Can photocatalytic and magnetic nanoparticles be a threat to aquatic detrital food webs?

Can photocatalytic and magnetic nanoparticles be a threat to aquatic detrital food 1 webs? 2 3 Arunava Pradhan1,2#*, Marta Fernandes1,2,3#, Pedro M. Martins2,3, Cláudia Pascoal1,2, 4 Senentxu Lanceros-Méndez3,4,5, Fernanda Cássio1,2 5 #Equal contribution of authors 6 7 1Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, 8 4710-057-Braga Portugal 9 2 Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, 10 Campus of Gualtar, 4710-057 Braga, Portugal 11 3 Centre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 12 4 Basque Center for Materials, Applications, and Nanostructures, UPV/EHU Science Park, 13 48940 Leioa, Spain 14 5 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain 15 16 *Corresponding author: 17 Arunava Pradhan 18 E-mail: [email protected], [email protected], 19 Tel.: +351253604045 20 21 22 ABSTRACT 23 Freshwaters are likely to serve as reservoirs for engineered nanomaterials (ENMs) due to 24 their accelerated production and usage, increasing the relevance of assessing their impacts on 25 aquatic biota and the ecosystem processes they drive. Stream-dwelling microbes, particularly 26 fungi, and invertebrate shredders play an essential role in the decomposition of organic matter 27 and transfer of energy to higher trophic levels. We assessed the impacts of two photocatalytic 28 (nano-TiO2 and nano-Er:TiO2) and one magnetic (nano-CoFe2O4) ENMs on detrital-based 29 food webs in freshwaters by exposing chestnut leaves, colonized by stream-dwelling 30 microbes, to a series of concentrations (0.25–150 mg L–1) of these ENMs. Microbial 31 decomposition and biomass of fungal communities, associated with leaves, were not affected 32 by the ENMs. However, the activities of antioxidant enzymes of microbial decomposers were 33 stimulated by ENMs in a concentration-dependent way, suggesting oxidative stress in stream 34 microbial communities. The stronger responses of these stress biomarkers against nano-TiO2 35 suggest a higher toxicity of this ENM comparing to the others. To determine whether the 36 effects could be transferred across trophic levels, the invertebrate shredder Sericostoma sp. 37 was exposed to ENMs (1 and 50 mg L–1) for 5 days either via contaminated water or 38 contaminated food (leaf litter). Leaf consumption rate by shredders decreased with increasing 39 concentrations of ENMs via food or water; the effects were more pronounced when exposure 40 occurred via contaminated food. Overall, the tested photocatalytic and magnetic ENMs can 41 be harmful to microbes and invertebrates that drive detrital food webs in streams at predicted 42 environmentally relevant concentrations. 43 Keywords: photocatalytic and magnetic nanoparticles, stream microbial decomposers, stress 44 biomarkers, invertebrate shredders, trophic interactions 45 46 47 1. INTRODUCTION 48 Recent developments in nanotechnology led to an increased worldwide production and 49 application of engineered nanomaterials (ENMs) (Stark et al. 2015). The TiO2 nanoparticles 50 (nano-TiO2) are among the most extensively used ENMs with a wide range of applications as 51 in supercapacitors, photocatalysis, sensors, personal care products, biomedicine, dye52 sensitized solar cells, lithium batteries, paints and food products (Chen and Mao 2007; Weir 53 et al. 2012; Tian et al. 2014). In July 2016, the European Commission allowed the application 54 of nano-TiO2 as a UV-filter in sunscreens at a concentration up to 25% (European 55 Commission, 2016), which may further enhance the commercial use of these ENMs in 56 Europe. The estimated global production of nano-TiO2 was about 104 tonnes per year, and 57 might even be higher in Europe (Piccinno et al. 2012). 58 Nanoparticles of TiO2 are often applied in wastewater effluent treatments and chlorine-free 59 disinfection due to their photocatalytic properties (Rickerby 2014). However, the nonporous 60 structure of the bare nano-TiO2 and their aggregation capacity in water may limit the 61 photocatalytic and adsorption of organic contaminants in aquatic environments. Nano-TiO2, 62 doped with rare earth metals, like erbium (nano-Er:TiO2), can enhance the photocatalytic 63 performance because of the vacant f-orbitals of Er3+ that allow intermediate energy states 64 (reducing the band gap), improving the adsorption of various molecules (e.g. amines, 65 alcohols, aldehydes, amines thiols) from contaminants onto the nanoparticle surface (Gomez 66 et al. 2012; Martins et al. 2014). On the other hand, due to suitable physicochemical and 67 magnetic properties, cobalt-ferrite nanoparticles (nano-CoFe2O4) undergo increasing 68 applications in biomedical engineering, including drug delivery, magnetic separation and 69 purification, biosensor, magnetic resonance imaging, cancer therapy and hyperthermia 70 (Cardoso et al. 2018; Srinivasan et al. 2018). Nanoparticles of CoFe2O4 have potential to 71 remove anionic dyes (Yavari et al. 2016) and to treat metal-rich industrial effluents or 72 wastewaters (Srivastava et al. 2016). 73 Due to the vast applications and use of these ENMs, they are likely to be present in 74 significant amounts in aquatic environments. Indeed, nanoparticles of TiO2 were detected in 75 groundwater and drinking water as a consequence of their release from house facades into the 76 nearby stream or from urban runoffs (Kaegi et al. 2008; Kiser et al. 2009; Westerhoff et al. 77 2011). Adverse effects of TiO2 on aquatic organisms including bacteria, microalgae, 78 invertebrates and vertebrates have been reported (Federici et al. 2007; Li et al. 2014b; 79 Schaumann et al. 2015; Girardello et al. 2016). Although bare or Er-doped nano-TiO2 are 80 expected to be biocompatible to humans (Martins et al. 2014; European Commission 2016), 81 the impacts of nano-Er:TiO2 on aquatic organisms are unknown. On the other hand, the few 82 ecotoxicological studies with nano-CoFe2O4 showed toxicity against plant-pathogenic fungi 83 (Sharma et al. 2017) and to freshwater algae and fish (Ahmad et al. 2015a; Ahmad et al. 84 2015b). 85 In forest streams, plant litter breakdown is a key ecosystem process driven by microbes, 86 predominantly fungi, and invertebrate shredders that transfer nutrients and energy from plant 87 litter of riparian trees to higher trophic levels (Graça 2001). Invertebrate shredders generally 88 prefer to feed on leaf litter colonized by stream microbial communities because microbial 89 activities and biomass improve leaf litter quality and its palatability (Graça 2001). However, 90 the knowledge on the impacts of nano-TiO2, nano-Er:TiO2 and nano-CoFe2O4 on detritus91 based food webs is lacking. 92 The current study aims to evaluate the effects of nano-TiO2, nano-Er:TiO2 and nano-CoFe2O4 93 on stream microbial decomposer communities and invertebrate shredders using the detrital 94 model system, which has proven sensitive to various contaminants (Pradhan et al. 2011; 95 Pradhan et al. 2015a; Tlili et al. 2016). We hypothesized that ENMs would i) reduce 96 microbial decomposition and the biomass of leaf-associated fungi; ii) induce oxidative stress 97 in microbial decomposer communities; and iii) decrease leaf litter consumption by 98 invertebrate shredders, mainly when animals were exposed via contaminated food. 99 100 2. MATERIALS AND METHODS 101 2.1. Synthesis and physicochemical characterization of ENMs 102 Titanium dioxide nanoparticles (nano-TiO2 P25; ~21 nm, ≥99.5%, CAS No. 13463-67-7) and 103 Cobalt ferrite nanoparticles (nano-CoFe2O4; 35-55 nm, 98%, density: ~5.3 g cm–3, CAS No. 104 12052-28-7) were purchased from Evonik (Evonik Industries AG, Essen, Germany) and 105 Nanoamor (Nanostructured & Amorphous Materials Inc, Katy, USA), respectively. 106 The TiO2 nanoparticles doped with erbium (nano-Er:TiO2) were synthesised according to 107 Gomez et al. (2012) and Martins et al. (2014). Briefly, titanium(IV) isopropoxide (97%, 108 Sigma-Aldrich) was mixed (in 1:15 v:v ratio) with analytical grade absolute ethanol 109 (Panreac). Afterwards, acetic acid (1:10 v:v; Panreac) and Er(III) nitrate pentahydrate (14.7 110 mg; Sigma-Aldrich) were added to get Er:TiO2 atomic ratios of 0.005 (0.5% Er). Deionised 111 water (5 mL) was added after 5 min (under magnetic stirring) and the solution was shifted to 112 a Teflon-lined steel autoclave, heated in a microwave oven (15 min, 120°C). The produced 113 ENMs were centrifuged (4536 × g, for 20 min) to remove debris and resuspended in absolute 114 ethanol (for 3 min) in sonication bath (42 kHz, 100 W, Branson 2510, Danbury, CT, USA). 115 This process was repeated twice and the nano-Er:TiO2 were placed overnight (at 80°C) in 116 oven (Gomez et al. 2012; Martins et al. 2014). 117 The morphology of the primary particles was monitored by transmission electron microscopy 118 (TEM, Tecnai T20, FEI). The ENMs were sonicated for 5 min to achieve a homogeneous 119 dispersion; a drop of the solution was placed on a copper grid and dried at room temperature 120 (RT). The crystallinity of the ENMs was assessed by X-ray powder diffraction (XRD) with 121 Philips X'Pert instrument equipped with Cu Kα radiation (λ = 1.54178Å) at 40 kV/50 mA. 122 The hydrodynamic diameter and zeta potential (ζ) of the ENMs were determined using 123 Zetasizer (NANO ZS-ZEN3600, Malvern Instruments Limited, UK), in backscatter mode 124 (173°). The analyses were performed at 25°C by dispersing 10 mg of ENMs in 100 mL of 125 ultra-pure water (to avoid multicasting). The suspension was sonicated for 30 min, and 126 aliquots were used to estimate the mean hydrodynamic diameter from the intensity-weighted 127 distributions (Zeta-average), as well as the polydispersity index (PdI), and Zeta-potential 128 values (Zetasizer 6.20 software). 129 2.2. Stream microbial colonization on leaves 130 Leaves of Castanea sativa (L.) (chestnut) were collected during autumn and air-dried at RT. 131 Chestnut is one of the dominant riparian plant species in Northwest Portugal. The chestnut 132 leaves were cut into discs (12-mm) and placed into fine-mesh (0.5-mm) bags (to minimize 133 the access of benthic macroinvertebrates), and immersed for 12 days in Algeriz Stream 134 (41°35'24.56"N, 8°22'36.96"W) to allow colonization by stream-dwelling microbes. The 135 stream was situated in a low populated area. At the sampling site, the width and depth of the 136 stream were 0.5–0.8 m and 0.3–0.4 m, respectively; the geological substratum was composed 137 mostly of sand and pebbles. 138 The physicochemical properties of stream water, measured in situ using multiparametric field 139 probes (Multiline F/set 3 no. 400327, WTW, Weilheim, Germany), were: pH, 6.4 ± 0.2; 140 temperature, 13.5 ± 0.2°C; dissolved oxygen, 9.2 ± 0.1 mg L–1; and conductivity, 31 µS cm–1. 141 Concentrations of NO3––N (0.14 ± 0.01 mg L–1; HACH, programme 355), PO43––P (0.02 ± 142 0.001 mg L–1; HACH kit, programme 480) and NH3–N (0 mg L–1; HACH kit, programme 143 385) were determined with a HACH DR/2000 (HACH, Loveland, CO, USA) in the 144 laboratory. 145 2.3. Exposure in microcosms 146 After 12 days, leaf bags were retrieved from the stream and taken to the laboratory where the 147 leaf discs were carefully washed and allocated to 150-mL Erlenmeyer flasks (microcosms). 148 Stock suspensions (1500 mg L–1) of nano-TiO2, nano-Er:TiO2 and nano-CoFe2O4 were 149 prepared in mineral water followed by sonication (Pradhan et al. 2011). Composition of the 150 mineral water was: pH 5.8 ± 0.2, silica 9.5 ± 2 mg L–1, sodium 4.1 ± 0.4 mg L–1, potassium 151 0.6 ± 0.1 mg L–1, calcium 1.3 ± 0.3 mg L–1, chloride 4.1 ± 0.5 mg L–1, sulphate 1 ± 0.2 mg L– 152 1, and bicarbonate 8 ± 0.6 mg L–1 (Fastio®, Gerês Mountain, Portugal). A gradient of 153 concentrations of each type of ENMs (0.25, 1, 10, 50 and 150 mg L–1) was prepared by 154 diluting the stock suspension with mineral water to get 90 mL of final volume in each 155 microcosm. Mineral water without ENMs was used as controls. Three replicates were used 156 per treatment. All microcosms were incubated at 14°C for 21 days under shaking (140 rpm), 157 and water suspensions were renewed every 7 days. The experiment was performed in the 158 absence of light, because TiO2 is photosensitive, especially reactive to ultraviolet radiation 159 (Rickerby 2014; Li et al. 2014b). 160 2.4. Loss of leaf mass 161 The mass loss of chestnut leaves was estimated by weighing (up to 0.001 mg) lyophilized 162 (Christ alpha 2–4, B. Braun, Germany) leaf discs before and after the microbial colonization 163 in Algeriz Stream, and after the microcosm experiment. Initial leaf mass was determined by 164 immersing 3 leaf bags in the stream for 30 min, and the leaf discs were subsequently 165 lyophilized and weighed. 166 2.5. Fungal biomass 167 To determine fungal biomass, ergosterol, a sterol present in fungal cell membranes, was 168 quantified by ultra-high-performance liquid chromatography (UltiMate 3000, Thermo 169 Scientific UHPLC system) using a LiChrospher 100 RP18 (5 μm) column (Merck) in 6 170 lyophilized chestnut leaf discs per replicate. Lipid extraction was carried out from the 171 chestnut leaf discs by heating (80°C, 45 min) in KOH-methanol (0.8%), before purified by 172 solid-phase extraction and eluted in isopropanol (Sigma-Aldrich, analytical grade). Ergosterol 173 peaks were monitored at 282 nm and eluted (at 1.4 mL min−1) with methanol (Sigma-Aldrich, 174 HPLC-grade). The concentrations of ergosterol from the samples were computed using a 175 standard curve (Sigma-Aldrich) in isopropanol. The extracted ergosterol was converted to 176 fungal biomass considering the factor of 5.5 μg of ergosterol per mg dry biomass (Gessner 177 and Chauvet 1993). 178 2.6. Activities of antioxidant enzymes 179 For determining the activities of antioxidant enzymes (glutathione peroxidase: GPx, 180 glutathione S-transferase: GST, and catalase: CAT) in microbial communities on chestnut 181 leaves, 15 leaf discs from each microcosm were retrieved, washed thrice with ultrapure water, 182 and frozen in liquid nitrogen (to prevent biological activities). Leaf discs were homogenised 183 (Utratratrax T 25, IKA, Staufen, Germany) using potassium phosphate (K-phosphate, 0.1 M, 184 pH 7.4) buffer (1:10 w:v) and PMSF (phenylmethylsulfonyl fluoride as protease inhibitor, 1 185 mM) at 4°C. The leaf homogenates were centrifuged (10,000 × g, 20 min, 4°C) and the 186 supernatants (cell-free extract: CFE) were separated and frozen at –80°C in several aliquots 187 till the measurement of the activities of antioxidant enzymes. 188 Protein concentration was measured in the CFE according to Bradford (1976) in 96-well flat189 bottomed microplates and expressed per unit mass of leaves. The activities of the antioxidant 190 enzymes were measured in CFE using a spectrophotometer (SpectraMax Plus 384 Microplate 191 Reader, Molecular Devices) and normalized to the protein concentration. The activity of 192 GST was determined by measuring the formation of 1-glutathione-2,4-dinitrobenzene 193 resulting from the conjugation of GSH with the substrate 1-chloro-2,4-dinitrobenzene 194 (CDNB) (Habig et al.; Barros et al. 2019a). The cell-free extract was added to the reaction 195 mixture (1:3 v:v) containing K-phosphate (0.1 M, pH 6.5) buffer, GSH (1.5 mM) and CDNB 196 (1.5 mM). The GST activity was computed from the slope of absorbance curve (at 340 nm, ԑ 197 = 9.6 mM–1 cm–1). 198 For CAT activity, the CFE was added to a reaction mixture (1:11 v:v) containing K199 phosphate (0.05 M, pH 7.0) buffer and H2O2 (30 mM). The CAT activity was calculated from 200 the slope of decrease in absorbance (at 240 nm, ԑ = 0.04 mM–1 cm–1) due to the dismutation 201 of H2O2 (Claiborne 1985; Barros et al. 2019a). 202 For activity of GPx, the CFE was added to a reaction mixture (1:29 v:v) containing K203 phosphate (0.05 M, pH 7.0) buffer, EDTA (1 mM), GSH (reduced glutathione, 1 mM), NaN3 204 (1 mM), NADPH (reduced nicotinamide adenine dinucleotide phosphate, 0.24 mM), H2O2 205 (0.25 mM) and GR (0.2 U). H2O2 served as substrate and NaN3 prevented CAT activity. 206 When GR reduced the GSSG (oxidized glutathione) to GSH, the oxidation of NADPH was 207 monitored from absorbance (at 340 nm, ԑ = 6.2 mM–1 cm–1) and the GPx activity was 208 computed from the slope (Flohé and Günzler 1984; Barros et al. 2019a). 209 2.7. Invertebrate collection and exposure to nanoparticles 210 Sericostoma sp. (Latreville) is an invertebrate shredder (Trichoptera, Sericostomatidae) 211 common in low-order streams in Southwest Europe (Bonada et al. 2008; Varandas and Cortes 212 2010) with good water quality. Early-stage larvae (1.1 ± 0.1 cm) of Sericostoma sp. were 213 collected in upstream of the Cávado River (Northwest Portugal) and brought to the laboratory 214 in a cold box. Shredders were placed in aquaria with mineral water (Fastio®, Gerês 215 Mountain, Portugal) and sterilized (121°C, 20 min) sand and maintained under aeration at 216 16°C, with a photoperiod (12h/12h: light/dark). Shredders were allowed to feed on chestnut 217 leaves for 28 days before the feeding experiment. To assess the potential effects of nano218 TiO2, nano-Er:TiO2 and nano-CoFe2O4, the shredders were exposed to contaminated water or 219 contaminated chestnut leaves for 5 days in microcosms. For exposure via water, the 220 microcosms with mineral water (Fastio®) were supplemented with nano-TiO2, nano-Er:TiO2 221 In our study, the possible action mechanisms of nano-TiO2 might have involved the following 372 steps: i) interaction and adsorption of nanoparticles to microbes, ii) release of Ti4+ ions from 373 surface of the outer membrane-localized nanoparticles and internalization of the ions by the 374 cells, iii) partial internalization of the nanoparticles, iv) release of Ti4+ ions in acidic condition 375 of the lysosome-like organelles, and v) reduction of Ti4+ to Ti3+ by peroxides via pseudo376 Fenton-type reaction and reoxidation (Ti4+ + H2O2 → Ti3+ + OH− + •OH; Ti3+ + O2 → Ti4+ + 377 •O2−), resulting in ROS generation that induced oxidative stress (Dodd and Jha 2011; Dalai et 378 al. 2012; Pradhan et al. 2015b; Liu et al. 2017). Similar mechanisms are expected for nano379 Er:TiO2; but their relatively lesser stability and higher agglomeration compared to the bare 380 nano-TiO2 might have contributed to induce less oxidative stress in microbial decomposer 381 communities. Moreover, the doping with Er might have decreased the surface release of Ti4+ 382 ions. On the other hand, relatively greater primary particle size and higher agglomeration of 383 nano-CoFe2O4 might have led to the less negative effects of these nanoparticles. These 384 magnetic nanoparticles might have been attached to microbial cells, and Co2+ and Fe3+ ions 385 released from the surface of the nanoparticles could be internalized by the cells where the 386 ions might have undergone pseudo-Fenton-type reactions to generate ROS and induce 387 oxidative stress (Novak et al. 2013; Ahmad et al. 2015b; Pradhan et al. 2015b). Co2+ ions 388 appeared to be more toxic than nano-CoFe2O4, and intracellular accumulation of Co2+ have 389 been shown while nano-CoFe2O4 were not retained in vivo (Novak et al. 2013). 390 Our results also showed that photocatalytic and magnetic ENMs can affect stream 391 invertebrate shredder performances. Negative effects of nano-TiO2 on freshwater 392 invertebrates were reported earlier (Menard et al. 2011; Girardello et al. 2016). Changes in 393 the feeding activity of invertebrates may have dramatic ecological consequences and have 394 often been used to assess sublethal effects of nano-metal oxides (Buffet et al. 2011; Pradhan 395 et al. 2012; Pradhan et al. 2015a). In the present study, the feeding rate of Sericostoma sp. on 396 microbially colonized leaves in the absence of ENMs was within the conventional range 397 (0.04-0.5 mg leaf mass mg–1animal mass day–1) documented for invertebrate shredders in 398 streams (Arsuffi and Suberkropp 1989). The feeding rate decreased significantly upon 399 exposure to all ENMs, even at the lowest concentration (1 mg L-1) via contaminated food or 400 water. The lowest observed effect concentration on shredder feeding rate in our study was 401 similar to the hazard concentration (HC50: 1.1 mg L-1) of nano-TiO2 estimated for freshwater 402 secondary consumers, predominantly invertebrates (Semenzin et al. 2015). 403 The reduced feeding rate of the shredders probably resulted from the food avoidance 404 behaviour (Wilding and Maltby 2006; Pradhan et al. 2012; Pradhan et al. 2015a). In our 405 study, the effects of ENMs on feeding rate via contaminated leaves were more pronounced 406 than via contaminated water, which was probably due to the decreased quality and 407 palatability of the chestnut leaves after 21 days of exposure to the ENMs. The exposure to 408 ENMs might have led to high adsorption and accumulation of metals and/or nanoparticles to 409 leaves (Pradhan et al. 2012) and aquatic fungi (Barros et al. 2019b). Indeed, an earlier study 410 on trophic transfer of nano-TiO2 in freshwaters demonstrated that, comparing to aqueous 411 exposure, the dietary intake could constitute the main route of ENM exposure to higher 412 trophic levels (Zhu et al. 2010). In addition to the decrease in food quality, the aqueous or 413 dietary exposure of shredders to ENMs probably led to their accumulation in the gut, 414 inducing oxidative stress to the invertebrate shredders (Pradhan et al. 2015a; Girardello et al. 415 2016). 416 In our study, the adverse effects of ENMs on microbial decomposers and invertebrate 417 shredders in stream detrital food web were observed even at concentrations predicted to be 418 environmentally relevant (Gottschalk et al. 2013; Xia et al. 2017). On the other hand, the 419 effects of ENMs at higher concentrations may mimic the conditions of wastewaters, mine420 drainage streams or accidental spills and, therefore, are also relevant to be considered for 421 environmental safety. 422 423 5. CONCLUSIONS 424 Overall, the responses of enzymatic biomarkers revealed that nano-TiO2, nano-Er:TiO2, and 425 nano-CoFe2O4 induced oxidative stress in microbial decomposer communities involved in the 426 decomposition of plant litter in streams. The effects increased in a dose-dependent manner for 427 all ENMs, although the effects of nano-TiO2 were the most pronounced. All three ENMs 428 were able to decrease the feeding rate of the invertebrate shredder Sericostoma sp. via 429 aqueous and dietary exposure. The effects on the feeding rate were stronger when the 430 shredders were exposed to ENMs via contaminated food (leaves). To our knowledge, our 431 study is the first to show the harmful effects of erbium-doped nano-TiO2 and nano-CoFe2O4 432 on microbial decomposer communities and invertebrate shredders with a key role in detrital 433 food webs in streams. Our study also provided evidence that photocatalytic and magnetic 434 ENMs can induce negative effects even in the absence of light at predicted environmentally 435 relevant concentrations. These findings pinpoint that stream detrital food webs may have 436 potential for ecological risk assessment of emergent contaminants in complex realistic 437 environments. 438 439 Declaration of competing interests 440 The authors declare that they have no known competing financial interests or personal 441 relationships that could have appeared to influence the work reported in this paper. 442 Acknowledgement 443 This research study was supported by the strategic programme UID/BIA/04050/2019, 444 UID/FIS/04650/2019 and the project Emergemix (PTDC/BIA-BMA/30922/2017) funded by 445 national funds through Portuguese Foundation for Science and Technology (FCT) I.P. and by 446 ERDF via COMPETE2020 – Programa Operacional Competitividade e Internacionalização. 447 448 449 REFERENCES: 450 Ahmad F, Liu X, Zhou Y, Yao H. 2015a. An in vivo evaluation of acute toxicity of cobalt 451 ferrite (CoFe2O4) nanoparticles in larval-embryo Zebrafish (Danio rerio). Aquat 452 Toxicol 166:21–28. 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