TXRF analysis of earthworm coelomic fluid extracts: a useful tool to assess the bioavailability of As in soils

Arsenic (As) is a metalloid often associated to mining and industrial sites and its presence can cause health problems to living organisms and human beings. In order to assess As bioavailability in soils, tests which use earthworms as sentinel organisms are usually done. The evaluation of As concentration in the earthworm is commonly done after the whole digestion of the organism, which can lead to the overestimation of the concentration due to the possible presence of soil residues in the intestine. However, As is mainly accumulated by earthworms in the coelomic cavity , in particular in the coelomic fluid. This fluid can be easily extracted without killing the organism and analysed. Due to its small amount (less than 100 microliters), total x-ray fluorescence spectroscopy (TXRF) has demonstrated to be a powerful method for As detection in coelomic fluid extracts [1]. For these reasons, the present work aims at developing a new method to assess As bioavailability in contaminated soils by analyzing the As concentration in coelomic fluid extracts with TXRF. Six natural As polluted soils and two control soils were characterized by XRPD and WDXRF, while the As mobility was assessed by a sequential extraction procedure. Then, 10 sexually mature earthworms were exposed to each soil for 14 days. The elemental distribution inside the earthworm was studied by μXRF on thin sections, which localized As only in the coelomic cavity. After 24h without nourishment, three earthworms per soil were washed with distilled water and coelomic fluid extracts were collected from each of them applying a voltage of 5 V for 3 seconds. Ten microliters of extract were mixed with 80 µl of PVA and 10 µl of Y standard solution (10 mg/l). In order to compare the As concentration in the coelomic fluid extracts with the As concentration in the earthworm body, additional three earthworms per soil were depurated, dryed at 60 °C and pulverized. Slurry suspensionswere prepared using 100 mg of powder, 5 ml of Triton X-100 and 10 µl of Y standard solution (1000 mg/l). In both cases, 10 µl of sample were pipetted onto a quartz reflector and were left drying at 50 °C. TXRF analysis were performed with a S2 Picofox spectrometer (Bruker) using an acquisition time of 1000 s. Results revealed that As (both in fluids and whole bodies) increased with increasing mobile As in the soil. hHowever, As saturation (in both fluid and body) was observed when the mobile As fraction exceeded 200 mg/kg. Finally, the As concentration in coelomic fluids was positively correlated with that in the whole body. These results show that TXRF is a powerful tool to determine As concentration in earthworm coelomic fluids and that it can be used for As bioavailbility studies in soils. [1] I. Allegretta, C. Porfido, O. Panzarino, M.C. Fontanella, G.M. Beone, M. Spagnuolo, R. Terzano, Spectrochimica Acta Part B 130, 2017, 21-25.