Phytotoxicity of ionic, micro- and nano-sized iron in three plant species

https://doi.org/10.1016/j.ecoenv.2015.07.024Get rights and content

Highlights

  • Nanoremediation activities use nano-zerovalent iron (nZVI).

  • Knowledge of nZVI effects on macrophytes are still scarce.

  • Standard phytotoxicity tests (72 h exposure) showed no adverse effects of nZVI.

  • Micro-sized iron showed no significantly different effects from nZVI.

  • Apparent uptake of ZVI detected by microscopy.

Abstract

Potential environmental impacts of engineered nanoparticles (ENPs) can be understood taking into consideration phytotoxicity. We reported on the effects of ionic (FeCl3), micro- and nano-sized zerovalent iron (nZVI) about the development of three macrophytes: Lepidium sativum, Sinapis alba and Sorghum saccharatum. Four toxicity indicators (seed germination, seedling elongation, germination index and biomass) were assessed following exposure to each iron concentration interval: 1.29–1570 mg/L (FeCl3), 1.71–10.78 mg/L (micro-sized iron) and 4.81–33,560 mg/L (nano-iron). Exposure effects were also observed by optical and transmission electron microscopy. Results showed that no significant phytotoxicity effects could be detected for both micro- and nano-sized zerovalent irons, including field nanoremediation concentrations. Biostimulation effects such as an increased seedling length and biomass production were detected at the highest exposure concentrations. Ionic iron showed slight toxicity effects only at 1570 mg/L and, therefore, no median effect concentrations were determined. By microscopy, ENPs were not found in palisade cells or xylem. Apparently, aggregates of nZVI were found inside S. alba and S. saccharatum, although false positives during sample preparation cannot be excluded. Macroscopically, black spots and coatings were detected on roots of all species especially at the most concentrated treatments.

Introduction

Engineered nanomaterials (ENMs) are highly preferred for a broad spectrum of applications due to their unique properties. Engineered nanoparticles (ENPs) have a promising use in many areas including catalysis, optics, biology, agriculture, and microelectronics (Wu et al., 2012, Libralato et al., 2013, Corsi et al., 2014; Libralato, 2014; Minetto et al., 2014). Further applications are currently focused on environmental remediation due to their likely performance in contamination removal and toxicity mitigation (Gavaskar et al., 2005, Tratnyek and Johnson, 2006). Iron-based ENPs stimulated research for engineering applications especially for treating polluted water and groundwater (Tang and Lo, 2013) including both inorganics and organics (Crane and Scott, 2014, Mar Gil-Díaz et al., 2014; Zeino et al., 2014). Their impact is still highly empirical and limited to nanoparticles' elemental composition, size and stability showing both positive and negative effects. The high reactivity of iron-based ENPs, and in particular of nano-zerovalent iron (nZVI), in association with their high specific surface area made them suitable to immobilise and degrade contaminants in soils (Chang et al., 2007, Machado et al., 2013). Thus, the use of nZVI for soil clean-up purposes could pose potential hazards for macrophytes and soil organisms (Ma et al., 2010a, Ma et al., 2010b, Ma et al., 2010a, Ma et al., 2010b). In all ecosystems, plants are the basic component playing a crucial role in the fate and transport of ENPs in the environment through plant uptake and bioaccumulation (Monica and Cremonini, 2009).

Although in remediation activities, terrestrial macrophytes could be directly exposed and potentially affected by nZVI, effect data are still scarce despite the current use of this technique (Li et al., 2015). Zhu et al. (2008) found that Cucurbita maxima grown in an aqueous medium absorbed, translocated and accumulated Fe3O4 ENPs, but this event did not occur with Phaseolus limensis under the same testing conditions. This suggested that the biological effect could be species-dependent. Lee et al. (2010) showed that Fe3O4 ENPs in Arabidopsis thaliana did not significantly affect seed germination and the number of produced leaves, while the root elongation was negatively influenced at all exposure concentrations (400, 2000, and 4000 mg Fe3O4/L). Kim et al., 2014, Kim et al., 2014 investigated the effect of nZVI on A. thaliana root elongation showing an enhanced growth by 150–200% at 0.5 g/L compared to the blank. Further studies on A. thaliana evidenced that nZVI triggered high plasma membrane H+-ATPase activity resulting in a 5-fold higher stomatal opening than in unexposed plants (Kim et al., 2015). Mushtaq (2011) observed that concentrations of Fe3O4 ENPs within 100–5000 mg/L were able to significantly reduce Cucumis sativus root development compared to controls suggesting the presence of stressing conditions. Phytotoxic effects of Fe3O4 ENPs were assessed in lettuce (Lactuca sativa), radish (Raphanus sativus) and cucumber (C. sativus) (Wu et al., 2012) evidencing median effective concentrations (EC50) of more than 5000 mg/L for lettuce and radish, and of 1682 mg/L for cucumber, respectively. For all species, the germination index was significantly different from standard conditions showing seedling inhibition effects. Seeds of Linum usitatissimum, Lolium perenne and Hordeum vulgare were used to investigate the potential inhibition effects of nZVI (El-Temsah and Joner, 2012). Concentration of 2 and 5 g/L of nZVI completely inhibited seed germination, while no detrimental effects on plants were observed at concentrations <250 mg/L. Pereira et al. (2013) observed changes in root and shoot lengths, number of lateral roots, photosynthetic pigments, and internal CO2 concentration in four rice cultivars when nano-iron exposure in the growth medium increased from 4 to 9 mM. Similarly, other authors found differences in plant growth, nutrient uptake, and lateral roots morphology in Ipomoea pescaprae and Canavalia rosea when exposed to bulk FeSO4 (Siqueira-Silva et al., 2012). Ma et al. (2013) showed that concentrations higher than 200 mg/L of nZVI reduced plant growth and biomass in Typha latifolia and hybrid poplar (Populous deltoids×Populous nigra). Trujillo-Reyes et al. (2014) investigated the effects of Fe3O4 ENPs in L. sativa. No physiological change was detected compared to negative controls. Iron ions or ENPs (10 and 20 mg/L) had low or no negative effect on cell membrane integrity and chlorophyll content. Mukherjee et al. (2014) studied ZnO iron doped (Fe@ZnO) ENPs toxicity in Pisum sativum (L.) analysing seed germination, uptake, chlorophyll and H2O2 content and enzymatic activity. No signs of necrosis, stunting, chlorosis or wilting were found, while variations were observed concerning physiological and biochemical responses in terms of plant growth, chlorophyll content and induction of reactive oxygen species (ROS). Li et al. (2015) observed that Arachis hypogaea seeds exposed to nZVI (0.0024 and 0.0048 mg/L) produced significantly longer seedling compared to negative controls suggesting that nanoparticles may have penetrated the peanut seed coat increasing the water uptake and thus stimulating germination.

This short overview indicated that phytotoxicity data about nZVI are still scarce on macrophytes that are key direct biological targets in case of nanoremediation activities, thus, not sufficient for a sound environmental hazard assessment and most data are based just on nominal concentrations. The aim of this research was to understand the potential effect of nZVI compared to its ionic and micro-sized form considering three well-known testing species (Lepidium sativum, Sinapis alba and Sorghum saccharatum) (Baudo, 2012) and four endpoints (germination, seedling elongation, germination index and biomass).

Section snippets

Materials and reagents

Commercially available materials were purchased for the experiments: FeCl3·6H2O (iFe) (Sigma-Aldrich, USA), micro-sized iron (mFe) (Aldrich Chemistry, Germany) and nano-sized zerovalent iron (nFe) (American Elements, USA). Boric acid (Sigma-Aldrich, USA) was used as reference toxicant. Concentrated HCl (34–37%, SpA) and HNO3 (67–69%, SpA) were purchased from Romil. All reagents used during the experiment were of analytical grade.

Stock solutions and suspensions of 10 g/L and all treatments

Physico-chemical characterisation of powders, solutions and suspensions

Microscopy analyses of mFe and nFe (Appendix A Supplementary material, Appendix A Supplementary material) revealed that primary particle sizes were 59±48 μm and 25±10 nm, respectively. Average hydrodynamic diameter of nFe obtained from DLS in ultrapure water after 10 min of contact time was 289±47 nm between 4.81 and 992 mg/L of nFe. After 24 h of contact time and resuspension by handshaking for 1 min, it was 208±43 nm (n=7). Zeta-potential values were all >30 mV for suspensions presenting a

Generalised discussion

Results from all endpoints evidenced that nZVI at concentrations used for field activities (2340 and 33,560 mg/L) was not phytotoxic. Germination, SEI, GI and biomass did not show significant toxic effects compared to negative controls with no apparent difference in the sensitivities between dicotyledons (L. sativum and S. alba) and monocotyledon (S. saccharatum), nevertheless the high variability of SEI data. Conversely, biostimulation phenomena frequently occurred at higher mFe and nFe levels

Conclusions

According to the considered standard endpoints (inhibition of germination, seedling elongation, and biomass production and germination index) and experimental scenarios, ionic iron, and micro- and nano-sized iron particles showed no significant adverse effects to L. sativum, S. alba and S. saccharatum. Neither nZVI field exposure concentrations (2340±27 mg/L and 33,560±153 mg/L) evidenced adverse effects significantly different from negative controls. Moderate biostimulation was observed at the

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