Martijn J Martena
email: martijn.martena@vwa.nl

Jacqueline C A Van Der Wielen

Ivonne M C M Rietjens

Walther N M Klerx

Henk De Groot

Erik J M Konings

H N De 5 Groot

Ailing Yihao

Arsenolite Angong

Niuhuang San

-Wan

Realgar Niuhuang Cinnabar

Shen An

Nao Bu

Pian

Zi Yang

Xin Wan

Cinnabar Baochi

San Cinnabar Baochiwanying-San $

Cinnabar Baolong

Wan * Cinnabar

Bao Ning

Bao Ying

Cinnabar Bingpen

San Cinnabar

Bushen Yinao

Pian Cinnabar

Ding Xian

Ershiwuwei Shanhu

Ershiwuwei Songshi

Wan Cinnabar Feierwanyaopian

$ Geng

Yi Wan

Cinnabar Hongling

San Cinnabar

Hua Realgar

Chun Chong

Dan

Hu Cinnabar

Bao Po

Wan Long

Po Lung

Yuen Med Pills

Cinnabar Jian

Nao Wan

Cinnabar Jierezhenluesan

$ Cinnabar

Liu Shen

Wan * Realgar

Meihua Dianshe

Realgar Niuhuang Baolong

Realgar Niu

Huang Cheng

Qi Tang

Huang Niu

Ya Wan Chiang

High

Niu Huang

Ching Hsin

$ Niuhuang

Jiedu Pian

Realgar Niuhuang

Jiedu Wan

Qianjin San

Cinnabar Niuhuangqingfei-San $ Cinnabar

Huang Qing

Wan Zhenjing

Peaceful

Cinnabar Pinggan

Shuluo Wan

Cinnabar Po

Lung Yuen

Med Pills

Cinnabar Qianjinsan

Qi Li

Cinnabar Qizhen

Realgar Renqing

Changjue Cinnabar

Renqing Mangjue

Cinnabar San

Li Hui

Chun Dan

Shayao Cinnabar

Dan She

Pi Chen

San

Cinnabar Sheng

Tie Luo

Yin Shixiang Fansheng

Aristolochia Radix

Shugan Wan

Cinnabar Shuzheng

Realgar Suhexiang

Wan / Su

He Xian

Yingerbaofeining $ Cinnabar

Yingerle $ Cinnabar

Yixian Wan Cinnabar

Realgar Yi

Yuan San

Cinnabar Zaizao

Zhi Bao

Dan Cinnabar

Realgar Zhou

Che Wang

Calomel Zhuhuang

Chuihou San

Realgar Zhu

Sha An Shen

Ding / Zi

Jin Ding

Cinnabar Zixue

Xue Zi

Tzuhsueh Tan

Monitoring of mercury, arsenic and lead in traditional Asian herbal

Keywords: Heavy metals -arsenic, Heavy metals -mercury, Lead Food Types: Dietary supplements Ayurveda, Traditional Chinese Medicine, Traditional Tibetan Medicine, herbal remedies, dietary supplements, heavy metals, bhasmas, mercury, arsenic, lead 15 Ayurveda, Traditional Chinese Medicine, Traditional Tibetan Medicine, herbal remedies, dietary supplements, heavy metals, bhasmas, mercury, arsenic, lead

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In Asian traditional medicine systems such as Traditional Chinese Medicine (TCM), Traditional Tibetan Medicine (TTM) and the Indian traditional medicine systems Ayurveda, Unani-Tibb and Siddha, traditional herbal preparations (THPs) play an important role (Bajaj & Vohora, 2000;[START_REF] Aziz | Kushta(s): unique herbo-mineral preparation s used in South Asian traditional medicine[END_REF]Patwardhan et al., 2005;[START_REF] Sallon | Mercury in traditional Tibetan medicine -panacea or problem?[END_REF]. In the Netherlands Asian THPs can be brought 5 on the market as food supplements, which do not require prior safety evaluation. Asian THPs have been reported to contain herbal toxic principles, undeclared synthetic drugs and significant amounts of mercury, arsenic and lead [START_REF] Espinoza | Arsenic and mercury in traditional Chinese herbal balls[END_REF][START_REF] Johanns | An epidemic of epileptic seizures after consumption of herbal tea[END_REF]Ernst, 2002b;Blok-Tip et al., 2004;[START_REF] Saper | Heavy metal 5 content of ayurvedic herbal medicine products[END_REF]Mino & Yamada, 2005;MHRA, 2005;Rietjens et al., 2005;[START_REF] Martena | Heavy metal ingredients of traditional Asian herbal preparations[END_REF][START_REF] Martena | Enforcement of the ban on Aristolochic acids in Chinese traditional herbal preparations on the Dutch market[END_REF]. Use of Indian, Tibetan and Chinese THPs has resulted in lead poisonings 10 worldwide and several mercury and arsenic intoxications as well [START_REF] Dunbabin | Lead poisoning from Indian herbal 10 medicine (Ayurveda)[END_REF][START_REF] Kang-Yum | Chinese patent medicine as a potential source of mercury poisoning[END_REF][START_REF] Kew | Arsenic and mercury intoxication due to Indian ethnic remedies[END_REF][START_REF] Thatte | The flip side of Ayurveda[END_REF][START_REF] Keen | Indian herbal remedies for diabetes 10 as a cause of lead poisoning[END_REF]Prpic-Maijc et al., 1996;[START_REF] Wu | Lead poisoning caused contaminated Cordyceps, a Chinese herbal medicine: two case reports[END_REF][START_REF] Li | Mercury intoxication presenting with tics[END_REF]Moore & Adler, 2000;[START_REF] Van Vonderen | Severe gastrointestinal symptoms due to lead poisoning from Indian traditional medicine[END_REF][START_REF] Auyeung | Three patients with lead poisoning 15 following use of a Chinese herbal pill[END_REF][START_REF] Tait | Severe congenital lead poisoning in a preterm infant due to a herbal remedy[END_REF]CDC, 2004;[START_REF] Lee | Hemolytic anemia as a sequela of arsenic intoxication following long-term ingestion of traditional Chinese medicine[END_REF][START_REF] Schilling | Bleiintoxikation durch Einnahme ayurvedische 15 Arzneimittel[END_REF][START_REF] Khandpur | Chronic arsenic toxicity from Ayurvedic medicines[END_REF]. In Ayurveda, Siddha, Unani-Tibb, TTM and TCM ingredients high in the metals mercury and lead or the metalloid 15 arsenic are added to THPs for therapeutic purposes [START_REF] Kang-Yum | Chinese patent medicine as a potential source of mercury poisoning[END_REF][START_REF] Thatte | The flip side of Ayurveda[END_REF]Bajaj & Vohora, 2000;[START_REF] Aziz | Kushta(s): unique herbo-mineral preparation s used in South Asian traditional medicine[END_REF][START_REF] Sallon | Mercury in traditional Tibetan medicine -panacea or problem?[END_REF]. In TCM tranquilizing and detoxifying effects are attributed to mercury [START_REF] Kang-Yum | Chinese patent medicine as a potential source of mercury poisoning[END_REF] and nearly 10% of the formulas in the Pharmacopoeia of China incorporate the mercury containing mineral cinnabar (Young et al., 2002;Huang, 2006). In 25% of the 634 formulas for Ayurvedic THPs in the official Ayurvedic Formulary of 20 India, mercury, lead and arsenic compounds are listed as ingredients (Ministry of Health, 2000;Ministry of Health, 2003). An Ayurvedic discipline called 'Rasa shastra' is dedicated to the study of the therapeutic use of mercury and other metals or metalloids [START_REF] Dash | Alchemy and metallic medicines in Ayurveda[END_REF]. In most cases high levels of metals or metalloids in Ayurvedic THPs result from intentional incorporation of certain metallic preparations called 'bhasmas' in the products. The metals and metalloids in bhasmas are 25 claimed to be detoxified by elaborate processing steps including many cycles of heating and subsequent cooling in herbal mixtures and animal products such as cow's urine or ghee (Thatte et Ravinarayan & Skandhan, 1995;Ministry of Health, 2000;Ministry of Health, 2003;[START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF]. Comparable preparations called kushtas and parpams are in use in Unani-Tibb and Siddha, respectively (Bajaj & Vohora, 2000;[START_REF] Aziz | Kushta(s): unique herbo-mineral preparation s used in South Asian traditional medicine[END_REF].

Metals and metalloids can exist in different defined chemical species and the toxicity of these species can vary significantly [START_REF] Who | Elemental speciation in human health risk assessment[END_REF]. Available reports consistently point to the presence of 5 inorganic metal or metalloid compounds in Chinese, Tibetan and Indian THPs including Ayurvedic THPs. Naga bhasma, a widely used Ayurvedic lead preparation (Ministry of Health, 2000), contains lead sulfide (PbS); other lead preparations contain lead oxide (PbO) or lead sulfate (PbSO 4 ) (Ravinarayan & Skandhan, 1995;[START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF]. In TCM, lithargyrum or 'mi tuo seng' (PbO) is used (Bensky & Barolet, 1990). Important Ayurvedic mercury preparations are 'parada' (purified 10 mercury), 'hingula' (cinnabar, HgS), 'kajjali' (HgS) and 'makaradhvaja' (HgS) [START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF]Himalaya, 2008). In TTM processed mercury preparations (tsothel) are used as well, which mainly consist of mercuric sulfide (HgS) and smaller amounts of mercuric sulfite (HgSO 3 ) and mercuric sulfate (HgSO 4 ) [START_REF] Sallon | Mercury in traditional Tibetan medicine -panacea or problem?[END_REF]. In TCM mercury is used as cinnabar (zu sha) and as calomel (qing fen, Hg 2 Cl 2 ) (Bensky & Barolet, 1990;[START_REF] Kang-Yum | Chinese patent medicine as a potential source of mercury poisoning[END_REF]Bensky & Gamble, 1993; 15 Mino & Yamada, 2005;[START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF]. In both Chinese and Ayurvedic THPs, realgar (As 4 S 4 ), orpiment (As 2 S 3 ) and arsenolite (As 2 O 3 ) are used but in Ayurveda these minerals are processed before use (Bensky & Barolet, 1990;Bensky & Gamble, 1993;Mino & Yamada, 2005;[START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF]Liu et al., 2008b). In certain kushtas (used in Unani-Tibb) As 2 O 3 was found but kusthas can contain other inorganic arsenicals as well [START_REF] Aslam | Heavy metals in some Asian medicines and cosmetics[END_REF][START_REF] Kew | Arsenic and mercury intoxication due to Indian ethnic remedies[END_REF][START_REF] Aziz | Kushta(s): unique herbo-mineral preparation s used in South Asian traditional medicine[END_REF].

20

In 2004 the Dutch Food and Consumer Product Safety Authority (VWA) received reports of two cases of lead poisoning linked to the use of Ayurvedic THPs from, respectively, India and Nepal (VWA, 2005;[START_REF] Kanen | Chronic lead intoxication associated with Ayurvedic medication Ned[END_REF]. These cases prompted the VWA to intensify investigations into metal and metalloid contents of Asian THPs. The objective of this investigation was to study the metal and metalloid contents of Asian THPs on the Dutch market and assess the related risks by

Material and methods

Sampling

VWA inspectors sampled 292 Asian THPs for oral use on the Dutch market between December 2004 and June 2007. The aim of the sampling plan was to collect commercially available Asian THPs that could potentially contain significant amounts of mercury, arsenic or lead. Samples were of Ayurvedic, 5Chinese and Tibetan origin and were directly collected from importers, producers and vendors throughout the Netherlands. The locations were selected from the VWA inspection database or identified by an internet search. No THPs were purchased via Internet. Per product, one or more units of at least 20 g were sampled. Criteria for inclusion were an apparent relation to Ayurveda, TCM or other traditional Asian medicine systems and the presence of ingredients used traditionally in these 10 medicine systems. THPs lacking such features were excluded. For instance, a food supplement made in China with the contemporary ingredient spirulina or an American supplement with the traditional ingredient gingseng but lacking a link with TCM, would be excluded. Sampling was aimed at multi ingredient THPs with herbs and minerals but some single herb preparations were also included. The labeling of each potential sample was scanned for ingredients listed in table 1 reported to contain 15 mercury, arsenic or lead (Bensky & Gamble, 1993;Kang-Yum & Oransky, 1999;Ministry of Health, 2000;MCA, 2001;Ernst, 2002b;Ministry of Health, 2003;Liu et al, 2008b). We also compared the product name to a list of names of classical formulas for Ayurvedic THPs (Table 2) and a list of classical formulas for Chinese THPs (Table 3) that are reported to include mercury, arsenic or lead compounds. Table 2 was compiled from the official Ayurvedic Formulary published by the Indian 20 government (Ministry of Health, 2000;Ministry of Health, 2003) by selecting from this source the names of formulas for THPs for oral use that included ingredients with lead, mercury or arsenic. Table 3 was compiled from the Pharmacopoeia of the People's Republic of China (English edition of 1997) by the British Medicine Control Agency supplemented with names of formulas for Chinese THPs which were described in a TCM handbook to contain ingredients with lead, mercury and arsenic 25 (Bensky & Barolet, 1990;MCA, 2001). Also included in table 3 were Chinese THPs reported to contain mercury, arsenic or lead [START_REF] Kang-Yum | Chinese patent medicine as a potential source of mercury poisoning[END_REF][START_REF] Espinoza | Arsenic and mercury in traditional Chinese herbal balls[END_REF][START_REF] Auyeung | Three patients with lead poisoning 15 following use of a Chinese herbal pill[END_REF] Mino & Yamada, 2005;Huang et al., 2006;Liu et al., 2008b). THPs with ingredients listed in table 1 or product names listed in tables 2 or 3 were sampled preferentially but other THPs were sampled as well.

ICP-MS determination of metals and metalloids

5

A routine method for the determination of metals and metalloids in herbal preparations with Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was developed and validated for several elements including arsenic, lead, and mercury. Routinely cadmium, zinc, copper, nickel, chromium and aluminum were monitored as well. Samples were completely homogenized by grinding them to powder with a grinder (Retsch). To 20 -100 mg (depending on the elements to be measured) of the 10 powdered sample 3 ml nitric acid (HNO 3 , 65%) and 1 ml hydrogen peroxide (H 2 O 2 , 30%) were added.

A destruction step was then performed using a microwave oven (Ethos Plus Microwave Labstation, Milestone) in which the samples were treated for 25 minutes at 200ºC. The solution produced by the destruction process was then quantitatively transferred with de-ionized water from the destruction vessel to a 50 ml volumetric flask. Before filling the flask to the 50 ml mark with de-ionized water, 15 500 µl aureumchloride (AuCl, 100 mg/L) solution was added. Throughout the analysis Milli-Q grade de-ionized water was used. The sample solutions were diluted (up to 10000 times) when necessary to fit within the calibration curve and measured with ICP-MS (Thermo Electron PQ Excell) using the 'Collision Cell Technology mode' (CCT-mode) with a helium/hydrogen flow (95/5%). Using this gas mixture in the CCT-mode most interferences were reduced [START_REF] Van Der Wielen | Determination of interferences in the analysis of elements in foodstuffs with ICP-MS using Collision Cell Technology (CCT)[END_REF]. In order to 20 test if each destruction vessel was clean prior to use, test runs were performed on each vessel with only 3 ml HNO 3 (65%) and 1 ml H 2 O 2 (30%) before each series of samples. This was repeated after each series in order to confirm that no metals or metalloids were left in the vessels. For the in house validation of the method, first the limits of detection and quantification (LOD and LOQ) were determined. The LOD was defined as three times the residual standard deviation of the standard 25 calibration curve. The LOQ was defined as twice the LOD. The range of the calibration curve was used to calculate the range of application. Samples were diluted and reanalyzed when concentrations appeared to exceed the highest concentration of the calibration curve. The accuracy of the method was determined by analysis of a Certified Reference Material (CRM)(IRMM; BCR 482: trace elements in Lichen). The analytes of this CRM had to be within the 95% confidence interval of the certified values. Furthermore the relative standard deviation (RSD r ) was measured by replicate analysis (n=6) of the CRM under repeatability conditions. The RSD r is a measure of the degree of agreement of results obtained by the same analyst with identical reagents, equipment and instruments within a short 5 period of time under conditions kept as constant as possible. The CRM was also used as quality control sample in each series of measurements. The expanded measurement uncertainty (in mg/kg) was determined as the reproducibility from measurements (n>10) of this CRM, analyzed by different persons on different days.

Table 4 lists the method performance characteristics as the LOD, LOQ, range of application, 10 accuracy, RSD r and the measurement uncertainty (in mg/kg). According to the AOAC guidelines for single laboratory validation of chemical methods for botanicals and dietary supplements, the accuracy has to be between 75 and 120% at the measured concentrations (AOAC, 2002). The values for the accuracy of the method for mercury, arsenic and lead as listed in table 4, fitted well within these limits as did the other elements that can be analyzed with this method. The RSD r for all elements was found 15 to be below 10% with mercury displaying the highest value of 9.5%. These values are also in accordance with this AOAC program that requires the RSD r to be below 10% at the measured concentrations. For the elements Al, Ni, Cu and Zn the limits of detection and quantification were about 10 times higher than that of mercury, which has an LOD of 0.03 mg/kg. Addition of AuCl to the sample and flush solutions helped to overcome initial problems with persistent mercury residues in the 20 system because of the capacity of mercury to complex with gold (Falciani et al., 2000).

Estimation of the weekly mercury, arsenic or lead intake and selection of safety limits

At the time of completion of this study neither Dutch nor European food law contained maximum levels for mercury, arsenic or lead in food supplements. We based our enforcement actions therefore 25 on the legal requirement that only safe foods are to be placed on the market. In order to assess the safety of a THP we estimated the associated intake of mercury, lead and mercury and subsequently compared the estimated intakes of these elements to established safety limits. The estimated mercury, arsenic or lead intake per week was calculated from the analytically determined concentrations of these elements, the unit dose weight and the highest recommended daily dose stated on the label of the THP. In a few cases where no recommended dose could be identified on the label or from other sources a default value was used of, depending on the dose form, 1 pill or 1 g of powder per day. We did not consider background exposure from other sources such as foods, water or ambient air in our 5 risk assessment. For each of these 3 elements we selected an established safety limit by reviewing risk assessments by international and national bodies (Table 5). We preferentially considered safety limits for inorganic mercury, arsenic and lead compounds because available sources on the speciation of these elements in Chinese, Ayurvedic and other Asian THPs consistently indicate that mercury, arsenic and lead are present as inorganic compounds [START_REF] Aslam | Heavy metals in some Asian medicines and cosmetics[END_REF]Healy & Aslam, 1986;10 Bensky & Barolet, 1990;[START_REF] Kang-Yum | Chinese patent medicine as a potential source of mercury poisoning[END_REF]Bensky & Gamble, 1993;[START_REF] Kew | Arsenic and mercury intoxication due to Indian ethnic remedies[END_REF][START_REF] Delhi | Lead preparations in Ayurvedic medicines[END_REF]Ravinarayan & Skandhan, 1995;Ministry of Health, 2000;[START_REF] Aziz | Kushta(s): unique herbo-mineral preparation s used in South Asian traditional medicine[END_REF]Ministry of Health, 2003;Mino & Yamada, 2005;[START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF][START_REF] Sallon | Mercury in traditional Tibetan medicine -panacea or problem?[END_REF][START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF]Himalaya, 2008). From the selected safety limit we calculated a tolerable weekly intake (TWI) limit for a 60 kg adult. For mercury we selected the TDI for inorganic mercury of 2 µg/kg 15 bw/day set independently by the Dutch National Institute for Public Health and the Environment (RIVM) and the International Program on Chemical Safety (IPCS) [START_REF] Baars | Re-evaluation of human-toxicological maximum permissible risk levels[END_REF]IPCS, 2003).

This TDI is equivalent to a TWI limit of 0.84 mg per week for a 60 kg adult (Table 5). For arsenic we selected the TDI for inorganic arsenic set by RIVM of 1.0 µg/kg bw/day, equivalent to a TWI limit of 0.42 mg per week for a 60 kg adult (Table 5) [START_REF] Baars | Re-evaluation of human-toxicological maximum permissible risk levels[END_REF]. For lead we selected the provisional 20 tolerable weekly intake (PTWI) limit for lead from all sources of 25 µg/kg bw/wk established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), equivalent to a TWI limit of 1.5 mg per week for a 60 kg adult (Table 5) (JECFA, 1993;JECFA, 2000).

In agreement with European Regulation (EC) No. 333/2007 we established for enforcement purposes for mercury, arsenic or lead each, the estimated weekly intake level at which the TWI limit is 25 exceeded beyond reasonable doubt (European Commission, 2007). We derived these VWA action limits by adding to the selected TWI limit for the element, the analytically obtained expanded measurement uncertainty for the estimated weekly intake (in mg/wk). For the estimated lead intake of a THP, a VWA action limit of 1.85 mg per week was derived. The VWA action limit for the estimated weekly intake of mercury was 1.31 mg per week and for arsenic the VWA action limit was a maximum estimated weekly intake of 0.510 mg per week. Recently the European Commission established for food supplements maximum product levels of 0.10 mg/kg for mercury and 3.0 mg/kg for lead, which will enter into force on the 1 st of July 2009 (European Commission, 2008). No limit 5 has yet been proposed for arsenic in food supplements. We also applied these EU maximum levels to our data to estimate the effect of this new legislation on the availability of Asian THPs with mercury or lead on the Dutch market. Furthermore, we applied safety limits used in several pertinent studies on mercury, arsenic or lead in Asian THPs to our data in order to explore how the selection of other relevant limits would have affected the outcome of the study.

10

Results

Mercury, arsenic or lead contents of THPs

In total 186 (64%) of 292 THPs used in Ayurveda, TCM and TTM contained arsenic, mercury or lead.

Figure 1 shows for lead, mercury and arsenic separately, how the THPs are distributed over increasing 15 concentration ranges of these elements. The labeling of less than 20% of the THPs did contain indications that matched an entry of tables 1, 2 or 3, which would suggest the presence of ingredients with mercury, arsenic or lead levels. The remaining THPs were sampled according the general criteria of the defined sampling protocol to test the presence of mercury, arsenic or lead in Asian THPs prevalent on the Dutch market.

20

Lead was present in 123 (42%) THPs and levels ranged from 0.5 to a maximum of 60,000 mg/kg that was found in the Ayurvedic THP vasant yog. The average lead level in the positive samples was 1,000 mg/kg; the median level was 13 mg/kg and the 95 th percentile 573 mg/kg. Mercury was found in 131 (45%) THPs in levels from 0.2 up to 171,000 mg/kg and the highest concentration was found in the Ayurvedic THP yogendra ras. The average mercury level in the positive samples was 25 11,800 mg/kg, the median level was 50 mg/kg and the 95 th percentile was 86,000 mg/kg. In 105 (36 %) of the 292 collected THPs arsenic was present in levels ranging from 0.2 up to 89,800 mg/kg, the latter in the Ayurvedic THP swashanti yog. The average arsenic level in the positive samples was 2,300 mg/kg; the median level was 7.6 mg/kg and the 95 th percentile for arsenic of 7,600 mg/kg. In a few cases low levels of cadmium were found but the associated estimated intakes were not above international safety limits (data not shown). Table 6 shows for lead, arsenic and mercury separately the number of Ayurvedic, Chinese and Tibetan THPs in which these elements were detected above the 5 LOQ.

Estimated weekly metal or metalloid intakes in relation to the selected safety limits

In total 64 (22%) of 292 THPs were likely to result in weekly intakes of lead, arsenic or mercury separately or in combination, above the selected TWI limits when used according to recommendations. 10 Levels of these elements in 59 of these 64 THPs would result in intakes that also exceeded the VWA action limits; this signified that in total for 59 (20%) of the 292 THPs the estimated intakes of lead, arsenic or mercury would exceed the selected TWI limits beyond reasonable doubt (Table 7). In total 25 THPs from table 7 carried indications that according to table 1, 2 and 3 suggested that ingredients high in mercury, arsenic or lead, could be present. Use according recommendations of the Ayurvedic 15 THPs 'raja parvatini', 'vasant yog' and 'nature slim' would result in daily doses of lead, mercury and arsenic above all 3 VWA action limits simultaneously.

Figure 2 shows for lead, mercury and arsenic respectively the distribution of THPs over increasing ranges of weekly intake estimates in relation to the individual TWI limits for these elements. The median intake of lead with THPs positive for this metal was 0.11 mg/wk, the median 20 intake of mercury with THPs mercury containing this metal was 0.52 mg/wk and the median arsenic intake with THPs positive for this metalloid was 0.08 mg/wk. The lead intake estimates at proposed dose levels for 10 THPs exceeded the TWI limit of 1.5 mg lead per week (range 1.6 -192 mg/wk) (Figure 2) and the lead intake estimates for 8 of these also exceeded the VWA action limit of 1.85 mg/wk. Of these 10 THPs, 8 THPs would at their proposed dose levels also result in intakes above 25 TWI limits for arsenic or mercury or both as well. The estimated mercury intake at the proposed dose level of 55 THPs would be above the TWI limit of 0.84 mg inorganic mercury per week (range 0.92 -1747 mg/wk) (Figure 2) and mercury intake estimates of 50 of these were also above the VWA action limit of 1.31 mg/wk. Of 26 of these 55 THPs, the intake of arsenic or lead resulting from their proposed dose levels, would also be in excess of the TWI limits for one or both of these two elements.

We found 29 THPs where the proposed dose levels would result in arsenic intakes above the TWI limit for arsenic (range 0.44 -427 mg/wk) (Figure 2) and the proposed dose levels of 26 of these would result in intakes above the VWA action limit for arsenic of 0.510 mg/wk. Additionally, the 5 proposed dose levels of 24 of these 29 THPs would also give rise to mercury or lead intakes in excess of the TWI limits selected for these metals.

Table 8 shows for lead, mercury and arsenic separately, the number of Ayurvedic, Chinese THPs and Tibetan THPs for which use at their proposed dose levels would result in intakes above TWI limits for each of these elements.

10

In addition to the TWI limits applied above, we also compared our estimated intake data to other relevant safety limits for mercury, arsenic and lead as well as to the maximum levels for lead and mercury in food supplements recently established by the European Commission (European Commission, 2008). Table 9 shows the number of THPs where use according to recommendations would result in intakes of mercury, arsenic and lead above these other safety limits. When the newly 15 defined European maximum levels for lead and mercury were applied, the highest number of exceedances was seen: 101 (82%) of 123 of THPs positive for lead and 130 (99%) of 131 of the products containing mercury were above these limits (Table 9). of these elements at proposed use levels were all above selected safety limits. Lead was found in 13 THPs (19%), mercury and arsenic both in 6 preparations (9%). Saper et al. used a US Pharmacopoeia specification as a safety limit for lead that was considerably more conservative than the JECFA PTWI for lead (JECFA, 2000) used in our study which would explain why we found a lower percentage of 5 THPs with estimated lead intakes above the TWI limit (Tables 5, 8 and9 [START_REF] Saper | Lead, mercury, and arsenic in US-and Indian-manufactured Ayurvedic medicines sold via the Internet[END_REF]. [START_REF] Mcelvaine | Lead poisoning from the use of 15 Indian folk medicines[END_REF] studied metal and metalloid contents of 22 East Indian THPs sampled in India. Arsenic was found in 9 (41%) of the THPs, lead and mercury were each detected in 14 (64%) of the samples and high cadmium levels were shown in 2 products.

Maximum and median arsenic and mercury concentrations, considerably exceeded values found in our study but intake estimates were not provided. Garvey et al. (2001) sampled 54 Chinese and Southeast 20 Asian THPs in Vietnam, Hong Kong and the US and found in 36 (67%) THPs levels of arsenic, lead and mercury that would result in intake estimates above safety limits that were similar to limits selected for our study. In a study by [START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF] at least 160 (65%) of 247 Chinese THPs sampled on the Australian market were contaminated with mercury, lead or arsenic. Background exposure to mercury, arsenic or lead from other foods was subtracted from the TDI, thereby reducing 25 room for mercury, arsenic or lead intake from THPs, and a conservative JECFA PTWI for total mercury (JECFA, 1972) was applied. In total 3% of the Chinese THPs contained mercury, arsenic or lead levels resulting in intakes at the recommended dose that would exceed the selected TDI estimates [START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF]. In our study 5 (12%) of 42 Chinese THPs could result in estimated mercury, arsenic or lead intakes exceeding safety limits in a range of 1.1-fold to more than 2000-fold (Tables 7 and8).

In order to identify THPs possibly high in mercury, arsenic or lead beforehand, we screened the labeling for traditional names for Asian THPs or traditional names for ingredients as listed in tables 5 1-3, which can contain arsenic, lead or mercury. Of the 59 THPs in our study that would result in mercury, arsenic or lead intakes significantly above TWI limits, the labeling of 25 (42%) carried indications from table 1-3, which could suggest high levels of these elements (Table 7). Our sampling protocol, which includes tables 1-3, was therefore helpful in identifying products with significant mercury, arsenic or lead levels which carried traditional product names or ingredients on the labeling. 10 However, it proved not to be effective in identifying high-risk THPs with incomplete ingredient lists or modern product names. Our data suggests that consumers cannot rely on the labeling for avoiding THPs with high amounts of mercury, arsenic or lead and that there is a significant risk of inadvertent exposure to these elements from Asian THPs.

In order to estimate the risks associated with exposure to mercury, arsenic or lead from Asian 15 THPs appropriate safety limits have to be selected. Table 9 shows that the selection of a safety limit considerably influences the outcome of the study which should be considered when interpreting results of studies into mercury, arsenic or lead in THPs. It is pertinent that the differences in toxicity of the various species of an element are taken into account [START_REF] Who | Elemental speciation in human health risk assessment[END_REF]. Reports on the speciation of mercury, arsenic or lead in Asian THPs are scarce. Establishing the speciation of elements is less 20 straightforward for Ayurvedic THPs then for Chinese THPs because Ayurvedic THPs are subjected to more rigorous processing steps that include heat treatments. The ultimate mercury, arsenic or lead species in Ayurvedic THPs are therefore more likely to differ from the initial species in the starting materials.

Mercury from Ayurvedic THPs requires special consideration. Purified mercury (parada) is 25 used as the starting material for most mercury containing Ayurvedic THPs. Elemental mercury shows a low oral toxicity when compared to organic and inorganic mercury compounds (ICPS, 2003;Baldwin & Marshal, 1999;Neustadt & Piczenik, 2007). To test if Ayurvedic THPs contained any residual elemental mercury we performed a pilot study on 23 Ayurvedic THPs in which we had shown mercury to be present. No elemental mercury was detected in these products (data not shown), which indicates that all mercury detected relates to either inorganic or organic mercury. For the preparation of Ayurvedic THPs, mercury is mixed with sulfur until kajjali (black mercuric sulfide, HgS) is formed [START_REF] Dash | Alchemy and metallic medicines in Ayurveda[END_REF]Ministry of Health, 2000;Ministry of Health, 2003;Himalaya, 2008). Mercury reacts 5 with sulfur, which forms HgS (Fuhrmann et al., 2002;[START_REF] Baughman | Elemental Mercury Spills[END_REF]. The presence of HgS in Ayurvedic THPs was analytically confirmed for the important Ayurvedic mercury preparation Makaradhwaja [START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF], which is based on kajjali [START_REF] Dash | Alchemy and metallic medicines in Ayurveda[END_REF]. The preparation was shown to contain 85.3% mercury and 14.2% sulfur matching the stoichiometry of HgS [START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF]. Use of mercuric sulfide in an Indian ethnic remedy in a daily dose of 180-252 mg during 4 10 weeks resulted in adverse effects consistent with a mercury poisoning [START_REF] Kew | Arsenic and mercury intoxication due to Indian ethnic remedies[END_REF]. Inorganic mercury poisonings from oral use of Chinese THPs with calomel (mercurous chloride, Hg 2 Cl 2 ) (Kang-Yum & Oransky, 1992) and cinnabar (used long-term) have been recorded (Liu et al., 2008a). Studies on rats and guinea pigs have shown that mercuric sulfide and its natural form cinnabar are bioavailable and neurotoxic (Chuu et al., 2001;Young et al., 2002;Chuu et al., 2007). Because methylmercury is 15 primarily formed through methylation by microorganisms [START_REF] Satoh | Occupational and environmental toxicology of mercury and its compounds[END_REF]ICPS, 2003) and because methylmercury is not used in traditional medicines (Liu et al., 2008a) we considered the presence of this compound in toxicological relevant quantities to be unlikely. The JECFA PTWI for total mercury used by [START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF] is mainly applicable to diets with high consumption of fish with high levels in methylmercury (JECFA, 1989b) and does not necessarily apply to foods high in inorganic 20 mercury (JECFA, 1972). Therefore a TDI for inorganic mercury [START_REF] Baars | Re-evaluation of human-toxicological maximum permissible risk levels[END_REF]IPCS, 2003) was selected for the risk assessment of mercury from Asian THPs. Although this TDI is based on a NOAEL for inorganic mercuric chloride it also applies to other inorganic mercury compounds such as mercuric sulfide [START_REF] Baars | Re-evaluation of human-toxicological maximum permissible risk levels[END_REF]IPCS, 2003).

Arsenic is used in Chinese THPs in inorganic form (Bensky & Barolet, 1990;Bensky & 25 Gamble, 1993;Mino & Yamada, 2005;[START_REF] Cooper | Public health risks from heavy metals and metalloids present in traditional Chinese medicines[END_REF]. In the Ayurvedic Formulary 7% of the formulas contain realgar (manahsila, As 4 S 4 ) and orpiment (talaka, As 2 S 3 )(Ministry of Health, 2000;Ministry of Health, 2003). From talaka a bhasma is made. Talaka bhasma was reported to contain As 2 S 3 [START_REF] Kumar | Bhasmas: unique ayurvedic metallic-herbal preparations, chemical characterization[END_REF]. Furthermore arsenic trioxide (As 2 O 3 ) was found in two kushtas from India and Pakistan [START_REF] Aslam | Heavy metals in some Asian medicines and cosmetics[END_REF]. Arsenic trioxide is significantly more toxic than orpiment and realgar (Liu et al., 2008). Inorganic arsenic compounds are more toxic than several organic arsenic compounds, notably those compounds (eg. arsenobetaine) which can be found in seafood in considerable quantities and which are not of major toxicological concern (IPCS, 2001). A case of 5 arsenic poisoning involving Indian preparations has been reported in Italy (Muzi et al., 2001) and in the UK where use of Indian preparations resulting in intakes of up to 210 mg As 2 O 3 per day for more than 4 months, caused symptoms consistent with inorganic arsenic poisoning [START_REF] Kew | Arsenic and mercury intoxication due to Indian ethnic remedies[END_REF]). An Indian case of arsenic poisoning showed keratosis after 6 months of use of several Ayurvedic preparations with significant arsenic levels and after 18 months of use non-cirrhotic portal 10 hypertension was diagnosed [START_REF] Khandpur | Chronic arsenic toxicity from Ayurvedic medicines[END_REF]. Several chronic arsenic poisonings with Chinese THPs have been reported which resulted in cutaneous manifestations such as malignancies and kerotic plaques (Chan, 1994;[START_REF] Wong | Cutaneous manifestations of chronic arsenicism: review of seventeen cases[END_REF][START_REF] Lee | Hemolytic anemia as a sequela of arsenic intoxication following long-term ingestion of traditional Chinese medicine[END_REF]Hanjani et al., 2007). We selected a TDI for inorganic arsenic set by RIVM for the risk assessment of arsenic from Asian THPs. The TDI is based on the JECFA PTWI for inorganic arsenic (JECFA, 1989a) to which an extra uncertainty factor of 2 15 was applied for observation errors in epidemiological studies [START_REF] Baars | Re-evaluation of human-toxicological maximum permissible risk levels[END_REF].

Lead in Ayurvedic and Chinese THPs is mostly found in inorganic form. Reported forms are PbS and PbO, which can cause intoxications (Ravinarayan & Skandhan, 1995;[START_REF] Delhi | Lead preparations in Ayurvedic medicines[END_REF]ICPS, 1995;Healy & Aslam, 1986). A large number of lead poisonings with Ayurvedic THPs has been reported [START_REF] Dunbabin | Lead poisoning from Indian herbal 10 medicine (Ayurveda)[END_REF][START_REF] Kew | Arsenic and mercury intoxication due to Indian ethnic remedies[END_REF][START_REF] Sheerin | Simultaneous exposure to lead, arsenic and mercury from Indian ethnic remedies[END_REF][START_REF] Keen | Indian herbal remedies for diabetes 10 as a cause of lead poisoning[END_REF]Bayly et al., 20 1995;Prpic-Maijc et al., 1996;[START_REF] Van Vonderen | Severe gastrointestinal symptoms due to lead poisoning from Indian traditional medicine[END_REF]Anderson et al., 2001;[START_REF] Tait | Severe congenital lead poisoning in a preterm infant due to a herbal remedy[END_REF]CDC, 2004;[START_REF] Schilling | Bleiintoxikation durch Einnahme ayurvedische 15 Arzneimittel[END_REF][START_REF] Kanen | Chronic lead intoxication associated with Ayurvedic medication Ned[END_REF][START_REF] Van Schalkwyk | Ayurvedic medicine: patients in peril from 5 plumbism[END_REF]. These reports contradict the claim that lead in Ayurvedic THPs is detoxified. A case of lead poisoning with a Chinese THP has been recorded in Hong Kong [START_REF] Auyeung | Three patients with lead poisoning 15 following use of a Chinese herbal pill[END_REF]. The US Environmental Protection Agency (US EPA) did not establish an oral reference dose (RfD) for lead nor was the 25 Agency for Toxic Substances and Disease Registry (ATSDR) able to derive a minimal risk level (MRL) for lead, because both agencies could not define a clear threshold for some of the health effects linked to exposure to lead (US EPA, 2004;ATSDR, 2007). RIVM set a TDI for lead and lead compounds which is directly derived from the JECFA PTWI for lead from all sources [START_REF] Baars | Re-evaluation of human-toxicological maximum permissible risk levels[END_REF]. We selected therefore the PTWI set by JECFA (JECFA, 2000) for the risk assessment of lead from Asian THPs.

Our study shows that the mercury, arsenic and lead contents of Asian THPs are still a cause for concern and that Asian THPs should routinely be tested for metals and metalloids. The VWA will , 1993;Kang-Yum & Oransky, 1999;Ministry of Health, 2000;MCA, 2001;Ernst, 2002b;Ministry of Health, 2003;Liu et al, 2008b) Page * Calculated either by comparing the estimated intake of a element resulting from the recommended use of the THP with the safety limit referred to in the next column calculated for a 60 kg adult or by comparing a maximum product level to the element concentration.

25 comparing

 25 the estimated metal and metalloid intake resulting from use at the proposed dose levels to established toxicological safety limits. The current report presents the results of this study.

Discussion 20 Mercury

 20 , lead and arsenic were detected in 186 (64%) of 292 THPs used in Ayurveda, TCM and TTM sampled in the Netherlands. Use at the proposed dose levels of 59 (20%) of these 292 THPs would result in estimated mercury, arsenic and lead intakes significantly above established safety limits.Furthermore, 82% of the THPs with lead and 99% of the THPs containing mercury exceeded the recently established European maximum product levels for food supplements (Table9)(European 25Commission, 2008). Our results are in good agreement with several studies on mercury, arsenic and lead contents of Asian THPs sampled in Western and Asian countries.[START_REF] Saper | Heavy metal 5 content of ayurvedic herbal medicine products[END_REF] found or lead in 20% of 70 Ayurvedic THPs collected in Boston and the estimated intakes

  ). Relative to the results obtained bySaper et al., the subset of Ayurvedic THPs in our study included more THPs (46 (19%) of 242) with estimated mercury intakes above the TWI, which also exceeded the number of THPs with estimated arsenic or lead intakes above the TWI (22 (9%) and 10 (4%), respectively)(Table8). A similar distribution of mercury, arsenic and lead among Ayurvedic THPs is found in the official 10 Ayurvedic formulary, which includes more formulas with mercury (23%) than arsenic (7,3%) or lead (2,5%)(Ministry of Health, 2000; Ministry of Health, 2003). In 2008 Saper et al. published a study on mercury, arsenic and lead in Ayurvedic THPs purchased via the Internet in 2005. Lead, mercury and arsenic were found in 40 (20.7%) of 193 Ayurvedic THPs manufactured in the US and India. The intake estimates for mercury, arsenic and lead for all THPs that contained these elements, exceeded 15 selected standards

  fold to up to 2750-fold

5

  continue to monitor metals and metalloids in Asian THPs on the Dutch market and enforce the safety of these products.

  Bayly GR, Braithwaite RA, Sheehan TM, Dyer NH, Grimley C, Ferner RE. 1995. Lead poisoning from Asian traditional remedies in the West Midlands--report of a series of five cases. Hum ExpToxicol. 14,24-28 Bensky D and Barolet R.1990. Chinese Herbal Medicine: Formulas and Strategies. Eastland Press, 5 Incorporated. Seattle. Bensky D and Gamble A. 1993. Chinese Herbal Medicine: Materia Medica. Revised edition. Eastland Press, Incorporated, Seattle. 10 Blok-Tip L, Zomer B, Bakker F, Hartog KD, Hamzink M, ten Hove J, Vredenbregt M, de Kaste D. 2004. Structure elucidation of sildenafil analogues in herbal products. Food Addit Contam. 21:737-748 CDC. 2004. Lead poisoning associated with ayurvedic medications--five states, 2000-2003. MMWR Morb Mortal Wkly Rep. 53:582-584 15 Chan TY. 1994. The prevalence use and harmful potential of some Chinese herbal medicines in babies and children. Vet Hum Toxicol. 36:238-240 Chuu JJ, Liu SH, Lin-Shiau SY. 2001. Effects of methyl mercury, mercuric sulfide and cinnabar on 20 active avoidance responses, Na+/K+-ATPase activities and tissue mercury contents in rats. Proc Natl Sci Counc Repub China B. 25:128-136. Chuu JJ, Liu SH, Lin-Shiau SY. 2007. Differential neurotoxic effects of methylmercury and mercuric sulfide in rats. Toxicol Lett. 169:109-120. Zhuang ZX, Tai Y, Huang RF, Wang XR, Lee FSC. 2006. Direct analysis of mercury in Traditional Chinese Medicines using thermolysis coupled with on-line atomic absorption spectrometry. Talanta. 68:728-734. IPCS. 1995. Inorganic lead; Environmental Health Criteria 165. Geneva: World Health Organization. 5 IPCS. 2001. Arsenic and arsenic compounds (2nd edition); Environmental Health Criteria 224. Geneva: World Health Organization. IPCS. 2003. Elemental mercury and inorganic mercury compounds: human health aspects; Concise 10 International Chemical Assessment Document 50. Geneva: World Health Organization. JECFA. 1972. Evaluation of mercury, lead, cadmium and the food additives amaranth, diethylpyrocarbonate, and octyl gallate; WHO Food Additives Series, No. 4. Geneva World Health Organization. Mercury. 15 JECFA. 1989a. In: Evaluation of certain food additives and contaminants; WHO Technical Report Series, No. 776, Geneva: World Health Organization. Arsenic; p. 27-28. JECFA. 1989b. Evaluation of certain food additives and contaminants; WHO Technical Report Series, 20 No. 776. Geneva: World Health Organization. Methylmercury; p. 33-34. JECFA. 1993. Evaluation of certain food additives and contaminants; WHO Technical Report Series,No. 837. Geneva: World Health Organization. Lead;

25 JECFA

 25 . 2000. Evaluation of certain food additives and contaminants;WHO Technical Report Series, No. 896. Geneva: World Health Organization. Lead;

  Ministry of Health and Family Welfare, Department of Indian Systems of Medicine and Homoeopathy, Government of India. 2003. The Ayurvedic Formulary of India, Part I. Second revised English edition. Delhi: Controller of Publications. 5 Mino Y, Yamada Y. 2005. Detection of high levels of arsenic and mercury in some Chinese Traditional Medicines using x-ray fluorescence spectrometry. J Health Sci. 51:607-613. Moore C, Adler R. 2000. Herbal vitamins: lead toxicity and developmental delay. Pediatrics. 106:600-602. 10 Muzi G, Dell'omo M, Madeo G, Abbritti G, Caroli S. 2001. Arsenic poisoning caused by Indian ethnic remedies. J Pediatr. 139:169. Patwardhan B, Warude D, Pushpangadan P, Bhatt N. 2005. Ayurveda and traditional Chinese 15 medicine: a comparative overview. Evid. Based Complement. Alternat Med. 2:465-473. Prpic-Majic D, Pizent A, Jurasovic J, Pongracic J, Restek-Samarzija N. 1996. Lead poisoning associated with the use of Ayurvedic metal-mineral tonics. J Toxicol Clin Toxicol. 34:417-23. 20 Ravinarayan A, Skandhan KP. 1995. Lead preparations in Ayurvedic medicines. Postgrad Med. J. 71: 251. Rietjens IMCM, Martena MJ, Boersma MG, Spiegelenberg W, Alink GM. 2005. Molecular mechanisms of toxicity of important food-borne phytotoxins. Mol Nutr Food Res. 49:131-158.

Figure 1 .

 1 Figure legends

  al.,

	1993;
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

  .,

	2002;
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	6

Table 1 .

 1 Traditional names of lead, mercury and arsenic preparations used as ingredients of Asian

				Deleted: Levels of heavy metals in
				selected traditional Asian herbal
				preparations on the Dutch market and
				estimation of associated risks.
				Deleted: heavy metal
	THPs.
				Deleted: Heavy
	Element Pharmaceutical or traditional names of	Synonyms and related preparations or compounds	Deleted: metal
		preparation		Deleted: metal containing ingredient
	Lead	lithargyrum (lead monoxide)	A: girisindura, litharge, mrddara srnga,
			mrddarasrnga, muddarasankha, sindura
			C: mi tuo seng
		A: naga bhasma	A: ahi, bhujagalauha, sisaka,
	Mercury A: parada (purified mercury)	A: chapala, haraja; isa, mrtasuta, rasa, rasa
			sindura, rasaraja rasendra, rasesa, rasesvara,
			rasottama, suddhasuta, suta, sutaka,
		cinnabar (red mercury sulfide)	A: aruna, caliya, hingula, hingulotthaparada,
			hingula sambhava suta, suddha hingula
			C: zhu sha, chen sha, dan sha
		calomel (mercurous chloride)	C: qing fen, shui yin fen, gong fen, chen fen
		hydrargyri oxydum rubrum (red
		mercury oxide)
	Arsenic orpiment (arsenic trisulfide)	A : ala, haratala, haritala, tala, talaka
			C : cihuang
		realgar (arsenic sulfide or arsenic	A: manahsila, manayola, sila,
		disulfide)	C: xiong huang, xiong jing, yao huang
		arsenolite (white arsenic or arsenic	A: gauripasana, malla, svetamalla
		trioxide)	C: pishi
	A: traditional name used in Ayurveda

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	F o r P e e r R e v i e w	O n l y
		3

Table 2 .

 2 Formulas for Ayurvedic THPs for oral use possibly containing mercury, arsenic or lead.

	Ayurvedic THP	Synonyms	Ayurvedic THP	Synonyms	Ayurvedic THP	Synonyms
	agnitundivati	angitundi vati	caturbhuja rasa	chaturbhuj rasa	kasturyadi vayu gutika
	ajirna kantaka rasa		caturmukha rasa	chaturmukh ras	kravyada rasa
	amavatari rasa	amavatari ras; amvatari ras	cintamanicaturmukha rasa		krmi kuthara rasa	krimi kuthar rasa
	amlapittantaka rasa	amlapittantka rasa	ekangavira rasa	kangveer ras; ekangvir ras	krmimudgara rasa	krimimudgar ras
	anandabhairava rasa	anand bhairav ras;	gaganasundara rasa		kumarakalyana rasa	kumarkalyan rasa
		anandbhairav ras	gandamala kandana rasa		laghvananda rasa
	arogyavardhini gutika		gandhaka vati	gandhak bati (raj bati)	lakanatha rasa
	arsakuthara rasa astaksari gutika asvakancuki rasa balarka rasa bola parpati brhadagnikumara rasa brhanmaricadya taila brhat candrodaya	arsh kuthar ras ;arsha kuthar rasa ashwakanchuki ras balark rasa bol parpati	garbha cintamani rasa garbhapala rasa gorocanadi vati grahani kapata rasa guducyadi modaka gulma kalanala rasa haritala bhasma hemagarbha pottali rasa	garbhapal ras gorochandi vati grahani kapat ras gulma kalanal ras harital bhasma	laksminarayana rasa laksmivilasa rasa lauha parpati lila vilasa rasa mahagandhaka vati maha jvarankusa rasa maha laksmivilasa rasa	laxminarayan rasa laxmivilas ras; laxmi vilas rasa lilavilas rasa mahajwarankush ras; mahajvarankush rasa mahalaxmi vilas rasa;
	makaradhvaja		hemanatha rasa	hemnath ras		mahalaxmivilas ras;
	brhat garbhacintamani rasa brihat garbhachintamani rasa;	hingulesvara rasa			mahalakshmi vilas ras
	garbha chintamani ras vrihat brihat kasturi bhairav rasa brihat purnachandra rasa brhat sarvajvarahara lauha sarvajwarhar loha br. brhat kasturibhairava rasa brhat nrpavallabha rasa brhat purnacandra rasa brhat vangesvara rasa bangeshwar ras brihat brhat vatacintamani rasa brihat vata chintamani; brihat vatchintamani rasa brhat vidyadharabhra rasa brhat visamajvarantaka lauha brhatcchrngarabhra rasa candrakala rasa chandrakala ras (Tamra Yukta) candramrta rasa chandramrit ras candramsu rasa	hiranya garbha pottali rasa hrdanyarnava rasa icchabhedi rasa jalodarari rasa jvaraghni gutika jvarankusa rasa (ka) jvararyabhra kalakuta rasa kanakasundara rasa kancanabhra rasa kantavallabha rasa karpura rasa kasisadi ghrta kasturi bhairava rasa	hridayarnava ras ichhabhedi ras (jaypal yukta) khantavallabha rasa karpur ras kastoori bhairav ras	mahatarunarka rasa maha vata vidhvamsana rasa mahavatvidhvasan ras; mahavatvidhvansan rasa; mahavat vidhwans ras maha vatagajankus a rasa maha yogaraja guggulu maha yograj guggul makara dhvaja makardhwaj; makardhvaj rasa mallasindura mallasindur manikya rasa manmathabhra rasa maricadya taila mrtyunjaya rasa mrityunjai ras muktapancamrta rasa mukta panchamrit ras; moti

Page 32 of 43 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 5Table 3 .

 3 Formulas for Chinese THPs for oral use possibly containing mercury, arsenic or lead.

			Food Additives and Contaminants
	Product Name	Toxic substance	Product Name	Toxic substance	Product Name	Toxic substance
		F o r P F o r P e e r R e v i e w e e r R e	O n l y
			v i e
			w
			O n l
				y
				7

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Table 4 .

 4 Characteristics (limit of detection (LOD), limit of quantification (LOQ), range of application, accuracy and expanded measurement uncertainty) of the analytical method for Pb, As and Hg determined by in house validation.

								Deleted: Metal
	Element	LOD	LOQ	Range of	Accuracy 1	RSD r	2	Expanded Measurement
		(mg/kg)	(mg/kg)	application	(%)	(%)		Uncertainty 3 (mg/kg)
				(mg/kg)
	Pb	0.18	0.36	0.25 -12.5	103	5.1		3.4 (at level of 37.3 mg/kg)
	Hg	0.03	0.06	0.25 -12.5	99	9.5		0.047 (at level of 0.549
								mg/kg)
	As	0.06	0.13	0.25 -12.5	96	2.1		0.09 (at level of 0.89 mg/kg)

1 : Accuracy has to be between

75 and 120% (AOAC, 2002)

2 : Relative Standard Deviation (RSDr) may not exceed 10%

(AOAC, 2002)

3 : The expanded measurement uncertainty (mg/kg) was calculated as the reproducibility at the concentration level of the CRM.

Table 5 .

 5 Safety limits for lead, mercury and arsenic.

	Element	Safety limit	Safety limit set by	Safety limit set for	Tolerable weekly
					intake (mg/wk)*
	Lead	4.5 µg/d	US Pharmacopeia-	lead from a daily dose	0.0315
			specification 1	of calcium carbonate
		25 µg//kg bw/wk JECFA-PTWI 2	lead from all food	1.5
				sources
	Mercury	0.3 µg/kg bw/d	US EPA-RfD 3	mercuric chloride	0.126
		5 µg/kg bw/wk	JECFA-PTWI 4	total mercury from diets	0.3
				high in methylmercury
		2 µg/kg bw/d	IPCS-TDI 5 /RIVM-	inorganic mercury	0.84
			TDI 6
	Arsenic	0.3 µg/kg bw/ d	US EPA-RfD 3	inorganic arsenic	0.126
		1.0 µg/kg bw/d	RIVM-TDI 6	inorganic arsenic	0.42
		15 µg/kg bw/wk	JECFA-PTWI 7	inorganic arsenic	0.90
		3 µg/kg bw/d	ANZFA-TDI 8	inorganic arsenic	1.26

Table 6 .

 6 Number of Ayurvedic, Chinese and Tibetan THPs with lead, mercury or arsenic above the limit of quantification.

					Deleted: heavy metals
		# of Ayurvedic THPs # of Chinese THPs	# of Tibetan THPs
	Total of the THP type sampled	242	42	8
	Lead above LOQ*	105	13	5
	Mercury above LOQ	117	8	6
	Arsenic above LOQ	85	15	5
	Total of THPs with at least one element above LOQ	161	19	7	Deleted: metal

*LOQ: Limit of quantification.

Table 7 .

 7 Asian THPs resulting in estimated lead, mercury or arsenic intakes significantly above one or more TWI limits. : Estimated element intake values in bold are above the VWA action limit for that element at the proposed use level; values in italics are between the TWI limit and the VWA action limit.

					Deleted: heavy metal
	Asian THP	Type of	Arsenic	Mercury	Lead
		Asian THP*		mg/wk
	Abhrak Bashma	A	0.82	n.d.	1.3
	Abhrak Bhasma	A	1.0	n.d.	1.6
	Agni Vati	A	n.d.	72	n.d.
	Amrit Prash	A	0.1	5.1	0,04
	Amrit Ras	A	1.8	377	0.27
	Arjuna Hepatone	A	n.d.	198	1,0
	Arogya Vardhini Vati	A	n.d.	338	n.d.
	Arogyavardhini Vishisht	A	0.04	1.9	0.21
	Ayfer	A	0.05	0.12	4.0
	Ayu-Hirakam	A	0.21	2.6	0.14
	Bat Chintamani	A	2.2	471	0.26
	Bo Zi Yang Xin Wan	C	n.d.	1473	n.d.
	Chandraprabha	A	0.07	n.d.	15
	Diabetomed	A	1.4	3.5	0.41
	Ding Xin Wan	C	0.44	430	1.3
	Ere Forte	A	n.d.	12	0.34
	Femi-Smilin	A	7.5	0.73	0.33
	Gandhadi Yog	A	n.d.	11	n.d.
	Ghandramsu Ras	A	n.d.	132	0.14
	Guggulu	A	0.38	7.9	3.1
	Gurgum-8	T	2.9	317	0.04
	Guyu Depak	T	0.47	192	0.1
	Guyu Depak	T	0.94	239	0.15
	Heartina	A	262	340	n.d.
	Herba Figura	A	0.07	2.3	0.07
	Herbo Gastrol	A	n.d.	14	0.33
	Herbogastrol	A	n.d.	12	0.23
	Herbogastrol	A	n.d.	27	n.d.
	Hridaya Chintamani	A	2.6	396	n.d.
	Jeevani Vati	A	0.22	173	n.d.
	Kamachudamani Ras	A	7.7	22	0.27
	Liv.52	A	n.d.	n.d.	2.6
	Luxmi Vilas Classic	A	0.02	138	0.05
	Maha Manjishtadi Ghan Vati	A	0.04	4.2	0.09

Noten.d.: not detected. * THP types sampled: 'A' for Ayurvedic THPs, 'C' for Chinese THPs, 'T' for Tibetan THPs ** The daily dose assumed to be 1 pill (0.195 g)

Table 8 .

 8 Number of Ayurvedic, Chinese and Tibetan THPs where use at the proposed dose levels results in lead, mercury or arsenic intakes above TWI limits.# of Ayurvedic THPs # of Chinese THPs # of Tibetan THPs

					Deleted: heavy metal
	Total of THP in study	242	42	8
	Lead intake above TWI	10	n.a.	n.a.
	Mercury intake above TWI	46	5	4
	Arsenic intake above TWI	22	3	4
	Total of THPs with at least one element above TWI	50	5	4	Deleted: metal
	n.a.: not applicable.				Deleted: N

Table 9 .

 9 Lead, mercury and arsenic intakes with THPs compared to various safety limits for these elements and EU maximum product levels for lead and mercury.

	Element	# of THPs	# of THPs with	Risk assessment or risk	Limit recently
		positive for	estimated element	management body	applied for Asian
		element	intakes or element	responsible for limit and	THPs in:
			level above limit*	type of limit
	Lead	123	10	JECFA-PTWI 1	this study; Cooper et
					al., 2007
			99	US Pharmacopeia-	Saper et al., 2004
				specification 2
			101	EU-maximum level 3	this study
	Mercury	131	55	RIVM-TDI 4 / IPCS-TDI 5 this study
			68	JECFA-PTWI 6	Cooper et al., 2007
			91	US EPA-RfD 7	Saper et al., 2004
			130	EU-maximum level 3	this study
	Arsenic	105	29	RIVM-TDI 4	this study
			17	ANZFA-TDI 8	Cooper et al., 2007
			21	JECFA-PTWI 9	Mino and Yamada,
					2005
			39	US EPA-RfD 7	Saper et al., 2004

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Acknowledgements 10

The authors would like to thank the VWA inspectors specialized in health foods for the proficient sample collection and enforcement actions, Eddy van de Male and Elly Tissen for the analytical work and Edith de Haan and her group from TNO Zeist for the rapid determination of elemental mercury in selected Ayurvedic THPs.

European Commission. 2008. Commission Regulation (EC) No 629/2008 of 2 July 2008 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union. L 173/6. Falciani R, Novaro E, Marchesini M, Gucciardi M. 2000. Multi-element analysis of soil and sediment 5 by ICP-MS after a microwave assisted digestion method. J Anal At Spectrom. 15:561-565. FSANZ: The 20 th Australian total dietary survey [Internet]. 2002. Canberra: Food Standards Australia and New Zealand; [cited 2008 September 28] Available from: www.foodstandards.gov.au/newsroom/publications/20thaustraliantotaldietsurveyjanuary2003/ 10 Fuhrmann M, Melamed D, Kalb PD, Adams JW, Milian LW. 2002. Sulfur Polymer Solidification/Stabilization of elemental mercury waste. Waste Management. 22:327-333. Garvey GJ, Hahn G, Lee RV, Harbison RD. 2001. Heavy metal hazards of Asian traditional remedies. 15 Int J Environ Health Res. 11:63-71. Hanjani NM, Fender AB, Mercurio MG. 2007. Chronic arsenicism from Chinese herbal medicine. Cutis. 80:305-308. 20 Healy MA and Aslam M. 1986. Lead-containing preparations in the Asian community: a retrospective survey. Public Health. 100:149-151. Himalaya Herbal Healthcare: 'Shingraf, Hingula', in: Himalaya's Herbs and Minerals [Internet]. 2008. India: The Himalaya Drug Company; [cited 2009 January 18]. Available from: 25 http://www.himalayahealthcare.com/aboutayurveda/cahs.htm#shingraf Page 20 of 43 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants


Sumita Dixit

Shakendra K Purshottam

Subhash K Khanna

Mukul Das

Surveillance of quality of turmeric powders from city markets of India on the basis of curcumin content and presence of extraneous colours

Keywords: 2D-HPTLC, Turmeric powder, curcumin content, artificial color-metanil yellow, surveillance

Surveillance on quality of turmeric powders vis a

Introduction

Turmeric a bright yellow spice obtained from the rhizomes of Curcuma longa is an important medicinal plant, whose products are marketed as turmeric powder, turmeric oleoresin, and turmeric oil. India is the largest producer and consumer of turmeric, with a production of 721,539 tonnes in the year [START_REF] Tayyem | Curcumin content of turmeric and curry powders[END_REF]-07 (Spice Board India, 2008a). During the same period, the country exported 51,500 tonnes of turmeric, valued at Rs 16480 million to over 20 countries including Iran, Japan, Singapore, South Africa, Sri Lanka, UAE, UK and USA (Spice Board India, 2008 b;[START_REF] Mathai | Variability in turmeric (Curcuma species) germplasm for essential oil and curcumin[END_REF].

Curcumin, the principal curcuminoid of turmeric is responsible for its yellow color and serves as a measure of turmeric quality. Though dried or fresh rhizome of turmeric are usually free from adulteration, turmeric powder can be admixed with other curcuma species or their curcumin/volatile oil extracted matrices; foreign starches and artificial colors which serve as a cheaper substitute. The markets have both branded (packed) products by organized units and non-branded (loose) turmeric powders processed at retailers and small millers' level, meeting the requirements of populations of different economic strata. The rules specified in the 2008 Prevention of Food Adulteration Act (PFA) of India prescribe quality standards for turmeric powder and restrict that it should be free from extraneously added foreign matters and artificial colors (PFA, 2008). Mixing of other Curcuma species [START_REF] Sasikumar | PCR Based Detection of Adulteration in the market samples of Turmeric Powder[END_REF], extraneous starches (Govindrajan, 1980;[START_REF] Remya | Isolation and amplification of DNA from turmeric powder[END_REF]) and/ or artificial colors have been occasionally reported [START_REF] Khanna | Non-permitted colors in food and their toxicity[END_REF]1975;1987;[START_REF] Bis | Food quality surveillance on colors in eatables sold in rural markets of Uttar Pradesh[END_REF] and the admixed turmeric powders may show low curcumin content depending upon the extent of substitution [START_REF] Balasubrahmanyam | Packaging and storage studies on ground turmeric (C.longa L.) in flexible consumer packages[END_REF].

In general, major Indian regional trade types of turmeric show curcumin content of 2.2-6.0 % wherein the varieties of Kerala show higher curcumin than those of Andhra Pradesh [START_REF] Krishnamurthy | Color content of turmeric varieties and studies on its processing[END_REF][START_REF] Rao | Promising turmeric types of Andhra Pradesh[END_REF][START_REF] Mathai | Variability in turmeric (Curcuma species) germplasm for essential oil and curcumin[END_REF]. A field level study by in India, a vast majority had curcumin contents ranging from 2.15-8.6%, with an average of 4.79% [START_REF] Velayudhan | Curcuma Genetic Resources, National Bureau of Plant Genetic Resources[END_REF]. The Bureau of Indian Standards [START_REF] Bis | Food quality surveillance on colors in eatables sold in rural markets of Uttar Pradesh[END_REF], the apex standards setting body in India, suggests a minimum of 3% curcumin for powdered turmeric, whereas the mandatory PFA (2008) rules do not specify any minimum curcumin limit. In the export markets also, turmeric is valued based on its curcumin content but specifications of many importing countries including Germany, Spain, Netherlands, UK, and USA do not insist on any minimum curcumin requirement [START_REF] Esa | European Spice Association Quality Minima Document Rev. 1[END_REF]. Similarly, American Spice Trade Association (ASTA) Cleanliness Specifications for unprocessed Spices, Seeds and Herbs also have no mention of curcumin limits (ASTA, 2007).

Although methods to quantify the curcumin content in powdered turmeric are available, there has been no method to quantify the presence of extraneously added color (metanil yellow) and simultaneously measure the curcumin content in turmeric powders.

Recently, a 2D-HPTLC method was developed for identification and quantitation of extraneous colors along with curcumin content and offers quick screening of large numbers of turmeric powder samples [START_REF] Dixit | A simple 2-Directional High-Performance Thin-Layer Chromatographic method for the simultaneous determination of curcumin, metanil yellow, and Sudan Dyes in Turmeric, Chili, and Curry Powders[END_REF]. A few studies on limited number of samples for curcumin analysis in turmeric powders are reported [START_REF] Verghese | Curcuminoids, the magic dye of C. longa L. rhizome[END_REF][START_REF] Tayyem | Curcumin content of turmeric and curry powders[END_REF] but no organized survey on market samples has been attempted. Therefore, the present surveillance has been undertaken to study the quality of turmeric powders vis a vis curcumin content and presence of unwarranted extraneous colors from city markets of India, using the recent method of [START_REF] Dixit | A simple 2-Directional High-Performance Thin-Layer Chromatographic method for the simultaneous determination of curcumin, metanil yellow, and Sudan Dyes in Turmeric, Chili, and Curry Powders[END_REF]. Simple random selection technique has been followed for sample design. The country has been tentatively divided into four zones namely, North, South, East and West. Each zone included four states and from each state, four cities were selected. Two prominent market locations were chosen from each city and five loose, non-branded turmeric powder samples were randomly picked up from each market. The single product sampling design thus permitted collection of 10 samples from two market locations from each city; 40 samples from each state; 160 samples from each of the 4 state zones and 640 samples from the country, covering 16 major states. However, non-availability of loose turmeric powders in the south zone led to analysis of 612 samples only. One hundred branded packed turmeric powders comprising 10 samples each of the 10 reputed national brands available throughout the country, were collected and analyzed for comparison.

Materials and Methods

Sampling Design

Reagents

Curcumin (technical) was purchased from Aldrich Chemical Company, Milwaukee, WI, USA, was used because of the non-availability of Certified Reference Material.

Precoated HPTLC aluminium silica gel 60F 254 sheets for nano-TLC (Art 5548) were purchased from E. Merck, Darmstadt, Germany. Metanil Yellow (CI number 13065; CI name Acid Yellow 36) was the product from Lobachemie, Indoaustranal Co. Mumbai, India.

Chloroform and methanol were of GR grade and purchased from Merck Ltd, Mumbai, India.

Other chemicals used were of highest purity available commercially.

HPTLC instrumentation

The HPTLC system used was a product of M/s DESAGA GmbH, Weisloch, Germany and comprised of an applicator (AS-30), chromatographic chamber (DC-MAT) and densitometer (CD 60) with ProQuant software version 1.00.005.

Processing of samples

Turmeric powders were analyzed employing the method of [START_REF] Dixit | A simple 2-Directional High-Performance Thin-Layer Chromatographic method for the simultaneous determination of curcumin, metanil yellow, and Sudan Dyes in Turmeric, Chili, and Curry Powders[END_REF] which offers simultaneous measurement of curcumin and metanil yellow content. Turmeric sample (100 mg) was extracted four times using 95% ethanol, the extracts were pooled and made up to 20 ml. Standard curcumin and metanil yellow were treated similarly. Five µl of aliquots were applied onto pre-coated HPTLC plates and developed using chloroform: methanol (90:10, v/v) as solvent system. The resolved spots were read at 420 nm using CD 60 Desaga GmbH (Wiesloch, Germany) densitometer.

Quality Assurance

The identification of curcumin was based on its retention factor and spectral characteristics and matched with the composition of Indian turmeric [START_REF] Dixit | A simple 2-Directional High-Performance Thin-Layer Chromatographic method for the simultaneous determination of curcumin, metanil yellow, and Sudan Dyes in Turmeric, Chili, and Curry Powders[END_REF]. The quality assurance data showed a linear response for metanil yellow in the range of 100-500 ng and that of curcumin between 50-300 ng per spot. The LOD was 17. 4 and 42.9 ng/spot for curcumin and metanil yellow respectively, while the respective LOQ was 53 and 130 ng/spot.

Percentage recovery of metanil yellow varied in between 97 -103 with an RSD of 2.4-3.8% while the recovery of curcumin ranged from 87-99 %, with an RSD of 1.3-3.3 %.

Results

The curcumin content of the branded and non-branded turmeric powders has been shown in Table 1. Among 100 analyzed branded samples, the curcumin content ranged from 2.2-3.7%, with only 18% showing ≥ 3% curcumin. In the non-branded loose turmeric powders, none of the sample had ≥ 3% curcumin content which ranged between as low as 0.3-2.6%. None of the branded samples showed the presence of artificial color. In non- branded samples, turmeric powders dyed with artificially added color metanil yellow, showed a lower mean curcumin content of 0.9 % as against 1.8% for non-colored turmeric powders (Table 1).

The pattern of artificially colored turmeric samples, in the four state zones is shown in Table 2. Among 612 analyzed samples, 17.2 % were found to show the presence of artificial color -metanil yellow. None of the turmeric powders picked up from the south zone showed artificial color admixture. In the remaining 3 zones, the presence of color was noted in 12.6 -27.7 % samples and the proportion of color usage ranged from 1.0 to 8.5 mg g -1 (Table 2).

The results of individual states in terms of percentage of artificially colored turmeric samples indicated that no artificial coloration was seen in the states of Assam, Andhra Pradesh, Himachal Pradesh, Karnataka, Kerala and Tamilnadu. The maximum number of metanil yellow dyed samples was noted in the state of Maharashtra (59.5%) followed by Haryana (30.6%). In eight other states namely, Bihar, Gujarat, Madhya Pradesh, Orissa, Punjab, Rajasthan, West Bengal, and Uttar Pradesh, the extent of artificial coloration ranged between 5.7 to 26.8% of analyzed turmeric powder samples.

The break up of turmeric powder samples based on curcumin content indicated that none of the branded powders had less than 2% curcumin and a vast majority (72%) had 2.0-3.0 %, while 18% samples had more than 3.0% curcumin. Among non-branded samples, 10% showed ≤1% curcumin, while 61% fell in the range of 1.0-2.0 percent; followed by 28 percent having 2.0-3.0% curcumin. All branded turmeric samples were free from artificial coloration with metanil yellow. Among non-branded turmeric powders, a maximum proportion of 44% samples were found to contain metanil yellow in the range of 4.0-6.0 mg g -1 . In 30% samples, metanil yellow varied between 2.0-4.0 mg g -1 while 14% had < 2.0 mg g -1 metanil yellow. A set of 9.5% samples employed 6.0-8.0 and 2% had > 8.0 mg g -1 levels of the color.

Discussion

Though dried or fresh turmeric rhizome is usually free from adulteration, turmeric powder is more prone and can be admixed with certain other species of curcuma; low/curcumin depleted turmeric powders; foreign starches or artificial colors (Govindrajan, 1980;[START_REF] Remya | Isolation and amplification of DNA from turmeric powder[END_REF]. The food legislation in India allows the cultivation of Curcuma longa L. as the only species for the production of turmeric (PFA, 2008). However, there are reports that closely related curcuma species such as C. aromatica, C. amada. C. malabarrica and C. xanthorrhiza/ C.zedoaria or their left over matrices after extraction of oleoresins/ essential oils/starch/cosmetic/ medicinal ingredients, may partly find outlet in the retail level loose domestic turmeric powder market [START_REF] Govindarajan | Turmeric -Chemistry, Technology and Quality[END_REF][START_REF] Jyothi | Physicochemical and functional properties of starch from two species of curcuma[END_REF][START_REF] Lechtenberg | Quantitative determination of curcuminioids in curcuma rhizomes and rapid differentiation of Curcuma domestica Val. and Curcuma xanthorrhiza Roxb. by capillary electrophoresis[END_REF][START_REF] Remya | Isolation and amplification of DNA from turmeric powder[END_REF][START_REF] Sasikumar | PCR Based Detection of Adulteration in the market samples of Turmeric Powder[END_REF][START_REF] Sasaki | Rapid identification of Curcuma longa and C. Aromatica by LAMP[END_REF].

Curcumin, the principal curcuminoid of turmeric is responsible for its yellow color and serves as a measure of quality of turmeric. Since there is lot of demand for curcumin both in domestic and export markets, vending curcumin depleted turmeric powder becomes an alluring proposition whereby the consumers are being cheated and ignorantly get nutrient depleted turmeric powders. Our survey results show that curcumin content of branded turmeric powders ranged from 2.2 to 3.7 % while the non-branded loose powders showed as low as 0.3% to a maximum of 2.6% i.e. even samples with as low as 0.3% curcumin can pass in absence of any prescribed curcumin limits if these meets other specifications. The branded turmeric powders of organized sector analyzed in the survey showed no color adulteration and possessed comparatively higher curcumin contents. However, even these have scope for higher curcumin contents because the major Curcuma longa trade types of India have been reported to possess curcumin content ranging from 2.1-8.6%, with an average of 4.8 % [START_REF] Velayudhan | Curcuma Genetic Resources, National Bureau of Plant Genetic Resources[END_REF]. Assuming a 3% curcumin requirement as suggested by BIS, 72% of branded and all the non-branded turmeric powders in present study shall fall below the standard. With a tentative 2% curcumin limit, though all branded powders may comply but still 80% of non-branded samples shall not clear the quality standard.

Samples with low curcumin are the ones that presumably employ foreign starches and may use extraneous artificial color to compensate for the depleted curcumin look of the turmeric powder. The presence of non-permitted color-metanil yellow has been detected in over 17% of non-branded turmeric powder samples in this survey, though none of the branded samples employed artificial coloration. Fortunately, south zones states with higher education and awareness had scanty availability of loose turmeric powders and were devoid of any extraneous color admixture, compared to other three state zones. Very high levels (40-60 folds) of the color-metanil yellow were detected compared to 0.1 mg g-1 levels for the various permitted food colors allowed in specified food commodities under the Indian food rules (PFA, 2008). Metanil yellow has been shown to cause anti-testicular and other toxic manifestations in experimental animals [START_REF] Khanna | Toxicity studies on metanil yellow in rats[END_REF][START_REF] Singh | Safety evaluation studies on metanil yellow, (I) Acute and subchronic exposure response[END_REF][START_REF] Singh | Surveillance of non-permitted food colors in eatables[END_REF].

As part of our recommendations, we feel that the first and foremost step should be to fix realistic curcumin levels, otherwise curcumin rich turmeric powders may not be ensured as there is no obligation on the part of traders to stick to any minimum level. It is likely that majority of importing countries are not purchasing powdered turmeric but get whole rhizomes and hence they are also not insisting for any minimum curcumin levels in their advisories (ASTA, 2007;[START_REF] Esa | European Spice Association Quality Minima Document Rev. 1[END_REF]. Since the major problem is with loose turmeric powders grounded at small millers or at retail shop owner levels who indulge in malpractices due to economic gains, the food authorities may ensure availability of small temper proof packs or pouches for economically poor consumers and intensify vigilance so that consumers are ensured of curcumin rich products. Also, the labs should include DNA based amplification tests so that admixture of starch from non-curcuma or other than Curcuma 10 longa species could be detected. Among other stakeholders, the consumers are advised to purchase either branded turmeric powders avoiding loose powders or resort to the conventional household level grinding. The farmers may be encouraged to grow curcumin rich variants of turmeric so that there is no difficulty in offering high curcumin products for the markets.

  National Bureau of Plant Genetic Resources showed that among 21 morphotypes of C. longa

Table 1 : Curcumin content in branded versus non-branded turmeric powders

 1

	No. of samples	Curcumin content, %

** Values in parentheses represent the percentage.

Table 2 : Pattern of artificial colouration (metanil yellow) in loose non-branded turmeric powder samples from four state zones

 2

	Deleted: 3

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Table 1 : Quality assurance data on curcumin and metanil yellow measurement

 1

	Analyte	Matrix	LOD	LOQ	Recovery	RSD%	Measurement	Accreditation
			(ng spot -1 )	(ng spot -1 )	Range %	(n= 3 )	uncertainty
	Curcumin	Turmeric	17.39	52.71	86.7-98.7	1.26-3.32	0.72-2.49	No
		Powder
	Metanil	Turmeric	42.90	130.0	97.1-103.0	2.37-3.85	2.24-3.98	No
	Yellow	Powder
	Page 4: [2] Deleted		sumita		4/28/2009 3:10:00 PM

Table 4 : Bifurcation of turmeric samples based on curcumin and metanil yellow contents

 4 No. of samples with curcumin content,% No. of samples with metanil yellow content, mg g -1

	Turmeric Powder
	0.2-1.0 1.0-2.

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Acknowledgement

The authors are grateful to Director, IITR for his keen interest in the present study.

One of us (S.K.K) is thankful to Indian Council of Medical Research, New Delhi, for the award of Emeritus Medical Scientist scheme. Financial support of CSIR Net work project # 17 is gratefully acknowledged. The manuscript is IITR communication # 2695.