Assessment of synergistic and antagonistic interactions between volatile compounds thymol, carvacrol, and eugenol diluted in solvents against Rhipicephalus microplus in in vitro tests

The cattle tick Rhipicephalus microplus is prevalent in tropical and subtropical regions, causing substantial economic losses due to its resistance to conventional acaricides. There is an urgent need to identify safe and effective new acaricidal agents. Essential oils and their volatile compounds are promising alternatives. Ensuring the use of optimal solvents or surfactants that do not compromise the acaricidal activity of these compounds during testing is crucial. This study aims to evaluate how compounds thymol, carvacrol and eugenol interact with xylol, methanol, ethanol, acetone, isopropyl alcohol, glycerol, dimethyl sulfoxide, castor oil, propylene glycol, vaseline, and Tween 80® to enhance (or to worse) their acaricidal efficacy against R. microplus. Larval mortality time were compared against one negative control (soybean oil) and two positive controls (commercial pour-on products). The experiments were conducted in 48-well polyethylene plates, with around 100 larvae immersed in 200 μl of each solvent at 100, 50, 25, 12.5, 6.25, 3.125 and 1.56% and diluted in soybean oil or water, according to solubility. Each volatile compound (Thymol, carvacrol and eugenol) was diluted in the tested solvents to assess larval mortality time. Xylol demonstrated the shortest larval mortality time, even at a minimum concentration (p < 0.05). In contrast, liquid vaseline exhibited the longest larval mortality time. When thymol, carvacrol, and eugenol were combined with xylol, they achieved the shortest larval mortality time. Conversely, when diluted in liquid vaseline they exhibited synergistic effects decreasing the mortality time. Tween 80® worsen the efficacy of thymol, carvacrol, and eugenol, resulting in prolonged larval mortality times. These findings emphasize the critical role of solvent selection, indicating the choice of solvent profoundly affects the formulation's effectiveness, directly influencing the activity of the active compounds.