Research PaperEmbryotoxicity of silver nanomaterials (Ag NM300k) in the soil invertebrate Enchytraeus crypticus – Functional assay detects Ca channels shutdown
Graphical abstract
Introduction
Global consumption of engineered silver nanomaterials (Ag NMs) is predicted to continue to increase at an exponential rate (Syafiuddin et al., 2017). Ag NMs used in healthcare, clothing industry and electronic and electrical devices is estimated to reach more than 60, 40 and 30 tons per year by 2022, respectively, (Syafiuddin et al., 2017). The terrestrial environment is one of the major sinks of NMs, including Ag NMs (Gottschalk et al., 2015). Among the main sources of entry to soil are sewage sludge application as agriculture fertilizer (Schlich et al., 2013) and washing of paints with rainwater and sedimentation (Topuz et al., 2015). Environmental estimations for Europe point at an annual increase of Ag NMs at levels of 1.2–2.3 ng/kg for sediments and soils (Sun et al., 2014). A literature search on studies with Ag NMs in soil invertebrates (for details, please see Table S1), covered earthworms (Gomes et al., 2015; Heckmann et al., 2011; Mariyadas et al., 2018; Schlich et al., 2013; Shoults-Wilson et al., 2011; Tsyusko et al., 2012; van der Ploeg et al., 2014; Kwak et al., 2014) nematodes (Roh et al., 2009; Schultz et al., 2016; Tyne et al., 2015; Rossbach et al., 2020), isopods (Tourinho et al., 2015), collembolans (Mendes et al., 2015; Waalewijn-Kool et al., 2014; Kwak and An, 2016), terrestrial snails (Chen et al., 2017; Radwan et al., 2019) and enchytraeids (Bicho et al., 2016; Gomes et al., 2017, Gomes et al., 2013; Maria et al., 2018; Mendonça et al., 2020; Ribeiro et al., 2015; Topuz and van Gestel, 2017). In these studies, several endpoints were assessed, i.e. survival, reproduction, fertility, growth, hatching success, bioaccumulation, trophic transfer, avoidance, burrowing and locomotion behaviour, feeding activity, oxidative stress biomarkers, gene expression, genotoxicity, immunotoxicity and cytotoxicity. Also various test setups were used, e.g. multigenerational, full life cycle, life-span and bioaccumulation. Despite the considerable number of studies and attention, data on embryotoxic effects with NMs in soil invertebrates are virtually absent in the literature. This is probably because few embryotoxicity assays have been developed for soil invertebrate species: one for the snail Helix aspersa (Druart et al., 2012, Druart et al., 2010) and one for the enchytraeid Enchytraeus crypticus (Gonçalves et al., 2015). Recently a related test, a novel egg life-stage test, was also developed for the collembolan Folsomia candida (Guimarães et al., 2019).
The exposure of E. crypticus to Ag materials (AgNO3 and Ag NM300K) in a full life cycle test (FLCt) (Bicho et al., 2016) showed an adverse effect on hatching success (day 11), which was confirmed at day 46 to be a hatching delay effect for silver nitrate (AgNO3) and mortality for Ag NM300K (ca. 15 nm, dispersed). The effects of Ag NM300K may have occurred during the embryonic development, hatching (inhibition) or the survival of the new hatched juveniles. Further, a transcriptomics study with E. crypticus (Gomes et al., 2017) identified transcytosis as a uniquely affected process from Ag NM300K exposure. Other studies have shown activation of transcytosis by Ag NMs, which e.g. crossed the blood–brain barrier in brain rat cells (Tang et al., 2010), or entered cells and undergo transcytosis in human nasal epithelium cells (Falconer et al., 2018). Transcytosis is a mechanism for transporting macromolecules across cells and the transferrin receptor (TfR) is one of the major receptors that mediates this mechanism (Gao et al., 2017). The primary role of TfR is to deliver iron into cells trough endocytosis of transferrin (Crichton, 1991). This receptor has been described in several species of invertebrates (Huebers et al., 1984; Lambert et al., 2005) and the role of transferrin as iron carrier has been shown in insect species (Lambert, 2012). In human medicine transcytosis has also been widely explored as a favourite intracellular transport for NMs as drug carriers (Neves et al., 2016; Reinholz et al., 2018). Additionally, effects of Ag in calcium (Ca) homeostasis have been have shown e.g. in studies in plant cells (Klíma et al., 2018) and rat cells (Haase et al., 2012; Ziemińska et al., 2014). Hence, evidences are that Ag can be transported across cells via transcytosis and also that Ag disturbs organisms` Ca metabolism. Here it is hypothesized that 1) Ag affects tissue differentiation during embryonic development; 2) if uptake of NMs occurred in cocoons, mechanisms of extracellular transport, like transcytosis will be activated and 3) Ag affects Ca metabolism during embryonic development. To test these hypotheses, an embryotoxicity test was performed, exposing E. crypticus cocoons to Ag NM300K (0–10–20–60–115 mg Ag/kg) and AgNO3 (0–20–45–60–96 mg Ag/kg) in LUFA 2.2 soil. Samples were collected at intervals between 3 and 7 days after cocoon laying, to cover all embryonic development. For this, histological and immunohistochemistry analysis were performed to assess tissue/organ effects. Two mechanisms were targeted for immunohistochemistry a) transcytosis, via the expression of the transferrin receptor (TfR) and b) Ca metabolism via the expression of L-type Calcium channels (LTCC).
Section snippets
Test organisms
Enchytraeus crypticus (Oligochaeta: Enchytraeidae) were used. Animals have been maintained at the University of Aveiro for more than ten years, cultures are maintained in laboratory under controlled conditions (20 °C, 16:8 light: dark photoperiod) in agar plates with a salt solution of CaCl2, MgSO4, KCl and NaHCO3 and fed ad libitum with oatmeal. Synchronized cultures were prepared as described in (Bicho et al., 2015). In short, adults with well-developed clitellum are transferred into new agar
Embryotoxicity test and histology
Values for soil pH did not change significantly with concentrations and time, being for Ag NMs: 5.32 ± 0.04 and AgNO3: 5.43 ± 0.06 (AV ± SE). The number of cocoons retrieved from the soil per sample was 100%, i.e. the 10 cocoons per replicate were all retrieved. However, due to the small size of cocoons (few μm) after histological procedures (paraffin embedding and transfer to paraffin blocks) 50% of the cocoons were fit for histological and immunohistochemistry analysis, i.e. 5 cocoons per
Discussion
This follow up exposure study of E. crypticus in an embryotoxicity test, which combined histology and immunohistochemistry tools, allowed inspection of when effects of Ag NM300K and AgNO3 occurred during the full life cycle. From Bicho et al. (2016) it was unidentified if the observed effects were initiated during embryonic development or in later stages of the life cycle, now we could confirm that effects actually did occur during embryonic development. In fact, for both Ag materials effects
Conclusions
Both Ag materials, Ag NM300K and AgNO3, affected the embryonic development of E. crypticus during blastula stage. The expression and localization of TfR in E. crypticus teloblasts cells was shown, although this transcytosis mechanism was not activated. Ag affected Ca metabolism during embryonic development, for AgNO3 LTCC expression was initially activated, compensating the impact, whereas for Ag NM300K, LTCC was not activated, hence no Ca balance occurred, causing irreversible consequences,
Funding
This study was supported by the European Commission Projects: BIORIMA - BIOmaterial RIsk MAnagement (H2020-NMBP2017, GA No. 760928), NanoInformaTIX - Development and Implementation of a Sustainable Modelling Platform for NanoInformatics (H2020-NMBP-14-2018, GA No. 814426) and NANORIGO - Establishing a Nanotechnology Risk Governance Framework (H2020-NMBP-TO-IND-2018, GA No. 814530). Further support within CESAM (UIDB/50017/2020+UIDP/50017/2020), to FCT/MEC through national funds, and the
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors acknowledge the support provided by V. Maria.
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