due to the occurrence of the industrialization and urbanization without environmental care , toxic elements such as lead ( pb ) , cadmium ( cd ) , and arsenic ( as ) , coming mainly from mining , industrial processes , pesticides , chemical fertilizers , and atmospheric deposition , have become a major source of environmental contamination .
toxic elements are considered highly hazardous to human health and they may cause acute or chronic poisoning . chronic exposure to lead has been associated with the induction of pathological changes and damage in organs and central nervous system , leading to lower intelligence quotient in children .
cadmium is highly toxic to the kidneys and this metallic element is considered as carcinogenic . besides
, cadmium may cause bone mineralization , osteoporosis being a critical effect resulting from this element exposure .
arsenic is also considered as carcinogenic , and the majority of its chronic exposure reports are focused on skin problems like pigmentation and keratosis [ 1 , 2 ] . because of the high soil mobility and availability of the total arsenic , cadmium , and lead derived from the human activities and natural sources , there is a general concern about their phytotoxicity and risks to organisms , as they are rapidly able to spread out at different levels in the food chain .
it has been shown that plants growing in soils contaminated with toxic elements are not capable of preventing their uptake and accumulation in the plant tissue , but are capable of restricting them only .
thus , the foods contaminated with metals have turned out to be serious problem due to the potential bioaccumulation in biosystems through contaminated water and soil .
this circumstance is associated with the fact that some toxic elements are slowly eliminated from the human body , and they tend to accumulate in different tissues such as liver , muscles , and bones , threatening the human health .
rice ( oryza sativa l. ) is one of the most consumed cereals in the world , and it is part of the staple diet of the world population ; and it is considered as the most important source of nutrients for billions of people around the world .
rice provides 20.0% of energy and 15.0% of the daily requirement of protein for adults .
brazil is the largest non - asian rice producer in the world , and the average consumption of this cereal per person is nearly 25.0  kgyear [ 1 , 5 ] . according to the brazilian ministry of agriculture - livestock and food supply ,
brazil imported 372,567 tons of rice and exported 961,473 tons of the cereal in 2015 .
high concentrations of toxic elements are found in rice when compared to other plants grown under the normal conditions .
many toxic elements ' accumulation in rice is associated with the plant characteristics and its cultivation , as it is usually grown in flooded or very humid areas , which optimize the transfer of such elements from the soil to the plant . only fish and seafood
may carry higher concentrations of arsenic than rice ; however , while arsenic in rice occurs mainly as inorganic arsenic species , which are very toxic , arsenic in fish and seafood occurs primarily as organic species , which are less toxic [ 8 , 9 ] . in this context , the scope of this study was to investigate a suitable method for rice sample preparation , as well as to validate a method for monitoring the concentration of total arsenic , cadmium , and lead , in rice using inductively coupled plasma mass spectrometry ( icp - ms ) .
the proposed method was applied for analyzing different rice types ( polished , brown , and parboiled ) coming from various brazilian regions in order to investigate whether they are in accordance with the national legislation , aiming at public health promotion .
high purity deionized water ( resistivity 18.2  m  cm ) from a millipore water purification system ( bedford , ny , usa ) was used throughout this study .
lead ( pb ) , cadmium ( cd ) , arsenic ( as ) , germanium ( ge ) , indium ( in ) , and rhenium ( re ) at 1000  g  ml standard solutions , produced according to the iso guide 34 , were acquired from inorganic ventures ( christiansburg , va , usa ) . all of the employed reagents were of analytical grade .
suprapur hno3 ( 65.0% ) and h2o2 ( 30.0% ) used in the digestion procedures were acquired from merck ( darmstadt , hesse , germany ) .
instrumental daily performance solution was purchased from perkinelmer ( shelton , ct , usa ) .
certified reference material irmm 804 rice flour was acquired from the joint research centre institute for reference materials and measurements ( geel , antwerp , belgium ) .
external calibration was performed using a five - point analytical curve , prepared by diluting the individual arsenic , cadmium , and lead standards with 5.0% ( v / v ) hno3 .
analytical curve concentrations ranged from 1.0 to 40 , 1.2 to 24 , and 1.0 to 20  g  l for arsenic , cadmium , and lead , respectively . a multielement ( germanium for arsenic , indium for cadmium , and rhenium for lead ) internal standard solution with 5  g  l of each element in 0.2% ( v / v ) hno3 solution was prepared by serial dilution of 1000  g  ml monoelement stock solutions .
internal standard solution was added in - line to the analyzed solutions through a mixing tee , used to blend in the internal standards with the samples after the peristaltic pumping and before the nebulizer .
all of the measurements were conducted using an icp - ms ( elan drc ii , perkinelmer ) instrument , equipped with a glass meinhard ( golden , co , usa ) nebulizer and a cyclonic glass spray chamber .
a standard 2.0  mm i d quartz injector and pt sampler ( 1.10  mm orifice diameter ) and skimmer ( 0.9  mm orifice diameter ) cones were used .
standard , blank , and sample solutions were delivered using a s10 ( perkinelmer ) autosampler .
l of mg , in , u , ce , and ba ( perkinelmer ) .
according to the instrument manufacturer , the following parameters were evaluated during the daily performance analysis : sensitivity ( mg , in , and u ) , doubly charges ( ba ) , and oxides ( ce ) formation .
sample preparation procedure is a critical point for the success of the analysis , being considered an important source of error in analytical method development . in the present study
different sample preparation procedures were investigated in order to check their suitability for rice decomposition aiming at determining the total arsenic , cadmium , and lead concentrations by means of icp - ms . a rice package ( 1  kg ) of a given brand randomly selected
was acquired in a market located in so paulo city , sp , brazil . at first
, the samples were weighed using an analytical balance ( shimadzu , kyoto , japan ) and then grinded in an ika analytical mill ( staufen , baden - wrttemberg , germany ) , except when performing the procedure carried out in the muffle furnace , where the entire grain was ashed . for investigating the analytes recovery for the various sample preparation procedures ,
the samples were spiked using arsenic , cadmium , and lead aqueous solutions . for each sample preparation procedure , three independent replicates of the sample were analyzed , and the respective procedural blanks were considered in the final results .
samples were fortified in order to get final concentrations ranging from 8.0 to 30.0  g  l ( within the calibration curves concentration range ) for all the analyzed elements in the final sample solutions .
laboratory standard procedure for food samples mineralization was performed using a fornitec ( so paulo , sp , brazil ) muffle furnace . in a porcelain capsule ,
2.0  g of the sample was weighted and 2  ml of 60.0% mg(no3)2 was added .
the samples were initially ignited in a bunsen burner , and then they were taken into a muffle furnace with a heating ramp of 150c until reaching 420c , maintaining this temperature for 4  h. after cooling , 1  ml of hno3 was added and allowed to dryness on a heating plate .
samples returned to the muffle furnace at 420c until reaching the complete destruction of the organic matter .
samples were spiked in order to obtain a final concentration of 9.6  g  l for arsenic , cadmium , and lead in 50  ml of at 5% ( v / v ) hno3 . for the metallic block ,
1  g of the sample was weighted in a polytetrafluorethylene ( ptfe ) flask .
hno3 ( 1  ml ) and h2o2 ( 2  ml ) were used as reagents , and an overnight predigestion step was performed .
digestion was performed in a marconi ( piracicaba , sp , brazil ) metallic block at 100c for 5 hours .
samples were spiked in order to get a final concentration of 16.0 , 9.6 , and 8.0  g  l of arsenic , cadmium , and lead , respectively , in 25  ml of deionized water . for digesting on the hot plate , 0.5  g of the sample
was weighted in an erlenmeyer flask and 1  ml of hno3 and 2  ml of h2o2 were added .
the mixture was left overnight and then heated on a hot plate ( marconi ) at 130c for 2  h. samples were spiked in order to get a final concentration of 8.0  g 
l of the elements in the sample solution ( volume completed to 25  ml by adding deionized water ) . for the digestion assisted by microwave radiation ,
the method published by batista and coworkers   was performed with some modifications . in the present study ,
0.5  g of rice sample was weighted and transferred to the ptfe flask specific for the microwave oven used ( ethos one from milestone , sorisole , bergamo , italy ) .
two ml of hno3 , 2  ml of h2o2 , and 4  ml of h2o were added to the sample in the flask after an overnight predigestion step .
the following program was run : 1000  w at 100c ( 5  min .
samples were spiked in order to reach a final concentration of 30.0 , 10.0 , and 10.0  g  l of arsenic , cadmium , and lead , respectively , in the final sample solution ( volume completed to 25  ml by adding deionized water ) .
for the method validation , the sample solution obtained from digestion in microwave oven was selected due to the lesser time consumption involved and the lower blank values .
the analytical method validation was performed by considering the limit of detection ( lod ) , limit of quantification ( loq ) , linearity , precision ( repeatability ) , and relative bias .
lods and loqs were established as three and ten times , respectively , the standard deviation of six rice samples independently digested considered as having approximately 1  g 
l of arsenic and concentration of cadmium and lead < loq in the final sample solution .
the final values of both parameters were calculated taking into account the samples dilution factor and the weight .
linearity was established by preparing the calibration curves of all of the target elements by employing an unweighted least - squares linear regression .
relative bias and repeatability of the method were evaluated by analyzing seven replicates of the certified reference material irmm 804 rice flour , for determining arsenic , cadmium , and lead .
relative bias was obtained as the percentage difference between obtained and certified values , while the repeatability was calculated as relative standard deviation ( rsd% ) . to evaluate the analytical method performance for determining the arsenic , cadmium , and lead in different rice types samples consumed by the brazilian population ,
37 rice samples from different brands and lots were collected from various states of the country ( cear , esprito santo , gois , paraba , rio grande do sul , rondnia , and so paulo ) .
samples were collected by the health surveillance system from 2013 to 2015 and sent to the inorganic contaminants laboratory - adolfo lutz institute .
they consisted of 27 polished rice samples , two parboiled rice samples , and eight brown rice samples .
expanded uncertainty estimation ( uc ) for the obtained results was calculated according to the criteria described in eurachem / citac . for this evaluation ,
the following parameters were considered : analytical curve uncertainty associated with the linear least - square fitting procedure ; volumetric flasks calibration and temperature effect on the volumetric measurement ; and analytical balance calibration . expanded uncertainty
was determined by multiplying the coverage factor ( k ) and the combined uncertainty ( uc ) : uc = k  uc , considering the coverage factor k = 2 , for a confidence level of 95.45% .
the well - known polyatomic interference caused by arcl was considered in the total arsenic determination by using arithmetic correction . according to t - test ( p = 0.005 , n = 4 )
similar result was found by cai and coworkers   for citrus leaves standard reference materials digested with hno3 and h2o2 , reagents also used in the present study .
this result can be explained by the fact that only 2% of the total chlorine contents in rice plants are present in the edible part . 

table 2 shows the analytes recovery in the spiked samples submitted to the different preparation procedures for determining total arsenic , cadmium , and lead in rice .
recoveries varied from 81.0 to 115.0% , indicating that results for all elements determined in the sample submitted to different preparation procedures were within the percentages recommended by fda , which comprise the range between 80.0 and 120.0% .
this finding highlights the possibility of using all of the sample preparation procedures employed in the present study , including arsenic determination whose volatility is well known .
soylak and coworkers   also compared various sample preparation procedures for determination of trace elements in turkey spices .
recoveries ranged from 95.0 to 97.0% for muffle oven , 99.0 to 95.0% for hot plate , and 98.0 to 103.0% for microwave oven ; and these results are comparable to those found in the present investigation .
the results found in the present study are in agreement with the data reported by wei et al . , where acid digestion assisted by microwave radiation was carried out , reaching recoveries of 97.0 , 106.0 , and 103.0% for total arsenic , cadmium , and lead , respectively .
nowadays , researches on sample preparation procedures have advanced , leaning more and more to the microwave technology , becoming difficult to find present - day publications using other procedures .
the advantages of microwave technology are undeniable , but the use of less resourceful instruments may eventually be appropriate , providing good results for the analytes under study .
the overall performance of the proposed method for total arsenic , cadmium , and lead determination in rice is summarized in tables 3 and 4 .
lods and loqs results were considered acceptable as the values were below the limits established by the brazilian legislation for arsenic , lead , and cadmium for rice and its derivatives : 0.30  mg  kg , 0.20  mg  kg , and 0.40  mg  kg , respectively .
the linearity of the calibration curve was evaluated by investigating the correlation coefficient ( r ) of the calibration curves .
the r values were higher than 0.998   for all of the analytes , as shown in table 3 , and the regression analyses showed that the linear correlation between concentration and signal intensity was significant ( p < 0.05 ) .
relative bias ( table 4 ) was assessed through the difference between the mean value of obtained results and the certified values .
for all elements , the rsd ( repeatability ) was in compliance with the european commission decision 2002 , and it did not exceed 10.0% .
therefore , as expected , the use of microwave oven for rice acid digestion was appropriate for determining the total arsenic , cadmium , and lead concentrations in rice using icp - ms .
the above was method applied for quantifying the concentration of total arsenic , cadmium , and lead in 37 rice samples ( polished , parboiled , and brown rice ) .
results are summarized in table 5 . according to table 5 , of 37 analyzed samples ,
35 showed total arsenic concentration higher than the loq , ranging from 0.061 to 0.660  mg  kg . in two polished rice samples ,
the average total arsenic concentration in parboiled rice was lower than that found in other rice types ( 0.071  mg  kg ) , followed by polished rice ( 0.130  mg  kg ) and brown rice , which had the highest arsenic concentration ( 0.224  mg  kg ) .
in one brown rice sample , collected in the state of so paulo , the total arsenic concentration was 0.660  mg  kg .
this value was higher than that established by the brazilian legislation   and the codex alimentarius , which is 0.30  mg  kg .
total arsenic concentrations were in agreement with the maximum level established for total arsenic in raw rice .
total arsenic mean concentrations found in the present study were in agreement with those cited in the literature ; and the respective authors pointed out the evident occurrence of the highest arsenic concentrations in brown rice .
this occurs owing to the fact that rice plants concentrate this element in the outer layer of the grain , in the region corresponding to the pericarp .
a great amount of arsenic is removed during the rice polishing [ 21 , 5052 ] .
besides , variation in the arsenic concentration may occur due to different cultivars , geography , environment , water quality , and growth conditions [ 21 , 24 , 25 , 29 , 30 , 37 , 51 , 5356 ] .
the results obtained in the present work are comparable to others in investigations performed in brazil , as shown in table 6 .
usually , the data from several studies presented the same tendency , the total arsenic concentration being higher in brown rice , followed by polished and parboiled rice .
the results found in this study on the total arsenic contents were also in agreement with those previously reported for rice analyzed in other countries ( table 7 ) .
the exception is for arsenic concentration found in rice cultivated in bangladesh , one of the countries that have been mostly vulnerable to arsenic exposure , causing a serious public health problem [ 33 , 34 , 53 ] . in the 1970s ,
the plumbing pipes contaminated by arsenic were installed in the country , and it caused the contamination of the water consumed by the population . in consequence , it originated the contamination water used for rice irrigation .
with respect to cadmium , only one polished rice sample had cadmium concentration above the loq ( 0.042  0.008  mg  kg ) . in the other rice samples , cadmium concentration was below the lod in 17 samples , while in 19 samples it was below the loq .
in none of the analyzed samples , cadmium concentrations exceeded the maximum value of 0.40  mg  kg allowed by the brazilian legislation   and the codex alimentarius .
thus , referring to cadmium contamination , the polished , parboiled , and brown rice samples were considered as satisfactory for consumption .
cadmium concentration found in the analyzed samples were similar to those reported in other studies carried out by brazilian investigators , as outlined in table 6 . in all of the investigations ,
the cadmium levels were lower than those recommended by the brazilian legislation . the same is observed when the data on rice from other countries are compared ( table 7 ) .
however , higher cadmium concentrations in rice have been reported , which can be attributed to the soil contaminated by metals in mining and industrial areas [ 31 , 56 , 58 , 59 ] .
as for lead , in 14 polished rice and one parboiled rice samples lead concentration was below the lod , and 11 polished rice and four brown rice samples had lead concentrations below the loq . in one parboiled rice ( 0.127  mg  kg ) , two polished rice ( 0.0870.115  mg  kg ) , and four brown rice ( 0.0650.124  mg  kg ) samples , the lead concentrations were above loq , with mean values of 0.101  mg  kg for polished rice and 0.104  mg  kg for brown rice . in all of the analyzed samples , the results indicated that the lead concentrations were lower than the maximum value allowed , which is 0.20  mg  kg , according to the brazilian legislation   and european community . thus , regarding to lead , the brazilian rice evaluated in this study was shown to be suitable for consumption .
the lead concentrations found in brazilian rice ( table 6 ) and other countries ( table 7 ) were comparable , not exceeding the maximum value established by the brazilian legislation .
higher lead contents found in some investigations might indicate the soil contamination by fertilizers , industrial , and/or mining activities [ 31 , 56 , 59 ] .
regarding the sample preparation , all of the procedures ( ashing in muffle furnace , acid digestion under heating on metallic block or hot plate , and acid digestion assisted by microwave radiation ) led to good results ( recoveries ranging from 80.0 to 120.0% ) .
this information is important for laboratories that execute rice analyses , as the sample preparation may be cheaper .
the parameters obtained for validation have demonstrated that the method fits for its purpose , which is to quantify the total arsenic , cadmium , and lead in complying with the brazilian legislation limits . regarding the parboiled , polished , and brown rice samples analyzed , the concentrations of total arsenic , cadmium , and lead were lower than the established limits according to the brazilian legislation , codex alimentarius , and european community , except for total arsenic in one sample .
the other analyzed rice samples were considered satisfactory for human consumption regarding the investigated elements .
the data obtained indicated that only the consumption of brown rice can represent risk to brazilian population .
therefore , it is necessary to establish programs to focus attention on this type of rice aiming at promoting the public health , since rice might be an important route for human exposure to toxic elements , particularly in populations following diets based on this cereal , as is the case of the brazilian population .
 this study is aimed at investigating a suitable method for rice sample preparation as well as validating and applying the method for monitoring the concentration of total arsenic , cadmium , and lead in rice by using inductively coupled plasma mass spectrometry ( icp - ms ) . 
 various rice sample preparation procedures were evaluated . 
 the analytical method was validated by measuring several parameters including limit of detection ( lod ) , limit of quantification ( loq ) , linearity , relative bias , and repeatability . regarding the sample preparation , 
 recoveries of spiked samples were within the acceptable range from 89.3 to 98.2% for muffle furnace , 94.2 to 103.3% for heating block , 81.0 to 115.0% for hot plate , and 92.8 to 108.2% for microwave . 
 validation parameters showed that the method fits for its purpose , being the total arsenic , cadmium , and lead within the brazilian legislation limits . 
 the method was applied for analyzing 37 rice samples ( including polished , brown , and parboiled ) , consumed by the brazilian population . 
 the total arsenic , cadmium , and lead contents were lower than the established legislative values , except for total arsenic in one brown rice sample . 
 this study indicated the need to establish monitoring programs for emphasizing the study on this type of cereal , aiming at promoting the public health . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusions