Evaluation of direct and indirect photodegradation of mianserin with high-performance liquid chromatography and short-term bioassays

Ecotoxicology and Environmental Safety 115 (2015) 144–151

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Evaluation of direct and indirect photodegradation of mianserin with high-performance liquid chromatography and short-term bioassays Milena Wawryniuk n, Agnieszka Pietrzak, Grzegorz Nałęcz-Jawecki Department of Environmental Health Sciences, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, Warsaw PL-02097, Poland

art ic l e i nf o

a b s t r a c t

Article history: Received 28 July 2014 Received in revised form 5 February 2015 Accepted 6 February 2015

The widespread use of pharmaceuticals has lead to their detection in surface and ground waters. In the last year antidepressants in particular have shown very high growth dynamics of consumption and numerous research shows that these pharmaceuticals are detected in the environment and even in drinking water. Drugs and their metabolites can be subject to two types of photoreaction, direct and indirect photodegradation. These pharmaceuticals even at low concentration can have adverse effects on aquatic life, and the resulting photoproducts can be more toxic than parents compounds. The aim of this study was to evaluate the direct and indirect photodegradation of mianserin. The kinetics of the process and the identification of photoproducts were investigated by HPLC-PDA and HPLC–MS/MS, respectively. Ecotoxicity of mianserin before and after irradiation was assessed with a battery of assays with bacteria, protozoa and crustacea. The results show that mianserin was not toxic to Vibrio fischeri (Microtox), but its toxicity to protozoan Spirostomum ambiguum (Spirotox) and crustacean Thamnocephalus platyurus (Thamnotoxkit F™) was comparable to other antidepressants. On the basis of the results of the toxicity and HPLC before and after irradiation it can be seen that the decrease toxicity of mianserin was related only to a decrease of its concentration. The photoproducts had no impact to toxicity. The direct photodegradation of mianserin was more effective in UV/vis light than vis light. However the presence of humic acid in the indirect photodegradation increases the rate of degradation without regard to the kind of used light. & 2015 Elsevier Inc. All rights reserved.

Keywords: Acute toxicity Antidepressants Phototoxicity Humic acids HPLC–MS/MS

1. Introduction About 3000 different substances are used in human medicine in the European Union (Fent et al., 2006). The authors reviewed that the most commonly consumed human pharmaceuticals are non-steroidal anti-inflammatory drugs, antibiotics, lipid regulators, cardiovascular drugs, neuroactive compounds and steroids. The widespread use of pharmaceuticals has lead to their detection in surface and ground waters. Data from many countries show the magnitude of the problem of the presence of pharmaceuticals in the aquatic environment (Fent et al., 2006). For example, the amount of pharmaceuticals in Germany that is prescribed but not used and therefore disposed of totals approximately 4500 t annually (Scheytt et al., 2006). About 1500 t of pharmaceuticals were used in 2003 in Italy (Castiglioni et al., 2006). Drugs and their metabolites have been constantly introduced into the environment making them pseudopersistent pollutants, which leads to continuous exposure of aquatic n

Corresponding author. E-mail address: [email protected] (M. Wawryniuk).

http://dx.doi.org/10.1016/j.ecoenv.2015.02.014 0147-6513/& 2015 Elsevier Inc. All rights reserved.

organisms over their whole life cycle (Minagh et al., 2009). Pharmaceuticals even at low concentration can have an adverse effect on aquatic life (Santos et al., 2010). It is also important that pharmaceuticals do not occur individually, but as mixtures together with their metabolites, products of transformation and other pharmacologically active compounds. Chemicals dissolved in natural waters are subject many types of chemical reaction, such as hydrolysis, sorption, photolysis and oxidation (Wang and Lin, 2014). Solar radiation is one of the important abiotic factor influencing the decomposition of chemical compounds present in the environment. Drug substances and drug products, which are found in the environment, may be decomposed under exposure to light, which is confirmed by numerous studies (Calza et al., 2008; Kwon and Armbrust, 2004; Kim and Tanaka, 2009). There are two types of photoreaction, direct and indirect photodegradation (Wang and Lin, 2014). In the first case, the chemicals absorb sunlight directly and are transformed to products when unstable excited states of the molecule decompose. In the second case, photodegradation occurs when chemicals present in natural water, such as dissolved organic matters (DOMs), absorb light and form reactive oxygen species (ROS) that may oxidize a target compound. DOMs are significant photosensitizers of indirect photolysis. ROS are

M. Wawryniuk et al. / Ecotoxicology and Environmental Safety 115 (2015) 144–151

molecules and ions of oxygen that have an unpaired electron, for example triplet-state DOMs, singlet oxygen, hydroxyl and carbonate radicals, thus rendering them extremely reactive. Sunlight photolysis could increase the toxicity of pharmaceuticals (Wang and Lin, 2014). For example, research of acute toxicity on Vibrio fischeri, showed that during irradiation of amiloride more toxic compound(s) were created (Calza et al., 2008). Guidelines for the photodegradation of chemicals in laboratory conditions were developed by the United States Environmental Protection Agency (USEPA) and they also apply to studies of photodegradation of pharmaceuticals (USEPA 1998a, 1998b). Drugs acting on the central nervous system (CNS), especially antidepressants have very high growth dynamics of consumption. Antidepressant pharmaceuticals are used to treat the symptoms of depression, but can also be used for sleep and eating disorders, drug and alcohol abuse, panic, chronic pain and post-traumatic stress disorders (Santoke et al., 2012). In the global burden of disease (GBD) in 2000, depressive disorders were the third leading cause of burden after diarrhoeal diseases and lower respiratory infections (Ferrari et al., 2013). According to the latest Eurobarometer report on mental health, 6% of Polish and 7% of European citizens were treated for mental disorders with antidepressants in 2009 (Giebułtowicz and Nałęcz-Jawecki, 2014). In therapeutics, the selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, fluvoxamine, paroxetine and sertraline are the most widely used antidepressants (Santos et al., 2010). Mianserin is atypical, tetracyclic antidepressant, the activity of which is attributed mainly to presynaptic alfa2-adrenoreceptor blocking and to serotonin receptor antagonism (Sfair et al., 2012b). Its sales volume in Poland is as high as 799 kg/a, which corresponds to a predicted environmental concentration (PEC) equal to 29 ng/l (Giebułtowicz and Nałęcz-Jawecki, 2014). This value is higher than the threshold value proposed by the European Medicines Agency (EMA, 2006), above which the ecotoxicological tests should be performed for the pharmaceutical. Due to their high sales volumes and resistance to biodegradation in wastewater treatment plants and in freshwaters, antidepressants were detected in rivers in many countries at concentrations up to several hundred ng/l. In a EU-wide monitoring survey venlafaxine, citalopram, mianserin and fluoxetine were found in 99%, 83%, 28% and 22% of municipal effluent samples, respectively (Loos et al., 2013). Due to high octanol/water partition coefficient, some antidepressants e.g. sertraline, imipramine, paroxetine, nortriptyline, fluoxetine and mianserin are expected to cause a pharmacological effect in fish (Fick et al., 2010). In the prioritization scheme for environmental risk assessment Roos et al. (2012) ranked mianserin very high due to a high PBT (persistency/bioaccumulation/ecotoxicity) index. Studies have also confirmed that SSRIs can be bioaccumulative in aquatic organisms, and the mixture of SSRIs produce toxic effects that are additive (Silva et al., 2012). Whereas, data on the lesser known antidepressants, but equally often prescribed are hardly ever found in the literature. Giebułtowicz and Nałęcz-Jawecki (2014) studied the presence of 21 antidepressant pharmaceuticals in the Vistula and Utrata rivers in Poland. They reported that the antidepressants, including citalopram, mianserin, sertraline, clomipramine, moclobemid, venlafaxin, fluoxetine, mirtazepin, and tianeptin, were detected in the Vistula river. Furthermore, the first five of these pharmaceuticals, mianserin among others to be precise, were also detected in tap water in Warsaw. These data have shown that antidepressants are present in the aqueous environment and are not indifferent to the organisms, therefore new research should be concentrated on this problem. The purpose of this study was to evaluate the direct and indirect photodegradation of mianserin. The kinetics of the process and the identification of photoproducts were investigated by HPLC-PDA and HPLC–MS/MS, respectively. Ecotoxicity of

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mianserin before and after irradiation was assessed with a battery of assays with bacteria, protozoa and crustacea.

2. Materials and methods 2.1. Chemicals Mianserin hydrochloride was extracted with methanol from MIANSECs 30 (Jelfa SA, Jelenia Gora, Poland). Mianserin hydrochloride coated tablets were claimed to contain 30 mg of the drug and the following inactive ingredients: ludipress, magnesium stearate, colloidal anhydrous silica, ethylcellulose, macrogol 6000, titanium dioxide, indigo carmine. The extract was filtered using a 0.2 mm filter. Its purity and identify were confirmed by HPLC–MS/ MS and was compared to a purchased authentic standard (SigmaAldrich, Poznan, Poland). The stock solution (1000 mg/l) was made up in methanol and stored at 4 °C in dark glass bottles. Working solutions of mianserin (20 mg/l) was prepared ex tempore by dilution of the stock solution with water or synthetic humic water. The chemical structure and relevant data for mianserin are shown in Table 1. Humic acids (sodium salt) were received from SigmaAldrich (Poznan, Poland). Deionized water was obtained by using Milli-Q water system (Milipore, U.S.). The high-performance liquid chromatography-grade solvent (acetonitrile) was provided by Merck (Darmstadt, Germany). Reagent grade trifluoroacetic acid was provided by J.T. Baker (Deventer, Netherlands). 2.2. Preparation of synthetic humic water Synthetic humic water (HA) was made according to the United States Environmental Protection Agency Guidelines (USEPA, 1998b). In brief, 20 g of humic acid was extracted with 1 l of 0.1% NaOH solution by stirring for 1 h at room temperature. This mixture was decanted off and filtered through coarse filter paper. The pH was adjusted to 7.0 with dilute H2SO4 and the solution was filter sterilized through a 0.2 mm filter. Pre-aging was performed by exposing to direct sunlight. Before use this mixture was diluted 10-fold with 0.01 M phosphate buffer to produce a pH 7.0 solution with an absorbance of 5.00
10  2 at 370 nm. 2.3. Instruments 2.3.1. Liquid chromatography with photodiode array detector (HPLCPDA) The HPLC analyses were performed using a LC-10AT Shimadzu spectrophotometer equipped with a SPD-M10A diode-array detector and a SCL-10A system controller. The degradation products Table 1 The chemical structure and relevant data for mianserin. Name

Mianserin

Formula Chemical structure

C18H20N2

Molar mass (g/mol) λmax (nm)

264.37 279

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A

B

Fig. 1. Absorption spectrum (A) and chromatogram (B) of mianserin and its photoproducts in aqueous solution after 2 h UV/vis light irradiation acquired with HPLC-PDA. Peaks: Photoproduct I (RT¼ 1.9 min), mianserin (RT¼4.0 min), photoproduct II (RT ¼4.3 min), photoproduct III (RT¼ 4.7 min).

were separated using a Merck 3 mm C18 55 mm
4 mm analytical column. Injection volume was 20 ml and the flow rate of the mobile phase was held at 1 ml/min. The gradient programme was as follows: 0.05% trifluoroacetic acid in water/0.05% trifluoroacetic acid in acetonitrile 75/25–0/100 in 10 min. Mianserin was detected at 279 nm and the retention time (RT) equal to 4.0 70.1 min. The absorption spectrum of mianserin is shown in Fig. 1. 2.3.2. Liquid chromatography with mass spectrometer detector (HPLC–MS/MS) The qualitative analysis of mianserin and their photoproducts were performed using Agilent 1260 Infinity HPLC (Agilent Technologies, Santa Clara, CA, U.S.) connected to a QTRAPs4000 (AB SCIEX, Framingham, Massachusetts, U.S.) mass spectrometer equipped with a Turbo Ion Spray source that was operated in

positive mode. The curtain gas, ion source gas 1, ion source gas 2 and collision gas (all high purity nitrogen) were set at 35 psi, 60 psi, 40 psi and “medium” instrument units, respectively, and the ion spray voltage and source temperature were set at 5000 V and 600 °C, respectively. Chromatographic separation was achieved with a Kinetex 2.6 mm RP-18 column 100 mm
4.6 mm (Phenomenexs). The column was maintained at 40 °C at flow rate of 0.5 ml/min. The gradient programme was: 0.2% formic acid water/0.2% formic acid in acetonitrile 80/20–5/95 in 15 min. Injection volume was 10 ml. The target compounds were analysed first in Q1 mode, then in MS2 (Product Ion Scan) mode and EPI (Enhanced Product Ion Scan). Quantitative analysis was performed under MRM (Multiple Reaction Monitoring) mode and the two most abundant fragmentation products (selected as quantifier and qualifier) were recorded for each compound.

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The HPLC–MS/MS analysis of UV/vis irradiated mianserin aqueous solution was performed in 3 steps. Firstly, the total ion current (TIC) was recorded in a range m/z 100–350 Da. The peaks, the intensity of which changed during the irradiation were selected, and selected ion current (SIC) was extracted from the raw data (Fig. S1 in the Supplementary material). Finally, the MS/MS spectra were acquired for the selected ions.

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eggs, during 24-h of exposure. Thamnotoxkit F™ was performed according to the standard guideline ISO 14380 (2011) in 24-well plates with 10 organisms per well, five concentrations and three replicates per concentration. As a diluent, EPA medium hard water was used (Nałęcz-Jawecki and Persoone, 2006). Lethal responses of the crustaceans were observed after 24 h incubation at 25 °C and EC50 values were calculated with the graphical interpolation method.

2.4. Photodegradation experiments Irradiation was performed using a Suntest CPS þ (Klimatest, Warsaw, Poland). Full-spectrum light (UV/vis) and visible light (vis) irradiation was provided by the use of appropriate filters. Direct and indirect photodegradation experiments were conducted at a concentration of mianserin of 20 mg/l. Samples were prepared in 50 ml screw-capped tubes of quartz glass and irradiated for 3 h in a temperature controlled chamber with a 1500 W xenon lamp. The fluence rates was set to 750 W/m2, which corresponds to the dose of 2700 kJ/m2 per hour. The temperature measured during the experiments was 30–35 °C. The CPS þ microprocessor controller, along with the sensors for irradiance, fluence rates and temperatures, allow for programming and controlling of test conditions in pre-programmed steps for different test segments. All test and control solutions were analyzed by HPLC before irradiation and after 1, 2 and 3 h of irradiation.

2.5.4. Data treatment The toxicity value EC50 express the concentration of the tested sample causing a 50% toxic effect, sublethal and lethal. For pure compound, the values are expressed in mg/l. For the samples after irradiation the measured EC50M value is expressed in a percent of the initial concentration equal to 20 mg/l mianserin. Then, the values were transformed to measured toxicity units (TUM):

TUM = 1/EC50M⁎100 The predicted toxicity units (TUP) were calculated on the basis of mianserin concentration: TUP ¼ C / EC50 C – concentration of the mianserin in the irradiated sample. EC50 – toxicity value of the mianserin.

2.5. Toxicity measurements 3. Results and discussion Toxicity and phototoxicity of samples was tested by using Microtoxs, Spirotox and Thamnotoxkit F™. Table 2 presents the main characteristics of the bioassays used in this study. Bioassays were performed before and after 2 h of irradiation. 2.5.1. Microtoxs Microtoxs determines the acute toxicity by measuring the changes of light produced naturally in samples exposed to bioluminescent bacteria V. fischeri under standard conditions (Johnson, 2005). Samples were investigated in a medium containing 2% sodium chloride, in four dilutions and luminescence was registered after 15 min and 30 min of incubation at 15 °C. Light measurements of each cuvette was measured with Microtoxs M500 (Modern Water, New Castle, U.S.) and the MicrotoxOmni software was used for calculation of the EC50. 2.5.2. Spirotox Spirotox is a 24-h acute toxicity test (Nałęcz–Jawecki, 2005). The test organism is a large ciliated protozoan Spirostomum ambiguum. As a diluent, a Tyrod solution is used. The test was performed in 24-well plates with 10 organisms per well, five concentrations and three replicates per concentration. Morphological deformations and lethality were observed after 24 h incubation at 25 °C. On the basis of all endpoints EC50 values were calculated with the graphical interpolation method. 2.5.3. Thamnotoxkit F™ This test is based on the measurement of lethality of the crustaceans Thamnocephalus platyurus, hatched from dormant

3.1. Identification of photoproducts The HPLC–MS/MS analysis of UV/vis irradiated mianserin aqueous solution was performed in 3 steps. Firstly, the total ion current (TIC) was recorded. TIC of the positive mode scanned in a range m/z 100–350 Da of sample irradiated with UV/vis revealed 4 peaks with m/z 251 (10.7 min), 265 (11.6 min) and 281 (14.3 and 14.5 min) (Fig. S1). Similar peaks were detected by the PDA detector (Fig. 1). Structures of these detected components were determined based on HPLC–MS/MS analysis in the positive mode. The spectrum of molecular ion 265 Da was identical with that of the mianserin standard (Fig. S2). Sfair et al. (2012b) received a similar spectrum of mianserin in a range m/z 100–300 Da with the fragment ion peaks 222, 208 (most intensive), 161, 146 and 118. On the basis of the spectrum the possible fragmentation for mianserin is shown in Fig. S2. The m/z 251 corresponds probably to N-desmethylmianserin. The fragmentation spectrum of this compound (Fig. S3) is very close to the spectrum of mianserin, as the fragment ion peaks are separated by 14 Da. It suggested cleavage of primary or secondary amines with increased length of alkyl chain(s) (Figs. S2 and S3). The ion 281 is by 16 Da heavier than mianserin. It is suspected to be hydroxymianserin or mianserin oxide. According to Sfair et al. (2012b) m/z 281 could be identified as oxide formation of members in condensed aromatic ring (hydroxymianserin) or oxidation of nitrogen in the tetracyclic ring (mianserin N-oxide). The presence of fragment ion 253 Da in the spectrum of the compound (Fig. S4) suggests the first possibility.

Table 2 Characteristics of the bioassays used in this study. Test

Species

Taxon

Exposure time

Assessment endpoint

Measurement endpoint

Microtox Spirotox Thamnotoxkit F™

V. fischeri S. ambiguum T. platyurus

Bacteria Protozoa Crustacean

15 min and 30 min 24 h 24 h

Luminescence Lethality Lethality

EC50 EC50 EC50

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A

B

C

D

Fig. 2. Change in parent compound (●), photoproduct I (■), photoproduct II (▲) and photoproduct III (♦) during the irradiation in aqueous solutions (UV/vis-A, vis-B) and in presence of HA (UV/vis-C, vis-D).

3.2. Photodegradation kinetics Mianserin was irradiated in aqueous solutions (direct photodegradation) and in the presence of humic acid (indirect photodegradation). The photodegradation kinetics of the mianserin is presented in Fig. 2. The decrease in the concentration of this pharmaceutical was exponential. Mianserin in aqueous solution degraded rapidly under the UV/vis light. After 2 and 3 h of the irradiation UV/vis light, the concentration of mianserin decreased by 37% and 56%, respectively. The mianserin photodegradation rate for a pseudo-first order kinetics was 0.246 h  1, and the half-life was 2.82 h. Irradiation with vis light did not affect to degradation before 2 h. The concentration decreased only by 14% after 3 h. This suggests that the UV light is necessary for effective degradation of mianserin in aqueous solution. Mianserin does not absorb light above 400 nm, as can be seen in Fig. 1, therefore it is resistant to the visible part of sunlight. Mianserin in presence of HA was decomposed faster than in aqueous solutions under the influence of UV/vis and vis light. The concentration of mianserin decreased to only 25% of the initial level during 3 h of exposure. Humic acid addition during UV/vis and vis treatment can significantly promote the degradation of mianserin. In this case, the kind of used light was irrelevant. The photodegradation rate constant and the half-life of mianserin in presence of HA were 0.450 h  1, 1.54 h and 0.438 h  1, 1.58 h, for UV/vis and vis irradiation, respectively. Consequently, mianserin in presence of HA was degraded 1.8-fold faster than it was in the aqueous solution. Humic acid are known to be sensitizers in the photoreactions of organic chemicals by generation of various active oxygen species. These photosensitized reactions can result in accelerated photodegradation of compounds that are stable to

sunlight in pure water (Kwon and Armbrust, 2004). This relationship can be seen between the irradiation of vis light of mianserin in water and in presence of HA (Fig. 2). During irradiation of mianserin three degradation products were formulated, as can be seen in the chromatogram (Fig. 1). Photoproduct I – N-desmethylmianserin (RT ¼1.9 min) was formed in the largest quantity through the experimental period 3 h, indicating its stability to further photodegradation (Fig. 2). The quantity of photoproduct I increased with the decrease of the concentration of mianserin irrespective of irradiation used. Only in the case of UV/vis irradiation in presence of HA can there be seen a decrease in the quantity of the photoproduct I after 2 h of irradiation. This may suggest that it was susceptible only to indirect photodegradation. Photoproducts II (RT ¼ 4.3 min) and III (RT ¼4.7 min) were formed more slowly and in smaller amounts than the photoproduct I. It is likely that the photoproduct I is more hydrophilic, whereas photoproducts II and III are less hydrophilic than the parent compound based upon retention times on the analytical column and mobile phase composition. By comparing direct and indirect photodegradation of mianserin it was found that indirect photolysis of this pharmaceutical was more effective than direct photolysis. This can be explained by the fact that direct photodegradation occurs only for chemicals which absorb the light in the region 4 290 nm (Rúa-Gómez and Püttmann, 2013; Kwon and Armbrust, 2004; USEPA, 1998a). Mianserin had absorption maxima below 290 nm, and do not absorb visible light. Therefore, the direct photodegradation of mianserin with vis light was not observed in our study. The study of photodegradation of mianserin was performed by Sfair et al. (2012a). They observed 50% degradation of mianserin after 90 min of UV-C radiation. To date there have been no studies

M. Wawryniuk et al. / Ecotoxicology and Environmental Safety 115 (2015) 144–151

A

149

B

Fig. 3. Ecotoxicity data (EC50) for mianserin in aquatic solutions ( ) and in presence of HA ( ) (Spirotox – A, Thamnotoxkit F™- B) before (dark) and after 2 h of irradiation (UV/vis and vis).

on photodegradation of mianserin by simulated sunlight. While, similar studies were conducted for other antidepressants. Kwon and Armbrust (2004) studied hydrolysis and photolysis of paroxetine in aqueous buffer solutions, in lake water and in synthetic humic water. Paroxetine was degraded completely within 4 days by simulated sunlight in all aqueous solutions. Two photoproducts were detected, the first of them was degraded by photolysis, but the second product was stable and accumulated. In another study of these authors, fluvoxamine was not photodegradated for 30 days, but it was isomerized to its (Z)-isomer by simulated sunlight (Kwon and Armbrust, 2005). Hörsing et al. (2012,) showed that 92% of citalopram was removed within the first 7 min of UV irradiation, while after 30 min this pharmaceutical was not detected in the solution. No photoproducts were detected in the UV treated samples, which may be justified by its rapid removal during irradiation. 3.3. Toxicity of mianserin and other antidepressants to aquatic organisms The effects of mianserin were investigated using three aquatic test systems. The most sensitive organism was the protozoan S. ambiguum and the least sensitive assay was the marine bacterium V. fischeri. The highest mianserin concentration used in the Microtoxs (20 mg/l) resulted in too small inhibition of luminescence both after 15 min and 30 min to calculation an EC50. Therefore, the results for this test were not discussed. Fig. 3 shows the toxicity of mianserin on two of the test species. The EU-Directive 93/67/EEC (Cleuvers, 2003) classifies substances according to their EC50-value in three classes: very toxic to aquatic organisms (EC50 o1 mg/l), toxic to aquatic organisms (1 mg/ l oEC50 o10 mg/l) and harmful to aquatic organisms (10 mg/ l oEC50 o100 mg/l). EC50s in two of the three tests were in the range between 1 and 10 mg/l. By this classification mianserin was toxic to aquatic organisms used in these tests. Humic acid, in used concentration, was not toxic to organisms and their addition to mianserin solutions had no effect on the toxicity of mianserin (Fig. 3). All published reports only describe the influence of humic acid on the photodegradation of chemicals, while the influence on the toxicity of xenobiotics has so far not been investigated. The acute toxicity of mianserin has not been previously published for these organisms, though the toxicity of other antidepressants has been reported very often to different kind of bioassays. Microtoxs with the luminescent bacteria V. fischeri is widely used as a screening test in environmental studies of pharmaceuticals (Wang and Lin, 2014). It was the least sensitive assay from the 3 tests used in our study. Similar results obtained

Minagh et al. (2009), who found that V. fischeri was the least sensitive organism to sertraline with 30 min-EC50 equal to 7.30 mg/l. Nałęcz–Jawecki et al. (2008) also concluded that the toxicity of antidepressants to V. fischeri was much lower than to protozoa, rotifer and crustacean. The toxicity of antidepressants on protozoan S. ambiguum, have been studied by Nałęcz-Jawecki et al. (2008). The lethal and sublethal effects of selected antidepressants amitriptyline, imipramine, clomipramine, fluoxetine and their N-desmethylated metabolites nortriptyline, desipramine, norclomipramine and norfluoxetine were observed. The toxicity of antidepressants was homogenous with 24h-EC50 in a range 0.30–0.60 mg/l, and was comparable to our results for mianserin. The tricyclic antidepressants have a similar structure to mianserin. The studies on the influence of antidepressants on crustacea are the most common. Minguez et al. (2014a) studied the toxicity of mianserin to Daphnia magna. Its 48h-EC50 (7.81 mg/l) is 5-fold higher than the 24h-EC50 for T. platyurus obtained in our study. The acute toxicity of the other antidepressants to daphnids (D. magna and Ceriodaphnia dubia) was comparable to that of T. platyurus. The toxicity of sertraline to T. platyurus was 0.60 mg/l (Minagh et al., 2009), very similar to our results of mianserin for this crustacean. Nałęcz-Jawecki et al. (2008) found that T. platyurus was 2–3 fold less sensitive for antidepressants than S. ambiguum with 24h-EC50 from 0.40 to 1.70 mg/l, which is comparable to our findings for mianserin. In another study, Christensen et al. (2007) tested the toxicity of five selective serotonin reuptake inhibitors (SSRI)-citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline to D. magna. The 48h-EC50s ranged from 0.92 to 20.0 mg/l for sertraline and citalopram, respectively. Henry et al. (2004) studied the acute and chronic toxicity for the same SSRIs to C. dubia. This crustacean was 7-fold more sensitive than D. magna with 48hEC50s ranged from 0.12 to 3.90 mg/l for sertraline and citalopram, respectively. The same result of acute toxicity of sertraline for C. dubia was received by Lamichhane et al. (2014). C. dubia and D. magna acute toxicity tests of fluoxetine were performed by Brooks et al. (2003). Average 48h-EC50s for these organisms were 0.23 and 0.82 mg/l, respectively. In a study on vertebrates, van der Ven et al. (2006) demonstrated that mianserin has estrogenic activity and produces endocrine disruption in zebrafish. Brooks et al. (2003) also performed the acute toxicity of fluoxetine on Pimephales promelas, with the 48h-EC50 ¼0.705 mg/l, close to the toxicity of crustaceans. Several SSRIs have been found to be directly related to gonadal maturation, induction of parturition, metamorphosis and spawning in aquatic organisms. For example, concentration as low as 32.0 ng/l induced spawning in male Dreisseena polymorpha (Calisto and Esteves, 2009). In a seperate report, Minguez et al.,

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A

B

C

D

Fig. 4. Ecotoxicity data (TUM- , TUP- ) for mianserin in aquatic solutions (Spirotox-A, Thamnotoxkit F™-B) and in presence of HA (Spirotox-C, Thamnotoxkit F™-D) before (dark) and after 2 h of irradiation (UV/vis and vis).

(2014b) studied in vitro cytotoxicity and immunomodulatory properties of amitriptyline, clomipramine, citalopram and paroxetine on primary cultures of abalone hemocytes (Haliotis tuberculata), after 48 h-exposure. They demonstrated that amitriptyline was the most potent and citalopram the least potent drug in altering the immune function in H. tuberculata. 3.4. Influence of mianserin's irradiation on its toxicity Mianserin after exposure to UV/vis and vis light was also nontoxic to V. fischeri. After irradiation with UV/vis for 2 h, the toxicity of mianserin aqueous solutions decreased 1.6 and 2.2 fold for S. ambiguum and T. platyurus, respectively. Vis light influenced on the toxicity of samples only for the crustacean, and the EC50 value increased 1.7 fold (Fig. 3). The decrease of toxicity in aqueous solutions was proportional to the decrease of concentration of mianserin determinate with HPLC-PDA. In the presence of HA, the toxicity of solutions decreased significantly more than aqueous solutions. Irrespective of used light, the EC50 values increased 2.7 and 3.8 times, in Spirotox and Thamnotoxkit F™, respectively (Fig. 3). The predicted toxicity units of mianserin (TUP) was calculated on the basis of the concentrations of the drug measured with HPLC-PDA. However, no standards were available for mianserin photoproducts, so their levels were investigated only semiquantitatively with the comparison of the area of the chromatographic peaks. TUM of the irradiated samples were compared with TUP of mianserin calculated on the HPLC-PDA values (Fig. 4). The TUM and TUP were similar in almost all cases (Fig. 4). Thus, the toxicity of the irradiated solutions was caused mainly by mianserin. Moreover, in the Thamnotoxkit F™ the measured toxicity

was significantly lower than the predicted toxicity, especially in the vis irradiated aqueous solution. The presented data suggest that the biological activity of mianserin photoproducts was much lower than of mianserin, or the activity was not additive. No ecotoxicological data of photoproducts of mianserin and other antidepressants has been published so far. Sfair et al. (2012a) studied the cytotoxicity of mianserin in vitro after irradiation by UV-C. Their analysis demonstrated that mianserin in initial concentrations of 500 and 200 mg/l had potential cytotoxic effect. Nałęcz–Jawecki et al. (2008), with the use of a battery of bioassays, found that desmethyl derivatives of antidepressants fluoxetine, clomipramine, imipramine and amitriptyline have similar biological activity than the parent compounds. In this study, in spite of the high level of N-desmethylmianserin, especially in the irradiated HA solutions of mianserin, the toxicity decreased proportionally to the level of the drug. More research is needed to show if this photoproduct was not toxic to bioassays or the toxicity of the antidepressant and its photoproduct(s) e.g. N-desmethyl derivative is additive.

4. Conclusions In this study, the direct and indirect photodegradation of mianserin was assessed by the use of analytical and biological methods. The results show that mianserin was not toxic to V. fischeri, but its toxicity to protozoan and crustacean was comparable to other antidepressants. On the basis of the toxicity and HPLCPDA data before and after irradiation it can be seen that the decrease of toxicity of mianserin was related only to the decrease of its concentration. The photoproducts had no impact to toxicity.

M. Wawryniuk et al. / Ecotoxicology and Environmental Safety 115 (2015) 144–151

Comparing both types of photodegradation it can be deduced that UV light is necessary in the photodegradation of mianserin, and the presence of humic acid significantly increase this process. This research shows the significance of investigating the photodegradation of antidepressants in different variants of irradiation and in a solution with the addition of substances which can generate ROS and modified the photodegradation of the photostable pharmaceuticals. Although mianserin does not create more toxic photoproducts, comprehensive evaluation of phototoxicity of pharmaceuticals should be continued.

Acknowledgments The authors would like to thank K. Sikorska for technical support during mass spectrometry analysis.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.02. 014.

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