Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide induces DNA damage and mutagenic effects in the guppy (Poecilia reticulata)

https://doi.org/10.1016/j.etap.2020.103521Get rights and content

Highlights

  • The co-exposure of γ-Fe2O3 NPs and GBH induced toxic effects in guppy.

  • Co-exposure caused DNA damage (genotoxicity) in the guppy.

  • Co-exposure induced nuclear alterations (mutagenicity) in the guppy.

  • γ-Fe2O3 NPs affect GBH toxicity in freshwater fish.

Abstract

Iron oxide nanoparticles (IONPs) have been tested to remediate aquatic environments polluted by chemicals, such as pesticides. However, their interactive effects on aquatic organisms remain unknown. This study aimed to investigate the genotoxicity and mutagenicity of co-exposure of IONPs (γ-Fe2O3 NPs) and glyphosate-based herbicide (GBH) in the fish Poecilia reticulata. Thus, fish were exposed to citrate-functionalized γ-Fe2O3 NPs (0.3 mg L−1; 5.44 nm) alone or co-exposed to γ-Fe2O3 NPs (0.3 mg L−1) and GBH (65 and 130 μg of glyphosate L−1) during 14 and 21 days. The genotoxicity (DNA damage) was analyzed by comet assay, while the mutagenicity evaluated by micronucleus test (MN test) and erythrocyte nuclear abnormalities (ENA) frequency. The co-exposure induced clastogenic (DNA damage) and aneugenic (nuclear alterations) effects on guppies in a time-dependent pattern. Fish co-exposed to NPs and GBH (130 μg glyphosate L−1) showed high DNA damage when compared to NPs alone and control group, indicating synergic effects after 21 days of exposure. However, mutagenic effects (ENA) were observed in the exposure groups after 14 and 21 days. Results showed the potential genotoxic and mutagenic effects of maghemite NPs and GBH co-exposure to freshwater fish. The transformation and interaction of iron oxide nanoparticles with other pollutants, as herbicides, in the aquatic systems are critical factors in the environmental risk assessment of metal-based NPs.

Introduction

The iron oxide nanoparticles (IONPs) are particles with small diameters (1–100 nm), large surface area to volume ratio, magnetic properties, and biocompatibility (Hammad et al., 2017). Among the IONPs, the maghemite (γ-Fe2O3 NPs) has been used in biomedical applications from hyperthermia treatment to magnetic resonance imaging (Huber, 2005; Roca et al., 2009; Teja e Koh, 2009), as well as in wastewater treatment and nanoremediation due to its capacity to interact with organic and inorganic pollutants, changing its bioavailability and ecotoxicity (Adeleye et al., 2016; Alabi et al., 2019; Berg et al., 2010; Gupta et al., 2018; Mohmood et al., 2015; Srikanth et al., 2015, 2014; Xu et al., 2012; Yousef et al., 2019).

Despite its wide application, the citrate-functionalized γ-Fe2O3 NPs (3.97 ± 0.85 nm) at 0.3 mg L−1 caused inflammatory and immune cellular responses, mutagenic and genotoxic effects on erythrocytes of the guppy (Poecilia reticulata) during 21 days of exposure (Qualhato et al., 2018, 2017). Furthermore, the exposure of zebrafish (Danio rerio) embryos to uncoated γ-Fe2O3 NPs (20−30 nm; 1–100 μg L−1) for 120 h also increased mortality, hatching inhibition, decreased the heartbeat rate and growth, changed the motor capabilities, caused metabolism disruption and mitochondrial impairments (Huang et al., 2019), while the adult zebrafish exposed to meso-2,3-dimercaptosuccinic acid-functionalized γ-Fe2O3 NPs (5.7 nm; 4.7–74.4 mg L−1) for 96 h showed oxidative stress, DNA damage, lipid peroxidation, and changes in the gene expression patterns (Villacis et al., 2017). Moreover, Capoeta fusca exposed to Fe3O4 NPs (20−30 nm; 1–100 mg L−1) for 28 days showed gills and intestine histopathological alterations and a high Fe uptake by body tissues (Sayadi et al., 2020). Despite these ecotoxicological studies with IONPs alone, on aquatic environments, IONPs could interact with other pollutants, leading to potential changes in their bioavailability and ecotoxicity (Canesi et al., 2015; Naasz et al., 2018).

IONPs have been applied for remediation of chlorinated solvents (i.e., oxidation and adsorption of trichloroethylene present in water and groundwater) (Gui et al., 2012; Salih et al., 2012) and metal/metaloids (i.e. lead and arsenate in aqueous solution; Co, Ni, Cu, Zn, As, Ag, Cd, Hg, and Tl in river) (Cheng et al., 2012; Kilianová et al., 2013; Warner et al., 2012). On the other hand, the knowledge about its co-exposure and interactive effects with herbicides in aquatic ecosystems remains scarce. Among the pesticides, glyphosate is a post-emergent and non-selective herbicide of main concern and was detected in aquatic systems in several concentrations, reaching over 1 mg L−1 (Ronco et al., 2016; Ruiz-Toledo et al., 2014; Tzaskos et al., 2012). It is estimated that approximately 747 million kg of glyphosate-based herbicide (GBH) was applied in 2014 (Benbrook, 2016). After application, the GBH can reach the aquatic systems by runoff and leaching, inducing high ecotoxic impact in different taxonomic groups, such as bacteria, microalgae, protozoa (Tsui and Chu, 2003), crustaceans (de Melo et al., 2019; Tsui and Chu, 2003) and fish (Rocha et al., 2015; dos Santos et al., 2017).

The GBH induced oxidative stress, protein expression alterations, and histopathological changes on gills and liver of several fish species, as P. reticulata exposed to Roundup Transorb® at 1.82 mg L−1 of its active ingredient (N-(phosphonomethyl)-glycine) for 24 h (Rocha et al., 2015; dos Santos et al., 2017) and Prochilodus lineatus exposed to Roundup® at 7.5 and 10 mg L−1 for 6, 24 and 96 h (Langiano et al., 2008). The Roundup® at 10 mg L−1 promoted neurotoxicity (acetylcholinesterase inhibition) in P. lineatus juveniles after 96 h of exposure (Modesto and Martinez, 2010). The GBH also induced genotoxic (DNA damage) and mutagenic damages (nuclear alterations) on erythrocytes of Corydoras paleatus exposed to Roundup® at 6.67 μg L-1 for 3, 6 and 9 days (De Castilhos Ghisi and Cestari, 2013), as well as in the gills of P. reticulata after exposure to Roundup Transorb® at 1.4, 2.83, 4.24 and 5.65 μL L−1 for 24 h. In addition, Roundup® at 10 mg L-1 induced genotoxicity, mutagenicity and reduced cell viability in erythrocytes and gill cells of P. lineatus exposed for 6, 24 and 96 h. Furthermore, the exposure to glyphosate at 50, 300 and 1800 μg L-1 induced mortality and complete loss of sperm motility of Astyanax lacustris (Gonçalves et al., 2018), as well as the glyphosate at 15 and 150 μg L-1 induced behavioral changes on D. rerio larvae after 24 h of exposure (Faria et al., 2020). On the other hand, the potential genotoxic and mutagenic effects of the GBH after co-exposure with nanomaterials to fish species remain unknown.

The P. reticulata is recommended as a model organism by international organizations (APHA, 1989; OECD, 2019) and represent a suitable model to assess the toxic effects of nanomaterials and herbicides (De Souza Filho et al., 2013; Qualhato et al., 2017; dos Santos et al., 2017; Vajargah et al., 2019). In addition, the comet assay associated with micronucleus test (MN test) and other nuclear alterations has been indicated as a suitable analysis of nanogenotoxicity (Rocha et al., 2015; Qualhato et al., 2017).

Accordingly, the present study aimed to analyze the effects of co-exposure of maghemite NPs and GBH on erythrocytes of the guppy by genotoxic and mutagenic assays. Thus, the hypothesis that the co-exposure of maghemite NPs and GBH induces genotoxic and mutagenic effects in guppies was tested. Although Qualhato et al. (2017) demonstrated that the exposure to isolated IONPs can induce DNA damage and nuclear alterations on P. reticulata after long-term exposure (14 and 21 days), knowledge about the genotoxicity and mutagenicity of maghemite NPs co-exposure with other pollutants in aquatic organisms remains limited. Here, we analyzed the toxicological impact of environmental mixtures of maghemite NPs and GBH, the world's most widely used herbicide. In this sense, the present study provided experimental subsidies to understand the aneugenic and clastogenic effects in freshwater fish after long-term exposure to co-exposure of IONPs and herbicides.

Section snippets

Nanoparticles and herbicides

Citrate-functionalized maghemite NPs were produced by alkaline co-precipitation based on the method previously described by Ali et al. (2016) and Unal et al. (2010) with modifications by Qualhato et al. (2017). Afterward, these NPs were characterized by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Electrophoretic Light Scan (ELS), X-ray diffraction and Vis-near-IR spectroscopy (Qualhato et al., 2017). The maghemite stock suspension presents the total iron

Nanoparticles and herbicide

TEM analysis showed that the prepared citrate-functionalized maghemites were round and crystalline (Fig. 2A–B) with DTEM of 5.44 ± 1.99 nm (Fig. 2C). The ELS and DLS results demonstrated that these NPs have a negative surface charge (-19.5 ± 6.5 mV) and a Dh of 89.6 ± 46.45 nm in reconstituted water (Fig. 2D), confirming the aggregation of maghemite in the exposure medium. The presence of the GBH in the exposure medium changed the zeta potential and hydrodynamic diameters of NPs. The

Conclusion

The hypothesis that the co-exposure of IONPs and GBH induces genotoxic and mutagenic effects in guppies was confirmed. Overall results showed that the maghemite NPs (alone or associated with GBH and regardless the exposure time) induced DNA damage and different ENA in the guppy P. reticulata, confirming the genotoxic and mutagenic potential of this mixture to the aquatic organism. Results indicated that further studies are necessary for better understanding more questions about the use of

Conflict of Interest

The authors declare no conflict of interest.

CRediT authorship contribution statement

Nicholas Silvestre de Souza Trigueiro: Methodology, Data curation, Writing - review & editing. Bruno Bastos Gonçalves: Methodology, Formal analysis. Felipe Cirqueira Dias: Methodology, Formal analysis. Emília Celma de Oliveira Lima: Methodology, Writing - review & editing. Thiago Lopes Rocha: Conceptualization, Supervision, Data curation, Writing - review & editing. Simone Maria Teixeira Sabóia-Morais: Conceptualization, Supervision, Data curation, Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This study was funded by the Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG; edital nº 04/17 (Proj. 201710267001261) – Programa Pesquisa para o SUS: Gestão Compartilhada em Saúde ‒ FAPEG/SES-GO/CNPq/MS-DECIT/2017 – PPSUS/GO), by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors also acknowledge CRTI, LabMic-UFG and Central Analítica IQ-UFG for their collaboration in the

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