Xenometabolomics in plasma of anurans inhabiting sites affected by sewage discharges

SALGADO COSTA, C.; BAHL, F.; NATALE GS; VENTURINO A; MARINO DJM; RODRIGUEZ-MOZAZ, S.; SANTOS L

Montevideo

Congreso; SETAC Latin America 15th Biennial Meeting; 2023

SETAC LA

A thorough assessment of organisms exposed to complex chemical mixtures such as sewage effluents can benefit from analytical non-target approaches, which do not require pre-selection of target compounds. Moststudies in the field of biomonitoring of aquatic organisms use invasive techniques that sacrifice them. In recent years, priority has been given to techniques that aimed to replace, reduce and/or refine practices (the 3Rs approach). In this regard, biofluid extraction, such as plasma, is a less invasive approach because individuals can be safely returned to their environment after sample collection. The aim of this study was to perform a non-target xenometabolomics study on the plasma of adult individuals of Leptodactylus luctator (Anura: Leptodactylidae) inhabiting wastewater effluent channels. Two sites with apparently different levels of anthropogenic disturbance were selected in northeastern Buenos Aires Province (Argentina): a reference site in Punta Indio and a site with direct discharge from a secondary wastewater treatment plant in Chascomús. Adult individuals of both sexes (n = 12) were collected at both sites in December 2022. Individuals were anesthetized and blood samples were collected through the femoral vein with heparinized syringes and then centrifuged at 7000 rpm for 15 min to collect plasma. Each plasma sample was divided into two replicates and deuterated atrazine was added (final concentration in plasma 50 μg/L). Proteins were then precipitated with acetonitrile (3:1, v/v) and centrifuged at 7000 rpm for 15 min. Procedural controls were prepared in duplicate with 0.7% saline solution. Instrumental analysis was performed using LC-MS Orbitrap, operating both in positive and negative mode. Preliminary results indicate a clear separation of individuals from both sites. This study will provide valuable information on the health status of the organisms, as plasma contains many endogenous metabolites that may be altered by exposure to xenobiotics, as well as xenobiotics and their metabolites that may be accumulated and distributed to other tissues in exposed organisms. This is the first non-target xenometabolomics study in adult anurans environmentally exposed to various contaminants.