Differential alteration in reproductive toxicity of medaka fish on exposure to nanoscale zerovalent iron and its oxidation products

Highlights

• nZVI is oxidized to nano iron oxide and iron ion in oxygenic water.

• Exposure of nano iron oxide (nFe₃O₄) inhibited medaka egg production.

• nFe₃O₄–induced reproductive toxicity is associated with oxidative stress in ovary of medaka fish.

Abstract

Nanoscale zerovalent iron (nZVI) is a redox-active nanomaterial commonly used in remediation of soil and groundwater pollution and wastewater treatment processes. A large quantity of nZVI (e.g., >100 mg/L) accidentally released from in situ sites to nearby oxygenized aquifers could be rapidly oxidized to iron oxides (e.g., Fe₃O₄ or Fe₂O₃) and ions (e.g., Fe²⁺), for acute hypoxia effects to aquatic life. However, we do not know the ecotoxicological fate of nZVI and its oxidation products at lower, environmentally concentrations in surface water receiving waterborne transportation or effluent discharge in terms of exposure to aquatic vertebrate species. This study assessed the causal effect on reproductive toxicity in medaka adults (Oryzias latipes) of carboxymethyl cellulose-stabilized nZVI (CMC-nZVI), Fe²⁺ and iron oxide nanoparticles (nFe₃O₄) with 21-day aqueous exposure at 5 and 20 mg/L (Fe-equivalent). Such concentrations did not significantly change the dissolved oxygen, oxidation-reduction potential or pH values in the 3 iron solutions during the fish exposure period. Neither CMC-nZVI nor Fe²⁺ treated adults showed altered daily egg production (fecundity) and oxidative stress responses in observed tissues, as compared to controls. However, the fecundity in nFe₃O₄ (20 mg/L)-treated pairs was significantly decreased, with increased incidence of abnormal immature oocytes in the ovary. As well, nFe₃O₄ treatment suppressed activities of the antioxidants superoxide dismutase and expression of glutathione peroxidase (gpx) in the brain and ovary. Although nFe₃O₄ or Fe²⁺ treatments inhibited mRNA expression of hepatic estrogen receptor (er-α) in females, plasma levels of sex hormones and (Na, K)-ATPase activity in gills of treated fish did not differ from controls for both sexes. Hence, oxidation products (e.g., nFe₃O₄) from nZVI at lower milligram-per-liter levels may be potent in inducing nanoparticle-specific reproductive toxicity in medaka fish by inducing oxidative stress in female gonads.

Main finding

nZVI oxidation product nFe₃O₄ at lower mg/L induces nanoparticle-specific reproductive toxicity in medaka fish.

Introduction

As the industry worldwide invests vast amounts of money in nanotechnology, the number of engineered nanomaterials and their applications has been growing exponentially. Metallic nanoparticles (NPs) such as silver, titanium dioxide or iron oxides have been widely used for biomedical purposes, commercial products and industrial applications (Chae et al., 2009; Costa and Fadeel, 2015; Liu et al., 2013). Iron-based NPs have received much attention in biomedical or industrial fields and also for pollution remediation because of their special physicochemical properties of large surface area, high activity and homogenous composition and structures as well as relatively less toxicity as compared with other metallic NPs (Cundy et al., 2008; Grieger et al., 2010; Liu et al., 2013).

Nanoscale zero-valent iron (nZVI; Fe⁰_(s)) is a redox-active nanomaterial widely used for soil and groundwater remediation and wastewater treatment processes in both in or ex situ applications (Cundy et al., 2008). To prevent particle agglomeration, they are often surface-modified with stabilizers such as tetrapolyphosphate (Yoon et al., 2018) or carboxymethyl cellulose (He and Zhao, 2007). nZVI with surface modification is more dispersed in solution and has higher mobility for practical use in the real situation than without modification.

Large quantities of nZVI accidentally released from in situ sites to nearby oxygenized aquifers may result in acute hypoxia effects and mass mortality in aquatic life (Chen et al., 2011; Chen et al., 2012). In field-scale studies, nZVI in soil migrates only a few meters away from infection sites (Elliott and Zhang, 2001; Krol et al., 2013). The release of nZVI from soil or groundwater remediation seems low in most cases (Lefevre et al., 2016). However, recent pilot studies demonstrated the high efficiency of nZVI for removing As and Cu in wastewater (Li et al., 2014a; Li et al., 2014b). nZVI has smaller aggregation and magnetite force in high oxidative water, so it has higher mobility than in anaerobic water (Jiang et al., 2015). Therefore, the potential risk of increased nZVI exposure from effluent of nZVI-related wastewater treatment plants needs to be further evaluated before it is widely applied in water or wastewater treatment.

nZVI has a core-shell structure with an Fe⁰ core surrounded by iron oxides (e.g., Fe₃O₄ or Fe₂O₃) under aerobic conditions (Liu et al., 2017). Once nZVI enters the oxygenic surface water via water transportation within aquifers or effluent discharge from wastewater treatment plants, it can rapidly oxidize to iron oxides (e.g., Fe₃O₄ or Fe₂O₃) or ions (e.g., Fe²⁺) (Liu et al., 2017). Several studies indicated that iron oxides including nFe₃O₄ derived from nZVI could be stably present in the aqueous media (Chen et al., 2012; Liu et al., 2017), thus increasing exposure risk to aquatic organisms.

Another possible source of iron oxide nanoparticles includes magnetic iron oxide NPs (e.g., nFe₃O₄), which are used as sorbets, co-precipitants or contaminant immobilizing agents in soil and surface water pollution sites (Lakshmanan et al., 2013; Mohan and Pittman, 2007). Also, magnetic iron oxide (e.g., Fe₃O₄/γ-Fe₂O₃) nanocrystals that possess super-paramagnetic properties are widely used as catalysis agents, sensors and in medical diagnosis and therapy (Liu et al., 2013). Current studies mostly focused on ecotoxicological fate of nZVI; however, iron oxide related NPs (e.g., nFe₃O₄) are less studied. Although evidence of the occurrence of iron NPs in the aquatic environment is still uncertain, these iron oxide NPs with relatively stable properties are likely retained in surface aquifers for some time (Chen et al., 2012; Liu et al., 2017); hence, in terms of risk of exposure and toxicity, the aqueous fate and ecotoxicity of iron oxide NPs (e.g., nFe₃O₄) either from manufacturing wastewater discharge or oxidation products from nZVI-related pollution are important issues in aquatic ecosystems.

nZVI and related iron species can induce oxidative stress and acute mortality among prokaryotes and eukaryotes (Liu et al., 2017; Yoon et al., 2018). Cellular reactive oxygen species (ROS) were induced in E. coli exposed to different surfaces modified by nZVI (100 mg/L) for 24 h, but the ROS level did not change in B. subtilis with the same exposure, so ROS-induced oxidative stress is highly related to nZVI toxicity in E. coli (Yoon et al., 2018). ROS-induced differential oxidative stress responses with different nZVI species were also observed in water flea (Daphnia magna) (Yoon et al., 2018). Our previous studies showed greater mortality and oxidative response with stabilized than uncoated nZVI (100 mg/L) in medaka larvae (Oryzias latipes), although the bioconcentration and oxidative stress were higher with uncoated nZVI and iron oxide nFe₃O₄, which were easily aggregated and then settled down in the bottom of dosing tanks, for increased exposure to embryos and larvae of medaka (Chen et al., 2011; Chen et al., 2012; Chen et al., 2013).

For reproductive toxicity, recent studies showed that a 30-day exposure to nZVI (100 mg/kg soil) significantly inhibited reproduction in 2 earthworm species (Eisenia fetida and Lumbricus rubellus) (El-Temsah and Joner, 2012; Liang et al., 2018). Caenorhabditis elegans with 48 h exposure of CMC-nZVI, nFe₃O₄ and Fe²⁺ showed inherited reproductive toxicity from the parent to 2 generations (Yang et al., 2016). Besides soil-dwelling organisms, mediterranean mussel (Mytilus galloprovincialis) showed enhanced sperm toxicity with both uncoated and modified nZVI exposure (Kadar et al., 2013). Thus, nZVI-related iron NPs can induce reproductive toxicity in invertebrates, but none of these studies assessed the reproductive effects on aquatic vertebrates, such as fish.

Medaka is one of most popular laboratory fish models used around the world. Unlike zebrafish, medaka has a defined XY system of sex chromosomes, similar to humans, so it is a useful model for studying sex determination and reproductive regulation (Matsuda, 2003). Also, medaka is recommended as a suitable fish model for reproductive toxicity tests by Organisation for Economic Co-operation and Development guidelines (OECD, 2012). Medaka reaches sexual maturation at about 2–4 months post-hatching. A pair of medaka adults can continuously reproduce 20-40 eggs per day, which is convenient to obtain experimental materials during experimental periods (Kinoshita, 2009). In this study, we treated sexually mature adults of medaka pairs with fully characterized solutions of stabilized CMC-nZVI, uncoated nFe₃O₄ and Fe²⁺ for 21 days at sub-lethal and low mg/L levels (5 and 20 mg/L). The tested concentration are based on the total iron concentration measured in the monitoring wells after in situ injection of nZVI ranging from 40 to 370 mg/L (Wei et al., 2010) and our preliminary acute toxicity tests. We evaluated the causal effect on reproduction, including egg number, fertility, hatchability and possible mechanisms in terms of oxidative stress responses and sex hormone-related endpoints. These results were further compared with our previous studies with medaka larvae exposed to same iron species at higher concentrations (e.g., 100 mg/L) (Chen et al., 2011; Chen et al., 2012; Chen et al., 2013).