Assessment of agglomeration, co-sedimentation and trophic transfer of titanium dioxide nanoparticles in a laboratory-scale predator-prey model system

www.nature.com/scientificreports OPEN received: 30 December 2015 accepted: 20 July 2016 Published: 17 August 2016 Assessment of agglomeration, co-sedimentation and trophic transfer of titanium dioxide nanoparticles in a laboratory-scale predator-prey model system Govind Sharan Gupta1,2, Ashutosh Kumar1, Rishi Shanker1 & Alok Dhawan2 Nano titanium dioxide (nTiO2) is the most abundantly released engineered nanomaterial (ENM) in aquatic environments. Therefore, it is prudent to assess its fate and its effects on lower trophiclevel organisms in the aquatic food chain. A predator-and-prey-based laboratory microcosm was established using Paramecium caudatum and Escherichia coli to evaluate the effects of nTiO2. The surface interaction of nTiO2 with E. coli significantly increased after the addition of Paramecium into the microcosm. This interaction favoured the hetero-agglomeration and co-sedimentation of nTiO2. The extent of nTiO2 agglomeration under experimental conditions was as follows: combined E. coli and Paramecium > Paramecium only > E. coli only > without E. coli or Paramecium. An increase in nTiO2 internalisation in Paramecium cells was also observed in the presence or absence of E. coli cells. These interactions and nTiO2 internalisation in Paramecium cells induced statistically significant (p < 0.05) effects on growth and the bacterial ingestion rate at 24 h. These findings provide new insights into the fate of nTiO2 in the presence of bacterial-ciliate interactions in the aquatic environment. Engineered nanomaterials (ENMs) are used in diverse applications, owing to their unique optical, chemical, mechanical, thermal, magnetic and catalytic properties1. Currently, more than 1800 nano-based consumer products derived from 45 different ENMs are manufactured globally2. ENMs can enter into the environment at various stages in their life cycle: production, manufacturing, transportation, consumer use and product disposal3–5. Nano titanium dioxide (nTiO2) is one of the most abundant materials in products such as cosmetics, paints, food additives, pharmaceuticals, electronics, and textiles as well as in construction and wastewater treatment6–8. Moreover, the unique photocatalytic and UV-reflecting properties of nTiO2 have enhanced the exponential growth of low-cost and safer consumer products9–11. Risk assessment studies have predicted nTiO2 to be the most abundant ENM in the environment [21–10000 ng/L in surface water, 1–100 μg/L in waste water treatment plant (WWTP) effluent, 100–2000 mg/kg in WWTP sludge]12. Aquatic environments act as a sinks for chemicals as well as emerging metal pollutants such as ENMs13. Aquatic bodies contain a dominant and ubiquitous community of bacteria (~106 cells/ ml) as well as the bacterial predators ciliated protozoans (102–104 cells/ml)14,15. ENMs affect the organisms within and across trophic levels in the aquatic food chain. Poor water solubility and long-term persistence of ENMs in aquatic systems16,17 facilitate their bioaccumulation and biomagnification in aquatic organisms such as bacteria, ciliated protozoans, rotifers, algae, crustaceans, zebrafish, and mussels18–24. The accumulation of ENMs can also affect the growth, reproduction, ingestion and digestion behaviour of aquatic organisms18,20,21. Factors such as surface interactions (adsorption or hetero-agglomeration), internalisation, oxidative stress, membrane damage and mitochondrial perturbations have been reported to be responsible for the acute toxicity of ENMs in microorganisms, cell lines 1 Division of Biological & Life Sciences, School of Arts & Sciences (Formerly, Institute of Life Sciences), Ahmedabad University, University Road, Navrangpura, Ahmedabad - 380009, Gujarat (India). 2Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31-M.G. Marg, Lucknow - 226001, U.P. (India). Correspondence and requests for materials should be addressed to R.S. (email: rishi. ) or A.D. (email: ) Scientific Reports | 6:31422 | DOI: 10.1038/srep31422 1 www.nature.com/scientificreports/ Groups 1h 24 h nTiO2-UZ 395 ±  8 299 ±  44 nTiO2-MZ 411 ±  6 318 ±  41 nTiO2-LZ 411 ±  9 402 ±  28 Ec + nTiO2-UZ 699 ±  9** 425 ±  10 Ec + nTiO2-MZ 521 ±  6** 530 ±  4* Ec + nTiO2-LZ 601 ±  6** 814 ±  15** Pm + nTiO2-UZ 742 ±  8** 734 ±  6** Pm + nTiO2-MZ 657 ±  11** 790 ±  19** Pm + nTiO2-LZ 781 ±  10** 1211 ±  47** Ec + Pm + nTiO2-UZ 1036 ±  27** 1138 ±  46** Ec + Pm + nTiO2-MZ 967 ±  6** 1400 ±  68** Ec + Pm + nTiO2-LZ 875 ±  17** ND Table 1. Hydrodynamic diameter (d-nm) of nTiO2 in the microcosm, reflecting agglomeration. Pm, Paramecium, Ec, E. coli. ND, not detected. Values represented are the mean ± SE of three independent experiments. *p < 0.05 was considered significant compared with the control. and eukaryotic organisms25. The surface interactions of ENMs with microbial cells, the first step in ENM toxicity, are predominantly governed by charge interactions between ENMs and microbes26,27. ENMs with positive surface charges have been found to have higher toxicities than ENMs with negative charges. This finding has been attributed to the negative charges of cell surfaces28,29. In the natural environment, bacterial cells are ubiquitously present and have a high ratio of surface area to their volume; thus, the cells interact with and absorb high levels of ENMs15,30. Additionally, the presence of exopolymeric substances (EPS) on the outer membranes of bacterial cells complements the adsorption of ENMs from the aquatic environment15,31,32. Ciliated protozoans such as Tetrahymena secrete mucus from their mucous membranes under stress conditions, and this surface coating affects the fate of ENMs in the medium33. To understand the actual behaviours and toxicities of ENMs in aquatic systems, it is necessary to study the surface interactions, such as adsorption and hetero-agglomeration, of ENMs with microorganisms. For instance, the physical properties of E. coli cells are affected by exposure to hematite nanoparticles (NPs)34. The adsorption of ENMs on the E. coli surface is dependent on size: large hematite NPs adsorb faster than smaller NPs do32. In another study conducted in Paramecium multimicronucleatum, nTiO2 has been found not to cause toxicity, owing to weak surface interaction energy35. Furthermore, different pH and ionic strength conditions play roles in the hetero-agglomeration and co-sedimentation of discharged oxide nanoparticles with chlorella cells36. Studies examining the surface adsorption and hetero-agglomeration of ENMs with biotic factors have been limited to the single organism level. No studies have examined the adsorption, hetero-agglomeration and co-sedimentation of ENMs in the presence of a predator-prey interaction model of a real-world environmental situation. Such an interaction model, involving two organisms in lower trophic levels of the food chain, can be (...truncated)