Geophagy and potential health implications: geohelminths, microbes and heavy metals

Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 787–795

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Transactions of the Royal Society of Tropical Medicine and Hygiene journal homepage: http://www.elsevier.com/locate/trstmh

Geophagy and potential health implications: geohelminths, microbes and heavy metals Ruth Kutalek a,∗ , Guenther Wewalka b , Claudia Gundacker c,d , Herbert Auer e , Jeff Wilson f , Daniela Haluza g , Steliana Huhulescu b , Stephen Hillier f , Manfred Sager h , Armin Prinz a a

Medical University of Vienna, Center for Public Health, Department of General Practice and Family Medicine, Unit Ethnomedicine and International Health, Waehringerstrasse 25, 1090 Vienna, Austria Austrian Agency for Health and Food Safety AGES, Institute for Medical Microbiology and Hygiene, Vienna, Waehringerstrasse 25A, 1090 Vienna, Austria c Medical University of Vienna, Center for Public Health, Department of Ecotoxicology, Waehringerstrasse 10, 1090 Vienna, Austria d Medical University of Vienna, Department for Medical Genetics, Waehringerstrasse 10, 1090 Vienna, Austria e Medical University of Vienna, Center for Pathophysiology, Infectiology and Immunology, Department of Medical Parasitology, Institute of Specific Prophylaxis and Tropical Medicine, Kinderspitalgasse 15, 1090 Vienna, Austria f Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8QH, United Kingdom g Medical University of Vienna, Center for Public Health, Institute of Environmental Health, Kinderspitalgasse 15, 1090 Vienna, Austria h Austrian Agency for Health and Food Safety AGES, Competence Centre Elements, Spargelfeldstrasse 191, 1226 Vienna, Austria b

a r t i c l e

i n f o

Article history: Received 4 February 2010 Received in revised form 1 September 2010 Accepted 1 September 2010

Keywords: geophagy soil microbes parasites heavy metals Africa

a b s t r a c t The practice of geophagy (soil-eating) is widespread among pregnant and breast-feeding women in sub-Saharan Africa. To assess some of the potential risks accompanying the consumption of geophagic material, we analysed contamination with bacteria, fungi, and geohelminths as well as heavy metals (lead, mercury and cadmium) in 88 African geophagic soil samples, which were purchased in Central, West and East Africa, Europe and the United States. Median microbial viable counts of positive samples were 440 cfu/g (maximum 120 000 cfu/g). The median metal concentrations were 40 mg/kg lead (up to 148 mg/kg), 0.05 mg/kg mercury (up to 0.64 mg/kg), and 0.055 mg/kg cadmium (maximum 0.57 mg/kg). No geohelminth eggs were found in these samples. Our results suggest that geophagic soil samples can be highly contaminated with microbes and may contain high levels of lead. Geophagy, however, is not a cause of adult helminth infection. The periodic consumption of geophagic materials at high dosages might be problematic particularly during pregnancy. © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

1. Introduction Geophagy, ‘the habit of eating earth including clay and other types of soil’,1 has been observed in many parts of the world,2–4 but is especially widespread in sub-Saharan Africa (Supplementary Figure 1). Soil or clay is mostly consumed by pregnant or breast-feeding women and also

∗ Corresponding author. Tel.: +43 1 4277 63436; fax: +43 1 4277 9634. E-mail address: [email protected] (R. Kutalek).

by children.5–8 Little data is available on the percentage of women consuming clay or on the amount consumed. Several studies, however, suggest that in African regions where geophagy occurs, between 46% and 73% of pregnant or breast-feeding women consume soil regularly. The amounts differ considerably, with average values from 1100 g/day (and more) being reported.5,9–11 The practice of geophagy is deeply embedded in cultural traditions, and in many cultural contexts earth eating is seen as normal. Geophagy clearly also has biological components. A great deal of misunderstanding surrounds this issue because

0035-9203/$ – see front matter © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2010.09.002

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Figure 1. Geophagic samples were collected in Tanzania, Kenya, Uganda, Rwanda, Swaziland, Democratic Republic of Congo, Sudan, Burkina Faso and Senegal, and from ethnic shops in Austria, Belgium, the United Kingdom, and the United States.

the assumption is that it is pathological and because it is largely practiced in disadvantaged populations or countries.12 Various hypotheses on geophagy have been forwarded and there are conflicting views as to whether soil eating is beneficial to health or not. Several studies point to positive effects: geophagic material is a source of mineral nutrients – especially of iron13,14 – helps against gestational nausea15 and detoxifies foods, for example, by adsorption of various plant secondary compounds.16 The consumption of clay increases food efficiency,17 which might be important in poor countries. Furthermore, smectite and other clays have long been used in pharmaceutical preparations and to treat acute gastroenteritis.18 Other studies report negative effects, e.g., an association of geophagy with iron and other mineral nutrient deficiencies,19 anaemia20,21 and geohelminth infections6,8,9 . Moreover, geophagic materials can contain toxic heavy metals, especially lead.22–25 The heavy metals lead, mercury and cadmium are ubiquitous and persistent environmental pollutants that primarily affect the kidneys and the brain. Exposure to these metals during pregnancy and early childhood can impact children’s renal systems and brain development. This implies the need to control and regulate the potential sources of lead, mercury and cadmium contamination. Geophagic earth may vary widely in its physical, mineralogical, and chemical properties.26 In Africa, it is either procured from local sources, such as termite mounds, house walls and from natural exposures or it is purchased in local markets where it has often been traded over long distances.15 It is ingested as a drink or powder, gnawed or crushed. Through international migration, geophagy is also present in typical immigrant countries. Here, the clay is mostly imported from African and Asian countries and sold in ethnic stores and markets in urban areas.13,22 Therefore, the consumption of soil is increasingly seen as an international public health issue especially regarding pregnant and breast-feeding women.23–25,27,28 We collected African geophagic soil samples from a wide geographical range and analysed their contamination with geohelminths, bacteria and fungi, as well as their content of toxic heavy metals (lead, mercury and cadmium).

To our knowledge, no studies are available on the microbial contamination of geophagic soil. Only a few studies deal with their heavy metal contents (usually analysing low sample numbers)22,23,25 or the presence of geohelminths in geophagic soils consumed by adults.29 Our study is unique for its combined focus on geohelminths, potentially pathogenic microorganisms and heavy metals. The broad interdisciplinary approach and the variety and amount of African geophagic clays analysed are also a first. 2. Materials and methods 2.1. Sample collection Geophagic material was purchased from markets or local shops in Tanzania (Arusha, Moshi, Njombe, Zanzibar), Uganda (districts Migi, Mubende, Mukono, Wakiso), Kenya, Rwanda, Swaziland (Nhlangano), Democratic Republic of Congo (Isiro, Kinshasa, Kisangani), Sudan (Tuok Nyekok), Burkina Faso and Senegal (Fimla, Joual, Dakar) and from ethnic shops in the United Kingdom (London), United States (New York), Belgium (Brussels) and Austria (Vienna) (Figure 1). All 88 samples originated from Africa, although specific information on the physical geographical sources was lacking. Shopkeepers were asked to wrap the substances in materials (mostly paper and plastic bags) they use for their customers. All samples were numbered and put into polyethylene bags. 2.2. Mineralogy The bulk mineralogy and clay mineral composition of 29 samples of geophagic materials were assessed by X-ray powder diffraction analysis. These samples were considered to be representative of the samples set as a whole based on their physical appearance and their geographical location. 2.3. Helminths As geophagic samples are stored in shops or on markets for an unknown period of time, fresh soil material was not available. To detect geohelminths about 3 g of each of

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the 88 geophagic samples were dispersed in 10 ml sodium acetate-acetic acid-formalin (SAF) solution according to Marti and Escher.30 The SAF supernatant was removed and the sediment was examined microscopically (100-400x magnification). This method has been successfully used when analysing palaeo-parasitological material.31

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(n = 12). The limit of detection (LOD) was determined by the concentration equivalent to the threefold standard deviation of the signal of the blank solution and was 0.27 ␮g/kg lead, 0.02 ␮g/kg cadmium, and 0.20 ␮g/kg mercury. Recoveries of reference material were 104% (lead), 84% (cadmium), and 95% (mercury). In six samples, mercury concentrations were below LOD.

2.4. Microbiology 2.6. Statistical analysis From newly broken surfaces of 88 geophagic samples material was scraped with sterilised knives and pulverised in sterilised mortars. Then, 0.3 g powder of each sample was suspended in 2.7 ml buffered pepton water (bioMérieux, Marcy-l‘Etoile, France) and suspensions were plated in volumes of 1 ml and 0.1 ml onto different agar media. The cut off for viable counts of bacteria and fungi was 10 colony forming units (cfu) per g. For investigation of total viable counts of aerobic bacteria, especially potentially pathogenic bacteria, Columbia-agar with 5% sheep blood (bioMérieux) was used.32 Colimycin-nalidixicacid-agar was used for aerobic growth of gram-positive bacteria,32 and bromthymol-blue-lactose-agar for aerobic growth of gram-negative bacteria.33 Plates were incubated aerobically at 35 ◦ C for 24 h and thereafter at 22 ◦ C for an additional 48–72 h. Fungi were investigated by plating 1 ml and 0.1 ml of suspensions onto sabouraud-glucose-gentamycinchoramphenicol-agar (bioMérieux)34 and incubating aerobically at 30 ◦ C for up to 10 days. Quantitative and qualitative analysis involved counting cfu of morphologically different bacteria and fungi. Bacteria were determined to genus level by means of gram stain, oxidase reaction, catalase reaction and, if necessary, by API identification system (bioMérieux). The identification was done mostly to genus level by macroscopic attributes, e.g., speed of growth, pigmentation and texture of air mycel, and microscopic attributes, e.g., types of conidia, size, colour and septation of fungal filaments, and by lactophenol-cotton-blue stained preparations. 2.5. Lead, mercury and cadmium analyses Between 100 and150 mg of the 88 geophagic samples and reference materials (light sandy soil [trace elements] BCR 142R, Institute for Reference Materials and Measurements, Geel, Belgium) were digested with a mixture of 2 ml 65 vol% HNO3 suprapur (Merck, Darmstadt, Germany) and 0.75 mL 30% H2 O2 (Merck) in a microwave oven (MLS, mls 1200 mega, Leutkirch, Germany). The sample solutions were volumetrically filled up to 10 ml with deionised water. Metal concentrations were analysed by AAS using a Hitachi Z 8200 Polarized Zeeman Atomic Absorption Spectrophotometer (Hitachi, Tokyo, Japan). Lead and cadmium concentrations were determined by graphite furnace AAS (GF-AAS). Mercury contents were analysed by cold vapour AAS (CV-AAS) in combination with an amalgamation unit (Uwe Binninger Analytik, Schwaebisch Gmuend, Germany) and a hydride generation system (Hitachi; HFS-3). All samples were measured in duplicate (RSD<10%) by the working curve method. Quality assurance was achieved by measuring blank test solutions (n = 12) and reference material

The data were analysed statistically, using the SPSS (Statistic Package for Social Science, SPSS Inc., Chicago, IL, USA) 17.0 software. Histograms of microbial counts were checked for normality. Based on the spread of the data, a log transformation was performed. Log transformation was used to improve interpretability and visualisation of the data. All reported P-values were two-sided, and all associations were considered significant when P-values were < 0.05. Because metal levels were not normally distributed (Lilliefors test P<0.001), the non-parametric Mann Whitney U test was applied. 3. Results 3.1. Mineralogy Many of the soils showed some rudimentary manufacturing. Some samples were mixed with herbs (especially the Ugandan samples), shaped in certain forms, or made into a powder. They were mostly air dried and occasionally baked, smoked or salted. Most samples consisted of the raw material which can usually be described as a weakly bedded, fine grained mudstone or shale. Both manufactured and raw materials showed a variety of colours, ranging from white to light red as assessed by the Munsell Color chart for soils. The bulk samples were usually dominated by quartz or by a kaolin-type mineral, which was also dominant in most clay (<2 ␮m) fractions. Some clay fractions, however, were dominated by a mica-type mineral and others by a swelling smectitic clay mineral (Supplementary Table 1). 3.2. Helminths Neither helminth eggs nor larvae were detected in any of the samples. In one sample (No. 63), an unidentified fragment of an arthropod, probably the larva of an insect (ca. 500 ␮m long), was found. 3.3. Microbiological contamination All but three of the 88 geophagic samples contained aerobic bacteria. Total viable counts of positive samples ranged between 10 and 120 000 cfu per gram with a median of 440 cfu/g. The total viable counts were highest in samples from East Africa (median: 2050 cfu/g) followed by samples from Central Africa (median: 700 cfu/g), West Africa (median: 130 cfu/g) and imported specimens (median: 65 cfu/g), but the differences were not significant (Figure 2A). Spore-forming bacteria of the genus Bacillus were most frequent and occurred in all of the 85 geophagic samples with

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Bacteria 35

(A)

bacterial contamination (Table 1). The Enterobacteriaceae occurring in two samples in viable counts of 1600 to 26 000 cfu/g belonged to the genera Enterobacter, Klebsiella and Pantoea which can be summarised as coliform bacteria. Contamination with fungi was found in 65 of the 88 samples. Total viable counts ranged between 10 and 100 00 cfu/g, with a median for all samples of 30 cfu/g. Values were highest in specimens from East Africa (median: 60 cfu/g) followed by imported specimens (median: 50 cfu/g), specimens from West Africa (median: 40 cfu/g) and specimens from Central Africa (median: 30 cfu/g) (Figure 2B). Moulds of the genus Penicillium were found most frequently (in 58 samples) (Table 2). Aspergillus flavus which potentially produces aflatoxins occurred in five samples in low numbers. About one fourth of the samples (all of them from Congo) were reportedly baked. When differentiating between baked (n = 21) and unbaked samples (n = 65), fewer viable counts were found in baked material (mean value: 1463.8 cfu/g, SD: 3618.2) than in unbaked material (mean value: 13 788 cfu/g, SD: 28 521.5).

<10 cfu/g

30

10-99 cfu/g 100-999 cfu/g

25

1000-9999 cfu/g 10 000-99 999 cfu/g

20

>100 000 cfu/g

15 10 5 0 Total

Central Africa

East Africa

West Africa

imported

Fungi 40

(B)

35 1-9 cfu/g

30

10-99 cfu/g

25

100-999 cfu/g

3.4. Lead, mercury and cadmium in the geophagic samples

1000-9999 cfu/g

20 15

The median lead concentration was 40 mg/kg (range: 3–148 mg/kg), the median mercury content 0.053 mg/kg (range: 0.001–0.637 mg/kg), and the median cadmium concentration 0.055 mg/kg (range: 0.007 and 0.573 mg/kg) (Table 3). When we tested for region-specific differences, the levels of all three metals were higher in samples from Central Africa than in samples from West Africa and East Africa, respectively (P < 0.05). The exposure assessment in Tables 4 and 5 is based on median, minimum and maximum concentrations, respectively. We assumed a daily consumption of 1, 10, 30 and 100 g of geophagic materials, variable bioavailability of

10 5 0 Total

Central Africa

East Africa

West Africa

imported

Figure 2A. Total viable counts of aerobic bacteria in 88 geophagic samples from different regions ranged between 10 and 120 000 colony forming units (cfu) per gram with a median of 440 cfu/g. Total viable counts were highest in samples from East Africa. Figure 2B. Total viable counts of fungi in 88 geophagic samples from different regions ranged between 10 and 10 000 colony forming units (cfu) per gram with a median of 30 cfu/g. Values were highest in samples from East Africa.

Table 1 Groups of bacteria found in geophagic samples from different regions (n = number of samples collected in each region). Groups of bacteria

Central Africa n = 34

East Africa n = 31

West Africa n = 17

Imported specimens n = 6

Total n = 88

Bacillus spp. Corynebacterium spp. Coagulase negative Staphylococci Micrococcus spp. Acinetobacter spp. Enterobacteriaceae Pseudomonas spp.

32 1 4 1 2 0 0

31 9 5 1 4 1 0

17 8 4 10 2 1 1

5 1 0 2 0 0 0

85 19 13 13 6 2 1

Table 2 Groups of fungi found in geophagic samples from different regions (n = number of samples collected in each region). Groups of fungi

Central Africa n = 34

East Africa n = 31

West Africa n = 17

Imported specimens n = 6

Total n = 88

Penicillium spp. Aspergillus spp. Alternaria spp Cladosporium spp. Streptomyces spp. Acremonium spp. Paecilomyces spp. Rhizopus spp. Chrysonilia spp. Nigrospora spp. Scedosporium spp. Candida spp.

28 7 3 4 1 1 1 0 0 0 0 0

13 10 5 3 3 2 1 1 0 1 1 0

14 6 4 3 0 0 0 1 2 0 0 1

3 1 1 0 0 0 0 0 0 1 0 0

58 24 13 10 4 3 2 2 2 2 1 1

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Table 3 Lead, mercury and cadmium contents of geophagic samples n All Mean ± SD 88 Median Range Central Africa Mean ± SD 34 Median Range West Africa Mean ± SD 17 Median Range East Africa Mean ± SD 31 Median Range Imported (Europe, UK, USA) Mean ± SD 6 Median Range

Lead (mg/kg)

n

Mercury (mg/kg)

n

Cadmium (mg/kg)

47 ± 35 40 3–148

82

0.20 ± 0.24 0.050 0.001–0.637

88

0.073 ± 0.078 0.055 0.007–0.573

57 ± 37 59 3–137

34

0.42 ± 0.21 0.53 0.002–0.637

34

0.097 ± 0.093 0.093 0.014–0.573

39 ± 24 34 6–114

15

0.006 ± 0.004 0.005 0.002–0.014

17

0.051 ± 0.094 0.027 0.017–0.413

32 ± 23 29 6–105

28

0.048 ± 0.045 0.035 0.001–0.230

31

0.065 ± 0.046 0.056 0.007–0.206

92 ± 50 89 20–148

5

0.048 ± 0.026 0.0047 0.017–0.074

6

0.044 ± 0.024 0.0051 0.008–0.068

metals (3%, 10%, 30%), body weight of 60 kg for adult women (Table 4), body weight of 3 kg for newborns (Table 5), and 100% placentar transfer (see Discussion). These exposure scenarios were selected because geophagic material consumption varies substantially in a range from 1–100 g/day (and more) being reported.5,11 Also, the amount of bioaccessible (the amount of a contaminant that is released from the matrix [e.g. soil] during the digestion in the gastrointestinal tract) or bioavailable (the fraction of an orally administered dose that reaches the systemic circulation) lead, mercury, and cadmium in geophagic samples is highly variable depending on the sample matrix, chemical form of the metal, soil pH, stomach and intestinal pH, soil-to-solution ratio, and fasting conditions.35–39 4. Discussion Microbial contamination of geophagic material was unexpectedly high, especially in unbaked material. The median lead content was very high, that of cadmium and mercury relatively low. We found no contamination of geophagic material with helminths. 4.1. Helminths Our findings that geophagic materials purchased on markets are not contaminated with helminths support the studies by Vermeer and Frate40 and Young et al.29 Geophagy has long been speculated to be a risk factor for soil-transmitted helminth infection,9,20 especially among children.6,8 Children, however, are less choosy and are more likely to use unsafe soil which might contain a higher helminth density. Past studies differ greatly in their sampling and methods. The only study that determined the parasite content of earth consumed by adults found no helminths.29 Even though Toxocara canis and T. cati infection is sometimes associated with geophagy and Toxocara contamination in soil is quite widespread,41 we found no

eggs or larvae. Furthermore, our study showed a wide geographic distribution of uncontaminated clay. 4.2. Microbial contamination Microbial contamination of geophagic material was unexpectedly high, especially in unbaked material. This can be explained mainly by the long survival time of spores of the bacteria genus Bacillus under dry conditions. The European Pharmacopoeia42 sets a limit of 1000 aerobic cfu for clays used for ingestion in pharmaceutical preparations. Current FAO standards43 for dry foodstuff, which does not undergo germ-reducing procedures before consumption, require total viable counts of aerobic mesophilic bacteria to be below 100 000 cfu/g, and four out of five samples should have viable counts below 10 000 cfu/g. In our study, two out of 88 samples exceeded values of 100 000 cfu/g; 10 samples reached counts between 10 000 and 99 999 cfu/g. In addition, a second criterion in FAO standards limits the amount of coliform bacteria to 100 cfu/g. Both samples containing coliform bacteria exceeded this amount and had total viable counts >10 000 cfu/g. The microbiological investigation of the samples showed that two of 88 samples exceeded limits for total viable counts of aerobic mesophilic bacteria of the genus Bacillus and two further samples contained high numbers of coliform bacteria; accordingly, four samples would be rejected if examined as foodstuff. Some of the most frequently found aerobic spore-forming bacteria of the genus Bacillus could potentially cause gastrointestinal irritation when germinating in the upper gastrointestinal tract by producing toxins. The presence of coliform bacteria indicates faecal contamination and the potential presence of diarrhoeal pathogens. Limits for fungal contamination, especially moulds, are not included in the FAO standards. According to the German Society for Hygiene and Microbiology (DGHM)44 standards, however, mould contamination is allowable up to 10 000 cfu/g for instant products. None of the geophagic clay

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Table 4 Daily lead, mercury and cadmium intake through ingestion of geophagic materials for a person weighing 60 kg in various exposure scenarios. Daily ingestion: 1 g 3% BA

MED MIN MAX MED MIN MAX

Daily ingestion: 10 g 30% BA

Pb intake (mg/kg body weight/day) 0.000020 0.000067 0.000200 0.000002 0.000005 0.000015 0.000074 0.000247 0.000740 Hg intake (␮g/kg body weight/day) 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000001 0.000003 Cd intake (␮g/kg body weight/day) 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000001 0.000003

Daily ingestion: 30 g

Daily ingestion: 100 g

3% BA

10% BA

30% BA

3% BA

10% BA

30% BA

3% BA

10% BA

30% BA

0.000200 0.000015 0.000740

0.000667 0.000050 0.002467

0.002000 0.000150 0.007400

0.00060 0.00005 0.00222

0.00200 0.00015 0.00740

0.00600 0.00045 0.02220

0.00200 0.00015 0.00740

0.00667 0.00050 0.02467

0.02000 0.00150 0.07400

0.000000 0.000000 0.000003

0.000001 0.000000 0.000011

0.000003 0.000000 0.000032

0.00000 0.00000 0.00001

0.00000 0.00000 0.00003

0.00001 0.00000 0.00010

0.00000 0.00000 0.00003

0.00001 0.00000 0.00011

0.00003 0.00000 0.00032

0.000000 0.000000 0.000003

0.000001 0.000000 0.000010

0.000003 0.000000 0.000029

0.00000 0.00000 0.00001

0.00000 0.00000 0.00003

0.00001 0.00000 0.00009

0.00000 0.00000 0.00003

0.00001 0.00000 0.00010

0.00003 0.00000 0.00029

Pb: lead; Hg: mercury; Cd: cadmium BA: Bioavailability; MED: Median; MIN: Minimum; MAX: Maximum; mg: milligrams; ␮g: micrograms Metal intakes exceeding the provisional tolerable daily intake (PTDI) are marked in bold. The PTDI was calculated from the provisional tolerable weekly intake (PTWI) (i.e., 0.025 mg/kg body weight/week (Pb), 5 ␮g/kg body weight/week (Hg), 7 ␮g/kg body weight/week (Cd)), which is defined as the estimate of the amount of a substance that can be ingested over a lifetime without appreciable risk to health.

Table 5 Daily lead, mercury, and cadmium intake through ingestion of geophagic materials for a newborn weighing 3 kg in various exposure scenarios. Daily ingestion: 1 g 3% BA MED MIN MAX MED MIN MAX MED MIN MAX

Pb (mg/kg) in soil 40 3 148 Hg (␮g/kg) in soil 0.050 0.001 0.637 Cd (␮g/kg) in soil 0.055 0.007 0.573

10% BA

Daily ingestion: 10 g 30% BA

Pb intake (mg/kg body weight/day) 0.000400 0.001333 0.004000 0.000030 0.000100 0.000300 0.001480 0.004933 0.014800 Hg intake (␮g/kg body weight/day) 0.000001 0.000002 0.000005 0.000000 0.000000 0.000000 0.000006 0.000021 0.000064 Cd intake (␮g/kg body weight/day) 0.000001 0.000002 0.000006 0.000000 0.000000 0.000001 0.000006 0.000019 0.000057

Daily ingestion: 30 g

Daily ingestion: 100 g

3% BA

10% BA

30% BA

3% BA

10% BA

30% BA

3% BA

10% BA

30% BA

0.004000 0.000300 0.014800

0.013333 0.001000 0.049333

0.040000 0.003000 0.148000

0.01200 0.00090 0.04440

0.04000 0.00300 0.14800

0.12000 0.00900 0.44400

0.04000 0.00300 0.14800

0.13333 0.01000 0.49333

0.40000 0.03000 1.48000

0.000005 0.000000 0.000064

0.000017 0.000000 0.000212

0.000050 0.000001 0.000637

0.00002 0.00000 0.00019

0.00005 0.00000 0.00064

0.00015 0.00000 0.00191

0.00005 0.00000 0.00064

0.00017 0.00000 0.00212

0.00050 0.00001 0.00637

0.000006 0.000001 0.000057

0.000018 0.000002 0.000191

0.000055 0.000007 0.000573

0.00002 0.00000 0.00017

0.00006 0.00001 0.00057

0.00017 0.00002 0.00172

0.00006 0.00001 0.00057

0.00018 0.00002 0.00191

0.00055 0.00007 0.00573

Pb: lead; Hg: mercury; Cd: cadmium BA: Bioavailability; MED: Median; MIN: Minimum; MAX: Maximum; mg: milligrams; ␮g: micrograms Metal intakes exceeding the provisional tolerable daily intake (PTDI) are marked in bold. The PTDI was calculated from the provisional tolerable weekly intake (PTWI) (i.e., 0.025 mg/kg body weight/week (Pb), 5 ␮g/kg body weight/week (Hg), 7 ␮g/kg body weight/week (Cd)), which is defined as the estimate of the amount of a substance that can be ingested over a lifetime without appreciable risk to health.

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MED MIN MAX

Pb (mg/kg) in soil 40 3 148 Hg (␮g/kg) in soil 0.050 0.001 0.637 Cd (␮g/kg) in soil 0.055 0.007 0.573

10% BA

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samples exceeded this limit. The low counts of the potentially aflatoxin-producing Aspergillus flavus in the samples indicate a low threat for human health. These microbiological results indicate no concrete health hazard following the consumption of the geophagic clay products. They do, however, give cause for concern because, contrary to most food purchased on markets (which is either cooked or peeled before consumption), geophagic material is typically consumed uncooked. 4.3. Levels of lead, mercury and cadmium Lead and cadmium are less bioavailable than mercury.35 Bioaccessibility of lead in various soil types amount to 3–83%,38,45 with the value being 3% in geophagic soils.13 Mercury bioaccessibility ranges between 5% and 100% depending on its chemical form (mercuric sulfide: 1%, total mercury: 5%, mercuric chloride: 100%),36 and that of cadmium is certainly beyond 30%.39 Lead intake through geophagic soil consumption by a person weighing 60 kg exceeds the PTDI of 0.0036 mg/kg body weight when more than 10 g are consumed per day and when bioavailability of lead is relatively high (>10%) (Table 4). The exposure assessment shows that newborns (3 kg body weight) are at a higher risk of exceeding the PTDI of lead (Table 5). Nonetheless, the PTDI of 0.714 ␮g/kg body weight (mercury) and 1.0 ␮g/kg body weight (cadmium) is not exceeded by any of the exposure scenarios assumed here. The daily ingestion of geophagic clay does not contribute to unsafe mercury and cadmium intakes, neither in adults nor newborns. The risk of prenatal cadmium exposure is even lower than estimated here: this metal is trapped by placenta tissues, with very little reaching the fetus.46 In the case of lead, however, adults and particularly newborns are at risk to exceed critical levels. Lead exposure of pregnant and breast-feeding women gives cause for concern. This metal easily crosses the placenta and is excreted into breast milk. The fetus is particularly sensitive to lead, which is a potent neurotoxin. Adverse effects on child neurodevelopment and physical growth may occur even at relatively low exposure levels.47 Children are also known to consume geophagic clays.6–8,25 Two factors should be considered when interpreting our exposure assessment. First, consumption habits are known to vary substantially: thus, we might have overestimated the risk by assuming daily consumption. Nonetheless, representative data on consumption habits are not yet available. Second, the percentage of lead that is bioavailable to humans in the soils analyzed here is highly speculative. In one scenario the metal is embedded within the structure of the constituent minerals. If such, host minerals are insoluble, then the metal would probably not be bioavailable and would simply pass through the body. If, however, the metal results from the external environment, then it will probably be adsorbed at the surfaces of the fine-grained minerals in the geophagic materials. In this scenario, bioavailability will depend, amongst other things, on the adsorption mechanism involved. Again, metals such as lead potentially precipitate in forms that are so insoluble as to render them completely non-available. Finally, the pH and particle size distribution of the geophagic materi-

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als, as well as age, nutritional status, dietary composition and body weight of the exposed individual needs to be taken into account.26,48 These issues would require further detailed research. Our exposure assessment refers to the provisional tolerable weekly intake (PTWI) levels set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), which are appropriate guideline values with regard to metal toxicity. To date, there is no consensus on which regulatory guidelines should be applied to geophagic soil samples. The UK Food Standards Agency23 and other authors13,22 find the application of food quality standards for geophagic material appropriate because it is consumed in large quantities.27 According to EC Commission Regulation (No. 466/2001)49 the highest concentration of lead, cadmium and mercury permitted in specific foodstuffs should not exceed 1 mg/kg. The European Pharmacopoeia43 has set a maximum level of 25 mg/kg lead for clays for internal use. These clays, however, are used in much lower quantity than geophagic material. Metal levels in geophagic samples differed greatly according to sampling site. The regional differences in the lead, mercury, and cadmium levels between samples collected, but, as already mentioned, not necessarily originating, from Central Africa and samples collected from West Africa and East Africa are significant. They show that pregnant women consuming geophagic soils in Central Africa are at higher risk than pregnant women consuming geophagic soil in other regions. Many of the samples we examined are clearly rock samples, not soils, and have presumably been sampled mainly from natural outcrop exposures (Supplementary Table 1). This calls for consideration of the mean lead, mercury, and cadmium contents that such materials (clays and shales) contain in their natural state. The mean cadmium content of shales is 1 ppm, that of mercury ∼0.4 ppm. The levels of these two metals in our samples were much lower. For lead, however, the mean content in clays and shales is ∼22 ppm.50 The levels in our samples are therefore twice as high as those occurring naturally in shales and clays. It is not known at present whether this enrichment is natural or due to contaminating sources from the atmosphere or from surface waters. 5. Conclusions Geophagic soil samples can be contaminated with pathogenic microbes. This gives cause for concern because these materials are usually consumed uncooked. Furthermore, the high lead contents of some soil samples are problematic. When pregnant females regularly consume highly contaminated soils, the vulnerable fetus might be exposed to undesirably high lead levels. Note, however, that lead is one of the least bioavailable metals and probably only a fraction will become bioaccessible. This calls for cautiously interpreting the risk. We found cadmium and mercury in low to very low concentrations in geophagic materials. Thus, it is unlikely that geophagy contributes to toxic cadmium and mercury exposure levels. To date, we cannot satisfactorily estimate the health risks associated with geophagy. Most of the exposure factors and individual risk factors are poorly known. Data are

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required with regard to: (1) consumption habits, particularly of pregnant and lactating women, and children, (2) nature and provenance of the geophagic materials and the ways in which they are prepared or manufactured and consumed, (3) bioaccessibility and bioavailability of lead, cadmium, and mercury (including the particularly neurotoxic compound methyl mercury) in geophagic soils and (4) prenatal metal exposures of children born to mothers who practice geophagy. Because of the frequency and nearly worldwide distribution of earth consumption, as well as the perceived benefits of geophagy (e.g., reduced nausea during pregnancy), it is unlikely that consumers will refrain from the practice. Regulations will be difficult to apply because geophagic materials are often not sold openly, especially in Europe.27 Overall, our current knowledge suggests that the periodic consumption of geophagic materials should be discouraged, particularly during pregnancy. Authors’ contributions: RK and AP designed the study, oversaw sample collection and drafted the article; GW and SH examined the samples, wrote the microbiology methods, and interpreted the results on microbiology; CG examined the samples, wrote the methods on heavy metal analyses, and interpreted the results on heavy metals; HA examined the samples, wrote the methods on helminth analyses and interpreted the results on geohelminths. JW and SHi examined the samples, wrote the methods on mineralogy and interpreted the results; DH wrote the methods on statistical data and interpreted the results statistically; MS helped with the interpretation of results and critically revised the article. All of the authors revised the manuscript critically for important intellectual content and approved the final version submitted for publication. AP is guarantor of the paper. Acknowledgements: We thank all the persons who helped in obtaining geophagic samples and with laboratory work, especially Ingrid Gruener. SH acknowledges support from the Scottish Executive Environment and Rural Affairs Department (SEERAD). We thank Manfred Maier, Sera L. Young and Horst Aspöck for valuable comments on a previous draft of the manuscript. Funding: Medical University of Vienna, the Austrian Agency for Health and Food Safety AGES, and the Macaulay Land Use Research Institute, Aberdeen, United Kingdom. Conflicts of interest: None declared. Ethical approval: Not required. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.trstmh.2010.09.002.

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