The effects of photon-upconversion nanoparticles on the growth of radish and duckweed: Bioaccumulation, imaging, and spectroscopic studies

Highlights

• UCNPs/SiO₂-COOH are stable, showing no tendencies to aggregate/agglomerate.

• UCNPs/SiO₂-COOH can become adsorbed onto plants (R. sativus roots/L. minor fronds).

• UCNPs/SiO₂-COOH can translocate from roots to leaves/fronds (R. sativus/L. minor).

• UCNPs/SiO₂-COOH show significant toxicity to plants at 100 μg/mL and 1000 μg/mL.

• LIBS can be used as a fast tool to acquire Y, Yb, and Er distribution maps in plants.

Abstract

In this study, radish (Raphanus sativus L.) and common duckweed (Lemna minor L.) were treated with an aqueous dispersion of carboxylated silica-coated photon-upconversion nanoparticles containing rare-earth elements (Y, Yb, and Er). The total concentration of rare earths and their bioaccumulation factors were determined in root, hypocotyl, and leaves of R. sativus after 72 h, and in L. minor fronds after 168 h. In R. sativus, translocation factors were determined as the ratio of rare earths concentration in hypocotyl versus root and in leaves versus hypocotyl. The lengths of the root and hypocotyl in R. sativus, as well as the frond area in L. minor, were monitored as toxicity endpoints. To distinguish rare earth bioaccumulation patterns, two-dimensional maps of elemental distribution in the whole R. sativus plant and L. minor fronds were obtained by laser-induced breakdown spectroscopy with a lateral resolution of 100 μm. Moreover, the bioaccumulation was inspected using a photon-upconversion laser microscanner. The results revealed that the tested nanoparticles became adsorbed onto L. minor fronds and R. sativus roots, as well as transferred from roots through the hypocotyl and into leaves of R. sativus. The bioaccumulation patterns and spatial distribution of rare earths in nanoparticle-treated plants therefore differed from those of the positive control. Overall, carboxylated silica-coated photon-upconversion nanoparticles are stable, can easily translocate from roots to leaves, and are expected to become adsorbed onto the plant surface. They are also significantly toxic to the tested plants at nominal concentrations of 100 and 1000 μg/mL.

Introduction

Lanthanide-doped photon-upconversion nanoparticles (UCNPs), a new type of luminescent nanomaterials, display several notable features such as large anti-Stokes shifts, sharp emission bands, long-lived luminescence, low background interference, and high resistance to photobleaching (Chatterjee et al., 2008; Haase and Schäfer, 2011; Li et al., 2015; Liu et al., 2015). Photon-upconversion nanoparticles are most commonly composed of NaYF₄ nanocrystals and doped with Er³⁺ and Yb³⁺ in different ratios; such NPs were also used in the present study. Photon-upconversion nanoparticles have become widely used as luminescent labels because of their unique optical properties, which could lead to the replacement of traditional organic dyes and quantum dots (Liu et al., 2015). In comparison to the latter, UCNPs offer better detection sensitivity, reduced background autofluorescence of biological tissues, and deeper tissue penetration (Chatterjee et al., 2008; Wang and Liu, 2009). The UCNPs can, nevertheless, be released into the environment with the disposal of the products containing these nanoparticles or during their use as luminescent labels. Consequently, they can be hazardous for living organisms, and it is important to evaluate their possible environmental risks. However, the use of UCNPs is still in its infancy, and the information about their pathways to the environment, expected environmental concentrations, and toxic effects is still unknown.

At present, only a limited number of phytotoxicity studies have researched the toxic effects of UCNPs and the possibility of their translocation or biotransformation in plants, namely in mung bean (Vigna radiata; Peng et al., 2012), soybean (Glycine max; Yin et al., 2015), pumpkin (Cucurbita maxima; Nordmann et al., 2015), moth orchids (Phalaenopsis sp.), and thale cress (Arabidopsis thaliana; Hischemöller et al., 2009). These preliminary studies have shown that low concentrations of UCNPs (≤50 μg UCNPs/mL) promoted plant growth without any toxic effects, whereas germination rate and plant growth decreased at higher concentrations (≥50 μg UCNPs/mL; Peng et al., 2012; Yin et al., 2015). However, the possibility that the observed negative effects of UCPNs were caused by free rare-earth ions (Y³⁺) cannot be excluded, even though the release of Y³⁺ from polyethyleneimine-covered NaYF₄:Yb,Er UCNPs was found to be less than 0.05% at a relatively high concentration (1000 μg UCNPs/mL) after a prolonged period of 12 days (Yin et al., 2015).

In our study, the possible toxic effects of released free ions were limited by coating UCNPs with carboxylated silica shell (UCNPs/SiO₂-COOH). This shell can be easily silanized with various functional groups (e.g., −NH₂, −COOH, −SH), which make UCNPs/SiO₂ excellent platforms for theranostics and molecular labeling. The functionalized silica shell enables UCNPs to be used in bioconjugation processes, improves their chemical stability, and decreases their cytotoxicity by limiting the release of rare-earth ions (Liu et al., 2015; Sedlmeier et al., 2016; Hlaváček et al., 2016, 2017; Farka et al., 2017). To the best of our knowledge, no phytotoxicity studies with UCNPs/SiO₂ have been conducted in any aquatic or terrestrial plant, and only a very limited number of UCNPs studies have been carried out with terrestrial plants in the hydroponic environment. Furthermore, the toxicity of Y, Yb, and Er ions has also not been tested in duckweed; so far, there is only one study reporting on the Y phytotoxicity for fringed water lily (Nymphoides peltata) as a representative of higher aquatic plants (Fu et al., 2014). Considering the possible negative effects of UCNPs, it is very important to address this information shortage in plant ecotoxicology.

In the present study, various short-term toxicity assessments were conducted in two plant species. The first one was common duckweed (Lemna minor L., Lemnoideae, Araceae), which is a well-known metal bioaccumulator and a bioindicator for the detection and monitoring of metal pollution (Garnczarska and Ratajczak, 2000). The utilization of L. minor as a model aquatic organism in similar studies with different types of NPs, for example, ZnO NPs, CuO NPs or Ag NPs, was summarized by Modlitbová et al. (2018). For toxicity tests with L. minor, our laboratories have successfully implemented a novel microbioassay (Kalčíková et al., 2018) based on the standard OECD (Organization for Economic Co-operation and Development) L. minor toxicity test (Test No. 221: Lemna sp. Growth Inhibition Test using Steinberg medium). This microbioassay further improves the usefulness, sensitivity, low cost, and simplicity of the OECD test by implementing multiwell culture plates (Kalčíková et al., 2018). Its main advantages are reduced solution volumes, less consequent waste, and a decreased number of test organisms.

As the second test species, we selected the common radish (Raphanus sativus L.), which is a widespread crop plant commonly used in toxicity testing of heavy metals, such as cadmium (Vitoria et al., 2003) and lead (Lane and Martin, 1977), and organic compounds, such as the antibiotic enrofloxacin (Migliore et al., 2003). Recently, R. sativus has also been used in toxicity, uptake, and distribution testing of NPs, such as CeO₂ NPs (Trujillo-Reyes et al., 2013), CuO NPs (Atha et al., 2012), Ag NPs (Zuverza-Mena et al., 2016), Au NPs (Zhu et al., 2012), and a mixture of TiO₂ NPs and Cd salts (Manesh et al., 2018). R. sativus has already been used in phytotoxicity testing of rare-earth elements (YCl₃) in artificial soil (Thomas et al., 2014). However, the results were too variable to allow any firm conclusion, which highlights the need for future studies.

Laser-induced breakdown spectroscopy (LIBS) has been shown as a suitable method for the determination of elemental distribution in plants at a lateral resolution comparable to the one used in this study (Kaiser et al., 2012; Santos et al., 2012). However, LIBS has only rarely been used for the detection of rare-earth elements. Abedin et al. (2011) simultaneously analyzed the concentration of eight elements in monazite sands, including Y, Yb, and Er. Ytterbium in coal ash was also detected by LIBS (Phuoc et al., 2016). In both articles, the analysis was performed directly on pellets. To the best of our knowledge, no study has attempted to detect Y, Yb, and Er in biological matrices. However, LIBS has recently made progress at the detection of various types of NPs in different matrices, for example, quantum dots applied onto filter paper (Škarková et al., 2017) or Gd NPs in mammal organs (Sancey et al., 2014). Nevertheless, LIBS has only rarely been used for the detection of NPs in plant tissues (Krajcarová et al., 2017).

The first goal of this work was to assess the toxicity of NaYF₄:Yb³⁺,Er³⁺-SiO₂-COOH NPs (UCNPs/SiO₂-COOH) for L. minor and R. sativus in short-term toxicity tests (168 h and 72 h, respectively) by monitoring several macroscopic endpoints (frond area, root length, hypocotyl length). A mixture of Y, Yb, and Er chlorides served as a positive control. The following task was to determine the concentration of Y, Yb, and Er by inductively-coupled plasma optical emission spectroscopy (ICP-OES) in various parts of both plants to distinguish how rare-earth elements from different sources accumulate in L. minor and R. sativus organs. In this part, a mixture of Y, Yb, and Er chlorides served as a reference compound to test the hypothesis that plants can bioaccumulate a larger amount of rare-earth elements in ionic form than in the form of UCNPs. The third objective was to inspect plants by photon-upconversion laser microscanner (Sedlmeier et al., 2016) to detect UCNPs adsorbed on the plant surface and UCNPs transferred from roots through hypocotyl into leaves (R. sativus). Finally, we investigated dried and epoxide-fixed plant tissues by LIBS to distinguish the bioaccumulation patterns of NPs from those of salts. In this study, LIBS was shown to be beneficial for the mapping of rare-earth elements distributed in large-scale samples (analytical spot size compared to the sample size) in a short time with sufficient resolution. We conclude with a consideration of possible hazardous effects of UCNPs/SiO₂-COOH on R. sativus and L. minor as a crop plant and bioindicator species, respectively.