Exposure and risk characterizations of ochratoxins A and aflatoxins through maize (Zea mays) consumed in different agro-ecological zones of Ghana

Mycotoxin contamination of foodstuffs is a serious food safety concern globally as the prolonged ingestion of these toxins has the tendency to worsen the risk of hepatocellular carcinoma. This study aimed at estimating ochratoxin A (OTA) and aflatoxin (AF) levels above international (European Food Safety Authority, EFSA) and local (Ghana Standards Authority, GSA) standards as well as the health risks associated with the consumption of maize (n = 180) sampled from six (6) regions representing the agro-ecological zones of Ghana. OTA and AF were measured with High-Performance Liquid Chromatography with a Fluorescence detector. Out of the 180 samples analyzed for total aflatoxins (AFtotal), 131/180 tested positive and 127 (70.50%) exceeded the limits of EFSA and ranged 4.27–441.02 µg/kg. While for GSA, 116 (64.44%) of samples exceeded this limit and ranged between 10.18 and 441.02 µg/kg. For OTA, 103/180 tested positive and 94 (52.22%) of samples between the range 4.00–97.51 µg/kg exceeded the tolerable limit of EFSA, whereas 89 (49.44%) and were in the range of 3.30–97.51 µg/kg exceeded the limits of GSA. Risk assessment values for total aflatoxins (AFtotal) ranged between 50 and 1150 ng/kg bw/day, 0.4–6.67, 0–0.0323 aflatoxins ng/kg bw/day and 1.62–37.15 cases/100,000 person/year for Estimated Daily Intake (EDI), Margin of Exposure (MOE), Average Potency, and Cancer Risks respectively. Likewise, ochratoxin (OTA) values were in the ranges of 8.6 × 10–3–450 ng/kg bw/day, 0.05–2059.97, 0–0.0323 ochratoxins ng/kg bw/day and 2.78 × 10–4–14.54 cases/100,000 person/year. Consumption of maize posed adverse health effects in all age categories of the locations studied since the calculated MOE values were less than 10,000.


Materials and methods
Sample collection. To collect a representative data set, we first obtained the list of villages in each district from the Regional Directorate of the Ministry of Agriculture. From each district, an average of 5 villages (Table 1) was then randomly selected. The maize sellers in each market were conveniently sampled where about one kilogram (1 kg) of raw maize samples were purchased concurrently from July to December 2020. Five hundred (500) grams each of maize samples were fetched and kept in sterile bags in ice chests and sent to the laboratory within the same day in a vehicle where they were stored in a deep freezer at − 20 °C until ready for chemical analysis 30 . Determination of Ochratoxins A. Chemicals and standards. The analytical standard of OTA was supplied by Sigma-Aldrich (St. Louis, MO, USA). All solvents used for the preparation of the mobile phase were Table 1. Geographical locations and some attributes of the origin of samples. www.nature.com/scientificreports/ HPLC grade and obtained from Merck (Darmstadt, Germany). Methanol and hexane used for extraction were of analytical grade supplied by Sigma-Aldrich. All homogenized mixtures and eluates were filtered through Whatman no. 4 and 0.45 mm membrane filters respectively (Whatman plc, Maidstone, UK). De-ionized water was obtained with a Millipore Elix Essential purification system (Bedford, MA, USA). OCHRA PREP immunoaffinity columns were supplied by R-Biopharm, Rhone limited, and used for SPE and cleanup. These columns have a concentration capacity of 100 ng/mL with at least 90% recovery. Phosphate-buffered saline (PBS) was prepared by dissolving PBS tablets (Sigma-Aldrich) in distilled water. Sodium chloride (≥ 99.0%) was sourced from Sigma-Aldrich. Six-point calibration was made using the pure Ochratoxin A standard at concentrations of 5 µg/kg, 10 µg/kg, 15 µg/kg, 20 µg/kg, 25 µg/kg and 30 µg/kg. Linearity was accepted at 0.99 or 99% for the regression curve (CEN official method EN14123 33 ).
Determination of Ochratoxin. Ochratoxin A was determined based on CEN official method EN14123 (2007) 32 . About 500 g each of maize was sampled by thoroughly mixing and heaping the whole batch into a cone. Using cardboard, the heap was divided into four equal parts. Two opposite parts were mixed, and the remaining two parts were packed, and the process repeated until a representative 500 g sample was achieved and ground into fine maize powder and groundnut slurry. Exactly 25 g of powdered or slurred samples were extracted with 5 g Sodium Chloride and 200 mL methanol in distilled water in a ratio of 4:1, respectively. Hexane (100 mL) was added to the groundnut mixture and the samples homogenized for 3 min (i.e., 3000 rpm for 2 min and at 3500 rpm for 1 min). The groundnut mixture generated two organic layers (the hexane upper layer and methanol lower layer). The lower methanol layer of the groundnut mixture and the maize mixture was filtered through Whatman number 4 filter paper. Ten milliliters (10 mL) of filtrate were used for Ochratoxin A solid-phase extraction and cleanup. Exactly 150 mL of phosphate buffer saline (PBS) was added to 10 mL of filtrate and the mixture stirred. Immuno-affinity columns specific for Ochratoxin A were pre-conditioned and antibodies in the column were activated by eluting 10 mL of phosphate buffer saline through columns at a flow speed of 3 mL/min. Exactly 50 mL of the 160 mL filtrate-PBS mixture was loaded onto pre-conditioned immune-affinity columns specific for Ochratoxin A and allowed to drain by gravity. The columns were washed three times with 5 mL PBS and allowed to elute at a flow rate of 5 mL/min. Using a vacuum pump, the air was blown through the columns to get rid of all wash solvent molecules. Ochratoxin A was eluted in two steps into a 5 mL volumetric flask by first eluting with 1 mL of methanol (highest grade) followed by another 1 mL of methanol after one minute. Air was blown through the column to collect all eluates. Aqueous acetic acid (1%) was used to make up the volume of eluate to 4 mL and eluate vortexed, after which 2 mL was pipetted into HPLC vials for quantification.
HPLC parameters. Agilent high-performance liquid chromatography system (HPLC 1260 infinity series) with a quaternary pump and fluorescence detection was used for OTA quantification. Data acquisition and quantification were done using Chem station (Open Lab edition). The Agilent HPLC equipped with a fluorescence detector was set at an excitation wavelength of 333 nm and an emission wavelength of 467 nm and the column compartment temperature regulated at 30 °C. The mobile phase was a mixture of 5 mM sodium acetate with acetic acid (pH 2.4): methanol: acetonitrile at ratios of 40:30:30, respectively, and an isocratic delivery mode employed at a flow rate of 1 mL/min with an injection volume of 10 µl. The run time was set at 10 min (CEN official method EN14123 33 ).
Aflatoxins determination. Extraction of samples. AFB 1 , AFB 2 , AFG 1 , and AFG 2 were extracted from the samples according to the European Committee for Standardization (CEN) official method EN14123 33 for aflatoxin extraction. Methanol in water (200 mL) (8 + 2) and 5 g NaCl were used to extract 25 g of sample. Hexane (100 mL) was added to samples containing more than 50% fat. The mixture was homogenized for 3 min at 3000 rpm (2 min) and 3500 rpm (1 min). The extracts were filtered and 10 mL of the filtrate added to 60 mL of phosphate buffer saline (PBS) for solid-phase extraction using a preconditioned immune-affinity column specific for, AFB 1 , AFB 2 , AFG 1 , and AFG 2 . The 70 mL filtrate-PBS mixture was loaded onto the preconditioned column and allowed to elute by gravity at a flow rate of 1 mL/min. This was followed by a cleanup with 15 mL distilled water at a flow rate of 5 mL/min. Aflatoxins were eluted in two steps into a 5 mL volumetric flask with 0.5 mL followed by 0.75 mL of methanol (HPLC grade) and allowed to elute by gravity. Deionized water was used to make up the volume of eluate to 5 mL and eluate vortexed and 2 mL pipetted into HPLC vials for quantification.

Limit of detection/quantification (LOD/LOQ).
Limits of detection and quantification (LOD/LOQ) of the HPLC were estimated by making a calibration curve around the standard used for spiking, 5 µ/kg (the lowest concentration range of the calibration curve). Blank did not produce any signal, so the LOD and LOQ were calculated as; Measurement accuracy. Spiking of pure aflatoxin standard solution was done to ensure the measurement accuracy of the analysis. Three levels of spiking were done at the lower, mid, and upper concentration range of the calibration curve concentrations (5 ppb, 15 ppb, and 30 ppb). Spike volumes of pure standards were calculated as; Spike volumes were distributed evenly on aflatoxin free sample (blank) and the spiked sample analyzed for percentage recovery which was calculated as; Measurement precision. Repeatability and intermediate precision analyses of an internal reference material (IRM) were used to ensure the measurement precision of the method. For repeatability analysis, 10 parallel extractions of the IRM were done by the same analyst at the same time using the same HPLC and the relative standard deviation between the results was calculated. For intermediate precision, 10 extractions of the IRM were done on different days by different analysts, and the relative standard deviation between the results was calculated. The relative standard deviations were calculated as; [Standard deviation/mean] * 100 (CEN official method EN14123 33 ).
Required performance criteria for accuracy and precision. Repeatability: Relative standard deviation among repeatable results should be less than 15%.
Intermediate Precision: Relative standard deviation among results obtained under intermediate precision conditions should be less than 20%.
Recovery: Percent recovery of the measurement procedure should be in the range of 80-120%. Limit of Detection: The limit of detection should be less than 1 ug/kg for all aflatoxins. Limit of Quantification: The limit of quantification should be less than 3 ug/kg for all aflatoxins. Linearity: Linearity of the regression curve should be 0.99 (B1, B2, G1) and 0.98 (G2).
(1) LOD = 3 * standard deviation/slope.    Risk assessment of exposure to total aflatoxins via consumption of maize. Exposure estimation. Estimated Daily Intake (EDI) was considered by using the mean quantities of mycotoxins (ochratoxins or aflatoxins) derived from the cereal samples, the number of samples consumed daily, and the average body weight. The EDI for mean aflatoxin was premeditated according to the following formula (5) and expressed in μg/kg of body weight/day (μg/kg bw/day) 34,35 .
The daily intake of maize in Ghana according to MOFA-IFPRI (2020) in Kortei et al. 2 is approximately 0.107 kg/day (39.3 kg/year).
The different age categories according to EFSA 36 and their corresponding estimated average weights in Ghana used in this study were done as follows; Infants-2.9 (2.5-3.2) kg 37,38 , Toddler-9.8 (7-12.6) kg 39 Margin of exposure characterization for aflatoxins and ochratoxins. Genotoxic compounds such as aflatoxins and ochratoxins have their risk assessments fittingly computed based on the Margin of Exposure (MOEs) approach, which was estimated by dividing the Benchmark dose lower limit (BMDL) for aflatoxins is 400 ng/kg bw/day by toxin exposure 23,45 .
A public health alarm is raised in instances where MOEs are less than 10,000 for both ochratoxin A and aflatoxins.
Estimated liver cancer risk due to consumption of maize. The ingestion of aflatoxins, likewise ochratoxins, can be linked to the onset of liver cancer 48,49 . Therefore, liver cancer risk estimation for Ghanaian adult consumers was calculated for aflatoxins 45 . This involved estimating the population cancer risk per 100,000, which is a product of the EDI value and the average hepatocellular carcinoma (HCC) potency figure from individual potencies of Hepatitis B surface antigen (HBsAg) (HBsAg-positive and HBsAg-negative groups).
Thus, the cancer risk (cancers per year per 100,000 population per ng aflatoxins/ochratoxins /kg bw/day) was estimated using the following formula in Eq. (8) 2,45 : The use of plants in the present study complies with international, national, and/or institutional guidelines. Statistical analysis. The ochratoxins and aflatoxin concentrations were calculated using regression analysis from the curves generated from the standards of ochratoxins/aflatoxins with Excel for Microsoft Windows (version 10). SPSS 22 (Chicago, USA) was used in the analysis of data. Descriptive analysis was performed to

Results
Occurrence of aflatoxins and ochratoxins A. Samples tested produced good linearity or coefficients of correlations (R 2 > 0.990) within the tested range. For the recovery analysis, samples previously tested to guarantee the nonappearance of the studied mycotoxins were used in the validation procedure. For ochratoxins, the limit of detection was 0.83 µg/kg while the Limits of Detection for AFB 1 and AFB 2 , likewise AFG 1 and AFG 2 , ranged between 0.13 and 0.15 µg/kg. The limit of Quantification for ochratoxins was 2.49 µg/kg. Aflatoxins ranged between 0.39 and 0.45 µg/kg, respectively, for both ( Table 2). Out of a total of one hundred and eighty (180) samples tested, 131 tested positive. The general trend of occurrence of aflatoxins was in the decreasing order of AFB 1 > AFB 2 > AFG 1 > AFG 2 and were in the ranges of 0-337 µg/kg, 0-101.00 µg/kg, 0-24.80 µg/kg, and 0-5.51 µg/kg respectively. The aggregated aflatoxins (AFtotal) were in the range of 0-441.02 µg/kg. While for ochratoxins (OTA), 103 samples tested positive. OTA levels were also observed to be lesser than AFB 1 , AFB 2 but more than AFG 1 and AFG 2 and ranged between 0 and 97.51 µg/kg. There were significant (p < 0.05) differences observed in all categories of the tested samples. For the Upper East region representing the Sudan Savanna zone ( Fig. 1), the range of values was 0-106.18 µg/kg for Total aflatoxins. 32.84, 30.35, and 668.51 µg/kg were recorded from the summary statistics as mean, median, and variance, respectively, while 0.83 and 0.89 were recorded as skewness and kurtosis respectively which implied a symmetrical and normally distributed data for Total Aflatoxins (AFtotal) (the distribution is not outside the range of normality) (  75 represented the mean, median, and variance. Skewness and kurtosis were 0.77 and − 0.50, respectively, and the implied distribution produced fewer and less extreme outliers than did the normal distribution or were fairly symmetrical and light-tailed. There was a significant (p < 0.01) correlation between AFB 1 and AFtotal in this data set ( Table 4). The Northern Region (Guinea Savanna) zone recorded a range of values of 0-285.31 for Total aflatoxins. Values of 48.93, 25.09, and 4214.84 µg/kg were recorded for mean, median, and variance, respectively, while the skewness and kurtosis were 2.14 and 5.35 respectively and showed that the data set of Total aflatoxins (AFtotal) obtained in this zone was asymmetrical and heavy-tailed (Table 5). Ochratoxins (OTA), were within the range of 0-46.77 µg/kg. Values of 14.14, 6.70, and 232.47 µg/kg were recorded as mean, median, and variance, respectively, while 0.85 and − 0.58 were obtained as skewness and kurtosis respectively which suggested a fairly symmetrical distribution and light-tailed. There were significant (p < 0.01) correlations established between AFB 1 and AFtotal as well as AFtotal and OTA (Table 6). Total aflatoxins for the Ashanti region representing Transitional zones were within the range of 0-230.00 µg/kg, while the mean, median, and variance recorded were 62.37, 46.77 and 4247.89 µg/kg respectively. The data set showed symmetrical and light-tailed as the skewness and kurtosis were 1.12 and 0.71, respectively (Table 7). Ochratoxins were also in the range of 0-76.05 µg/kg. Mean, median, and variance were 11.52, 0.00, and 403.94 µg/kg, respectively, with Skewness and Kurtosis of 1.70 and 2.5, suggesting the distribution is not outside the range of normality (Table 7). Significant (p < 0.01) correlations were established between AFB 1 and AFtotal, and likewise AFtotal and OTA (Table 8). For Eastern Region representing the Semi-deciduous zones recorded a range of 0-441.02 µg/kg. Mean, median, and variance of 53.11, 4.83, and 10,840.01 µg/ kg, respectively, were recorded. The data set for the Semi-deciduous zone was asymmetrical and light-tailed (2.48 and 6.24 for skewness and kurtosis, respectively, for Total aflatoxins (AFtotal) ( Table 9). Ochratoxins levels ranged between 0 and 97.51 µg/kg. Mean, median, and variance recorded were 17.75, 4.10, and 827.54 µg/kg, respectively. The data set was symmetrical and light-tailed (Skewness and Kurtosis were 1.78 and 1.97 respectively). Significant (p < 0.01) correlations were recorded between AFB 1 and AFtotal, and likewise AFtotal and OTA (  53 and Ghana Standards Authority (GSA) 2,54 regulatory limits for total aflatoxins (AFtotal) used were 4 and 10 µg/kg respectively. While ochratoxins (OTA) limits used were 4 and 5 µg/kg, respectively, for the two institutions 55 (Table 15) (Table 17).

Discussion
The discrete incidence of ochratoxins and aflatoxins in foodstuffs is quite common in cereals and is a worldwide problem during pre-and post-harvest stages 56 . However, the concomitant occurrence of these mycotoxins has not been researched adequately in Africa. In this study, a range of 0-97.51 μg/kg (mean 48.76 μg/kg) of ochratoxins was obtained and were comparable to other previous studies globally. Nalle 64 reported ochratoxins (OTA) levels that ranged from 2.14 to 214 μg/kg which was detected in 71% of commercially grown maize samples in Pakistan. Makun et al. 65 reported that samples of maize showed the highest levels of OTA were in the range of ND to 139.2 μg/kg in Nigeria. Conversely, Iram et al. 66 did not detect ochratoxins in maize samples obtained from Punjab, Pakistan. Adebajo et al. 67 also detected OTA in cornbased snacks in Nigeria only at toxicologically significant levels. The co-occurrence of different mycotoxins in one commodity created by fungal genera is common 68 . The permissible limit of OTA recognized by FAO/WHO Joint Committee Experts on Food Additives is 100 ng/kg/week and 14 ng/kg/day, whereas European Food Safety reestablished 120 ng/kg of OTA, which is nearly 17.1 ng/kg 47 . Ochratoxins have been implicated in a variety of adverse health effects both in humans and in animals suggested to be reached from renal, neuro-, immuno-, and embryo toxicity to muta-or teratogenicity 69 . OTA was proven as a renal carcinogen in rodents 70 , nevertheless its transferability to humans is still not clear 71 . Its carcinogenicity has been adopted to be related with genetic changes leading to a new assessment so that OTA can also be considered as genotoxic/mutagenic 72 . The high presence of OTA might be attributed to other fungal species that have not yet been explored or due to other pieces. Aflatoxin level recorded in this study were in the range of 0-441.02 μg/kg, which is within the range of the values reported by Adebajo et al. 67 74 reported greater quantities of aflatoxin > 1000 ppb in maize samples from contamination due to aflatoxin of commercial maize products during an outbreak of acute aflatoxicosis. Dadzie et al. 73 recorded Total aflatoxins levels in the samples per community were in the range of limit of detection (LOD) with 692 ng/g, 23 ng/g, 945 ng/g, and 112 ng/g for Fumesua, Wenchi, Ejura, and Akomadan, respectively. Lower aflatoxin levels have been reported in other studies. de Souza et al. 75 reported quantities as low as 0-16 in Brazilian maize and maize-based foods. Total aflatoxin values of 50.234, 70.102, and 30.943 ng/g were, respectively, obtained from three composite samples taken from the Ejura market was reported by 76 . Danso et al. 77 reported aflatoxin levels of 2.9-3.4 ppb in all markets in the minor season maize samples, but levels ranging from 38.2 to 64.0 ppb were found in the major season samples. Total aflatoxin levels of 82.9 ppb, 48.9 ppb, and 48.9 ppb were recorded for maize samples stored in polypropylene sack, hermetic bags, and local crib respectively by 78 .
The variation in contamination levels as seen in the different agro-ecological zones could be attributed to speckled infection levels of the toxigenic fungus genus, Aspergillus (esp. flavus), owing to their ubiquitous nature, infect maize grains in the field even before harvest 79,80 . Ghana is reliant on rain-fed agriculture which is coupled with high temperatures and unavailability of regular rains, the crop is left under stress which predisposes the crop to fungal invasion 81 during the growth cycle. This may explain the comparatively greater quantities of aflatoxins on maize obtained from the Easter Region since the region is endowed with sufficient rainfall and relatively high temperatures which induce aflatoxin production. Additionally, the different water activity levels of the grains due to the drying process used before and during storage could account for the different mycotoxin levels.
There were significant correlations between AFtotal and OTA. Likewise, AFB 1 and AFtotal in this study. These observations corroborate the findings of some previous researchers 63,64,66,82 . The co-existence of two or more fungi and their subsequent mycotoxin production in an environment suggests a possible non-antagonistic metabolite interaction, Moreover, the probable effect of combined exposure to aflatoxins with other mycotoxins Table 16. Evaluation of risk for Total Aflatoxins via consumption of maize. Margin of Exposure-MOE. Mean aflatoxins-Upper East = 32.84 µg/kg, Brong = 48.93 µg/kg, Ashanti = 62.37 µg/kg, Eastern = 53.14 µg/ kg. Central = 30.68 µg/kg, Western = 62.12 µg/kg. Daily intake of maize for infants and toddlers were halved (0.5 × 0.107 kg/day). Daily intake of 0.107 kg/day was used for children, adolescents and adults. Benchmark Dose Lower limit = 400 ng aflatoxins/kg bw/day.  82 reported that among food products analyzed in Italy, dried vine fruits were mainly contaminated with OTA and less with AFs. Discovered from the pertinent literature, there is an inverse correlation between AFB 1 and OTA. To buttress this claim 83 observed, no AFB 1 was found in dried vine fruits, while OTA was detected at high levels. In addition, Dimitrokallis et al. 84 reported that OTA inhibits AFB 1 production by Aspergillus species in a related study. The different categorizations of the biosphere (agro-ecological zones) presage different growth conditions for the proliferation of fungi. Brzonkalik et al. 85 as well Garcia et al. 86 emphasized mycotoxin production depends on species or/and strain, which is affected by the growth substrate and environmental conditions. de Souza et al. 75 and Kortei et al. 88 also explained co-existence of fungal strains on a substrate can affect both the level of mycotoxin production and the toxicity of the contaminated grains resulting in additive and synergistic effects when tested Nonetheless, data on multiple mixtures is very rare 87 . In this study the relatively high aflatoxin (AF) and ochratoxin (OTA) concentrations in maize grains obtained in the Eastern Region is expected as it geographically falls in a semi-deciduous zone with mean rainfall and temperatures of 1400-1900 mm and 25.9 °C respectively suggesting favorable environmental conditions for the growth of these toxigenic fungi 88,89 implicated. Pitt and Hocking 90 noted that Penicillium spp. and Aspergillus spp. grow well between 0 and 30 °C and have been found to produce greater quantities of ochratoxin A and aflatoxins respectively, this may explain the high incidence of this mycotoxin. Furthermore, Magan et al. 89 also emphasized fungal contamination in cereals is influenced by two main factors. Firstly, by initial high moisture content in crops or late harvesting of crops in rural areas, and secondly, the lack of poor storage facilities characterized by poor ventilation, high temperatures, and humidity.
Regular monitoring and pre-and post-harvest control measures can be used to control mycotoxins by enhancing the resistance of the crop to incursive fungi through plant breeding or genetic engineering, which are laborious and time-consuming. Effective, sustainable, and universally applicable preharvest intervention strategies  96 reported EDI values of range 0.02-0.04 respectively, for the age ranges of 3-6, 7-17, 18-59, and above 60 years for maize and products. All their computed MOE values were below the safe threshold of 10,000 and so the risk analysis results showed that most of the lower bound MOE values ranged from 10 to 100, indicating a concern for risk management. Age-group analysis suggested close attention were paid to the 3 ~ 6 years of age group, whose MOE value was the lowest. Their results reflected that preschool children might have the highest risk of being exposed to AF. Their results agreed with our findings. The MOE values (995-860 at mean and 336 at 95th percentile exposure) and cancer potency estimates, based on the current exposure levels indicated a potential health concern for Turkish adults was reported by 97 . Li et al. 98 pointed from a Chinese survey data, that the average daily intake of AFB 1 from maize in the high-risk area was 184.1 µg, and the probable daily intake is estimated to be 3.68 µg/kg bw/day. Chun et al. 99 estimated excess cancer risk values for liver cancer incidence by ingestion of these foods for AFB 1 were calculated to be 5.78 × 10 -6 for individuals negative for hepatitis B and 1.48 × 10 -4 mg/kg bw/day for individuals positive for hepatitis B in Korea.
In this study, EDI were slightly greater compared with other EDI values reported globally. This implies a significant impact of aflatoxins on the nutritional status of humans and animals. Omari et al. 54 observed that aflatoxin exposures were to some extent linked to nutrient deficiency in humans following a suggestion that aflatoxin exposure expedites intestinal damage resulting in a decline in nutrient absorption.
A strong association between anaemia and aflatoxin has been reported in Ghana showing that aflatoxin exposure may contribute partly to the high iron deficiency prevalent in children in developing countries including Ghana. The consumption of aflatoxins at high levels in a single dose or repeatedly for a brief period induces acute intoxication, henceforward labeled aflatoxicosis, in humans and animals with typical symptoms, including jaundice, lethargy, nausea, edema, hemorrhagic necrosis of liver tissues, bile duct hyperplasia, and eventually death (10-60%) subsequent to severe liver damage 100 . Although there is no consensus on the specific dose of aflatoxins that triggers acute toxicity in humans, it is well established that such a dose is highly variable depending on many factors, including the age, gender, health and nutritional status, presence or absence of underlying factors (e.g., chronic viral hepatitis, alcoholism, smoking, cirrhosis, exposure to hepatotoxic microcystins); and it is lowest in youngsters, as substantiated by the highest death rates of this age-group in aflatoxicosis outbreaks. A scoping review by Wu 100 have shown the presence of these aflatoxins appeared in greater proportion in kwashiorkor of children and in different organs (brain, heart, kidney, liver, and lung) and biological samples (serum, stool, urine). This observation is particularly worrying since the Eastern Region of Ghana, is a major crop producing zone where most of the foodstuffs (maize, groundnuts etc.) used as ingredients for the production of local weaning formulas for humans and then animal feeds for poultry and livestock are produced due to favorable climatic conditions. Various interventions have been established to combat aflatoxin biosynthesis and accumulation, ranging from preharvest to dietary interventions. Simply avoiding or reducing the consumption of foods that are frequently contaminated with aflatoxin has shown effectiveness in reducing liver cancer mortality in one population 100 . Advocacy on strict compliance with good agricultural practice (GAP), good manufacturing practice (GMP), as well as good hygiene practice (GHP), which are critical ingredients to alleviate the formation of aflatoxins in the field as well as during storage of foodstuffs, must be strengthened. By impeding aflatoxins formation in foods, there is the protection of both public health and the prevention of economic losses. Monitoring foods prone to fungal infection and the presence of mycotoxins regularly is cautious to assess the public level of awareness.

Conclusion
Based on the results obtained in this study, per the permissible limits set by the European Food Safety Authority (EFSA) (2 and 4 µg/kg) and the Ghana Standards Authority (GSA) (5 and 10-15 µg/kg), it can be construed that out of the 180 samples analyzed for total aflatoxins (AFtotal), 68% exceeded the limits of EFSA whereas 58% exceeded GSA limits. In the case of AFB 1 , 71% of the samples exceeded EFSA limits while 64% exceeded GSA limits. For OTA, 94 (52.22%) of samples exceeded the tolerable limit of EFSA, while 89 (49.44%) exceeded the limits of GSA.
Health risk assessment for ochratoxin A as well as aflatoxin exposure via maize consumed in different regions of Ghana by infants, children, and adolescents, and adults showed a significant adverse health risk in all age Scientific Reports | (2021) 11:23339 | https://doi.org/10.1038/s41598-021-02822-x www.nature.com/scientificreports/ categories of humans since all calculated values for MOE were less than 10,000 for both aflatoxins and ochratoxins. This study scratches the surface of a dire situation that calls for attention by all stakeholders involved in mitigating the harmful effects associated with these hazardous mycotoxin exposures to humans. Presently, the prime challenge of mycotoxins proliferation in our foods is the link with climate change. There is now a widespread consensus that the world is warming at an unparalleled rate and this is expected to seriously affect our crop production as well as the phyllosphere microflora of these crops. The irrepressible growth of A. flavus under extreme heat and dry condition is an expected and emerging dilemma mainly in many parts of the world (Serbia, Hungary etc.) where there were very low reports of mycotoxin contamination. However, surges in maize contamination were observed after prolonged hot and dry weather. Due to this, the world's largest agricultural food exporters such as Brazil, Argentina and some parts of Asia to include China and India have been identified as hot spots for impacts of climate change. From a food security viewpoint, a more precise forecast of impacts of climate change on mycotoxins need to be addressed to prevent conceded food sustainability which possibly results in negative social consequences.
Notwithstanding, some possible routes to improve the situation is the utilization of enzymatic biotransformation (purified enzymes) to degrade mycotoxins with much better precision in feeds/foods. Again the inclusion of binding agents or enterosorbents in the diet of humans or animals has been given considerable attention as a strategy to reduce foodborne exposures to mycotoxins by decreasing the mycotoxin availability thereby reducing its absorption. Recently, materials such as mycosorb (a new product which acts by binding pathogens and mycotoxins without affecting gut bacteria) and aluminosilicates have been reported to be adequately efficient in this regard. Furthermore, the use of biofuels and fermentation by-products such as distillers dried grains (DDGs) are some promising ways to drastically reduce the impacts of these mycotoxins on the gut and health of livestock and poultry. Lastly, a more practical means of curbing mycotoxicity is the diversification of our diets which include the non-adherence to a few food classes (esp. cereals and legumes) but an expanded one to include different classes is a more promising approach.