Effects of post-harvesting practices on heavy metal levels of mopane caterpillar ( Gonimbrasia belina ) products and associated risk assessment

Mopane caterpillars ( Gonimbrasia belina ) are one of the most consumed edible insects in Southern Africa, and they contribute nutritional benefits to human health. However, their contribution to the food and nutritional security can be hindered by food safety concerns due to heavy metal contamination associated with harvesting, processing and poor post-harvest practices. This study investigated the effectiveness of post-harvest practices (degutting, charcoal roasting and sun drying, boiling, frying and open pan roasting) in reducing the heavy metal contaminants (cadmium (Cd), mercury (Hg), lead (Pb), nickel (Ni), aluminium (Al), zinc (Zn), copper (Cu) and iron (Fe)) in mopane caterpillar sourced from Gwanda district in Zimbabwe. The post-harvest practice categories used in this study included: unprocessed (ungutted and naturally degutted samples), processed (manually degutted, and charcoal roasted and sun-dried), and cooked (boiled and salted; boiled, salted and roasted, and boiled, salted and fried) samples. An atomic absorption spectrometry (AAS) was used for quantification of metals and the values detected were used to assess health risks to consumers using a quantitative risk assessment calculation method. The unprocessed and manually degutted samples showed higher levels Cd and traces of Pb, while charcoal roasting and sun drying resulted in elevated levels of Al, Zn, Cu and Fe. Hg and Ni were not detected in any of the unprocessed, processed or cooked samples. Cooking processes resulted in reduction of the concentrations of detected metals to levels within the permissible values. The health risk assessment disclosed noncarcinogenic risks based on the high hazard index (HI) values (HI > 1) in unprocessed, processed, boiled and salted, and boiled, salted and roasted samples, indicating that caution should be taken. The overall findings from the study shows that by cooking mopane caterpillars through boiling and frying, health risks associated with heavy metal contamination can be reduced.


Insect consumption and nutritional benefits
Utilisation of edible insects as a protein substitute for meat has been promoted highly worldwide due to efficacy reasons and the nutritional benefits associated with the products.Mopane Gonimbrasia belina caterpillars are one of the most commercialised edible insects in Zimbabwe and Southern Africa as a whole (Dube et al., 2013;Kwiri et al., 2014;Manditsera et al., 2018).G. belina are the larval stage of the emperor moth, which bears spines on the body surfaces.G. belina constitutes approximately 55% crude protein value, and contain substantial amounts of potassium, sodium, and calcium (Musundire et al., 2016).As they are a bivoltine species, they emerge in November to December and February to March.This results in the insects being preserved by drying and stored for consumption in the months that they are not usually found unprocessed.

Mopane caterpillar harvesting and post-harvest activities
In Zimbabwe G. belina is abundantly found in Gwanda district which is located in the agro-ecological 4 and 5a zones characterised by low annual rainfall of 450-650 mm, respectively, and temperature range of max 27-30 °C (Manatsa et al., 2020).The main economic activities of the district are mineral mining and agriculture (Food and Nutrition Council and World Food Programme, 2022).With mining activities having a potential to result in contamination of soil, ground and surface water from chemicals such as lead, mercury and cadmium, which can affect human health (Mathe and Phiri, 2016;Stephens and Ahern, 2001), there is a need to assess the heavy metal content of food produced in such areas.Mining practices in Gwanda district, such as the arseniferous gold mining, and nickel and arsenic mines in the western Gwanda and Lower Gwanda greenstone belt (Ashton et al., 2001), have potential to negatively impact the mopane caterpillar breeding environments as these chemical hazards in the environment have potential to be transferred to the mopane value chain activities.Mopane caterpillars are herbivorous, feeding on the mopane tree Calophospermum mopane and burrowing into the soil just before they pupate, at which point they are usually collected for consumption.Presence of heavy metals in the ecosystem has potential for biomagnification if picked up by the primary producers such as plants.Nakayama et al. (2012) presented evidence that shows that biomagnification results in herbivores accumulating higher levels of heavy metals than soil and food plants.This was supported by the work of Imathiu (2020), which showed the ability of edible insects to bioaccumulate heavy metals in their system due to breeding in contaminated environments.Concerns for bioaccumulation of metals then arise especially for mopane caterpillar harvested from such mineral rich areas in which heavy metal deposits can occur.Greenfield et al. (2014), revealed that mopane caterpillar can accumulate elevated levels of heavy metals in their bodies if they are exposed and forage on mopane woodland leaves from polluted areas.Use of contaminated or untreated water for the washing of manually degutted mopane caterpillar can also result in contamination of the degutted insects through residue accumulation on the surface of the edible insects.

Food safety concerns
Rising concerns over food safety is increasingly putting pressure on risk assessment linked to heavy metals.Some of the negative health risks associated with contaminated food include kidney, cardiovascular, bone and nervous system (Anwar et al., 2016).Heavy metals have the potential to create congenital disabilities, low birth weight of born babies (<2.5 kg) and premature births (<37 weeks of completed gestation) (Balalimood et al., 2021;Jyothi, 2021).Other metals such as Cu (Pudpong and Chantangsi, 2015), Ni (Genchi et al., 2020), and Zn (Plum et al., 2010), are required in the human diet in optimum amounts for the essential body function.Cadmium (Cd) (Balali-mood et al., 2021;Jaishankar et al., 2014), Pb (Jaishankar et al., 2014;Jan et al., 2015), As (Tchounwou et al., 2012(Tchounwou et al., , 2019)), and hexavalent chromium [Cr(VI)] (Jaishankar et al., 2014;U.S. Department of Health and Human Services, 2012) may cause carcinogenic effects even in trace quantities, thus their presence in human food is undesirable.
Most research done on heavy metal contamination of edible insects focused mostly on reared insects and other insect species such as termites and grasshoppers (Imathiu, 2020;Kapaale et al., 2022;Musundire et al., 2021;Poma et al., 2017).Among insect-related heavy metals are mercury, cadmium, lead and zinc (Banjo et al., 2010(Banjo et al., , 2013;;EFSA Scientific Committee, 2015;Greenfield et al., 2014;Purschke et al., 2017).Insects collected from natural forests are usually considered to be clean and free of chemicals (Durst et al., 2008).However, due to their lack of controlled environments during their production and growth phase, as well as during their processing, wild harvested edible insects may pose a larger risk for heavy metal contamination (Greenfield et al., 2014;Kapaale et al., 2022).The studies revealed that the sources of these heavy metals include residues from mining factories, refineries and possible wrong application of chemical pesticides near their habitats, the food eaten by the worms and pollutants settling on leaves.Some of the edible insect harvesting sites can be close to agricultural areas as well as mining activities and hence the risk of chemical contamination becomes higher (Greenfield et al., 2014;Kapaale et al., 2022).
Limited research is present to establish the levels of heavy metals present in mopane caterpillar as a result of harvesting, processing and cooking methods.The current study investigates the effects of harvesting and post-harvest practices on heavy metal contamination levels in mopane caterpillar and investigation on the non-carcinogenic risk associated with consumption of mopane caterpillar products.Specifically, we sought to identify the heavy metals that were present in unprocessed, processed and cooked mopane caterpillars; determine the effects of the processing and cooking methods on the levels of the heavy metals and evaluate the health risks to consumers associated with the detected levels.

Study site
Gwanda District, has a total surface area of 46,276 square kilometres in size, and lies in Matabeleland South Province of Zimbabwe.The district consists of 24 wards which are divided into three administrative constituencies namely Gwanda North (wards 1-11), Central (13,14,21,22,23) and Gwanda South (12,15,16,17,18,19,20 and 24, with a total population of 115,778.There are about 26,933 households with an average household size of about 4.3 persons.The area is located in the agroecological region IV, in which the mean annual rainfall lies between 400 mm and 600 mm, and is considered arid according Zimbabwe's agro-climate zones.The region is best suited for livestock rearing and wildlife land use patterns.The mopane woodlands thrive in this environment (Ghazoul et al., 2006).The Gwanda rural district was selected as the study area based on known ecological patterns of mopane caterpillar and suitability of the area as the breeding site (Ghazoul et al., 2006) for mopane caterpillar.Three wards namely, Ward 13 (Sifanjani), Ward 14 (Sengezane and Zhokwe) and Ward 21 (Timber farm) which lie between S 21°04.110E 028°59.737,S 21°20.466E 028°5 8.416 and S 20°57.029E 029°20.475,S 20°59.019E 28°58.152(Figure 1) were selected for this study.

Selection of participating households for sample collection
The detailed sampling protocols utilised for selection of wards and households used in the study are illustrated in Figure 2. Households for study were drawn from a list of the wards as reported by the Zimbabwe Forestry Commission Gwanda Division.The households' lists were used to select insect collectors and households using stratified sampling which was based on insect collection, processing and cooking activities.Samples were collected from willing participants using this criterion.

Sampling and sample preparation
Gonimbrasia belina samples were collected from identified households at different stages along the value chain and the factors (harvesting, processing, cooking practices) influencing the heavy metal concentration of the G. belina products were investigated.One sample was collected at harvesting (fresh ungutted and fresh naturally degutted samples) stage, which was then exposed to the different processes: processing (by manually degutting and charcoal roasting then sun-drying samples) and cooking (by boiling and salting; boiling, salting and frying, and boiling, salting and roasting samples) (Figure 3).The fresh specimens collected at the harvesting stage were the ungutted (A) and naturally degutted (B) mopane caterpillar.Processed samples were then manually degutted (C) and further processed by collectors by charcoal roasting followed by sun drying (D).Cooked mopane caterpillar samples were collected from willing participants after being cooked in the three ways: boiled and salted (E), boiled and salted then roasted (F) and boiled, salted and fried (G).All cooked samples were boiled for 10 minutes and drained, and then boiled for another 30 minutes in salted water.
The cooking processes were done at household level of willing participants.Samples collected from the insect collectors were taken to the households for further processing.No spices or food additives were added.Roasting of mopane caterpillar was done in a flat metal pan with raised edges and using charcoal fitted metal grids at 200 °C for 10-15 minutes depending on the dryness of the roasted mopane caterpillar.Frying of mopane caterpillar samples was carried out in a stainless frying pan containing vegetable cooking oil.Frying was done for 5 minutes.The study was repeated over the two seasons when mopane caterpillars are harvested in this region.Each sample portion was 500 g with a further subsample of 30 g taken at laboratory level.Samples were transported in zip lock bags and cool conditions from the field to the laboratory.The three subsamples of 30 g of each sample were pulverised (Stoops et al., 2016), then stored in polyethylene zip lock bags at −20 °C.

Chemical analysis
The pulverised samples which were packed in zip lock bags and labelled into three replicates were then taken for heavy metal analysis.Heavy metals (toxic metals -Cadmium (Cd), Mercury (Hg), Lead (Pb), and potentially harmful elements -Nickel (Ni), Aluminium (Al), Zinc (Zn), Copper (Cu) and Iron (Fe)) were determined by wet ashing using nitric acid and perchloric acid and then read on an Atomic Absorption Spectrophotometer (AAS) (PinAAcle 900F, Perkin Elmer, Waltham, MA, USA).Duplicates of each individually packed sample were weighed to 0.5 g into digestion flasks, and treated with concentrated 10 ml of nitric acid and 5 ml of per-chloric acid was added.A blank sample was prepared using 10 ml of nitric acid and 5 ml of perchloric acid in an empty digestion flask (Rashid et al., 2016).Digestion of organic material was done by heating the digestion flask with sample and acids until a clear liquid was produced.The solution was cooled and filtered using Whatman No. 42 filter paper before being transferred quantitatively to a clean 50 ml volumetric flask.The filtrate was diluted to the 50 ml mark by addition of deionized water, before samples were analysed using the AA spectrophotometer.Quantification was possible by use of a calibration curve drawn using certified standard solutions.

Quality control and assurance during analysis
The following measures were taken to ensure reliability of the study results throughout the study analysis processes: only good or high purity reagents and chemicals were used; glassware was thoroughly cleaned using detergents and then rinsed several times with distilled water to remove detergent traces.During AAS  detection and quantification, blank solution readings were done three times for the two blank solutions prepared, to allow for standard deviation considerations for noise generation levels.Standard calibration curves were used in the conversion of absorbance to concen-trations reported in the results, with use of certified standard solutions.Detection limits of metals were: Cd < 0.002 mg/kg; Pb < 0.050 mg/kg; Ni < 0.020 mg/kg; Al < 0.0005 mg/kg; Fe < 0.010 mg/kg; Zn < 0.050 mg/kg; Cu < 0.020 mg/kg.

Health risk assessment
Health risks associated with long term consumption of mopane caterpillar contaminated with heavy metals were assessed through calculation of the average daily potential dose (ADD), target hazard quotient (THQ), hazard index (HI), and non-carcinogenic risk (NCR).
The parameters used to characterise the ADD are shown in Table 1.
Using the parameters given in Table 1, the ADD values were calculated using the following equation 1 The symbol C represents the metal concentration in the mopane caterpillar samples, IR is the ingestion rate or amount of food containing the contaminant that an individual consumes, EF is the exposure frequency, ED is the exposure duration pegged at 70 years, BW is the body weight which in this case is given as that of an adult consumer and AT is the average time of exposure for non-carcinogens (days in a year (365 days) ⁎ number of exposure years (assuming 70 years)).Health risk assessment in relation to both non-carcinogenic and carcinogenic effects was done based on the ADD calculation and defined toxicity according to the relationships given by the HQ, HI and NCR (Integrated Risk Information System | US EPA, 2011; Wongsasuluk et al., 2014).The average daily potential dose of the different metals was used to determine the associated human health risks.It was computed by taking into consideration the mean concentrations of the Cd, Pb, Hg, Ni, Al, Cu, Zn and Fe in the various samples under study.
The HQ estimated value is based on non-carcinogenic risks or effects associated with consumption of the contaminated samples.The target hazard quotient is the ratio of the calculated daily dose of the contaminant to the oral reference dose (RfD) considered to be detrimental.Oral reference dose for the analysed heavy metals and minerals used in the determination are shown in Table 2.  (Ortiz et al., 2000).
Hazard quotient was computed as (Equation 2): where ADD is the average daily potential dose and RfD is the reference dose.The ADD values was calculated from the given values, for each metal, which was then used for the HQ and HI calculations.For the HQ values less than 1 (HQ < 1), the daily exposure at this level is unlikely to cause deleterious effects and the effects can be said to be negligible.For HQ values greater than 1 (HQ > 1), noncarcinogenic effects can occur with a probability that will tend to increase as the HQ value increases.
A Hazard index is used to estimate potential human health risk when there is contamination by more than one heavy metal in the consumed food.It is computed as the sum of the calculated HQs for the different heavy metals.

Statistical analysis
Effects of post-harvest activities namely harvesting, processing and cooking practices on the heavy metal concentration of mopane caterpillars were tested using one-way Analysis of Variance (ANOVA).This was coupled with a Tukey post-hoc test in order to identify the significant differences between different samples.The ANOVA tests were preceded by Levene's test, to test for equality of variances.A Pearson correlation was conducted to identify presence of any relationships between the parameters analysed.This analysis also related post-harvest practices and the heavy metal concentration, and the dynamic concentration relationships between heavy metals.

Heavy metal concentration in unprocessed and processed mopane caterpillar
Heavy metal concentration for mopane caterpillar samples sampled at different stages of the value chain are presented in Supplementary Table S1.Lead (Pb), and cadmium (Cd) were detected in the fresh samples of mopane caterpillars in significantly lower values compared to processed samples.Cooked samples did not have detectable levels, (P ≤ 0.05) (Supplementary Table S1).Mercury (Hg) and nickel (Ni) were below detectable levels in all samples showing they were in very low concentrations or not present in the mopane caterpillar samples.Processing insects through charcoal roasting and sun drying resulted in the significant decline of Cd concentration (P < 0.05).Ungutted, naturally degutted and manually degutted samples showed the highest levels of Cd with manually degutted insects showing the highest concentration at 5.88 mg/kg, fresh ungutted samples having 5.28 ± 1.476 mg/kg and fresh naturally degutted samples having 4.59 mg/kg, all being above the WHO/FAO stipulated limit (Supplementary Table S1).Each post-harvest stage resulted in a significant reduction of Cd levels.For example, a significant reduction from 5.88 mg/kg to 0.04 mg/kg occurred between fresh (ungutted and degutted) samples and processed samples (charcoal roasted and sun-dried).
Traceable Lead (Pb) levels were detected in fresh manually degutted mopane caterpillar (0.004 mg/kg), fresh ungutted (0.003 mg.kg) and naturally degutted samples (0.002 mg/kg), whilst all processed and cooked samples were below detectable levels (Supplementary Table S1).The level of lead found in the fresh was below maximum limit (Supplementary Table S1).
Other elements with potential human health effects assessed in this study included Aluminium (Al), Copper (Cu), Zinc (Zn) and Iron (Fe).Charcoal roasting coupled with sun drying had significant effects on Al in insect samples.Fresh samples had undetectable levels of Al, with the processed samples showing significantly high levels of 481.80 mg/kg compared to 176.63 mg/kg in boiled, salted and fried samples.The levels of Al found in the processed and cooked mopane caterpillars was over 200-and 50-fold higher than the permissible levels, respectively.
Copper concentration was also significantly affected by processing.Increasing concentrations for this element were observed with increased handling processes.
Copper concentration in the mopane caterpillar samples was found to be 28.32 mg/kg for ungutted samples (A), 21.95 mg/kg for fresh naturally degutted mopane caterpillars (B) and 18.43 mg/kg for the fresh manually degutted samples (C).However, after processing by charcoal roasting and sun drying, the concentration increased to 33.12 mg/kg, which was then reduced after cooking.Boiling in salted water reduced the Cu level to 25.83 mg/kg, boiling then frying reduced it to 21.32 mg/kg and after boiling then oven roasting the samples, the Cu levels dropped to 30.56 mg/kg.There was a significant difference (P < 0.05) in the Cu levels between all samples, though all levels recorded were below the WHO/FAO stipulated limit (Supplementary Table S1).
The concentration of zinc and iron in cooked mopane caterpillar samples was found to be significantly higher than in fresh samples.From Supplementary Table S1, the average iron concentration in fresh ungutted, fresh naturally degutted and fresh manually degutted sam- ples was found to be 69.13,49.27 and 62.37, respectively.After exposing the mopane caterpillar to charcoal roasting and sun drying processing, the Fe levels increased to 415.73 mg/kg.The employment of the three cooking processes, boiling in salted water, boiling in salted water then frying and boiling in salted water then oven roasting, resulted in the processed mopane caterpillar to have reduced levels of Fe.Boiling reduced the Fe concentration to 179.55 mg/kg, boiling and frying reduced the Fe level to 185.71 mg/kg and boiling and oven roasting the mopane caterpillars reduced the Fe levels to 287.25 mg/kg.The average fresh mopane caterpillar concentration of zinc was found to be in the range 22.71-41.46mg/kg, which was within the tolerable level of 60 mg/kg (Supplementary Table S1).Fresh ungutted mopane caterpillar had 26.41 mg/kg of Zn, fresh naturally degutted mopane caterpillar had 41.46 mg/kg and the fresh manually degutted mopane caterpillar showed the lowest level of Zn at 22.71 mg/kg.Processing of the mopane caterpillar by charcoal roasting and sun drying resulted in an increased level of Zn to 109.07 mg/kg, which was the highest level found in any of the samples.However, after employing them into the three cooking methods, boiling in salted water reduced the Zn concentration to 61.65 mg/kg, boiling in salted water then frying reduced it to 52.46 mg/kg and boiling then oven roasting reduced it to 89.65 mg/kg.Results also showed that cooking by boiling, boiling then frying, and boiling then oven roasting the processed mopane caterpillar reduced the levels of Al, Zn, Cu and Fe.

Correlation of heavy metal parameters
The degree of interrelation and association of the heavy metals under study was assessed using Pearson correlation analysis.Cadmium and lead were shown to have a strong positive correlation whilst both had a strong neg-ative correlation with aluminium (Table 3).Aluminium strongly correlated positively with zinc and iron.The metals that had strong positive correlations demonstrated a potential for mutual associations and similar pollution or contamination sources.For those that showed a strong negative correlation, there is demonstration of no mutual associations being present.This is realised in the case of cadmium versus aluminium, were activities that promoted occurrence of aluminium were responsible for limiting presence of cadmium.

Health risk assessment
In order to assess and estimate the exposure level of the contaminant, exposure routes to the consumer are used to detect the health risk.Different pathways of human exposure to toxins are present including ingestion, inhalation and direct contact.The order of heavy metals with respect to ADD in fresh ungutted mopane caterpillar samples is Fe > Zn > Cu > Cd > Pb = Ni = Hg = Al.However, for fresh manually degutted and fresh naturally degutted samples, ADD of heavy metals was found in the order of Fe > Cu > Zn > Cd > Pb = Ni = Hg = Al (Table 4).For the processed and cooked mopane caterpillar samples, the order of heavy metals with respect to ADD was Al > Fe > Cu > Zn > Cd = Hg = Pb = Ni.The results revealed that ADD of all the heavy metals analysed (Cd, Pb, Hg, Ni, Al, Fe, Zn, and Cu) for the fresh, processed and cooked mopane caterpillar samples were within the provisional maximum tolerable daily intake (PMTDI) values.With Pb, Hg, and Ni the ADD levels remained at 0.000 mg/kg/day for all the fresh, processed and cooked samples.The highest and lowest ADD of Cd, Al, Zn, Cu and Fe in mopane caterpillar were 0.000 mg/kg/day for processed and cooked samples and 0.004 mg/kg/day for the fresh ungutted and fresh manually degutted samples, 0.000   4).It is worth noting that the ADD of all metals was highest in processed mopane caterpillar samples except Cd which was highest in the fresh mopane caterpillar samples.The Hazard index (HI) depicts a value calculated by taking into consideration all HQ for the different heavy metals quantified in the mopane caterpillars.The values of HI ranged from 0.89 for boiled, salted and fried insects to 9.29 for fresh manually degutted insects (Table 5).With the exception of the boiled, salted and fried mopane caterpillar, all other unprocessed and processed insects had HI values greater than 1.This indicates that the contribution of heavy metals along the mopane caterpillar value chain can result in an aggregated health risk via their consumption.
In fresh mopane caterpillar (ungutted and degutted samples) intake HQ of heavy metals followed a different trend to the ADD which was Cd > Zn > Cu > Fe > Pb > Ni = Al = Hg.However, the sequence of HQ of heavy metals in processed and cooked mopane caterpillar samples followed a pattern in the order of Zn > Fe > Al > Cu > Cd > Hg = Pb = Ni and Zn > Ni > Cu > Co (Table 5).The HQ of Cd was highest in fresh mopane caterpillar samples, which exceeded the safe value of 1. HQ of all analysed heavy metals in processed (charcoal roasted and sun-dried samples) and all cooked was under the safe value of 1, indicating that the mopane caterpillar consumption of these heat-treated processed products will not have any significant non-carcinogenic effects on humans.The HI value reflects the cumulative effects of various heavy metals in mopane caterpillar consumption.Based on the HI values calculated for the different mopane caterpillar samples, they are not risk free with the exception of cooked samples that were boiled, salted and fried (Table 5).The HI values were highest in the fresh manually degutted samples at 9.29 and lowest in the boiled, salted and fried samples at 0.86.Hence, there is an urgent need to lower the heavy metals concentration in these sampling sites; otherwise, it may pose serious health hazards to humans in the future.

Heavy metal concentration in unprocessed and processed mopane caterpillar
This study was carried out to determine the extent of the effects of different practices on heavy metals levels in fresh ungutted (A), naturally degutted (B), manually degutted (C) and charcoal roasted then sun dried (D) mopane caterpillars.Fresh mopane caterpillars which have not been heat-treated showed high levels of toxic heavy metal Cd and trace levels of Pb.Manually degutted fresh mopane caterpillar samples were shown to have the highest levels of Cd.The levels of Cd found in the fresh mopane caterpillar samples were higher than those found in mopane caterpillar harvested from the wild in a study by Greenfield et al. (2014) which ranged from 2.93-4.02mg/kg.This study showed even higher Cd levels when compared to other types of edible insects, as can be seen in the study by Poma et al. (2017) who studied Greater wax moth larvae, Locusta, Mealworm, and buffalo worm and found that the unprocessed edible insects had Cd levels ranging from <0.03-0.06mg/kg ww and Hyun et al. (2012) found that in edible grasshoppers from Korea Cd was at a mean level of 0.02 mg/kg.The higher level of Cd shows how the environment associated with mopane caterpillar harvesting and processing can be contaminated by metal residues from the mining or other activities done in the area.According to Imathiu (2020), the studies on safety of edible insects harvested from natural forests with regard to toxic heavy metals is very limited.The accumulation of these toxic heavy metals is a result of bioaccumulation caused by pollution and human activities (Uddin et al., 2021).These high concentrations thus require establishment of measures to regulate or stop the contamination or pollution of the collection and production area.
In the fresh mopane caterpillar samples, the Cd and Pb concentration was reduced when the fresh samples were processed and cooked by boiling (E), boiling then frying (F) and boiling and roasting (G) (P > 0.05).From the results obtained, the average Cd concentration in the fresh mopane caterpillar was high above the recommended levels, however, after employing the processing and three cooking methods, charcoal roasting and sun drying the mopane caterpillar decreased the Cd concentration (P < 0.05), which was further decreased by cooking the mopane caterpillar to undetectable levels.The reduction of Pb from the levels found in the fresh unprocessed samples (A and B) to the non-detectable levels in the processed samples can be attributed to the reduction of water in the mopane caterpillar as a result of the charcoal roasting process and sun drying.This process could invariably reduce the Pb in the processed mopane caterpillar samples since Pb is found mostly in the cytoplasm of the cell (Joyce and Emikpe, 2016), which was reduced during charcoal roasting.The drop in heavy metals concentration in cooked samples can also be attributed to effects of the boiling process (E), which would allow for the metals to leach out and possibly through conversion of the metals to other compounds.Joyce and Emikpe (2016) as well as Phrukphicharn et al. (2021) stated that heavy metals are elaborated through various processes such as evaporation of water and solubilisation of the metal, hence, this might have contributed to the reduction of the toxic heavy metals during processing (D) and cooking practices (E, F, G).A study by Lee et al. (2019), showed that by boiling food (noodles) and discarding the water, the heavy metal content of the food reduces.This was attributed to how heavy metals were water soluble, and how boiling would allow for the leaching to occur.According to a study by Kananke et al. (2015) on effects of processing on heavy metal concentrations, combining boiling and frying during cooking of food results in lowered levels of Ni, Cd, Cr, Pb, and Cu.This study supports the results found for cooked samples (boiled, fried and roasted), as the Cd was below detectable levels.From their study heavy metals were shown to be elaborated through various means such as evaporation of water, solubilisation of the metal or element and combining with other components present in the food such as carbohydrates, proteins or lipids (Kananke et al., 2015), as a result of the cooking methods employed (boiling, steaming or frying).The drop in concentration of heavy metals in the processed samples was supported by the work of Ali et al. (2011) and also Eboh and Mepba (2006) on fish meat studies who attributed the reduction of heavy metal concentration to heat effects and the likelihood of the heavy metals being converted to other compounds.
Both Hg and Ni were below detectable levels in the fresh unprocessed, processed and cooked mopane caterpillars.The Ni levels found were lower than levels found in the Greater wax moth larvae, Locusta, mealworm and buffalo worm in the study by Poma et al. (2017) which ranged from <0.03-0.28mg/kg ww.Insects collected from natural forests are usually considered to be clean and free of chemicals (Durst et al., 2008).Although the insects were gathered from natural habitats, they showed the presence of toxic heavy metals such as Cd and Pb in high (over 10 times more than maximum recommended) and trace amounts, respectively.Heavy metals such as Pb, Hg, As, and Cd are capable of inducing toxicity even at low levels of exposure (Jan et al., 2015).Bioaccumulation of the heavy metals can result in consumers due to consumption of contaminated insects, leading to adverse health conditions.Cd is a residual or zinc by-product that can be found mostly in foods such as mushrooms, shellfish, freshwater fish, dried algae, and potable water, among others (AGQ Labs USA, 2019).Presence of such toxic heavy metals in the insects is a reflection of its habitat and their presence in the insects denotes contamination in the environment.Azam et al. (2015) conducted a study on heavy metal contamination of edible insects and found Cd in the insects, suggesting that insects can be used as indicators of heavy metal contamination in the environment.
Al was not detected in fresh mopane caterpillar but was found in high levels in the processed mopane caterpillar.The low levels in the fresh samples is a contradiction to the levels found in mopane caterpillar in the study by Greenfield et al. (2014), which ranged from 40.02-41.43mg/kg.The increase in Al in the charcoal roasted and sun-dried mopane caterpillar can be attributed to the effects of direct roasting of mopane caterpillar with charcoal contact and use of wire mesh.Studies by Elemo et al.(2021) as well as Semwal et al. (2006), show that continuous use of stainless steel and aluminium cookware and utensils can result in leaching of Al, Cu, Zn into the food resulting in increased levels of the minerals.As can be seen from the results, the processing of the mopane caterpillar with metal utensils and cookware resulted in the increased levels of the elements.According to Ramos et al. (2019), Vincevica-Gaile et al. (2021), and Wang and Dibdiakova (2014) as well mineral elements including Al can be present in wood ash as both oxides or in trace contents as the element.With direct contact of the mopane caterpillars with the charcoal, traces of the ash or charcoal will be present in the charcoal roasted samples.Aside from charcoal roasting another source of aluminium might be the other equipment and utensils that might be used for mopane caterpillar processing, including the wire mesh used during charcoal roasting processes.According to a study by Turhan (2006), using high aluminium packages and equipment to cover food during high heat treatments or processes can result in increased levels of aluminium in baked food.
Copper concentrations in the fresh, processed and cooked mopane caterpillar was found to be in a range well within the permissible level.However, the levels of the Cu after processing increased significantly (P < 0.05) and the cooking processes reduced the levels.The levels of Cu in the fresh mopane caterpillar were found to be higher than that of other edible insects including edible termites ranging 0.08-0.16mg/kg (Kapaale et al., 2022), locust ranging 3.47-5.31mg/kg ww (Poma et al., 2017), showing how mopane caterpillars are a good source of Cu in the diet.However the levels of Cu in the unprocessed and fresh mopane caterpillar was different from the reported values of Greenfield et al. (2014) which ranged from 43.48-67.73mg/kg, in the different sampling areas.The Cu level increased in the mopane caterpillars after processing by charcoal roasting and sun drying.A similar trend to Al is seen in the samples after exposing the mopane caterpillars to different cooking conditions.The increased level of Cu from the processing decreased significantly (P < 0.05), reducing the Cu concentration to a more acceptable level.A study on field cricket and Dune cricket by Soren et al. (2021) showed that with roasted crickets, they contain again a lower level of Cu (0.085 mg/kg and 0.065 mg/kg, respectively) as compared to the high level in the roasted mopane caterpillar.Copper is a mineral with crucial roles in the human diet, including to help with collagen development as well as for iron absorption.However, care should be taken to consume it in the correct amounts because of the severe health disorders it provokes such as anaemia, stomach problems, and harm to the kidneys and the liver (de Romaña et al., 2011).It can be categorised as a heavy metal when in high doses.Copper is a major concern because it is prevalent in homes and cookware, making exposure to copper at times higher than normal.It is necessary in small amounts for human health, growth, and sense of taste.Possibly safe when taken in larger doses for a short period, however, taking doses higher than 40 mg daily might decrease how much copper the body absorbs (Singh et al., 2010).According to Rahman et al. (2014) and also Sarker et al. (2022), consuming excess levels of Cu can result in other side effects such as stomach aches and liver damage.The results showed that the levels of copper in both unprocessed and processed samples was within the allowed dose, thus it was not an element of major concern in mopane caterpillar consumption.
Iron concentration showed a significant difference (P < 0.05) between the fresh ungutted and fresh manually degutted mopane caterpillar to the fresh naturally degutted mopane caterpillars.The naturally degutted fresh samples had the lowest level of Fe.Charcoal roasting and sun drying the samples increased the Fe concentration significantly (P < 0.05), while cooking by boiling, frying and open pan roasting reduced Fe concentration levels.Though the average iron concentration for processed mopane caterpillar was high, it was still within the permissible level for human consumption.This increase of Fe could be attributed to the interactions between the mopane caterpillar and the metal grid which is often made of iron (Joyce and Emikpe, 2016), and the metals present in the charcoal used in direct contact with the mopane caterpillar.Therefore, during charcoal roasting, some of the Fe particles can get into the mopane caterpillar.The results for cooked samples are in line with Fe levels found in mopane caterpillar samples cooked in the similar way in a study by (Hlongwane et al., 2022), though the zinc levels were found to be lower in the research.In their study, Fe in cooked mopane caterpillar ranged from 149.77 to 191.44 mg/kg, whilst Zn ranged from 133.53 to 135.30 mg/kg.This is contradictory to the study by Manditsera et al. (2019), which reported that cooking in any way will not have a significant effect on the iron and zinc levels of the edible insects.The discrepancy could be due to the ash roasting and sun drying processes done for the mopane caterpillar.However, the losses can be attributed to boiling time, chemical form of the element and the food matrix in which the elements are found.Other studies conducted on tree locusts (El-Lahamy et al., 2019;El Hassan et al., 2008), showed that both zinc and iron levels remained stable after boiling.According to Payne et al. (2015), cookware can have an effect on the levels of some metals and minerals found in edible insects.Da Silva et al. (2017) reported that for most minerals, losses are incurred during the boiling process, as they are lost into the boiling water.

Health risk assessment
Food safety analysis was mainly affected by the high HQ for Cd concentrations in the fresh samples (Table 5).According to Huang et al. (2008) and also Lim et al. (2008), if there are HQ values greater than 1, there are potential health risks associated with consumption of the food product under study.However, considering that mopane caterpillars are rarely consumed in their fresh unprocessed state, the values will not be a major risk.As a food that undergoes several processes before consumption, this high HQ will be of no consequence as long as the mopane caterpillars are processed or cooked before consumption.Results of the HQ of all metals under study were below 1, suggesting an acceptable and tolerable level of non-carcinogenic adverse health risk, with the exception of Cd (Table 5).
The study showed that the HI was high (HI > 1) showing that the heavy metal contaminants in mopane caterpillars are extremely high, with the exception of mopane caterpillar that undergo boiling and frying before consumption.The values indicate that there is a significant non-carcinogenic health risk to mopane caterpillar consumers if the proper processing and cooking conditions are not met.The health risks perceived from long-term exposure from consumption of mopane caterpillars with high levels of metals can be more severe for special populations.Special populations include pregnant women, people with weak constitution and who are sensitive.Aside from the mopane caterpillars other consumed, supplementary and complementary foods such as vegetables (Ametepey et al., 2018), fish (Wang et al., 2005) and meat (Barone et al., 2015;Bortey-Sam et al., 2015;Zhang et al., 2009;Zheng et al., 2007) can also contribute to more elevated levels of heavy metals in the consumer's diet.

Conclusions
In conclusion, the different post-harvest practices employed in mopane caterpillar value chain had effects on the various heavy metals' concentrations.The application of heat in the processing and cooking practices reduced the levels of the toxic heavy metals (cadmium and lead) found in the fresh mopane caterpillar.For aluminium, iron (Fe), zinc (Zn) and copper (Cu), their concentration is affected by the processing and different cooking methods.Charcoal roasting with sun drying practices increased their levels, which would be reduced to permissible levels by the cooking practices employed.Boiling, then frying the mopane caterpillars have the capacity to reduce the concentration of the heavy metals to an acceptable level considering it was the method that produced the best results.These findings will help to inform measures in consumer safety.
On the issue of food safety, significant differences were found in the content of these heavy elements in mopane caterpillar from the different levels of mopane caterpillar value chain.The average concentrations of Cd, Pb, Al, Zn, Cu, and Fe were statistically significantly different for the fresh, processed and cooked mopane samples.However, HQ calculations showed that the HQ in all metals is far less than 1 in all the mopane caterpillar samples, with the exception of Cd in fresh mopane caterpillar.If all other routes of entry of heavy metal and their combined effects are considered the potential health risks for residents might actually be higher.The hazard index exceeded 1 for all unprocessed and processed insects, with the exception of boiled, salted, fried insects, suggesting potential for future health risks.However, from the study the consumers can be recommended to employ boiling and frying cooking practice as the process reduced the heavy metal content to an acceptable level, thus yielding a safer product.Based on the findings of this study, development of proper and ideal processing and cooking procedures can be done to help reduce the heavy metal levels in the mopane caterpillars and thus prevent any potential health risks.

Figure 1
Figure 1 Geographic location of study area.

Figure 2
Figure 2 Sampling framework for the selection of 4 sampling points from different wards in Gwanda district.

Figure 3
Figure 3 Sampling points in the mopane caterpillar value chain.

Table 3
Correlation matrix for heavy metals in unprocessed and processed mopane caterpillar ⁎⁎ Correlation is significant at the 0.01 level (2-tailed); ⁎ Correlation is significant at the 0.05 level (2-tailed); aCannot be computed because at least one of the variables is constant.

Table 4
Average daily potential dose (ADD) (mg/kg/day) for unprocessed and processed mopane caterpillars