Ecotoxic effects of loratadine and its metabolic and light-induced derivatives

Article abstract of DOI:10.1016/j.ecoenv.2018.11.116

Loratadine and desloratadine are second-generation antihistaminic drugs. Because of human administration, they are continuously released via excreta into wastewater treatment plants and occur in surface waters as residues and transformation products (TPs). Loratadine and desloratadine residues have been found at very low concentrations (ng/L) in the aquatic environment but their toxic effects are still not well known. Both drugs are light-sensitive even under environmentally simulated conditions and some of the photoproducts have been isolated and characterized. The aim of the present study was to investigate the acute and chronic ecotoxicity of loratadine, desloratadine and their light-induced transformation products in organisms of the aquatic trophic chain. Bioassays were performed in the alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and in two crustaceans, Thamnocephalus platyurus and Ceriodaphnia dubia. Loratadine exerted its acute and chronic toxicity especially on Ceriodaphnia dubia (LC50: 600 μg/L, EC50: 28.14 μg/L) while desloratadine showed similar acute toxicity among the organisms tested and it was the most chronically effective compound in Ceriodaphnia dubia and Pseudokirchneriella subcapitata. Generally, transformation products were less active in both acute and chronic assays.

Full text of DOI:10.1016/j.ecoenv.2018.11.116

EcotoxicologyandEnvironmentalSafety170(2019)664–672
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety
journal homepage: www.elsevier.com/locate/ecoenv
Ecotoxic effects of loratadine and its metabolic and light-induced derivatives
⁎
Maria Rosaria Iescea, , Margherita Lavorgnab, Chiara Russob, Concetta Piscitellib,
⁎
Monica Passanantic, Fabio Temussia, Marina DellaGrecaa, Flavio Cermolaa, Marina Isidorib,
a Dipartimento di Scienze Chimiche, Università Federico II, Via Cintia, 4, I-80126 Napoli, Italy
b Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università della Campania “Luigi Vanvitelli”, Via Vivaldi 43, I-81100 Caserta, Italy
c Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, Gustaf Hällströmin katu 2a, FI-00014, University of Helsinki, Finland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Loratadine and desloratadine are second-generation antihistaminic drugs. Because of human administration,
they are continuously released via excreta into wastewater treatment plants and occur in surface waters as
residues and transformation products (TPs).
Antihistaminic drug
Loratadine
Desloratadine
Acute toxicity
Chronic toxicity
Photoproducts
Loratadine and desloratadine residues have been found at very low concentrations (ng/L) in the aquatic
environment but their toxic effects are still not well known. Both drugs are light-sensitive even under en-
vironmentally simulated conditions and some of the photoproducts have been isolated and characterized. The
aim of the present study was to investigate the acute and chronic ecotoxicity of loratadine, desloratadine and
their light-induced transformation products in organisms of the aquatic trophic chain. Bioassays were performed
in the alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and in two crustaceans,
Thamnocephalus platyurus and Ceriodaphnia dubia. Loratadine exerted its acute and chronic toxicity especially on
Ceriodaphnia dubia (LC50: 600 µg/L, EC50: 28.14 µg/L) while desloratadine showed similar acute toxicity among
the organisms tested and it was the most chronically effective compound in Ceriodaphnia dubia and
Pseudokirchneriella subcapitata. Generally, transformation products were less active in both acute and chronic
assays.
1. Introduction
and Kronberg, 2009; Radjenović et al., 2009; Valcarcel et al., 2011) and
due to their low polarity and scarce volatility, they may represent a
Drugs are continuously released as mixtures of parent compounds
and metabolites and enter the aquatic environment through hospital
and municipal wastewaters. Here, these mixtures may undergo trans-
formations due to redox or light-induced reactions, hydrolysis, and
other reactions leading to transformation products, in some cases more
harmful than parent compounds (DellaGreca et al., 2014; Passananti
et al., 2015; Isidori et al., 2016). The importance of these events in the
breakdown of drugs has stimulated a large number of research studies
concerning kinetics, degradation mechanism, isolation and toxicity of
the transformation products (Lambropoulou and Nollet, 2014). Gen-
erally, the most commonly occurring drugs in the aquatic systems are
the most administered. However, some classes of drugs highly utilized
by patients are not detected in the waters because rapidly degraded,
while in some cases drugs less utilized are detected at high con-
centrations because resistant to biodegradation. Among the most ad-
ministered drugs, antihistamines are detected in surface waters because
of their poor removal by conventional wastewater treatments (Kosonen
hazard for the aquatic ecosystem (Berninger and Brooks, 2010;
Kristofco and Brooks, 2017).
Among antihistamines, ranitidine, difenidramine, cimetidine and
loratadine are the most detected in the effluents of sewage treatment
plants and the detection of loratadine in surface waters has exceeded
therapeutic hazard values (THVs) showing the need of understanding
the aquatic toxicology, hazards and risks associated with this drug
(Kristofco and Brooks, 2017). Loratadine is a second-generation anti-
histaminic drug so called because it causes less sedation and drowsiness
than the first-generation antihistamines used to treat allergic reactions;
it was approved by US Food and Drug Administration in 1993. Lor-
atadine is a selective inverse agonist of peripheral H1-receptors (Witiak,
1970; Peyrovi and Hadjmohammadi, 2015). It is mainly metabolized
through the hepatic system to desloratadine, which is a pharmacolo-
gically active compound, deriving from the loss of carbamate moiety
(Yumibe et al., 1996). Forty percent and 42% of the ingested loratadine
dose is excreted unchanged in urine and the feces, respectively
⁎ Corresponding authors.
E-mail addresses: iesce@unina.it (M.R. Iesce), marina.isidori@unicampania.it (M. Isidori).
https://doi.org/10.1016/j.ecoenv.2018.11.116
Received 22 May 2018; Received in revised form 12 November 2018; Accepted 25 November 2018
0147-6513/©2018ElsevierInc.Allrightsreserved.

M.R. Iesce et al.
EcotoxicologyandEnvironmentalSafety170(2019)664–672
(Ramanathan et al., 2007). It has been detected not only in surface
waters in Europe (in some Spanish river samples) in the low con-
centration range of 3.96–17.1 ng/L (López-Serna et al., 2012) but also
in wastewater effluents in Europe, North-America and Asia-Pacific with
a maximum concentration of 58.5 ng/L (Kristofco and Brooks, 2017).
Desloratadine has been detected in Europe with a maximum con-
centration of 81 ng/L (Kristofco and Brooks, 2017). Both drugs have
also been recovered in lower amounts in marine waters of Mediterra-
nean coasts (Moreno-González et al., 2015). Based on the anti-
histamines consumption data, loratadine and desloratadine should
occur in wastewaters at higher concentrations. However, loratadine has
low affinity for suspended matter (octanol/water partition coefficient
log P equal to 5 for loratadine and 3.2 for desloratadine; El-Awady
et al., 2013) and therefore does not accumulate appreciably in sedi-
ments and remains in the water column (Moreno-González et al., 2015).
Loratadine is known as photolabile, in fact it is stated that the drug
should be stored protected from light (Parfitt, 1999). Its UV spectrum
shows an absorption band at λ 280 nm with a tail up to 300 nm, hence
the drug is able to adsorb sunlight at ground level and to undergo light-
induced transformations in the aquatic compartment. While its photo-
stability has been investigated, no data on photoproducts identification
are reported (Abounassif et al., 2005). In this context, we have ex-
amined the photochemical behaviour of loratadine and desloratadine in
aqueous medium under UV-B and sunlight irradiation in order to isolate
and fully characterized the photoproducts. For this purpose, con-
centrated solutions, far from environmental concentrations, were used
(DellaGreca et al., 2014). The ecotoxic effects of the parent drug, its
metabolite and its transformation products were evaluated in producers
and primary consumers.
Then, the same ratio was maintained constant for 24 min; finally, the
initial ratio (30% acetonitrile and 70% water) was reached in two
minutes.
In other cases HPLC analysis was performed under isocratic condi-
tions and H2O/CH3CN 4:6 v/v was used as eluent.
GC-MS analyses were performed on a 6890 MSD quadrupole mass
spectrometer (Agilent technologies) equipped with a gas chromato-
graph by using
a
Zebron ZB-5HT Inferno (5%-Phenyl-95%-
Dimethylpolysiloxane) fused silica capillary column (Column 30 m x
0.32 mm x 0.10 µm) from Phenomenex. The injection temperature was
250 °C, the oven temperature was held at 50 °C for 3 min and then in-
creased to 150 °C at 12 °C/min, increasing to 230 °C at 18 °C/min, to
280 °C at 10 °C/min and finally to 300 °C at 30 °C/min and held for
3 min. Electron Ionization mass spectra were recorded by continuous
quadrupole scanning at 70 eV ionization energy, in the mass range of
m/z 30–600.
2.2.3. Irradiation apparatus
The photoreactor (Multirays, Helios Italquartz) was equipped with
six 15 W lamps with a maximum at 310 nm (UV-B). Open quartz tubes
(1 cm optical path) and open and closed pyrex tubes (20 cm x 1 cm,
25 mL) were used.
2.3. Chromatographic separation materials
Analytical and preparative Thin Layer Chromatography (TLC) was
made on Kieselgel 60 F254 plates with 0.2 mm, 0.5 or 1 mm layer
thickness, respectively (Merck).
2.4. Experiments
2. Materials and methods
2.4.1. Stability in aqueous solution in the dark
2.1. Chemicals
Loratadine (1) solutions (1 ×10-4 M) in H2O/CH3CN (9:1, v/v) at
pH 4, 7 and 9 were prepared. The acid and alkaline solutions were
made using NaOH 2 M and HCl 2 M to adjust pH level. All solutions
were kept in the dark and analysed by HPLC (isocratic conditions) at
12 h and 48 h.
Loratadine (99.4%, CAS Number: 79794–75-5) and desloratadine
(99.6%, CAS Number: 100643–71-8) were purchased by Kemprotec. All
chemicals were used without further purification unless otherwise in-
dicated. Solvents (acetonitrile, methanol and diethylether) were of
HPLC grade and were purchased from Sigma Aldrich. Water was of
Milli-Q quality and was obtained from a Milli-Q gradient system
(Millipore).
2.4.2. Kinetic constant and quantum yield determination
Kinetics data were obtained by irradiating the drug (1 ×10-4 M solu-
tion in H2O/CH3CN 9:1, v/v) in open quartz tubes and monitoring the
solution at fixed time intervals by HPLC using the proposed gradient
elution. The time evolution was fitted with a pseudo-first order equation
C0 = Ct x e-Kt where C0 is the initial drug concentration, Ct the con-
centration at time t and k the pseudo-first order degradation rate constant.
The incident photon flux (4.98 ×1021 photons m−2 s−1) in solution,
used to calculate the quantum yield of loratadine, was calculated using
p-nitroanisole/pyridine actinometer (Dulin and Mill, 1982).
2.2. Apparatus
2.2.1. Spectroscopic techniques
Nuclear magnetic resonance (NMR) spectra were recorded on a
Varian Inova-500 instrument operating at 499.6 and 125.6 MHz for 1H
and 13C, respectively, and referenced with CDCl3. The carbon multi-
plicity was evidenced by DEPT experiments. The proton couplings were
evidenced by 1H–1H COSY experiments. The heteronuclear chemical
shift correlations were determined by HMQC and HMBC pulse se-
quences.
2.4.3. Irradiation experiments
Two solutions of loratadine in H2O/CH3CN (9:1, v/v, 1 ×10-4 M)
and H2O/CH3CN (75:25, v/v, 1 ×10-4 M) were irradiated in open
quartz tube and analysed by HPLC at selected times. The photoproducts
were identified by HPLC comparing their Rt values with those of stan-
dard compounds which were isolated and characterized by performing
preparative photochemical experiments (see below). An aliquot of the
H2O/CH3CN (75:25, v/v, 1 ×10-4 M) was analysed by GC-MS after
6 min irradiation.
IR spectra were recorded on a Jasco FT/IR-430 instrument equipped
with single reflection ATR using CHCl3 as solvent.
UV–Vis spectra were recorded with a Varian Cary 300 UV–Vis
spectrophotometer or on a PerkinElmer Lambda 7 spectrophotometer.
2.2.2. Chromatographic analysis
HPLC experiments were carried out on an Agilent 1100 HPLC
system, equipped with an UV detector set at 254 nm, using a RP-18
column (Gemini, 5 µm, 110 A, 250 mm × 4.6 mm). at a flow rate of
0.8 mL min-1.
2.4.4. Preparative experiments for photoproducts isolation
The photoproducts were isolated by means of preparative TLCs of
irradiation mixtures obtained by appropriate experiments. Their
structures were determined by spectroscopic analyses (Hesse et al.,
2008). The presence of functional groups was deduced by IR spectra
and identification of all different protons and carbons was obtained by
NMR spectra.
The analysis of the solutions used for determining the kinetic con-
stant of loratadine photodegradation was carried out using the gradient
elution as follows: at initial time 30% acetonitrile and 70% water for
7 min, followed by an increase of acetonitrile up to 70% in two minutes.
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M.R. Iesce et al.
EcotoxicologyandEnvironmentalSafety170(2019)664–672
2.4.4.1. Isolation of isoloratadine 2. Loratadine (35 mg) was dissolved in
92 mL of H2O/CH3CN (75:25 v/v, 1 ×10-3 M) and divided in four
closed quartz tubes. Each solution was saturated with argon and
irradiated by UV-B lamps. After 20 min of irradiation the solvents
were evaporated under vacuum and the residue was analysed by 1H
NMR and separated by preparative TLC. Elution with Et2O gave a
fraction consisting of 3 and 4 (2 mg), isoloratadine 2 (6 mg), loratadine
1 (3 mg) and an intractable polar material (11 mg).
2.4.4.5. Irradiation of desloratadine 5. 1 × 10−3
M solution of
desloratadine 5 was prepared by dissolving 5 mg in 16 mL of H2O/
CH3CN 7:3 v/v. The solution was irradiated by UV-B lamps and
analysed by 1H NMR.
2.5. Toxicity testing
Samples were dissolved in dimethylsulphoxide (DMSO, 3% v/v),
stored in the dark at 4 °C, further diluted in deionized water (Elix 10,
Millipore, Milan, Italy) and sonicated for 30 min to obtain stock solu-
tions. The test solutions were prepared by mixing the appropriate vo-
lumes of the stock solutions and ISO test media. Toxicity assays were
performed in the following organisms: the green alga
Pseudokirchneriella subcapitata, the planktonic rotifer Brachionus calyci-
florus abundant in freshwaters, the anostracan crustacean
Thamnocephalus platyurus, highly sensitive in acute toxicity testing and
the cladoceran crustacean Ceriodaphnia dubia, worldwide distributed
and often employed in acute and chronic toxicity testing.
Ethyl
4-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyr-
idin-11-yl)-5,6-dihydropyridine-1(2 H)-carboxylate (2): EI-MS m/z 382/
384; UVλmax (CH3OH) nm 266 (log ε 3.8); IRνmax (CHCl3) 1690 (-N-CO-
O-), 1606 (stretching vibrations of aromatic rings), 1371 (C-O
stretching) cm-1; 1H NMR (500 MHz, CDCl3) δ 8.39 (1 H, d, J = 4.0 Hz,
H-2), 7.42 (1 H, d, J = 7.3 Hz, H-4), 7.20–7.12 (4 H, m, H-3, H-7, H-9
and H-10), 4.84–4.80 (2 H, m, H-11 e H-3’), 4.10 (2 H, q, J = 7.0 Hz,
CH2O), 3.88 (2 H, m, H-2’), 3.50–3.45 (4 H, m,), 2.88–2.74 (2 H, m),
1.94–1.71 (2 H, m,), 1.23 (3 H, t, J = 7.0 Hz, CH3). 13C NMR (126 MHz,
CDCl3) δ 157.0 (C-1a), 155.5 (CO), 146.8 (C-2), 141.8 (C-6a), 138.5 (C-
4), 135.9 (C-10a), 135.0 (C-4a), 133.1 (C-7), 133.0 (C-8), 131.1 (C-4’),
129.8 (C-10), 126.3 (C-9), 122.4 (C-3), 121.1 (C-3’), 62.3 (C-11), 61.2
(CH2O), 43.4 (C-2’), 40.5 (C-6’), 31.3 (x2, C-5 e C-6), 28.0 (C-5’), 14.9
(CH3).
2.5.1. Determination of drugs concentration in test samples
The concentrations of drugs were measured (n = 1) using the solid
phase extraction (SPE) coupled with HPLC. Each test solution con-
taining drugs at the beginning of each toxicity test and after 24 h, 48 h
and 72 h passed through a C18 Sep-Pak® light column (Waters) used as
a solid phase extraction cartridge, previously conditioned with 5.0 mL
methanol followed by 5.0 mL water. The cartridge was then eluted with
5 mL methanol. The eluate was evaporated to dryness under reduced
pressure and the residue was suspended in 1.0 mL acetonitrile. Portions
of 200 μL volume were then injected into the HPLC system.
2.4.4.2. Isolation of compounds
3 and 4. Loratadine (50 mg) was
dissolved in 130 mL of H2O/CH3CN (75:25, v/v, 1 ×10-3 M) and
irradiated by UV-B lamps. The irradiation mixture was analysed at
different time by HPLC. After 40 min of irradiation the solvents were
evaporated under vacuum, and the residue was analysed by 1H NMR
and separated by preparative TLC. Elution with Et2O gave a fraction
consisting of 3 and 4 in ca. 3:1 molar ratio (11 mg), tricycle 3 (2 mg),
isoloratadine 2 (2 mg), loratadine 1 (5 mg) and an intractable polar
residue (8 mg).
2.5.2. Acute toxicity tests
B. calyciflorus organisms were hatched from cysts (MicroBioTest
Inc., Nazareth, Belgium) in synthetic moderately hard freshwater
8-Chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine (3):
EI-MS m/z 229/231; UVλmax (CH3OH) nm: 279 (log ε 3.1); IR νmax
(CHCl3) 1580 (stretching vibrations of aromatic ring), 1070 (aryl C-
halogen stretching) cm-1; 1H NMR (500 MHz, CDCl3) δ 8.35 (1 H, d, J =
4.9 Hz, H-2), 7.40 (1 H, dd, J = 7.5, 1.4 Hz, H-4), 7.20 (1 H, d, J =
8.1 Hz, H-10), 7.16 (3 H, m, H-4, H-7 and H-9), 4.35 (2 H, s, H-11), 3.33
(4 H, brs, H-5 and H-6);13C NMR (50 MHz, CDCl3) δ 156.9 (C-1a), 146.5
(C-2), 140.8 (C-6a), 137.7 (C-4), 136.0 (C-10a), 133.8 (C-4a), 132.3 (C-
8), 130.7 (C-10), 128.9 (C-7), 126.3 (C-9), 122.0 (C-3), 35.7 (C-11),
31.3 (C-5 and C-6).
(80–100 mg/L CaCO3, pH 7.5
0.3) at 25
1 °C under continuous
illumination (3000–4000 lx) for 16–18 h prior to test initiation, as re-
ported in the ASTM E 1440-1491 (2004) guidelines. Six replicates with
five rotifers/well, less than 2 h old, were performed for each con-
centrations (0.3 mL of test solution for each test well in 36-well plates,
MicroBioTest Inc., Nazareth, Belgium) of each compound.
The T. platyurus test was performed in according to ISO 14380
(2011) using larvae hatched from cysts (Thamnotoxkit F, MicroBioTest
Inc., Nazareth, Belgium) in 20–22 h before the assay in the standard
freshwater (dilution 1:8 with deionized water) at 25 °C under con-
tinuous illumination (3000–4000 lx). Tests were performed in 24-well
plates with 10 crustaceans/well (1.0 mL of test solution), in three re-
plicates.
Spectral data of piperidinone 4 were deduced by those of the mix-
ture of 3 and 4 after the signals of tricycle 3 were subtracted; it was
identified by comparison of its signals with those reported in literature
(Hirsch and Havinga, 1976).
Ethyl 4-oxopiperidine-1-carboxylate (4) (in mixture with 3 in ca. 3:1
molar ratio): EI-MS m/z 171; 1H NMR (500 MHz, CDCl3) δ 4.19 (2 H, q,
J = 7.1 Hz, CH2O), 3.76 (4 H, t, J = 6.1 Hz, H-2 and H-6), 2.45 (4 H, t,
J = 6.1 Hz, H-3 e H-5), 1.29 (3 H, t, J = 7.1 Hz, CH3). 13C NMR
(125 MHz, CDCl3) δ 207.1 (C-4), 155.0 (CO), 61.8 (CH2O), 43.0 (C-2
and C-6), 41.1, (C-3 and C-5), 14.6 (CH3).
The C. dubia test was performed over 24 h of exposure using young
organisms less than 24 h old following test conditions reported in EPA-
600–4-90 (US EPA, 1993) with slight modifications. Neonates of at least
third generation coming from a healthy mass culture (starting organ-
isms were purchased from Aquatic Research Organisms, Inc., Hampton,
NH, USA) were maintained at 25
1 °C in synthetic medium (hard-
ness 250 mg/L expressed as CaCO3) with a 16:8 h light: dark cycle
(600 lx) Tests were performed in 24-well plates with 10 crustaceans per
well (1.0 mL of test solution), in three replicates.
2.4.4.3. UV-B irradiation experiments for mechanistic purposes. Two
1 × 10−3 M solutions of loratadine in pure CH3CN were prepared by
dissolving 5 mg in 13 mL. A solution was irradiated in open quartz tubes
and the other one in closed quartz tubes after saturating with argon.
After 15 min the solvent was evaporated and each residue analysed by
1H NMR.
For each test considered above, both a negative control (only test-
medium) and a solvent control (DMSO 1% v/v related to the maximum
concentration of compounds tested equal to 100 mg/L) were per-
formed. The plates were incubated in darkness at 25 °C for 24 h.
The end-point considered was mortality, and the concentration re-
sulting in a 50% effect in 24 h-exposure was indicated as Median Lethal
Concentration (LC50).
A similar procedure was used for two 1 × 10−3 M solutions of
loratadine in methanol and for two solutions of loratadine in H2O/
CH3CN (7:3 v/v).
In acute assays, compounds were tested for a maximum of eight
dilutions depending on the respective sensitivity of the organisms (100-
31.25-9.76-3.15-0.98-0.31-0.09 mg/L) starting from the highest con-
centration of 100 mg/L with a geometric progression of 3.2.
2.4.4.4. Irradiation of isolaratadine 2. A 1 × 10-4
M solution of
compound 2 in H2O/CH3CN (9:1 v/v) was irradiated in open quartz
tubes with UV-B lamps and analysed by HPLC and 1H NMR.
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M.R. Iesce et al.
EcotoxicologyandEnvironmentalSafety170(2019)664–672
5
6
5
6
4
7
7
4
4a
6a
Cl
O
Cl
5
6
11
11
4
10a
7
1a
9
10a
9
2
4a
6a
N
N
hν
6
Cl
3'
5'
5'
6'
11
3'
2'
+
aqueous solution
+
N
10a
9
1a
2
N
N
N
O
O
3
O
O
4
O
O
loratadine 1
2
5
6
7
4
4a
6a
Cl
11
1a
9
10a
2
N
hν
Cl
unidentified products
+
5'
6'
3'
2'
aqueous solution
N
N
H
3
desloratadine 5
Fig. 1. UVB irradiation of loratadine 1 and desloratadine 5.
2.5.3. Chronic toxicity tests
tested for a maximum of nine dilutions (1000-312.5-97.66-30.52-9.54-
2.98-0.93-0.29-0.09 µg/L) starting from the highest concentrations of
1000 µg/L with a geometric progression of 3.2. For the P. subcapitata
growth inhibition test, compounds were tested for a maximum of ten
The B. calyciflorus chronic test was based on the offspring reduction
over 48 h exposure (ISO, 20666, 2008) and was performed on young
organisms less than 2 h old. Cysts were hatched as previously described
for the acute test. Tests were performed in 48-well plates with one
rotifer/well (0.9 mL of test solution prepared in moderately hard dilu-
tion water, ASTM E1440-91), in six replicates. The organisms were fed
with a fresh suspension (0.1 mL) of 107 cells/mL of the unicellular alga
P. subcapitata. Plates were incubated in darkness at 25 °C.
dilutions
(10000-3125-976.56-305.17-95.37-29.80-9.31-2.91-0.91-
0.28 µg/L) starting from the highest concentration of 10000 µg/L with a
geometric progression of 3.2.
2.5.4. Ecotoxicological data analysis
The chronic test in C. dubia was performed with female neonates
< 24 h old from at least the third generation of a stock culture main-
tained in synthetic water with ISO medium were individually exposed
to 25 mL of test solution in beakers over 7 days (ISO, 20666, 2008).
Tests were conducted in semi-static conditions (all test media were
exchanged five times per week) and, from the fourth day-exposure
onward, the offspring produced by each parent organism were counted
and removed daily. The organisms were fed daily with 200 μL of a
combination of yeast Saccharomyces cerevisiae, alfalfa and flake food in
addition to the unicellular green alga P. subcapitata (108 cells/mL). Ten
For each kind of assay, three independent experiments were per-
formed. For each independent experiment, the effect percentages were
calculated comparing each specific negative control. For each assay, the
effect percentages coming from three independent experiments were
pooled using Prism5 software (Graphpad Inc., CA, USA) to estimate the
concentrations giving x% effect (L(E)Cx) by non-linear regression (log
agonist vs. normalized response-variable slope). The LC50 value, cor-
responding to the 50% of mortality for each test-organism, was the test
parameter for acute tests, whereas EC50, EC20, and EC10 were the
concentrations giving 50%, 20% or 10% of the effect used in chronic
tests to evaluate the inhibition of reproduction or the inhibition of the
algal growth.
replicates per concentration were incubated at 25
light: dark cycle (600 lx).
1 °C with a 16:8 h
The P. subcapitata growth inhibition test was performed according
to OECD 201 (2011) with slight modifications reported by Paixao et al.
(2008). The single samples were incubated with 104 cells/mL of algal
suspension in 96-well microplates in six replicates under continuous
3. Results
The SPE coupled with HPLC analysis revealed a non-appreciable
difference between nominal and actual concentrations: the actual
concentrations of tested chemicals diverged from the nominal con-
centrations by 5% after 24 h, around 10% after 48 h, and around 15%
after 72 h. According to Li (2012), when the actual concentrations are
at least 80% of the nominal concentrations, the measured and the ex-
pected concentrations are considered to be very close and no sig-
nificantly different, so that in the present study the effective con-
centrations are reported as nominal concentrations.
illumination at 25
1 °C on a microplate shaker (450 rpm). The plates
were read at 450 nm (SpectraFluor, Tecan, Switzerland) immediately
before the test and after 24 h, 48 h and 72 h.
For all chronic tests, a negative control (test-medium control) was
used to the test series. Only for P. subcapitata growth inhibition test, the
% DMSO exceeded the maximum percentage recommended in toxicity
testing (0.01%). Thus, for this kind of assay, a solvent control (DMSO
0.1% v/v related to the maximum concentration of compounds tested
equal to 10000 µg/L) was performed. The number of the offspring or
the algal growth outputs were compared to the values obtained for the
negative control in order to determine the chronic effective percentages
and to evaluate the chronic Effective Concentrations (ECx).
3.1. Photochemical behaviour of loratadine
Loratadine 1 is slightly soluble in water, hence acetonitrile was
In B. calyciflorus and C. dubia chronic assays, compounds were
chosen as co-solvent to obtain clear solutions (Fig. 1). Preliminary
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EcotoxicologyandEnvironmentalSafety170(2019)664–672
11 and H-3’ (singlet + multiplet, respectively) are observed at δ 4.80 as
expected due to the shift of the double bond. The shift of the double
bond produces, in the 13C NMR spectrum, the disappearance of the
singlet carbon signal at δ 133.3 (C-11 of 1) and the appearance of a
doublet carbon signal at δ 120.8 (C-3’ of 2).
The structure of photoproduct 3 (Rt 10.2 min) was confirmed by the
presence in the mass spectrum of molecular peak at 229/231 m/z cor-
responding to the molecular formula C14H12ClN2. The 1H NMR spec-
trum shows the presence of six aromatic protons in the δ range
8.40–7.10, of signals at δ 4.35 and at δ 3.33 due to di-benzylic me-
thylene proton H-11 and to benzylic methylene protons H-5 and H-6,
respectively.
Piperidinone 4 was obtained by TLC in mixture with compound 3
(ca 1:3 molar ratio) and its data were deduced by comparison with
those reported in literature (Hirsch and Havinga, 1976). It was not
observed by HPLC analysis since it is transparent at the selected wa-
velength (254 nm) of the detector. Its presence in the irradiation mix-
ture was confirmed by GC-MS (Fig. 2) and 1H NMR analysis of the crude
irradiation mixture.
NMR analysis was particularly useful to examine the irradiation
mixtures since loratadine and its photoproducts 2–4 have characteristic
identifiable signals.
Aqueous solutions of the drug were also exposed to sunlight in
Naples (Italy) in July 2017, under environmental-like conditions. As
expected, degradation was slower. HPLC analysis showed a decrease of
drug concentration to approximately 50% after 2 days and a complex
mixture of photoproducts. The chromatographic and spectroscopic
analysis of the irradiation mixture showed the presence of photo-
products 2–4.
Fig. 2. HPLC (a) and GC-MS (b) profiles of irradiation mixtures of loratadine 1
solutions (1 ×10-4 M) in H2O/CH3CN 9:1 v/v (a) and 75:25 v/v (b) after 2 min
(a) and 6 min (b) of UVB irradiation.
3.2. Mechanistic interpretation
In order to gain more mechanistic information on photoproducts
formation, UV-B irradiation experiments were performed under various
conditions (in different solvents such as acetonitrile, methanol, water/
acetonitrile; in the presence and absence of oxygen) and the reactions
were monitored by HPLC and 1H NMR. The experimental conditions
and the results are reported in Table S2.
experiments were carried out in the dark using 1 × 10-4 M solutions in
H2O/CH3CN 9:1 v/v. The drug was stable after 48 h even when tested in
acidic (pH 4) and alkaline (pH 9) solutions. These pH ranges are usually
considered in environmental analysis (Valenti et al., 2009).
Loratadine solution was then irradiated in a photoreactor with UV-B
lamps. HPLC analysis showed the formation of photoproducts already
after 2 min (Fig. 2): compound 2 at Rt16.2 min and compound 3 at
Rt10.2 min (Fig. 1). The photoproducts were identified comparing their
Rt values with those of standard compounds which were isolated and
characterized by performing preparative photochemical experiments.
Kinetic experiment under these conditions showed that loratadine
has a half-life of 137.4 s and a polychromatic quantum yield of
5.89 × 10-4 (Table S1).
Accordingly with previous data (Abounassif et al., 2005), loratadine
degraded faster in solutions containing water (after 15 min of irradia-
tion only 5% photodegradation in methanol or acetonitrile vs. 55% in
H2O/CH3CN 7:3 v/v, Table S2 runs c, e, g). In all irradiation conditions,
especially under argon, photoproduct 2 was present while the forma-
tion of compounds 3 and 4 was observed only in aqueous solution.
Control experiments showed that isoloratadine 2 was photolabile and
converted to unidentified material after 20 min of UV-B irradiation.
On the basis of literature data, a plausible mechanistic interpreta-
tion is reported in Fig. 3. Compound 2 should derive from a 1,3-hy-
drogen shift, probably via a radical pair, from an excited triplet state of
loratadine 1, as suggested by the quenching with oxygen. The radical
recombination can give loratadine 1 or its isomer 2 (Turro et al.,
2010a). Addition of water to give intermediate 6 and β-cleavage of the
alkoxy radical intermediate 7 should give products 3 and 4. β-Cleavage
of alkoxy radicals to give ketones and stable radicals is well known
(Turro et al., 2010b).
Preparative experiments to isolate and characterize the photo-
products were carried out by UV-B irradiation of 1 × 10-3 M solutions
of the drug in H2O/CH3CN 75:25 v/v. HPLC analysis confirmed the
trend observed in dilute conditions and revealed the presence of the
peaks of products 2 and 3 together with other minor products. After
40 min of irradiation, TLC on silica gel afforded three photoproducts:
compounds 2 and 3 and a new product 4. Structures 2–4 (Fig. 1) were
assigned on the basis of spectral data. In particular, 1D and 2D NMR
spectroscopy was used because it is a powerful technique for identifi-
cation and structure elucidation of small organic molecules
(Elyashberg, 2015; Fuloria and Fuloria, 2013).
In all the experimental irradiation conditions, desloratadine 5 was
not observed. This result is not surprising considering that the carba-
mate function is quite photostable and it does not absorb light in the
UV-C and UV-B regions (Iesce et al., 2006). However, control experi-
ments showed that when a 1 × 10-3 M solution of desloratadine 5 in
H2O/CH3CN 7:3 v/v was irradiated by UV-B lamps as reported above
for loratadine, it was photodegraded within 60 min and gave a complex
mixture of products among which the sole identifiable product was
tricycle 3.
The mass spectrum of photoproduct 2 (Rt 16.5 min) shows a mole-
cular peak at 382/384 m/z corresponding to the molecular formula
C
22H23ClN2O2, hence suggesting that it is a loratadine isomer. The mass
spectrum evidences a peak at m/z 154, absent in the mass spectrum of
loratadine, attributable to the tetrapyridine fragment C8H12NO2. The
1H NMR spectrum shows significant differences only in the aliphatic
proton region. In particular, two overlapping signals due to protons H-
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M.R. Iesce et al.
EcotoxicologyandEnvironmentalSafety170(2019)664–672
Cl
Cl
Cl
hν
N
H
N
N
H
H
N
N
N
O
O
O
O
O
N
O
2
loratadine 1
hν
H2O
Cl
Cl
N
O
H
OH
H
H
H
-H.
H
H
3 + 4
hν
N
N
O
O
O
O
7
6
Fig. 3. Suggested mechanistic pathways for photoproducts 2–4.
3.3. Ecotoxicological experiments
as isoloratadine 2 and the mixture of tricycle 3 and piperidinone 4
showed different acute effects. In fact, albeit isoloratadine was more
effective in C. dubia (LC50 = 1.19 mg/L), the mixture of tricycle 3 and
piperidinone 4 caused 50% mortality at units of mg/L not only in cla-
doceran crustaceans but also in rotifers and it is more lethal than parent
loratadine 1 for B. calyciforus (Table 1). To the best of our knowledge,
scientific data on the aquatic toxicity of the compounds here tested is
rather scarce. Nevertheless, there are several data on diphenhydramine
(DPH), the same histamine H1-receptor antagonist as loratadine.
Goolsby et al. (2013) found that the diphenhydramine (DPH) was
acutely toxic in C. dubia with an LC50 value equal to 3.94 mg/L, while
Kristofco et al. (2015) found that DPH caused a 50% immobilisation in
the cladoceran crustacean Daphnia magna at 374 µg/L after 48 h-ex-
posure and the 50% mortality in the fish Danio rerio at 45.5 mg/L after
72 h exposure. In the 2011, Berninger et al. found a median acute effect
in D. magna at 0.37 mg/L after DPH exposure and from units to dozens
of mg/L in the fish Pimephales promelas. Differently from vertebrates
which are known to possess some degree of genetic homology for DPH
targets like histamine-H1 receptors, with a similarity from 40% to 70%
(Gunnarsson et al., 2008; Berninger et al., 2011), the effects of the
antihistamines in invertebrates may likely be related to other me-
chanisms of actions affecting histamine ion channel transporters, as
suggested by Haas et al. (2008) and Berninger et al. (2011). Regarding
transformation products, they were found to be slightly toxic for all
aquatic organisms tested excepted isoloratadine 2 in C. dubia. Although
acute toxicity data are generally very far from those of environmental
concern and from the water solubility of chemicals, they are still re-
levant regarding the assessment of environmental risk since chronic
data are often lacking.
Tests were performed with loratadine 1, and its photoisomer 2 and
the mixture of compounds 3 and 4 (in ca. 3:1 molar ratio) obtained by
preparative experiments (see 2.4.4). We also examined desloratadine 5
and its photodegradation mixture (DPM) obtained as reported in
2.4.4.5.
3.4. Acute toxicity results
In order to verify that the acute effects were not DMSO-dependent, a
solvent control was performed for each kind of assay, at the highest
tested percentage (1% v/v), and referred to the highest tested con-
centration of 100 mg/L, observing no significant difference with the
negative control, with a survival higher than the 90% both in negative
and in solvent controls (Table S3) as recommended by test validity
criteria. The parent compound loratadine, its metabolite desloratadine,
the transformation products and the degradation mixture of deslor-
atadine were found to cause mortality in both crustaceans and rotifers
and the LC50 values, obtained after 24 h exposure (coming from three
independent experiments pooled using Prism5) are reported in Table 1.
In addition, LC50 values espressed as mean
SD of the indipendent
experiments are reported in Table S4. Loratadine was able to cause the
50% of mortality in C. dubia at hundreds of µg/L, differently from the
effects found at dozens of mg/L in the rotifers and in the anostracan
crustaceans. On the other hand, all the aquatic organisms showed the
same sensitivity to the metabolite, with LC50 values found at units of
mg/L, while DPM was more active in the rotifer B. calyciflorus (LC50
equal to 2.02 mg/L) than in crustaceans. Transformation products such
Table 1
LC50 values in mg/L obtained in acute toxicity testing, with 95% confidence intervals.
Compound
C. dubia
B. calyciflorus
T. platyurus
Loratadine 1
0.60 (0.37–0.98)
1.19 (0.59–2.40)
4.42 (2.73–7.18)
1.52 (0.92–2.59)
11.14 (7.94–15.65)
17.00 (11.56–24.99)
43.42 (24.10–78.23)
1.77 (1.12–2.78)
1.21 (0.94–1.56)
2.02 (1.43–2.86)
50.70 (19.76–130.10)
18.34 (10.28–32.70)
21.61 (13.85–33.73)
6.22 (4.18–9.27)
Isoloratadine 2
Tricycle 3 and piperidinone 4
Desloratadine 5
Desloratadine photodegradation mixture (DPM)
41.51 (26.02–66.23)
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EcotoxicologyandEnvironmentalSafety170(2019)664–672
3.5. Chronic toxicity results
The chronic toxicity data for the five samples, reported as EC50,
EC20, and EC10 values (coming from three independent experiments
pooled using Prism5) and expressed in µg/L, are shown in Table 2. In
addition, EC50 values espressed as mean
SD of the indipendent ex-
periments are reported in Table S5. In order to verify that the chronic
effects were not DMSO-dependent, a solvent control was performed
only for P. subcapitata growth inhibition assay, at the highest tested
percentage (0.1% v/v), and referred to the highest tested concentration
of 10000 µg/L, as explained above. No significant difference with the
negative control was found, with a growth higher than the 90% both in
negative and in solvent controls (Table S3) as recommended by test
validity criteria. Loratadine 1 was the most chronically active com-
pound in the rotifer (EC50 = 51.32 mg/L), while its metabolite de-
sloratadine 5 was the most active both in C. dubia, with a median ef-
fective concentration at few units of µg/L, and in the green alga P.
subcapitata (EC50 = 220.20 µg/L), as also depicted in Fig. 4, where the
concentration/effect curves of samples are reported for each aquatic
organism. As shown in Fig. 4, the parent compound was less active in C.
dubia than its metabolite but more active than the transformation
products. Furthermore, DPM determined a median chronic toxic effect
at hundreds or thousands µg/L, in the case of aquatic consumers and
producers, respectively (Table 2). In fact, as also reported in Fig. 4,
differently from consumers (C. dubia and B. calyciflorus), in producers
(P. subcapitata) there is a slow increase in concentration/effect re-
lationship with an evident response only at the highest concentrations.
Watanabe et al. (2016) tested the diphenhydramine histamine H1-
receptor antagonist in the alga P. subcapitata finding an EC50 value
equal to 1240 µg/L, the same order of magnitude of the EC50 obtained
in this study.
Isidori et al. (2009) tested the ranitidine, a histamine H2-receptor
antagonist in the consumers C. dubia and B. calyciflorus finding a
median offspring reduction in the order of thousands of µg/L, therefore
underlining a higher sensitivity of these aquatic organisms to histamine
H1- than to histamine H2-receptor antagonists. The environmental
chronic toxicity of the histamine H1-receptor antagonist such as lor-
atadine and its derivatives towards the aquatic tested organisms is
observable already at few units-dozens µg/L (EC20 values, Table 2),
however these residues occur in surface waters at ng/L levels which are
too low to cause an immediate threat to exposed organisms but can
pose delayed long term effects interfering with organism metabolic
pathways.
The EC10 and EC20 values of loratadine and EC10, EC20 and EC50
values of desloratadine (Table 2) are lower than their respective water
solubility equal to 11 µg/L and 3950 µg/L, making the results of this
study interesting to understand the behaviour of these drugs in real
water samples. At the best of our knowledge, no data of photoproducts
water solubility is available.
4. Conclusion
Loratadine 1 is transformed either under UV-B or by sunlight ex-
posure. The reactive site is the double bond while the carbamate moiety
is unreactive. Transformation products derive by photoisomerization
and water photoaddition followed by a cleavage reaction. Photolability
is also observed in desloratadine 5 but this drug leads to a complex
photodegradation mixture.
The toxic effects of loratadine 1 and desloratadine 5 occur in both
acute and chronic assays at concentrations higher than their environ-
mental occurring concentrations differently affecting the organisms
selected from two trophic levels. However, the environmental trans-
formations of the parent compounds, here simulated by the UV-irra-
diation treatments, lead to the formation of a bioactive mixture of re-
sidues and transformation products, which could represent a harmful
combination to some of the organisms tested. In order to define water
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EcotoxicologyandEnvironmentalSafety170(2019)664–672
Fig. 4. Concentration/chronic effect curves (non-
linear regression: log agonist vs. normalized response-
variable slope) of Loratadine 1, Isoloratadine 2,
Tricycle
3 and piperidinone 4, Desloratadine 5,
Desloratadine photodegradation mixture (DPM) in C.
dubia, B. calyciflorus and P. subcapitata. Bars indicate
standard deviations of three independent experiments.
quality criteria protective for all aquatic species, further toxicity studies
towards other aquatic species are needed especially to increase species
sensitivity diagrams used in EU and North American approach to anti-
histamine management to derive water quality benchmark, and to
broaden knowledges of mechanisms involved in the different biological
responses of the organisms.
Sci. Technol. 16, 815–820.
El-Awady, M., Bela, F., Pyell, U., 2013. Robust analysis of the hydrophobic basic analytes
loratadine and desloratadine in pharmaceutical preparations and biological fluids by
sweeping—cyclodextrin-modified micellar electrokinetic chromatography. J.
Chromatogr. A 1309, 64–75.
Elyashberg, M., 2015. Identification and structure elucidation by NMR spectroscopy.
Trends Analyt. Chem. 69, 88–97.
Fuloria, N.K., Fuloria, S., 2013. Structural Elucidation of Small Organic Molecules by 1D,
2D and Multi Dimensional-Solution NMR Spectroscopy. J. Anal. Bioanal.Tech. S11
(001). https://doi.org/10.4172/2155-9872.S11-001.
Acknowledgement
Goolsby, E.W., Mason, C.M., Wojcik, J.T., Jordan, A.M., Black, M.C., 2013. Acute and
chronic effects of diphenhydramine and sertraline mixtures in Ceriodaphnia dubia.
Environ. Toxicol. Chem. 32, 2866–2869.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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