Phytotoxicity of multi-walled carbon nanotubes on red spinach (Amaranthus tricolor L) and the role of ascorbic acid as an antioxidant

Abstract

Carbon nanotubes (CNTs) are a novel nanomaterial with wide potential applications; however the adverse effects of CNTs following environmental exposure have recently received significant attention. Herein, we explore the systemic toxicity and potential influence of 0–1000 mg L⁻¹ the multi-walled CNTs on red spinach. The multi-walled CNTs exposed plants exhibited growth inhibition and cell death after 15 days of hydroponic culture. The multi-walled CNTs had adverse effects on root and leaf morphology, as observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Raman spectroscopy detected the multi-walled CNTs in leaves. Biomarkers of nanoparticle toxicity, reactive oxygen species (ROS), and cell damage in the red spinach were greatly increased 15 days post-exposure to the multi-walled CNTs. These effects were reversed when the multi-walled CNTs were supplemented with ascorbic acid (AsA), suggesting a role of ROS in the multl-walled CNT-induced toxicity and that the primary mechanism of the multi-walled CNTs’ toxicity is oxidative stress.

Introduction

Carbon nanotubes (CNTs) are increasingly used as key-materials to meet numbers of nano-technological demands [1], [2]. CNTs can be divided into multi-walled CNTs and single-walled CNTs based on the rolled layer of graphene. The multi-walled CNTs consist of multiple rolled layers of graphene while the single-walled CNTs having a single rolled sheet of graphite. The multi-walled CNTs have been produced worldwide (Belgium, China, Japan, Germany, etc.,) in industrial quantities and numbers of products containing the multi-walled CNTs have already appeared in the markets. Concerning the possible human exposure, it is important to consider the uptake of the multi-walled CNTs in media and organisms that are routinely consumed by humans (e.g., plant, vegetables, and fish). While the tremendous positive impacts of nanotechnology are widely publicized, studies of potential threats or risks to human health and the environment are just beginning to emerge [3]. After penetrating the body through the food web, CNTs may be harmful to humans [4]. The unique nanometer-scale structure of CNTs is based on a graphene cylinder that is typically a few nanometers in diameter and can range in length from a few micrometers to millimeters [5]. Ingestion of the CNTs can result in adverse biological effects [6]. Parallel to CNTs, the so-called zero-dimensional nano-particles, such as carbon black, silica, titanium oxide, alumina, iron oxide, and zirconium oxide had been also investigated [7], [8], [9]; they were found to be toxic to plants but with relatively lower toxicities [10], [11], [12], [13].

Plants and plant cells showed high tendencies to accumulate CNTs [14], [15], making plants as an important link in the pathway by which CNTs enter the food chain and biological cycles [16]. Torney and co-wokers [17] demonstrated that CNTs can assist the delivery of biological molecules into plant cells. Abundant studies [18], [19], [20], [21] have demonstrated that CNTs can cause pronounced toxicity to plants. Tan et al. [22] and Canas et al. [23] described seed germination and growth inhibition induced by CNTs in selected plants and plant cells. The higher sensitivity to CNTs may be a universal biological phenomenon, as it has also been observed in human [24], animal [25], and bacteria [26] systems. The threshold at which symptoms of toxicity become established differs widely among plant species, and the impact of nano-particles on different plant species can vary greatly, depending on the plant growth stage, the method and the duration of exposure, as well as on the nano-particle size, concentration, chemical composition, surface structure, solubility, shape, and aggregation [27]. The varying experimental conditions used in different studies make it difficult to rigidly classify plants into tolerance groups. Some broad generalizations are possible, but there is a vital need to examine the possible toxicity of CNTs to diverse crop species.

Reactive oxygen species (ROS) generation and oxidative stress have been suggested as primary mechanisms by which CNTs alter plant cell growth [20], [28]. ROS generation and oxidative stress can lead to cell membrane, mitochondria, and DNA damage [29], which could ultimately impact the whole organism in terms of development, reproduction, and viability. Response to oxidative stress involves the induction of antioxidant molecules and detoxification enzymes [30].

We conducted a preliminary screening to determine the multi-walled CNTs’ phytotoxicity toward seven plant species (chili, cucumber, lady's finger, lettuce, red spinach, rice, and soybean) [31]. The vegetative plant red spinach was selected for further study, since its roots and leaves both displayed toxic symptoms and its small seed size results in a relatively large surface area to volume ratio, which is conductive to higher sensitivity to toxicants [32]. The present study investigated the adverse impact of the multi-walled CNTs on red spinach and discovered these effects to be mediated by oxidative stress, underscoring the importance of nanomaterial presentation to the phytotoxic response. We also showed, for the first time in an in vivo study, the alleviation of the multi-walled CNTs toxicity by treatment with the antioxidant ascorbic acid (AsA).