| Summary: | Due to the increasing incidence of SARS-CoV-2 on a global scale a pandemic was declared at the beginning of 2020. Among the various mitigation measures proposed by all governments and entities responsible for human health, the use of disinfectants was one of the most consensual. Disinfectants are now applied in homes, outdoors, in hospitals, and for personal use. Given the increased demand, it is expected that they may reach aquatic environments in greater quantities. Assessing the possible effects of these compounds becomes even more imperative, as the use of disinfecting agents is expected to grow by 10% by 2027. Among the most commonly used and most frequently encountered classes of disinfectants in the environment, we identify cationic and anionic surfactants, and those whose active substances are natural compounds. For the scope of this work, benzalkonium chloride (BAC), a cationic surfactant belonging to the family of quaternary ammonium compounds, the salt of sodium dodecylbenzene sulfonic acid (SDBS), an anionic surfactant, and thymol (THY), a plant-derived compound with multiple applications, were thus selected. Although there are some studies on the properties and effects of these active ingredients on aquatic organisms, their adverse effects on aquatic species remain largely unknown. In this sense, this study aimed to evaluate, in five-day-old larvae of the species Danio rerio, through acute exposures, embryonic, behavioral (total activity, thigmotactic behavior and erratic movements) and metabolic biomarkers, such as the activity of phase I enzymes (CYP 1A1 and CYP 1A2), the phase II metabolism isoenzymes, glutathione-S-transferases (GSTs) and, finally, antioxidant defense enzymes (catalase, CAT; glutathione peroxidase, GPx). The concentrations of the 3 agents to which the animals were exposed were taken from levels of these compounds already reported in various aquatic matrices (effluents, municipal waters, and rivers) to obtain greater relevance of the data. Exposure of animals to BAC resulted in increased mortality at 48h after exposure to a concentration of 2.5 mg/L, and was also responsible for an increased number of malformations, such as pericardial edema and tail malformations. Behaviorally, total activity, thigmotactic behavior and the number of erratic movements (class I) were also increased in animals exposed to a concentration of 2.5 mg/L. This overall increase in activity may be related to changes in the ionic balance and alterations in the balance of cholinergic pathways. Metabolic biomarkers reported an increase in their activity, in the case of CYP 1A1 and CAT (in animals exposed to 0.5 mg/L), and an inhibition of CYP 1A2, GPx and GSTs in animals exposed to the remaining concentrations (0.1; 0.5 and 2.5 mg/L). These results suggest that, in a first step, BAC is metabolized by CYP 1A1, leading to the production of reactive oxygen species (ROS), increasing CAT activity. The possible continued production of ROS led to the inhibition of GPx and GSTs. SDBS exposure was responsible for increased mortality at 96h (animals exposed to 5 mg/L) but no predominant malformation was observed. In behavioral terms, there was an increase in total activity and in the number of erratic movements (class I) which is presumably due to the changes in acetylcholinesterase (AChE) levels observed in other studies. In the enzymatic biomarkers an increase in CAT activity was observed which translates to a possible activation of antioxidant defense. In contrast, a general inhibition of the enzymes GPx and GSTs was observed, which may stem from increased ROS. The general increase in ROS levels, in a direct or indirect way, may promote enzyme denaturation, given their interaction with proteins, lipids, nucleic acids, leading to oxidative damage. The inhibition of CYP 1A1 and CYP 1A2 reported in our study is possibly due to the specificity of the compound being metabolized by other isoenzymes. Finally, THY exposure also led to increased mortality at 96h, and a high incidence of pericardial edema. Regarding swimming activity and thigmotactic behavior (parameter of distance traveled in the peripheral zone) a decrease was observed. As for erratic movements, a significant increase was observed for animals exposed to all concentrations. Changes in the balance of ionic currents of potassium (K+) and sodium (Na+) ions and in AChE levels reported in previous studies, may translate the mechanisms underlying these changes. Inhibition of the enzymatic activity of CYP 1A1, CYP 1A2, CAT and GSTs were also verified. THY is an antioxidant compound, which promotes its action either by destroying hydrogen peroxide (via antioxidant enzymes), or by neutralizing peroxide radicals (by transferring H to ROO*). However, THY, in large amounts can also act as a pro-oxidant. Given these properties the data reported in the present study suggest that this decrease in overall enzymatic activity is due to the antioxidant properties of this active ingredient. We thus conclude that the disinfectants tested at environmentally relevant concentrations cause adverse effects in the model organism studied here. The observed effects raise further concerns given that the production and consumption of these compounds will continue to increase.
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