Introduction
The novel mechanical, thermal, optical, and antimicrobial properties of nanomaterials (NMs) make them valuable tools in many sectors such as the food industry and also in the aquaculture activity . However, t here is great concern regarding the impacts of engineered NMs , mainly metallic nanoparticles (NPs), on environmental and human health. NPs are present in the marine environment, and background levels of TiO2 and Ag NPs have been reported in molluscs [1,2]. Therefore, studies regarding controlled exposition of NPs to cultured molluscs and fish are important for elucidating the bioaccumulation rate of these new emerging pollutants and the impact on the aquaculture sector.
Transmission electronic microscopy (TEM) and single particle-inductively coupled plasma – mass spectrometry (sp -ICP-MS) has been for studying and quantifying the presence of TiO2 and Ag NPs in mussels (Mytilus sp) and turbot (Scophthalmus maximus) after controlled exposure in an aquaculture facility. This communication summarises the preliminary results regarding the bioaccumulation rate of these new pollutants in tissues from mussels (digestive gland, mantel and gill) and turbot (muscle, kidney, intestine and liver).
Methods and results
Mussel and turbot tissues were subjected to microwave assisted acid digestion followed by ICP-MS for assessing total Ti and Ag contents; whereas, an assisted enzymatic hydrolysis procedure and sp-ICP-MS was used for TiO2 and Ag NPs quantification/characterization. The collected tissues follow a standard procedure for TEM analysis. Shortly, fixation with Karnovsky fixative, post-fixation with osmium tetroxide and finally embed and include on resin .
ICP-MS have shown low bioaccumulation of TiO2 NPs when exposing mussels to P25 TiO2 (25 nm) at 0.1 mg L-1 [total Ti increase from 0.03 (day 0) to 0.27 µg g-1 (day 28)] and at 1.0 mg L-1 [total Ti increase from 0.03 (day 0) to 9.0 µg g-1 (day 28)]. The presence of TiO2 NPs were not detected by sp-ICP-MS since the low NPs size (25 nm) but the measurement of dissolved Ti plus TiO2 NPs (sp-ICP-MS) and total Ti (ICP-MS) were in good agreement.
Regarding Ag NPs (PVP-15 nm Ag) , exposes at 0.1 mg L-1 showed an increase on the total Ag from 0.003 µg g-1 (time 0) to 1.5 µg g-1 , value which reached a plateau at sampling day 21st. Low bioaccumulation was also observed for experiments at 1.0 mg L-1 ( the maximum total Ag concentration was closed to 1.5 µg g-1, value which was obtained after sampling day 7th , being then constant until the end of the experiment). Ag bioaccumulation in mussels appears to be independent on the dose at longer exposure times since the maximum Ag concentrations measured in the tissues has been the same for both Ag NPs concentrations. This finding could be related with the dark scum observed in some tanks after long exposure times and/or high Ag NPs concentrations (the analysis of the dark scum revealed the presence of silver). Similar findings have been found when assessing Ag NPs instead of total Ag: Ag NPs are accumulated at a maximum concentration of 2.37×108 Ag NPs g-1 after 28 days (0.1 mg L-1 Ag NPs) and 2.56×108 Ag NPs g-1 after 28 days (1.0 mg L-1).
Digestive gland, mantel and gill from mussels were analysed by TEM after 0, and 28 days of exposure . Neither Ag NPs nor TiO2 NPs were observed in any tissue.
Regarding turbot, both TiO2 and Ag NPs were found to be poorly accumulated and negligible total Ti and Ag concentrations were detected in the muscle . Most of Ti was detected in the faeces, which concentrations ranging from 1.6 (equivalent dose 0) to 40 µg g-1 (equivalent dose 1.5 mg Kg-1 per fish and day). It seems that Ti is eliminated in faeces. An analysis of the faeces by energy dispersive X-ray spectrometry (EDX)-scanning electr on microscopy (SEM) confirmed the massive elimination of the TiO2 NPs by this route. However, liver was found to be a target organ for TiO2 and Ag NPs, and little bioaccumulation rates were observed: total Ti concentration up to 0.25 µg g-1 (dose of 1.5 mg kg-1, day 90) and total Ag concentration up to 6.0 µg g-1 (dose of 1.5 mg kg-1 , days 30, 45 and 90). Ag was also found to be accumulated in kidney [Ag concentration up to 0.25 µg g-1 (dose of 1.5 mg kg-1 , days 30, 45 and 90)]. Because of the low concentrations of total Ti and Ag in turbot tissues, the levels of TiO2 and Ag NPs in were found un-detected in cultured turbot by sp-ICP-MS . TEM analysis of muscle, liver, kidney and intestine after 0 and 90 days of exposure showed that there was not significant bioaccumulation because no NPs were visualized in any tissue, which is in agreement with the sp-ICP-MS results. However, some ultrastructural changes were observed in some of the tissues (e.g. kidney) indicating some damaged that is currently being investigated.
References
[1] M.V. Taboada-López, et al. Ultrasound assisted enzymatic hydrolysis for isolating titanium dioxide nanoparticles from bivalve mollusc before sp-ICPMS. Analytica Chimica Acta, 1018 (2018) 16–25.
[2] M.V. Taboada-López , et al. Determination and characterization of silver nanoparticles in bivalve molluscs by ultrasound assisted enzymatic hydrolysis and sp -ICPMS. Microchemical Journal, 148 (2019) 652–660.
Acknowledgments
The authors wish to acknowledge the financial support of the European Union (Interreg Atlantic Area, project NANOCULTURE, reference EAPA_590/2018) , and the Ministerio de Economía y Competitividad (project INNOVANANO, reference RT2018-099222-B-100).