Arsenic, cadmium, and lead in rice and rice products on the Austrian market

ABSTRACT Fifty-one rice samples, i.e. 25 rice varieties, 8 rice products, and 18 rice containing baby foods from the Austrian market were surveyed for arsenic, cadmium, and lead. Inorganic arsenic (iAs) is most toxic to human health, and its mean concentrations in rice were 120 µg kg−1, 191 µg kg−1 in rice products, and 77 µg kg−1 in baby foods. The average dimethylarsinic acid and methylarsonic acid concentrations were 56 µg kg−1 and 2 µg kg−1, respectively. The highest iAs concentration was found in rice flakes (237 ± 15 µg kg−1), close to the Maximum Level (ML) set by the EU regulation for husked rice (250 µg kg−1). The levels of cadmium (12 to 182 µg kg−1) and lead (6 to 30 µg kg−1) in the majority of rice samples were below the European ML. Upland grown rice from Austria showed both, low inorganic arsenic (<19 µg kg−1) and cadmium (<38 µg kg−1) concentrations.


Introduction
Rice (Oryza sativa) consumption is one of the major pathways of arsenic (inorganic and organic) exposure in the human body and poses a hazard to more than half of the world's population (Lai et al. 2015). Rice, especially in low-and lower-middle-income countries, is a dominant diet component, but high consumption is also recorded in developed countries, for consumers suffering from allergies or plant-based dieters, due to substitution of, for example, wheat products to ricebased products (Šlejkovec et al. 2021). Furthermore, babies and young infants can be exposed to a particularly high arsenic load due to their low body weight in combination with a high intake of rice-based food. A study of Gibson and Gage (1982) showed that the arsenic concentration in the hair of a one-year-old weaned toddler was 10-fold greater than in a one-yearold, breast-fed baby, which could be attributed to the consumption of pre-cooked, milled rice as the main carbohydrate source to weaning babies.
Common forms of arsenic in soil and water available for plant uptake are arsenate (AsV), arsenite (AsIII), methylarsonic acid (MA), and dimethylarsinic acid (DMA) (Bentley and Chasteen 2002). Arsenic is mainly located in rice bran, which results in a higher arsenic concentration in brown (husked) rice, than in white polished rice, due to removal of the bran during the milling process (Meharg et al. 2008;Zhao et al. 2010). A high arsenic load can also be expected in parboiled rice, as a result of the parboiling procedure where arsenic is diffusing under pressure from the bran into the endosperm of white rice (Zhao et al. 2010;Rahman et al. 2019).
Elevated arsenic exposure in humans has been a health concern for several decades (Gibson and Gage 1982;Hojsak et al. 2015). According to the European Union (EU) and the International Agency for Research on Cancer (IARC), arsenic is classified as "toxic" and a group one carcinogen. The World Health Organisation (WHO) set inorganic arsenic among its priority contaminants (Šlejkovec et al. 2021). Consumption of inorganic arsenic is of particular concern, since long-term ingestion is associated with skin lesions, cancer, cardiovascular diseases, abnormal glucose metabolism, diabetes, and neurotoxicity, according to the European Food Safety Authority (EFSA 2010). Thus, the inorganic arsenic content of rice available for human consumption needs to be regulated and controlled. The EU Commission Regulation 2015/1006 lists MLs for iAs as non-parboiled milled rice (polished or white rice) of 200 µg kg −1 , parboiled rice, and husked rice of 250 µg kg −1 , rice waffles, rice wafers, rice crackers, and rice cakes of 300 µg kg −1 . Organic species (DMA, MA) show lower acute toxicity than iAs, however, these are also classified as possible cancerogenic by the IARC, as cited by the German Federal Institute of Risk Assessment (2014). The Austrian market offers a large number of rice commodities, such as different rice varieties, rice products, and rice-based baby foods.
These originate from various countries, including Austria, Italy, and other European countries, as well as Asian countries. Many of these products are labelled with the EU organic agriculture seal or the Fairtrade seal, which does not give any information about the arsenic concentration.
Besides arsenic (As), cadmium (Cd) and lead (Pb) are of toxicological importance in rice. Cadmium is a toxic, carcinogenic element and an environmental pollutant. Cd is taken up by plants and affecting human health through food uptake, especially through rice consumption (Satarug et al. 2003;Jorhem et al. 2008;Kato et al. 2019;Shi et al. 2020). Studies have shown that upland (aerobic) rice cultivation resulted in higher Cd content when compared to lowland (anaerobic) rice growth conditions (Hu et al. 2013). The maximum concentrations of Cd and Pb in rice have been set to 150 and 200 µg kg −1 respectively by the EU (European Regulation 2021, 2006. In most countries, including Europe, the common agricultural rice growing practice is under lowland and continuous flooding , thereby establishing anaerobic conditions in the soil. Only in some areas, especially in cool temperate regions, rice is grown under upland aerobic conditions. In Austria, the upland (aerobic) agricultural practice has proven to be able to successfully grow rice the furthest north in Europe since 2015, with approximately 200 tons per annum. Rice plants take up arsenic from the soil and groundwater, especially in flooded systems water mobilises arsenic in the soil and hence increasing the accumulation of arsenic in rice (Takahashi et al. 2004;Tuli et al. 2010). Generally, water management, genotype, and the environment are factors that are related to the content of arsenic in rice (Tenni et al. 2017). The agricultural practice of upland (aerobic) growth conditions has a lower environmental impact due to lower water usage (no continuous flooding) and no/low methane production compared to lowland (anaerobic) growth conditions. Rice paddies are one of the main sources of methane, which has an impact as greenhouse gas (Schimel 2000).
The aim of this study was to carry out a survey of a variety of rice and rice products on the Austrian market and analyse the arsenic, arsenic species, cadmium, and lead levels.

Samples
Fifty-one samples were purchased in a variety of food shops, covering a range of samples normally consumed in Austria. The selection basis was widespread availability, common store-named products, and Austrian grown rice. Although for many rice products and baby foods, the manufacturer's information about the origin of the rice was not provided (Supplementary material) most rice varieties (72%) were purchased from the two main supermarket chains in Austria, and most rice products and baby foods (61%) were purchased from the main drugstore chains in Austria. Additional samples were purchased from local organic food shops and Asian food shops. The samples included white/polished, brown/unpolished, parboiled, and glutenous/waxy rice. Additionally, rice containing baby foods were collected. The samples were divided into six subgroups: brown/unpolished (N = 9), parboiled (N = 3), glutenous/waxy (N = 6), white/polished (N = 7), rice products, such as flakes, cakes, and drinks (N = 8) and rice containing baby foods (N = 18). Dry samples (at least half or more of a consumer package) were homogenised with a ZM 300 mill (Retsch GmbH, Haan, Germany). Liquid samples were freeze-dried (Christ Gamma 1-16 LSC, Wien, Austria) and homogenised with a pestle and mortar.

Digestion for total As, Cd, and Pb determination
All 51 rice samples were digested by weighing 200 mg (to 0.1 mg) milled rice in 3 mL concentrated sub boiled HNO 3 and 2 mL MilliQ water in a microwave (UltraCLAVE III, MLS GmbH, Germany). Subsequently, the digested solutions were diluted to 30 mL. The same preparation was applied to the CRMs NIST1568b and ERM-BC211 rice flour.

Extraction for arsenic speciation analysis
Rice samples with a total arsenic level >100 µg kg −1 were chosen for speciation analysis, which resulted in 32 out of 51 rice samples. The extraction was carried out with 150 mg (weighed to 0.1 mg) milled rice in 10 mL 1% (v/v) HNO 3 and 2% (v/v) H 2 O 2 for 60 min in a water bath at 90°C. The extracts were centrifuged (Rotina 420 R, Tuttlingen, Germany) at 4000 rpm for 10 min, and the supernatant was removed for analysis. Before measurement the extracts were centrifuged again at 4000 rpm for 10 min. For speciation analysis, a mild extraction method is mandatory to obtain both the inorganic and organic arsenic species, where H 2 O 2 reduces AsIII to AsV and the overall inorganic arsenic concentration (iAs) is measured as AsV.

Quantification
For total element determination, multielement calibration standards at a suitable concentration range were prepared in 10% HNO 3 for external calibration. Continuous internal standards 103 Rh and 193 Ir (200 µg kg −1 in 2% HNO 3 ) were used. Extracted solutions were also measured for total As to determine extraction efficiencies. For this calibration standards in 1% (v/v) nitric acid were prepared. For speciation analysis, mixed standards with dimethyl arsenic acid (DMA), methyl arsenic acid (MA), and inorganic arsenic as AsV were prepared in water from 1 g As kg −1 stock solutions. The ratio between species was 0.75 to 0.25 to 1, respectively.

Quality control and quality assurance
Certified reference material (CRM) NIST SRM 1568b rice flour was used for quality control of total element analysis. For speciation analysis, ERM-BC211 rice flour served as the CRM. Both reference materials were prepared and measured in three replicates in the same way as the samples. Extraction efficiency and column recovery were calculated from total As and the sum of the eluted species from the extract versus total arsenic in the digest . LODs were calculated using the three σ-criteria with σ as the standard deviation (SD) of the slope from the linear regression. Limits of detection (LODs), recoveries, and R 2 for total As, Cd and Pb are given in Table 1.
With an R 2 of 0.999 for all elements and relative standard deviations (RSDs) ranging from 2 to 5%, the precision of the measurements is acceptable. As and Cd measurements resulted in a recovery of 98% and 100%, respectively, while Pb had a recovery of 146% for NIST SRM 1568b. The latter can be explained by the low Pb concentration in the reference material. CRM values, column recoveries (CRs), extraction efficiencies (EEs), and LODs of each species from the speciation analysis are given in Table 2. With a CR ranging from 76% to 99% and an EE ranging from 67% to 107% the values were acceptable.

Determination of total As, Cd, and Pb by ICP-MS
Total arsenic, cadmium, and lead concentrations were determined with a 7900 ICP-MS system (Agilent Technologies, Inc., Santa Clara, CA, USA) at three gas modes (no gas, hydrogen, helium), RF Power 1550 V, Carrier gas flow rate 1.12 L min −1 , Nebuliser pump 0.1 rps and spray chamber temperature 2°C for single quad scans. The instrument was optimised daily. Arsenic was measured at the mass-to-charge ratio (m/z) 75, Table 1. LOD, recovery, and R 2 of total As, Cd, and Pb determination as well as measured and certified concentrations in the CRM (SD from three replicates). cadmium at m/z 113, and lead at m/z 207. Alternatively, an Agilent 8900 ICP-MS/MS was used for total arsenic determination in extracts at similar settings, only differing for a carrier gas flow rate of 1.05 L min −1 . In this case, As was measured using oxygen in the reaction cell as 75 As 16 O+ at m/z 91.

Arsenic speciation analysis by HPLC-ICP-MS
Speciation analysis was done on an Agilent HPLC 1260 Infinity system coupled to an Agilent 7700 ICP-MS. The column was directly connected to the ICP-MS nebuliser via a short length of PEEK-tubing and As was measured at m/z 75 in no gas mode. Three arsenic species (AsV, MA, and DMA) were analysed. Settings of the applied configurations are given in Table 3.

Origin of the samples
The rice varieties in this study were from different growing areas: 5 samples originated from Austria, 10 from Italy, another 5 from elsewhere in Europe and 9 were grown in Asia. For 22 out of the 51 samples, no origin was documented. Mean arsenic concentration was the lowest (5 µg kg −1 ) in rice grown in Austria. The highest mean arsenic concentration of 186 µg kg −1 was found in a sample of Italian origin, whereas rice from other countries in Europe and Asia showed mean arsenic concentrations between 79 and 86 µg kg −1 (Figure 1).

Total arsenic concentration in rice
The mean total arsenic content in the rice varieties was 134 µg kg −1 . Fifty percent of the samples (between the 25th and 75th percentiles) had an arsenic concentration ranging from 84 to 180 µg kg −1 (Figure 2), representing the "normal" range for As levels in rice of 82-202 µg kg −1 (Zavala and Duxbury 2008). Rice from upland (aerobic) dry cultivation in Austria, samples E-02, E-03, E-15 and E-16, showed all a low arsenic concentration, with a mean value of 5 µg kg −1 . In contrast, rice from Italy, samples E-01 and E-21, showed high levels of arsenic, with a total As concentration of 343 ± 11 µg kg −1 and 261 ± 3 µg kg −1 respectively. The As concentration in sample E-21 is similar to reported As concentration from Italian grown rice (Tenni et al. 2017). Similarly to Zavala and Duxbury (2008), we found that Italian rice contained higher  , glutinous/waxy rice (N = 6), brown/ husked rice (N = 9) and parboiled rice (N = 3). The lowest mean As concentration (81 µg kg −1 ) was measured in the white/polished rice varieties, followed by higher mean values in parboiled rice (134 µg kg −1 ), brown/husked rice (144 µg kg −1 ) and glutinous/waxy rice varieties contained the highest amount of As (171 µg kg −1 ), Figure 3. These findings correspond to the literature, where arsenic is found to accumulate predominantly in the bran (Zhao et al. 2010).

Total arsenic in rice products
The mean As concentration of the rice products was 137 µg kg −1 (Figure 4), comparable to As levels in the rice varieties mentioned above. However, within the range of the rice products, major differences were found. The As concentration in 50% of the samples was less than 10 µg kg −1 or below the LOD, while the other 50% had a mean As concentration of 237 µg kg −1 , this included two rice cakes (O-32, O-33) and two rice flakes samples (E-26, O-28) (Figure 4), of unspecified origin. In a previous study, rice cakes and flakes were also shown to have a high arsenic load (Rahman et al. 2019). In contrast, rice cakes made from rice grown in Austria had low total As concentrations of 5 to 19 µg kg −1 . Three liquid samples (rice drinks and syrup; Figure 4) contained less than 10 µg kg −1 , which was due to the low rice content in these products of 13 and 14%.

Total arsenic in rice-based baby foods
In rice-based baby foods, a mean As concentration of 135 µg kg −1 was found. Fifty percent of the samples, between the 25th and 75th percentile, ranged from 46 to 139 µg kg −1 (Figure 5). Taking into account that infants are 2-to 3-fold more exposed to arsenic than adults, according to the risk assessment of EFSA, most of the samples for babies, and young infants had a concerning As concentration. In this study, the highest total As concentrations were found in samples O-47 of 444 ± 28 µg kg −1 and O-48 of 464 ± 19 µg kg −1 . Both samples were rice cakes for children, with no specific origin. Due to the high total As concentration, these samples might pose a health risk. In regards to the arsenic concentration, sample A-37, a rice roll snack for children from Taiwan, with a total As level of 286 ± 13 µg kg −1 was also concerning. The rice snack additionally contained seaweed, which is also known for higher arsenic content (van Hulle et al. 2002). Low As levels were determined in the rice drink and syrup rice samples (O-39 to O-44), as these samples had less than 20% rice content ( Figure 5).

Cd and Pb in all rice samples
Cadmium concentrations ranged between 12 and 82 µg kg −1 in 25 out of 51 samples. Two samples, which originated from Italy, exceeded the maximum level of 150 µg kg −1 (European Regulation 2021) with a total cadmium concentration of 180 ± 10 µg kg −1 and 168 ± 1 µg kg −1 . The lead concentration in 25 out of 51 samples was below the LOD of 3 µg kg −1 rice. In the other 26 samples the Pb concentration ranged between 6 and 32 µg kg −1 (Figure 6), so all samples were below the EU regulation of 200 µg kg −1 (European Regulation 2006).   These results were similar to findings from rice surveys on the Slovenian (Šlejkovec et al. 2021) and Swedish market (Jorhem et al. 2008). In both studies the Cd levels ranged from <1 to 80 µg kg −1 , whereas Pb was only reported for the Swedish survey (<2-16 µg kg −1 ). Austrian rice, grown under upland (aerobic) conditions and with no continuous flooding, was low in As (<19 µg kg −1 ) and Cd (<38 µg kg −1 ), which is a relevant result, due to ongoing worldwide investigation of As and Cd levels in rice in regard to food safety. Low levels of both As and Cd have also been reported by Kato et al. (2019) for rice cultivated under upland (aerobic) growth conditions in Brazil. The findings of low As and Cd content in rice grown under upland conditions in Austria and Brazil are contradictive to other studies, regarding upland growth conditions. In a study conducted in Japan (Arao et al. 2009) it was found that water management of flooding and dry treatment times decreased arsenic content, but increased cadmium levels in rice. Similarly, from rice growth experiments in China with different water management (aerobic to flooded), the aerobic and intermittent watering mostly led to higher Cd concentration and decreased As concentration in rice (Hu et al. 2013). The inversely correlated As and Cd levels in rice, due to aerobic and flooded water management, were also reported in a recent study by Linam et al. (2022).

Arsenic speciation in rice
In all samples, DMA, MA, and iAs were detected. AsIII levels were below the LOD, which proves that all AsIII was successfully converted into AsV when using 2% H 2 O 2 during sample extraction. Therefore, the AsV values reflect the sum of AsIII and AsV, so all inorganic As. An example of a chromatogram can be seen in Figure 7. Only sample A-37 was an outlier among the samples, where a fourth arsenic containing compound eluting immediately before DMA was observed. This sample was a rice roll with seaweed flavour. Based on the elution behaviour of this unknown species and the fact that algae are present the product, the species was classified as an arseno sugar (van Hulle et al. 2002). However, for this study the identity of the species was not further investigated. In all samples, the MA concentration was negligible. Inorganic arsenic was in most of the samples (88%) the dominant species, with an iAs content ranging from 62 to 88%. The mean iAs concentration was 120 µg kg −1 for the different rice varieties, 190 µg kg −1 in rice products, and 77 µg kg −1 in rice-based baby foods. Sample E-01 Figure 8. Speciation analysis of brown/unpolished rice, parboiled rice, white/polished rice, and glutenous/waxy rice with a total arsenic concentration greater than 100 µg kg −1 . The letter indicates the origin with E=Europe, A=Asia and O= other/not specified. The ML for iAs of each category is represented as the dash line. Figure 9. Speciation analysis of rice flakes, cakes and rice containing baby foods with a total arsenic concentration greater than 100 µg kg −1 . The letter indicates the origin with E=Europe, A=Asia and O= other/not specified. The ML for iAs of each category is represented as the dash line.
(organic brown rice from Italy) exceeded the ML for iAs in husked rice (250 µg kg −1 ) by 0.2%. Samples E-26 and O-28 exceeded the ML for iAs in white/polished rice (200 µg kg −1 ) by 12 and 2%, respectively (Figures 8 and 9). In rice-based baby food samples, both iAs and DMA concentrations were of concern regarding to food safety ( Figure 9). In four baby food samples, DMA was the major component with a DMA/total As ratio ranging from 52 to 84%. Samples O-47 and O-48, rice cakes for toddlers, contained 327 ± 7 µg As kg −1 and 354 ± 10 µg As kg −1 DMA, respectively (Figure 9). Although, DMA is less toxic than AsV, DMA has been classified by the IACR as "possibly carcinogenic to humans" due to increased tumour incidence in the bladder and lungs of rats and mice upon repeated ingestion (German Federal Institute of Risk Assessment 2014).
In several countries in the Americas, inorganic arsenic levels in rice and rice products ranged from 30 to 130 µg kg −1 . Only two infant rice products were included in this market survey, and both had total As levels of 100 µg kg −1 (García-Rico et al. 2020), so were compliant with the EU legislation for infant foods (EU 2015). Lin et al. (2021) reported results from a survey of 258 rice samples originating from the Yangtze River Delta in China, of which 19 exceeded the level of 200 μg kg −1 for total arsenic in rice. Upon measurement of the iAs concentrations in these 19 samples the iAs concentrations were below the Chinese standard of 200 μg kg −1 for iAs in rice. In a rice survey from Australia and New Zealand, two rice products for infants and young children exceeded the EU ML of 100 iAs µg kg −1 (Ashmore et al. 2019).
In our study sample O-51 (chocolate puffed rice) would exceed the ML for iAs in rice-based baby and young infant food (100 µg kg −1 ) by 12%, if it was declared to be intended as food for young infants. Nevertheless, some rice-based products like O-51 (chocolate puffed rice), even when not officially classified as baby and young infant food and henceforth do not fall into the respective ML category of 100 µg kg −1 , might regularly be consumed by young children for breakfast. This observation was also made in a recent scientific report by EFSA, where rice-based food products indicated for children played a relevant role in the dietary exposure to iAs (Arcella et al. 2021). Further examples are samples O-28 and E-35 (both rice flakes), which are not subject to the ML for baby and young infant foods, though are mostly consumed by babies at weaning age (Gibson and Gage 1982), especially when considering that rice product E-35 was not located in the baby food section, but presented in the general supermarket section. Rice flake sample O-28 had a concentration of 226 ± 13 µg kg −1 iAs. If it would have been classified as baby food, the product would more than two times exceed the ML of 100 µg kg −1 . Consumption of 20 g of these rice flakes three times a day by a one-year-old baby, with an average bodyweight of 9.6 kg, would result in an intake of 1.5 µg iAs per kg body weight per day. A daily intake of 1.5 µg iAs per kg body weight is within the range of values for the 95% lower confidence limit of the benchmark dose of 1% extra risk (BMDL 01 ) identified by the CONTAM Panel of 0.3 to 8 μg kg −1 bw per day (EFSA 2010). Therefore, in the future, it would be useful to investigate, by means of a separate survey, if parents are aware of regulated arsenic levels in rice in regard to product classification.

Conclusion
A survey of rice and rice-based products in a city in Austria in 2022 showed that some products in local supermarkets exceeded the limits for iAs in rice, five years after the introduction of regulation (EU) 2015/ 1006. The findings are highlighting the importance for arsenic speciation analysis, because iAs has the highest toxicity among all arsenic species. Furthermore, it has been shown that rice grown under upland (aerobic) agricultural conditions in Austria, had both low iAs and low Cd levels, which is in favour of food safety and for sourcing rice for infant foods. To better understand the low levels of iAs and Cd in rice grown in Austria, it would be worthwhile to compare the soil and rice cultivar conditions in Austria, with other rice growing areas in the world as to optimise rice production to the benefit of consumers.

Acknowledgments
This work was done as part of a research ERASMUS+EU internship at the University of Graz, Austria. The authors report there is no funding associated with the work featured in this article.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.