Environ Sci Pollut Res DOI 10.1007/s11356-014-3059-5

RESEARCH ARTICLE

Biochemical and standard toxic effects of acetaminophen on the macrophyte species Lemna minor and Lemna gibba Bruno Nunes & Glória Pinto & Liliana Martins & Fernando Gonçalves & Sara C. Antunes

Received: 23 January 2014 / Accepted: 19 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Acetaminophen is globally one of the most prescribed drugs due to its antipyretic and analgesic properties. However, it is highly toxic when the dosage surpasses the detoxification capability of an exposed organism, with involvement of an already described oxidative stress pathway. To address the issue of the ecotoxicity of acetaminophen, we performed acute exposures of two aquatic plant species, Lemna gibba and Lemna minor, to this compound. The selected biomarkers were number of fronds, biomass, chlorophyll content, lipid peroxidation (TBARS assay), and proline content. Our results showed marked differences between the two species. Acetaminophen caused a significant decrease in the number of fronds (EC50 =446.6 mg/L), and the establishment of a dose-dependent peroxidative damage in L. minor, but not in L. gibba. No effects were reported in both species for the indicative parameters chlorophyll content and total biomass. However, the proline content in L. gibba was substantially reduced. The overall conclusions point to the occurrence of an oxidative stress scenario more prominent for L. minor. However, the mechanisms that allowed L. gibba to Responsible editor: Henner Hollert B. Nunes : G. Pinto : F. Gonçalves : S. C. Antunes Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal B. Nunes (*) : G. Pinto : F. Gonçalves Departamento de Biologia da Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal e-mail: [email protected] L. Martins Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP), Porto, Portugal S. C. Antunes Departamento de Biologia da Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal

cope with acetaminophen exposure were distinct from those reported for L. minor, with the likely involvement of proline as antioxidant. Keywords Aquatic plants . Pharmaceutical drugs . Paracetamol . Oxidative stress . Biomarkers

Introduction The presence of a large variety of pharmaceuticals in the aquatic environment has been an interesting matter of scientific debate. Despite their presence in extremely low levels, pharmaceuticals are prone to elicit significant adverse effects in exposed biota and may pose ecological issues that were only recognized and enlightened in recent years (Boxall et al. 2012). Drugs do not follow the trends observed for common anthropogenic contaminants; in fact, drugs pose new challenges to the environment since they are continuously released in amounts comparable to those of pesticides, remain biologically active in the wild, exert effects in extremely low levels, are designed to face metabolic activities of living organisms, exhibit lipophilicity and promptly traverse biological membranes, are resistant to common procedures of water treatment, and can be effective on a multiplicity of organisms that share pharmacological similarities (Nunes et al. 2004, 2005; Rodrigues et al. 2012). Additionally, drugs in the environment can assume distinct chemical forms as a result of the concomitant human and microbial metabolism (Peréz and Barceló 2007). Nowadays, it is common knowledge among the scientific community that pharmaceuticals are present not only in sewage treatment effluents (Ferrari et al. 2003; Metcalfe et al. 2003a, b) but also in surface waters (Jones et al. 2002; Sanderson et al. 2004; Roberts and Thomas 2006), and even in drinking water (Benotti et al. 2009; Boleda et al. 2011; Bull et al. 2011), in concentrations that usually range from the

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nanograms per liter to the micrograms per liter (Ternes 1998; Peréz and Barceló 2007). The calculation of predicted environmental concentrations for drugs that are not routinely monitored also show that the levels in the aquatic compartment are in the same order of magnitude (Garcia et al. 2013). Despite being reported in low levels, some drugs can in fact elicit considerable toxicological effects (Daughton and Ternes 1999; Fent et al. 2006), especially if one considers synthetic estrogens (Corcoran et al. 2010). Among pharmaceuticals whose presence has been already reported in the aquatic compartment, acetaminophen assumes a particularly significant role. This substance is used worldwide (Lourenção et al. 2009; Yang et al. 2008; Solé et al. 2010). Despite being considered a safe drug in human therapeutics (Xu et al. 2008), due to its antipyretic/analgesic properties, it is not exempted of adverse effects. The environmental concerns regarding its use are related to the fact that it was already detected in concentrations of up to 6 μg L−1 in European STP effluents (Ternes 1998), up to 10 μg L−1 in natural waters in the USA (Kolpin et al. 2002), and above 65 μg L−1 in the Tyne River, UK (Roberts and Thomas 2006). The toxicity of acetaminophen frequently results from overdose, and has been documented in distinct animal models (Prescott 1980; Jaeschke et al. 2003; Hinson et al. 2004; Jaeschke and Bajt 2006; Brind 2007; Xu et al. 2008), including aquatic organisms such as clams (Brandão et al. 2011; Antunes et al. 2013). Acetaminophen metabolism described so far in animal models involves a first step of conjugation with co-factors, forming the non-toxic conjugated metabolites, acetaminophen glucuronide and acetaminophen sulfate (Patel et al. 1993; Klaassen 2001; Jaeschke and Bajt 2006; Xu et al. 2008). This step corresponds to a detoxification process that accounts for approximately 90 % of the acetaminophen ingested. Under normal conditions, a portion of about 10 % of the acetaminophen suffers oxidation via cytochrome P450 enzymes (primarily CYP 2E1, 1A2, and 3A4), thus producing the toxic metabolic intermediate N-acetyl-p-benzoquinoemine (NAPQI), which is highly reactive and electrophilic (Xu et al. 2008). NAPQI is usually (in common dosages) detoxified by conjugation with intracellular glutathione (Prescott 1980; Patel et al. 1993; Klaassen 2001; Xu et al. 2008). However, in high drug load and/or low intracellular glutathione reserve, the NAPQI is not adequately excreted and is free to exert multiple toxic effects, such as covalent modifications of thiol groups on cellular proteins (Xu et al. 2008), DNA and RNA damage, and oxidation of membrane lipids, resulting in necrosis and cellular death (Prescott 1980; Jaeschke et al. 2003; Hinson et al. 2004; Jaeschke and Bajt 2006). Plants are an important component of freshwater ecosystems since several ecological services rely upon their presence. As summarized by Davy et al. (2001), plants have an important contribution to primary productivity and are directly involved in the production and release of molecular oxygen.

Additionally, plants are involved in hydrological processes, such as water flow, and are important sources of habitat and nutrients, including for detritivor organisms. Aquatic plants are also physically important for the stabilization of bottom sediments. Aquatic plants have also been shown to be able to absorb and metabolize considerable amounts of pharmaceuticals under realistic conditions (Li et al. 2012; Matamoros et al. 2012). Considering that one of the most important organelles present in plant tissues (the chloroplast) shares considerable homologies (mostly in terms of cellular receptors) with bacterial cells (Brain et al. 2008), it is not surprising to observe that particular pharmaceutical compounds may elicit deleterious effects in plants. However, the number of ecotoxicological studies using plant species (namely, aquatic) is still scarce. The majority of ecotoxicological studies with aquatic plants employ growth inhibition as effect criteria, as summarized by Brain et al. (2008), but does not give further insights concerning the mechanisms of plant toxicity that are potentially involved. Thus, it is of paramount significance to address the issue of toxic outcomes in plants, also taking into consideration the probable mechanisms of aggression/ defense; for this reason, it is noteworthy to mention the urgent need for establishing reliable biomarker-based analyses to study plant toxicity, especially in the case of pharmaceuticals exposure. To attain this specific objective, the here-presented study reported a series of data concerning the potential deleterious effects of a commonly used pharmaceutical (acetaminophen) on several physiological markers of two aquatic plant species, Lemna gibba and Lemna minor. To the best of our knowledge, no previous study has ever focused on the potential effects of paracetamol on plants from an ecological or toxicological standpoint; additionally, no data are available concerning the toxic response in plants caused by paracetamol, but we can assume that some similarities must exist, at least in terms of oxidative stress (Lushchak 2011), and occurrence of oxidative metabolism with the involvement of cytochrome p450 (Huber et al. 2012). Both plants are standard species described in ecotoxicity testing guidelines (Stewart et al. 1999; OECD 2006) and are also recommended in international policy acts (e.g., USA) as test organisms (Lewis and Wang 1999). Both species are naturally present in Portuguese freshwaters in which they represent the lower level of aquatic food chains playing a key role in ecosystem balance. Unspecific markers, such as chlorophyll content, were determined to assess the overall physiological status of both species following exposure to acetaminophen. Additionally, and considering the already described pro-oxidative effect of acetaminophen in animals, we assessed the extent of lipid peroxidation of membrane lipids by means of quantifying the malondialdehyde (MDA) content (Buege and Aust 1978). MDA is one of the most unspecific markers of toxicity by oxidative stress due to the fact that all lipidic biologic

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membranes are likely to be target of oxidative attack, resulting in the formation of MDA. The content of the amino acid proline was also assessed since this compound is involved in a large number of responses to environmentally stressful conditions exclusively in plants, including oxidative stress homeostasis (Szabados and Savouré 2009).

Material and methods Lemna spp. assay L. minor and L. gibba were obtained from laboratory cultures, reared under controlled conditions (temperature 20±2 °C; photoperiod 16h L :8h D ; light intensity, 10,000 lx) in Steinberg medium, according to the guideline OECD 221 (OECD 2006). Initially, testing procedures were aimed to determine the EC50 values for both species; this was mandatory to select for all subsequent testing of sublethal concentrations of acetaminophen. Final tests were performed with a range of acetaminophen concentrations (22.5, 45.0, and 90.0 mg L−1) for both species and in agreement with the previously obtained data concerning EC50 determinations. L. minor and L. gibba were exposed in five replicates with a final volume of 100 mL of Steinberg medium per replicate adequately supplemented with acetaminophen. The assay was started placing nine to ten fronds of each species of Lemna per vessel. In the control replicates, L. minor and L. gibba were exposed only to the Steinberg medium. Assays were conducted under controlled conditions similar to those described above (culture conditions). Lemna spp. fronds were exposed to acetaminophen for 7 days; after this period, at the end of the assays, the number of fronds and the fresh weight (fresh biomass) of each replicate were recorded. For the quantification of photosynthetic pigments (total chlorophylls), one entire specimen per replicate was collected. The remaining exposed individual of each replicate was stored at −80ºC for determination of specific biochemical endpoints: malondialdehyde and proline contents. Photosynthetic pigments (chlorophylls) Total chlorophylls were determined spectrophotometrically according to the method described by Lichtenthaler (1987). Pigments were extracted from one frond of Lemna spp. per replicate (about 5 mg) in 1 mL of 96 % ethanol. The extracts were then left to incubate at −4 °C overnight. The next day, samples were thoroughly vortexed for about 30 s and centrifuged for 5 min at 4,000 rpm and at 4 °C. The obtained supernatants were used to quantify total chlorophylls (chl a+ chl b) through spectrophotometry by measuring absorbances of the extracts at wavelengths of 470, 648.6, and 664.2 nm in a

Thermo Scientific Vis Spectrophotometer 10S TM. The extraction solution was used as blank. Malondialdehyde content The content of malondialdehyde (MDA) in samples of plant tissue was determined by the thiobarbituric acid method as described by Elkahoui et al. (2005) to indirectly assess the occurrence of lipid peroxidation in plant cells, as the MDA is an end product of this process. Samples of leaves from one plant per replicate, with an estimated weight of about 0.5 g, were homogenized in 5 mL of 0.1 % trichloroacetic acid (TCA) (Riedel-de Haën). The homogenates were then centrifuged for 5 min at 10,000 rpm and at 4ºC. Aliquots of 1 mL of the supernatants were transferred to falcon tubes to which were added 4 mL of 20 % TCA solution, also containing 0.5 % of thiobarbituric acid (TBA) (≥98 %, Sigma-Aldrich). The tubes were placed in a water bath at 95 °C for 30 min. After cooling, the tubes were centrifuged for 10 min at 10,000 rpm and at 4ºC. The specific and the non-specific absorbance of the supernatant were measured at 532 and 600 nm, respectively. Distilled water was used as blank. Measurements were performed in a Thermo Scientific TM 10S Vis spectophotometer. The concentration of MDA was calculated subtracting the non-specific absorbance (measured at 600 nm) and using the molar extinction coefficient ε= 155 mM−1 cm−1. Proline content The proline content of plant fronds was determined according the method described by Bates et al. (1973). From each plant (one plant per replicate), about 100 mg of leaves were homogenized in 1.5 mL of 3 % sulfosalicylic acid (≥99 %, Sigma). After centrifugation of the extracts at 4,000 rpm, 100 μL of the supernatants were transferred to new tubes and mixed with 2 mL of glacial acetic acid (pro-analysis, Panreac) and 2 mL of ninhydrin (Riedel-de Haën). The mixture was incubated in a boiling water bath for 1 h. After this period, tubes were placed on ice, and 1 mL of toluene (99.9 %, Merck) was added to each cooled tube to allow development of color. Absorbance of the chromophore solution was measured at 520 nm in a Thermo Scientific TM 10S UV/Vis spectrophotometer. The content of proline in samples was then extrapolated from a calibration curve obtained measuring the absorbance of proline standard solutions of known concentration (0.2, 0.1, 0.05, 0.025, and 0.0125 mg mL−1). Statistical analysis EC50 values and corresponding 95 % confidence limits for each concentration were determined by nonlinear regression analysis, fitting a logistic equation to the data using technique

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of least squares. The software Statistica 11.0 was used for this purpose. Biochemical parameters were statistically analyzed with one-way ANOVA, followed by Dunnett’s multicomparison test to discriminate significant differences between toxicant concentrations and the control treatment. The adopted level of significance was of 0.05.

Results EC50 values Our data allowed calculating a value of EC50 of 446.6 mg L−1 (95 % confidence interval, 360.1–580.2) for L. minor. No value was possible to calculate for L. gibba, allowing the conclusion that under the proposed conditions defined by the adopted guideline, no acute toxicity occurred (>1 g L−1). Number of fronds and biomass Exposure of L. minor to increasing concentrations of acetaminophen caused a significant, dose-dependent reduction of the number of fronds [F(3, 8) =31.7; p=8.63E-05], with a calculated NOEC value of 22.5 mg L−1 and a LOEC value of 45.0 mg L−1 (Fig. 1). The biomass was also slightly impacted; however, no significant effects were reported [F(3, 8) =6.20; p=0.018], and it was thus impossible to calculate a NOEC value (Fig. 1). Nevertheless, no effects were observed after exposing L. gibba to acetaminophen for both parameters, number of fronds [F(3, 8) =0.20; p=0.89], and biomass [F(3, 8) =0.44; p=0.73] (Fig. 1). No LOEC values were calculated for all endpoints, and NOEC values were only obtained for the highest concentration tested. Photosynthetic pigments (chlorophylls) Figure 2 shows variations on the content of total chlorophyll. Our data showed no variation in terms of total chlorophyll Fig. 1 Effects of acetaminophen on standard endpoints in L. minor (black bars) and L. gibba (white bars). For each parameter, mean and standard error are shown. *stand for statistical differences in relation to the control, within each species

Fig. 2 Effects of acetaminophen on total chlorophyll in L. minor (black bars) and L. gibba (white bars). For each parameter, mean and standard error are shown. *stand for statistical differences in relation to the control, within each species

[F(3, 8) =1.66; p=0.25] for L. minor. Similarly, no effects following exposure of L. gibba to acetaminophen were observed in terms of total chlorophylls [F(3, 8) =1.37; p=0.32]. No LOECs values were calculated for both species, and NOEC values are coincident with the highest concentration tested.

Lipid peroxidation (MDA content) Exposure of L. minor to acetaminophen caused a significant, dose-dependent increase in lipid peroxidation parameters, with a significant increase in MDA content [F(3, 8) =48.39; p=1.79E-05], with a NOEC value of 22.5 mg L−1 and a LOEC value of 45.0 mg L−1 (Fig. 3). However, a similar pattern of response was not elicited by this substance on the other tested species since no significant effects were reported to L. gibba [F(3, 8) =46.34; p=0.79]. No LOEC values were calculated for L. gibba, and NOEC values were coincident with the highest concentration tested.

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Fig. 3 Effects of acetaminophen on cell damage endpoints (MDA and proline content) in L. minor (black bars) and L. gibba (white bars). For each parameter, mean and standard error are shown. *stand for statistical differences in relation to the control, within each species. Cell damage: MDA and proline

Proline levels A strong disparity was again reported when comparing both species for proline content (Fig. 3). L. minor, after being exposed to acetaminophen, had unchanged levels of proline [F(3, 8) =2.28; p=0.15], while L. gibba showed to be highly responsive, with a significant reduction of proline levels [F(3, 6) =55.42; p=9.12E-05]. For L. minor, a NOEC value was reported at the lowest concentration tested (22.5 mg L−1), and a LOEC value was calculated at a concentration of 45.0 mg L−1. No LOEC values were observed for L. gibba, and NOEC values were coincident with the highest concentration tested.

Discussion Responsiveness of both species to acetaminophen was clearly distinct, while values reported in the literature for L. gibba are in line with the here-obtained indications (EC50 >1 g L−1; Brain et al. 2004; Crane et al. 2006); the calculated value of EC50 for L. minor is much lower despite not having noteworthy relevance in ecological terms. This comparison allows the conclusion that acetaminophen poses a negligible threat to both species in terms of growth inhibition if exposures occur at the already reported environmental levels of acetaminophen in the water compartment. Effects of acetaminophen in terms of total chlorophyll content presently serving as unspecific marker of toxicity showed that both species were similarly not affected. According to several studies (Eaton et al. 1995; Dogan et al. 2009; Yan and Zhou 2011), the use of alterations of chlorophyll content can serve as indicator of the overall physiological status of a photosynthetic organism and have been frequently used in ecotoxicological studies for the

assessment of toxicity of distinct organic chemicals. It is thus possible to conclude that acetaminophen did not challenge the production of photosynthetic pigments, and probably, did not impair photosynthesis efficacy, as total biomass did not significantly decrease. The study conducted by Aarti et al. (2006) showed that oxidative stress caused by methyl viologen is capable of conditioning the biosynthesis of chlorophyll in the plant species Cucumis sativus. It is thus possible to suggest that despite the likely occurrence of oxidative stress following exposure to acetaminophen, none of the Lemna species tested had their chlorophyll biosynthetic pathway compromised. Lipid peroxidation was a distinctive and sensitive parameter that responded differently between the two species. L. minor seemed to be more responsive to the pro-oxidative effect of acetaminophen when compared to L. gibba. This result for L. minor shows that acetaminophen, as described for other toxicological models (namely, animals) is prone to be metabolized via the oxidative pathway in plants, with the formation of an oxidant metabolite that exerted lipoperoxidative effects reflected by a significant rise in MDA levels. The study by Huber et al. (2009) showed that acetaminophen in plants (namely, cells of Armoracia rusticana) could undergo an oxidative metabolic pathway, similar to the one already described for mammals, and with the involvement of an activation phase, probably mediated by cytochrome P450 which resulted in the formation of reactive species (including NAPQI). Furthermore, and taking into consideration that peroxidative damage occurred probably due to the presence of NAPQI, it is also possible to suggest that the antioxidant defense mechanisms of L. minor were ineffective to cope with the oxidative aggression. The combination of high MDA levels and lower amounts of chlorophyll was also reported for other compounds, including pesticides such as hexaconazole (Huang et al. 2012), suggesting that a deleterious effect of the establishment of oxidative stress

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conditions in plants may imply a reduction in the chlorophyll content, which in turn can compromise the efficacy of the photosynthetic process. In our study, such association did not occur, signifying that the levels/duration of exposure were not challenging for the chlorophyll content. The absence of photosynthetic impairment was reinforced by data concerning the parameters number of fronds and biomass, which were also assessed in both species. These parameters showed again an interesting species-specific response. L. minor was shown to be more responsive than L. gibba; a significant reduction in the number of fronds of L. minor, despite no significant effects in terms of biomass reduction, showed that acetaminophen could compromise the cellular division and, consequently, growth. There is abundant evidence that reactive oxygen species (ROS) play roles in cell growth and that spatial regulation of ROS production is an important factor controlling plant fitness (Gapper and Dolan 2006). It was interesting to notice that the reduction of number of fronds in L. minor was followed by an increase in MDA content. The lack of apparent effects in L. gibba was most probably counteracted by extensive modifications in proline biosynthesis/storage, as shown by the significant decrease of this compound concentration in analyzed tissues. In fact, acetaminophen exposure was responsible for a sensitive modification in proline levels, reinforcing the possibility of oxidativebased effects. Proline is an amino acid involved in several functions of plant physiology, including regulation of oxidative stress, and chemically induced abiotic stress (Misra and Gupta 2005; Jaleel et al. 2007; Szabados and Savouré 2009). However, the most frequent pattern of response to oxidative stress in plants involves an increase in proline levels, as shown for Oryza sativa (Thounaojam et al. 2012). It can be hypothesized, based on the here-obtained results, that the physiological role attributed to proline by L. gibba is more related to its scavenging properties more than being a mere intermediate in regulatory processes. In fact, proline can directly scavenge free radicals, as shown by Bohnert and Jensen (1996). This direct antioxidant effect could lead to the ultimate decrease of biologically available proline observed in our study. Mattioli et al. (2009) proposed an alternative possibility, involving proline in the energetic metabolism of plant cells. According to this augment, proline may provide energy to sustain metabolically demanding periods of plant reproduction/development. A similar strategy is used in animal tissues; in this case, proline is used to fuel the initial, more energy-demanding phase of the flight of many insects (Micheu et al. 2000). From our results, it is possible to conclude that L. minor was systematically more sensitive than L. gibba to paracetamol exposure. Major alterations in all parameters were clearly established for L. minor, while the same toxicity outcomes were almost not observed in L. gibba. An ecological comparison between these two aquatic macrophyte species showed that L. gibba is more resistant than L. minor to adverse

environmental conditions, a factor which allows the out competition of the later when in the presence of L. gibba (Rejmánková 1975). Despite the absence of physiology data that sustain this observation, the effectiveness of L. gibba to cope with paracetamol clearly surpassed the performance of L. minor; it is possible to hypothesize that a more effective defense mechanism (probably also involving the antioxidant defense) against harsh environmental conditions is a key element that explains the competitive advantage of L. gibba in comparison with L. minor. In conclusion, our study was a significant contribution in terms of knowledge with regard to the performance of nontarget organisms imposed on such aquatic plants under acetaminophen exposure. However, despite the undisputable validity of the obtained results, one major conclusion points to the need of redefining new strategies of ecotoxicity testing. Well-established testing guidelines do not possess adequate sensitivity to address exposure to specific compounds (e.g., pharmaceuticals) that are not capable of eliciting evident toxicity that challenges survival of test organisms. On the contrary, these types of substances are more prone to exert subtle chronic deleterious effects, which can remain unnoticed unless a more biochemistry-oriented approach is adopted. If this orientation is not to be followed, alterations exerted by realistic levels of exposure to these substances may remain uncharacterized, and the consequent toxicity may not be adequately assessed. Acknowledgments This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEst-C/MAR/LA0017/2013. Glória Pinto was hired under the programme Ciência 2008 (FCT, Portugal) and Bruno Nunes under the programme Investigador FCT co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and European Social Fund (EU).

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