A Market Basket Survey of Horticultural Fruits for Arsenic and Trace Metal Contamination in Southeast Nigeria and Potential Health Risk Implications

Background. Elevated arsenic and trace metal contamination of the terrestrial food chain represents one of the most significant environmental risk exposures for human populations in developing countries. Metalloid and metal contamination in horticultural crop produce such as fruit is a public health concern in Nigeria. Local fruits are cheap sources of vitamins and minerals for the resident population and pose an important dietary threat of metal(loid) toxicity through consumption. Objectives. Market basket investigation of five locally grown (guava, pineapple, orange, and pawpaw) and imported (apple) fruits was conducted to measure the total concentrations of arsenic (As), mercury (Hg), copper (Cu), and lead (Pb) present in these fruits from southeastern Nigeria (Awka, Anambra). Methods. Fruits were analyzed for As and the three metals using atomic absorption spectrophotometry. Moisture content of fruits was determined and used to transform metal concentrations in dry weight to wet weight and compared to Codex food grade standards and assorted (sub)tropical fruits, edible and inedible peels. Results. The mean ± standard deviation of elemental concentrations in dry weight ranged from 20.0±0.71–96.84±0.00 μg g−1 for As, 0.02±0.02 – 0.89±0.33 μg g−1 for Hg, 0.11±0.01 - 0.18±0.40 μg g−1 for Cu, and <0.001 – 0.03±0.05 μg g−1 for Pb. The As concentrations (wet weight) in fruits were ~32–166 orders of magnitude higher than Codex Alimentarius Commission (Codex) maximum As food grade levels. Guava and apple methyl Hg concentrations were ~6–~1 orders of magnitude higher than Codex maximum levels, while the content of Cu and Pb in fruits were within acceptable standard limits. Conclusions. The significant concentrations of As and Hg in the examined fruits indicate a potential public health threat. Efforts are needed to initiate and sustain continued monitoring of trace elements in fruits and food sold to consumers due to variation in contaminating sources to ensure food safety. Although a great deal of information exists on Hg toxicity, research on metalloids such as As remains limited in Nigeria and no reliable guidelines exist. Further research is recommended to determine the ecotoxicity of As in Nigeria.


Introduction
Food systems in developing countries are not always as organized as in developed countries due to problems associated with a burgeoning population, rapid urbanization, and shortfall of resources needed to handle food processing. 1 In addition, environmental dispersion of contaminants from uncoordinated industrialization and waste management systems further compromise food quality and safety, and raise the probability of dietary risk exposure among consumers. [1][2][3][4][5] In addition to the fecal and microbial food contamination often related to poor hygiene and sanitation, risks of metal and metalloid (generally referred herein as metals except as otherwise indicated) ingestion through food consumption and associated health Background. Elevated arsenic and trace metal contamination of the terrestrial food chain represents one of the most significant environmental risk exposures for human populations in developing countries. Metalloid and metal contamination in horticultural crop produce such as fruit is a public health concern in Nigeria. Local fruits are cheap sources of vitamins and minerals for the resident population and pose an important dietary threat of metal(loid) toxicity through consumption. Objectives. Market basket investigation of five locally grown (guava, pineapple, orange, and may depend on the species, soil characteristics, metal properties, and environmental conditions. 21-23 Beyond dietetic metal exposure, air and water are significant random pathways of exposure reported in many case studies. 2-4,24, 25 The several mutually reinforcing physical and chemical properties of metals nevertheless enhance ease of bioaccumulation and biomagnification in food webs. 26- 31 The growing concern of entrance and transference of metals into terrestrial food webs stems from the potential for adverse effects on the health status of both humans and animals continuously exposed to such harmful contaminants via food consumption. 6 A number of studies have been conducted on trace metals linked to the incidence of gastrointestinal cancer, and cancer of the pancreas, urinary bladder or prostate. 9,32-34 It is therefore crucial to determine the concentrations of metals present in food as a practical step for effective risk assessment. 6 While trace metals (often regarded as heavy metals) have been studied extensively and reliable guidelines developed in many jurisdictions, information on metalloid ecotoxicity is relatively limited and not fully understood. 35 One of the few environmentally and biologically important metalloids, arsenic (As) has been widely studied due to its public health implications and identified multiple pathways of human exposure. 36-39 The carcinogenicity of As in humans is well known. Inorganic As is a class one human carcinogen whose chronic ingestion may cause cancers of the bladder, kidney, liver, lung, prostate and skin. 40,41 Environmental pollution resulting from industrial activities such as mining and smelting and production processes can increase the dispersion of As and contamination of agronomic crops through water and atmospheric deposition. 20,35,42-44 Farm irrigation using As-contaminated groundwater has been shown to elevate As concentrations in rice grains. 45 Food standards have not been widely developed for As in many countries, although efficient screening exercises and risk communication strategies are in place in most developed countries to safeguard consumers from As exposure. 20, 46 No such safeguards are in place in many developing African countries.
In Nigeria, there are poor food quality controls and existing mechanisms are not very effective at protecting consumers. Deleterious effects of food contaminants have been underreported in the country as a result of inadequate risk assessment, communication and resource deficits. 47 Contamination of metals in commonly consumed horticultural fruits and effects on human health have not been previously investigated. In this study, we investigated As and trace metals (Hg, Cu, and Pb) contamination in orange (Citrus sinensis), pawpaw (Carica papaya), guava (Psidium guajava), apple (Malus domestica), and pineapple (Ananas comosus) commonly supplied in the Awka region where there has been a history of metal smelting activities. 48 This market basket survey aimed to collate elemental concentration data and compare to internationally acceptable standards for use in preliminary exposure assessments for the local population.

Location Description and Sample Collection
Samples of locally supplied fruits were collected from the largest market in Awka South, which serves the entire Local Government Area and surrounding towns of Awka North. Awka South is in Anambra State of southeastern Nigeria. It is located within ~6.642° N and ~7.067° E ( Figure 1) and experiences two distinct seasons brought about by two predominant winds that rule the area: the southwestern monsoon winds from the Atlantic Ocean and the northeastern dry winds from across the Sahara Desert. Seven months of heavy tropical rains (April-October) are followed by 5 months of dryness (November-March). Awka South is generally hot and humid with a temperature range of 27-28°C from July through December but rising to 35°C between February and April. 2, 49 It has an estimated population of over 400,000 inhabitants based on 2017 population projection. 50 The region is comprised of Awka city (capital of Anambra State), Amawbia, Ezinato, Nibo, Nise, Umuawulu, Isiagu, Okpuno, and Mbaukwu. Awka city is the location of many commercial and social facilities and has a history of indigenous technology and crafts, including wood processing, metal smelting and blacksmithing. The uncoordinated waste management systems that characterize agriculture, wood processing and industrial

Figure 1 -Map of Anambra State and the capital Awka
for each of the market basket survey fruit (orange, pawpaw, guava, apple, and pineapple). It was not determined if use of adulterants had any effects on apples imported into the country; thus, such effects were deemed to be negligible during preparation and analysis.

Sample Preparation
Fruit samples were taken to a laboratory in an air-tight container to prevent further atmospheric deposition that might interfere with elemental analysis. Samples were neither washed nor peeled prior to preparation. The intent was to reflect dietary exposures and common consumer food handling strategies, where fruits are purchased from an open market and consumed without washing. Samples were finely diced in a food processor, which was thoroughly cleaned with 10% extra and rinsed in distilled water after processing each sample. Ground samples were then frozen until further processing.

Analytical Determination of Arsenic, Mercury, Copper, and Lead in Fruit Samples
All samples were analyzed for total As, Hg, Cu, and Pb using established methods with modification. 46,51 Analytical grade reagents were used throughout the metal determination with purity in the range of 98-99.99%. Samples were oven dried at 55°C. Approximately 0.5 g each of the dried fruits was accurately weighed out into 50 ml polyethylene centrifuge tubes; 5 ml of concentrated nitric acid was added to each sample and then left overnight. We digested the samples in a pre-programmed microwave digestion system at a changing temperature and time: 5 minutes ramp to 60°C and then held at 60°C for 10 minutes (400 W); 5 minutes ramp to 75°C and then held at 75°C for 10 minutes (400 W); and 5 minutes ramp metal activities in the area have led to increased environmental pollution through indiscriminate disposal and dumping of tailings. 48 Although there has been a gross decline of these metal processing activities due to rapid technological development, unregulated artisanal production still exists. 48 The city is also one of the most important industrial, economic, cultural, political, and commercial centres in southeast Nigeria. It continues to undergo heavy urbanization and infrastructure development. The industrial facilities in Awka South are scattered at the city centre and surrounding suburbs.
Local fruits were purchased from a wide variety of retailers at the market (known as Eke Awka), including fruits sold openly on market tables and shelves. 20 All fruits were locally supplied except for apples, which are largely imported in Nigeria from South Africa. Ten samples were purchased

Figure 2 -Arsenic concentration of the fruits sampled in the Awka market basket survey. Values represent mean As concentration ±standard deviation, n = 3. Mean values bearing different superscripts are significantly different (Kruskal-Wallis with Bonferroni correction, P < 0.05). Codex indicates Codex
food grade standard. 55 to 100°C and then held at 110°C for 30 minutes (400 W). All microwave digestions were carried out in triplicate with a reagent blank. After cooling, digested samples were diluted to 50 ml and total As, Hg, Cu, and Pb analysis was performed by atomic absorption spectrophotometry at a commercial laboratory.

Quality Control
Each analytical batch of ten runs was accompanied by an acid blank, and three certified reference materials (CRM): apple leaves, (National Institute of Standards and Technology (NIST) Standard Reference Material 1515); pine needles, (NIST Standard Reference Material 1575a); and rice flour, (NIST Standard Reference Material 1568a) to monitor for instrument accuracy and method extraction efficiency. Mixed element internal standards including all examined metals were measured as part of the analytical quality control. Thus, a total of 25 independent digests including reagent blanks and CRMs were analyzed. Mean recoveries were in an acceptable range (75-98.5%) compared to the CRM theoretical or certified values for the elements. All glassware used in the analyses were cleaned with a detergent-free solution, soaked in acid (10% hydrochloric acid) and then rinsed with metal-free distilled water. Metal concentrations were reported in µg/g dry weight at instrumental detection limit of 0.001 µg/g.

Moisture Content Determination
We determined the percentage (%) moisture contents of all the fruits examined to enable transformation of metals concentrations from dry weight (dw) to wet weight (ww) basis, and facilitate the comparison of our data with those of Codex Alimentarius Commission (Codex) guideline values (As = 0.50 µg/ g ww, Hg = 0.10 µg/ g ww, Cu = 2.00 µg/ g ww, and Pb = 0.10 µg/ g ww  52 We compared the concentrations of the metals in fruits using the Kruskal-Wallis method, a nonparametric analysis of variance test based on rank transformation, with Bonferroni correction and Monte Carlo approximation for unbiased estimate of the exact P value (based on 100,000 random tables from the reference set using a starting seed of 2,000,000). 53 Metal concentration values less than the maximum detection level were replaced with 0 prior to analysis.

Figure 4 -Copper concentration of the fruits sampled in the Awka market basket survey. Values represent mean Cu concentration ± standard deviation, n = 3. Codex indicates Codex food grade standard. 55
subsequently used to transform metal concentrations from dry weights to wet weights (Figures 2-5). The mean, minimum, and maximum total As, Hg, Cu and Pb concentrations (dw) for the independent fruit samples collected from Awka market are presented in Table 1 Table 1). Comparatively, in wet weight, the level of As was significantly higher (P < 0.05) than the Codex maximum level for As in fruits ( Figure 2).
The Codex provides a guideline level for methyl Hg of 0.10 µg/g ww. Based on the supposition that methyl mercury is ~83% of total mercury, as presented in Table 1, the concentration of methyl Hg in our samples was calculated in wet weight and compared with the Codex guideline level, and guava and apple methyl Hg concentrations were ~6 and ~1 orders of magnitude higher than the Codex maximum guideline level, respectively ( Figure 3). 54 The amounts of Cu and Pb in the sampled fruits were within the Codex standards (Figures 4 and 5). Apple, pineapple, orange, and paw paw had Pb concentrations of <0.001 µg/g.

Discussion
The present study investigated concentrations of As, Hg, Cu, and Pb in commonly consumed horticultural fruits to assess food quality and make recommendations based on an explicit understanding of human health risks. Metal contamination of edible fruits poses a potential health risk to both humans and animals. Awka South was

Figure 5 -Lead concentration of the fruits sampled in the Awka market basket survey. Values represent mean Pb concentration ±standard deviation, n = 3. Mean values bearing different superscripts are significantly different (Kruskal Wallis with Bonferroni correction, P < 0.05). Codex indicates Codex food grade standard. 55
selected for the current survey because of the open market system in the area, and because physical structural changes are rapidly taking place within its urban ecology, such as continuous road and building construction. These activities are, in many cases, not rigorously planned. Dust pollution and fog are common weather events that characterize the region, and markets are indiscriminately located along roads. Thus, monitoring of food contamination by metals and hazardous substances is necessary for the assessment and reduction of dietary exposures. 6,7,19,23,46,51 The examined fruits had significantly higher total As concentrations (~32-166×) than the Codex maximum food grade level (0.5 µg/g ww). While fruit Cu and Pb concentrations were below the Codex standards, we found that Hg in guava and apple were 6.1 and 1.3 times higher than safe levels for consumption (0.1 µg/g ww), respectively. 55 Fruits are capable of accumulating metals in their internal tissues, which are often species-specific and regulated by factors such as ecological setting and soil type. 56-59 In some cases, surface contamination of fruits with hazardous substances are enabled by poor handling and processing. 6 Our observation of relatively high As and Hg concentrations compared to Codex standards is particularly striking, and appears to indicate similar sources of contamination of these fruits. We postulate that surface/ atmospheric deposition of soil and dust might be the primary mechanism of contamination. 20, 60 Awka has a high population density (~1283.2 inhabitants/km 2 as of the 2006 census) and it is near the metropolitan area of Onitsha which has a much higher population density (2017 projected population of 1,318,660 at +9.03%/ year) and industrial activities. 50 There is scanty vegetation cover Table 1

-Average (minimum-maximum) Arsenic, Mercury, Copper and Lead Concentrations in Fruits Collected for the Awka Market Basket Survey
in Awka, which is subject to wind erosion and consequent agricultural contamination. 2-5,24,25,61 Despite poor handling of fruits at the market where they are sold, wood processing is also a common activity within the vicinity and could contribute to discrete As environmental load and the resultant high concentrations measured in fruits. Arsenate is used in wood preservation and it is converted to more toxic arsenite by incineration of wood waste. 62 With the indiscriminate waste disposal system in Awka, airborne pollutants may deposit on unprotected food displayed for sale. Previous studies have demonstrated that direct atmospheric deposition of As was the dominant pathway of contamination in leafy vegetables grown proximal to a wood preservation factory. 62 Arsenic is a Research well known toxic element to humans with high levels of exposure. 40 About 30%-95% of As is readily absorbed by humans when ingested, and children are more susceptible than adults to toxicity due to the lack of hepatic detoxification enzymes. 63,64 Mercury is one of the most toxic elements among the studied trace metals and exposure to high levels could permanently damage the brain, kidneys and developing fetus. 65 Concentrations of total Hg reported in fruit samples are expected to be mostly composed of methyl Hg. 54, 66 The effects of public health exposure to methyl mercury are critical. Epidemiological studies on daily oral ingestion of methyl Hg concentrations as low as 0.86 µg/g/day from fish alone in expectant mothers have been correlated with neurophysiological disorders in their children by 7 years of age. 67 The highest Hg concentration in both dry weight and wet weight was found in guava and the lowest found in orange. Although atmospheric and surface deposition is speculated to be a primary vehicle of metal mobilization, we cannot rule out the possibility that these metals were also present in the fruit samples as a result of direct uptake from soil, which has been previously reported. 68,69 Furthermore, we did not wash or peel the fruit samples prior to analysis, which may have raised the observed metal concentrations levels, as processing prior to analytical determination appears to reduce metal concentrations in some studies. 20, 70 The lower concentrations of Cu and Pb compared to the Codex guidelines indicates that fruits sold at the market may not have been contaminated with these metals at the time of sampling.
There is lack of information on food trace metal contents in Awka and as this is the first market basket survey of fruits in this area, comparative analysis is difficult. However, the Hg concentrations measured in our work were higher than values (0.022-0.03 µg/g dw) reported in fruits collected from a Saudi Arabian market, but the fruit Cu and Pb concentrations (3.57-7.30 µg/g dw, and 2.62-6.70 µg/g dw, respectively) were ~33 and ~34× the highest Cu and Pb concentrations measured in our work, respectively. 15 Market basket survey of some trace metals in fruits has been previously conducted in Owerri town, an area 117-121 km from Awka South, where a maximum Pb concentration of 4.33 µg/g dw was reported. 71 The Pb level was ~21 orders of magnitude higher than the average Pb measured in our work. Interestingly, our present data on Cu and Pb concentrations in dry weight were ~11× and ~2× the maximum concentrations (0.02 and 0.13 µg/g, respectively) measured in fruits purchased from markets in the most densely populated and industrially active Nigerian city of Lagos. 72 The observed difference in total trace metal concentrations between the published studies indicates variation in metal contaminations in response to spatial characteristics and other factors such as pollutant mobilization, deposition and or contamination rates. Although Cu is an essential element needed for biological mechanisms in both humans and animals, the measured concentrations of Pb in the examined fruits are of concern due to possible bioaccumulation and dietary toxicity. 73 The

Conclusions
The present study shows that the concentrations of common metalloids and trace metals in fruits is varied and highlights the potential impact of anthropogenic activities on metal mobility, deposition, and food contamination. The elevated As and Hg concentrations measured in the fruits indicate potential dietary exposure to metals, and may be of great public health concern. Although Pb concentrations were lower than Codex standards, there is a need to investigate whether these low exposures may contribute to risks of adverse health effects given recent studies indicating health effects at relatively low concentrations. This is a pilot-based screening; larger-scale monitoring is necessary to assess fruits not included in the current survey and correlate contamination levels with metal concentrations in farm soil and atmospheric dust. Food intake assessment methods based on a more comprehensive determination of food types and consumption patterns of the local population need to be implemented and individual consumption data generated to facilitate the risk assessments of metals. Although the present investigation did not examine the effects of adulterants on imported apples, the screening system should be expanded to monitor and control their use on fruits, as there is evidence of adverse health effects associated with adulterants in the food industry. 77 Further studies are needed to continue to improve the database on food metal contents for comparative studies. Moreover, an analytical approach for As and Hg speciation in fruits is necessary for effective food safety assessment. The development of a more robust risk communication framework is needed for better information on specific dietary exposures and food contamination.