The Enchytraeus crypticus stress metabolome – CuO NM case study

Abstract

The stress metabolome provides a thorough insight into the signals and hence mechanisms of response of organisms. This is an excellent tool to advance the understanding of interactions, especially for substances like nanomaterials (NMs), for which there is an urgent need for alternative methods for hazard assessment. The metabolome of Enchytraeus crypticus was studied for the first time. The case study, CuO NM (and CuCl₂) covered exposure along a time frame [0–7–14 days (d)] and two reproduction effect concentrations (EC10 and EC50). High-performance liquid chromatography-mass spectrometry based method (HPLC-MS) was used, with reversed phase (RP) separation and mass spectrometric detection in positive and negative modes. Metabolite profiling of Cu materials yielded 155 and 382 metabolite features in positive and negative modes, respectively, showing an expression related with time, material, and ECx. The number of differentially expressed metabolites (DEMs) decreased with exposure time (14 d) for CuO NM, whereas for CuCl₂ EC50 it increased. Overall, almost all DEMs are down-regulated for CuO NM and up-regulated for CuCl₂ (both modes). Early effects were mainly related to amino acids and later to lysophospholipids (down-regulation). Furthermore, the underlying mechanisms of CuO NM toxicity (e.g. neurotransmission, nucleic acids generation, cellular energy, and immune defense) differ from CuCl₂, where later metabolomic responses are mostly linked to the metabolism of lipids and fewer to amino acids. This study reports a large scale metabolome profiling for E. crypticus and identifies potential markers of Cu materials, which can help to align intelligent testing strategies and safer-by-design materials.

Keywords:

• Non-target metabolomics
• HPLC-MS system
• Enchytraeidae
• engineered nanomaterials

Acknowledgements

The authors acknowledge the laboratory assistance provided by M. J. Ribeiro.

Disclosure statement

The authors declare that they have no competing interests.

This article does not contain any studies with human participants performed by any of the authors. According to Directive 2010/63/EU of the European Parliament and of the Council of 22/9/2010, on the protection of animals used for the scientific purpose, invertebrates like pot worms (E. crypticus) are permitted biological models for scientific experimentation and consequently free of Ethical Statement. The scientific and social relevance is related to education and scientific knowledge, its applicability to environmental protection and to evaluate the risks to human health.

Additional information

Funding

This study was supported by the European Commission by the EU FP7 project SUN (G.A. No. 604305) and by the Foundation for Science and Technology (FCT)-FSHE/POCH post-doctoral grant to V.L. Maria (SFRH/BPD/95093/2013). Thanks are also due to CESAM (UID/AMB/50017-POCI-01-0145-FEDER-007638), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete for 2020.