Chemical Characterization and Quantification of Titanium Dioxide Nanoparticles (TiO2-NPs) in Seafood by Single-Particle ICP-MS: Assessment of Dietary Exposure

The significant increase in the production and variety of nanoparticles (NPs) has led to their release into the environment, especially into the marine environment. Titanium dioxide nanoparticles (TiO2-NPs) are used in different industrial sectors, from the food industry to several consumer and household products. Since the aquatic environment is highly sensitive to contamination by TiO2-NPs, this work aimed to give a preliminary assessment of the contamination of packaged seafood, where the food additive TiO2 (E171) is not to be intentionally added. This allowed providing a chemical characterization and quantification of TiO2-NPs in processed canned fish products belonging to different trophic positions of the pelagic compartment and in canned clam. The new emerging technique called single-particle inductively coupled plasma mass spectrometry (spICP-MS) was applied, which allows the determination of nanoparticle number-based concentration, as well as the dissolved titanium. This study highlights how processed food, where the pigment E171 was not intentionally added, contains TiO2 in its nanoparticle form, as well as dissolved titanium. Processed clam represented the seafood with the highest content of TiO2-NPs. In pelagic fish species, we found progressively higher levels and smaller sizes of TiO2-NPs from smaller to larger fish. Our results highlight the importance of planning the characterization and quantification of TiO2-NPs in food both processed and not, as well as where the pigment E171 is intentionally added and not, as it is not the only source of TiO2-NPs. This result represents a solid step toward being able to estimate the real level of dietary exposure to TiO2-NPs for the general population and the related health risks.


Introduction
In Earth science, nanoparticles (1-100 nm) are important components of the biogeochemical system, but human impact on the environment has altered their natural cycle [1] by introducing engineered nanoparticles with several physicochemical characteristics and applications [2]. Nanoparticles represents a blooming industrial sector destined to increase because of the numerous investments that it is able to attract. Due to their peculiar characteristics (e.g., different optical properties, greater environment. According to the literature, very few studies reported the levels of nanoparticles in marine organisms [25][26][27][28][29]. With this study, we wanted to provide a chemical characterization and quantification of TiO 2 -NPs (<100 nm), TiO 2 total particles (TiO 2 -Ps-Tot, all particle sizes), dissolved Ti, and total Ti in processed canned seafood products belonging to different trophic positions. Our choice to use canned seafood for this evaluation study was because it is a fundamental component in human nutrition, representing an important source of unsaturated fatty acids, protein, and different micronutrients [30].
Furthermore, we also provided data related to the estimated daily intake for both adults and children, which may be useful to risk assessors for developing a provisional tolerable daily intake for TiO 2 -NPs.

Alkaline Digestion and Single-Particle Inductively Coupled Plasma Mass Spectrometry (spICP-MS) Analysis
Different brands of canned tuna (five), mackerel (four), anchovy (three), and clam (three), among the best-selling and low-cost brands, were purchased from the main Italian supermarket chains in the city of Catania, (Italy), in the period between June and July 2019, and stored at −80 • C until analysis. For each brand of seafood product, we also chose to purchase three different batches, which were extracted and processed in triplicate. Thus, we performed a total of 45 extractions for canned tuna, 36 for canned mackerel, 27 for canned anchovy, and 37 for canned clam.
Assessment of TiO 2 was performed using a NexION ® 350D (Perkin Elmer, Waltham, MA, USA) with the Syngistix Nano Application software (Perkin Elmer, Waltham, MA, USA). The instrumental operating conditions for the determination of TiO 2 are listed in Table 1. The dwell time used was chosen on the basis of other studies [27,31]. The new emerging technique of single-particle inductively coupled plasma mass spectrometry (spICP-MS) allows the determination of particle number-based concentration, with rapid simultaneous characterization of the elemental composition, number of particles, size and size distribution, and dissolved concentration. Furthermore, by modifying the integration window, it is possible to collect data related to a specific size distribution. Accordingly, for each sample, we captured data on the total TiO 2 particles (Ps-Tot) and TiO 2 nanoparticles (NPs < 100 nm) only.
To support the quality of spICP-MS measurements, the particle size distribution of the powder TiO 2 -NPs standard was assessed as follows: a TiO 2 -NP stock suspension (215 ng/L or 4.5 × 10 5 particles/mL) was prepared in ultrapure water and dispersed for 30 min at 37 • C using an ultrasonic bath, to maximize a homogeneous dispersion. The transport efficiency (TE%) was calculated with the certified reference material PELCO (Ag-NPs, 39 ± 5 nm, 110 ng/L, monitoring m/z 107) under the same instrumental conditions as the samples, obtaining a value of TE% 2.54. This solution gave a TiO 2 -NP concentration of 4.6 × 10 5 ± 0.16 × 10 5 particles/mL (n = 10), in agreement with the particle concentration prepared. TiO 2 -NPs showed a size range of 44-85 nm with a mean size of 66.2 ± 3.0 (n = 10) and a modal size of 56.6 ± 4.4 (n = 10). The results obtained were in compliance with the size of the TiO 2 -NP standard supplied by the manufacturer (60 nm).
Before performing the spICP-MS analysis, an alkaline digestion of the samples was performed using the method described by Gray et al. (2013) [32]. Approximately 0.5 g of wet sample tissue was weighed in DigiTUBEs (SCP Science, Baie D'Urfé, Québec, Canada) and mixed with 5 mL of tetramethylammonium hydroxide (TMAH, 20% v/v), which is a strong organic base capable of digesting tissues and releasing nanoparticles without altering them. A vortex was used, at first, to prevent the tissues from sticking to the walls of the container used for digestion. The TiO 2 extraction was obtained through sonication for 30 min at 37 • C using an ultrasonic bath to accelerate tissue breakdown and prevent particle aggregation. Subsequently, the samples were left to digest for another 24 h at room temperature. At the end of digestion, samples were diluted with MilliQ water (Millipore, Bedford, MA, USA) to 1% TMAH concentration before analysis and 0.1% Triton X-100 to allow the detection of single particles.
All samples and calibration solutions were sonicated for 30 min before being analyzed. To avoid contamination between samples, the system was rinsed with nitric acid (2%, v/v) prior to the measurement. The TiO 2 -NP standard was also used for the determination of the transport efficiency within the 3-8% range, in agreement with the TE obtained with an Ag certified reference material.
The limit of detection (LOD) and the limit of quantification (LOQ) were calculated by analyzing 10 alkaline extract blanks, in the same analytical condition of the samples, on the basis of the mean ± 3 SD and the mean ± 10 SD criteria of the number of particles/mL obtained, respectively. The LOD was 1.3 × 10 3 particles/mL, while the LOQ was 2.5 × 10 3 particles/mL. Referring to the sample weight and digestion volume used, these values were equivalent to 2.6 × 10 5 /g and 5.0 × 10 5 /g, respectively.
where 3σ blank is three times the standard deviation of the counts/dwell time of alkaline blanks (1% TMAH), R is the slope of the calibration curve of ionic Ti solutions, f a is the mass fraction of the analyzed metallic element in the TiO 2 NPs, and ρ is the density of the TiO 2 NPs. For each batch of analysis, a quality control was performed with analytical recovery before and after spiking with 60 nm TiO 2 -NPs at a concentration of 5 µg/L, corresponding to a concentration of 1.1 × 10 6 parts/mL. The values (range 90-95%) were calculated for the whole size distribution by dividing the TiO 2 -NP concentration by the TiO 2 -NP concentration found in the solution of TiO 2 -NPs used for spiking and multiplying by 100.

Acid Digestion and ICP-MS Analysis of Total Ti
For the determination of total titanium, the same samples were processed by acid digestion. Aliquots of 0.5 g of wet samples were weighed in Teflon reactors using an analytical balance (Mettler Toledo) and then digested in a microwave oven (Ethos, TC, Milestone, Sorisole (BG), Italy), by adding 6 mL of 67% super-pure nitric acid (HNO 3 ; Carlo Erba, Italy) and 2 mL of 30% hydrogen peroxide (H 2 O 2 ; Carlo Erba, Italy) for 1 h at 80 • C. After acid digestion, all samples were diluted to 50 mL with ultrapure water and were filtrated through a 0.45 µm membrane filter before analysis. Total Ti was quantified with the same inductively coupled plasma mass spectrometer (ICP-MS NexION ® 350D, Perkin Elmer, Waltham, MA, USA) in standard mode, using the standard addition technique covering the concentration from 0 to 50 µg/L. Instrumental condition for Total Ti determination are showed in Table 2. A single-element standard solution of Ti (1000 mg/L in 5% di HNO 3 , 0.5% HF) was purchased from CPAchem. Standards for instrument calibration were prepared in the same acid matrix, and yttrium (Y) was used as an internal standard to verify the accuracy.
To verify if acid digestion of the samples allowed the total dissolution of TiO 2 particles and then the quantification of total Ti, we conducted a preliminary acid digestion of 4 µg/L ionic Ti (n = 6), canned clam (n = 6), and canned tuna (n = 6). The quantification of TiO 2 particles was detected using Syngistix Nano Application software, and the resulting background signals, expressed as particles/mL, are shown in Table 3. Table 3. Descriptive statistics concerning the quantification of TiO 2 particles (Ps-Tot) expressed as particles/mL detected with Syngistix Nano Application software after acid digestion of the samples. The threshold value of TiO 2 particles was estimated as the mean + 3 SD given by ionic Ti, which resulted in 1029 particles/mL. Digested samples of canned clam and canned tuna did not show background values of TiO 2 particles higher than the calculated threshold, demonstrating the effectiveness of the acid digestion in dissolving all TiO 2 particles.
The LOD and LOQ were calculated by analyzing 10 acid extract blanks according to the mean ± 3 SD and mean ± 10 SD criteria, respectively. They resulted 0.06 and 0.16 mg/kg, respectively.
For each batch of analysis, a quality control was performed with analytical recovery after spiking with 20 µg/L of ionic Ti.
Accuracy was tested using a laboratory-fortified matrix with a seafood sample (use of ionic Ti at 20 µg/L) processed at each batch of digestion, obtaining a recovery range of ionic Ti from 92-115%.

Estimated Meal Intake
An exposure assessment derived from the consumption of selected seafood products was conducted for TiO 2 -NPs (TiO 2 particles <100 nm), TiO 2 -Ps-Tot (TiO 2 total particles) and dissolved Ti according to the method described in a previous paper [34]. Briefly, the estimated meal intake (EDI) (mg/kg body weight (BW) per day) was determined according to Equation (2).
where C is the TiO 2 -NPs, TiO 2 -Ps-Tot, or dissolved Ti (mg/kg wet weight), M is the meal size (0.227 kg for adults and 0.114 kg for children) [34], and BW is the body weight, considered as 16 kg for children (6 years) and 70 kg for adults (70 years) [35].

Statistical Analysis
The statistical software package IBM SPSS 20.0 (IBM, Armonk, NY, USA) was used for statistical analysis. One-way ANOVA and a post hoc Tukey test were performed to evaluate differences in TiO 2 and dissolved Ti levels among canned products. We processed a total of 45 samples of canned tuna, 36 of canned mackerel, 27 of canned anchovy, and 37 of canned clam.

Results
The total TiO 2 particles present (Ps-Tot size), as well as the level of Ti in its dissolved form, were analyzed using the NexION ® 350D (Perkin Elmer, Waltham, MA, USA) with the Syngistix Nano Application software, by processing the samples with ultrasound-assisted alkaline digestion. In a separate working session, we also determined the level of total Ti measured with ICP-MS in standard mode, by processing the samples digested with acid. As shown in Table 4, the quantification of dissolved Ti did not differ significantly from the total Ti. The slightly higher result of total Ti can be related to the acid dissolution of the Ti bonded in the particles, as supported in the experimental data shown in Table 3. As shown in Table 4 and Figure 1, canned anchovy and canned clam revealed the highest mean diameters of Ps-Tot size, close to 140 nm, significantly higher than the mean diameters found in canned tuna and canned mackerel, close to 100 nm. The modal size was found to be significantly higher in canned anchovy versus the other seafood products, followed by canned mackerel, canned clam, and canned tuna. With regard to the Ps-Tot level, the samples of canned clam had the highest levels of TiO 2 -NPs compared to the fish species, with a mean value of 0.326 mg/kg. Among fish species, canned tuna had levels of TiO 2 -NPs significantly higher than canned mackerel and anchovy, with a mean value of 0.117 mg/kg. Considering the ionic Ti determined using the single-particle technique, canned clam and canned anchovy had higher levels than the other seafood products, with mean values of 1.31 and 1.22 mg/kg, respectively.  For each sample, by reducing the integration window of particle size, we selected the particle size distribution <100 nm to quantify the nanoparticles only. The analyses carried out showed the presence of TiO 2 -NPs, with a diameter lower than 100 nm, in all the seafood samples. As shown in Table 5 and Figure 2, with regard to NPs size, canned anchovy and canned clam revealed the highest mean diameters, close to 90 nm, higher than the mean diameters found in canned tuna and canned mackerel (both below 80 nm). The modal size was found to be significantly higher in canned anchovy versus the other seafood products with a mean value of 93 nm, followed by canned mackerel, canned clam, and canned tuna. With regard to the NP level, the samples of canned clam had the highest levels of TiO 2 -NPs compared to the fish species, with a mean value of 0.112 mg/kg. Among fish species, canned tuna had levels of TiO 2 -NPs fourfold higher than canned mackerel and anchovy, with a mean value of 0.038 mg/kg. Furthermore, the percentage of TiO 2 -NPs in the TiO 2 total particles was close to 60% in all seafood products except for canned anchovy, which showed a percentage close to 30% (Table 5 and Figure 2).

Discussion
Our results are the first to provide a quantitative analysis of TiO 2 -Ps-Tot in packaged pelagic fish and to deepen findings related to bivalve mollusks. It is important to specify that, in all the products we chose, the use of the additive E171 was not indicated on the label, indicating that it was not intentionally added during food processing. Thus, the presence of titanium dioxide in particle or nanoparticle form may be attributed to contamination within the food industry, in addition to the bioaccumulation of organisms in their natural environment. As shown in Table 5 and Figure 2, our findings highlighted how the longest-lived and larger-sized organisms, such as tuna and mackerel, had a lower TiO 2 nanoparticle diameter than anchovy, a small pelagic fish, and clams, as well as the highest percentage of NPs in the total particles. Accordingly, it seems that nanoparticles of smaller diameter have a higher potential of bioaccumulation and biomagnification along the food chain in aquatic systems. This environmental behavior of TiO 2 -NPs seems to be promoted by their poor water solubility and long-term persistence in aquatic systems [36].
Nevertheless, both TiO 2 -Ps-Tot and TiO 2 -NP concentrations were found to be significantly higher in clam, surely due to their filter-feeding behavior, making it a great biological model to test for the bioavailability of contamination at the sediment-water interface. The sedimentation capacity of TiO 2 -NPs is strictly related to the availability and characteristic of natural organic matter (NOM). NOM can act to maintain smaller particle sizes and more negative surface charges on the NPs [37], reducing the rate of sedimentation of metal oxide NPs [38,39]. As experimentally proven, the low NOM content and high ionic strength of seawater promote a high rate of sedimentation of metal oxide nanoparticles [38]. In this way, NPs are less mobile and their interaction with filter-feeders and sediment-dwelling organisms is favored [40], supporting our findings in stating that benthic mollusks are more exposed to TiO 2 -NP bioaccumulation.
The first occurrence of data related to the single-particle characterization and quantification of TiO 2 -Ps-Tot in shellfish was reported in [26,27], although the authors did not reduce the particle acquisition spectrum to the nano-fraction of 100 nm in diameter. Taboada-López et al. (2018) processed different shellfish species collected both fresh and frozen at the supermarket, by applying an ultrasound-assisted enzyme digestion and spICP-MS analysis. The levels of dissolved Ti they found in fresh clam species (0.802-1.31 mg/kg wet weight) are comparable to ours (1.31 ± 0.21 mg/kg wet weight), while they found a slightly lower particle concentration (4.16-7.56 × 10 7 parts/g vs. 10.5 ± 1.52 × 10 7 parts/g). Furthermore, Taboada-López et al. (2018), by analyzing the variegated scallop, found a significantly higher concentration of TiO 2 particles and dissolved Ti in frozen samples than fresh ones, suggesting contamination by the food industry. Overall, the mean size of TiO 2 -NP diameter given for these shellfish was in the range of 60-84 nm versus a mean size of 89.8 ± 6 nm found in our packaged clams. Xu et al. (2018) also performed an enzyme digestion and spICP-MS analysis in shellfish collected from an offshore aquaculture farm. In clams, they found comparable concentrations of dissolved Ti (1.11 ± 0.35 mg/kg wet weight) and mean size of TiO 2 -NPs (70.9 ± 12.4 nm), but a lower concentration of TiO 2 -NPs (2.10 ± 0.31 × 10 7 parts/g). Furthermore, they did not find any significant differences with the other shellfish species analyzed.
We also provided a first estimate of TiO 2 -NPs, TiO 2 -Ps-Tot, and dissolved Ti intake derived from a meal consisting of the different packaged seafood, both for adults of 70 years and children of 6 years. Results were significantly higher in children than in adults, due to the low body weight of the considered age class.
According to a carcinogenicity study with TiO 2 (as E171) in mice and in rats, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) chose the lowest no observed adverse effects levels (NOAEL), which was 2250 mg TiO 2 /kg BW/day [5], but it may contain up to 3.2% of its weight in nanoparticles. For these reasons, E171 as a food additive is not considered as a nanomaterial according to the EU recommendation on the definition of a nanomaterial [5]. In our samples, we found a high occurrence of TiO 2 in its nanoparticle structure, with the lowest in canned anchovy (32%) and the highest in canned tuna (59%); thus, we cannot refer to the established NOAEL for toxicological evaluation.
Most studies available evaluated the oral intake of total TiO 2 and TiO 2 -NPs for TiO 2 added during the production process in food, toothpaste, and supplements [8,11,41]. Bachler et al. (2015), by developing a physiologically based pharmacokinetic (PBPK) model, calculated the dietary intake of total titanium for the German population as between 0.5 and 1.0 mg/kg BW for all age groups except the age group "other children", who had the highest titanium intake of all age classes (approximately 2.0 mg/kg BW). Furthermore, by assuming that 10% of the total titanium intake is in the nano-range, which is a conservative estimation according to the ratio of ionic to particulate titanium intake (1:19) and the amount of TiO 2 -NPs in E171 (approximately 11% by weight) [11], they calculated the 95th percentile of TiO 2 -NPs dietary intake as below 170 ng/g for all age groups. Moreover, Rompelberg et al. (2016) estimated the long-term intake of TiO 2 -NPs for the Dutch population as ranging from 0.19 µg/kg BW/day for the elderly to 0.55 µg/kg BW/day for 7-69-year-olds and 2.16 µg/kg BW/day for young children. The results found in the literature are significantly lower than the potential exposure calculated in this study. Nevertheless, considering that the presence of E171 as a food additive in canned fish is not expected, our results give an estimate of the dietary intake probably related to TiO 2 -NP bioaccumulation in fish and seafood species through environmental contamination. However, we cannot be sure regarding the potential total amount of TiO 2 -NPs which can be accumulated in human tissue after oral ingestion, considering that part of the compound can be altered in the gastrointestinal environment by extreme pH and co-ingested food [42], although intestinal epithelial cells are affected at a functional level [43]. Lastly, a study limitation was the inability to discern the origin of TiO 2 -NPs, i.e., natural or anthropogenic. Taking into account the numerous applications of TiO 2 -NPs, a proper improvement of the wastewater depuration process could be a step to prevent the contamination of the water bodies.

Conclusions
This study provided, for the first time, an evaluation of the estimated daily intake of TiO 2 -NPs starting from quantitatively measured data. Our findings highlighted how processed seafood, where the pigment E171 is not intentionally added, may contain amounts of TiO 2 in its particle and nanoparticle form, as well as dissolved titanium. In particular, our results revealed that the consumption of selected types of foods could be an important route for the uptake of TiO 2 , especially for a vulnerable group such as children. Processed clam was the seafood with the highest content of TiO 2 -NPs, while, among pelagic fish species, we found progressively higher levels and smaller sizes from small to large fish. Nevertheless, seafood consumption represents only a small part of the human total diet; thus, to provide a realistic exposure assessment, it is important to carry out TiO 2 -NP characterization and quantification in a large number of food items, both processed and not, as well as where the pigment E171 is intentionally added and not, as it is not the only source of TiO 2 -NPs. This information would be a solid step toward being able to actually estimate the TiO 2 -NP dietary exposure of populations and the related health risks. The new emerging technique of spICP-MS, which is able to distinguish between the particulate and the dissolved fraction, is crucial for a better understanding of the fate and transport characteristics of the particles and ionic forms, as well as their possible (independent or synergistic) environmental and (eco) toxicological impacts.

Conflicts of Interest:
The authors declare no conflict of interest.