Aflatoxin contamination is one of the major threats to food safety and human health. However, limited information is available on its toxicological impact on the soil ecosystem. The soil preparation (addition of toxins), livestock farming, standard toxicity tests and genetic studies of the earthworms were carried out in the NWU laboratories in Potchefstroom. The analysis of the soil samples using the ELISA was performed in the laboratories of the campus of the University of South Africa, Florida.
The reference style used in this thesis follows the requirements of the journal Mycotoxin Research. B SHOWS CELLS IN THE 30% WATER HOLDING CAPACITY TREATMENT AND A SIGNIFICANTLY (P < 0.001) HIGHER PERCENTAGE OF CELLS WITH DNA DAMAGE REPRESENTED BY THE CHARACTERISTIC COMET TAILS. AN * INDICATES A SIGNIFICANT DIFFERENCE (P < 0.05) BETWEEN THE AFLATOXIN CONCENTRATION AND ITS CONTROL TREATMENT AT THE SAME MOISTURE AND TEMPERATURE CONDITIONS.
DIFFERENT ALPHABETICAL LETTERS INDICATE SIGNIFICANT (P < 0.05) DIFFERENCES WITHIN THE SAME FAUNAL GROUP AT THE SAME SPIKE. DIFFERENCES BETWEEN EARTHWORM (EW) AND NON-EARTHWORM GROUPS UNDER THE SAME ENVIRONMENTAL CONDITIONS ARE INDICATED IN BOLD. THE ASTERISK (*) INDICATES THE LEVEL OF SIGNIFICANCE * P < 0.05; ** P < 0.01.
Introduction
Literature Review
Aflatoxin
Aflatoxin can be cytotoxic, genotoxic, mutagenic and immunosuppressive and is classified as a group 1 carcinogen (Ostry et al. 2017). Matrix interference is possible, especially with plasma samples, and confirmatory LC analyzes are often required (Yao et al. 2015). Indicators are selected based on their relevance to a particular soil hazard, ecosystem function or service (Bünemann et al. 2018).
Therefore, a combination of biological (bioanalyzer), chemical and physical indicators are often used as tools to monitor the effects of toxins in the ecosystem and greatly improve the quality assessment (Bünemann et al. 2018). Therefore, biomarkers are considered as an early warning of toxicological effects within an ecosystem (Lionetto et al. 2012). Hackenberger et al. 2018) have been developed over the past ten years and have become a focal point in climate change-related studies.
Any impairment of coelomocyte functioning can negatively affect the health of the organism (Lionetto et al. 2012). Genotoxic biomarkers are often more sensitive when evaluating contaminants in the soil than whole organism responses such as survival, growth and reproduction (Smit et al. 2009).
Aflatoxins in the soil ecosystem: An overview of its occurrence, fate and
Ecotoxicological effects of aflatoxins on earthworms under different
- Experimental design
- Introduction of aflatoxin into the soil
- Aflatoxin Concentrations
- Earthworm survival, weight and reproduction test
- Comet assay
- Statistical analysis
- Aflatoxin concentrations in the soil
- Earthworm survival, weight change and reproduction
- Genotoxicity - Comet Assay results
- Interaction effect of climate conditions on the toxicity of aflatoxin
- Aflatoxin concentration
- Earthworm survival, growth and reproduction
- Genotoxicity
- Temperature and Moisture interaction with aflatoxin toxicity
CONCENTRATION (C), MOISTURE (WHC), AND TEMPERATURE ON SOIL DAMAGE AND PROPAGATION AFTER 30 DAYS OF EXPOSURE, DF - DEGREES OF FREEDOM.
Biological control of aflatoxins by earthworms
Experimental design
A laboratory experiment was conducted at North-West University, South Africa and University of South Africa science laboratories to assess the degradation of AFs in the presence and absence of earthworms at two different temperatures (21 and 26 °C ) and soil moisture. (30% and 50% water holding capacity (WHC) for four weeks. Two different AF concentrations (10 µg/kg and 100 µg/kg) were used to enrich the soil based on concentrations measured in environmental samples from previous studies (Table The experiment used a standardized soil medium, based on the guidelines of the Organization for Economic Co-operation and Development (OECD, 2016) to overcome some of the complexity and heterogeneity of the soil environment.
Four treatments were prepared for each concentration group based on a combination of air temperature and soil moisture (as groundwater holding capacity) (González-Alcarez et al. 2018). In each treatment, three replicates contained earthworms, and three replicates were prepared without earthworms (Table 5.1). 69 | P a g e Table 5.1: The laboratory experiment was designed to include a combination of two temperature and two moisture treatments at two aflatoxin concentrations.
Soil spiking with aflatoxin
Enzyme-linked immunosorbent assay (ELISA)
The soil samples were crushed and 2 g of homogenate was mixed with 10 ml of 70% methanol in a 50 ml centrifuge tube. The tube was mixed in a shaking incubator for 30 minutes and centrifuged at 4 x 1000 rpm for 10 minutes. To obtain a tenfold dilution, 0.5 ml of the supernatant was mixed with 0.5 ml of deionized water.
When the optical density values were outside the detection limits of the kit, usually due to high concentrations of AF in the sample, a further 10-fold dilution was made by mixing the supernatant mixture with 35% methanol to give a 100-fold dilution (10-fold of the first step x 10 times from the second step). Equal amounts of horseradish peroxidase conjugate and antibody working solution were added to each well and incubated for 30 min at 25°C. After incubation, the liquid in the wells was carefully discarded and the plate was washed five times with phosphate buffer and aqueous wash solution (1:19).
Tetramethylbenzidine (TMB) reagent was added and the plates were incubated for 15 min to allow color development.
Concentration calculation (4PL)
71 | Page Figure 5.1: Standard curves based on a four-parameter logistic (4PL) model generated from absorbance values of standards.
Statistical analysis
72 | Page At 72 h after 10 µg/kg soil spray, lower soil AF concentrations were detected in the earthworm-free treatment at 21 °C and was statistically significant (p < 0.001) at increased moisture (50% WHC). ) group. In contrast, significantly (p <0.05) lower soil AF concentrations were detected in the earthworm treatment at 26 °C. At the added concentration of 100 µg/kg, higher AF concentrations were initially detected in all earthworm-treated soils than in earthworm-free soils.
The higher concentrations in the samples were possibly due to the bioturbation activity of the earthworms in the early stages before AF degradation. However, after 32 days, the mean AF concentration in the earthworm groups at 26 °C was lower than in the non-earthworm groups, indicating improved degradation. In contrast, mean AF concentrations in the earthworm treatments at 21°C remained higher than in the non-earthworm treatments, and t-tests showed statistically (p < 0.05) higher concentrations under standard conditions (21°C and 50% WHC).
The lowest degradation potential was observed at 26 °C with 30% WHC and was statistically (p = 0.03) different from the soil with the highest AF degradation at 26 °C with 50% WHC in the earthworm and non-earthworm treatments. Results confirmed the main effects observed in the one-way analysis and indicated significant interaction effects between the different environmental conditions (Table 5.3). The main effect of moisture (WHC) was significant (p < 0.05) in the lower concentration (10 µg/kg).
A significant (p <0.01) interaction effect between moisture (WHC) and temperature (T) was reported at the lower increased concentrations (AF10), but it was dependent on the third variable because it was only observed in the no-earthworm group. . 75 | Page However, excluding the results without earthworms and focusing only on soil treated with earthworms, the combination of increased moisture and temperature produced a significant (p = 0.031, F-value = 5.10) interaction effect with earthworm treatment at 100 µg/kg , which explains the maximum degradation rate for the earthworm treatment at 26 °C at 50% WHC. The AF degradation processes observed in soil without earthworms indicate degradation solely due to microbial activity in the soil, while soil treated with earthworms indicates individual and combined effects of earthworms and soil microorganisms.
76 | P a g e the highest reproductive output was observed in the soil with the highest % reduction in AF concentrations. The greatest degradation potential was observed in treatments with increased moisture (50% WCH), possibly due to increased burrowing activity (Wen et al. 2020). The interaction of temperature and soil moisture in earthworm treatments at increased concentrations further emphasizes the importance of climatic factors in the performance of soil organisms.
General conclusions and recommendations
Several soil microbes (bacteria, fungi and yeasts) have been found to effectively degrade aflatoxins in the soil environment, but the potential of other soil organisms to regulate aflatoxins remains unclear and warrants further investigation. In this phase of the study, the possible effects of aflatoxins on earthworms in a soil medium were investigated. The null hypothesis that aflatoxin causes no difference in 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 show any negative effect on survival . growth or reproduction under standard test conditions (Chapter 4).
However, the interaction of moisture with temperature significantly influenced the exposure effect outcomes because statistically significant (p < 0.001) negative effects in earthworm reproduction and DNA damage were indicated. Previous research reports the role of earthworms 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 was to investigate whether the presence of earthworms improves the aflatoxin degradation potential in soil.
83 | P a g e earthworms do not significantly reduce aflatoxin concentrations in soil was accepted because observations found no significant differences in percent reduction between soil treated with earthworms and soil not treated with earthworms under standard test conditions. The null hypothesis that soil climatic conditions do not significantly affect their degradation ability was rejected because enhanced aflatoxin degradation was observed in soil treated with earthworms at elevated temperatures. Aflatoxin degradation was also significantly enhanced in soil treated with earthworms with added moisture at low concentrations.
Interactive effects between temperature and moisture were not observed in soil treated with earthworms, but an interaction of temperature with increased concentrations was shown. Aflatoxin concentrations were monitored in soil treated with earthworms and soil not treated with earthworms, and at higher concentrations increased degradation was observed, but the rate of degradation was slower in soil with low moisture. . This research highlighted several gaps in knowledge about the consequences of aflatoxin contamination in the soil environment.
Future studies with truly contaminated farmland will be valuable in predicting the effect of aflatoxins in the natural environment. Furthermore, it will be valuable to characterize the soil microbiome associated with aflatoxin degradation under the same climate variables and in the presence and absence of earthworms. 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.
