Eco-design of nanostructured cellulose sponges for sea-water decontamination from heavy metal ions

Eco-design of nanostructured cellulose sponges for sea-water decontamination from heavy metal ions

Journal Pre-proof Eco-design of nanostructured cellulose sponges for sea-water decontamination from heavy metal ions Andrea Fiorati, Giacomo Grassi, A...

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Journal Pre-proof Eco-design of nanostructured cellulose sponges for sea-water decontamination from heavy metal ions Andrea Fiorati, Giacomo Grassi, Aurora Graziano, Giulia Liberatori, Nadia Pastori, Lucio Melone, Lisa Bonciani, Lorenzo Pontorno, Carlo Punta, Ilaria Corsi PII:

S0959-6526(19)33879-X

DOI:

https://doi.org/10.1016/j.jclepro.2019.119009

Reference:

JCLP 119009

To appear in:

Journal of Cleaner Production

Received Date: 9 August 2019 Revised Date:

4 October 2019

Accepted Date: 21 October 2019

Please cite this article as: Fiorati A, Grassi G, Graziano A, Liberatori G, Pastori N, Melone L, Bonciani L, Pontorno L, Punta C, Corsi I, Eco-design of nanostructured cellulose sponges for seawater decontamination from heavy metal ions, Journal of Cleaner Production (2019), doi: https:// doi.org/10.1016/j.jclepro.2019.119009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Eco-design of nanostructured cellulose sponges for sea-water decontamination from heavy metal ions Andrea Fiorati1, Giacomo Grassi2, Aurora Graziano1, Giulia Liberatori2, Nadia Pastori1, Lucio Melone1, Lisa Bonciani3, Lorenzo Pontorno3, Carlo Punta1,* and Ilaria Corsi2,* 1

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Department of Chemistry, Materials, and Chemical Engineering “G. Natta” and INSTM Local Unit, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133, Milano, Italy

Department of Physical, Earth and Environmental Sciences and INSTM Local Unit, University of Siena, Siena, Italy 3

BIOCHEMIE LAB S.r.l., Via di Limite 27G, 50013 Campi Bisenzio (FI), Italy

Corresponding authors* Phone: +390223993026; e-mail: [email protected] (C.P.) Phone: +390577232169; e-mail: [email protected] (I.C.)

Abstract The growing human activity on the sea coasts is more and more associated to a progressively increasing of seawater pollution. Ship operational discharges (such as bilge water), together with local accidents or illegal activities, often lead to detect ppm concentrations of heavy metal ions in marine waters. For this reason, highly efficient remediation technologies are required to guarantee the abatement of these contaminants. The use of engineered nanomaterials (ENMs) is emerging as a valuable alternative for environmental remediation. However, the concerns related to ENMs potential ecotoxicity still limit their use in real scenarios, in spite of the good to excellent remediation performances. The correct choice of the starting material and of the synthetic protocol could provide new safe-by-design solutions for this purpose. Following this approach, we herein report the eco-design strategy used for the development of eco-friendly cellulosebased nanostructured sponges (CNS). The latter resulted to be effective sorbent units for heavy metals removal from seawater. The materials were obtained following a two-step protocol, consisting first in the production of TEMPO-oxidized cellulose nanofibers, followed by their cross-linking in the presence of branched polyethyleneimine. CNSs herein described exhibit high performances in removing a wide range of heavy metal ions (Zn(II), Cd(II), Cr(III), Hg(II), Ni(II), and Cu(II)) from artificial sea water (ASW) in a 1

concentration range of 1-250 ppm. Environmental safety of materials (ecosafety) was investigated by using a standardized ecotoxicity bioassay as algal growth inhibition test (OECD 201) coupled with an in vivo exposure study using a filter-feeder marine bivalve species in which immune cells viability (neutral red retention time) and genotoxicity (micronucleus test) were investigated. The results in terms of eco-safety evaluation led to the optimization of the material synthetic strategy (eco-design) and allow to combine the best decontamination efficiency with no risk for aquatic biota.

Keywords Eco-design, sea water remediation, nanocellulose, nanosponges, polyethyleneimine.

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Graphical abstract

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Highlights •

Synthesis of cellulose nanosponges is simple, cheap, and scalable



Eco-design of materials follows a step by step environmental safety assessment



Materials show superb adsorption performances of transition metals from sea water



Ecotoxicological validation in realistic scenarios limits risk for aquatic biota

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1. Introduction Heavy metals pollution in marine coastal and off-shore waters is still posing a serious threat to both humans and wildlife by reducing aquatic ecosystem services, and consequently the benefits derived from them. As underlined by descriptor 8 of the Marine Strategy Framework Directive (MSFD, Directive 2008/56/EC), a Good Environmental Status should be reached in European Commission marine waters by 2020 (ECC 2008a) by limiting concentrations of contaminants at levels not giving rise to pollution effects and thus protecting the resource base upon which marine-related economic and social activities depend. Although introduced in marine waters at low concentrations by a variety of sources and activities, including sewage and industrial effluents, ship operational discharges, produced waters and coastal landfill sites, such chronic exposure led to bioaccumulation in edible marine species (e.g., shellfish and fish), posing at risk top predators, including humans (Brand et al., 2018; EEA 2011; USEPA 2010). Frequently Cu, Cd, Cr, As, Pb and Zn exceed upper limits of water quality standards (µg/L) (e.g. Environmental Quality Standard Directive 2013/39/EU) in marine coastal waters moderately affected by human activities (e.g. estuaries and harbours). Pb and Zn are the most abundant and in concentration often within one order of magnitude higher than 10 µg/L (Meng et al., 2008; Lin et al., 2013; Wang et al., 2015; Burton et al., 2017). However, they reach far higher concentrations up to mg/L in sea waters receiving discharges from land filling, desalination plants, fishing boats, oil spills and solid rubbish and in waters from ship operational discharges as bilge water (EPA 1999). In order to cope with their increasing production volumes and illegal/accidental release into the sea, a variety of strategies, including treatment technologies, have been developed to meet discharge limits and reach sea water quality standard with no risks to human and marine wildlife (Andrade 2009). The several approaches reported in the literature for water decontamination from inorganic contaminants include coagulation, precipitation, electrochemical treatment, membrane filtration, adsorption/ion exchange systems, and bioremediation. However, most of these technologies are known to be expensive, partially effective at low contaminant concentration, and time-consuming (Seidel et al., 2004; Hokkanen et al., 2013; Piccin et al., 2017). Moreover, the synthesis of sorbent materials such as activated carbons, zeolites, and

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synthetic polymeric resins, which have been already applied successfully in several wastewater treatments, usually requires high energy consumption to provide quite low final yields (Piccin et al., 2017). The efficiency of decontamination of all these solutions is often even considerably reduced when they are applied to the treatment of sea water, mainly due to the high salinity of the medium, which makes inefficient most of the technologies previously discussed. For this reason, the issue of sea water decontamination is even more critical and urgent. The call for a continuous improvement and innovation in environmental remediation has led to explore the potentials of nanotechnologies in environmental remediation, opening the way to the emerging technology of nanoremediation (Ali 2012; Canesi et al., 2016; Mahfoudhi and Boufi, 2016). The nanometric size of engineered nanomaterials (ENMs) guarantees a higher and more reactive surface area compared to bulk structures of conventional approaches. Therefore, ENMs, being more efficient and less costly, apply to be a valuable alternative to standard remediation procedures, both from an economical and an environmental point of view. However, those ENM’s properties which make them suitable for pollutants removal, raises concerns in terms of environmental and human safety based on evidences of ecotoxicity to aquatic wildlife (Karn et al., 2009; Holland 2011; Sanchez et al., 2011). In order to overcome these limits, a safer-by design concept should be followed in design and developing innovative, green and environmentally safe (nano)-solutions, capable to set at minimum any toxicological effects for humans and wildlife, while maintaining high efficiency in the remediation action (Corsi et al., 2018a). To reach these goals, we envisioned that a possible strategy to design a suitable nano-sorbent material should take into account the following guidelines: i) Correct choice of the starting building blocks, possibly derived from sustainable and renewable resources; ii) Switching from nano-sized to nano-structured microdimensioned systems, in order to overcome the potentials risks associated to the use of ENMs, while maintaining the advantages obtained by operating at the nano-scale; iii) Ecologically oriented design of the new material (eco-design), which means a step by step environmental safety assessment since the very beginning of the design and synthesis of the nano-sorbents (Corsi et al., 2018b). 6

Possible scenarios of material interactions with natural ecosystems should be included in the design process and, in order to limit any potential ecotoxicity, more than one trophic level should be included (Corsi et al., 2014, 2018a, b). A more ecologically oriented hazard assessment of ENMs entering the marine environment, has already been proposed and should be extended to materials for environmental application as remediation (Holden et al., 2016). In this context, we considered cellulose as a sustainable and renewable raw starting material, being the most abundant and cheap biopolymer on Earth. It has been widely reported that it is possible to cleave the hierarchical structure of native cellulose by promoting nanofibrillation and consequent production of nanocellulose (NC), in the form of cellulose nanocrystals (CNCs) or cellulose nanofibers (CNFs). These non-toxic (DeLoid et al., 2019) building blocks are suitable for the fabrication of a wide range of ENMs, which have found consistent application in different fields, including environmental remediation (Mahfoudhi and Boufi, 2017; Bejoy et al., 2018; Mohammed et al., 2018; Das et al. 2018). Among the several consolidated mechanical and chemical approaches for the production of CNFs, we considered the 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO)-mediated oxidation by NaClO/NaBr system as the most advantageous technique for the first step in the design of a scalable and ecosafe material for water treatment (Isogai et al., 2011; Pierre et al., 2017) In fact, according to this protocol, the alcoholic groups in the C6 position of the glucopyranosic units are partially converted to the corresponding carboxylic acids (1-1.7 mmol g-1). This chemical transformation allows to achieve the physical nano-defibrillation of the cellulose hierarchical structure at basic pH, thanks to the simple electrostatic repulsion between deprotonated carboxylic units on CNFs backbone. Moreover, the presence of carboxylic functional moieties facilitates the cross-linking of CNFs in the presence of suitable molecules, favouring that switch from nano-sized to nanostructured systems previously recommended (Corsi et al., 2018a; Corsi et al. 2018b) According to this approach, in 2015 we reported a two-step thermal protocol for the cross-linking of TEMPO-oxidized and ultra-sonicated CNFs (TOUS-CNFs) in the presence of branched polyethyleneimine 25 kDa (bPEI) in water (Figure 1). The new cellulose nano-sponges (CNS) showed superb performances as heavy metal and organic sorbent systems from MilliQ water (Melone et al., 2015a), but also a high versatility for a wider range of applications (Riva et al., 2019; Fiorati et al., 2017; Melone et al., 2015b) 7

Herein we report the results obtained by applying CNS to solve the stringent issue of sea water decontamination from heavy metal ions. Supported by a step by step ecosafety assessment, we re-designed the sorbent material in order to ensure high remediation efficiency and, at mean time, to make the new nanotechnology free from any potential negative effect on marine ecosystems.

2. Materials and Methods Cellulose from cotton linters was provided by Bartoli Spa paper mill (Capannori, Lucca). The MilliQ water was produced by a Millipore Milli-Q® Integral Water Purification System. All the other reagents were purchased from Sigma-Aldrich. Artificial sea water (ASW) was prepared according to a standard procedure (LaRoche et al., 1970) by dissolving in 60 L of MilliQ water the following salts: NaF (0.144 g), SrCl2·6H2O (3.4 g), H3BO3 (1.2 g), KBr (4.02 g), KCl (27.96 g), CaCl2·2H2O (43.98 g), Na2SO4 (159.6 g), MgCl2·6H2O (199.8 g), NaHCO3 (7.98 g) and NaCl (1659 g). The solution was maintained uncovered for a few hours under stirring, in order to reach the CO2 dissolution equilibrium. The obtained ASW was filtered at 0.45 µm and stored at 4-6 °C. For algal growth inhibition test ASW was enriched with macronutrients, trace metals and vitamins based on f/2 phytoplankton standardized growth medium formulation and filtered (0.2 µm) before use (OECD 201).

2.1. Synthesis of TOUS-CNF TOUS-CNF were obtained following the procedure previously described in literature, but on a larger scale (Melone et al., 2015a). Briefly, 100 g of cotton linters paper were minced in a 1 L flask with a domestic mixer, by gradually adding deionized water. At mean time, TEMPO (2.15 g, 13.8 mmol) and KBr (15.42 g, 129 mmol) were dissolved in 2 L of deionized water in an 8 L flask under magnetic stirring. The cellulose pulp was added to the catalytic solution and deionized water was added in order to obtain a total volume of 5.7 L. While maintaining the solution under magnetic stirring, a pH-meter and two dropping funnels were installed above the 8 L flask, one containing NaClO (12.5 % w/w aqueous solution - 437 mL), and the other one NaOH (4 M aqueous solution - 250 mL). NaClO solution was slowly added, while the pH value was maintained in the range of 10.5–11 by using the sodium hydroxide solution. The reaction was left stirring 8

overnight (12-16 hrs) and then acidified to pH 1–2 with concentrated HCl (37 % w/w aqueous solution). The white precipitate was filtered on a sintered glass funnel and washed extensively with deionized water (5 x 2 L). Final washes were performed with acetone (2 x 0.5 L) in order to remove water and allow rapid drying of nano-cellulose (84 g, 84 % yield). The amount of carboxylic groups was determined by titration with a NaOH solution and phenolphthalein as colorimetric indicator. TEMPO-oxidized cellulose is then suspended in water and 1 equivalent of NaOH respect to carboxylic acids is added. The dispersion was ultra-sonicated at 0 °C (Branson Sonifier 250 equipped with a 6.5 mm probe tip working at 20 kHz in continuous mode, with an output power 50 % the nominal value (200 W)) until a clear solution is obtained, indicating the complete defibrillation in TOUS-CNF.

2.2. General procedure for cellulose nanosponge (CNS) preparation The preparation of original CNS (α-CNS) was performed following the procedure already described in literature (Melone et al., 2015a) (Scheme 1, steps 1-3). Briefly, TOUS-CNFs (3 % w/v) were dispersed in a water solution containing the double amount in weight of branched-polyethylenimine (bPEI, 25 kDa) respect to cellulose nanofibers. The mixture was sonicated at 0 °C for 10 min. The obtained viscous gels were transferred into a 24-well plate, frozen at -80 °C and freeze-dried for 48 h, affording the corresponding xerogels which, in turn, were treated in oven (103 °C, 16 h). The resulting sponge-like materials were removed from the molds and washed with methanol under reflux in a Soxhlet apparatus for 24 h, dried and stored in dry environment. A-CNS were prepared according to the same protocol, but the resulting dry xerogels were ground in a mortar before washing, affording a homogeneous fine powder. Moreover, after the optimization of the purification protocol, CNS washing was performed by maintaining ground powder under stirring in deionized water at 30 °C (6x150 mL, 1 h contact time for each cycle). Finally, for formulations B-H, citric acid (CA) was added in the bPEI/TOUS-CNF hydrogels before the thermal treatment and in the ratios according to Table 1. Grinding and purification were conducted as for A-CNS

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Table 1 List of bPEI/TOUS-CNS batches under investigation. Sample α-CNSb A-CNS B-CNS C-CNS D-CNS E-CNS F-CNS G-CNS H-CNS a

bPEI (g) 2 2 2 1 1.5 1 1.5 1 1.5

TOUS-CNFs (g) 1 1 1 1 1 1 1 1 1

CA (%)a 18 18 18 24 24 30 30

mol% of CA relative to primary amino groups in 25 kDa bPEI; The sample was not ground.

b

2.3. Adsorptions experiments of metal ions from mono-contaminated ASW solutions Metal ion adsorption equilibrium experiments were performed ASW by mono-contaminating the solution with fixed amounts of metal ion chlorides. Each test was repeated 3 times in order to ensure statistical significance to the analysed results. For each test, 12 mg of the selected CNS (±0.2 mg) were put into a Falcon test tube and then dispersed in 15 mL of mono-contaminated ASW solution (mass of sorbent material per volume of solution 0.8 mg mL-1). The test tubes were sealed and left at 25 °C and shacked for 24 hours. Solutions were filtered and analysed by Agilent Technologies Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 7900, equipped with a MicroMist nebulizer and a spray chamber in a Peltier-cooled sample introduction system, to increase stability and consistency, followed by a Shield Torch System and Temperature-controlled collision/reaction octopole ion guide cell, to provide interference removal in helium collision mode. All analytical operations were carried out in compliance with method EPA 6020B 2014. Instrument Calibration was carried out by standard solutions used as reference material. These solutions were purchased by qualified suppliers (Exaxol Italia) containing the analytes in a suitable concentration range. The adsorption capacity at the equilibrium (q) was calculated according to Eq. 1

=





(1)

where C0 and Ceq are the initial and the final pollutant concentration (mg L-1), respectively, V is the volume of the solution (L), and m represents the mass of the adsorbent material (g).

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The experimental data for the construction of adsorption isotherms were obtained from batch conditions equilibrium adsorption experiments, conducted as previously described, by varying the initial pollutant concentration (C0) from 0 to 250 mg ml-1. The scanning electron microscopy (SEM) analysis has been conducted by Colorobbia Italia, using a Zeiss instrument Gemini Supra 40 model (accelerating voltage 20 kV, spot size 60 µm), with an energy-dispersive electron probe X-ray (EDX) (Oxfod x-act). Samples were prepared by cutting an horizontal section of αCNS, after metal adsorption, at half height. They were fixed to aluminium stubs using graphite powder (Agar scientific, G3300 Leit C – conducting carbon cement) and heated in oven at 50 °C for 30 minutes, after which a coating with graphite has been realized by using an Emitech K450 apparatus.

2.4. Ecosafety assessment approach α-CNS were initially tested with the marine microalga Dunaliella tertiolecta, following OECD 201 guidelines with minor modifications (OECD 2011). Briefly, α-CNS chunks (Scheme 1, inset 3) were statically pre-wetted in ASW, by a 24 h incubation at a concentration of 5 mg mL-1. ASW was then recovered, supplemented with f/2 nutrients, 0.2 µm sterile-filtered and used for growth inhibition assays. Subsequently, to enforce an eco-design approach, the formulation of CNS was screened for potentially toxic chemicals. In particular, bPEI was also tested in the range between 0.25 and 1000 µg mL-1 to determine the 50% effect concentration (EC50) on algal growth by using same algal species. The intrinsic ecosafety of two nanosponge batches (A-CNS and B-CNS) was evaluated accordingly. Attention was paid in mimicking the envisaged real usage scenario of CNS in the decontamination of polluted sea waters. In particular, CNS samples were incubated with ASW at a concentration of 1.25 g L-1 for 2h at room temperature, under vigorous magnetic stirring, in order to mimic the treatment process usually applied to remediate contaminated waters. Mixtures were then filtered at 0.45 µm to remove CNS powder and the conditioned ASW was supplemented with f/2 nutrients and sterile filtered at 0.2 µm. The obtained medium was used for toxicity tests both in its undiluted form and after serial dilutions (1:20, 1:10, 1:5 and 1:2) with standard f/2 algal growth medium. Exponentially growing D. tertiolecta cells were incubated with the prepared test media and ASW as a control, at an initial density of 104 cells mL-1, for 72 h. Cell number were estimated by microscope counting through a Burker chamber and growth rates were calculated by the following equation: 11

=

lnNt- lnN0

(2)

0

Where µ is the estimated growth rate (hr-1), t0 and t are the initial time of the test and any time after the beginning of the exposure, respectively, and N0 and Nt are the estimated cell densities at the aforementioned time points. The experiments were performed in three biological replicates and growth inhibition is reported as percent effect with respect to control (ASW only groups). B-CNS and C-CNS ecosafety was also tested by an in vivo study using marine mussel's cells responses, ASW was again used as control. Adult mussels of Mytilus galloprovincialis (medium length of the valves 6.5±0.5 cm) were purchased from an aquaculture farm (Arborea, OR, Italy), and placed for 48 hours in natural sea waters (NSW) (salinity 40±1%, pH 8±0.1 and temperature 18±1°C) taken from a pristine area in Tuscany (Italy) for acclimation period. Physical-chemical parameters of the waters were checked daily and temperature was maintained constant (18 ± 1°C). Mussels were divided in glass tanks in number of 6 individuals for each experimental group and run in triplicate tanks, using the ratio 1 mussel: 1 L of exposure medium. B-CNS and C-CNS were tested using the same procedure applied for algal growth inhibition assay. CNS samples were incubated with ASW at a concentration of 1.25 g L-1 for 2 h at room temperature, under vigorous magnetic stirring, then first filtered at 0.45 µm to remove CNS powder and at 0.2 µm before adding mussels. Solutions were renewed every 24 h and mussels were not fed during experiment. After 48 h mussels were removed from tanks and before sacrifice, the haemolymph was withdrawn with a sterile syringe without needle from the posterior adductor muscle and filtered through a sterile gauze and pooled. Hemocytes were analysed for lysosomal membrane stability (LMS) by the Neutral Red Retention Time assay (NRRT) (Lowe et al., 1995), and chromosomal damage detected by Micronucleus Test (MN) (Venier et al., 1997). The hemolymph was withdrawn from mussel's adductor muscle with a 1 mL sterile syringe pre-loaded with a saline solution to prevent clotting (20 mM HEPES, 436 mM NaCl, 53 mM MgSO4, 12 mM KCl, 10 mM CaCl2). NRRT assay was conducted using haemocytes monolayers, placing 200 µL of haemocytes suspension in a coverslip and incubating it in a humid chamber for 1 h in the dark at 18°C. Cells were then incubated for 15 min at 18°C with 200 µL of NR dye solution (final concentration 0.1 mg mL-1 from a stock 12

solution of 20 mg mL-1 NR in DMSO). Excess dye was removed using physiological saline solution and slides sealed for optical microscopy analysis. Slides were checked every 15 min under an optical microscope (Olympus BX51) 80X and the percentage of cells showing loss of the NR dye from lysosomes was counted. At least 100 cells were observed at each time-point. The end-point of the assay is defined as the time at which 50% of the cells show signs of lysosomal leaking, so the cytosol becoming red and the cells roundedshaped. For each exposure group three independent slides made by three replicates of composed by pooled haemolymph were analysed. For MN assay haemolymph was withdrawn from mussel's adductor muscle using a sterile syringe preloaded with a solution 10 mM EDTA in physiological saline. A volume of 400 µL of haemolymph was placed in a cold slide and allowed to adhere for 1 h. Then, the slides were located in cold methanol at -20°C for 20 min and placed in an Hellendal containing 6% Giemsa dye in MilliQ water for 20 min. MN frequency analysis every 1000 cells was performed with the 80X Optical Microscope (Olympus BX51). We analysed three replicates of three different slides composed by pooling haemolymph taken from at least five animals for each experimental group. 2.5. Statistical analysis Algal growth rate data were tested for significant (p< 0.05) differences between CNS exposed and control groups (ASW) with one-way analysis of variance (ANOVA), followed by Dunnet’s multiple comparison test. The EC50 value, derived from bPEI growth inhibition test, was calculated using the GraphPad Prism software package, version 6. NRRT and MN frequency data were analysed by GraphPad Prism software package, version 6. In order to compare all treatment respect to the control group one-way analysis of variance (ANOVA), plus Bonferroni post-test were performed; p< 0.05 was set as significant cut-off.

3. Results and Discussion 3.1. Synthesis and adsorption performances in sea water of first class of CNS Preliminary experiments to verify the heavy metals absorption efficiency of CNS in sea water were first conducted by using the xerogel formulation previously developed for the analogous tests in MilliQ water. 13

TOUS-CNFs were produced from cotton linters as original source, with a medium content of carboxylic units of 1.4 mmol g-1. The CNS synthetic procedure is described in Scheme 1. Briefly, aqueous suspensions of TOUS-CNFs 3.5 % w/v and bPEI in the 1:2 weight ratio were first freeze-dried in a 24-well plate, promoting the formation of xerogels, and then thermally heated in an oven at slightly 100 °C. The thermal treatment favoured the formation of amide bonds between the carboxylic groups of TOUSCNFs and the primary amines of bPEI. The resulting materials consisted into cylindrical bPEI/TOUSCNF2:1 (α) CNS, whose micro-porosity derived from the ice-templating action of water, while nanoporosity associated to the cross-linking was recently highlighted by means of a Small Angle Neutron Scattering (SANS) study of the water nano-confinement geometries in the network (Paladini et al., 2019)

Scheme 1. (1) TOUS-CNFs, bPEI and CA (the latter limited to the samples B-H) are homogeneously dispersed in water; (2) the mixture is placed in molds and frozen. The water is then removed by freezedrying; (3) the resulting xerogel undergoes a thermal treatment to promote the formation of amide bonds and consequent reticulation. The material is finally washed in order to remove non-reticulated components. The obtained cellulose nanosponges (CNS) can be ground in order to obtain a homogeneous powder (4).

Besides the synthesis of original α-CNS, an analogous material A-CNS was prepared by simply grinding α-CNS in a mortar before use, in order to obtain a homogeneous fine powder (Scheme 1 (4)). In fact, preliminary adsorption experiments, conducted on mono-contaminated solutions of transition metal salts (150 ppm) in MilliQ water, showed in all cases a higher decontamination efficiency for the powder, compared to the same amount in weight of the corresponding whole sponge (Fig. 1 A). The scanning 14

electron microscopy with energy-dispersive electron probe X-ray (SEM-EDX) analysis, conducted on a horizontal section of the dry α-CNS after adsorption tests from Cd(II) and Zn(II) MilliQ water solutions (Fig. 1, B and C respectively), on one side confirmed the complete penetration of the material by the aqueous medium, but it also revealed a non-homogeneous distribution of the ions. The concentration of the metal analytes drastically decreased by moving from the external surface to the inner core of the sponge, indicating that the adsorption efficacy was limited by the diffusion kinetic of the solution in the CNS.

Fig. 1. A) CNS adsorption capacity at the equilibrium (q) from mono-contaminated MilliQ water solutions of transition metal salts. 12 mg of CNS in 15 mL of 150 ppm metal ion solutions. A comparison between whole α-CNS (blue bars) and same amount of A-CNS in the powder form (orange bars). B) SEM-EDX image of αCNS cross section after Cd(II) adsorption from 50 ppm MilliQ water solution. Green dots represent metal ions. C) SEM-EDX image of α-CNS cross section after Zn (II) adsorption from 50 ppm MilliQ water solution. Green dots represent metal ions.

The first adsorption tests in artificial sea water (ASW) were conducted by contacting 15 mL of monocontaminated solutions of different heavy metal chlorides (CdCl2, CrCl3, CuCl2 and ZnCl2) with 12 mg of ground A-CNS, in batch conditions. All the solutions were prepared in order to have a 150 ppm initial concentration of each metal ion. Despite the high salinity of the medium, a good to excellent adsorption capacity at the equilibrium (qeq) was measured for all the analytes under investigation (Cd(II): 84 mgg-1; Cr(III): 94 mgg-1; Cu(II): 194 mgg-1; Zn(II): 125 mgg-1) (Fig. 2). Surprisingly, in all the cases the adsorption efficiency was even better than when operating in MilliQ water under the same experimental conditions (Melone et al., 2015b). We ascribed this behaviour simply to the slight basic pH (8) of sea water which, 15

preventing the protonation of amino groups, allowed them to completely exploit their chelating action towards transition metal ions. Moreover, A-CNS exhibited also a significantly higher adsorption performance in comparison with simple TOUS-CNF (Cd(II): 13 mg•g-1; Cr(III): 40 mg•g-1; Cu(II): 84 mg•g1

; Zn(II): 32 mg•g-1), once again confirming the crucial role of the polyamine cross-linker in capturing the

heavy metal ions. 3.2. Ecosafety evaluation of first class of CNS With these promising results in hand, we also wanted to verify indirectly the ecosafety of our sorbent materials. Initially, we performed a toxicity assessment of α-CNS. In order to overcome the difficulty of testing the toxicity of a bulk, non-soluble material, we incubated α-CNS with ASW for 24 hours in order to assess the release of toxic compounds. This also allowed us to relate our tests to an envisaged usage scenario of CNS, where the material itself is not released in aquatic media but rather the treated water is re-immitted in the environment. ASW conditioned with α-CNS caused a remarkable inhibition of growth rate of the exposed microalgal cultures of 74.6 ± 29%. The observed high level of decreased cell viability confirmed that the materials synthesized at this stage were unsuitable for an environmental use, due to lack of ecosafety. Moreover, this preliminary result provided an important input to target the synthetic process of CNS in order to “correct” this issue. In particular, such consistent effects strengthened the hypothesis of a potential release from the tested material of a toxic compound used in the nanosponge synthesis. Therefore, we focused on bPEI, as a constitutive ingredient of CNS formulation. bPEI is essential for the reticulation of cellulose nanofibers and the formation of the final nanostructured material. It is well known that synthetic macromolecules bearing a high number of positively charged moieties can exert cytotoxic effects (Parhamifar et al., 2010). Polycations, including bPEI, have successfully been employed in DNA delivery applications to different cell lines. Nonetheless, a wide range of cytotoxic effect is often associated with such applications (Kafil and Omidi, 2011; Fischer et al., 2003), such as lysosomal membrane destabilization, DNA damages, induction of necrotic and apoptotic events, with the magnitude of those being directly proportional to the molecular branching and charge density. Moreover, bPEI toxicity as a nanoparticles capping agent has been recently reported towards human cells and anaerobic bacteria (Choi et al., 2017; Gitipour et al., 2016). Hence, the inherent toxicity of bPEI towards the model marine microalga D. tertiolecta was evaluated via growth inhibition assays. A consistent growth rate reduction was observed for 16

culture exposed to 5 µg mL-1, while amounts of bPEI higher than 25 µg mL-1 caused a complete disruption of cell growth (Fig. S3 in Supporting information). The obtained EC50 value, computed from growth inhibition data, was 2.75 µg mL-1, with 95% confidence values of [2.43–3.05]. Such pronounced bPEI toxicity towards D. tertiolecta could be ascribed to the interaction of charged moieties with the negatively charged algal cell wall. It has already been reported that bPEI coated silver NPs dramatically enhances their association with algal cells (Malysheva et al., 2016). Hence, we hypothesized that membrane damage and loss of cell mobility could be the underlying causes of the bPEI-induced toxicity towards D. tertiolecta.

3.3. Synthesis and adsorption performances in sea water of second class of CNS. In order to overcome the issue of bPEI partial release and its ecotoxicological side-effect, we embraced two complementary routes to re-design the CNS material and optimize its purification. First of all, we considered to optimize the purification protocol for inhibiting the potential hydrolysis of amide bonds in the last step of CNS production. For this reason, the washing phase was modified by substituting methanol with water and by operating at milder conditions (30 °C). By considering final use of xerogels in powder form, and assuming that the washing would have been more efficient if performed on ground specimens, this new protocol was applied on A-CNS. Moreover, from a synthetic point of view we envisioned that the best way to reduce bPEI release was to strengthen its cross-linking with TOUS-CNF by increasing the overall carboxylic content in the final hydrogel formulation. This could also minimize the amounts of polyamine required to produce the desired material. As it is well known (Isogai et al., 2011) that C6 alcoholic groups of cellulose can be oxidized only to a limited extent selectively to the corresponding carboxylic acids, and only when a large excess of oxidant is used, in 2017 we reported an alternative route to increase the carboxylic content, based on the addition of citric acid (CA) to the bPEI/TOUS-CNF hydrogel before thermal treatment (freeze-drying followed by heating) (Fiorati et al., 2017). In that case, our main purpose was to enhance the mechanical stability of the xerogel, which was then applied in drug delivery. We considered that this approach could be suitable to increase immobilization of bPEI in the nano-structured network. As a result, we first synthesized the new sample B, analogous to the A-CNS, but with an 18% content in mmol of CA relative to mmol of primary amino groups present on bPEI. In line with our previous 17

work (Fiorati et al., 2017), the FTIR analysis confirmed the increasing of amide bond formation by increasing the CA content (see also Fig. S4). The adsorption efficiency of B-CNS in ASW was compared with that of A-CNS for the same set of metal ions (Cd(II), Cr(III), Cu(II) and Zn(II)). Results are reported in Fig. 2. In all the cases the new B-CNS showed decontamination performances comparable with those of the original A material, proving that the introduction of CA in the nano-structured network does not negatively affect its activity. Finally, a wider range of CNS (C-H, Table 1) was prepared by varying the amount of both bPEI and CA. Elemental analysis of the new materials allowed to verify how the content of bPEI in the final CNS was just slightly affected by decreasing the stoichiometric amount of the polyamine in the synthetic procedure up to 1:1 w/w ratio respect to TOUS-CNF (see SI for detailed data). This behaviour would confirm once again the positive effect of CA in fixing the highest amount of bPEI even at the lower concentration of the polymer. Results reported in Fig. 2 on the absorption performances of all the new CNS towards a wider range of metal ions (including Ni (II) and Hg(II)), confirmed the almost steady trend.

qeq [mg g-1] 220 200 180

A-CNS

160

B-CNS

140

C-CNS

120

D-CNS

100

E-CNS

80

F-CNS

60

G-CNS

40

H-CNS

20 0 Cd(II)

Cr(III)

Cu(II)

Hg(II)

Ni(II)

Zn(II)

Fig. 2. A-H-CNS adsorption capacity at the equilibrium (q) from mono-contaminated ASW water solutions of transition metal chloride salts. 12 mg of CNS in 15 mL of 150 ppm metal ion solutions. 18

3.4. Ecosafety evaluation of second class of CNS In order to verify the efficacy of our ecosafe design, we carried out a new algal (D. tertiolecta) growth inhibition assays to test first the two batches A-CNS and B-CNS. We firstly incubated CNS with ASW, with the aim to mimic a decontamination process. After CNS removal the obtained ASW was used for growth inhibition assays for both undiluted and after dilution with standard growth medium. The results of the tests are reported in Fig. 3.

% of control 120 100 80 60

A-CNS

40

B-CNS

20 0 ASW

1:20

1:10

1:5

1:2

Undiluted

Fig. 3. Estimated growth rates of D. tertiolecta cultures incubated with A-CNS- and B-CNS-conditioned ASW at different dilutions.

Overall, we observed very mild effects on D. tertiolecta growth for all the tested conditions with a maximum growth inhibition, for undiluted CNS-conditioned ASW, of 11.7% and 9.3% for A- and B-CNS, respectively. These encouraging results highlighted how the formulation and the synthetic and post-synthesis treatment of CNS resulted in a substantially non-toxic material yet showing outstanding heavy metal removal efficiencies. The observed negligible effect on D. tertiolecta growth can certainly be ascribed to a very slight bPEI release by CNS during ASW conditioning yet kept to a minimum thanks to the optimized synthesis and washing of the materials. It is also worth pointing out that, in real environmental scenarios, the chemistry of natural sea water to be treated is certainly more complex than standard ASW. Hence, it can be envisaged that the minimal quantity of bPEI released by CNS treatment would be complexed by natural organic matter, organic 19

and inorganic colloids and particulate matter, naturally present in sea water, thus lowering the associated risks. To further support our preliminary conclusions, we also conducted in vivo exposure tests with mussels, by incubating B-CNS with ASW. After CNS removal the obtained ASW was tested on mussels. Lysosomal membrane stability (LMS) measured with NRRT assay and micronucleus (MN) frequency resulted significantly altered. In mussels exposed to B-CNS more than 50 % of the cells showed destabilized lysosomes with dye released on cytosol associated with alterations in cell morphologies (data not shown) (Fig. 4). Lysosomes are cytoplasmic, single membrane organelles involved in the degradation of the material taken up into the cell by endocytosis but most important they are able to accumulate a wide number of different classes of chemical contaminants (Moore et al., 2016; Moore, 1985; Viarengo et al., 1985; Viarengo, 1989). They are extremely sensitive to minimal concentrations of toxic chemicals that penetrate into the cells and the change of LMS was reported to be extremely rapid in mussels exposed even to nano-molar concentrations of both organic and inorganic contaminants (Marigómez et al., 2005; Izaguirre et al., 2009). The NRRT assay is considered a reliable tool to assess LMS which represents as a general stress biomarker as well as a prognostic indicator for putative pathologies and as such is an integrated pathophysiological indicator of mussel’s health status (Moore et al., 2016; OSPAR, 1997; UNEP/RAMOGE, 1999; MSFD Directive 2008/56/EC). According to standardized protocols set up for mussels haemocytes, if more than 50% of the cells show a clear cytosol and any signs of lysosomal destabilization, the organism can be considered in a good health status and no pathologies or cell damages are expected (ICES, 2010; Davies et al.; 2012). On the opposite, if there is evidence of dye loss into cytosol and/or lysosomal abnormalities in more than 50 % of the haemocytes, organism is facing a stress condition and further pathologies or damage can be associated. Therefore, based on our findings, B-CNS could not be considered ecosafe, probably still suffering for potential leaking of the higher concentration of bPEI used during synthesis. For this reason, we decided to conduct the same analysis on C-CNS, where the content of bPEI in starting formulation is halved. Indeed, LMS and MN frequency resulted significantly lower in haemocytes of 20

mussels exposed to C-CNS compared to those exposed to B-CNS (p<0.05). A significant improvement of LMS was observed in mussel's haemocytes exposed to C-CNS which show more than 50 % of stable lysosomal membranes with cells, with clear cytosols and no further sign of lysosomes abnormalities. Similar findings were obtained from genotoxic damage recorded by micronucleus assay (MN), which show lower frequencies of MNs in haemocytes of mussels exposed to C-CNS compared to those exposed to B-CNS (p<0.05). In particular, MN frequencies observed in haemocytes of C-CNS exposed mussels are similar to those recorded in controls suggesting the complete absence of any DNA damage caused by exposure to CCNS batch (Fig. 5). Micronucleus are formed during the cell division process and they can occur upon genotoxicants exposure either as chromosome breakage or missegregation during mitosis (Bolognesi and Fenech, 2012). Mussel’s haemocytes are primarily exposed to waterborne toxic compounds and due to their role as a defense system, they can quickly respond to genotoxic compounds by an increase frequency of MN formation (Venier et al., 1997).

Fig. 4. Neutral red retention time expressed as % of destabilized mussel’s haemocytes exposed to B-CNS, CCNS and ASW (control) after 48 h. The values are expressed as means ± sd (** p<0.001; *** p<0.0001).

21

Fig. 5. Micronucleus frequency of mussel’s haemocytes exposed to B-CNS, C-CNS and ASW after 48 h. The values are expressed as means ± sd (** p<0.001).

The absence of differences in MN frequencies between C-CNS exposed mussels and controls further confirm C-CNS batch more ecosafe than B-CNS. These results, combined with the adsorption efficiency reported in Figure 2, suggest the C-CNS (bPEI/TOUS-CNF/CA 1:1:18%) as the best compromise between bPEI minimum content and decontamination efficiency in ASW. 3.5. Equilibrium adsorption isotherms. A comparison of the adsorption isotherms of A-CNS and C-CNS for Zn(II) and Cd(II) cations at 25 °C is reported in Fig. 6a and b, respectively. The adsorption data obtained were interpolated using the Langmuir model (Eq. 3), with a nonlinear regression approach.

=

(3)

Here qeq represents the adsorption capacity at the equilibrium (mg g-1), qm the maximum adsorption capacity (mg g-1), which is associated to the curve plateau reached when the material is completely saturated, Ceq the equilibrium concentration (mg L-1), and kL the Langmuir constant, which is directly correlated to the slope of curve (Langmuir, 1918; Piccin et al., 2017).

22

Fig. 6. Comparison between A-CNS and C-CNS adsorptions isotherms for Zn(II) (a) and Cd(II) (b).

The higher initial slope of the Langmuir adsorption isotherm for C-CNS, for both Zn(II) and Cd(II), compared to A-CNS, revealed how the optimized new material was even more efficient in removing these contaminants at low cation concentrations. In fact, for all solutions with concentrations ranging from 1 to 45 ppm, which are typical in waters from ship operational discharges as bilge water, the heavy metal ion abatement was higher than 90 %. Moreover, while the contact time was fixed to 24 h, in order to be sure to reach the equilibrium concentration, we could verify how fast the decontamination process is, as in all cases, after less than 2 hours, the adsorbed amount was always higher than 90 % of the equilibrium value.

Table 2 Computed Langmuir parameters.

2+

Zn Cd2+

qm 138.9 100.2

A-CNS kL R2 0.07401 0.9620 0.06078 0.9806

SSE 2498 531.6

qm 108.6 101.0

C-CNS kL R2 0.3844 0.9852 0.5058 0.9904

SSE 595.8 361.6

Thus, by using a multi-trophic ecotoxicological approach which mimic a more realistic natural exposure scenarios (phytoplankton and filter-feeders), we were able to identify the best ecosafe CNS for sea water decontamination from heavy metal ions with no risks for marine biota, combining ecosafety with a good to excellent decontamination efficiency.

23

4. Conclusions We showed here the eco-design of CNS for heavy metals removal from sea water that fulfill all requirements for their application in real environmental scenarios. We progressively re-design the CNS by reducing the amount of bPEI, which was considered responsible for the observed ecotoxicity of first CNS formulations. Furthermore, we confirmed the positive effect of CA in fixing the highest amount of bPEI even at the lowest concentration of the polymer, which was proved not toxic for marine algae and bivalves. Such reduction did not influence the CNS adsorption efficiency, so maintaining the desired properties of heavy metal removal from artificial sea water. Finally, we were able to develop an environmentally safe CNS nanostructured material which can be broadly employed in heavy metals removal from sea water without compromising their performances and with no risks for humans and the environment. The unique chemical step in the synthetic protocol, which is the TEMPO mediated oxidation of cellulose, is a metal-free process easily scalable and widely used in industry. We have recently found by Life Cycle Analysis (LCA) how this step has a very low impact on the overall process (Bartolozzi et al, 2019). Moreover, recent studies conducted in our laboratories reveal how CNS can be easily regenerated with a mild acidic treatment, and recycled several times after conditioning in slight basic medium. All these promising aspects will be object of future contributions. We envision the use of CNS powder as filler of floating pillows, which can be positioned as an active barrier towards the contamination diffusion, acting horizontally and vertically. Further technological development is under investigation.

Acknowledgements This work was supported by the project NANOBOND (Nanomaterials for Remediation of Environmental Matrices associated to Dewatering, Nanomateriali per la Bonifica associata a Dewatering di matrici ambientali) POR CReO FESR Toscana 2014-2020 - 30/07/2014- LA 1.1.5 CUP 3389.30072014.067000007.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: