Elsevier

Environmental Pollution

Volume 174, March 2013, Pages 157-163
Environmental Pollution

Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish (Danio rerio)

https://doi.org/10.1016/j.envpol.2012.11.017Get rights and content

Abstract

Nanoparticles (NPs) can be ingested by organisms, and NPs with antimicrobial properties may disrupt beneficial endogenous microbial communities and affect organism health. Zebrafish were fed diets containing Cu-NPs or Ag-NPs (500 mg kg−1 food), or an appropriate control for 14 d. Intestinal epithelium integrity was examined by transmission electron microscopy, and microbial community structure within the intestine was assessed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA. No lesions were observed in intestinal epithelia; however, presence of NPs in diets changed intestinal microbial community structure. In particular, some beneficial bacterial strains (e.g., Cetobacterium somerae) were suppressed to non-detectable levels by Cu-NP exposure, and two unidentified bacterial clones from the Firmicutes phylum were sensitive (not detected) to Cu, but were present in Ag and control fish. Unique changes in zebrafish microbiome caused by exposure to Ag-NP and Cu-NP indicate that NP ingestion could affect digestive system function and organism health.

Highlights

► Zebrafish ingest Cu- and Ag-nanoparticles (NPs) in diet. ► No effect of Cu-NPs or Ag-NPs on intestinal epithelial integrity. ► Cu-NPs and Ag-NPs alter endogenous microbiota of zebrafish.

Introduction

Increased production and incorporation of nanoparticles (NPs) into consumer products will ultimately lead to release of NPs into the environment and potential for unintended NP exposure in humans and other organisms (Nowack, 2007). Some NPs have antimicrobial properties and such properties have been previously established and described for Ag-NPs and Cu-NPs (Sondi and Salopek-Sondi, 2004, Cioffi et al., 2005, Yoon et al., 2007, Ruparelia et al., 2008). The mechanisms behind the anti-microbial activity are partly described and their efficacy varies depending on particle size, particle shape and bacterial species (Panacek et al., 2006, Pal et al., 2007, Ruparelia et al., 2008). As a result NPs have been included in the formulations of consumer products for disinfection purposes [e.g., Ag-NPs (Cho et al., 2005)]. The ability to kill or impede the growth of pathogenic bacteria is an important aspect of human and veterinary medicine; however, if unintended exposure to NPs occurs and affects beneficial bacteria there is potential for negative effects on animal health.

Perhaps the most environmentally relevant route of NP exposure is through the diet, either via ingestion of food contaminated by NPs, or by consumption of food items (e.g., prey) that have accumulated NPs (Petersen and Henry, 2012). Filter-feeding aquatic invertebrates can accumulate NPs from the aqueous phase (Petersen et al., 2009) and substantially increase dietary exposure of consumers (e.g., organisms that eat invertebrates) to NPs (Park et al., 2010). NP contamination of food via deliberate close association between food and NPs is an issue of considerable concern for the food packaging industry, which has embraced incorporation of some NPs into packaging materials to maintain freshness and inhibit growth of microorganisms (Xu et al., 2010). The potential for food items to become contaminated by NPs used in packaging has been recognized and is a concern for human health that requires further investigation (Xu et al., 2010).

Previous research on the toxicity of ingested NPs has focused on determining whether or not NPs are absorbed across the gut, and the consequences for systemic toxicity [e.g., humans (Panessa-Warren et al., 2006), fishes (Ramsden et al., 2009, Fraser et al., 2011)]. Experiments have documented absorption of some NPs (e.g., Ag-NPs) across epithelial membranes (e.g., Scown et al., 2010); however, accumulation appears to be minimal in internal tissues, and whether the accumulation is because of NPs or metal ions is unresolved (e.g., Ag-NPs, Scown et al., 2010; TiO2-NPs, Ramsden et al., 2009; Cu-NPs, Al-Bairuty et al., 2012). A neglected area of research in both animals and humans are the effects of ingested NPs on the major physiological functions of the gastrointestinal tract (GIT). For nutrition, these functions are gut motility, the secretion of fluids into the digestive tract, digestion, and the absorption of nutrients. Accumulation of some NPs within the gut lumen can influence gut motility in organisms (e.g., Heinlaan et al., 2011), and some lesions in epithelial mucosa of the gut associated with dietary NP-exposure have been reported (Smith et al., 2007, Federici et al., 2007).

The microbial community within the gut is an important contributor to organism health and the ingestion of NPs with antimicrobial properties may disrupt this community. The intestinal microbiome in organisms including humans is now recognized to form a complex symbiotic relationship that influences metabolism, physiology and gene expression (Kinross et al., 2009). A previous study reported that NPs may influence the gut microbiota of vertebrates: specifically, increased populations of lactic acid bacteria were detected in quail (Coturnix coturnix japonica) after ingestion of Ag-NPs (Sawosz et al., 2007). This preliminary study is interesting but the results are limited due to the culture-based microbiological approach used and the lack of a suitable Ag control (i.e. the experimental design does not allow for the differentiation of an Ag effect and an Ag-NP effect).

The zebrafish (Danio rerio) has become an important model species for the study of microbial communities in vertebrate intestines (Rawls et al., 2004, Rawls et al., 2007), and this model has also been useful for assessing the toxicity of NPs (Henry et al., 2007, Park et al., 2010, Park et al., 2011). Therefore, the primary objective of the present study was to make an initial assessment of the effect of dietary Cu-NPs or Ag-NPs on gut microbial communities in adult zebrafish. In addition to evaluation of the gut microbiota, the presence of lesions in the intestinal epithelial mucosa after exposure to NPs was assessed by electron microscopy. While uses of Cu-NPs are emerging they do not currently present substantial exposure risk in organisms; however, Cu is an important environmental toxicant and selection of Cu-NPs for this study was based on the interest to compare results with Ag-NPs, evaluate differences between nano and bulk forms of Cu, and to enable comparison with previous studies of Cu-NPs (e.g. Griffitt et al., 2007, Shaw et al., 2012).

Section snippets

Experimental diets

A basal diet was formulated to provide ca. 40% protein and 6% lipid (Table 1). This diet was used for the control group. Treatment diets containing NPs (Fig. 1) were prepared by adding the unmodified powdered form of each NP (no stabilizing coats or surface functionalization) to the basal ingredients of the diet to ensure even distribution of NPs within the feed pellets (see below) at 500 mg NP kg−1 feed. Selection of this exposure concentration was based on our previous experiments with

Survival and gross observations

During the experiment only one fish died (control group) and there were no clinical indications of disease, abnormal behaviour, or changes in feeding activity in any of the experimental fish. The fish from all treatments and controls showed normal feeding behaviour; all of the allocated food was consumed by fish within 5 min. No external or internal (peritoneum) gross lesions were observed in any fish (n = 150) euthanized and dissected for examination of intestine by TEM or evaluation of

Discussion

Dietary exposure of organisms to NPs is a likely outcome of the release of NPs into the environment, and the use of nanotechnology in food [review (Chaudhry et al., 2008)] will also result in exposure via ingestion. The present study is the first to report NP induced (as opposed to metal induced) changes in microbial communities in the GIT of any vertebrate animal after dietary NP intake. Zebrafish fed NPs had a feeding response that was consistent with fish fed the control diet suggesting that

Conclusions

The present study indicates that the microbial community of a vertebrate intestine can be disrupted by dietary exposure to NPs. The disruption of gut microbiota differed between Ag- and Cu-NPs and compared to the bulk metal controls, and indicated unique effects associated with ingestion of NPs. Although the presence and distribution of NPs in food pellets were documented, the forms of NPs during passage within the lumen of the gut are unknown (methods not available with current technology),

Funding

This work was supported by internal research funds within the Plymouth University. Dr. Imad Saoud was supported by the Arab Fund Fellowships Program-Distinguished Scholar Award.

Acknowledgements

The authors thank Prof. J. Rawls for his kind input and data sharing with regards to identification of phylotypes from the gut of zebrafish in studies conducted in the USA. S. McMahon provided much appreciated assistance with zebrafish husbandry.

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