Aquatic Toxicology 101 (2011) 459–465

Aquatic Toxicology 101 (2011) 459–465 Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Effects of a complex mixture of therapeutic drugs on unicellular algae Pseudokirchneriella subcapitata Candida Vannini a, Guido Domingo a, Milena Marsoni a,∗, Fabrizio De Mattia b, Massimo Labra b, Sara Castiglioni c, Marcella Bracale a a Department of Environment-Health-Safety, University of Insubria, Via G.B. Vico 46, I-21100 Varese, Italy b Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, I-20126 Milan, Italy c Department of Environmental Health Sciences, Mario Negri Institute for Pharmacological Research, Via La Masa 19, I-20156 Milan, Italy article info abstract Article history: Pharmaceutically-active compounds are regularly and widely released into the aquatic environment in Received 21 June 2010 an unaltered form or as metabolites. So far, little is known about their potential detrimental effects on Received in revised form algae populations which can ultimately impact nutrient cycling and oxygen balance. For our analysis, 20 September 2010 the common microalga Pseudokirchneriella subcapitata (P. subcapitata) was exposed to a mixture of 13 Accepted 31 October 2010 drugs found in Italian wastewaters and rivers. Traces of pharmaceuticals investigated were detected in treated algal cells, except for cyclophosphamide and ranitidine, indicating that these algae are able to Keywords: absorb pharmaceutical pollutants from the environment. The effects of the treatment were investigated Pharmaceutical pollution Pseudokirchneriella subcapitata by Amplified Fragment Length Polymorphism (AFLP) assessment of DNA damage and 2-DE proteomic AFLP analysis. While no genotoxic effect was detected, proteomic analysis showed that algae are sensitive to 2-DE the presence of drugs and that, in particular, the chloroplast is affected. LC–MS/MS analysis © 2010 Elsevier B.V. All rights reserved. 1. Introduction organisms (Richards et al., 2004; Cleuvers, 2004; Brain et al., 2004; Mimeault et al., 2005; Fent et al., 2006; Kim et al., 2009). Pomati Pharmaceutically-active compounds (PACs) are widespread in et al. (2006) designed a mixture of 13 PACs to mimic the combi- the environment due to their extensive use in human and veteri- nation and the experimental concentrations of drugs found in an nary medicine. PACs are frequently excreted in an unmetabolized Italian environment (Zuccato et al., 2000; Calamari et al., 2003). form or as a variety of different metabolites by humans and ani- This mixture is able to inhibit cell proliferation and activate stress- mals following medication. Moreover, improper disposal of expired response proteins in the human embryonic cell line HEK293 and in PACs and industrial emissions contribute to environmental pol- Zebrafish liver cells (Pomati et al., 2006, 2007). lution. More than 80 PACs from various therapeutic classes have The aim of this work was to explore the effects of the same mix- been detected up to ␮gl−1 levels in sewage, surface, and ground ture on the unicellular green alga Pseudokirchneriella subcapitata waters (Kummerer, 2009). Conventional sewage treatments do not (P. subcapitata). The rationale is that PACs can induce toxic effect remove PACs, and some pharmaceuticals have a long half-life and even in non target organisms, due to the evolutionary conserva- may therefore accumulate in the environment. tion of pathways and receptor targets among different organisms. PACs are specifically designed to elicit a biological response at Homologous targets for fluoroquinolone (chloroplast replication), very low levels and even small concentrations could have negative lincosamide (transcription and translation), sulphonamide (folate effects on target- and non-target organisms. Moreover, although biosynthesis) antibiotics have been identified in plants (Brain et al., PACs are designed to modify specific metabolic and molecular 2009). For other drugs as atenolol, carbamazepine, furosemide an targets in humans and animals, they often have important and indirect toxic effect on aquatic photosynthetic organisms has been unexpected side-effects as well. The effects of different PAC mix- shown (Escher et al., 2006; Isidori et al., 2006). tures have been reported on phyto-, zoo-plankton and other aquatic We chose P. subcapitata (Chlorophyceae; formerly known as Selenastrum capricornutum or as Raphidocelis subcapitata) because it is highly sensitive to the contamination of aquatic environments, ∗ and for this reason is recommended (by EPA and OECD) as a stan- Corresponding author at: Università ‘dell’ Insubria, Dipartimento Ambiente dard test organism in ecotoxicology. Moreover, this organism has Salute e Sicurezza, Via H. Dunant, 3, 21100, Varese, Italy. Tel.: +39 0332 421523; fax: +39 0332 421390. a modest DNA content and grows rapidly with a duplication time E-mail address: [email protected] (M. Marsoni). of approximately 18 h. In non-target organisms classic toxicity 0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.10.011 460 C. Vannini et al. / Aquatic Toxicology 101 (2011) 459–465 tests that use organism survival as the sole endpoint may overlook and constant light irradiance (4000 ± 500 lx). Erlenmeyer flasks sublethal effects, such as subtle alterations of genome or proteome containing 150 ml of solution were inoculated to obtain an initial profile, which could have long-term ecological importance. For concentration of 2 × 105 cell ml−1. Only cultures in logarithmic this reason, to have a more complete picture of the ecological phases were used for inoculation and for each experiment six risk, we analysed the potential physiological and genetic toxicity flasks were inoculated for untreated (control) and treated cells. of the mixture. We selected the Amplified Fragment Length Algae were treated with 1× and 10× PAC mixtures after 15 h from Polymorphisms (AFLP) approach as a suitable methodology for the inoculation to allow cells to recover from the effect of the subcul- evaluation of DNA damage. The specific changes in total soluble ture before exposing them to the PAC treatment. Cell proliferation protein pattern induced by the PAC mixture were detected through was recorded over three time points in a 72 h exposure period 2-DE and MS/MS analysis, which is an appropriate approach for during which cells were subjected to a single initial treatment positive identification of proteins from an organism whose genome with PAC mixture. Algal density was determined at 750 nm using has yet to be sequenced. an UV–Visible recording spectrophotometer (JASCO, V-560, UV/VS spectrophotometer). Measures were repeated at least three times. 2. Materials and methods After 72 h algal cells were collected and immediately used for pigment analysis. Cells for uptake, AFLP and 2-DE analysis were − ◦ 2.1. Preparation of PAC solution washed three times with MilliQ water and stored at 80 C until their use. The list of PAC used in this study is reported in Table 1. Pharma- ceuticals were all purchased from Sigma–Aldrich Co. (Dorset, UK), 2.3. PAC presence in algal cells apart from ciprofloxacin and ranitidine that were obtained from ICN Biochemicals (Meckenheim, Germany) and GlaxoSmithKline The presence of PACs in the algal cells exposed to the selected (Philadelphia, PA), respectively. Stock solutions were prepared in mixture of pharmaceuticals was evaluated by successive ultra- methanol as reported by Pomati et al. (2006), mixed and diluted sonic solvent extraction (USE), solid phase extraction (SPE) and in methanol to a concentration 100 times higher than the corre- high pressure liquid chromatography tandem mass spectrometry sponding test level and stored at −20 ◦C. Appropriate aliquots of (HPLC–MS–MS) analysis. The method for USE was adapted from this concentrated test solution were evaporated to dryness and Ternes et al. (2005), while SPE and HPLC–MS–MS analysis were suspended in algal culture media, to final concentrations mimick- performed using a multiresidue method described in detail in a ing those measured in the environment (1× solution reported in previous publication (Castiglioni et al., 2005). Algal pellets from − ◦ Table 1; Pomati et al., 2006). A mixture 10 times more concen- 80 C were lyophilised, weighed and minced in liquid nitrogen to trated (10×) was also assayed. The pharmaceuticals considered break cell walls and be able to recover also the pharmaceuticals in this investigation are mostly hydrophilic, since they have log K localized within the wall or inside the cells. Algal samples (about value lower than 2 (except bezafibrate and ibuprofen) and water 20 mg dry weight) were spiked with 20 ng of deuterated standards solubility always higher than 15 mg l−1 (Zuccato et al., 2004). The (salbutamol-d3, atenolol-d7, ciprofloxacin-d8, carbamazepine- composition and the purity of the stock solutions were checked d10, ibuprofen-d3) and were extracted successively with 10 ml before use by mass spectrometric analysis using an analytical methanol (two times), 10 ml 2% ammonia solution in methanol method described in detail in a previous publication (Castiglioni and 10 ml acetone. In each extraction step, the samples were et al., 2005). ultrasonicated for 10 min and then centrifuged at 6000 rpm. The supernatants were pooled, evaporated to a volume of about 1 ml, and diluted with MilliQ water to a final volume of 50 ml for solid 2.2. Algal culture conditions and growth inhibition

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