Despite the well-documented effects of human-induced environmental changes on the morphology, physiology, behaviour and life history of wild animals, next to nothing is known about how anthropogenic habitats influence anti-predatory chemical defence, a crucial fitness component of many species. We investigated the amount and composition of defensive toxins in adult common toads (Bufo bufo) captured in natural, agricultural and urban habitats, and in their offspring raised in a common-garden experiment. We found that, compared to toads captured from natural habitats, adults from both types of anthropogenic habitats had larger toxin glands (parotoids) and their toxin secretion contained higher concentrations of bufagenins, the more potent class of bufadienolide toxins. Furthermore, urban toads had lower concentrations of bufotoxins, the compounds with lower toxicity. None of these differences were present in the captive-raised juveniles; instead, toadlets originating from agricultural habitats had smaller parotoids and lower bufotoxin concentrations. These results suggest that toads’ chemical defences respond to the challenges of anthropogenic environments via phenotypic plasticity. These responses may constitute non-adaptive consequences of pollution by endocrine-disrupting chemicals as well as adaptive adjustments to the altered predator assemblages of urban and agricultural habitats.

Essential oils obtained by hydrodistillation (HD) and microwave-assisted HD (MWHD) of Origanum onites aerial parts were analyzed by GC and GC/MS. Thirty-one constituents representing 98.6% of the water-distilled oil and 52 constituents representing 99.6% of the microwave-distilled oil were identified. Carvacrol (76.8% HD and 79.2% MWHD) and thymol (4.7% HD and 4.4% MWHD) were characterized as major constituents in both essential oils. Separation of carvacrol and thymol was achieved by overpressured layer chromatography. HPTLC and TLC separations were also compared. Essential oils were evaluated for antifungal activity against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, C. fragariae, and C. gloeosporioides using a direct overlay bioautography assay. Furthermore, main oil components carvacrol and thymol were then evaluated for antifungal activity; only carvacrol demonstrated nonselective antifungal activity against the three Colletotrichum species. Thymol and carvacrol were subsequently evaluated in a 96-well microdilution broth assay against Phomopsis obscurans, Fusarium oxysporum, three Colletotrichum species, and Botrytis cinerea. No activity was observed against any of the three Colletotrichum species at or below 30 μM. However, thymol demonstrated antifungal activity and produced 31.7% growth inhibition of P. obscurans at 120 h and 0.3 μM, whereas carvacrol appeared inactive. Thymol and carvacrol at 30 μM showed 51.5 and 36.9% growth inhibition of B. cinerea at 72 h. The mechanism of antibacterial activity was studied in a bioautography-based BioArena system. Thymol and carvacrol showed similar inhibition/killing effect against Bacillus subtilis soil bacteria; the action could be enhanced by the formaldehyde generator and transporter copper(II) ions and could be decreased in the presence of L-arginine, a formaldehyde capturer. Results indicated that Origanum essential oils and its major components thymol and carvacrol appear to generate antimicrobial activity through a mechanism of action where formaldehyde and its reaction products are produced.

In this work, a rapid screening method for arsenic speciation, based on the use of polyethyleneimine (PEI) cellulose thin layer chromatographic (TLC) plates for separation and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for the detection of the As species, has been implemented. The TLC plates were developed with a mixture consisting of acetone : acetic acid : water (2 : 1 : 1 v/v/v). The procedure proposed has been shown to be capable of the separation of picograms (an LOD of 3 pg has been calculated) to nanograms levels of arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), while arsenobetaine (AsB) and arsenocholine (AsC) practically remained on the sample application point of the TLC plate, allowing their separation from the rest of the compounds but not enabling separating them from one another. The separation procedure only requires approximately 20 min (up to 8 samples can be simultaneously separated) while the LA-ICP-MS protocol only needs a couple of minutes per chromatographic lane. The use of the dynamic reaction cell pressurized with H₂ permits one to overcome the possible overlap of the signals of ⁷⁵As⁺ and ⁴⁰Ar³⁵Cl⁺, ensuring reliable monitoring of the analyte species. The different As species found in NIST SRM 1568a rice flour, after prior enzymatically-assisted extraction with an ultrasonic processor, could be satisfactorily detected, suggesting that the method may be of interest for the fast and qualitative monitoring of As species in biological samples.