Chemosphere 99 (2014) 96–101
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Physicochemical properties and biodegradability of organically functionalized colloidal silica particles in aqueous environment Mandy Schneider a, Fabian Meder b, Annette Haiß a, Laura Treccani b, Kurosch Rezwan b, Klaus Kümmerer a,⇑ a b
Institute of Sustainable and Environmental Chemistry, Leuphana University of Lüneburg, Scharnhorststraße 1, DE-21335 Lüneburg, Germany Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, D-28359 Bremen, Germany
h i g h l i g h t s Investigation of the biodegradability of organically surface functionalized silica. None of the functionalized particles met the biodegradability threshold value. Biodegradability of organic groups was reduced compare to unbound chemicals. Increased residence time of attached organic groups in the aquatic environment.
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Article history: Received 7 June 2013 Received in revised form 7 October 2013 Accepted 13 October 2013 Available online 8 November 2013 Keywords: Organic functionalization Colloidal silica particles Biodegradation Physicochemical characteristics Environmental fate
a b s t r a c t Engineered sub-micron particles are being used in many technical applications, leading to an increasing introduction into the aquatic environment. Only a few studies have dealt with the biodegradability of non-functionalized organic particles. In fact the knowledge of organically surface functionalized colloids is nearly non-existent. We have investigated the biodegradability of organically surface functionalized silica (SiO2) particles bearing technically relevant groups such as amino-, carboxyl-, benzyl-, sulfonate-, chloro-, and phosphatoethyl-derivatized alkyls. Essential physicochemical properties including zeta potential, isoelectric point, morphology, surface area, porosity, surface density, and elemental composition of the particles were investigated, followed by biodegradability testing using the Closed Bottle Test (OECD 301D). None of the particles met the biodegradability threshold value of 60%. Only a slight biodegradation was revealed for SiO2-Benzyl (13.7 ± 6.7%) and for SiO2-3-Chlorpropane (10.8 ± 1.5%). For the other particles biodegradability was below the normal background fluctuation of 5%. The results were different of those obtained from structurally similar chemicals not being functionalized on the particle surface and from general rules of structure-biodegradation prediction of organic molecules. Therefore, our results suggest that the attachment of the organic groups heavily reduces their biodegradability, increases their residence time and possibility for adverse effects to environmental species. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Engineered sub-micron particles (ESPs), like colloids and nanoparticles are becoming a key component for a variety of technical applications. Consequently, over the next decades the introduction of ESP in the environment via industrial processes and consumer products will increase, while their environmental fate is still largely unknown. Colloidal silica (SiO2) particles are widely used in paints and food technology, cosmetics, as well as environmental and biomedical applications (Duguet et al., 2009; Knopp et al.,
⇑ Corresponding author. Tel.: +49 4131 677 2894; fax: +49 4131 677 2848. E-mail addresses: [email protected] (M. Schneider), meder@ uni-bremen.de (F. Meder), [email protected] (A. Haiß), [email protected] (L. Treccani), [email protected] (K. Rezwan), [email protected] (K. Kümmerer). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.10.031
2009; Dickinson, 2012). They are promising adsorbents for the removal of pathogens or potentially toxic substances from wastewater (Andrzejewska et al., 2007; Majewski, 2007; Cademartiri et al., 2010; Majewski and Keegan, 2012)l or employed as drug-delivery vehicles and imaging vectors in biomedical fields (Knopp et al., 2009). For these applications, the particles can be surface functionalized with organic end groups to receive tailored physicochemical surface properties. Organosilane molecules, containing charged functional groups such as positively charged amino groups, or negatively charged carboxyl and sulfonate groups, are often used to electrostatically stabilize the particles in a specific medium (Duguet et al., 2009; Bertazzo and Rezwan, 2010; Meder et al., 2012). Moreover, the attached end groups can provide reactive groups for example for bioconjugation of proteins and drugs or for catalysis (Gill et al., 2007; Hermanson, 2008). Hydrophobic functional groups stabilize particles in hydrophobic media
M. Schneider et al. / Chemosphere 99 (2014) 96–101
(e.g. oil–water emulsions) and provide interaction sites for polymers and biomolecules. Therefore, the functional groups can essentially influence charge, aggregation and stability of the particles in aqueous environments. These parameters can also strongly influence the biodegradability of the organically functionalized particles. An important point for the assessment of the risk connected to the introduction of chemicals into the environment is knowledge on biodegradation. Compounds which are not completely mineralizable will remain as mother compound or transformation products in the environment with an increasing possibility for adverse effects to environmental species in soils and waters. Furthermore for organically functionalized particles biodegradation is of special interest, because it will alter the properties such as water solubility or bioavailability of such particles. To the best of our knowledge, no studies have been published that investigate in parallel the physicochemical properties and the ready biodegradability of organically modified SiO2 colloids. Generally only a few publications are available studying the biodegradability of different types of ESPs (Nowack and Bucheli, 2007; Brar et al., 2010; Hartmann et al., 2011; Kümmerer et al., 2011). For example, some biodegradation was reported for starch, cellulose nanoparticles and their macroscopic counterparts and no biodegradability was found for functionalized and non-functionalized fullerenes as well as carbon nanotubes (Kümmerer et al., 2011). The lack of data for the environmental fate and biodegradability makes it impossible to set limit values of engineered (nano) particles in surface waters (Baun et al., 2009) and to conduct a risk assessment. This is not only an increasing problem for ‘‘Registration, Evaluation and Authorization and Restriction of Chemicals’’ (REACH), but also for the ‘‘Water Framework Directive’’ (WFD) which should guarantee to maintain a good chemical and ecological status of surface waters. Therefore the aim should be a fast and full (bio-) degradation and mineralization of ESPs including their surface functionalities after their release into the environment. Such data can then be used for a proper, sustainable design of new compounds (Boethling et al., 2007; Kümmerer, 2007; Anastas, 2008) which would help to avoid typical follow-up problems and guarantee a greater acceptance (Kümmerer et al., 2011). According to the principles of green (Anastas, 2008), sustainable chemistry, and pharmacy (Kümmerer, 2007) the focus of interest in this study was to investigate the biodegradation of organically surface functionalized SiO2 particles. Consequently we focused on technically highly relevant alkylamino, alkylcarboxyl, alkylsulfonate, benzyl-, alkylchloro- and phosphoatoethyl- functionalizations (Table 1) which are widely used to adjust surface charges of SiO2 particles and the rheological properties of their suspensions. Furthermore, they provide important reactive groups for adsorption and conjugation mechanisms in biomedical, biotechnological and environmental applications. After the surface functionalization the particles were characterized and the ready biodegradability was analyzed with the Closed Bottle Test (CBT), which is a test also suitable for poorly soluble substances.
2. Materials and methods 2.1. Surface functionalization of particles Silica particles with a narrow size distribution (Angströmsphere™, purity > 99.9%, lot No. 100624-04, d50 = 184 nm) were purchased from Fibre Optics Center Inc., USA and calcinated at 800 °C for 4 h to remove any organic residues from the manufacturing process. The precursors for the surface functionalization were 3-aminopropyltriethoxysilane (99% in ethanol, Lot. 92196EJ-109, Sigma–Aldrich, Germany), 3-(triethoxysilyl)propylsuccinicanhydride (>94%, Lot. 1034945), 3-(trihydroxysilyl)-1-
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propanesulfonic acid (30–35% in H2O, Lot. 1070635), benzyltriethoxysilane (97%, Lot. 1106735), 3-(chloropropyl)triethoxysilane (97%, Lot. 1070827), diethylphosphatoethyltriethoxysilane (92%, Lot. 1089841) from ABCR GmbH, Germany. For all experiments double deionized water with a conductivity of 0.04 lS cm1 obtained from a SynergyÒ apparatus (Millipore, Germany) was used. The surface functionalization was conducted as described by Meder et al. (2012). In short, suspensions of 15 g silica particles in 50 mL water were sonicated for 10 min with an ultrasound horn (Branson SonifierÒ 450, USA, output: 150 W, pulse rate: 0.5 s) to break the agglomerates before precursor addition. The precursor solutions were prepared by mixing it with 50 mL water to a final concentration of 0.146 M (corresponding to 486 lmol per gram of the unmodified silica particles). The precursor solution were then added to the silica suspensions, stirred for 60 min at 40 °C and then heat treated at 120 °C for 90 min. Afterwards the particles were centrifuged at 1500g for 30 min and washed three times in 100 mL water to remove any unbound precursors. Particles were freeze dried for 120 h at 20 °C in vacuum using a freeze dryer (P8KE-80, Piatkowski Forschungsgeräte, Germany) and subsequently heat treated at 70 °C for 2 h to induce the evaporation of potentially remaining ethanol of the silane precursors and used for further experiments. 2.2. Physicochemical characterization of functionalized particles Elemental analysis was carried out by Mikrolabor Pascher, Germany. Phosphor and sulfur concentrations were analyzed after acid pressure hydrolysis of the samples via ICP-AES in an ICAP 6500 (Thermo, Germany). Chloride concentration was determined in an ion chromatograph 761 Compact IC (Metrohm, Germany) after combustion. Carbon concentrations were measured by conductometric carbon dioxide determination after combustion in a selfmade apparatus (Mikrolabor Pascher, Germany). The average and maximal error were calculated from two different measurements. Nitrogen adsorption isotherms were recorded at 196 °C with an automated surface area analyzer BELSORP-mini (Bel Inc., Japan). Specific surface areas were calculated from the adsorption isotherms using the Brunauer, Emmet, and Teller (BET) equation (Brunauer et al., 1938) porosity and pore size distribution were determined using the model of Barrett et al. (1951). All samples were gassed out at 120 °C under vacuum for 3 h before the measurement. The particle size was measured by dynamic light scattering (DLS) using an Ultrafine Particle Analyzer (UPA 150, Microtrac Inc., USA) in water (pH 7.4 ± 0.3 and ionic strength of 14 mM KCl) at a particle content of 0.1 vol% to assure a reliable signal. Average and standard deviation were calculated from 9 single measurements of the d50 (intensity), representative particle size distribution curves were revealed. Zeta potential measurements were carried out using an electroacoustic spectrometer (DT1200, Dispersion Technology Inc., USA). Zeta potential/pH-titrations were performed with the integrated titration unit using 1 M HCl and 1 M KOH respectively. All measurements were conducted with suspensions containing 1 vol% of particles. TEM and HR-TEM were recorded using a Titan 80-300 ST microscope (FEI™, the Netherlands) equipped with a Cs-corrector for the imaging lens using a 300 kV electron beam and a vacuum at 1.3107 mbar. 200 mesh cupper grids S162 covered with a formvar film (PLANO GmbH, Germany) were used as sample holders. 2.3. Biodegradability testing of functionalized particles In accordance with the Organization of Economic Cooperation and Development (OECD) 301D guideline, the CBT was used for
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Table 1 Scheme of particle functional surface groups and physicochemical characteristics: Elemental composition, surface density, surface area, particle size, isoelectric point, zeta potential (at pH 7.4 and an ionic strength of 14 mM). Scheme of functionalized particles
Elemental analysis (wt%)
Surface density (lmol g1)
IEP
Zeta potential pH 7.4; I = 14 mM (mV)
Surface area (m2 g1)
Particle size d50 (nm)
Sample name and abbreviation
60.01
0
3.8
37.4
19.4
184 ± 11
SiO2; SiO2
–
–
74
10.1
+39.8
17.6
227 ± 18
–
–
–
27
3.7
43.3
18.3
192 ± 13
0.09 ± 0
0.02
–
–
25
2.6
18.8
19.3
198 ± 10
0.315 ± 0.007
–
–
–
37
5.2
17.4
18.8
220 ± 23
SiO2-3 Aminopropane; SiO2-NH2 SiO2-pentane-3,5dicarboxylic acid; SiO2COOH SiO2-Propane-3-sulfonic acid; SiO2-SO3H SiO2-Benzyl; SiO2-Benz
0.145 ± 0.007
–
–
0.12
40
4.4
24.8
19.1
192 ± 7
0.28 ± 0
–
0.1
–
39
4.4
22.6
19.1
193 ± 10
C
S
P
Cl
60.03
60.003
60.003
0.265 ± 0.007
–
0.23 ± 0
testing ready biodegradability under aerobic conditions. The CBT is a screening test with low bacterial density (approximately 104–106 cells L1) and low concentration of the test substance corresponding to 5–10 mg L1 theoretical oxygen demand (ThOD). It can be assumed that substances classified as ‘‘readily biodegradable’’ are biodegradable in sewage treatment plants and therefore do not reach or accumulate in the aquatic environment (Nyholm, 1991). The biodegradation of the test substance, which is the only carbon source for the microorganisms, was monitored by using the biological oxygen demand over 28 d as end-point. The test was prepared in accordance with the OECD 301 D guideline (1992) at room temperature (20 ± 1 °C), at a pH of 7.4 ± 0.2, in the dark and in duplicates. The biodegradation test was performed twice and four different test series were included. All bottles contained the same mineral salt medium prepared according to the guideline and were inoculated with two drops of an effluent from a municipal sewage treatment plant (250 000 population equivalents) in Lüneburg (Germany). The first series, referred as ‘‘blank’’, contained only inoculum and mineral salt medium. The ‘‘quality control’’ was additionally prepared with readily biodegradable sodium acetate in a concentration corresponding to 5 mg L1 ThOD without considering a possible nitrification and was performed to monitor the activity of the microorganisms. The ‘‘test series’’ included one type of surface functionalized SiO2 particles as only organic compound while the ‘‘toxicity control’’ series contained additionally sodium acetate each in a concentration of 5 mg L1 ThOD. According to the test guideline a biodegradation of at least 25% based on total ThOD within 14 d is required in the toxicity control so that toxic effects of the test substance against the microorganisms can be excluded. The ThOD for the functional groups was calculated for 1 g of each particle type, based on the carbon amount of the functionalized particles as declared in Table 1. The biodegradation of the reference and the test substances were monitored by the biological oxygen demand of the microorganisms during the whole test at desired intervals using sensor spots in the bottles and an oxygen electrode (Oxi 196 with EO 196-1.5 WTW Weilheim, Germany). The test is only valid if the biodegradation value of the quality control has reached more than 60% after 14 d. Further validity criteria are: the difference between the duplicates must be less than 20%; the oxygen consumption in the inoculum blank should not exceed 1.5 mg L1 after 28 d and the residual concentration of oxygen in the test vessels should not fall below 0.5 mg L1 at any time. A test compound is classified as ‘‘readily biodegradable’’ if biodeg-
SiO2-3-Chloropropane; SiO2-Cl SiO2-Ethane-2diethylphos-phonate; SiO2-DPEt
radation exceeds 60% within a 10-d window after the oxygen consumption reached 10% ThOD (OECD, 1992).
3. Results and discussion 3.1. Physicochemical and morphological properties of surface functionalized particles Table 1 shows the physicochemical properties of the seven different silica particles used in this study. The surface functionalization is confirmed by the significantly increased carbon content after surface functionalization, as well as by the presence of specific elements such as chlorine, sulfur, and phosphorous for SiO2-3-Chloropropane, SiO2-Propane-3-sulfonicacid and SiO2Ethane-2-diethylphosphonate, respectively. The carbon concentration was transferred into an average surface density of functional groups in lmol g1 which were found to be between 25 and 74 lmol g1 and below a theoretical monolayer of silane molecules expected at 160 lmol g1 (Bartholome et al., 2003; Meder et al., 2012). The slight variation of the final functional group surface densities despite constant precursor concentration might rise from the different chemical composition and functional end group of the precursor molecules, which influence the precursor deposition (Bartholome et al., 2003). In particular, electrostatic attraction or repulsion, respectively between the pre cursor functional end groups (e.g. ANHþ 3 or ASO3 ) and the negatively charged silica surface can be supportive/unsupportive mechanism in the functionalization process (Xu et al., 1997). The silane precursors bind via their silanol groups to the hydroxyl groups of the SiO2 particles. Thereby the functional group is in general exposed to the particle surface and it was suggested not to interact with the particle surface (Plueddemann, 1991). However, interactions between the functional groups and the particle surface, e.g., between the positively charged amino group and the negatively charged silica surface, cannot be completely excluded, which may influence their bioavailability (Vandenberg et al., 1991). Nevertheless, the isoelectric point (IEP) and the zeta potential clearly indicated a variation of the particle surface chemistry in correlation to the exposure of the functional groups on the particle surface and properties i.e. the acid dissociation constants of the functional groups (Meder et al., 2012). The IEP of SiO2-3-Aminopropane yielded 10.1 and is close to the acid dissociation constant pKa (pKa = 10.6) of the alkylamino group (Meder et al., 2012) and, in addition, the positive zeta potential of these particles strongly
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decreased the IEP of the SiO2 particles as expected from their acidic character (pKa = 1.5 and 4.1, respectively) (Meder et al., 2012). The hydrophobic surface groups introduced on SiO2-Benzyl, SiO2-3-Chloropropane, and SiO2-Ethane-2-dietehylphosphonate can neither be deprotonated nor protonated as function of the pH and are itself not expected to change the zeta potential and the IEP significantly. Nevertheless, the slight increase of the IEP as well as the decrease of the zeta potential for these particles when compared to native SiO2, may indicate the reduction of the hydroxyl groups of SiO2 during the binding of the precursor molecules, which determine the IEP and zeta potential of native SiO2. Changes of the particle size and morphology after surface functionalization were not detected as specific surface area, particle size (d50), transmission electron microscopy (TEM), and high resolution TEM (HR-TEM) images of SiO2 and SiO2-3-Aminopropane (Fig. 1a–d) and do not show significant differences after surface functionalization. Nitrogen adsorption isotherms indicate a mesoporosity of the SiO2 particles, which was not significantly influenced by the surface functionalization (Fig. 1e). Altogether while the particle morphology is unchanged, the zeta potential and the IEP measurements as well as the elemental analyzes confirm the variation of the particle surface chemistry and the attachment of the functional molecules. 3.2. Ready biodegradability of surface functionalized particles
Fig. 1. (a and b) TEM images of non-functionalized particles SiO2 and amino propyl functionalized SiO2 (SiO2-NH2). (c and d) HR-TEM images of non-functionalized SiO2 and SiO2-NH2 particle surface. The surface functionalization does not significantly vary the particle size and shape. Nitrogen adsorption isotherms do not show any significant variation of the particles mesoporosity (2–50 nm pores) after surface functionalization.
indicates the availability of positively charged amino groups (ANHþ 3 ) on the particle surface. Sulfonate and carboxyl groups
In accordance to the OECD, all tests fulfilled the above-mentioned criteria of validity. Moreover, none of the tested particle was toxic against the microorganisms in the inoculum as shown by a biodegradation value in all toxicity controls of at least 33.15% (SiO2-Ethane-2-diethylphosphonate particles) after 14 d. Table 2 shows the biodegradation results of the functionalized SiO2 particles. As expected, the SiO2 particles alone were not readily biodegradable due to their inorganic nature. In addition, none of the surface functionalizations on the SiO2 particles reached the biodegradability threshold of 60%, irrespective of the type of the organic group. The highest biodegradation was measured for SiO2-Benzyl and SiO2-3-Chloropropane with 13.7 ± 6.7% and 10.8 ± 1.5%, respectively. In contrast, all the other functionalized particles showed no biodegradation (values below 5% are due to background and natural fluctuations). With regard to other biodegradation studies, our data show that biodegradability of the organic moiety is heavily influenced by their attachment to the particle surface. Some of the obtained results were in accordance with established general rules used for structure-biodegradation prediction of organic molecules (Loonen et al., 1999; Boethling et al., 2007). It is known that increasing chlorination reduce biodegradability. We have found that the presence of one chlorine atom in a short alkyl chain is still biodegradable (10.8 ± 1.5%). In contrast no biodegradation was measured for SiO2-Pentane3,5-dicarboxylicacid. This is contrary to the statements in the literature for the molecules not attached to particles. Herein structure-biodegradation models predicted that carboxyl groups favor biodegradability (Loonen et al., 1999). Furthermore, data from previous CBTs of structurally related substances with carboxyl groups such as valproic acid showed a ready biodegradability with values of 72.7% in the test vessels and 77.9% in the toxicity control (test concentration of 2.4 mg L1 [corresponding to 5 mg L1 ThOD]; K. Kümmerer and coworkers, unpublished results). But in another study with COOH-functionalized multi-walled carbon nanotubes no biodegradation was reported (Kümmerer et al., 2011). This supports the assumption that the attachment to the particle surface hinders the biodegradability of the organic groups. In our study SiO2-SO3H particles were also not biodegradable, while for substances containing sulfonate residues e.g. linear
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Table 2 Test concentrations of the organically functionalized particles and the comparison of their biodegradation values with those of non-bound substances.
a b c d
Sample name
Carbon amount by elemental analysis (mg g1)
Test concentration CBT (mg L1)
Corresponding ThODa (mg L1)
Biodegradation (% ThODa) (n = 4)
Biodegradation of non-bound chemicals (% ThODa)
SiO2 SiO2-NH2 SiO2-COOH SiO2-SO3H SiO2-Benz SiO2-Cl SiO2-DPEt
0.30 2.65 0.90 0.90 3.25 1.45 2.80
726.4 1527.7 842.4 1362.0 753.3 1360.9 703.8
0.0 15.3 5.7 5.2 7.9 8.3 8.1
0 2.8 ± 1.9 2.9 ± 2.6 0.4 ± 5.2 13.7 ± 6.7 10.8 ± 1.5 2.2 ± 1.9
– n.a.b 72.7 (valproic acid) 50.0 (alkylbenzene sulfonatec) 93.0 (toluene d) n.a.b n.a.b
Theoretical oxygen demand. No data available. Sütterlin et al., 2008. Lapertot and Pulgarin, 2006.
alkylbenzene sulfonate, a slight biodegradation (50%) was reported (Sütterlin et al., 2008). Particles functionalized with benzyl groups showed only a slight biodegradation of 13.7 ± 6.7 in contrast to 93% biodegradation of toluene molecules not attached to the particle surface (Lapertot and Pulgarin, 2006). Taken together, the organic residues attached on the particle surface were not or only low biodegradable in comparison to structurally very similar compounds not functionalized on the particle surface. In other words, the SiO2 functionalities seem to be prohibitive for biodegradation. In general bioavailability may play an important role for biodegradability. However, biodegradation may also be mediated by so called exoenzymes which are present outside of the bacterial cell. Here the work should be intensified to get more insight into the microbial biodegradation processes. In this context, it should be considered that we did not include the agglomeration behavior of the particles in our observations. The agglomeration can have a strong effect on the biodegradability, because it can influence the particle size, their bioavailability as well as the accessibility of the functional groups. A further parameter is the stability of the surface functionalization although hydrolysis processes are unlikely because silane functionalizations on SiO2 particles were assumed to be mainly stable in the aqueous environments (Xu et al., 1997; Szczepanski et al., 2006). Further studies will be necessary to investigate how particle characteristics, such as particle agglomeration, dissolution of particles and detachment of surface functionalization, accessibility of the functional groups at the particle surface, chemical structure of the functionalizations on the particle surface and their binding to the particle surface influence the biodegradability of the organic residues. Additional tests, e.g. for inherent biodegradability, are required to get a better data basis for the future design of particles which should be fast and fully degradable when they reach the aquatic environment. 4. Conclusion The physicochemical properties of six differently surface functionalized SiO2 particles were characterized in detail and it was revealed that none of the surface functionalized particles was readily biodegradable. The results are a first hint that at least ready biodegradable chemicals reduce their biodegradability when engineered on the surface of sub-micron or nanoparticles. Therefore, it can be assumed that they are available in the aquatic environment for a longer period than their non-bound counterparts with an increased risk to environmental species. Acknowledgements We thank Ms. Evgenia Logunova and Ms. Natalie Höhn for the performance of the CBT. The work was funded by the European Re-
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Nyholm, N., 1991. The european system of standardized legal tests for assessing the biodegradability of chemicals. Environ. Toxicol. Chem. 10 (10), 1237–1246. http://dx.doi.org/10.1002/etc.5620101002. OECD (Organization of Economic Cooperation and Development), 1992. Guideline for Testing Chemicals, Test guideline 301 D: Ready Biodegradability – Closed Bottle Test. OECD, Paris. Plueddemann, E.P., 1991. Silane Coupling Agents. Springer, Berlin Heidelberg. Sütterlin, H., Alexy, R., Coker, A., Kümmerer, K., 2008. Mixtures of quaternary ammonium compounds and anionic organic compounds in the aquatic environment: elimination and biodegradability in the closed bottle test monitored by LC–MS/MS. Chemosphere 72 (3), 479–484. http://dx.doi.org/ 10.1016/j.chemosphere.2008.03.008. Szczepanski, V., Vlassiouk, I., Smirnov, S., 2006. Stability of silane modifiers on alumina nanoporous membranes. J. Membr. Sci. 281 (1–2), 587–591. http:// dx.doi.org/10.1016/j.memsci.2006.04.027. Vandenberg, E.T., Bertilsson, L., Liedberg, B., Uvdal, K., Erlandsson, R., Elwing, H., Lundström, I., 1991. Structure of 3-aminopropyl triethoxy silane on silicon oxide. J. Colloid Interface Sci. 147 (1), 103–118. http://dx.doi.org/10.1016/ j.memsci.2006.04.02. Xu, Z., Liu, Q., Finch, J., 1997. Silanation and stability of 3-aminopropyl triethoxy silane on nanosized superparamagnetic particles: I. Direct silanation. Appl. Surf. Sci. 120 (3–4), 269–278. http://dx.doi.org/10.1016/S0169-4332(97)00234-1.
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Physicochemical properties and biodegradability of organically functionalized colloidal silica particles in aqueous environment Mandy Schneider a, Fabian Meder b, Annette Haiß a, Laura Treccani b, Kurosch Rezwan b, Klaus Kümmerer a,⇑ a b
Institute of Sustainable and Environmental Chemistry, Leuphana University of Lüneburg, Scharnhorststraße 1, DE-21335 Lüneburg, Germany Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, D-28359 Bremen, Germany
h i g h l i g h t s Investigation of the biodegradability of organically surface functionalized silica. None of the functionalized particles met the biodegradability threshold value. Biodegradability of organic groups was reduced compare to unbound chemicals. Increased residence time of attached organic groups in the aquatic environment.
a r t i c l e
i n f o
Article history: Received 7 June 2013 Received in revised form 7 October 2013 Accepted 13 October 2013 Available online 8 November 2013 Keywords: Organic functionalization Colloidal silica particles Biodegradation Physicochemical characteristics Environmental fate
a b s t r a c t Engineered sub-micron particles are being used in many technical applications, leading to an increasing introduction into the aquatic environment. Only a few studies have dealt with the biodegradability of non-functionalized organic particles. In fact the knowledge of organically surface functionalized colloids is nearly non-existent. We have investigated the biodegradability of organically surface functionalized silica (SiO2) particles bearing technically relevant groups such as amino-, carboxyl-, benzyl-, sulfonate-, chloro-, and phosphatoethyl-derivatized alkyls. Essential physicochemical properties including zeta potential, isoelectric point, morphology, surface area, porosity, surface density, and elemental composition of the particles were investigated, followed by biodegradability testing using the Closed Bottle Test (OECD 301D). None of the particles met the biodegradability threshold value of 60%. Only a slight biodegradation was revealed for SiO2-Benzyl (13.7 ± 6.7%) and for SiO2-3-Chlorpropane (10.8 ± 1.5%). For the other particles biodegradability was below the normal background fluctuation of 5%. The results were different of those obtained from structurally similar chemicals not being functionalized on the particle surface and from general rules of structure-biodegradation prediction of organic molecules. Therefore, our results suggest that the attachment of the organic groups heavily reduces their biodegradability, increases their residence time and possibility for adverse effects to environmental species. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Engineered sub-micron particles (ESPs), like colloids and nanoparticles are becoming a key component for a variety of technical applications. Consequently, over the next decades the introduction of ESP in the environment via industrial processes and consumer products will increase, while their environmental fate is still largely unknown. Colloidal silica (SiO2) particles are widely used in paints and food technology, cosmetics, as well as environmental and biomedical applications (Duguet et al., 2009; Knopp et al.,
⇑ Corresponding author. Tel.: +49 4131 677 2894; fax: +49 4131 677 2848. E-mail addresses: [email protected] (M. Schneider), meder@ uni-bremen.de (F. Meder), [email protected] (A. Haiß), [email protected] (L. Treccani), [email protected] (K. Rezwan), [email protected] (K. Kümmerer). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.10.031
2009; Dickinson, 2012). They are promising adsorbents for the removal of pathogens or potentially toxic substances from wastewater (Andrzejewska et al., 2007; Majewski, 2007; Cademartiri et al., 2010; Majewski and Keegan, 2012)l or employed as drug-delivery vehicles and imaging vectors in biomedical fields (Knopp et al., 2009). For these applications, the particles can be surface functionalized with organic end groups to receive tailored physicochemical surface properties. Organosilane molecules, containing charged functional groups such as positively charged amino groups, or negatively charged carboxyl and sulfonate groups, are often used to electrostatically stabilize the particles in a specific medium (Duguet et al., 2009; Bertazzo and Rezwan, 2010; Meder et al., 2012). Moreover, the attached end groups can provide reactive groups for example for bioconjugation of proteins and drugs or for catalysis (Gill et al., 2007; Hermanson, 2008). Hydrophobic functional groups stabilize particles in hydrophobic media
M. Schneider et al. / Chemosphere 99 (2014) 96–101
(e.g. oil–water emulsions) and provide interaction sites for polymers and biomolecules. Therefore, the functional groups can essentially influence charge, aggregation and stability of the particles in aqueous environments. These parameters can also strongly influence the biodegradability of the organically functionalized particles. An important point for the assessment of the risk connected to the introduction of chemicals into the environment is knowledge on biodegradation. Compounds which are not completely mineralizable will remain as mother compound or transformation products in the environment with an increasing possibility for adverse effects to environmental species in soils and waters. Furthermore for organically functionalized particles biodegradation is of special interest, because it will alter the properties such as water solubility or bioavailability of such particles. To the best of our knowledge, no studies have been published that investigate in parallel the physicochemical properties and the ready biodegradability of organically modified SiO2 colloids. Generally only a few publications are available studying the biodegradability of different types of ESPs (Nowack and Bucheli, 2007; Brar et al., 2010; Hartmann et al., 2011; Kümmerer et al., 2011). For example, some biodegradation was reported for starch, cellulose nanoparticles and their macroscopic counterparts and no biodegradability was found for functionalized and non-functionalized fullerenes as well as carbon nanotubes (Kümmerer et al., 2011). The lack of data for the environmental fate and biodegradability makes it impossible to set limit values of engineered (nano) particles in surface waters (Baun et al., 2009) and to conduct a risk assessment. This is not only an increasing problem for ‘‘Registration, Evaluation and Authorization and Restriction of Chemicals’’ (REACH), but also for the ‘‘Water Framework Directive’’ (WFD) which should guarantee to maintain a good chemical and ecological status of surface waters. Therefore the aim should be a fast and full (bio-) degradation and mineralization of ESPs including their surface functionalities after their release into the environment. Such data can then be used for a proper, sustainable design of new compounds (Boethling et al., 2007; Kümmerer, 2007; Anastas, 2008) which would help to avoid typical follow-up problems and guarantee a greater acceptance (Kümmerer et al., 2011). According to the principles of green (Anastas, 2008), sustainable chemistry, and pharmacy (Kümmerer, 2007) the focus of interest in this study was to investigate the biodegradation of organically surface functionalized SiO2 particles. Consequently we focused on technically highly relevant alkylamino, alkylcarboxyl, alkylsulfonate, benzyl-, alkylchloro- and phosphoatoethyl- functionalizations (Table 1) which are widely used to adjust surface charges of SiO2 particles and the rheological properties of their suspensions. Furthermore, they provide important reactive groups for adsorption and conjugation mechanisms in biomedical, biotechnological and environmental applications. After the surface functionalization the particles were characterized and the ready biodegradability was analyzed with the Closed Bottle Test (CBT), which is a test also suitable for poorly soluble substances.
2. Materials and methods 2.1. Surface functionalization of particles Silica particles with a narrow size distribution (Angströmsphere™, purity > 99.9%, lot No. 100624-04, d50 = 184 nm) were purchased from Fibre Optics Center Inc., USA and calcinated at 800 °C for 4 h to remove any organic residues from the manufacturing process. The precursors for the surface functionalization were 3-aminopropyltriethoxysilane (99% in ethanol, Lot. 92196EJ-109, Sigma–Aldrich, Germany), 3-(triethoxysilyl)propylsuccinicanhydride (>94%, Lot. 1034945), 3-(trihydroxysilyl)-1-
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propanesulfonic acid (30–35% in H2O, Lot. 1070635), benzyltriethoxysilane (97%, Lot. 1106735), 3-(chloropropyl)triethoxysilane (97%, Lot. 1070827), diethylphosphatoethyltriethoxysilane (92%, Lot. 1089841) from ABCR GmbH, Germany. For all experiments double deionized water with a conductivity of 0.04 lS cm1 obtained from a SynergyÒ apparatus (Millipore, Germany) was used. The surface functionalization was conducted as described by Meder et al. (2012). In short, suspensions of 15 g silica particles in 50 mL water were sonicated for 10 min with an ultrasound horn (Branson SonifierÒ 450, USA, output: 150 W, pulse rate: 0.5 s) to break the agglomerates before precursor addition. The precursor solutions were prepared by mixing it with 50 mL water to a final concentration of 0.146 M (corresponding to 486 lmol per gram of the unmodified silica particles). The precursor solution were then added to the silica suspensions, stirred for 60 min at 40 °C and then heat treated at 120 °C for 90 min. Afterwards the particles were centrifuged at 1500g for 30 min and washed three times in 100 mL water to remove any unbound precursors. Particles were freeze dried for 120 h at 20 °C in vacuum using a freeze dryer (P8KE-80, Piatkowski Forschungsgeräte, Germany) and subsequently heat treated at 70 °C for 2 h to induce the evaporation of potentially remaining ethanol of the silane precursors and used for further experiments. 2.2. Physicochemical characterization of functionalized particles Elemental analysis was carried out by Mikrolabor Pascher, Germany. Phosphor and sulfur concentrations were analyzed after acid pressure hydrolysis of the samples via ICP-AES in an ICAP 6500 (Thermo, Germany). Chloride concentration was determined in an ion chromatograph 761 Compact IC (Metrohm, Germany) after combustion. Carbon concentrations were measured by conductometric carbon dioxide determination after combustion in a selfmade apparatus (Mikrolabor Pascher, Germany). The average and maximal error were calculated from two different measurements. Nitrogen adsorption isotherms were recorded at 196 °C with an automated surface area analyzer BELSORP-mini (Bel Inc., Japan). Specific surface areas were calculated from the adsorption isotherms using the Brunauer, Emmet, and Teller (BET) equation (Brunauer et al., 1938) porosity and pore size distribution were determined using the model of Barrett et al. (1951). All samples were gassed out at 120 °C under vacuum for 3 h before the measurement. The particle size was measured by dynamic light scattering (DLS) using an Ultrafine Particle Analyzer (UPA 150, Microtrac Inc., USA) in water (pH 7.4 ± 0.3 and ionic strength of 14 mM KCl) at a particle content of 0.1 vol% to assure a reliable signal. Average and standard deviation were calculated from 9 single measurements of the d50 (intensity), representative particle size distribution curves were revealed. Zeta potential measurements were carried out using an electroacoustic spectrometer (DT1200, Dispersion Technology Inc., USA). Zeta potential/pH-titrations were performed with the integrated titration unit using 1 M HCl and 1 M KOH respectively. All measurements were conducted with suspensions containing 1 vol% of particles. TEM and HR-TEM were recorded using a Titan 80-300 ST microscope (FEI™, the Netherlands) equipped with a Cs-corrector for the imaging lens using a 300 kV electron beam and a vacuum at 1.3107 mbar. 200 mesh cupper grids S162 covered with a formvar film (PLANO GmbH, Germany) were used as sample holders. 2.3. Biodegradability testing of functionalized particles In accordance with the Organization of Economic Cooperation and Development (OECD) 301D guideline, the CBT was used for
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Table 1 Scheme of particle functional surface groups and physicochemical characteristics: Elemental composition, surface density, surface area, particle size, isoelectric point, zeta potential (at pH 7.4 and an ionic strength of 14 mM). Scheme of functionalized particles
Elemental analysis (wt%)
Surface density (lmol g1)
IEP
Zeta potential pH 7.4; I = 14 mM (mV)
Surface area (m2 g1)
Particle size d50 (nm)
Sample name and abbreviation
60.01
0
3.8
37.4
19.4
184 ± 11
SiO2; SiO2
–
–
74
10.1
+39.8
17.6
227 ± 18
–
–
–
27
3.7
43.3
18.3
192 ± 13
0.09 ± 0
0.02
–
–
25
2.6
18.8
19.3
198 ± 10
0.315 ± 0.007
–
–
–
37
5.2
17.4
18.8
220 ± 23
SiO2-3 Aminopropane; SiO2-NH2 SiO2-pentane-3,5dicarboxylic acid; SiO2COOH SiO2-Propane-3-sulfonic acid; SiO2-SO3H SiO2-Benzyl; SiO2-Benz
0.145 ± 0.007
–
–
0.12
40
4.4
24.8
19.1
192 ± 7
0.28 ± 0
–
0.1
–
39
4.4
22.6
19.1
193 ± 10
C
S
P
Cl
60.03
60.003
60.003
0.265 ± 0.007
–
0.23 ± 0
testing ready biodegradability under aerobic conditions. The CBT is a screening test with low bacterial density (approximately 104–106 cells L1) and low concentration of the test substance corresponding to 5–10 mg L1 theoretical oxygen demand (ThOD). It can be assumed that substances classified as ‘‘readily biodegradable’’ are biodegradable in sewage treatment plants and therefore do not reach or accumulate in the aquatic environment (Nyholm, 1991). The biodegradation of the test substance, which is the only carbon source for the microorganisms, was monitored by using the biological oxygen demand over 28 d as end-point. The test was prepared in accordance with the OECD 301 D guideline (1992) at room temperature (20 ± 1 °C), at a pH of 7.4 ± 0.2, in the dark and in duplicates. The biodegradation test was performed twice and four different test series were included. All bottles contained the same mineral salt medium prepared according to the guideline and were inoculated with two drops of an effluent from a municipal sewage treatment plant (250 000 population equivalents) in Lüneburg (Germany). The first series, referred as ‘‘blank’’, contained only inoculum and mineral salt medium. The ‘‘quality control’’ was additionally prepared with readily biodegradable sodium acetate in a concentration corresponding to 5 mg L1 ThOD without considering a possible nitrification and was performed to monitor the activity of the microorganisms. The ‘‘test series’’ included one type of surface functionalized SiO2 particles as only organic compound while the ‘‘toxicity control’’ series contained additionally sodium acetate each in a concentration of 5 mg L1 ThOD. According to the test guideline a biodegradation of at least 25% based on total ThOD within 14 d is required in the toxicity control so that toxic effects of the test substance against the microorganisms can be excluded. The ThOD for the functional groups was calculated for 1 g of each particle type, based on the carbon amount of the functionalized particles as declared in Table 1. The biodegradation of the reference and the test substances were monitored by the biological oxygen demand of the microorganisms during the whole test at desired intervals using sensor spots in the bottles and an oxygen electrode (Oxi 196 with EO 196-1.5 WTW Weilheim, Germany). The test is only valid if the biodegradation value of the quality control has reached more than 60% after 14 d. Further validity criteria are: the difference between the duplicates must be less than 20%; the oxygen consumption in the inoculum blank should not exceed 1.5 mg L1 after 28 d and the residual concentration of oxygen in the test vessels should not fall below 0.5 mg L1 at any time. A test compound is classified as ‘‘readily biodegradable’’ if biodeg-
SiO2-3-Chloropropane; SiO2-Cl SiO2-Ethane-2diethylphos-phonate; SiO2-DPEt
radation exceeds 60% within a 10-d window after the oxygen consumption reached 10% ThOD (OECD, 1992).
3. Results and discussion 3.1. Physicochemical and morphological properties of surface functionalized particles Table 1 shows the physicochemical properties of the seven different silica particles used in this study. The surface functionalization is confirmed by the significantly increased carbon content after surface functionalization, as well as by the presence of specific elements such as chlorine, sulfur, and phosphorous for SiO2-3-Chloropropane, SiO2-Propane-3-sulfonicacid and SiO2Ethane-2-diethylphosphonate, respectively. The carbon concentration was transferred into an average surface density of functional groups in lmol g1 which were found to be between 25 and 74 lmol g1 and below a theoretical monolayer of silane molecules expected at 160 lmol g1 (Bartholome et al., 2003; Meder et al., 2012). The slight variation of the final functional group surface densities despite constant precursor concentration might rise from the different chemical composition and functional end group of the precursor molecules, which influence the precursor deposition (Bartholome et al., 2003). In particular, electrostatic attraction or repulsion, respectively between the pre cursor functional end groups (e.g. ANHþ 3 or ASO3 ) and the negatively charged silica surface can be supportive/unsupportive mechanism in the functionalization process (Xu et al., 1997). The silane precursors bind via their silanol groups to the hydroxyl groups of the SiO2 particles. Thereby the functional group is in general exposed to the particle surface and it was suggested not to interact with the particle surface (Plueddemann, 1991). However, interactions between the functional groups and the particle surface, e.g., between the positively charged amino group and the negatively charged silica surface, cannot be completely excluded, which may influence their bioavailability (Vandenberg et al., 1991). Nevertheless, the isoelectric point (IEP) and the zeta potential clearly indicated a variation of the particle surface chemistry in correlation to the exposure of the functional groups on the particle surface and properties i.e. the acid dissociation constants of the functional groups (Meder et al., 2012). The IEP of SiO2-3-Aminopropane yielded 10.1 and is close to the acid dissociation constant pKa (pKa = 10.6) of the alkylamino group (Meder et al., 2012) and, in addition, the positive zeta potential of these particles strongly
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decreased the IEP of the SiO2 particles as expected from their acidic character (pKa = 1.5 and 4.1, respectively) (Meder et al., 2012). The hydrophobic surface groups introduced on SiO2-Benzyl, SiO2-3-Chloropropane, and SiO2-Ethane-2-dietehylphosphonate can neither be deprotonated nor protonated as function of the pH and are itself not expected to change the zeta potential and the IEP significantly. Nevertheless, the slight increase of the IEP as well as the decrease of the zeta potential for these particles when compared to native SiO2, may indicate the reduction of the hydroxyl groups of SiO2 during the binding of the precursor molecules, which determine the IEP and zeta potential of native SiO2. Changes of the particle size and morphology after surface functionalization were not detected as specific surface area, particle size (d50), transmission electron microscopy (TEM), and high resolution TEM (HR-TEM) images of SiO2 and SiO2-3-Aminopropane (Fig. 1a–d) and do not show significant differences after surface functionalization. Nitrogen adsorption isotherms indicate a mesoporosity of the SiO2 particles, which was not significantly influenced by the surface functionalization (Fig. 1e). Altogether while the particle morphology is unchanged, the zeta potential and the IEP measurements as well as the elemental analyzes confirm the variation of the particle surface chemistry and the attachment of the functional molecules. 3.2. Ready biodegradability of surface functionalized particles
Fig. 1. (a and b) TEM images of non-functionalized particles SiO2 and amino propyl functionalized SiO2 (SiO2-NH2). (c and d) HR-TEM images of non-functionalized SiO2 and SiO2-NH2 particle surface. The surface functionalization does not significantly vary the particle size and shape. Nitrogen adsorption isotherms do not show any significant variation of the particles mesoporosity (2–50 nm pores) after surface functionalization.
indicates the availability of positively charged amino groups (ANHþ 3 ) on the particle surface. Sulfonate and carboxyl groups
In accordance to the OECD, all tests fulfilled the above-mentioned criteria of validity. Moreover, none of the tested particle was toxic against the microorganisms in the inoculum as shown by a biodegradation value in all toxicity controls of at least 33.15% (SiO2-Ethane-2-diethylphosphonate particles) after 14 d. Table 2 shows the biodegradation results of the functionalized SiO2 particles. As expected, the SiO2 particles alone were not readily biodegradable due to their inorganic nature. In addition, none of the surface functionalizations on the SiO2 particles reached the biodegradability threshold of 60%, irrespective of the type of the organic group. The highest biodegradation was measured for SiO2-Benzyl and SiO2-3-Chloropropane with 13.7 ± 6.7% and 10.8 ± 1.5%, respectively. In contrast, all the other functionalized particles showed no biodegradation (values below 5% are due to background and natural fluctuations). With regard to other biodegradation studies, our data show that biodegradability of the organic moiety is heavily influenced by their attachment to the particle surface. Some of the obtained results were in accordance with established general rules used for structure-biodegradation prediction of organic molecules (Loonen et al., 1999; Boethling et al., 2007). It is known that increasing chlorination reduce biodegradability. We have found that the presence of one chlorine atom in a short alkyl chain is still biodegradable (10.8 ± 1.5%). In contrast no biodegradation was measured for SiO2-Pentane3,5-dicarboxylicacid. This is contrary to the statements in the literature for the molecules not attached to particles. Herein structure-biodegradation models predicted that carboxyl groups favor biodegradability (Loonen et al., 1999). Furthermore, data from previous CBTs of structurally related substances with carboxyl groups such as valproic acid showed a ready biodegradability with values of 72.7% in the test vessels and 77.9% in the toxicity control (test concentration of 2.4 mg L1 [corresponding to 5 mg L1 ThOD]; K. Kümmerer and coworkers, unpublished results). But in another study with COOH-functionalized multi-walled carbon nanotubes no biodegradation was reported (Kümmerer et al., 2011). This supports the assumption that the attachment to the particle surface hinders the biodegradability of the organic groups. In our study SiO2-SO3H particles were also not biodegradable, while for substances containing sulfonate residues e.g. linear
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Table 2 Test concentrations of the organically functionalized particles and the comparison of their biodegradation values with those of non-bound substances.
a b c d
Sample name
Carbon amount by elemental analysis (mg g1)
Test concentration CBT (mg L1)
Corresponding ThODa (mg L1)
Biodegradation (% ThODa) (n = 4)
Biodegradation of non-bound chemicals (% ThODa)
SiO2 SiO2-NH2 SiO2-COOH SiO2-SO3H SiO2-Benz SiO2-Cl SiO2-DPEt
0.30 2.65 0.90 0.90 3.25 1.45 2.80
726.4 1527.7 842.4 1362.0 753.3 1360.9 703.8
0.0 15.3 5.7 5.2 7.9 8.3 8.1
0 2.8 ± 1.9 2.9 ± 2.6 0.4 ± 5.2 13.7 ± 6.7 10.8 ± 1.5 2.2 ± 1.9
– n.a.b 72.7 (valproic acid) 50.0 (alkylbenzene sulfonatec) 93.0 (toluene d) n.a.b n.a.b
Theoretical oxygen demand. No data available. Sütterlin et al., 2008. Lapertot and Pulgarin, 2006.
alkylbenzene sulfonate, a slight biodegradation (50%) was reported (Sütterlin et al., 2008). Particles functionalized with benzyl groups showed only a slight biodegradation of 13.7 ± 6.7 in contrast to 93% biodegradation of toluene molecules not attached to the particle surface (Lapertot and Pulgarin, 2006). Taken together, the organic residues attached on the particle surface were not or only low biodegradable in comparison to structurally very similar compounds not functionalized on the particle surface. In other words, the SiO2 functionalities seem to be prohibitive for biodegradation. In general bioavailability may play an important role for biodegradability. However, biodegradation may also be mediated by so called exoenzymes which are present outside of the bacterial cell. Here the work should be intensified to get more insight into the microbial biodegradation processes. In this context, it should be considered that we did not include the agglomeration behavior of the particles in our observations. The agglomeration can have a strong effect on the biodegradability, because it can influence the particle size, their bioavailability as well as the accessibility of the functional groups. A further parameter is the stability of the surface functionalization although hydrolysis processes are unlikely because silane functionalizations on SiO2 particles were assumed to be mainly stable in the aqueous environments (Xu et al., 1997; Szczepanski et al., 2006). Further studies will be necessary to investigate how particle characteristics, such as particle agglomeration, dissolution of particles and detachment of surface functionalization, accessibility of the functional groups at the particle surface, chemical structure of the functionalizations on the particle surface and their binding to the particle surface influence the biodegradability of the organic residues. Additional tests, e.g. for inherent biodegradability, are required to get a better data basis for the future design of particles which should be fast and fully degradable when they reach the aquatic environment. 4. Conclusion The physicochemical properties of six differently surface functionalized SiO2 particles were characterized in detail and it was revealed that none of the surface functionalized particles was readily biodegradable. The results are a first hint that at least ready biodegradable chemicals reduce their biodegradability when engineered on the surface of sub-micron or nanoparticles. Therefore, it can be assumed that they are available in the aquatic environment for a longer period than their non-bound counterparts with an increased risk to environmental species. Acknowledgements We thank Ms. Evgenia Logunova and Ms. Natalie Höhn for the performance of the CBT. The work was funded by the European Re-
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