Aflatoxins are well known fungal toxins produced by specific strains of soil fungi (Aspergillus sp). It commonly occurs in soil, compost and food storage systems. Due to its toxic and carcinogenic nature, extensive research has been done on the impact of aflatoxin contamination for the food and feed industries. Research has mainly focused on post-harvest contamination with little consideration at pre-harvest stages. Aflatoxin B1 has so far been regarded not to pose any long-term environmental risk because it has a short half-life in soil due to microbial degradation and adsorption to soil organic matter. Limited research has been done on this topic since the 1980s.
The aim of this study was to clarify the risk of aflatoxins in the soil ecosystem under different soil climate (temperature and soil moisture) conditions. In the first phase of the study, existing literature about the occurrence, fate and effects of aflatoxins in soil, was reviewed. Results from the review led to focused experimental work to test several hypotheses about the toxicological effects of aflatoxin exposure to soil organisms that contribute to essential ecosystem services in the soil.
Earthworms (Eisenia andrei) were used as bioindicator species, and established biomarkers were selected to conduct the toxicology tests in the laboratory.
Aflatoxins in the soil ecosystem: an overview of its occurrence, fate, effects and future perspectives
A detailed review of the literature was done to determine the current knowledge on aflatoxins’
occurrence, fate and effects specific to the soil environment. The research from this phase of the study was published.
Fouché TC; Claassens S; Maboeta MS (2020) Aflatoxins in the soil ecosystem: an overview of its occurrence, fate, effects and future perspectives. Mycotoxin Research. 36 (3):303-309 https://doi.org/10.1007/s12550-020-00393-w
From the review, it was concluded that:
i. Aflatoxin contamination is still a serious worldwide problem that results in significant income losses for producers of edible crops. More focused research is required into the prevention at pre-harvest stages for improved management at post-harvest stages.
ii. Low aflatoxin concentrations are generally detected in soil and considered low risk for environmental contamination. However, soil concentrations may significantly increase during drought stress and when contaminated crops are left to decompose or worked into the soil as suggested by regulations, it increases the natural concentrations and prolongs exposure due to the gradual release of the toxins, thus altering the natural ecological balance over time.
Insufficient information is available on the effects of aflatoxin exposure on soil organisms and it is currently unclear to what extent increased aflatoxin concentrations might endanger soil health.
The functional response of soil organisms after aflatoxin exposure and the consequences on the
81 | P a g e soil communities’ structure and functions are unknown. It is an area of research that should be explored in more detail.
iii. Climate change has resulted in aflatoxin becoming a bigger risk in areas where it already occurs.
Environmental factors such as changes in temperature, pH, drought conditions or periods of waterlogging may cause post-harvest aflatoxigenic fungi to become more prevalent at pre- harvest stages (Medina et al. 2017). Drought stress, in particular, causes increased aflatoxin production by fungi. In addition, studies show that changes in temperature and soil moisture might increase the risk of aflatoxin contamination in areas where it did not previously occur (Battilani et al. 2016). Therefore, it is recommended that future risk assessments of aflatoxin contamination take on a climatic approach and consider a wider geographic distribution.
iv. We do not yet have a comprehensive understanding of AFB1 transformation in natural environments. Original studies from more than ten years ago found that when AFB1 enters the soil, it is transformed by soil microbes into less toxic transformation products, namely AFB2, AFG1
and AFG2, in a relatively short time (Accinelli, 2008). However, subsequent studies found AFB2a
as the major transformation product (Starr et al. 2017) in addition to the other less toxic forms.
AFB2a has DNA-binding capacity and is more polar than the other transformation products, suggesting a higher possibility of reaction with other molecules and an increased leaching potential in natural settings. The genotoxic and cytotoxic potential of the transformation products may still threaten the functional ability of soil organisms even after microbial degradation (Krifaton et al. 2011). There is insufficient information about the genotoxic effects of aflatoxins on soil organisms.
v. Several soil microbes (bacteria, fungi and yeasts) have been identified to effectively degrade aflatoxins in the soil environment, yet the potential of other soil organisms to regulate aflatoxins remains unclear and warrant further research.
Ecotoxicological effects of aflatoxins on earthworms under different temperature and moisture conditions
Earthworms play an important role in supporting soil functions, soil biodiversity and providing, regulating, and maintaining various ecosystem services. Potential negative effects for earthworms will have negative effects on soil health. This phase of the study investigated the potential effects of aflatoxins on earthworms in a soil medium. Complementary investigations that included life-cycle and mechanistic type biomarkers across a range of soil climatic conditions were conducted to make better-informed conclusions about the possible risks of aflatoxin exposure to soil organisms. Work from this phase of the study was submitted to the journal “Toxins” for review in November 2021 accepted for publication in December 2021 and published online in January 2022.
Fouché TC, Claassen S, Maboeta MS (2022) Toxins 2022, 14(2), 75 https://doi.org/10.3390/toxins14020075
82 | P a g e The first objective was to assess if aflatoxin affects earthworms’ survival, growth, and reproduction using a standard OECD test. The null hypothesis that aflatoxindoes not cause a difference in the earthworm life-cycle processes could not be rejected based on the observation that earthworms exposed to aflatoxins at environmentally relevant concentrations (10 – 100 µg/kg) did not demonstrate any negative effects on survival, growth or reproduction during standard testing conditions (Chapter 4). However, previous studies observed significant negative effects on earthworm physiology during a contact paper test with higher concentrations. It was concluded that higher concentrations in a soil medium might still negatively affect earthworm demographic processes.
The second objective assessed the genotoxicity of aflatoxins to earthworms using the comet assay.
The null hypothesis that aflatoxin exposure at environmentally relevant concentrations does not cause significant DNA damage in earthworms was rejected. Significant DNA damage was indicated at higher exposure concentrations but not at low concentrations.
The third objective of the study evaluated if different temperatures (21 °C and 26 °C) and soil moisture conditions (30% and 60% of soil water holding capacity) affect the toxicity of aflatoxins to earthworms. The null hypothesis that soil climatic conditions do not significantly contribute to the toxicology of AFB1 to earthworms was rejected. Changes in soil climate conditions, especially a decrease in moisture, significantly affected the population performance in terms of reproduction and genotoxicity. Increased temperatures alone generally had a positive effect on the population performance. However, the interaction of moisture with temperature significantly affected the exposure effect outcomes because statistically significant (p < 0.001) negative effects were indicated in the earthworm reproduction and DNA damage. There was also a significant increase in DNA damage and decreased reproduction due to the interactive effect of decreased moisture with increased aflatoxin concentration.
Biological control of aflatoxins in soil by earthworms
Numerous researches have investigated the biological control of aflatoxins by soil microorganisms, and several bacterial and fungal species have been identified that effectively degrade aflatoxins in soil. However, the aflatoxin degradation potential of other soil organisms involved during decomposition is not frequently reported. The activity of earthworms affects many essential soil processes and act as essential biological regulators of plant pathogens. Previous research reports earthworms’ role in the degradation of another type of mycotoxin, deoxynivalenol (Wolfarth et al.
2016), but no literature is available on the role of earthworms in the degradation of aflatoxins in soil.
The first objective investigated if the presence of earthworms improves the aflatoxin degradation potential in soil. The percentage decrease in aflatoxin concentrations from the initial spiked concentrations were monitored over four weeks. The null hypothesis that the presence of
83 | P a g e earthworms does not significantly decrease the aflatoxin concentrations in soil was accepted because observations did not find significant differences in the percentage decreased between earthworm and non-earthworm treated soil at standard testing conditions. At more favourable conditions (increased temperatures and sufficient moisture), the presence of earthworms significantly improved aflatoxin degradation, possibly due to increased burrowing activity and the stimulation of specific soil bacteria.
The second objective investigated if different temperature and moisture conditions affect the earthworms’ ability to degrade aflatoxins in soil. The null hypothesis that soil climatic conditions do not significantly affect their degradation ability was rejected because improved aflatoxin degradation was observed in the earthworm treated soil at increased temperatures. There was also significantly improved aflatoxin degradation in the earthworm treated soil at increased moisture at the low concentrations. Interacting effects between temperature and moisture were not observed in the earthworm treated soil, but an interaction of temperature with increased concentrations was indicated.
Aflatoxin concentrations were monitored in the earthworm and non-earthworm treated soil, and increased degradation was observed at the higher concentrations, but the rate of degradation was slower in the soil with low moisture. During drought conditions, the soil environment does not only be have an increased risk of toxin production by fungi (Medina et al. 2017), but also has an increased risk of prolonged exposure due to the slower aflatoxin degradation rate.
Research highlights and key contributions of the study
This research highlighted several gaps in the knowledge about the consequences of aflatoxin contamination in the soil environment. Under changing climate conditions, aflatoxin contamination cannot be considered a low risk in soil ecosystems. Results indicated the influence of changes in temperature and moisture on the exposure effect outcomes of aflatoxin in soil and the importance of reviewing standard testing conditions to include broader environmental conditions. It highlighted the possible risk of environmentally relevant aflatoxin levels to the functional ability of important soil organisms, specifically earthworms. Finally, it highlighted the potential of earthworms to contribute to the biological control of aflatoxins under favourable environmental conditions.
84 | P a g e Recommendations for further research
Although ELISA is a reliable and recognised method for aflatoxin detection, more sensitive and reliable test methods, specific for detecting aflatoxins in soil (Albert et al. 2021), are recommended to confirm concentrations.
This study took a single-species-based ecotoxicological approach to assess the ecological risk of a natural toxin and interactions of two environmental conditions. Future studies using real contaminated agricultural soil will be valuable for predicting aflatoxins’ effect in the natural environment. Investigations that allow for the assessment of community-level effects due to species interactions are recommended and will add value to this body of knowledge. Further, it will be valuable to characterise the soil microbiome associated with aflatoxin degradation under these same climatic variables and in the presence and absence of earthworms.
The scope of this study did not allow the mechanisms of toxicity to be investigated, and the application of profiling based techniques (metabolomics) would complement and validate the biomarkers that were used. Therefore, further investigations are recommended to assess aflatoxin toxicity on a suite of earthworm metabolites involved in cellular processes such as growth, cellular proliferation, DNA stability, cell death, and the production of amino acids and proteins.
85 | P a g e References
Accinelli C, Abbas HK, Zablotowicz RM, Wilkinson JR (2008) Aspergillus flavus aflatoxin occurrence and expression of aflatoxin biosynthesis genes in soil. Can J Microbiol 54:371–379.
Albert J, More CA, Dahlke NRP, Steinmetz Z, Schaumann GE, Muñoz K (2021) Validation of a simple and reliable method for the determination of aflatoxins in soil and food matrices. ACS Omega. 6(29):18684- 18693 https://doi.org/10.1021/acsomega.1c01451
Battilani P, Toscano P, Van der Fels-Klerx HJ, Moretti A, Camardo Leggieri M, Brera C, Rortais A, Goumperis T, Robinson T (2016) Aflatoxin B1 contamination in maise in Europe increases due to climate change. Sci.
Rep. 6:24328. https://doi.org/10.1038/srep24328
Krifaton C, Kriszt B, Szoboszlay S, Cserháti M, Szucs Á, Kukolya J (2011) Analysis of aflatoxin B1-degrading microbes by use of a combined toxicity-profiling method. Mutat Res Genet Toxicol Environ Mutagen 726:1–
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Medina Á, González-Jartín JM, Sainz MJ (2017) Impact of global warming on mycotoxins. Curr Opin Food Sci 18:76–81
Starr JM, Rushing BR, Selim MI (2017) Solvent-dependent transformation of aflatoxin B1 in soil. Mycotoxin Res 33:197–205
Wolfarth F, Schrader S, Oldenburg E, Brunotte J (2016) Mycotoxin contamination and its regulation by the earthworm species Lumbricus terrestris in the presence of other soil fauna in an agroecosystem. Plant Soil.
402:331–342
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ANNEXURE A – NWU ETHICS APPROVAL
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ANNEXURE B- UNISA ETHICS APPROVAL
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ANNEXURE C – WATER HOLDING CAPACITY
Water holding capacity (WHC) calculation of OECD soil.
WHC (% of dry mass) =
S − D
D × 100
Where S = weight (g) of saturated soil and D = weight (g) of dry soil.
1. Sample 1: S = 3.371 g ; D = 2.256 g
∴3.371 − 2.256
2.256 × 100 = 49.42 % 2. Sample 2: S = 2.243 g ; D = 1.476 g
∴2.243 − 1.476
1.476 × 100 = 51.96 % 3. Sample 3: S = 3.800 g ; D = 2.523 g
∴3.8 − 2.523
2.523 × 100 = 50.61 % 4. Sample 4: S = 3.731 g; D = 2.508 g
∴3.731 − 2.508
2.508 × 100 = 48.76 %
• the mean WHC (%) per dry mass is =50.19 %
• In 600 g soil sample: 301 ml = 100% WHC
i. 30% WHC – representative of dry soil 301 x 0.30 = 90 ml / 600 g soil.
ii. 50% WHC – representative of standard soil moisture 301 x 0.50 = 150 ml / 600 g soil.
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ANNEXURE D- FRONTPAGE OF PUBLICATION IN MYCOTOXIN RESEARCH
https://doi.org/10.1007/s12550-020-00393-w