 Se. With practice these interferences can be removed, and the analyte sensitivity maintained. Figure 4.8 shows the optimization plots for the helium mode and hydrogen mode for measurement of arsenic and selenium. Arsenic optimization plots indicate that the background and analyte sensitivity is reduced with increasing the gas flow in both of helium and hydrogen mode. However, the helium mode reduces the background more quickly than the analyte when the gas flow is > 3.7 ml/min. Therefore, 4.7 ml/min of helium was chosen for arsenic analysis. 59

71 Figure 4.8 Optimization of the reaction/collision gas flow for arsenic and selenium determination. The left side was helium mode, and the right side was hydrogen mode. From the selenium optimization plots, 3.0 ml/min of hydrogen flow was chosen QA/QC issues in TFA extraction A reagent blank and matrix spike with Spex CertiPrep standards were used with TFA extraction and ICP-MS for QA/QC issues. 1 ppb of arsenic and 0 ppb of selenium were found in the reagent blank. Compared with the arsenic amount for the sample extracts, which are all around 20 ppb, it is necessary to subtract the reagent blank from sample extracts. There is no concern with the selenium reagent blank. The matrix spike recoveries confirm the TFA extraction, which is 114 % for arsenic and 90 % for selenium Efficiency of the TFA extraction One of objectives of this study is to find the arsenic and inorganic selenium species and evaluate the arsenic potential risk. Trifluoroacetic acid is a non oxygen strong acid. Diluted TFA can help arsenic and selenium species dissolve, and keep them intact. In this study, samples of Jasmine brown and white rice (Royal, Thailand), Basmati brown and white rice (Royal, India) and Nishiki brown and white rice (JFC, California US) were selected for speciation analysis. 2 M TFA (ph=0.45) was used for extracting these samples. The extraction efficiencies range from 104±7 to 135±13 % for arsenic and 76±7 to 121±24 % for selenium, obtained from the ratio of the total arsenic or selenium in TFA extracts to that in nitric acid digesting solution. The Table 4.3 and 4.4 present the results of total arsenic and selenium in TFA extracts and also the extraction efficiencies. 60

72 The values of arsenic or selenium were based on triplicates reported as t=±4.30 for two degrees of freedom and 95% confidence. The value of extraction efficiencies was obtained from the propagation of measurement uncertainties. The uncertain S x was calculated by following equation [51]. Table 4.3 The total arsenic in TFA extracts and the efficiencies of arsenic TFA extraction Table 4.4 The total selenium in TFA extracts and the efficiencies of selenium TFA extraction 61

73 The arsenic extraction efficiencies are higher than 100%, ranged from 104±7 to 135±13 %. The Basmati white rice and Nishiki white have higher recoveries than others, which are 135±13 % and 125±10 % respectively. The recoveries of others are in the range from 104±7 to 119±9 %, which is in the same range as matrix spike recovery (114 %). The higher recoveries may be caused by experimental error or TFA and rice matrix, because the polyatoms of 40 Ar 19 F 16 O and 56 Fe 19 F can interference 75 As in TFA matrix. TFA is an effective solvent for arsenic species but the extraction method need be developed in the further study. Some organic selenium species in Basmati rice might not be extracted by 2 M TFA, because there was a bit lower extraction efficiency for these rice samples, which was 76±7 % for Basmati white rice and 77±8 % for brown rice. Most of the inorganic selenium species should be removed from rice by this process due to the solubility of inorganic selenium compounds in acid solution, and the matrix recovery of inorganic selenium (90 %) [53]. 62

74 4.2.4 Arsenic speciation analysis in TFA extracts Some Arsenic species in TFA extracts were identified by HPLC-ICP-MS with a Luna SCX 100A column (Phenomenex, Torrance, CA) and a Hamilton PRP-X100 anion exchange column (Hamilton Company, Reno, NV). The chromatograms from both columns suggest arsenic as As (III), As (V) and DMA chemical forms are in the rice samples. The chromatograms of 12 ppb of arsenic species standard mixture, Nishiki white rice and brown rice (JFC California US) are shown in Figure 4.9. The left side of the chromatograms is from the Hamilton PRP-100 anion exchange column, and the right side is from the Luna SCX 100A column. The chromatograms indicate that both of the columns work for the separation of As (III), As (V) and DMA, because the chromatograms of 12 ppb of As (III), As (V), and DMA show good separation results. However, the peaks of As (III) and As (V) from Luna SCX 100A column have tails and the issues get worse in the sample chromatograms. The mergence of injection peak with the peak of As (V) in sample chromatogram can cause problem in quantification analysis. In addition, the better peak shapes ( tall and skinny ) are found in chromatogram from Hamilton PRP-100 anion exchange column. Over all, the results suggest that the Hamilton PRP-100 anion exchange column works better for arsenic speciation analysis in the study. 63

75 Figure 4.9 Comparison of chromatograms obtained from HPLC-ICP-MS with a Hamilton PRP-100 anion exchange column and Luna SCX 100A column 64

76 HPLC-ICP-MS with a Hamilton PRP-100 anion exchange column was selected for quantifying arsenic species. The DL and LOQ were determined and the retention time was checked with a 6 ppb of arsenic species standard before and after the measurement. A four-point-calibration curve was plotted with a series of 0 ppb, 3 ppb, 6 ppb and 9 ppb of arsenic species standards for quantitative analysis. The quantitative figures of merit are showed in Table 4.5. Table 4.5 Quantitative Figures of Merit Species DL (as 75 As, ppb) LOQ (as 75 As, ppb) Calibration Curve R 2 Value As (III) DMA As (V) The amounts of As (III), DMA and As (V) as 75 As in the TFA extracts were obtained by comparing their integration with calibration curves, and reported on a solid weight basis. The species analysis results are presented in Table 4.6. The chromatographic recoveries were calculated by the ratio of the sum of arsenic species to the total arsenic in TFA extracts. The most column recoveries are higher than 100% except for Basmati brown rice which has 89±2 %. The higher recovery is unexpected, because they should be lower than 100 % if there are polyatom interferences in the ICP-MS measurement for TFA total arsenic. The higher results may cause by some error or sample uneven. A QC/QA protocols need be considered for the arsenic specie quantification analysis in the 65

77 further study to make results more reliable and repeatable. From these data, the following comments are indicated. Table 4.6 The summery of arsenic speciation analysis 1. There is a difference in the distribution of arsenic species among varieties. The Figure 4.10 shows the profile for the sum of arsenic species in these rice samples. Basmati brown and white rice has the least arsenic compared with Jasmine and Nishiki brown and white rice. Brown rice contains more arsenic than white rice, but there is less difference between basmati brown and white rice. Nishiki brown rice had much more arsenic than the white rice counterpart. 66

78 Figure 4.10 The profiles for sum of arsenic species in the Jasmine, Basmati, and Nishiki brown and white rice 2. The organic arsenic, DMA is the most predominant arsenic species in Basmati brown and white rice. The toxic As (III) is about one fourth of total arsenic in both brown and white rice. By comparing the arsenic species in the brown rice and white rice, it is found that there is a little arsenic in rice bran, and most of it is inorganic arsenic (V). The results are strong similarity of Basmati brown rice (Royal India) compared to the white rice. Figure 4.11 shows the arsenic species distribution. Figure 4.11 The profile of arsenic species in Basmati brown and white rice, Royal, India 3. Inorganic As (III) is the predominant arsenic species in Nishiki brown and white rice. The rice bran of Nishiki rice, JFC California US, contains a high amount of 67

79 As (III). The brown rice contains much higher As (III) than its white one. Figure 4.12 shows the profiles of arsenic species distributions. Figure 4.12 The profiles of arsenic species in Nishikii brown and white rice, JFC California US 4. Jasmine brown rice has more As (III) and As (V) than Jasmine white rice. The milling process removes parts of As (III) and As (V). As (III) is the highest arsenic species in rice bran. The details for arsenic species distribution are presented in Figure The comparisons for Jasmine white rice, Nishiki white rice and Basmati brown and white rice are illustrated in Figure The graph suggests that there is no significant difference in the arsenic total amounts. However, the Basmati Brown and white rice, Royal India, had less As (III) and more DMA. 68

80 Figure 4.13 The profiles of arsenic species in Jasmine brown and white rice, Royal, Thailand Figure 4.14 Comparison of arsenic species among Jasmine white rice, Nishiki white rice and Basmati white and brown rice 69