Toxicity of silver nanoparticles on fertilization success and early development of the marine polychaete Hydroides elegans (Haswell, 1883)

The silver nanoparticles (AgNPs) possess bactericidal activity (Bryaskova et al., 2010; Lara Villegas, Vanesa Ayala-Nu, Carmen Ixtepan Turrent, & Padilla, 2010) with cytotoxic and genotoxic effect on microbes and cells (Arora, Jain, Rajwade, & Paknikar, 2008; Fenech, 2006). The commercial exploitation of AgNPs in the production of pharmaceutical and cosmetic products lead to its effluent waste disposed in aquatic system. The AgNPs are a cause of concern on risk of toxicity at different levels of the food chain and developmental processes of the aquatic organisms (Chen & Schluesener, 2008; Ahamed, Alsalhi, & Siddiqui, 2010; Benn & Westerhoff, 2008). The reported toxicity of AgNPs include developmental deformities in zebrafish, Danio rerio, (Bar-Ilan, Albrecht, Fako, & Furgeson, 2009) and reduced growth rate and reproductive capacity in soil nematode Caenorhabditis elegans (Roh et al., 2009). The AgNPs as contaminants effect a decline in proliferation of mouse spermatogonial stem cell (C18-4) (Braydich-Stolle et al., 2010) and cause mitochondrial dysfunction and induction of ROS which in turn set off DNA damage and chromosomal aberrations resulting in cell cycle arrest (Asharani, Lian Wu, Gong, & Valiyaveettil, 2008). The accumulation of silver and its toxicity vary according to the stability, agglomeration, aggregation, dispersion, sedimentation, dissolution, and mobility of AgNPs in the aquatic environment (Handy et al., 2008; Navarro et al., 2008; Fabrega, Luoma, Tyler, Galloway, & Lead, 2011). These processes are dependent on physicochemical properties of the particle that are in turn influenced by environmental parameters such as pH, temperature, ionic strength, and presence of ligands or natural organic matter. The oxidation of AgNp in the cell or the medium to Ag⁺ probably enhances toxicity (Dibrov, Dzioba, Gosink, & Hase, 2002). There has been a long debate on whether AgNP-associated toxicity is mediated by the release of Ag+ or by the particle itself. On the one hand, some studies support the theory that the toxic effect of AgNPs, whether coated or not, is derived from the release of Ag+ (Fabrega et al., 2011; Yang et al., 2012; Buffet et al., 2013), which causes cell damage via an increase in ROS production and an induction of apoptotic pathways (Hwang et al., 2008). On the other hand, some have concluded that the AgNP effect is related to the particle itself (Siller et al., 2013; Choi & Hu, 2008; Griffitt et al., 2012). Nevertheless, it is established that the toxicity of AgNPs is influenced by how well the particles are dispersed within the medium (Kim, Truong, Wehmas, & Tanguay, 2013).

In the marine aquatic system, the contaminants of sea water have a potent effect on survivability and reproductive efficiency of the animals (Rand, Wells, & Mcarty, 1995, His, Beiras, & Seaman, 1999). The sessile organisms are dependent on food, and dispersal of reproductive cells and development of egg on the surrounding medium and the appraisal on the toxic effect of contaminants are required to reduce the alterations in aquatic system. The sessile marine polychaete worm, Hydroides elegans, are found attached to hard substrates (Udhayakumar & Karande, 1996), and the present study attempted to demonstrate the effect of AgNPs as a contaminant on its reproductive efficiency. The AgNPs were found to impact survivability of germ cells, fertilization, and development of fertilized egg of H. elegans. The cytotoxicity of AgNPs in germ cells and developing eggs was directly related to the increasing concentration of AgNPs in the medium suggesting bioaccumulation of the contaminant.

Polychaetes are ecologically important marine organisms, comprising about 30 to 80% of the total benthic fauna regardless of the ocean depth (Hutchings, 1998). They are important fouling communities, often forming dense aggregates on submerged surfaces in coastal area and sand and are common receivers of environmental contaminants including heavy metals and other compounds.

Nanotechnology has extensive applications of metal conjugated nanoparticles, and silver nanoparticles with its cytotoxicity have evoked a study on its disposal in aquatic bodies as a pollutant (Saba et al., 2012). Investigations on human cell lines have demonstrated AgNPs with inhibitory effect on metabolic (Park, Neigh, & Vermeulen, 2011) and activating mutagenesis in DNA (Asharani et al., 2008). The present study has attempted to give a brief account on the pollutant effect of AgNPs on the germ cells, fertilization, and embryonic development of H. elegans.

AgNPs in its colloidal form was prepared and identified by UV-vis spectroscopy as a peak at 408 nM in agreement with the finding of Maribel, Guzman, Dille, and Godet (2009). Since the animal spawns in the sea, the sea water devoid of AgNPs was taken a control and the experimental sea water contained increasing concentrations of AgNPs (1, 3, 5, 7, and 9 nM) to compare and explain the influence of AgNPs on the germ cells and the developmental stages.

The EC₅₀ of developmental stages of H. elegans was determined which showed the tolerance range of AgNPs in embryo development, and based on EC₅₀ value, the variations in development were assessed in H. elegans. The germ cells, the sperm, and unfertilized egg showed high tolerance to AgNPs and with a survival rate reduced by 37 and 39% at EC₅₀. In control, 98.60 ± 0.20% of egg developed fertilization membrane, out of which only 36.20 ± 0.30% developed into larva at 3 nM concentration. To further increase in concentration, even fertilization membrane formation was reduced significantly. At higher concentration of AgNPs in seawater, the embryo did not develop normally and showed deformities and stopped development. Earlier studies on toxicity of H. elegans showed the EC₅₀ values for Pb, Ni, and Zn for the first cell division and for the formation of fertilization membrane (FM stage) were 30.37, 38.30, and 44.22 mg l⁻¹, respectively, when the gametes were mixed together in the toxicant (Gopalakrishnan, Thilagam, & Raja, 2007). Moreover, exposure of gametes before fertilization decrease the fertilization rate in H. elegans, and thus, indicating the fertilization membrane formed may prevent the toxicants entering into the oocytes (Gopalakrishnan et al., 2008). In the present finding, sperm at EC₅₀ showed decrease of 51% when compared to control (88%). The decreased fertilization rate with increasing concentration AgNPs suggested tolerance at concentration below EC₅₀ and sensitivity above it. Similarly, Gopalakrishnan et al. (2008) have reported a decrease in fertilization rate by 31% at 80 μg Hg and 42.3, 71.2, 78.9, and 78.2% at 100 μg Cd, Pb, Ni, and Zn, respectively, compared to the controls after a 20-min exposure of sperm to control seawater and to heavy metals. Vashchenko, Zhadan, Malakhov, and Medvedeva (1995) detected that mercury ions affected motility and lowered the fertilizing capacity of Strongylocentrotus intermedius sperm, although they observed a stimulating effect at 5.6 mgHg l⁻¹. To evaluate from the reported investigations, the toxicity of AgNPs was evident in sperm and egg viability, effective fertilization, and delayed fertilization showing that the metal toxicity effected gametes fertilization either by arresting energy producing process that slowed down fertilization. The formation of fertilization membrane and the first division showed retardation in development and time. At lower concentrations of AgNPs, the influence was less, but at concentration above EC₅₀, the effect was evidently clear. A study on in vitro toxicity in the germline C18-4 showed greater sensitivity to AgNPs than that in the BRL 3A liver cells, which are widely used in toxicity studies and comparable to that of cadmium (Braydich-Stolle, Hussain, Schlager, & Hofmann, 2005). This explains the sensitivity of the germ cells to contaminants in the form of nanoparticles.

In 2-cell stage, survivability was possible up to 5 nM of AgNPs, with EC₅₀ at 3 nM of 84.26 ± 0.3% and the cumulative time increased by 2.9 min. However, in 4-, 8-, and 16-cell stages, the development was arrested at 4 nM stage and at the blastula stage at 3 nM concentration. This sufficiently explains the bioaccumulation influence through the increasing developmental stages. The EC₅₀ at each successive stage decreased due to the accumulated AgNPs at each stage that fail to disintegrate or metabolize at each stage. Our observations are supported by earlier work done in sea urchin embryos for uptake of nickel chloride by Timourian and Watchmaker (1972).

Interestingly, the observation of cumulative time showed that the difference to the control increased with the development. The difference of cumulative time was 2.9 min for a fertilization membrane formation which increased progressively before an arrest of development, 14.98 min (2 nM) to 55.02 min at blastula stage.

The concordant study of DNA damage by comet assay revealed the increase in DNA damage with the developmental stages. This indicated impairment of division and transcription that explains the extended time of development and finally arrest in development. The mutagenic potential of silver nanoparticles was evaluated in embryonic fibroblasts (MEF-LacZ) and supports our findings (Park et al., 2011). The influence of AgNPs in the energy metabolizing process is evident from the fact that after EC₅₀ concentration at each developmental stage, the period of cumulative time relatively increased in comparison to the control and also time of development of the previous stage. The cumulative time at each developmental stage increased due to accumulation of nanoparticles that reduced its ability to resist the transformations in the cell.

The fertilization success significantly differed among the concentration of AgNPs treated. Previous report on the highest blastula development was recorded in nickel treatment and lower in mercury treatment among the metals studied. The abnormal development of embryo increased as the concentration of the metal increased in seawater and also more abnormal development of the embryo seen in mercury exposure compared to other metals even at low concentration (Gopalakrishnan et al., 2008). Moreover, Chan and Chiu (2015) reported the proof concept study of the effects of coated AgNPs on growth, development, metamorphosis, and settlement of marine larvae as well as silver accumulation and particle biodistribution using three model marine invertebrate species from different phyla.

In the present study, evaluation of AgNPs at various concentrations determined the effect on cumulative time and percentage of developmental stages of H. elegans. From the results obtained, it was observed that abnormalities in embryo increases as the concentration of the AgNPs increases. The exposure to nanoparticles in the aquatic environment due to waste disposal of the products consisting of nanoparticles, in various fields such as in medicine, cosmetic, drug delivery, tissue engineering, and therapeutic for treating cancer, probably are detrimental to organisms exposed to it. Thus, the effect of toxicity of the silver nanoparticles depends upon the concentration of the nanoparticles and also the resistance of the exposed species.

The experimental data presented clearly revealed the toxicity of silver nanoparticles to early life stages of a marine polychaete worm H. elegans. Moreover, this bioassay is relatively easy to conduct and provides concordant results. Furthermore, the availability of gametes throughout the year and ease of collection makes H. elegans a suitable species for routine laboratory toxicity testing. The present investigation evaluates the risk of AgNPs imposed on invertebrate organisms which is exposed to it continuously. Thus, minimized use of such products has to be implemented or alternative and safe methodology adopted while disposing the AgNPs directly or indirectly in the environment to prevent ecotoxicity and its adverse effect on other organisms including human being.