Induced toxicity in early-life stage zebrafish (Danio rerio) and its behavioral analysis after exposure to non-doped, nitrogen-doped and nitrogen, sulfur-co doped carbon quantum dots

Highlights

• Carbon quantum dots (CQDs) with different dopants are evaluated through the embryonic development of zebrafish.

• The toxic effects on zebrafish vary according to dopant type, concentration and time of exposure.

• CQDs provoke morphological, behavioral and growth rate alterations depending on the dopant type and concentration.

• Behavioral analysis shows that locomotor activity increases as the toxicity of the nanomaterial elevates.

Abstract

In this study, the effects of doping of CQDs with alternative functional groups (dopants) were evaluated through embryonic development of zebrafish (Danio rerio). The CQDs were synthesized using simple and low-cost sources: Non-doped (citric acid was used as the carbon source), nitrogen-doped (N-doped) and nitrogen, sulfur-co-doped (N,S-doped). The CQDs induced significant toxicity to zebrafish (>150 μg/mL) and the toxic effects were dose-dependent. The N,S-doped CQDs were the most toxic (LD50 = 149.92 μg/mL), followed by the N-doped CQDs (LD50 = 399.95 μg/mL) while the non-doped CQDs were the least toxic (LD50 = 548.48 μg/mL) of the three. The growth rate (GR) was affected following the toxicity pattern (GR_NS-doped<GR_N-doped<GR_non-doped <GR_blanc), which, in turn, greatly depends on the type of dopant. Morphological malformations, such as pericardial edema, yolk sac edema, tail and spinal curvature were observed to zebrafish embryos as the toxicity, concentration and exposure time to the nanomaterial increased. Behavioral analysis showed that locomotor activity increases as the toxicity of the nanomaterial rises. The differences in toxicity, growth rate and malfunctions of CQDs were attributed to their doping with different heteroatoms. The N,S-doped CQDs, unequivocally, exhibited the most pronounced effects.

Introduction

Nanomaterials are a class of materials with unique properties and promising characteristics (Zhang et al., 2016), offering several applications in the fields of medicine (bioimaging, drug delivery), technology (biosensing) and environmental sciences (energy conversion and energy storage) (Lim et al., 2014; Yang et al., 2009). Their nanoscopic scale (less than 100 nm in size) (Wang and Hu, 2014) and the important optical, electrical and conductive properties (sensing, photocatalysis, electrocatalysis, etc.) (Zheng et al., 2015) are some of their main characteristics.

In the most recent years, a specific class of nanomaterials, the so-called carbon quantum dots (CQDs), have drawn intensive attention and triggered substantial investigation. They consist of carbon, hydrogen, oxygen, nitrogen, sulfur and other common elements (Wang and Hu, 2014) and manifest superior merits including excellent biocompatibility both in vitro and in vivo, resistance to photobleaching, easy surface functionalization and bio-conjugation, outstanding colloidal stability, eco-friendly synthesis and low cost. All these properties endow them with the high potential of replacing the conventional fluorescent heavy metal-containing semiconductor quantum dots or organic dyes (Osborne, 2013). The wide range of applications and the benefits offered by certain classes of nanomaterials have raised concerns about their potential toxic effects on organisms. For instance, metal-based quantum dots (Ag, Cd, Ti quantum dots) due to their minuscule size and low stability tend to interact with other substances, thereby causing toxic effects (Osborne, 2013; Chatzimitakos et al., 2016). Studies have shown that heavy metal-based quantum dots cause oxidative stress to fish brain cells (Oberdörster, 2004), DNA damage to mice exposed to nanoparticles (Trouiller et al., 2009) and high lethality rate in invertebrates, such as Daphnia magna, at concentrations in the range of 5.5 mg/L to 143 mg/L (Lovern et al., 2007; Zhu et al., 2009). For these reasons, researchers turned their interest to alternatives, which are more eco-friendly and less toxic. More importantly, compared with traditional metallic quantum dots, CQDs are thought to be environmentally benign and safer for biological uses as most of them exhibit quite lower toxicity in cell lines and mice (Lim et al., 2014; Yang et al., 2009; Kang et al., 2015). An important issue when trying to scientifically approach CQDs is that due to their minuscule size they tend to interact with other chemical/biological factors and their mechanism of action remains completely unknown or obscure (Lim et al., 2014; Yang et al., 2009). Specifically, CQDs may potentially cause toxic responses to aquatic ecosystems, which act as a sink for most contaminants (Osborne, 2013) and aquatic organisms and are considered to be the final recipients (Daughton, 2004; Valavanidis and Vlachogianni, 2010). Most of the studies confirmed that no noticeable signs of toxicity of CQDs have been observed in in vitro cells and in vivo animal models (Yang et al., 2009; Kang et al., 2015).

Zebrafish is a vertebrate animal model suitable for ecotoxicological, drug and chemical toxicity studies because of its high growth rate, especially at early life stages, of the small size of its larvae (about 4 mm), easy and low-cost maintenance, short generation time (Lin et al., 2016) and of the ability to lay a large number of transparent eggs (approximately 300 eggs) every 2–3 days (Wixon, 2000). Zebrafish is, conceivably, a "high-throughput" model system for various screening tests (i.e. toxicological, pharmacological, etc.) as a great number of small molecules may, in fact, permeate the chorion (Kokel et al., 2010). Moreover, it is highly homologous to other mammals (Kang et al., 2015; Rihel, 2010) and it has been shown to share many similarities in bioprocesses and genome with humans (Spitsbergen and Kent, 2007). In addition, zebrafish is used as an experimental model organism in behavioral studies due to the high development rate and the capability to study its locomotor activity (Rihel, 2010; Wang et al., 2018; Pitt et al., 2018). Lastly, studies employing the zebrafish as a model organism, whereby major ethical issues with animal laboratory use can be overcome, have greatly increased in the past years (Wiecinski, 2012).

As the use of nanomaterials in both commercial goods and novel applications expands, the need for relevant, accurate and predictive nanotoxicological assessment also arises. Moreover, the impact of CQDs on the environment is foreseen to grow as their burden will increase, because of their intensive study with vast potential scope in various fields even for high-volume applications. In this study, the potential acute and developmental toxicity of three CQDs of different elemental composition is investigated on zebrafish larvae with the aim to examine the potential risks on aquatic organisms and the environment and to draw intensive attention and trigger further investigation. The toxicity assays focus on testing the toxic responses induced in several levels of an organism (acute toxicity tests, morphological malformations, behavioral analysis tests). Non-doped and doped with nitrogen and sulfur-nitrogen were the three main CQD variants. The studied nanomaterials are well known for their multiple properties and applications and share in common the carbon source deriving from citric acid.