Co-Occurrence of Ochratoxin a and Aflatoxins in Maize (Zea Mays) and Their Associated Health Risk Estimations in Some Selected Regions (Different Agro-ecological Zones) of Ghana


 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 (Upper East, Northern, Ashanti, Eastern, Central, and Western) representing the agro-ecological zones of Ghana. Ochratoxins, total aflatoxins (AFTotal), and the integral aflatoxins (AFB1, AFB2, AFG1, and AFG2) were measured with High-Performance Liquid Chromatography (HPLC) with a Fluorescence detector. Risk assessments were also conducted using models prescribed by the Joint FAO/WHO Expert Committee on Additives (JECFA). The general trend of occurrence of the aflatoxins was in decreasing order of AFB1> AFB2> AFG1>AFG2 and were in the ranges of 0-337µg/kg, 0-101.00µg/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 ochratoxins (OTA) levels ranged between 0-97.51 µg/kg. There were significant (p<0.05) differences observed in all categories of the tested samples. 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 10.18-441.02µg/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 the 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 50-1150 ng/Kg.BW/day, 0.4-6.67, 0-0.0323 ng aflatoxins kg−1bwday−1 and 1.62-37.15 for Estimated Daily Intake (EDI), Margin of Exposure (MOE), Average Potency, and Cancer Risks respectively. Likewise, ochratoxin (OTA) values were in the ranges of 8.6x10-3-450 ng/Kg.bw/day, 0.05-2059.97, 0-0.0323 ng ochratoxins kg−1bwday−1 and 2.78x10-4-14.54. It was deduced that the consumption of maize posed adverse health effects on all age categories of people from some of the locations studied because all calculated MOE values were less than 10,000 respectively for aflatoxins and ochratoxins.


Introduction
Maize (Zea mays L), a principal cereal extensively consumed 1 around the globe is exceedingly prone to fungal infection by many toxigenic fungal species subsequently leading to mycotoxins production 2,3 due to its ideal nutrient composition 4 . This situation is particularly distressing since maize (Zea mays) accounts for 40% of the cereal production in Sub-Saharan Africa (SSA), where more than 80% is used as food 5 . The crop provides at least 30% of the total calorie intake of people in Sub-Saharan Africa Maize is consumed as a staple in the African region where intake ranges from 52 to 450 g/person/day 1,6 and so is an easy channel for mycotoxin contamination.
Mycotoxins which are natural toxins from fungi, contaminate maize grains and render them potentially dangerous. Mycotoxins represent one of the main global foodborne risks for human health are In addition to setting regulatory limits for mycotoxins, it is also imperative to conduct health risk assessments in the population due to dietary exposure. A low-dose extrapolation approach introduced by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1997 and the margin of exposure (MOE) method proposed at the 64th JECFA meeting in 2005 27 were both recommended and have been widely used worldwide 28, 29 to assess the risk of dietary exposure to mycotoxins.
There is a paucity of data on ochratoxin A contamination in foodstuffs in Ghana. The objectives of this study, therefore, were to assess the levels of ochratoxin A and a atoxins combination in maize samples as well as their associated health risk estimations in six (6) different regions representative of the different agro-ecological zones of Ghana.

Sample collection
To collect a representative data set, we rst 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 (1kg) 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 Ochratoxin
Ochratoxin A was determined based on CEN o cial 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 ne 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 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 ltered through Whatman number 4 lter paper. Ten milliliters (10 ml) of ltrates were used for Ochratoxin A solid-phase extraction and cleanup. Exactly 150 ml of phosphate buffer saline (PBS) was added to 10 ml of ltrates and the mixture stirred. Immunoa nity columns speci c for Ochratoxin A were preconditioned and antibodies in the column activated by eluting 10 ml of phosphate buffer saline through columns at a ow speed of 3 ml min − 1 . Exactly 50 ml of the 160 ml ltrate-PBS mixture was loaded onto the pre-conditioned immunoa nity columns speci c 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 ow rate of 5 ml min − 1 .
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 ask by rst 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 a volume of eluates to 4 ml and eluates vortexed after which 2 ml was pipetted into HPLC vials for quanti cation.

HPLC parameters
Agilent high-performance liquid chromatography system (HPLC 1260 in nity series) with a quaternary pump and uorescence detection was used for OTA quanti cation. Data acquisition and quanti cation were done using Chem station (Open lab edition ) 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 ltered and 10 ml of ltrate added to 60 ml of phosphate buffer saline (PBS) for solid-phase extraction using a pre-conditioned immunoa nity column speci c for AFB 1 , AFB 2 , AFG 1 , and AFG 2 . The 70 ml ltrate-PBS mixture was loaded onto the pre-conditioned column and allowed to elute by gravity at a ow rate of 1 ml min − 1 . This was followed by a cleanup with 15 ml distilled water at a ow rate of 5 ml min − 1 . A atoxins were eluted in two steps into a 5 ml volumetric ask 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 a volume of eluates to 5 ml and eluate vortexed and 2 ml pipetted into HPLC vials for quanti cation.

Limit of detection/quanti cation (LOD/LOQ)
Limit of detection and quanti cation (LOD/LOQ) of the HPLC were estimated by making a calibration curve around the least standard used for spiking, 5 µ/kg (lowest concentration range of calibration curve). Blank did not produce any signal, so the LOD and LOQ were calculated as; LOD = 3 * standard deviation/slope.

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 measurement procedure should be in a range of 80-120%.
Limit of Detection: The limit of detection should be less than 1 ug/kg for all a atoxins.
Limit of Quanti cation: The limit of Quanti cation should be less than 3 ug/kg for all a atoxins.

Cancer Risk Characterization for A atoxins and Ochratoxins
Genotoxic compounds such as a atoxins and ochratoxins have their risk assessments ttingly computed based on the Margin of Exposures (MOEs) approach, which was estimated by dividing the Benchmark dose lower limit (BMDL) for a atoxins is 400 ngkg − 1 bwday − 1 by toxin exposure 23,45 .
Estimated Liver Cancer Risk due to Consumption of food samples.
The ingestion of a atoxins 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 a atoxins 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 gure from individual potencies of Hepatitis B surface antigen (HBsAg) (HBsAg-positive and HBsAg-negative groups).
The JECFA 50 49 were adopted for this calculation. Also, the average HBsAg + prevalence rate of 7.74% (adult-8.36%, 14.3%-adolescents, 0.55%-children) for Ghana 51 52 was adopted and 92.26% (100-7.74%) was extrapolated for HBsAg-negative groups. Hence the average potency for cancer in Ghana was estimated as follows according to Eq. (7) as prescribed by 49  Thus the Cancer risk was estimated using the following formula in Eq. (8) 2,45 : Cancer risk = Exposure (EDI) × Average potency (8) The use of plants in the present study complies with international, national and/or institutional guidelines.

Statistical Analysis
The ochratoxins and a atoxins concentrations were calculated using regression analysis from the curves generated from the standards of the ochratoxins/a atoxins with Excel for Microsoft Windows (version 10

Results
Samples tested, produced good linearity or coe cients of correlations (R 2 > 0.990) within the tested range. For the recovery analysis, samples previously tested to guarantee the nonappearance of 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 0.13-0.15 µg/kg. The limit of Quanti cation for ochratoxins was 2.49 µg/kg. A atoxins ranged between 0.39-0.45 µg/kg respectively for both ( Table 2).
OTA levels were also observed to be lesser than AFB1, AFB2 but more than AFG1 and AFG2 and ranged between 0-97.51 µg/kg. There were signi cant (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 a atoxins. 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 the skewness and kurtosis respectively which implied a symmetrical and normally distributed data for Total A atoxins (AFtotal) (the distribution is not outside the range of normality) (          The European Food Safety Authority (EFSA) 53 and Ghana Standards Authority (GSA) 2,54 regulatory limits for total a atoxins (AF Total ) 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 9) in this study. Locally, both toxins   Table 9).
Out of a total of 180 samples analyzed for total a atoxins (AF Total ), 127 (70.50%) surpassed the EFSA limit and were within the range of 4.    (Table 11).

Discussion
The discrete incidence of ochratoxins and a atoxins in foodstuff 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. Ochratoxins have been implicated in a variety of adverse health effects both in humans and in animals suggested to be reaching 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 variation in contamination levels could be attributed to speckled infection levels of the toxigenic fungus genera, Aspergillus (esp. avus) owing to their ubiquitous nature, infect maize grains on the eld 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 presence of a atoxins on maize grains before storage.
There were signi cant correlations between AFtotal and OTA. Likewise, AFB1 and AFtotal in this study.
These observations corroborate the ndings of some previous researchers 63,64,66,82 . The co-existence of two or more fungi and their subsequent mycotoxins production in an environment suggests a possible non-antagonistic metabolites interaction additionally, the probable effect of combined exposure to a atoxins with other mycotoxins in foodstuffs could be additive or antagonistic. were below the safe threshold of 10,000 and so 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 is paid to the 3 ~ 6 years of age group, whose MOE value was the lowest. Their results re ected those preschool children might have the highest risk of being exposed to AF. Their results agreed with our ndings.
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 AFB1 from maize in the high-risk area was 184.1 µg, and the probable daily intake is estimated to be 3.68 µg kg − 1 bw day − 1 .
Chun et al 99 estimated excess cancer risk values to liver cancer incidence by ingestion of these foods for AFB1 were calculated to be 5.78 × 10 − 6 for individuals negative for hepatitis B and 1.48 x 10 − 4 mg kg − 1 bw day − 1 for individuals positive for hepatitis B in Korea.
Various interventions have been established to combat a atoxin biosynthesis and accumulation, ranging from preharvest to dietary interventions. Simply avoiding or reducing consumption of foods that are frequently contaminated with a atoxin has shown effectiveness in reducing liver cancer mortality in one population 100 .
Advocacy on the strict compliance to good agricultural practice (GAP), good manufacturing practice (GMP) as well as good hygiene practice (GHP) which are critical ingredients to alleviate the formation of a atoxins in the eld as well as during storage of foodstuffs, must be strengthened. By impeding the a atoxins 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.

Declarations Data Availability
All data supporting this study are included in the article and its supplementary material.