R Mailler

J Gasperi
email: gasperi@u-pec.fr

D Patureau

E Vulliet

N Delgenes

A Danel

S Deshayes

V Eudes

S Guerin

R Moilleron

G Chebbo

V Rocher

Fate of emerging and priority micropollutants during the sewage sludge treatments: case study of Paris conurbation. Part 1: Contamination of the different types of sewage sludges, condensed and centrifuged waters

Keywords: Emerging pollutants, priority substances, sewage sludge, sludge treatment, residual water

    Fate of emerging and priority micropollutants during the sewage sludge treatment: Case study of Paris conurbation. Part 1: Contamination of the different types of sewage sludge

INTRODUCTION

The presence of micropollutants in wastewater has been well demonstrated in the literature [START_REF] Heberer | Occurrence, fate, and removal of pharmaceutical residues in 23 the aquatic environment: a review of recent research data[END_REF][START_REF] Loos | EU-wide monitoring survey on emerging polar organic contaminants in 57 wastewater treatment plant effluents[END_REF][START_REF] Luo | A review on the occurrence of micropollutants in the aquatic 2 environment and their fate and removal during wastewater treatment[END_REF][START_REF] Verlicchi | Occurrence of pharmaceutical 23 compounds in urban wastewater: Removal, mass load and environmental risk after a 24 secondary treatment-A review[END_REF]. A wide range of these compounds, particularly hydrophobic pollutants, are eliminated during primary and biological treatments in wastewater treatment plants (WWTP), through sorption to sewage sludges [START_REF] Clara | Occurrence of selected 31 surfactants in untreated and treated sewage[END_REF]Mailler et al., 2014c;[START_REF] Ruel | Occurrence and fate of relevant substances in wastewater treatment 55 plants regarding Water Framework Directive and future legislations[END_REF]. This results in the contamination of sewage sludges by various priority and emerging pollutants, such as alkylphenols (APs), phthalates (PAEs), polycyclic aromatic hydrocarbons (PAHs), pharmaceuticals and hormones (PPHs) or organotins [START_REF] Bergé | Meta-analysis of environmental contamination by alkylphenols[END_REF][START_REF] Bergé | Meta-analysis of environmental contamination by phthalates[END_REF][START_REF] Clarke | Review of 'emerging' organic contaminants in 37 biosolids and assessment of international research priorities for the 38 agricultural use of biosolids[END_REF][START_REF] Mailler | Priority and emerging 8 pollutants in sewage sludge and fate during sludge treatment[END_REF].

The production of sewage sludges is estimated to be about 11 million tons of dry matter (DM) in Europe, including 1 million for France only [START_REF] Kelessidis | Comparative study of the methods used 37 for treatment and final disposal of sewage sludge in European countries[END_REF]. These sludges are treated and conditioned before to be land farmed, incinerated or landfilled [START_REF] Fytili | Utilization of sewage sludge in EU application 55 of old and new methods-A review[END_REF]. In France (> 70% of the DM mass), as well as in Europe in general (> 50%), the main pathway for sludge management is land farming. In this context, the presence of micropollutants in sludge is of concern, especially considering the transfer from sludge to soil of some pollutants such as metals [START_REF] Chipasa | Accumulation and fate of selected heavy metals in a 29 biological wastewater treatment system[END_REF], organotins [START_REF] Craig | Organometallic compounds in the environment[END_REF] or polychlorobiphenyls (PCBs) (Stevens et al., 2002). To limit contamination of the environment by micropollutants, European (EC, 1986) and national (French order of 8 th January of 1998) regulations have been established to progressively forbid sludge disposal and regulate land farming. Such regulations concern principally heavy metals, PAHs and PCBs. In particular, the Urban Wastewater Treatment Directive (EC, 1986), amended by (91/271/EEC) (EC, 1991), states maximum thresholds and maximum annual flux to land farm for metals. Moreover, present regulations are currently discussed and other compounds could possibly be added to the watch lists.

However, the studies displaying micropollutant contents in sewage sludge are still scarce compared to those for wastewater, in particular for emerging compounds such as PPHs or perfluorinated acids (PFAs). In addition, the impact of sludge treatments on micropollutant concentrations is difficult to evaluate considering that most studies do not distinguish different types of sludge from a given sludge treatment plant (STP); they just aimed at characterizing the contamination of raw and/or final treated sludge disregarding the type of sludge treatment. Finally, data about French sludge are very partial since only a dozen references are today available.

In this context, the Paris public sanitation service (SIAAP), which treats wastewater and sewage sludge from about 9 million inhabitants, and the Water Environment and Urban Systems laboratory (LEESU) have decided to assess the contamination of Parisian sewage sludges by micropollutants and their fate during sludge treatments. This project aims at i) featuring micropollutant contaminations of the different types of sludges and ii) assessing the efficiency of 4 sludge treatments to remove/decrease these contaminations. Therefore, raw sludge, centrifuged sludge, digested sludge, thermally dried sludge and sludge cake were studied and centrifugation, anaerobic digestion, thermal drying and cooking + press filtration were considered.

The first part of this project was mainly focused on priority pollutants and pesticides and a paper was recently published on both sludge contamination and treatments. Results shown a partial removal of alkylphenols, di(2-ethylhexyl)phthalate (DEHP), organotins and PAHs by sludge treatments, in contrary to metals and PCBs which accumulate through treatment. In addition, this work revealed that pesticides and volatile organic products were absent from Parisian sludges (< LOD), or present at very low contents (< LOQ).

The second part of this project is focused on new or emerging pollutants by investigating the fate of a total of 70 compounds, including 58 new molecules, i.e. PPHs, PFAs, APs, PAEs or linear alkyl benzene sulfonates (LAS), and 12 molecules in common with the phase 1 of the project.

In addition to objectives established for phase 1, the phase 2 also aims at establishing mass balances at the scale of sludge treatments, i.e. centrifugation, digestion, thermal drying and sludge cake process, to have both a large (impact of the treatment at STPs scale) and a process engineering view (removals and transfers at processes scale). Therefore, micropollutants in condensed water from thermal drying and centrifuged water from centrifugation were investigated too, in order to precisely identify the transfer pathways of each pollutant. Such data are among the first to be published.

Results obtained from the second part of this project are divided in two papers. The removals and transfer pathways of micropollutants are assessed in the 2 nd part of this article; this 1 st part aims at describing the contamination of the different types of Parisian sewage sludges by emerging micropollutants and comparing it to the literature. Therefore, a mini review of available data is furnished. In addition, this present article aims at i) evaluating the quality of the different types of sludges commonly encountered regarding emerging pollutants and ii) assessing their possible transfer via centrifuged and condensed water.

MATERIAL AND METHODS

Sludge treatment plants (STPs) description

The three STPs monitored are the same as described in [START_REF] Mailler | Priority and emerging 8 pollutants in sewage sludge and fate during sludge treatment[END_REF]) -Seine Aval, Seine Centre and Seine Grésillons. They are supervised by SIAAP and treat sludge produced by three WWTPs fed with wastewater from the same catchment (downstream Paris conurbation). The characteristics of each WWTP and STP are given in supporting material -Table S1, as well as the complete layout of each STP (supporting material -Table S2).

The Seine Centre plant treats 240,000 m 3 of wastewater per day. Sludge produced is first centrifuged to achieve a volume reduction, resulting in a production of almost 21,000 tons DM of centrifuged sludge per year (SIAAP source). Then, sludge is incinerated producing ash and smoke, which is specifically treated to minimize odors. The Seine Aval plant receives 1,700,000 m 3 of wastewater per day (biggest in Europe) and produces more than 55,000 tons DM of treated sludge per year (SIAAP source). The first sludge treatment consists in a mesophilic (37°C) anaerobic digestion to transform an important part of organic matter (about 40%) into biogas and eliminating pathogens and parasites. Digested sludge is then dewatered by thickening, thermal conditioning (heat exchange and cooking at 195°C and 20 bars) and press filtration. These successive treatments allow reducing sludge volume/mass/loads by more than a factor 10 (i.e. DM, Table 1) and producing a dewatered cake called sludge cake which is used as agricultural fertilizer. The Seine Grésillons plant treats 100,000 m 3 of wastewater per day. Sludge treatment is performed by centrifugation and then thermal drying. The thermal drying process can operate at a wide range of temperature, but the facility used in this plant operates at a high temperature (260°C) compared to conventional dryers (generally 105°C [START_REF] Voulvoulis | Fate of organotins in sewage sludge during 26 anaerobic digestion[END_REF]). This allows reducing drastically the water content (Table 1) to obtain, after compacting, almost 8,000 tons DM of solid pellets per year (SIAAP source) which are stocked in big bags or silos before to be used in agriculture.

Sampling strategy

Different types of sludges have been monitored within the three studied STPs, as described in supporting material -Table S2: raw sludge (RS), centrifuged sludge (CS), digested sludge (DS), thermally dried sludge (TS) and sludge cake (SC). In addition, centrifuged (CW) and condensed water (TDW) were also analyzed to determine if a transfer occurs from sludge to water. The sampling strategy allows assessing both the contamination of final sludges and the fate of emerging micropollutants within STPs.

Sampling campaigns were performed in April-May 2013 and April 2014. Each sludge sampling point (supporting material -Table S2) was sampled seven times except the inlet and outlet of thermal drying which were sampled six times. Samples were all punctual due to technical issues, except sludge cake which is a composite sample of sludge produced within a week, respecting all the guidelines to avoid sample contamination. In addition, the sampled volume was 3 L for RS, CS and DS while it was 2 L for TS and SC. For digestion, DS was sampled 16 days after RS, to take the sludge retention time (SRT) into account. CW and TDW samples were also punctual and performed simultaneously with sludge samples in centrifugation and thermal drying.

General sludge quality parameters

The general quality of each type of sampled sludge, characterized by dry matter (DM, in % -1% = 10 g/L) and volatile matter (VM, in % DM), is displayed in Table 1 with min -max values and mean. Both parameters are commonly used in sludge management. In addition, total suspended solids (TSS) were measured in condensed and centrifuged waters (Table 1). Overall, removals of dry matter and volatile matter during anaerobic digestion are about 40% and 54% respectively, in good agreement with conventional anaerobic digestion removal [START_REF] Mailler | Priority and emerging 8 pollutants in sewage sludge and fate during sludge treatment[END_REF][START_REF] Moletta | Méthanisation de la biomasse (Biomass methanisation). 14 Techniques de l'ingénieur[END_REF]. This is due to the biotransformation of organic matter to methane. VM in sludge cake is low (38.5% DM) compared to digested sludge highlighting a removal of organic matter during the process (thermal conditioning + press filtration, supporting material -Table S2). This is most likely due to the solubilization of a fraction of the organic matter [START_REF] Neyens | A review of thermal sludge pre-treatment 28 processes to improve dewaterability[END_REF][START_REF] Valo | Thermal, chemical and thermo-20 chemical pre-treatment of waste activated sludge for anaerobic digestion[END_REF] during thermal conditioning (195°C and 20 bars). This fraction is then removed with water during press filtration. In contrary, VM is rather constant in both thermal drying and centrifugation, highlighting that VM is whether not or similarly removed (no change in percentage) as DM during these treatments. The general quality parameters show that Seine Aval STP is the only one in the studied STPs to significantly reduce the quantity of volatile matter in sludge (not considering incineration).

Regarding CW and TDW, the TSS concentrations indicate that thermal drying performs a far more efficient separation of dry matter and water, as the concentration of TSS in TDW do not exceed 100 mg/l. TSS concentrations are 5,000 times lower than in sludge entering the process.

CW present significant higher TSS concentration (831-1,488 mg/l), about 40 times lower than RS.

Considering the higher amount of particles in CW this could lead to transfers of pollutants from the sludge to the wastewater, as CW and TDW are reinjected in raw wastewater of the WWTPs.

Pollutants and analytical procedures

A total of 70 emerging micropollutants have been analyzed in all sampled sludges, including 58 new compounds and 12 in common with first part of the project. Table 2 gives the groups of pollutants and the main information about their respective analytical methods. Depending on the compound, three laboratories were involved in analyses: the Institute of Analytical Sciences (CNRS -Villeurbanne, France) for pharmaceuticals and perfluorinated compounds, the laboratory of Environmental Biotechnology (INRA -Narbonne, France) for hormones, LAS, alkylphenols, PAHs and PCBs, and the laboratory of the Police Prefecture of Paris (LCPP -Paris, France) for phthalates and octylphenol (OP). The complete list of monitored compounds and their limits of detection (LOD) and quantification (LOQ) are given in supporting material -Table S3. For the compounds analyzed by ISA, sludge samples are first lyophilized, grinded and sieved at 250 µm. Then, for tetracyclines and fluoroquinolones, a pressurized liquid extraction is performed with a mix of acetonitrile/methanol/nitric acid (20:20:80, v:v) on a part of the sample (0.5 g). The extract (20 mL) is mixed with ultrapure water (480 mL) before to be purified by solid phase extraction (SPE) on an Autotrace® device with StrataX® cartridges, eluted with 6 mL of methanol. In contrary, for perfluorinated acids and the rest of pharmaceuticals, a QuEChERS extraction is performed on a part of the sample (2 g) with EDTA, heptane and acetate buffer. Then, a purification step is performed by SPE. Both parts of the sample are then analyzed by LC-MSMS.

For the compounds analyzed by LBE, sludge samples are also lyophilized and grinded, before to be centrifuged. The sample is extracted by accelerated solvent extraction with hexane/acetone (50:50, v:v) for alkylphenols, PAHs and PCBs, methanol for LAS and methanol/acetone (50:50, v:v) for hormones. The extract is purified by SPE for hormones analysis. Finally, the three fractions of the sample are analyzed by LC-FLD for LAS, alkylphenols and PAHs, LC-MS for hormones and GC-ECD for PCBs.

For OP and phthalates, samples were frozen during 72 hours at a temperature of -18°C. After lyophylization, about 1 g of sample was extracted by sonication in 20 mL of mixture of methanol/dichloromethane (10:90, v:v). This step was repeated twice to ensure complete extraction of the compounds. Both extracts were then purified on an alumina column (1 g), previously conditioned with 6 mL of dichloromethane. The extracts were then concentrated under a stream of nitrogen to a final volume of 1 mL. The concentrate was taken up in 1 mL of mixture of methanol and dichloromethane containing the internal standards. All chemicals and solvents were pesticide residue grade. OP and phthalates were analyzed by GC-MS (simple Quad, Agilent Technologies). Pharmaceuticals and hormones (PPHs)

PPHs are compounds that are not expected to be sorbed to sewage sludge a priori, considering their hydrophilic nature. However, most of PPHs monitored were detected in each type of sludge. The PPHs contents for each type of sludge considered are provided in Figure 1. Statistical data such as Fischer, Student and Mann-Whitney tests, to compare homogeneity of RS and CS from STP to STP, are provided in supporting material -Table S6. Occurrences of the compounds are also furnished in supporting materials -Table S4.

In raw sludges (Figure 1) and based on all RS samples (14 samples), all PPHs monitored were detected. Moreover, acetaminophen, carbamazepine, ciprofloxacin, domperidone, escitalopram, lidocaine, norfloxacin, ofloxacin, propranolol, tramadol and verapamil have a frequency of detection higher than 50%, while the remaining compounds are detected scarcely (occurrence < 50%). Fluoroquinolones (ofloxacin, ciprofloxacin and norfloxacin) are predominant in sludge with high contents (> 3,000 µg/kg DM) while other PPHs lay in the 10-1000 µg/kg DM range. For these latter compounds, ciprofloxacin has the highest content, with a median value of 10,285 µg/kg DM. Sulfamethoxazole, acetaminophen or lidocaine are found in median around 100 µg/kg DM.

In addition, a statistical analysis performed on RS (N=14) demonstrated that PPHs contents are not significantly different (p-value > 0.05 -confidence level of 95%) between Seine Aval and Seine Centre (Fischer, Student and Mann-Whitney tests, see supporting material -Table S6). This indicates that sewage sludge is quite homogenous in Paris, despite different wastewater treatment processes. This is consistent as the different WWTPs studied treat wastewater from the same catchment. A similar trend is observed between CS from Seine Centre and Seine Grésillons. Finally, DEHP, APs, LAS and PAHs also have comparable contents in RS from Seine Aval and Seine Centre and in CS from Seine Centre and Seine Grésillons (supporting material -Table S6).

In treated sludges (CS, DS, TS and SC), the number of compounds detected and the occurrence are lower than in RS. The fluoroquinolones (n=3) and carbamazepine are the only compounds to be still frequently detected in all treated sludges.

In TS and SC, which are both final sludges that are actually used as agricultural amendment, most compounds are measured at lower contents than in RS. For instance, acetaminophen and ciprofloxacin were respectively measured at a median RS content of 90.9 and 12,858 µg/kg DM against 47.6 and 6,077 µg/kg DM in TS, and 65.4 and 7,438 µg/kg DM in SC. This highlights the positive impact of the STP on micropollutant contamination of the sludge, although they were not designed for that.

However, some compounds have similar or higher contents in TS or SC compared to RS. For instance, carbamazepine, domperidone, escitalopram, ofloxacin and propranolol have similar contents in TS and RS. Ofloxacin has also a comparable content in SC while loratadine is much more concentrated in cake (1,360 µg/kg DM -median) than in RS (38 µg/kg DM). These recalcitrant compounds are whether similarly or less removed than dry matter during the sludge treatment process, leading sometimes to content increase, even if the mass flux has actually decreased. The part 2 of this article will focus more particularly on the mass balance and transfer during treatment.

In DS, acetaminophen, carbamazepine, miconazole and fluoroquinolones have similar or higher contents than RS, resulting from a low biodegradability (no biodegradation or weaker than the dry matter). The remaining compounds are not or scarcely (< 3 times) detected, displaying a notable impact of this process on sludge contamination (content decrease).

In CS, no transformation of the compounds can theoretically occur as this process consists in a simple physical separation of sludge and water. However, most PPHs (n=10) have lower contents in CS comparatively to RS, indicating a removal. In contrary, azithromycin, domperidone, glybencyclamide, loratadine, miconazole, and fluoroquinolones have similar or higher contents in CS.

Finally, comparing DS and SC allows having an idea of the impact of cooking and press filtration on sludge contamination. A reduction of contamination is observed for several compounds, i.e. acetaminophen, carbamazepine, miconazole and the three fluoroquinolones. This reduction should be due to solubilization and removal of water [START_REF] Neyens | A review of thermal sludge pre-treatment 28 processes to improve dewaterability[END_REF][START_REF] Valo | Thermal, chemical and thermo-20 chemical pre-treatment of waste activated sludge for anaerobic digestion[END_REF]. Moreover, acetaminophen and carbamazepine are also removed during centrifugation. In contrary, the other pharmaceuticals are rather accumulated as their contents are higher after cooking and press filtration.

The variability of contents is high for most pharmaceuticals in RS with coefficients of variability (CV) between 50% and 150% (based on 14 RS samples), as a probable result of the variability of concentrations in wastewater. In treated sludge, the variability remains high, especially in CS (33-200%) and TS (25-170%), except for digestion which seems to have a buffer effect (6-100%).

Regarding the literature (supporting material -Table S3), several works have displayed their propensity to be present in the particulate fraction thanks to adsorption mechanisms (Carballa et In contrary, no or very few data were found for domperidone, escitalopram, glybencyclamide, ivermectine, lidocaine, loratadine, miconazole, tramadol and verapamil, whatever the type of sludge.

1 Perfluorinated acids (PFAs) Similarly to pharmaceuticals, the variability of content is relatively high for perfluorinated 13 compounds with CV between 60 and 120%. The variability for these compounds is similar in all 14 kind of sludges. Regarding the Parisian sewage sludge (Figure 2; supporting material -Table S4), the 13 monitored 30

PAHs

PAHs were detected in all analyzed samples. The PAHs pattern is similar in the different sludges

31

(Figure 2), even if some differences of contents can be observed from sludge to sludge. Thus, In the literature, most studies found these compounds at comparable contents, especially in

Alkylphenols (APs)

1 2

For APs, nonylphenols (NP), octylphenol (OP), nonylphenol monoethoxylate (NP1EO) and 3 diethoxylate (NP2EO), as well as the sum of analyzed nonylphenols ethoxylates (Σ NPEOs), were 4 monitored. OP, NP and NP1EO were detected in every sludge sample, contrary to NP2EO with an 5 occurrence below 50% in CS, TS and SC. In RS, NP, NP1EO and NP2EO had a comparable median content, respectively 1,380, 1,720 and 8 940 µg/kg DM (Figure 3). While the repartition and the contents are similar to RS in CS and TS,

9
this is slightly different for DS and SC. In DS and SC, the three compounds have a higher median 10 content (4,520, 3,380 and 1,300 µg/kg DM). This increase of contents results from dry matter and 11 water removals. OP has a comparable content whatever the sludge, with a median content varying 12 between 190 and 609 µg/kg DM depending on the sludge.

14

The variability of content observed is from relatively low to moderate for these compounds, with 15 CV of 20-60%. This is surprising for DS taking their biodegradability into account and the fact that NP and nonylphenols ethoxylates in sewage sludge have been well studied in the literature,

21
particularly in treated sludge (supporting material -Table S5). Contents available are rather 22 variable, even for one type of sludge, and the contamination of Parisian sludge is in the lower range.

23

In particular, several studies measured these compounds at much higher contents than what is

LAS 29 30

LAS analyses include analyses of C10, C11, C12 and C13 congeners, as well as the sum of LAS 31 content. LAS, whatever the chain length (C10-C13), were always detected in all types of sludge 32 (Figure 3). These compounds are measured at very high contents in sludge compared to all the 33 other emerging micropollutants, i.e. about 100-10,000 mg/kg DM.

35

In RS, C10 is always significantly less concentrated than the others with a median content of 193 The variability of contents is low for this group of compounds, with CV between 25 and 35% in RS, 47 25-50% in CS, 20-25% in DS and 10-40% in TS. In SC, this variability is even lower (< 15%).

49

LAS are studied for a couple of decades in sewage sludges and the literature available is quite large (supporting material -Table S5). About 20 references were found. In addition, the contents 12 13

Contents measured in RS for PCBs (supporting material -Table S4) lie in the 10-2,000 µg/kg DM For PPHs, acetaminophen, azithromycin, lidocaine, propranolol and tramadol were systematically

14
detected in CW (Table 3). In addition, domperidone, fluoroquinolones and verapamil, as well as 15

PFAs, were detected at least ones. These molecules were quantified at relatively low concentrations

16

(5-2,300 ng/L), in the same range as APs and PAHs, except fluoroquinolones and azithromycin 17 which are measured between 2,000 and 6,000 ng/L.

19

Regarding the partitioning of compounds in CW, three cases are encountered. First, acetaminophen, 20 lidocaine and tramadol were only detected in the dissolved phase. It could be assumed that these 21 compounds were initially present in the dissolved phase in RS and not sorbed to dry matter. This is 22 consistent considering their hydrophilic properties (log KOW < 2.5). The contamination of CW for 23 them could be hence due to their high affinity for dissolved phase. Finally, the remaining detected compounds were found in both dissolved and particulate phase.

31

Thus, for PFA, azithromycin, domperidone, propranolol, OP and PAEs, the contamination of CW is 32 due to both presence in water and TSS lost. 33 34 In TDW, regarding the low concentration of TSS ( The results from TDW confirm that a very efficient separation of particles and water (thermal 41 drying), doesn't lead to a complete prevention of the micropollutant transfers with the residual 42 waters, as a fraction of several pollutants is transferred with the dissolved phase.

44

These results about micropollutants in residual waters from sludge treatment are the first published The contamination of various types of untreated and treated sewage sludges by a large panel of 3 emerging pollutants (n=70) has been investigated in this paper. Parisian sludges, from two WWTPs 4 treating wastewater from the same catchment, were considered in this paper, which represents 15% 5 of the total mass (DM) of yearly sludge produced in France. 6 7 This study presents among the first data for several pharmaceutical compounds (PPHs), such as PFAs, displaying a good propensity to be eliminated (more than dry matter). These results have to 25 be confirmed by mass balances and flux calculations, and are the subject of a second article.

27

Then, this paper also presents interesting data of micropollutants in residual waters from two

  molecules: LAS = linear alkyl benzene sulfonates; PAHs = polycyclic aromatic hydrocarbons; PCBs = polychlorobiphenyls. b Number of compounds per group. c Methods used for extraction-purification-analysis. LG = lyophilized and ground; PLE = pressurized liquid extraction; SPE = solid phase extraction; ASE = accelerated solvent extraction; S = sonication; AC = alumina column purification; LC = liquid chromatography; GC = gas chromatography; GC-ECD = GC with pulsed flame photometric detector; d Laboratories involved and bibliographic reference for the analytical method. e Limits of detection in µg/kg DM.

  More information about the analytical methods used in this article are given in (Patureau et al., 2012) for LAS, nonylphenols, PCBs and PAHs, (Muller et al., 2010) for hormones, (Peysson and Vulliet, 2013) for pharmaceuticals and PFAs. RESULTS AND DISCUSSION 1.

Figure 1 .

 1 Figure 1. Pharmaceuticals and PFAs contents in Parisian sewage sludges 2

23 24 PAHs

 24 are cited in French sludge regulations (order of 8 th January of 1998). In this text, threshold 25 values are stated for fluoranthene (5,000 µg/kg DM), benzo[b]fluoranthene (2,500 µg/kg DM) and 26 benzo[a]pyrene (2,000 µg/kg DM), limiting the use of contaminated sludge for agricultural

25 28 PAHs. 29 1Figure 2 .

 28292 Figure 2. PAHs and PAEs contents in Parisian sewage sludges 2

16 NP1EO

 16 and NP2EO can be degraded in NP during such treatment (Ejlertsson et al., 1998; Lu et al., 17 2008). Moreover, very variable removals were observed previously on the same facilities (Mailler 18 et al., 2014b), inducing a much higher variability of content in DS, SC and TS.

24 observed

 24 in this study[START_REF] Aparicio | Limitation of the concentration of 47 organic pollutants in sewage sludge for agricultural purposes: A case study in 48 South Spain[END_REF]; Ghanem et al., 2007; González et al., 2010; Samaras et 25 al., 2013), while others at comparable levels (Jones et al., 2014; Mailler et al., 2014b; Nie et al., 26 2009). For OP, no comparison was found in the literature.

  , PAHs, APs, fluoroquinolones and verapamil were only detected in the particulate 26 phase, what is consistent as they are hydrophobic (log KOW > 4) or have a great affinity for sorption 27 on particles (Byrns, 2001; Gasperi et al., 2010; Golet et al., 2003; Zhou et al., 2013). For these 28 compounds, the contamination of CW is due to the fraction of TSS lost during centrifugation.

45 and

 45 are required to understand well the micropollutant transfers in STPs. A deeper analysis of the

8

  domperidone, lidocaine or tramadol. It also contributes to complete the database about sludge 9 contamination for different groups of interesting pollutants (perfluorinated acids, phthalates, PAHs, 10 LAS, alkylphenols and PCBs).

14 ( 1 -

 141 predominant pollutants with contents ranging from few dozens to several thousands of 13 mg/kg DM. They are by far more concentrated than DEHP (10-100 mg/kg DM), fluoroquinolones 100 mg/kg DM) and alkylphenols (APs; 2-20 mg/kg DM). Finally, the remaining compounds 15 (PPHs, PFAs, PAHs, PCBs and PAEs) are less concentrated in sludges, with contents varying 16 between 10 and 1000 µg/kg DM. In addition, a statistical analysis demonstrated the homogeneity of 17 contents for most compounds in raw sludge from Seine Aval and Seine Centre, and in centrifuged 18 sludge from Seine Centre and Seine Grésillons.

19 20 A

 1920 simple and quick assessment of sludge contents allows highlighting the accumulation of some 21 compounds in treated sludge, such as LAS, APs, PAHs, DEHP and PCBs. The increase of content 22 results from dry matter and water removal coupled with a resistance of these compounds to 23 treatment. In contrary, some compounds have lower contents in treated sludges, such as PPHs and24

28 important

 28 sludge treatment processes: centrifugation and thermal drying. The presence of different 29 types of molecules at relatively high concentrations in CW and TDW highlights the transfers that 30 occur within the dissolved phase and the particles released from sludge during these processes. An 31 extended investigation of this issue is realized in a second article, performing a complete analysis of 32 removals and mass flux calculations.

  This study has been conducted with the OPUR research program and the SIAAP supports. Authors 38 would like to thank the SIAAP, LBE and ISA technical teams for their active participation in 39 sampling and analyses.

Table 1 . Dry matter and volatile matter content of the studied sludges

 1

			Digestion		Thermal drying	Centrifugation	Cake
		RS	DS	Removal	CS	TS	RS	CS	SC
	DM	3.5 -4.6	2.1 -2.4	34% -50%	21.7 -47.7	77.9 -95.2	4.1 -5.1	24.3 -28.9	48.8 -57.5
	(%)	3.9	2.3	40%	29.7	89.9	4.7	25.9	52.1
	VM (% DM)	71.0 -80.3 75.9	57.4 -60.8 59.0	46% -60% 54%	51.0 -75.9 64.4	50.3 -75.8 63.6	69.2 -78.5 75.6	70.7 -80.3 77.7	37.3 -40.0 38.5
	TSS (mg/L)				Condensed water (TDW) Centrifuged water (CW) 8 -92 831 -1,488 57 1,220

Table 2 . Pollutants and analytical methods Groups a Total b Methods c Reference d LOQ e

 2

	Pharmaceuticals Perfluorinated acids	18 2	LG -PLE or QuEChERS -SPE -(LC-MSMS)	ISA (Peysson and Vulliet, 2013)	1 -50 4 and 30
	Hormones	4	LG -ASE -SPE -(LC-MS)	LBE	??
	LAS PAHs	4	LG -ASE -(LC-FLD)	(Patureau et al., 2012)	40
				(Muller et al.,
				2010)

  al., 2004;[START_REF] Giger | Occurrence and fate of antibiotics as trace 8 contaminants in wastewaters, sewage sludges, and surface waters[END_REF] Lindberg et al., 2005a). Some pharmaceuticals were monitored in sewage sludge in different countries and the data available are rather large. Some compounds are well documented such as acetaminophen, azithromycin, carbamazepine, fluoroquinolones, propranolol or sulfamethoxazole but not for all types of sludge and without a process engineering point of view. Azithromycin, when detected, was found at lower contents in RS than former studies(Clarke and Smith, 2011; Göbel et al., 2005). In contrary, ciprofloxacin (Golet et al., 2003; Li et al., 2013), ofloxacin (Jones et al., 2014), carbamazepine (Nieto et al., 2010), sulfamethoxazole (Li et al., 2013; Nieto et al., 2010) and propranolol (Jones et al., 2014; Nieto et al., 2010) are rather more concentrated in RS from this study, because of a higher contamination of wastewater and/or more efficient treatments in WWTP resulting in a greater sorption. In treated sludges, data from the literature are in accordance with what is observed in Paris for acetaminophen, azithromycin,

carbamazepine, propranolol and sulfamethoxazole (Clarke and Smith, 2011; Harrison et al., 2006; Narumiya et al., 2013; Peysson and Vulliet, 2013; Radjenović et al., 2009; Subedi et al., 2014), but fluoroquinolones are more concentrated than in former studies (Golet et al., 2003; Jia et al., 2012; Lindberg et al., 2005b).

  PFAs are emerging pollutants with a growing interest. They are classified as priority substances

	1
	2
	3
	4	since 2013 (EC, 2013). Similarly to fluoroquinolones, PFOS was always detected in all sludges
	5	contrary to PFOA with a lower occurrence (<50%). PFOA was never detected in TS, and only 2
	6	times in SC and DS. Their sludge contents are in the range of those from pharmaceuticals (Figure
	7	1). In RS, the median content of PFOA and PFOS are respectively 26 and 316 µg/kg DM. For both
	8	compounds, the contents measured in the different treated sludges were lower, indicating a partial
	9	removal during treatments, even during centrifugation. Thus, PFOS was measured at a median
	10	content of 293 µg/kg DM in CS, 49 µg/kg DM in DS, 55 µg/kg DM in TS and 157 µg/kg DM in
	11	SC.
	12

  [START_REF] Aparicio | Limitation of the concentration of 47 organic pollutants in sewage sludge for agricultural purposes: A case study in 48 South Spain[END_REF]) but higher contents were found in digested sludge from UK Thus, this molecule was measured at a median content of 41,500 µg/kg DM in RS, 13 70,600 µg/kg DM in CS, 58,100 µg/kg DM in DS, 90,200 µg/kg DM in TS and 68,300 µg/kg DM 14 in SC. BBP, DEP and DnBP are found in the range as PAHs, with median content of 44.9-

	2
	3
	4	4.	Phthalates (PAEs)
	5
	6	PAEs were only monitored in 2014 (n=3-7 campaigns, depending on the STP). 4 compounds were
	7	monitored including the di(2-ethylhexyl) phthalate (DEHP), benzylbutyl phthalate (BBP), di-n-
	8	butyl phthalate (DnBP) and diethyl phthalate (DEP). The 4 compounds were found in all samples
	9	from RS, CS, DS and TS but BBP was not detected in SC while DnBP and DEP were only detected
	10	once in this sludge.
	11
	12	DEHP is present at much higher contents in Parisian sludge than the three other compounds
	(Figure 2). 23
	24
	39
	40
	41	The variability of content observed for PAHs is moderate (50% < CV < 100%) in RS except for
	42	dibenzo[ah]anthracene for which it is high (CV=114%). Sludge treatments reduce this variability
	43	and the coefficients of variability of all PAHs are below 50% in DS, TS and SC.
	44
	45	The RS contents observed in Paris are lower than in China (Cai et al., 2007; Zeng et al., 2010),
		Korea (Ju et al., 2009) and UK (Jones et al., 2014) for all PAHs. Overall, the contents found in

32

anthracene and dibenzo[a]anthracene have significantly lower contents than the other PAHs, always 33 lower than 100 µg/kg DM. In contrary, phenanthrene, fluoranthene and pyrene are always the most 34 concentrated PAHs in sludge, with contents higher than 100 µg/kg DM. PAHs are rather more 35 concentrated in treated sludges, especially SC, as already observed in a former study (Mailler et al., 36 2014b). Concerning TS and SC, managed through land farming, the three regulated compounds 37 were never measured above the threshold values. Indeed, these molecules were always measured 38 below 800 µg/kg DM, whatever the sludge. 46 Parisian treated sludge in this study (sampling in 2013-2014) are very close from those found in 47 2011 (Mailler et al., 2014b). Furthermore, another recent French study measured comparable 48 contents in various French STPs (Besnault et al., 2015 (submitted)). This suggests that PAHs 49 contamination of sewage sludge is rather homogenous in France. The results are also comparable to those from (1 (Stevens et al., 2002). 15 144.6 µg/kg DM for DEP, 351.9-1,141.0 µg/kg DM for BBP and 109.3-290.6 µg/kg DM for DnBP.

16

The contents in DS and SC are always lower or similar than in RS for BBP, DEP and DnBP, 17 highlighting a removal close or higher than dry matter. In contrary, DEHP accumulates in sludge 18 throughout the treatment process, resulting in an increase of the content. 19 20

For PAEs, RS and CS are the only sludge with enough samples (n=6-7) to estimate the variability of 21 content. The latter is low (CV < 50%) in both sludges, except for BBP which is more variable (55-22 80%).

  Similarly to PAHs, the LAS pattern doesn't change with the sludge treatment; the 38 LAS proportions are stable, indicating that the 4 compounds are similarly impacted by the 39 treatments. The pattern observed is in accordance with most of former studies[START_REF] Aparicio | Limitation of the concentration of 47 organic pollutants in sewage sludge for agricultural purposes: A case study in 48 South Spain[END_REF]

	36
	37	mg/kg DM, while C11, C12 and C13 have close median contents of respectively 1,171, 1,484 and
	982 mg/kg DM. 40
	41	Carballa et al., 2007; Muller et al., 2007) with a predominance of C10 in comparison to C11, C12
	42	and C13. Moreover, LAS contents are rather similar or higher in treated sludge compared to RS,
	43	indicating that they are not notably eliminated by the processes or at least less than the dry matter.
	44	For instance, the median Σ LAS content varies from 4,053 mg/kg DM in RS to 6,224 mg/kg DM in
		CS, 5,161 mg/kg DM in DS, 7,400 mg/kg DM in TS and 8,821 mg/kg DM in SC.

1

  observed are rather variable from study to study, from a mean value of total LAS of 10.7 mg/kg DM 2 in China[START_REF] Zeng | Levels and 28 distribution patterns of short chain chlorinated paraffins in sewage sludge of 29 wastewater treatment plants in China[END_REF] to more than 10,000 mg/kg DM in UK[START_REF] Holt | Linear alkylbenzenes in sewage sludges and 26 sludge amended soils[END_REF] and Greece[START_REF] Pakou | On the fate of 39 LAS, NPEOs and DEHP in municipal sewage sludge during composting[END_REF]. However, most studies provided data that are consistent with what is 4 observed in Paris. Furthermore, no data were found for centrifuged and thermally dried sludge for

	3
	5
	6	this group of compounds.
	7
	8	7.	PCBs
	9
	10	PCBs (n=20) were only monitored in 2013 (N=3-4 campaigns). Similarly to PAHs, French
	11	regulation stated a threshold value (800 µg/kg DM) for the sum of 7 PCBs (PCB 28, 52, 101, 118,
		138, 153 and 180).

APs and LAS contents in Parisian sewage sludges

  14 range for individual PCBs. This range is similar to the one for PPHs, PFAs and PAHs. The 7 PCBs 15 from the French regulation are those with the highest contents, leading to a median value of the sum 16 of 518 µg/kg DM, what is below the French threshold value. They represent 60-75% of the total 17PCBs content in RS. In addition, PCB 101 is the only congener with a median content higher than An increase of PCBs content in sludge is observed during sludge treatments, with higher contents 22 observed in DS, CS and SC than in RS. For instance, PCB 101 median contents in CS, DS and SC 23 are respectively 132, 203 and 368 µg/kg DM. SC is the sludge with the highest PCB contents 24 resulting from the reduction of the mass of sludge in digestion and cooking followed by press 25 filtration. This has already been observed within the same STPs (Mailler et al., 2014b). Thus, the Σ All micropollutants monitored in sludges, except PCBs, were also monitored in 2014 in the

	1
	2	8.	Micropollutant concentrations in CW and TDW
	3
	4
	5	centrifuged (CW) and condensed (TDW) waters from centrifugation and thermal drying to identify
	6	possible transfer pathways.
	7
	8	Most pollutants (36 on the 50 monitored) were detected in CW except several PPHs, including
	9	carbamazepine, miconazole or sulfamethoxazole. LAS, APs, PAHs and PAEs were detected in all
	10	samples. Similarly to sludge, LAS are measured at very high concentrations, lying in the 50,000-
	11	250,000 ng/L range. DEHP, DEP and DnBP concentrations are also high (1,000-12,500 ng/L),
	12	while BBP, APs and PAHs are quantified at notably lower concentrations (1-1,000 ng/L).
	13
	18
	19	100 µg/kg DM (106 µg/kg DM), while other congeners 18, 33, 105, 128, 170, 187, 194, 195, 199,
	20	206 and 209 were measured at a median content below 20 µg/kg DM.
	21
	26
	27	7 PCBs content reaches a median value of 2,503 µg/kg DM in SC, what is significantly higher than
	28	the threshold value, and the total PCBs content reaches 3,368 µg/kg DM. In contrary, a decrease of
	29	content is observed for all PCBs after thermal drying. This phenomenon is stronger for the lighter
	30	congeners. This results in lower PCBs contents in TS than in the other sludges, in particular RS. In
	31	this type of sludge, the Σ 7 PCBs and Σ 20 PCBs median contents are respectively 304 and 523
	32	µg/kg DM.
	33
	34	Similarly to APs, PCBs are more concentrated in Parisian sludge than in the other studies available
	35	(Abad et al., 2005; Gibson et al., 2005; Ju et al., 2009; Muller et al., 2007; Stevens et al., 2002). In
	36	contrary, (Blanchard et al., 2004) measured the Σ 7 PCBs in French treated sludge between 120 and
	37	1,930 µg/kg DM. This could suggest a decrease of sludge contamination by these compounds in ten
	38	years, as it was previously observed in France for metals (Mailler et al., 2014a) or in Sweden for
	39	some polybromodiphenylethers and norfloxacin (Olofsson et al., 2012).
	40
	1
	Figure 3. 2
	3
	4
	5
	6

Table 1 )

 1 , only dissolved concentrations were

	36
	37	azithromycin (350-3000 ng/L), lidocaine (64-178 ng/L), tramadol (184-2315 ng/L) and verapamil
	38	(<LOQ-301 ng/L) were detected in TDW samples. In addition, NP (3400-5350 ng/L), OP (732-
	39	2133 ng/L) and PAEs (67-30119 ng/L) were also detected in all TDW samples.
	40

35

assessed. PAHs, LAS and most PPHs were not quantified in dissolved phase of TDW. However

Table 3 . Concentrations measured in centrifuged (CW) and condensed (TDW) water in 2014 campaigns 1

 3

	Pollutant	CW 1 Total concentration (ng/L) CW 2 CW 3	TDW 1 Dissolved concentration (ng/L) TDW 2 TDW 3
	Acetaminophen	233	274	424	<LOQ	<LOQ	<LOQ
	Azithromycin	5529	4733	359	350	2949	408
	Carbamazepine	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Cefoperazone	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Ciprofloxacin	<LOQ	<LOQ	2,283	<LOQ	<LOQ	<LOQ
	Domperidone	<LOQ	88	178	<LOQ	<LOQ	<LOQ
	Escitalopram	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Glybencyclamide	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Ivermectine	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Lidocaine	278	185	94	82	178	64
	Loratadine	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Miconazole	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Norfloxacin	<LOQ	<LOQ	2,066	<LOQ	<LOQ	<LOQ
	Ofloxacin	<LOQ	<LOQ	2029	<LOQ	<LOQ	<LOQ
	Propranolol	407	522	513	<LOQ	<LOQ	<LOQ
	Sulfamethoxazole	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
	Tramadol	942	1163	587	515	2315	184
	Verapamil	<LOQ	114	<LOQ	<LOQ	301	283
	PFOA	8	5	<LOQ	5	11	<LOQ
	PFOS	26	<LOQ	24	29	52	<LOQ
	OP	278	<LOQ	91	2133	1314	732
	NP	610	940	590	3400	3800	5350
	NP1EO	140	90	220	-	-	-
	NP2EO	80	80	230	-	-	-
	BBP	396	302	299	186	288	67
	DEHP	2939	2816	12493	30119	21630	4080
	DEP	1404	2101	1097	1165	1676	445
	DnBP	3229	3172		2560	2520	4932
	C10	69609	70660	65861	<LOQ	<LOQ	<LOQ
	C11	781899	203487	190669	<LOQ	<LOQ	<LOQ
	C12	124308	182687	171976	<LOQ	<LOQ	<LOQ
	C13	64088	113839	64789	<LOQ	<LOQ	<LOQ
	Σ LAS	1039904	570664	493294	-	-	-
	Fluorene	10	7	11	<LOQ	<LOQ	<LOQ
	Phenanthrene	45	38	37	<LOQ	<LOQ	<LOQ
	Anthracene	0,6	0,7	7	<LOQ	<LOQ	<LOQ
	Fluoranthene	117	47	607	<LOQ	<LOQ	<LOQ
	Pyrene	42	24	28	<LOQ	<LOQ	<LOQ
	Benzo[a]anthracene	14	9	12	<LOQ	<LOQ	<LOQ
	Chrysene	11	3	8	<LOQ	<LOQ	<LOQ
	Benzo[b]fluoranthene	<LOQ	<LOQ	5	<LOQ	<LOQ	<LOQ
	Benzo[k]fluoranthene	6	0,5	2	<LOQ	<LOQ	<LOQ
	Benzo[a]pyrene	4	2	5	<LOQ	<LOQ	<LOQ
	Dibenzo[a]anthracene	0,8	<LOQ	1,3	<LOQ	<LOQ	<LOQ
	Benzo[ghi]perylene	5	<LOQ	7	<LOQ	<LOQ	<LOQ
	Σ 13 PAHs	255	131	734	-	-	-
	*LOQ in dissolved phase
	Dissolved concentration in italics, particulate concentration underlined
	2
	3
	4

removal pathways (dissolved and/or particulate fraction) and mass balances will be performed in a 47 second article.


Christophe Massard

Daniel Bourdeaux

Vincent Raspal

Emmanuelle Feschet-Chassot

Yves Sibaud

Eric Caudron

Thierry Devers

Kolma Oscar Awitor
email: komla.awitor@udamail.fr

One-pot Synthesis of TiO 2 Nanoparticles in Suspensions for Quantification of Titanium Debris Release in Biological Liquids

Keywords: Titanium Dioxide Nanotube Layer, TiO 2 Nanoparticles, Debris Release, Biological Matrix, Inductively Coupled Plasma Atomic Emission Spectroscopy

In this work we have developed an analytical method to measure potential titanium debris released from TiO 2 nanotube layers devices immersed in biological fluids. This quantitative study is highly required to ensure both the security and non toxicity of the nanostructured surfaces used as future implantable medical devices in the living. A one-pot synthesis process is developed to produce high quality standard solutions of titanium dioxide nanoparticles in aqueous medium. The elaborated dispersion is then used to fabricate standard solutions in both aqueous and human blood plasma media. The synthesized nanoparticles dispersion was characterized by granulometry. The nanoparticles structure and morphology were then observed using Transmission Electron Microscopy (TEM). Thermogravimetric Analysis (TGA) was used to evaluate the concentration of TiO 2 in the suspension. A quantitative routine by the use of Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is developed. The quantification threshold of titanium species is found to be in the 30 -40 ppb range. None interference is detected between the particles and the human blood plasma. Using the established quantitative routine, the titanium species release from titania nanotube layers in human blood plasma is evaluated.

Introduction

Nanotextured materials with the required surface properties are promising candidates to develop the implantation of new kind of biocompatible platforms in the living. Titanium dioxide nanotube layer is an active field of research concerning antibacterial efficacy [START_REF] Ercan | Diameter of Titanium Nanotubes Influences AntiBacterial Efficacy[END_REF], orthopedic [START_REF] Oh | Significantly Accelerated Osteoblast Cell Growth on Aligned TiO2 Nanotubes[END_REF], and dental surgery [START_REF] Khaled | Reinforcement of Resin Based Cement with Titania Nanotubes[END_REF], drug delivery platforms [START_REF] Popat | Titania Nanotubes: A Novel Platform for Drug-Eluting Coatings for Medical Implants?[END_REF], tissue regeneration [START_REF] Kwak | Drug Release Behavior from Nanoporous Anodic Aluminum Oxide[END_REF][START_REF] Zhang | Nanotechnology and Nanomaterials: Promises for Improved Tissue Regene-ration[END_REF][START_REF] Sinn Aw | Polymeric Micelles in Porous and Nanotubular Implants as a New System for Extended Delivery of Poorly Soluble Drugs[END_REF][START_REF] Perry | Drug-Delivery Strategies by Using Template-Synthesized Nanotubes[END_REF][START_REF] Gultepe | Nanoporous Inorganic Membranes or Coatings for Sustained Drug Delivery in Implantable Devices[END_REF] and implants [START_REF] Minagar | A Review of the Application of Anodization for the Fabrication of Nanotubes on Metal Implant Surfaces[END_REF]. The behavior of the nanostructured implants in the living is currently not well known. Numerous studies relate the toxicity of titanium dioxide nanoparticles in the living [START_REF] Fabian | Tissue Distribution and Toxicity of Intravenously Administered Titanium Dioxide Nanoparticles in Rats[END_REF][START_REF] Wu | Toxicity and Penetration of TiO 2 Nanoparticles in Hairless Mice and Porcine Skin after Subchronic Dermal Exposure[END_REF][START_REF] Chen | In Vivo Acute Toxicity of Titanium Dioxide Nanoparticles to Mice after Intraperitioneal Injection[END_REF]. However, as far as we know, few studies are dedicated to the quantification of potential metallic release from the implanted device surface and their underlying potential toxicity. Works need to be done in order to ensure the biological security of such future nanostructured medical devices implanted in the living [START_REF] Feschet-Chassot | Tunable Functionality and Toxicity Studies of Titanium Dioxide Nanotube Layers[END_REF]. Debris from a likely wear in the living of these devices could be hazardous to human health. In this work, we develop a quantitative method to measure possible metallic debris released from the use of TiO 2 nanotube layers as future nanostructured medical implants. This quantitative method using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) requires well defined TiO 2 standard solutions commercially unavailable. So we have developed a onepot synthesis of nanoparticles dispersion in an aqueous medium. The physico-chemical properties of the nanoparticles in dispersion are investigated by granulometry, transmission electron microscopy and thermogravimetry. Different standard solutions are made by dilution from the native dispersion and assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Calibration lines are obtained and the method is checked in term of linearity, accuracy and repeatability. The possible influence of the human blood plasma matrix on the ICP analytical results is also investigated. To simulate the release, nanotube layers samples were immersed in human blood plasma under magnetic stirring at 37˚C, and the titanium species concentration in the liquid is followed by the established quantification method.

Experimental Section

Synthesis of Stable Suspension of TiO 2 Nanoparticles

The starting precursor solution was 5 mL titanium IV isopropoxide 97% supplied by Sigma Aldrich mixed with 15 mL isopropanol (from Rhônes Poulenc) under a vigorous stirring. A 200 mL HNO 3 0.01 M solution was used as an acid hydrolysis catalyst. The nanoparticles preparation started up with the quick addition of the hydrolysis solution heated to 80˚C in the precursor solution under vigorous stirring. The spontaneous hydrolysis of the titanium isopropoxide leads to a turbid white solution which was heated under reflux for almost 48 hours (peptidization process). After this peptidization step, some tartric acid powder supplied by Rhônes Poulenc was added to the white mixture in order to graft a carboxylic acid as surfactant on the oxide nanoparticles surface. The white, opaque resulting mixture was cooled down to room temperature. A few drops of Rhônes Poulenc triethylamine were added under stirring: the opaque, settling solution turned to a clear, soft blued aqueous suspension of TiO 2 nanoparticles.

Granulometry Study of the Suspension

Granulometry studies are achieved by using the Nano Sizer S90 model (Malvern Instrument ltd.). In order to define the influence of the pH of the carrying liquid on the nanoparticles aggregate states, different samples were analysed by varying the pH from 3 to 10. A first sample with the highly measurable concentration is prepared and then different amounts of acid or base are added in order to vary the pH.

Transmission Electron Microscopy Analysis of the Suspension

The morphology and the particles sizes were characterized using a Philips CM 20 transmission electron microscope (TEM). The accelerating voltage was 200 kV. The samples were dispersed in methanol by ultrasonication. A drop of the suspension was then laid on a carbon-coated grid and dried. Selected area electron diffraction (SAED) was performed to determine the crystallinity of the structure. The interplanar distances were evaluated from the SAED patterns using the following formula:

L Rd   (1)
where λL is a constant of the microscope, R is the ring radius, and d is the interplanar distance. The constant of the microscope was calculated by measuring the radius of a gold standard pattern which interplanar distances are well documented in scientific publications.

Thermogravimetric Analysis of the Nanoparticles Suspension

The thermogravimetric analysis was carried out using the TGA 4000 thermogravimetric analyser (Perkin Elmer instrument). The analysis was performed at a heating rate of 5˚C/min under a 40 mL/min N 2 gas flow. Prior to the TGA tests, a designated volume of 50 mL of the white, opaque acid tartric grafted mixture was centrifuged at 7000 rpm during 10 minutes. The clear supernatant was removed and the wet TiO 2 grafted tartric acid nanopowder was dried at 110˚C overnight. As a reference 20 mg TiO 2 nanopowder supplied by Sigma Aldrich has also been analyzed.

Synthesis of TiO 2 Nanotube Layers

The main goal of this part is to obtain TiO 2 nanostructured surface in order to study the potential titanium species release in biological fluids. To fabricate anodic TiO 2 nanotube layers, Ti foils, 99.6% purity, 0.25 mm thick, supplied by Good-fellow are used. The foils are successively degreased in trichloroethylene, methyl alcohol, acetone and deionized water baths under sonication for 5 minutes. The drying is made under nitrogen flush. The anodization process is carried out at 20˚C in 0.4 wt% HF aqueous solution, under an anodizing voltage maintained at 20 volts during 20 minutes. Just after the anodization, samples are rinsed in deionized water, flushed under nitrogen and dried in an oven at 100˚C for 10 minutes. Finally, the anodized foils are cooled down to room temperature in a desiccator.

TiO 2 Nanotube Layer Surface Characterization

The morphological study of the nano-structured surfaces was carried out using a Zeiss Supra 55 VP scanning electron microscope (SEM) with secondary emission and in lens detector. The accelerating voltage and working distance are respectively 5 kV and 3 mm.

Inductively Coupled Plasma Atomic Emission Spectroscopy

Inductively Coupled Plasma Atomic Emission Spectroscopy analysis was carried out using a Varian Liberty II apparatus under the operating conditions given in Table 1. To increase the results accuracy, three emitting wavelengths of the titanium are used for quantification at 334.941, 336.121 and 337.280 nm respectively.

Titanium Species release Quantification from TiO 2 Nanotube Layers

In order to quantify the potential titanium release in human blood plasma, 3 anodized samples (0.5 cm × 2 cm) are immersed in 25 mL of human blood under magnetic stirring at 70 rpm. Each closed vial is kept at 37˚C in a thermostatic bath. A non-anodized titanium foil is used as a reference and dipped in the same conditions. A volume of 3 mL of solution is collected after the 2-day stirring and assayed by ICP in order to obtain the kinetic of release.

Results and discussion

Synthesis of Homogeneous Suspension of TiO 2 Nanoparticles

Functionalized crystalline TiO 2 nanoparticles in suspension have been synthesized by a derivated sol-gel process at low temperature. Figure 1 shows the proposed method for a one-pot synthesis of homogeneous suspension of TiO 2 nanoparticles in an aqueous medium. The process begins by hydrolysis and condensation of titanium alkoxide specie, which the inorganic TiO 2 core is made of. This step was followed by grafting a carboxylic acid as surfactant on the oxide nanoparticle surface. Finally the organic treatment was carried out by adding triethylamine into the mixture. This chemical functionalization of the surface provides the required stable dispersion of the particles in the aqueous medium, leading to a soft blued aqueous suspension of TiO 2 nanoparticles. Different synthesis methods lead to nanopowders with sharp sized distribution. The main problem occurs at the dispersion step of these nanopowders into water or water based solutions. The aggregative state is so high that it leads to turbid, opalescent and fairly stable solutions. Such settling solutions are not suitable for developing rigorous quantitative measurements. On the other hand, the use of commercial dispersion is not appropriate: the chemical compatibility between the turbid commercial product and the aqueous medium is low, so the dilutions cannot be used for quantitative analysis. In order to cope with this key problem of the required stability of the standard solution, we used a one-pot synthesis of TiO 2 nanoparticles dispersion in an aqueous medium. This method solves the problem of the turbidity and settlement generated by the nanopowder dispersion in liquid. This gives rise to stable clear nanoparticles suspension, ready to induce rigorous standard solution for the development of the quantitative analysis contrary to those made from nanopowder or commercial formulation. The difference between the formulations is evidenced in Figure 2.

Figure 2(a) shows a try of TiO 2 nanopowder dispersion in H 2 O: without chemical functions grafted on the nanoparticles surfaces, the TiO 2 nanopowder settled quickly in the bottom of the tube: a white TiO 2 paste is formed, the clear supernatant does not carry nanoparticles in suspension. Figure 2(b) demonstrates the high quality of our synthesized suspension; the suspension is not turbid, soft blued, with high transparency thanks to the chemical surface functions which maintained the TiO 2 nanoparticles in a non aggregated state and highly dispersed in the carrying liquid. Figure 2(c) illustrates that the commercial suspension is totally turbid, the white color of the liquid shows that the TiO 2 nanoparticles are aggregated: such dispersions can not be used as standards.

Granulometry Analysis

The grain size distribution is essential in our studies in order to control the nanoparticles dispersion in the carrying liquid. Most of the physico-chemical properties of the nanoparticles are correlated to their size distribution. The results of the different samples analyzed by Dynamic Light Scattering (DLS) are reported in Table 2. The three samples exhibit good correlogram, so the results are consistent with the real aggregative state of our samples.

The main peak in the 100 nm range is the most important one in order to check the efficiency of the nanodispersion of the TiO 2 entities. We found that our synthesized dispersion is very pH-sensitive. For the two samples, P1 and P2, the percentage intensity of the main peak is close to 95%; so the effectiveness dispersion is for pH ranging from 3.7 to 5.4. Highest pH value seems to induce a decrease in the population in the 100 nm range. The dispersion quality in the 100 nm range is correlated to the pH modulation of the carrying liquid. We think that the Stern layer is modified by the pH modulation of the liquid; this parameter seems to be interesting in order to tailor the size distribution of the TiO 2 na oparticles in the liquid. n

Transmission Electron Microscopy

retical ones we can observe a high degree of consistency see Table 3. This comparison reveals the polycrystalline nature of the nanoparticles. The interplanar distances measured correspond to the TiO 2 anatase structure.

A representative TEM image and the associated SAED pattern of our as synthesized sample is shown in Figure 3.

The TEM image Figure 3(a) shows that most of the particles are elongated, some of them are spherical. It is possible to observe that the powder is composed of quiet heterogeneous sizes oscillating mainly between 30 -100 nm consistent with the granulometry result. A typical SAED pattern corresponding to the most intense spots is shown in Figure 3(b). If we compare the interplanar distances calculated from the SAED patterns and the theo-

Thermogravimetry Study

Table 4 gives the characteristics of the samples analysed by TGA. Sample S2 is made of TiO 2 nanopowder without organic components and used as a reference. Sample S1 is the organic functionalized TiO 2 nanopowder from the synthesis suspension. TGA measurements were carried out from 50˚C to 1000˚C at a heating rate of 5˚C/min. As far as the manufactured TiO 2 nanopowder is concerned, no significant weight loss is recorded in the 50˚C -1000˚C range. In comparison, the weight loss curve of the organic functionalized TiO 2 nanopowder shows two significant weight losses. The first one occurs in the 100˚C -150˚C range and is attributed to the desorption of volatile compound such as residual water and alcohol. The second weight loss in the 150˚C -450˚C range is correlated with the progressive thermal degradation of the grafted tartric acid on the TiO 2 nanoparticle surface. The pyrolysis of the organic surfactant is taken up to 450˚C. At the highest temperature, in our conditions, no more weight loss is recorded, so pure inorganic TiO 2 nanopowder is obtained. For the sake of comparison between the samples, the total estimated weight loss is around 25 wt%.

Considering the initial amount of the precursor and the total volume of liquid, we have estimated the theoretical suspension concentration. The quantification of the weight loss of the organic functionalized TiO 2 nanopowder leads to an estimation of the TiO 2 concentration in the TiO 2 peptidized dispersion of 5.7 mg/mL or 3400 ppm of titanium. The calculated result is in good agreement with the value given by the TGA evaluation.

ICP AES Results

The investigation of potential titanium species in biological liquid requires a low detection threshold in the ten part per billion range (ppb). With the ICP-AES apparatus, this threshold value can be reached. The MS-ICP is more accurate but the possible interaction between calcium ions and titanium species can degrade the measurement qualities. All the following calibration process, linearity, repeatability studies are developed using the International Conference on Harmonisation (ICH) requirements concerning the validation of analytical method [START_REF] Ich | Validation of Analytical Procedure, Text and Methodology Q2(R1)[END_REF]. The calibration line is made with deionized water as carrying liquid. The starting solution is the as-synthesized dispersion (3400 parts per million in titanium). A first dilution is made in deionized water to reach a concentration of 340 ppm. Then, the different standard solutions are made with dilution in deionized water or in human blood plasma respectively. The calibration batch is composed of five standard solutions in the required concentration range:  0.1 ppm, 0.25 ppm, 1.0 ppm, 2.0 ppm.

The control batch is composed of three control solutions:  0.15 ppm, 0.4 ppm, 1.5 ppm.

Linearity Results

The calibration line obtained by assaying the five standard solutions and the blank is presented in Figure 5.

The correlation coefficient calculated with the linear regression analysis is r 2 = 0.9998 better than the requested one r 2 = 0.999. The method is linear in the (0 -2) ppm range.

Repeatability Results

In order to check the repeatability of our method, the whole calibration procedure is repeated three different days in the same operating conditions. The results demonstrate a good linearity of the calibration line for the three different days. In every case, the correlation coefficient r 2 is better than r 2 = 0.999. The repeatability of our method's linearity is proven in the (0 -2) ppm range.

Fidelity and Accuracy Studies

The fidelity of the method concerns the variation in the results obtained by assaying the control solutions on three different days. Fidelity is estimated by the variation coefficient VC:

      standard deviation % average value VC  1 0 0   (2)
Accuracy of the method is defined as the difference between the experimental value and the theoretical one (relative error) RE:

     exp.value-theor. value % theor. value RE  1 0 0  (3)
The fidelity and accuracy valided the method if their value are respectively below 10%. The different results obtained are described in Table 5.

All the results for the fidelity and accuracy criteria are below 10%. These results ensure the validity of our method in the range of the concentration.

Quantification Threshold Study

It is essential for our method to determine the quantification threshold. Using the ICH prescription, the quantification threshold is reached when the variation coefficient VC defined in Equation ( 2) is above 15%. For the quantification threshold determination, a calibration batch composed of five standard solutions and one blank in the required concentration range is used:  0 ppm, 0.1 ppm, 0.25 ppm, 0.5 ppm, 1.0 ppm, 2.0 ppm. Five others samples used as standard solutions are assayed three times for the quantification threshold determination:  10 ppb, 20 ppb, 30 ppb, 40 ppb, 50 ppb.

The results are depicted in Tables 6 and7.

The estimation of the quantification threshold is in the (40 -50) ppb range in titanium species.

Biological Matrix Effect

The potential impact of the biological fluid on the titanium species quantification by ICP-AES must be studied to ensure accurate results using a calibration line. To measure the possible influence of human blood plasma on the titanium species measured by ICP-AES, a calibration line made by assaying five standard solutions in water is used. Six control solutions are prepared using human blood plasma as carrying liquid and assayed twice 8.

The deviation between the theoretical concentration of the control sample in human blood plasma and the measured value is for the whole experiments under 8% concerning the Relative Error (RE) in the concentration range studied. No influence of the human blood plasma on the titanium species quantification is highlighted, the results are in good agreement using a calibration line made with standard solution in water and control solutions diluted in human blood plasma. The calibration line built in water as carrying liquid is also validated to determine the titanium concentration in blood plasma. Furthermore, the repeatability of our method is also demonstrated, the Variation Coefficient (VC) is below the threshold of 10%.

Release Studies from the Nanotube Layers

Immersed in Human Blood Plasma TiO 2 Nanotube Layers Morphology Ordered titanium dioxide nanotube arrays, approximately 80 nm in diameter, are observed from anodized Ti foil made with an anodizing voltage maintained at 20 volts during 20 minutes as shown in Figure 6.

In order to test our quantification method, a preliminary study is proposed. This study is the starting point for further investigations in the field of the potential toxicity of metallic nanostructured devices implanted in the living. We report the results concerning three anodized samples and a naked Ti foil (0.5 cm × 2 cm) immersed in 25 mL of human blood at 37˚C under magnetic stirring at 70 rpm during 2 days as described in Figure 7.

A volume of 3 mL of each sample are assayed by ICP- AES at different times. The experimental results are shown in Table 9.

In every case, the experimental values are below the quantification threshold found in the 40 -50 ppb range. In our experimental conditions, it seems that no signifycant titanium release occurs for 2 days in immersion in human blood plasma.

Conclusion

We reported the establishment of a rigorous quantitative method for assaying a potential titanium species release from TiO 2 nanotube layers in biological fluid using ICP-AES. In order to induce accurate standard solutions needed for the calibration line, a one-pot synthesis of TiO 2 nanoparticles in suspension is developed. The physico-chemical properties of the synthesized nanoparticles were characterized and the dispersion is used to induce well-defined standard and control solution by dilution in deionized water and human blood plasma. Satisfying results are obtained as far as repeatability, fidelity, linearity and accuracy are concerned. No biological matrix influence is detected; this fact validates the method using calibration line in water for titanium quantification in human blood plasma. The detection threshold is observed in the (40 -50) ppb range in titanium species. To invest gate the potential titanium i

Figure 1 .

 1 Figure 1. Proposed method for one pot synthesis of homogeneous suspension of TiO 2 nanoparticles in an aqueous medium.

Figure 2 .

 2 Figure 2. Comparison between a try of TiO 2 nanopowder dispersion in H 2 O (a); our synthesized sample (b) and the commercial standard (c) both at 3400 ppm in titanium.

Figure 3 .

 3 Figure 3. TEM image of TiO 2 nanoparticles (a) and SAED pattern of the particles (b).

Figure 4

 4 Figure4shows the TGA curves.As far as the manufactured TiO 2 nanopowder is concerned, no significant weight loss is recorded in the 50˚C -1000˚C range. In comparison, the weight loss curve of the organic functionalized TiO 2 nanopowder shows two significant weight losses. The first one occurs in the 100˚C -150˚C range and is attributed to the desorption of volatile compound such as residual water and alcohol. The second weight loss in the 150˚C -450˚C range is correlated with the progressive thermal degradation of the grafted tartric acid on the TiO 2 nanoparticle surface. The pyrolysis of the organic surfactant is taken up to 450˚C. At the highest temperature, in our conditions, no more weight loss is recorded, so pure inorganic TiO 2 nanopowder is obtained. For the sake of comparison between the samples, the total estimated weight loss is around 25 wt%.Considering the initial amount of the precursor and the total volume of liquid, we have estimated the theoretical suspension concentration. The quantification of the weight loss of the organic functionalized TiO 2 nanopowder leads to an estimation of the TiO 2 concentration in the TiO 2 peptidized dispersion of 5.7 mg/mL or 3400 ppm of titanium. The calculated result is in good agreement with the value given by the TGA evaluation.

Figure 4 .

 4 Figure 4. TGA curves of the TiO 2 grafted carboxylic acid nanopowder from the synthesis (a) and pure TiO 2 used as a reference (b).

Figure 5 .

 5 Figure 5. Calibration line obtained from the assayed samples .

Figure 6 .Figure 7 .

 67 Figure 6. SEM top-view image of the TiO 2 nanotube layer after 20 minutes of anodization at 20 Volts.

Table 1 . Instrumental operating conditions.

 1

	ICP analytical parameters
	RF power	1.2 kW at 10 MHz
	Wavelength range	165 -782 nm
	Resolution	0.006 nm
	Plasma viewing	Axial
	Detector	PM (Photo multiplication)
	Gas flow controls
	Plasma gas	15 L/min
	Auxiliary gas	0.8 L/min
	Nebulizer gas pressure	260 kPa
	Sample uptake rate	1.2 mL/min
	Auto integration	5 sec

Table 2 . Granulometric results for the Samples P1-P3.

 2

	pH	θ (˚C)	η (cP)	n	Z average d(nm)	Size peak 1 d(nm)	Int. peak1 (%)
	3.7	25	0.88	1.33	48.1	62.2	94
	5.4	25	0.88	1.33	41.7	50.4	97
	6.5	25	0.88	1.33	39.9	41.3	86

Table 3 . Interplanar distances for the TiO 2 nanoparticles deduced from the SAED patterns, compared to the expected ones for ideal anatase phase.

 3

	Interplanar distance from the SAED pattern (Å)	3.50	2.40	1.90	1.70
	Theoretical distance for the anatase phase (Å)	3.51	2.33	1.89	1.66
	Corresponding miller indice	(101)	(112)	(200)	(211)

Table 4 . Samples characterisitics analysed by TGA.

 4

	Sample identification	Charateristics	Weight	Drying condition
	S1	Nanopowder from the synthesis	20 mg	110˚C overnight
	S2	Nanopowder from sigma aldrich	20 mg	none

Table 5 . Fidelity and accuracy results for the control sam- ples.

 5

	Theoretical concentration (mg/L)	Measured concentration (mg/L)	VC (%)	RE (%)
	0.15	0.15	8.4	7.0
	0.40	0.41	8.0	6.5
	1.50	1.47	7.0	5.0

Table 6 . Quantification threshold determination.

 6

	Theo.(ppb)	T1(ppb)	T2	T3	T4	T5
	10	8.31	7.53	9.76	6.96	19.32
	20	18.36	16.49	23.54	18.22	30.80
	30	26.62	23.57	35.95	26.24	43.27
	40	48.36	37.75	62.09	41.42	49.53
	50	67.74	57.12	52.49	62.95	65.41

Table 7 . Variation coefficient (VC) calculations.

 7

	Theo.(ppb)	average	Stdev.	VC (%)
	10	10.38	4.60	44.04
	20	21.48	5.20	24.31
	30	31.13	7.40	23.69
	40	47.83	8.40	17.47
	50	61.14	5.60	9.14

by ICP. The results are depicted in Table

Table 8 . Influence of the blood plasma matrix on the quantification of titanium species by ICP-AES.

 8

	Theo.	T1	T2	average	St	VC	RE
	(ppb)	(ppb)	(ppb)	(ppb)	dev.	(%)	(%)
	0.15	0.16	0.14	0.15	0.02	9.94	-0.04
	0.15	0.16	0.15	0.16	0.01	7.50	-3.71
	0.40	0.40	0.40	0.40	0.00	0.88	-0.37
	0.40	0.38	0.37	0.38	0.00	0.89	6.36
	1.50	1.38	1.46	1.42	0.06	3.86	5.41
	1.50	1.34	1.44	1.39	0.07	5.05	7.42

Table 9 . Results of the quantification of titanium species released in human blood plasma from anodized surfaces.

 9 species release, a preliminary study consisting of samples made of TiO 2 nanotube layers immersed in human blood plasma is performed. No significant titanium species release was measured during this test. This work opens the way to further promising investigations concerning the biological security of the future medical metallic devices implanted in the living.

	Sample ID	Titanium concentration (ppb)
	Ti foil	28.3
	NS1	46.6
	NS2	32.1
	NS3	28.5

Copyright © 2012 SciRes. ANP

Acknowledgements

The authors thank Dominique Jalabert, Laurianne Truffault and Caroline Andreazza for the HRTEM images (université d'Orléans). The authors acknowledge the University of Auvergne for its financial support.