Lc and nmr studies for identification and characterization of degradation byproducts of olmesartan acid, elucidation of their degradation pathway and ecotoxicity assessment

Article abstract of DOI:10.3390/molecules26061769

The discovery of various sartans, which are among the most used antihypertensive drugs in the world, is increasingly frequent not only in wastewater but also in surface water and, in some cases, even in drinking or groundwater. In this paper, the degradation pathway of olmesartan acid, one of the most used sartans, was investigated by simulating the chlorination process normally used in a wastewater treatment plant to reduce similar emerging pollutants. The structures of nine isolated degradation byproducts (DPs), eight of which were isolated for the first time, were separated via chromatography column and HPLC methods, identified by combining nuclear magnetic resonance and mass spectrometry, and justified by a proposed mechanism of formation beginning from the parent drug. Ecotoxicity tests on olmesartan acid and its nine DPs showed that 50% of the investigated byproducts inhibited the target species Aliivibrio fischeri and Raphidocelis subcapitata, causing functional decreases of 18% and 53%, respectively.

Full text of DOI:10.3390/molecules26061769

molecules
Article
LC and NMR Studies for Identification and Characterization of
Degradation Byproducts of Olmesartan Acid, Elucidation of
Their Degradation Pathway and Ecotoxicity Assessment
Giovanni Luongo 1 , Antonietta Siciliano 2 , Giovanni Libralato 2 , Sara Serafini 2, Lorenzo Saviano 2,
Lucio Previtera 3, Giovanni Di Fabio 1 and Armando Zarrelli 1,
*
1
Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy;
giovanni.luongo@unina.it (G.L.); difabio@unina.it (G.D.F.)
Department of Biology, University of Naples Federico II, 80126 Naples, Italy;
antonietta.siciliano@unina.it (A.S.); giovanni.libralato@unina.it (G.L.); sara.serafini@unina.it (S.S.);
lorenzosaviano@libero.it (L.S.)
2
3
Associazione Italiana per la Promozione delle Ricerche su Ambiente e Salute Umana, 82030 Dugenta, Italy;
previter@unina.it
*
Correspondence: zarrelli@unina.it; Tel.: +39-081-674472
Abstract: The discovery of various sartans, which are among the most used antihypertensive drugs
in the world, is increasingly frequent not only in wastewater but also in surface water and, in some
cases, even in drinking or groundwater. In this paper, the degradation pathway of olmesartan acid,
one of the most used sartans, was investigated by simulating the chlorination process normally
used in a wastewater treatment plant to reduce similar emerging pollutants. The structures of
nine isolated degradation byproducts (DPs), eight of which were isolated for the first time, were
separated via chromatography column and HPLC methods, identified by combining nuclear magnetic
resonance and mass spectrometry, and justified by a proposed mechanism of formation beginning
from the parent drug. Ecotoxicity tests on olmesartan acid and its nine DPs showed that 50% of
the investigated byproducts inhibited the target species Aliivibrio fischeri and Raphidocelis subcapitata,
causing functional decreases of 18% and 53%, respectively.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Luongo, G.; Siciliano, A.;
Libralato, G.; Serafini, S.; Saviano, L.;
Previtera, L.; Di Fabio, G.; Zarrelli, A.
LC and NMR Studies for
Identification and Characterization of
Degradation Byproducts of
Olmesartan Acid, Elucidation of Their
Degradation Pathway and Ecotoxicity
Assessment. Molecules 2021, 26, 1769.
https://doi.org/10.3390/molecules
26061769
Keywords: olmesartan acid; chlorination; hypochlorite; degradation byproducts; water treatment;
Aliivibrio fischeri; Raphidocelis subcapitata
Academic Editor: Svend
Borup Jensen
Received: 25 February 2021
Accepted: 19 March 2021
Published: 22 March 2021
1. Introduction
Keeping water clean and healthy is difficult, but not impossible. The important thing
is to define which substances actually constitute a danger and a risk to the health of humans
and other species, to establish which ones must leave production cycles to be eliminated
from the environment, and to determine which ones must be kept in concentrations below
certain hazardous limits. In water, new substances, in addition to being highly persistent
contaminants, carry possible health and environmental effects. Pollutants of emerging
interest are considered one of the most significant environmental problems in recent years.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
These include perfluoroalkyl substances (PFAS) [
mones [ ], psychoactive substances [ ], pesticides [
and drugs [ ]. These substances have the potential to cause adverse effects on the environ-
ment and human health, but are still largely not specifically regulated by legislation, and
their effects are not yet clear [ 15]. At the local level, pollution could affect different types
of substances—and also byproducts or reaction products—so much so that an aggregate
and cumulative risk assessment is required that takes into account the multiplicity of
exposures. The effluents of wastewater treatment plants (WWTPs), not normally designed
to eliminate emerging contaminants, are one of the main sources of contamination of sur-
face waters by micropollutants [16]. Hundreds of tons of pharmaceutical substances flow
1
], cyanobacteria [
2], mycotoxins [3], hor-
4
5
6], cosmetics, and industrial additives
7
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
8–
Molecules 2021, 26, 1769. https://doi.org/10.3390/molecules26061769
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1769
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to WWTPs every year, hence the need to implement WWTPs with advanced treatments,
tertiary wastewater treatments, capable of improving removal efficiencies. Advanced
wastewater treatment processes are the subject of numerous studies. Technologies such
as ozonation [17–19], membrane filtration [20], adsorption [21] and above all advanced
oxidation [22], are configured as systems capable of improving the removal of emerging
contaminants from wastewater.
Sartans, among the most marketed antihypertensive drugs in the world, are now
considered ubiquitous emerging contaminants in many countries [23]. In particular, olme-
sartan acid (OLM) is one of these [24]. It is marketed as olmesartan medoximil, the prodrug
from which the active pharmaceutical form, olmesartan acid, is released in the intesti-
nal mucosa and rapidly enters the bloodstream [25,26]. However, the olmesartan acid is
released as-is through feces and urine. In recent studies, carried out in some European
countries, it was found that the concentration of olmesartan acid in wastewater reaches
up to 1200 ng/L [23], but in surface waters ranges from 150 ng/L to 800 ng/L, with peaks
above 2
groundwater [27].
The advanced oxidation processes while being effective in the removal of microp-
µg/L in some German rivers [24]. However, the concentrations are lower in
ollutants, if not carried out properly, can lead to the formation of intermediate reaction
products, often more toxic than the starting compounds [28–33].
In this paper, the degradation byproducts (DPs) of OLM were investigated under the
same conditions as the chlorination process normally used in WWTPs to reduce similar
emerging pollutants [24,29], carrying out two different experiments, one at concentrations
comparable to those at which OLM is present in the wastewaters (10−5 M) and one at
concentrations at least 100 times higher in order to isolate and identify the DPs. The
structures of 9 isolated DPs, eight of which were isolated for the first time, have been
determined by combining nuclear magnetic resonance (NMR) and mass spectrometry (MS),
using matrix-assisted laser desorption/ionization as a source and a time-of-flight analyzer
(MALDI-TOF) for mass analysis. These methods were justified by a proposed mechanism
of formation.
The evaluation of OLM degradation and the formation of by-products was subjected to
ecotoxicological tests performed with Aliivibrio fischeri bacteria and Raphidocelis subcapitata
algae. In order to monitor the performance of OLM degradation, the ecotoxicological assays
represent an advantageous technique for providing information about environmental risk
regarding treated effluent discharge or even in subsequent/tertiary treatments.
2. Materials and Methods
2.1. Drug and Reagents
Olmesartan acid (99.5%) was purchased from Sigma Aldrich (Milan, Italy). All the
other chemicals and solvents were purchased from Fluka (Saint-Quentin Fallavier, France)
and were of HPLC grade and used as received. For the antimicrobial assessment, tryptic soy
broth (TSB, Difco, Becton-Dickenson Labs) was used. All chemicals were of analytical grade
and supplied by Sigma Aldrich. Double-distilled water (Microtech) was used to prepare
dilution water and the treatments. Microbial growth was measured with an automatic
plate reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA).
2.2. Chlorination Reaction
2.2.1. Apparatus and Equipment
Column chromatography (CC) was carried out with Kieselgel 60 (230–400 mesh,
Merck, Darmstadt, Germany). HPLC was performed on a Shimadzu LC-8A system using
a Shimadzu SPD-10A VP UV-VIS detector (Shimadzu, Milan, Italy). Preparative HPLC
was performed using an RP Gemini C18-110A preparative column (10 µm particle size,
250
×
21.2 mm i.d., Phenomenex, Bologna, Italy) with a flow rate of 7.0 mL/min. The 1H-
and 13C-NMR spectra were recorded with an NMR spectrometer operated at 400 MHz and
◦
at 25 C (Bruker DRX, Bruker Avance, Billica, MA, USA) and referenced in ppm to the

Molecules 2021, 26, 1769
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residual solvent signals (CDCl3, at δH 7.27 and δC 77.0). The proton-detected heteronuclear
correlations were measured using a gradient heteronuclear single-quantum coherence
(HSQC) experiment, optimized for 1JHC = 155 Hz, and a gradient heteronuclear multiple
bond coherence (HMBC) experiment, optimized for nJHC = 8 Hz. The MALDI TOF mass
spectrometric analyses were performed on a Voyager-De Pro MALDI mass-spectrometer
(PerSeptive Biosystems, Framingham, MA, USA).
2.2.2. Chlorination Experiments
A 10−5 M OLM solution was treated for 10 min with 10% hypochlorite (molar ratio
OLM/HClO 1:1 concentration, spectroscopically determined
λ
max
292 nm, ε
350 dm3/mol
cm) at room temperature [34], simulating the conditions used in a typical WWTP. The
experiment was conducted at pH = 10.5. The presence of OLM was quantified using a
Lambda 12 UV-Vis spectrophotometer (Perkin Elmer, Waltham, MA, USA). Absorbance
peaks were determined at 230 nm. The absorbance values were converted into a con-
centration using a calibration curve prepared from standard solutions with known OLM
concentrations. The pH of the solution, measured and recorded continuously using a
pH-meter, increased immediately from the initial pH of 8.0 to 10.5, and pH remained at this
value during the reaction. An aliquot of the solution was taken every 15 min, quenched by
sodium thiosulphate excess, filtered, dried by lyophilization, and the residue dissolved in
a saturated sodium bicarbonate solution and extracted with ethyl acetate. The course of
the reaction was monitored using HPLC. DP1–DP9 were isolated from the ethyl acetate
extract of the aqueous solution (Scheme 1 and Figure 1) and identified by comparing their
retention times with those of commercially available standard compounds, or isolated by
performing preparative experiments with a solution of OLM at a concentration higher than
10−3 M and treated with 6% hypochlorite at room temperature for 2 h. The DPs obtained
were isolated using column chromatography and HPLC and were completely characterized
using NMR and MS analyses.
2.2.3. Chlorination Procedure and Product Isolation
Olmesartan acid (1.0 g, 2.24 mmol) was dissolved in 1.0 L of phosphate buffer
(KH2PO4/K2HPO4 0.1 M) [35]. A sodium hypochlorite solution (about 6% active chlo-
rine, molar ratio OLM/HClO 1:20; concentration spectroscopically determined at
λmax of
292 nm, cm) was added drop-by-drop to this solution under magnetic
ε
= 350 dm3/mol
×
stirring at room temperature. The pH of phosphate buffer was adjusted at a value of 6.50
by adding a 10% H3PO4 solution, checked with a pH-meter. The reaction was stopped
after 2 h with an excess of sodium thiosulphate and concentrated by lyophilization. The
residue was dissolved in water and pH adjusted to 7.00, and this solution was extracted
using ethyl acetate (EA). The crude EA fraction (579 mg) was chromatographed on sil-
ica gel CC, eluting with gradient of methylene chloride:methanol:acetic acid (100:0:0.5
to 70:30:0.5, v/v/v) to yield 16 fractions. The fraction EA3 (16 mg), eluted with methy-
lene chloride/methanol/acetic acid (100:0:0.5, v/v/v), was analyzed via HPLC using
a reversed-phase column and eluting with water/acetonitrile (20:80, v/v) to yield DP6
(6.7 mg). The fraction EA4 (30 mg), eluted with methylene chloride:methanol:acetic acid
(98:2:0.5, v/v/v), was separated by semipreparative HPLC using a reversed-phase column
Kromasil 10
µ
m 100 Å C18 (250 × 10 mm) and elution with a gradient of CH3COONH4 (A,
pH 4.0; 10 mM) and methanol (B), starting with 70% B for 1 min and installing a gradient
to obtain 100% B over 20 min at a solvent flow rate of 4 mL/min to yield DP2 (7.8 mg).
The fraction EA6 (36 mg), eluted with methylene chloride:methanol:acetic acid (95:5:0.5,
v/v/v), was analyzed via preparative HPLC using a reversed-phase column Gemini 10 µm
C18 110 Å (250 × 21 mm) and elution with a gradient of CH3COONH4 (A, pH 4.0; 10 mM)
and methanol (B), starting with 60% B for 1 min and installing a gradient to obtain 100% B
over 30 min at a solvent flow rate of 7.5 mL/min to yield 5 subfractions.
The subfraction EA6.3 (7 mg) was re-chromatographed via HPLC using a reversed-
phase column Luna 5
µm 100 Å C18(2) (150
×
4.6 mm) and elution with a gradient of

Molecules 2021, 26, 1769
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CH3COONH4 (A, pH 4.0; 10 mM) and acetonitrile (B), starting with 60% B for 1 min and
installing a gradient to obtain 100% B over 30 min at a solvent flow rate of 1 mL/min to yield
DP8 (1.3 mg). The fraction EA9 (13 mg), eluted with methylene chloride:methanol:acetic
acid (90:10:0.5, v/v/v) was separated via preparative HPLC using a reversed-phase column
Gemini 10
µ
m C18 110 Å (250
×
21 mm) and elution with a gradient of CH3COONH4
(A, pH 4.0; 10 mM) and methanol (B), starting with 50% B for 1 min and installing a
gradient to obtain 100% B over 30 min at a solvent flow rate of 7.5 mL/min to yield
3 subfractions. The subfraction EA9.1 (15 mg) was re-chromatographed via HPLC using
a reversed-phase column Kinetex 2.6
µ
m 100 Å C18 (100
×
4.6 mm) and elution with a
gradient of CH3COONH4 (A, pH 4.0; 10 mM) and acetonitrile (B), starting with 40% B
for 1 min and installing a gradient to obtain 100% B over 30 min at a solvent flow rate
of 0.8 mL/min to yield DP9 (0.9 mg). The fraction EA10 (30 mg), eluted with methylene
chloride:methanol:acetic acid (85:15:0.5, v/v/v), was analyzed via semipreparative HPLC
using a reversed-phase column Kromasil 10
µ
m 100 Å C18 (250
×
10 mm) and elution
with a gradient of CH3COONH4 (A, pH 4.0; 10 mM) and methanol (B), starting with
35% B for 1 min and installing a gradient to obtain 100% B over 20 min at a solvent
flow rate of 4 mL/min to yield 4 subfractions. The subfractions EA10.2 (7 mg) and
EA10.3 (9 mg) were re-chromatographed by HPLC using a reversed-phase column Kinetex
2.6
µ
m 100 Å C18 (100
×
4.6 mm) and elution with a gradient of acetic acid/acetonitrile
(A, 1:99, v/v) and acetic acid/water (B, 1:99, v/v), starting with 75% B for 3 min and
installing a gradient to obtain 100% A over 35 min and returning to 75% B for 10 min,
at a solvent flow rate of 0.8 mL/min. They contained DP7 (2.6 mg) and DP5 (5.9 mg),
respectively. The fraction EA11 (63 mg), eluted with methylene chloride:methanol:acetic
acid (80:20:0.5, v/v/v), was analyzed via preparative HPLC using a reversed-phase column
Gemini 10
µ
m C18 110 Å (250
×
21 mm) and elution with a gradient of CH3COONH4
(A, pH 4.0; 10 mM)) and acetonitrile (B), starting with 20% B for 1 min and installing a
gradient to obtain 90% B over 25 min at a solvent flow rate of 8 mL/min to yield DP1
(7.1 mg). The fraction EA14 (120 mg), eluted with methylene chloride:methanol:acetic acid
(70:30:0.5, v/v/v), was re-chromatographed on silica gel CC by elution with a gradient of
chloroform:acetone:acetic acid (100:0:0.5 to 60:40:0.5, v/v/v) to yield 10 subfractions. The
subfraction EA14.7 (33 mg), eluted with chloroform:acetone:acetic acid (70:30:0.5, v/v/v),
was separated by semipreparative HPLC using a reversed phase column Kromasil 10
µm
100 Å C18 (250 10 mm) and elution with a gradient of CH3COONH4 (A, pH 4.0; 10 mM)
×
and methanol (B), starting with 0% B for 5 min and installing a gradient to obtain 100% B
over 20 min at a solvent flow rate of 3.5 mL/min to yield DP3 (11.6 mg) and DP4 (6.6 mg).
Scheme 1. The isolation of the different identified compounds.

Molecules 2021, 26, 1769
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Figure 1. Chemical structures of olmesartan acid and its degradation byproducts DP1–DP9.
2.3. Ecotoxicity Data
The acute bioluminescence test with Aliivibrio fischeri (NRRLB-11177) was carried out
in accordance with the ISO 11348-3:2007 [36] standard protocol, and the algal growth inhi-
bition test with Raphidocelis subcapitata was performed according to the ISO 8692:2012 [37
]
standard protocol. These organisms are established biological models and are included in
most regulations for the assessment of wastewater on an end-of-pipe basis.
The inhibition of bioluminescence in the presence of the OLM and its DPs was mea-
sured after 30 min of exposure. The toxic effect values are given by the ratio of the decrease
in bacterial light output emitted by the bacterium in the sample compared to the control.
To provide the relevant osmotic pressure for the test organisms, the salinity concentration
of the stock solution was adjusted by 2% for NaCl. The temperature during exposure was
15 ◦C according to the Microtox standard procedure.
The growth of algae exposed to the sample was compared with the growth of algae
in a negative control. For each sample, three replicates were inoculated with 104 algal
◦
cells L−1 in well plates and incubated for 72 h at 23
±
2 C under continuous illumina-
tion. The specific growth rate of R. subcapitata in each replicate was calculated from the
logarithmic increase in cell density in the interval from 0 to 72 h. R. subcapitata density
was determined by an indirect procedure using a spectrophotometer (Hach Lange DR5000,
Loveland, CO, USA) and a 1 cm cuvette.
3. Results and Discussion
3.1. Chlorination Experiments
The OLM chlorination experiments were performed by mimicking the conditions of a
typical WWTP, in which a 10−5 M solution of the drug was treated for 10 min with 10%
hypochlorite (OLM:hypochlorite molar ratio of 1:1; concn.) at room temperature [38,39].
Then, the tests are repeated at much higher concentrations of the contaminant (>10−3 M),
with a much lower ratio of OLM:oxidizing agent (1:5 or 1:6), so as to ensure the degra-
dation of the studied contaminant and the possibility of isolating sufficient quantities of
degradation byproducts for the subsequent structural identification.

Molecules 2021, 26, 1769
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The course of the reaction was monitored by HPLC, and the DPs obtained were iso-
lated by column chromatography and HPLC (Scheme 1) and completely characterized
using NMR and MS analyses (see Supplementary Materials). Finally, DP1–DP9 (Figure 1
)
were isolated relative percentages of 1.23, 1.35, 2.01, 1.15, 1.02, 1.15, 0.45, 0.23, and 0.15, re-
spectively. The proposed mechanism of their formation from OLM is shown in Figure 2a,b
.
Except for DP9, all other DPs were isolated for the first time.
3.2. Structure Elucidation of Degradation Byproducts DP1–DP9
In the OLM treatment at the buffered pH, the concentration of DP1–DP9 was at a
maximum after 2 h and in the range of 2.01 to 0.15%. The plausible mechanism of the
DPs formation from OLM is shown in Figure 2a,b. Olmesartan acid could undergo the
oxidation of the side-chain at carbon C23 to obtain intermediate I1, whose subsequent
decarboxylation at carbon C22 generates intermediate I2. The reaction of this with HClO
could generate intermediate I3 from which intermediate I4 can be obtained via nucleophilic
attack of the CH3O− ion [40
–42]. The oxidation at carbon C23 (intermediate I5) and C24
(intermediate I6) and the chlorination at carbon C22 could provide DP5. Intermediate I6,
via its reaction with HClO, provides intermediate I7, which due to the loss of HCl would
provide intermediate I8. From I8, the oxidation of DP4 could be obtained. Olmesartan acid
could undergo the hydrolysis of the N19-C23 and C20-N21 bonds to release intermediate
I9, from which intermediate I10 could then be obtained via oxidation of the side-chain,
precisely on the carbon adjacent to the carbonyl function. The complete oxidation of the
I10 side-chain would provide I11, and from the nitrogen oxidation of the latter, DP8 could
be obtained. The partial oxidation of the C22 carbon of the starting product could provide
intermediate I12, which via the decarboxylation at carbon C23 would provide intermediate
I13. The chlorination of this could provide DP1. DP9 could be obtained from different
precursors with the loss of all the substituents of the imidazole ring.
The intermediate I2 could be chlorinated at carbons C22 and C23 to provide the
intermediate I14, which could then provide DP2 via oxidation of the side-chain to carbon
C24. Intermediate I3, by replacing the chlorine with OH−, could provide the intermediate
I15 which then, via the oxidation and opening of the imidazole ring, could provide DP7
through intermediate I16. DP7 could then provide intermediate I17 via oxidation of the
adjacent carbon of the carbonyl function of the side-chain, and then lactone DP6 could be
obtained via its intramolecular reaction.
Intermediate I5 could provide the corresponding I19 by reaction with HClO. The loss
of HCl (intermediate I20) and subsequent oxidation at carbon C22 could yield DP3. The
oxidation of the latter to the C-24 carbon of the side chain could provide DP4.

Molecules 2021, 26, 1769
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(a)
Figure 2. Cont.

Molecules 2021, 26, 1769
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(b)
) Plausible mechanism for the formation of DP1, DP4, DP5, and DP8–DP9. (b) Plausible mechanism for the
Figure 2.
(a
formation of DP2–DP4, DP6–DP7.
3.3. Spectral Data
0
Olmesartan medoximil: (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 1-((2 -(2H-tetrazol-5-yl)-
[1,10-biphenyl]-4-yl)methyl)-4-(2-hydroxypropan-2-yl)-2-propyl-1H-imidazole-5-car boxy-
late. White powder. H- and 13C-NMR, see Table S1.
1
0
0
Olmesartan acid: 1-((2 -(2H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-4-(2-hydroxypropan-
2-yl)-2-propyl-1H-imidazole-5-carboxylic acid. H- and 13C-NMR, see [43]. MS-TOF (positive
1
ions): m/z calculated for C24H26N6O3 m/z 446.21 [M]+; found 447.25 [M + H]+ (63%).
DP-1: 1-((20-(2H-tetrazol-5-yl)-[1,10-biphenyl]-4-yl)methyl)-5-chloro-2-propyl-1H–imidazo
le-4-carboxylic acid. White powder. H- and 13C-NMR, see Table S2. MS-TOF (positive ions):
1
m/z calculated for C21H19ClN6O2 m/z 422.13 [M]+; found 426.16 [M + 3 + H]+ (6%), 425.16
[M + 2 + H]+ (30%), 424.16 [M + 1 + H]+ (15%), 423.15 [M + H]+ (90%).
DP-2: 5-(40-((4,5-dichloro-2-(1-methoxypropyl)-1H-imidazol-1-yl)methyl)-[1,10-biphenyl]-
2-yl)-2H-tetrazole. White powder. 1H- and 13C-NMR, see Table S3. MS-TOF (positive
ions): m/z calculated for C21H20Cl2N6O m/z 442.11 [M]+; found 448.13 [M + 5 + H]+ (1%),

Molecules 2021, 26, 1769
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447.16 [M + 4 + H]+ (15%), 446.16 [M + 3 + H]+ (15%), 445.16 [M + 2 + H]+ (61%), 444.17
[M + 1 + H]+ (21%), 443.15 [M + H]+ (095%).
0
DP-3: 1-((2 -(2H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-methoxy-2-propyl imidazolidi-
1
ne-4,5-dione. White powder. H- and 13C-NMR, see Table S4. MS-TOF (positive ions): m/z
calculated for C21H22N6O3 m/z 406.108 [M]+; found 407.21 [M + H]+ (81%).
0
DP-4: 1-((2 -(2H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-(1-hydroxypropyl)-2-methoxyi-
midazolidine-4,5-dione. White powder. H- and 13C-NMR, see Table S5. MS-TOF (positive
1
ions): m/z calculated for C21H22N6O4 m/z 422.17 [M]+; found 423.20 [M + H]+ (73%).
DP-5: 3-((20-(2H-tetrazol-5-yl)-[1,10-biphenyl]-4-yl)methyl)-5-chloro-2-(1-hydroxypropyl)-
2-methoxyimidazolidin-4-one. White powder. 1H- and 13C-NMR, see Table S6. MS-TOF
(positive ions): m/z calculated for C21H23ClN6O3 m/z 442.15 [M]+; found 443.19 [M + H]+
(80%), 445.18 [M + 2 + H]+ (25%).
DP-6: 3-((20-(2H-Tetrazol-5-yl)-[1,10-biphenyl]-4-yl)methyl)-5-ethyloxazolidine-2,4-dione.
1
White powder. H- and 13C-NMR, see Table S7. MS-TOF (positive ions): m/z calculated
for C19H17N5O3 m/z 363.13 [M]+; found 364.18 [M + H]+ (71%).
DP-7: ((20-(2H-Tetrazol-5-yl)-[1,10-biphenyl]-4-yl)methyl)(butyryl)carbamic acid. White
powder. 1H- and 13C-NMR, see Table S8. MS-TOF (positive ions): m/z calculated for
C19H19N5O3 m/z 365.15 [M]+; found 366.19 [M + H]+ (57%).
DP-8: ((20-(2H-Tetrazol-5-yl)-[1,10-biphenyl]-4-yl)methyl)(hydroxy)carbamic acid. White
powder. 1H and 13C-NMR, see Table S9. MS-TOF (positive ions): m/z calculated for
C15H13N5O3 m/z 311.10 [M]+; found 312.14 [M + H]+ (29%).
DP-9: 1H-imidazole. Identified by comparison with an authentic sample.
3.4. Ecotoxicity Data
Toxicity data were summarized in Figure 3, including the effects of R. subcapitata 72 h
and A. fischeri after 30 min. The analysis of toxicity data evidenced the presence of the three
main groups of samples considering a threshold value for the effects statistically different
from the control groups [44]: (i) no effect (
effect (inhibition < −10%); and (iii) toxic effects (inhibition > 10%).
−
10%
≤
inhibition
≤
10%); (ii) biostimulation
Figure 3. The toxicity data of olmesartan acid (OLM) and its byproducts (DP1–DP9) with 72 h
R. subcapitata (R_s) and A. fischeri (A_f).
After 72 h of exposure, OLM presented no effect on R. subcapitata. Comparing OLM
and DPs results, only DP6 showed no toxicity. Several samples presented negative growth
effects (DP1, DP2, DP3, DP7, DP8, DP9), and their toxicity values ranged from 18% to a
maximum of 53%. DP4 and DP5 showed stimulatory effects. Toxicity data from A. fischeri
confirmed that the parent compound OLM showed no toxic effect after 30 min of exposure.

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Five of nine isolated DPs (DP3, DP4, DP5, DP6 and DP9) showed no inhibitory effect.
The other DPs presented acute toxicity like DP1, DP2, DP7, and DP8 showing inhibition
effects from 20% to 34%. Comparing the results of the algal growth inhibition with bacteria
luminescence inhibition, it was evident that the response of A. fischeri was less sensitive,
but the toxicity trends were linearly correlated (Pearson correlation, r = 0.61, moderately
high correlation).
4. Conclusions
This paper investigated the fate of OLM following the degradation treatment by chlori-
nation. The reaction was carried out by simulating the conditions of a typical WWTP using
excess sodium hypochlorite. After the chlorination treatment, chromatographic techniques
were used to isolate nine degradation byproducts, which were fully characterized by MS
and NMR analyses and via comparison with a commercial standard. OLM underwent
complete mineralization in about 59% of cases, and in 21% of cases was recovered as is.
OLM transformed into the corresponding byproducts in 20% of cases, and about 9% of
these were identified. A possible mechanism for the degradation of OLM and its degrada-
tion byproducts has been hypothesized. Half of the investigated DPs possessed anywhere
from slightly to highly toxic effects on the target species Aliivibrio fischeri and Raphidocelis
subcapitata; the remaining DPs presented no such effects. According to the selected battery
of toxicity, the correlation of the results suggested that DPs acted very similarly in unicel-
lular organisms. Moreover, due to the highlighted ecotoxicological effects, DPs acting on
organisms of low levels of complexity could have negative effects also on the whole trophic
chain (biomagnification).
1
Supplementary Materials: The following are available online, Table S1: H, 13C and 2D NMR data
of Olmesartan medoximil in CD 3 OD; Table S2: 1H, 13C and 2D NMR data of DP-1 in CD 3 OD;
1
1
Table S3: H, 13C and 2D NMR data of DP-2 in CD 3 OD; Table S4: H, 13C and 2D NMR data of DP-3
in CD 3 OD; Table S5: H, 13C and 2D NMR data of DP-4 in CD 3 OD; Table S6: H, 13C and 2D NMR
1
1
data of DP-5 in CD 3 OD; Table S7: H, 13C and 2D NMR data of DP-6 in CD 3 OD; Table S8: H, 13
C
1
1
and 2D NMR data of DP-7 in CD 3 OD; Table S9: H, 13C and 2D NMR data of DP-8 in CD 3 OD.
1
Author Contributions: G.L. (Giovanni Luongo) performed the chlorination experiments; S.S., A.S.,
G.L. (Giovanni Libralato) and L.S. performed the acute and chronic toxicity tests; L.P. and G.D.F.
supervision and writing—review and editing; A.Z. designed the research study, supervision, writing—
original draft preparation and wrote the last version of manuscript. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was supported by AIPRAS-Onlus (Associazione Italiana per la Promozione
delle Ricerche sull’Ambiente e la Salute umana) for the grants in support of this investigation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of financial interests.
Sample Availability: Samples of the compounds are not available from the authors.
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