## 3 Results and Discussion
### Effects of Sub-Lethal Concentrations of NP/Derivatives on AF Production: A. parasiticus  
NPs/derivatives were also examined in _A. parasiticus_ at the same concentrations applied to _A. flavus_ (See above) (Table 1). As determined in _A. flavus_, most compounds moderately affected the growth of _A. parasiticus_ at test concentrations, except SLD, which prevented fungal growth at as low as 0.5 mM, indicating _A. parasiticus_ was more susceptible to SLD compared to _A. flavus_ (growth prevention at 1.0 mM). Similarly, _A. parasiticus_ was also slightly more susceptible to 4I3M (0.4 mM), THY (0.4 mM) and CARV (0.4 mM) compared to _A. flavus_ (viz., less fungal growth; See Supplementary Figure S2), indicating \"compound--fungal species specificity\" exists with the NP/derivatives treatments. As shown in Figure 2b, 16 out of 104 concentration points (marked as an upper arrow (\"\(\cdots\)\")) exhibited enhanced AF (B\({}_{1}\), B\({}_{2}\), G\({}_{1}\), G\({}_{2}\)) production compared to the untreated control (5 to 110% enhancement), whereas 2,3-D, 2,5-D, 2H4M, 4I3M and 4-MEC did not enhance AF production at any test concentrations. As determined in _A. flavus_, while CARV increased the production of AFB\({}_{2}\) and AFG\({}_{2}\) at 0.2 mM (29% and 20% enhancement, respectively), both 4I3M and CARV at 0.4 mM exerted the most potent anti-aflatoxigenic activity (94% to 99% inhibition); conversely, the structural analog THY at 0.4 mM exerted the highest level of AF (B\({}_{1}\), B\({}_{2}\) G\({}_{1}\), G\({}_{2}\)) enhancement compared to other treatments (62% to 110% enhancement). Therefore, analogous to _A. flavus_, the order of AF inhibition by THY derivatives was determined as: 4I3M > CARV > THY (high to low). THY and CARV have been used as food/feed additives. For example, the feed additive product Biomin(r)DC-P (trade name) is a mixture of five compounds including THY, CARV, D-carvone, methyl salicate and L-menthol, which are encapsulated with a hydrogenated vegetable oil [53]. The additive has been used in feed for chicken fattening, chickens reared for laying, etc. However, the safety of this additive for laying/breeding birds was not conclusive [41]. Since cereal grains are the main ingredients in animal feed, ensuring that feeds are free of risk factors, such as mycotoxins or mycotoxin triggering agents, is of high importance. As investigated in this study, THY or CARV could function as risk factors by exerting mycotoxin-enhancing capabilities; therefore, optimization of concentrations of ingredients such as THY or CARV for feed use is highly necessary. Of note, 4I3M is the only compound that did not enhance AF production in both _A. flavus_ and _A. parasiticus_. 4I3M is a synthetic derivative of THY and CARV, which was previously developed as an antimicrobial preservative or fungicide [54; 55]; 4I3M has recently been examined as an ingredient of oral care products [56; 57] that target either fungal or bacterial pathogens, including _Candida_ species, _Aggregtibacter actinomycetemitans_, _Staphylococcus aureus_, _Pseudomonas aeruginosa_, _Streptococcus pneumoniae_, _Klebsiella pneumoniae_, _Porphyromonas gingivalis_, etc. Compared to THY or CARV, 4I3M possesses color- or odor-neutral characteristics; hence, it is more appealing to consumers [58]. Of note, a previous study indicated that the structural analog THY affected fungal antioxidant defense system such as superoxide dismutases, glutathione reductase, etc. [59]. Therefore, we speculate that 4I3M might also affect a similar oxidative stress defense system in fungi. The determination of the precise mechanism of antifungal and/or anti-mycotoxicic activity of 4I3M warrants future in-depth study. Also interesting is that, except for THY and CARV, most AF types enhanced in _A. parasiticus_ by NPs/derivatives were AFG\({}_{1}\) or AFG\({}_{2}\) (Table 1) but not AFB\({}_{1}\) or AFB\({}_{2}\) (_A. flavus_ naturally does not produce AFG\({}_{1}\) and AFG\({}_{2}\)) [45; 46; 60]. [PERSON] et al.