separations

Article
Reusable Water Bottles: Release of Inorganic Elements,
Phthalates, and Bisphenol A in a “Real Use” Simulation
Experiment
Maria Luisa Astolfi 1 , Federica Castellani 2,3 , Pasquale Avino 4 , Arianna Antonucci 2 , Silvia Canepari 5 ,
Carmela Protano 2, * and Matteo Vitali 2

1 Department of Chemistry, Sapienza University, Piazzale Aldo Moro 5, I-00185 Rome, Italy;
marialuisa.astolfi@uniroma1.it
2 Department of Public Health and Infectious Diseases, Sapienza University, Piazzale Aldo Moro 5,
I-00185 Rome, Italy; federica.castellani@uniroma1.it (F.C.); arianna.antonucci@uniroma1.it (A.A.);
matteo.vitali@uniroma1.it (M.V.)
3 Department of Ecological and Biological Sciences, Tuscia University, Largo dell’Università snc,
I-01100 Viterbo, Italy
4 Department of Agricultural, Environmental and Food Sciences (DiAAA), University of Molise,
via De Sanctis, I-86100 Campobasso, Italy; avino@unimol.it
5 Department of Environmental Biology, Sapienza University, Piazzale Aldo Moro 5, I-00185 Rome, Italy;
silvia.canepari@uniroma1.it
* Correspondence: carmela.protano@uniroma1.it; Tel.: +39-064-9914-568





 Abstract: Reusable water bottles are growing in popularity; thus, possible chemical release from the
Citation: Astolfi, M.L.; Castellani, F.; internal surface into water should be carefully considered to control related health risks. We experi-
Avino, P.; Antonucci, A.; Canepari, S.; mentally evaluated the release into deionized water of 40 elements, six phthalates, and bisphenol-A
Protano, C.; Vitali, M. Reusable Water for 20 different reusable bottles by simulating the use in real world scenario. The 20 bottles, identified
Bottles: Release of Inorganic as those most purchased in Italy, were made of various materials (stainless steel, aluminum, plastic,
Elements, Phthalates, and Bisphenol
and silicone). The experiment was carried out for four consecutive weeks in duplicate for each
A in a “Real Use” Simulation
type of bottle. Our results showed the release, to various extents, of inorganic elements from all
Experiment. Separations 2021, 8, 126.
20 bottles, while the release of phthalates and bisphenol-A was never found. The elements most
https://doi.org/10.3390/
frequently released were Al, Sr, Mo, and Cr, while the highest concentrations were for Ca, K, Mg,
separations8080126
and Na; the release of toxic elements (such as Pb, Cd, Ni, Sb) also occurred. The comparison of our
Academic Editors: Angela Sorbo, results with regulatory limits on drinking water quality revealed no exceeding values except for
Claudia Zoani and Daniele Passeri Al. However, these releases represent a further intake, and the related risks cannot be neglected,
especially for highly susceptible populations. Thus, it is essential to correctly inform consumers both
Received: 3 August 2021 with dedicated interventions and exhaustive labelling.
Accepted: 19 August 2021
Published: 20 August 2021 Keywords: reusable water bottles; inorganic elements; phthalates; bisphenol-A; migration; drinking
water; consumers’ health risks
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Single-use plastics have taken on an important role in the modern society, significantly
improving the safety of medical devices, packaging technology, and the possibility of
preserving food but, at the same time, greatly increasing the amount of disposable products
Copyright: © 2021 by the authors. waste [1]. In many cases, however, as for bottled water, the increase in the quantity of
Licensee MDPI, Basel, Switzerland. plastic disposed of as waste depends on the poor compliance with recovery and recycling
This article is an open access article
of the community and individuals; a recent life-cycle analysis demonstrated that PET
distributed under the terms and
bottles’ production and use assure a lower environmental impact compared to polylactic
conditions of the Creative Commons
acid and to aluminium bottles [2]. For these reasons, reusable water bottles, as possible
Attribution (CC BY) license (https://
alternative to single-use plastic bottles, are growing in popularity in recent years because
creativecommons.org/licenses/by/
they appear environmentally friendly [3], economic as refillable multiple times, and socially
4.0/).

Separations 2021, 8, 126. https://doi.org/10.3390/separations8080126 https://www.mdpi.com/journal/separations

Separations 2021, 8, 126 2 of 13

appreciated [4,5]. On the other hand, the risk of bacterial and/or fungal proliferation inside
the bottle and of migration of chemicals from the internal surface in direct contact with
water to the water itself should be taken into consideration. On this specific topic, there is
vast international legislation [6–10] that deals with both safety and quality issues of food
contact materials (FCMs), among which these water bottles are included. In particular,
when marketed in the European Union (EU), they should be produced according to strict
quality standards, use only materials present in the so-called “positive lists”, report on the
label all the elements for traceability, and provide the consumer with specific instructions
for use, maintenance, and disposal. Additionally, the EC mark and the “fork and glass”
symbol should be present and clearly visible on each bottle [6,7].
The biological risk associated with the use/reuse of water bottles has been evaluated
by recent studies [11–14], demonstrating high bacterial and fungal growth when cleaning
and maintaining practices are not correctly adopted. To date, however, the same attention
has not been given to the chemical risk related to the possible release of elements and/or
compounds to the water from the internal surface of the bottles. Indeed, water bottles
can be made of stainless steel, aluminum (also recycled), plastic, or silicone; metallic
bottles usually present an internal lining that is in direct contact with water, and heavy
metals residual of the catalysts used in the manufacturing process, in particular Sb, may be
present [15]. Thus, a great number of chemicals, including inorganic elements (metals, semi-
metals, non-metals), phthalates (PAEs), bisphenol-A (BP-A), and lining compounds, can
be released into water, posing potential health risks for consumers. Among the inorganic
elements potentially involved in migration, Co, Cr, Cu, Mn, Mo, Ni, Se, and Zn are
essential to maintain proper metabolic activity in living organisms, but, at certain levels,
they also present toxicological properties [16,17]. Other elements, such as Al, As, Ba, Be,
Cd, Sn, Pb, Sb, and U are toxic and do not present any biological role [17,18]. PAEs and
BP-A are chemicals widely used as plasticizing agents, mainly in the manufacturing of
polyvinyl chloride (PVC) and polyethylene terephthalate (PET) [19,20]. PAEs can dissociate
or migrate from plastics, especially when stressed by thermal and/or mechanical factors
or when into contact with lipophilic substances [21]. Both PAEs and BP-A have been
associated with adverse human health effects [22]. In particular, they have been recognized
as endocrine disruptors [23] and may negatively influence human fertility [24]. BP-A is also
associated with several adverse outcomes in pregnant women, fetuses, and children [25,26].
As regards the internal lining of metallic bottles, information on the composition is usually
not disclosed, preventing evaluations of the possible release.
This paper aimed to a) evaluate the release of 40 inorganic elements (Al, As, B, Ba, Be,
Bi, Ca, Cd, Ce, Co, Cr, Cs, Cu, Fe, Ga, K, La, Li, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, Sb, Se, Si,
Sn, Sr, Te, Ti, Tl, U, V, W, Zn, and Zr) and seven organic compounds (di-methyl-phthalate
(DMP), di-ethyl-phthalate (DEP), di-iso-butyl phthalate (DiBP), di-butyl phthalate (DBP),
di-ethyl-exyl-phthalate (DEHP), di-octyl-phthalate (DOP), and BP-A) from 20 different
reusable water bottles (made of stainless steel, aluminum, plastic, or silicone) into deionized
water by simulating their use in a “real world” scenario for four consecutive weeks; and b)
investigate possible consumers’ health risks by comparing the results with the maximum
limits fixed by current legislation on drinking water.

2. Materials and Methods

2.1. Selection of Reusable Water Bottles and Experimental Planning
The 20 studied reusable water bottles were selected by a simple market analysis of the
best-sellers, both retail and online, for each material (glass excluded) in Italy in 2019. In
total, we purchased 40 different bottles (20 typologies in duplicate) of different capacities
(from 350 to 1000 mL) and different materials (stainless steel—1 bottle, aluminum also
recycled—12, plastics—6, and silicone—1) (Table 1).
Separations 2021, 8, 126 3 of 13

Table 1. Descriptive characteristics of the studied water bottles.

Material
Consumers’ Capacity
Code (as Declared on Made in
Age Group (mL)
the Packaging)
01A–01B adults aluminum not indicated 500
02A–02B adults aluminum not indicated 600
03A–03B children aluminum not indicated 500
04A–04B children aluminum not indicated 500
05A–05B children aluminum China 750
06A–06B adults aluminum not indicated 600
07A–07B adults stainless steel not indicated 350
08A–08B adults aluminum not indicated 1000
09A–09B adults aluminum not indicated 750
10A–10B adults aluminum China 800
11A–11B adults aluminum not indicated 750
12A–12B adults aluminum not indicated 500
13A–13B adults aluminum not indicated 750
14A–14B adults BP-A-free plastic not indicated 500
15A–15B adults plastic Austria 500
16A–16B adults plastic not indicated 800
17A–17B adults plastic Italy 550
18A–18B children plastic Turkey 500
19A–19B children plastic China 400
20A–20B adults silicone China 500

The selected water bottles were intended for adult and/or pediatric consumers and
were tested by simulating their typical use (preliminary cleansing and daily rinsing, filling,
and use). The simulation was carried out at room temperature (19–22 ◦ C) using organics-
free deionized water and lasted a total of four weeks. Deionized water was used to allow
the quantification, also in trace levels, of released elements/compounds that could have
been masked by the use of tap water. Indeed, tap water usually contains minerals at
various levels (from µg L−1 to mg L−1 ) and trace residues of organic compounds [27–32].
Additionally, the experiment should not be reproducible because the content of studied
elements widely vary in different drinking waters according to their geographical and
geological origin.
The experimental timetable is shown in Table 2.

Table 2. Study project and water bottle sampling plan (n = 20 double water bottles).

Rinsing and Filling

Day Sample Taking Time Starting from Filling (hour) Emptying (hour)
(hour)
1 0 0.5 1 5 8 12 24 24
2 +24 - - 5 - - - +29
3 +48 - - 5 - - - +53
4 +72 - - 5 - - - +77
5 +96 - - 5 - - - +101
6 +120 - - 5 - - - +125
7 +144 - - 5 - - - +149
8–13 every morning at 8:00 every evening at 20:00
14 +312 - - 5 - - - +317
15–20 every morning at 8:00 every evening at 20:00
21 +480 - - 5 - - - +485
22–27 every morning at 8:00 every evening at 20:00
28 +648 - - 5 - - - +653

Briefly, before first use, each bottle was rinsed five times with deionized water, emptied,
and left to dry upside down in a suspended position, preventing the neck and the inside of
the cap from touching any surface.
Separations 2021, 8, 126 4 of 13

Each day of the four weeks-long experiment, the bottles were rinsed and filled with
deionized water at 08.00 am, sampled or not according to the timetable shown in Table 2,
and emptied and left to dry upside down in a suspended position at 20:00. In particular,
the analyses were performed on samples taken six times during the first day, then once a
day during the following six days of the first week and, finally, once a day during the 14th,
21st, and 28th day.
30 mL of water were sampled at each fixed interval, 10 mL in polypropylene tubes
for elemental analysis, and 20 mL in class A borosilicate glass vials for PAEs and BP-A
determination. All samples were stored at −20 ◦ C and analyzed within one month of
sampling. Method blanks were treated as samples for subtraction of the background signal
from the reagents.

2.2. Instrument
A quadrupole inductively coupled plasma mass spectrometry (ICP-MS; 820-MS,
Bruker, Bremen, Germany) equipped with a collision–reaction interface (CRI) and a glass
nebulizer (0.4 mL min−1 ; MicroMistTM; Analytik Jena AG, Jena, Germany) were used for
multi-elemental analysis. CRI was used for As, Cr, Fe, Mn, Se, and V determination with
He and H2 (both 99.9995% purity; SOL Spa, Monza, Italy) as cell gases. ICP-MS operating
parameters were detailed previously [33–35].
A gas chromatography/ion trap mass spectrometry (GC/ITMS; Trace GC coupled
with a PolarisQ mass spectrometer, Thermo Finnigan, Bremen, Germany) equipped with a
home-made fused-silica capillary column with chemically bonded phase (SE-54, 5% phenyl-
95% dimethylpolysiloxane) was used for PAEs and BP-A determination, as previously
described [21]. The software used was Xcalibur 1.4.1.
An Arioso Power I RO-UP Scholar UV (Human Corporation, Songpa-Ku, Seoul, Korea)
system was used to produce organics-free deionized water.

2.3. Materials, Reagents, and Standard Solutions

All solutions were prepared with deionized water at 18.3 MΩ.cm resistivity. Reagents
used for the sample preparation were HNO3 (70%, super-pure), n-hexane of pesticide
grade, and NaCl of analytical grade (Carlo Erba Reagents S.r.l. Milan, Italy). Adequate
aliquots of a multi-element standard solution (1.000 ± 0.005 mg L−1 As, Al, Ba, Be, Bi, Cd,
Ce, Co, Cr, Cs, Cu, Ga, La, Li, Mn, Mo, Nb, Ni, Pb, Rb, Sb, Se, Sn, Te, Ti, Tl, U, V, W, and
Zr; 10.00 ± 0.05 mg L−1 Fe and Zn; 50.00 ± 0.25 mg L−1 P and Si; 55.00 ± 0.25 mg L−1 B
and Sr; 500.0 ± 2.5 mg L−1 K, Mg, and Na; 1000 ± 5 mg L−1 Ca and S in 5% HNO3 , VWR
International S.r.l., Milan, Italy) were used for preparing the ICP-MS calibration solutions.
Y (at 0.005 mg L−1 from 1000 ± 2 mg L−1 ; Panreac Química, Barcelona, Spain), Sc, Rh,
In, and Th (at 10 mg L−1 from 1000 ± 5 mg L−1 ; Merck KGaA, Darmstadt, Germany) in
2% HNO3 (v/v) were used as internal standards to monitor matrix effects and sensitivity
drifts of the ICP-MS instrument, as previously reported [36,37]. Before each analysis run,
the ICP-MS performance was tested using a multi-standard stock solution of Ba, Be, Ce,
Co, In, Pb, Mg, Tl, and Th (10.00 ± 0.05 mg L−1 ; Spectro Pure, Ricca Chemical Company,
Arlington, TX, USA). Water reference standard certificate (SRM 1643e trace elements in
water; National Institute of Standards and Technology, NIST; Gaithersburg) was used to
assess the elemental analysis accuracy.
PAES (di-methyl-phthalate (DMP), di-ethyl-phthalate (DEP), di-iso-butyl phthalate
(DiBP), di-butyl phthalate (DBP), di-ethyl-exyl-phthalate (DEHP), di-octyl-phthalate (DOP))
and BP-A standards were obtained from Merck Life Science S.r.l. (Milan, Italy). Anthracene,
purchased from LabService Analytical (Anzola Emilia, Bologna, Italy), was used as internal
standard. A detailed description of the method was previously reported [21].

2.4. Elemental Analysis

The determination of 40 elements’ (Al, As, B, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr, Cs, Cu,
Fe, Ga, K, La, Li, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, Sb, Se, Si, Sn, Sr, Te, Ti, Tl, U, V, W,
Separations 2021, 8, 126 5 of 13

Zn, and Zr) content in the water samples stored in the reusable bottles was carried out
using ICP-MS, in accordance with the official methods and current legislation [38]. Briefly,
each water sample (10 mL) was filtered and acidified with 0.1 mL concentrated HNO3 ,
filtered, and analyzed without further dilution. Blanks and calibration standards were also
prepared as the tested water samples.
All ICP-MS calibration curves were performed using at least five levels (including
zero) and showed a correlation coefficient of at least 0.999 and good linear relationships
across all concentration ranges studied, as verified by Mandel’s fit test. The detection limits
(LODs) for each element were calculated as three times the standard deviation of the blank
sample, which was the deionized water used for the experiment (10 replicates).
LODs values ranged from 0.0005 (Be) to 70 (Ca, K, and P) µg L−1 . The accuracy (as
trueness bias percentage), evaluated using SRM 1643e trace elements in water, resulted
in a range between 5 and 10% for all the analyzed elements, while the relative standard
deviation (repeatability) did not, in any case, exceed 10%.

2.5. PAEs and BP-A Analysis

Analytical determinations of six PAEs and BP-A in the water samples were performed
by dispersive liquid–liquid micro-extraction and GC-IT/MS analysis, as previously re-
ported [21]. Briefly, the micro-extraction was carried out using 0.2 mL n-hexane for 10 mL
water sample; the solution was then subjected to forced solvent dispersion by Vortex,
treated in an ultrasound bath for 6 min, and then centrifuged for 30 min at 4000 rpm. The
micro-extraction solvent was then accurately measured (final volume), and 1 mL was in-
jected into the GC-MS system. The calibration curves were performed in the concentration
range from 10 to 5000 µg L−1 . LODs ranged between 0.1 (DEP) and 1.2 µg L−1 (DOP).
The recoveries (%) calculated on spiked organics-free deionized water (at 20 µg L−1 for
each analyte) and on spiked real samples (at 20 and 500 µg L−1 ) resulted, respectively, in
the range 85.2 (DMP)–99.6% (DiBP), and 76.3 (DMP)–101.6% (DOP). The inter-day and
intra-day precision was always lower than 12.1%.

2.6. Data Elaboration

The results of the analytical determinations for each studied bottle were expressed,
after subtracting blank concentration, as the arithmetic mean of the pair. Additionally, in
order to compare the migration among the different bottles, we calculated the inner surface
in cm2 of each kind of bottle (neck excluded); thus, results were also expressed as µg cm−2
of each compound.

3. Results and Discussion

3.1. Main Results of the Experimental Tests
Table 3 reports the LODs together with the percentage of bottles in which the specific
analyte resulted <LOD.
Elements’ concentrations above the LODs in most water samples (>50%) occurred
for Cu, La, Li, Mg, Na, Pb, Rb, and Zn from metallic bottles and Al, Cr, Mo, and Sr from
both types of bottles (metallic and non-metallic). Ce, Nb, and Te levels were lower than
LODs for all the studied bottles. As many as 91% of analyses provided measurable Al
concentrations for metallic bottles, while for As, Cd, Se, Sn, Tl, U, and Zr, less than 10%
of analyses provided measurable levels. The comparison of our results on elemental
release with other studies is not possible because, in our knowledge, this is the first study
measuring the elemental release from reusable water bottles and similar experimental data
are not available.
Both PAEs and BP-A concentrations were always <LOD. This finding is in contrast
with those reported previously by other studies, that demonstrated the occurrence of release
phenomena from different reusable bottles (plastic baby bottles, plastic bottles, metallic
with internal lining) [21,39,40]. Indeed, several previous studies have proved general
population exposure to these additives by urinary biomonitoring studies [41,42]. Thus,
Separations 2021, 8, 126 6 of 13

over the years, BP-A and PAEs have received special attention, resulting in the reduction
or elimination of their use in water bottle manufacturing, as confirmed by our results.

Table 3. Limits of detection (LODs) (µg L−1 ) of studied compounds in water samples stored in
metallic (stainless steel and aluminum) and non-metallic bottles and % of bottles in which the specific
analyte was < LOD.

Metallic Water Bottles Non-Metallic Water Bottles

Compounds LOD
% < LOD % < LOD
Al 0.7 9 42
As 1 90 90
B 0.7 86 89
Ba 3 60 65
Be 0.0005 62 62
Bi 0.004 70 76
Ca 70 56 64
Cd 0.01 95 99
Ce 0.004 57 73
Co 0.02 75 94
Cr 0.07 31 35
Cs 0.008 100 100
Cu 0.5 43 72
Fe 2 56 77
Ga 0.004 52 91
K 70 61 63
La 0.002 34 50
Li 0.01 48 73
Mg 4 43 58
Mn 0.4 57 71
Mo 0.01 23 42
Na 10 38 68
Nb 0.005 100 100
Ni 0.9 86 88
P 70 56 52
Pb 0.04 39 59
Rb 0.02 34 61
Sb 0.009 53 76
Se 1 92 96
Si 20 56 61
Sn 0.01 96 95
Sr 0.07 20 35
Te 0.1 100 100
Ti 0.2 64 67
Tl 0.002 95 98
U 0.002 92 96
V 0.2 81 80
W 0.01 53 75
Zn 1 47 55
Zr 0.005 93 97
BP-A 1.1 100 100
DMP 0.7 100 100
DEP 0.1 100 100
DiBP 0.8 100 100
DBP 0.4 100 100
DEHP 0.6 100 100
DOP 1.2 100 100

Table 4 shows the maximum levels found for each studied compound in water samples
stored in metallic and non-metallic bottles and expressed both as µg L−1 and µg cm−2 . Cs,
Nb, Te, PAEs, and BP-A are not reported because all results were <LOD.
Separations 2021, 8, 126 7 of 13

Table 4. Maximum levels found for each studied element in water samples stored in metallic (stainless
steel and aluminum) and non-metallic bottles, expressed both as µg L−1 and µg cm−2 .

Metallic Water Bottles Non-Metallic Water Bottles

Elements Max Value Max Value Max Value Max Value
(µg L−1 ) (µg cm−2 ) (µg L−1 ) (µg cm−2 )
Al 211 0.74 30.7 0.12
As 6 0.0199 5 0.0151
B 2.61 0.0085 9.1 0.0201
Ba 79 0.268 51 0.171
Be 0.0352 0.000119 0.0648 0.000200
Bi 0.049 0.000181 0.038 0.000125
Ca 3400 12.5 1800 5.7
Cd 0.15 0.000542 0.13 0.000462
Ce 0.044 0.000141 0.022 0.000067
Co 0.51 0.00141 0.03 0.00010
Cr 1.46 0.00538 1.24 0.00384
Cu 10.5 0.0389 3.1 0.0098
Fe 23 0.085 18 0.055
Ga 0.188 0.00069 0.006 0.00002
K 664 2.45 587 1.95
La 0.026 0.000095 0.017 0.000058
Li 0.45 0.00145 0.13 0.00037
Mg 116 0.429 52 0.158
Mn 4.5 0.0165 1.5 0.0053
Mo 57.0 0.204 2.90 0.010
Na 664 1.96 111 0.44
Ni 14.3 0.0392 4.6 0.0153
P 832 2.976 1100 3.635
Pb 3.26 0.01015 0.46 0.00152
Rb 0.95 0.00352 0.17 0.00069
Sb 1.14 0.00337 0.041 0.00016
Se 4 0.01187 4 0.00999
Si 191 0.614 262 0.741
Sn 0.10 0.000384 0.15 0.000431
Sr 9.34 0.0345 1.98 0.0066
Ti 3.2 0.0109 2.0 0.0062
Tl 0.004 0.0000117 0.003 0.0000094
U 0.008 0.0000288 0.011 0.0000364
V 0.5 0.00187 0.5 0.00151
W 0.42 0.00149 0.06 0.00017
Zn 93 0.344 46 0.157
Zr 0.034 0.000122 0.038 0.000116

The highest values, ranging from tenth of mg to mg L−1 and mg cm−2 , were recorded
for Ca, K, Mg, and Na in both metallic and non-metallic bottles. Additionally, as expected,
Al showed high levels in metallic bottles when compared to other metals. Notice that,
even if in low levels, many other elements, including rare or toxic metals, were revealed in
metallic bottles, such as As, Cr, Mo, Cd, Ga, U, W, and Zr. A similar feature was found for
non-metallic bottles, although at lower concentrations, indicating that plastic and silicone
materials are also at risk for inorganic elements’ release into water. However, the levels
of each monitored analyte varied widely among the 20 different bottles. In particular,
while alkali and alkaline earth metals showed a lower variability with respect to the other
elements, some heavy metals, such as Al, Cr, Mo, Ni, Mn, and Sb, presented relevant
differences among the studied bottles, as evidenced in Figure 1.
Separations 2021, 8, 126 8 of 13

Figure 1. Mean levels of some elements in the water stored in each bottle over 24 h.

The great variability in released amounts among all the studied bottles is likely
due to the kind and quality of the materials of which they are made. This variability
occurs mostly when considering metallic bottles (bottles n. 1–13), stainless steel included
(bottle n. 7), while non-metallic ones (bottles n. 14–20) showed concentrations quite similar
to each other.
As regards the trend of the release phenomena measured over time, for the majority
of the elements, the highest concentrations were recovered during the first 24 h of the
experiment in all cases. As an example, Figure 2 shows the concentration trends over time
of some elements (Al, Ba, Co, Cr, Cu, Mo, Ni, and Sr) for the bottle that presented the
highest release for that specific analyte.
As evidenced in Figure 2, some elements, such as Ba, Ni, and Sn, were no more
released after the first 24 h, while the others continued to be, although at much lower
amounts with respect to the first day of the experiment.

3.2. Possible Consumers’ Health Risks from Reusable Water Bottles Use
The second aim of the study was to evaluate the possible consumers’ health risks
due to the use of reusable water bottles. For this purpose, we compared our results with
the regulatory limits fixed by current legislation on drinking water. In particular, we
considered the recent European Union Directive 2020/2184 on drinking water quality [43]
and, for parameters not included in this Directive, we referred to other international bodies
such as the WHO or Canadian or USEPA guidelines [44–49]. In Table 5, we reported our
results, expressed as the highest value obtained during the experiment for all the elements
found at concentrations >LOD and for which regulatory limits are currently fixed, along
with the limits themselves.
Separations 2021, 8, 126 9 of 13

Figure 2. 28 days release trend for Al, Ba, Co, Cr, Cu, Mo, Ni, Sn for the bottle that presented the highest release for the specific analyte.
Separations 2021, 8, 126 10 of 13

Table 5. Maximum levels found for each element in water samples stored in reusable water bottles
and regulatory limits fixed by current legislation, both expressed in µg L−1 .

Element Maximum Level (µg L−1 ) Limit (µg L−1 )

Al 211 200 [43]
As 6 10 [43]
B 9.1 1500 [43]
Ba 79 1300 [44]
Be 0.0648 12 [45]
Cd 0.15 5.0 [43]
Cr 1.46 25 [43]
Cu 10.5 2000 [43]
Fe 23 200 [43]
Mn 4.5 50 [43]
Mo 57.0 70 [46]
Na 664 200,000 [43]
Ni 14.3 20 [43]
Pb 3.26 5.0 [43]
Sb 1.14 10 [43]
Se 4 20 [43]
Sr 9.34 7000 [47]
Tl 0.004 2 [48]
U 0.011 30 [43]
V 0.5 140 [49]

As shown in Table 5, in one case, the highest Al concentration exceeded its regulatory
limit. Among the other studied elements, toxic heavy metals were, respectively, equal
to 3, 6, 65, and 71% of the fixed limits for Cd, Cr, Pb, and Ni. Other relevant results are
related to As (60%), Mn (9%), Mo (81%), and Sb (11%). Notice that these values refer to
the maximum concentration levels found across the experiment. However, this finding
cannot be neglected for two main reasons: first, the use of reusable bottles contributes
to the consumers’ total intake of many elements, some of them recognized as a risk for
human health. Secondly, these bottles are made for drinking water, which usually contains
variable amounts of minerals, including those released by the studied metallic and non-
metallic bottles. Thus, the final concentration will be determined by the sum of the two
contributions, which could also exceed the regulatory limit.
This study had some limitations. Firstly, we used organics-free deionized instead of
drinking water for the experiments, which does not fully represent a real world scenario;
however, in this way, we were able to measure even small amounts of released elements
and made the experiment reproducible. Second, the number of studied bottles is not very
high, even if they were the most popular on the market. Additionally, our experiment lasted
28 days, while generally, people use reusable water bottles for a longer time. Nevertheless,
the release phenomena decreased after the first days of use, becoming negligible at the end
of our study in all cases.
4. Conclusions
Our findings demonstrated that all the studied reusable bottles (aluminum, stainless
steel, plastic, and silicone) release inorganic elements at variable amounts, while they
do not release PAEs and BP-A at detectable levels. This result is of relevance for human
health, especially for highly susceptible populations such as infants, children, and pregnant
women. Thus, it is essential to perform some public health interventions aimed to inform
the consumers. First, the general population should be correctly informed to making an
informed choice when deciding to use a water bottle. In addition, these bottles should be
accompanied by an exhaustive label, reporting the maintenance criteria and the recommen-
dation to not use the bottle with beverages other than drinking water. Indeed, sparkling
water and carbonated soft drinks should increase release phenomena due to their lower
pH. Additionally, fat substances contained in fruit juices or alcohol should extract organic
compounds from the inner of the bottles.
Separations 2021, 8, 126 11 of 13

Author Contributions: Conceptualization, C.P. and M.V.; methodology, M.V. and C.P.; software,
M.L.A. and F.C.; validation, C.P. and M.V.; formal analysis, M.L.A., F.C., A.A. and P.A.; investiga-
tion, M.L.A., F.C., A.A. and P.A.; resources, M.V., C.P. and S.C.; data curation, C.P., S.C. and A.A.;
writing—original draft preparation, M.L.A., A.A., F.C. and P.A.; writing—review and editing, C.P.
and M.V.; visualization, C.P.; supervision, M.V. and C.P.; project administration, C.P., M.V., P.A.
and S.C.; funding acquisition, M.V. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was partially funded by Fondazione Acqua, Rome, Italy.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

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