Preliminary evaluation of Diopatra neapolitana regenerative capacity as a biomarker for paracetamol exposure

Preliminary evaluation of Diopatra neapolitana regenerative capacity as a biomarker for paracetamol exposure Rosa Freitas, Diogo Coelho, Adília Pires, Amadeu M. V. M. Soares, Etelvina Figueira & Bruno Nunes Environmental Science and Pollution Research ISSN 0944-1344 Volume 22 Number 17 Environ Sci Pollut Res (2015) 22:13382-13392 DOI 10.1007/s11356-015-4589-1

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Author's personal copy Environ Sci Pollut Res (2015) 22:13382–13392 DOI 10.1007/s11356-015-4589-1

RESEARCH ARTICLE

Preliminary evaluation of Diopatra neapolitana regenerative capacity as a biomarker for paracetamol exposure Rosa Freitas 1 & Diogo Coelho 2 & Adília Pires 1 & Amadeu M. V. M. Soares 1 & Etelvina Figueira 1 & Bruno Nunes 1

Received: 12 December 2014 / Accepted: 22 April 2015 / Published online: 5 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract An increasing number of studies established unequivocal relationships between exposure to pharmaceutical drugs and toxicity in wildlife. However, few studies investigated physiological alterations caused by such compounds in polychaetes. Thus, in this study, the effects of increasing concentrations of paracetamol were studied in the polychaete Diopatra neapolitana using tissue regenerative capacity as a biomarker. The obtained results revealed that individuals exposed to ecologically relevant concentrations (namely, 25 μg/L) of paracetamol exhibited significantly lower capacity to regenerate their body in comparison with control organisms. This study evidenced that paracetamol can induce significant physiological alterations in D. neapolitana resulting in an overall diminished regenerative capacity, which is of significance to a species with high ecological and economic relevance. Additionally, this study indicates the promise of D. neapolitana as a test organism in laboratory-based bioassays, but also as an adequate sentinel species to pharmaceutical drugs.

Keywords Polychaetes . Pharmaceutical drug . Acetaminophen . Body regeneration . Anthropogenic contamination

Responsible editor: Markus Hecker * Rosa Freitas [email protected] 1

Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal

2

Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal

Introduction There is an increasing concern regarding the large number of contaminants present in aquatic ecosystems. One group of chemicals that are of particular interest in this context is pharmaceuticals that are continuously released into aquatic ecosystems, and for many of which no water quality guidelines exist to date. In fact, over the last two decades, researchers have detected a variety of pharmaceuticals (and their residues and degradation products) in the aquatic environment, in great part due to the increasing sensitivity of analytical instruments with detection limits down to nanogram/liter (Kümmerer 2010). Despite their benefits in promoting human and animal health, as well as increasing livestock and aquaculture productivities, the presence of pharmaceuticals in the environment has been of growing concern due to the possible ecotoxicological risks they may pose to non-target aquatic organisms (HallingSørensen et al. 1998). Nevertheless, although recently an increasing amount of data has been published on the occurrence of pharmaceuticals in the aquatic environment (e.g., Carlsson et al. 2006; Daughton and Ternes 1999; Fatta-Kassinos et al. 2011; Fent et al. 2006; Heberer 2002; Monteiro and Boxall 2010), there is still a paucity of studies reporting on the potential biological effects of these compounds to non-target organisms (Carlsson et al. 2006; Fent et al. 2006; Halling-Sørensen et al. 1998; Li et al. 2014; Kümmerer 2009, 2010). Among the most frequently detected classes of pharmaceuticals in the environment are analgesic and antipyretic drugs such as paracetamol (also termed as acetaminophen) (Nikolaou et al. 2007). In fact, paracetamol is one of the most common drugs worldwide (Lourenção et al. 2009; Solé et al. 2010; Yang et al. 2008), used mainly therapeutically in humans (Xu et al. 2008). The environmental importance concerning the need to study paracetamol derives from its ubiquitous presence in the aquatic environment. Previous studies have found

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paracetamol at concentrations that vary from 6 μg/L in European sewage treatment plant effluents (Ternes 1998) to above 65 μg/L reported in the Tyne River, UK (Roberts and Thomas 2006). Considering its ubiquitous presence, toxicity, persistence and environmental fate, paracetamol has been considered a priority pharmaceutical in the aquatic environment (de Voogt et al. 2009). Paracetamol has been shown to exert toxic effects to common laboratory animal models, such as rodents (Moldéus 1978; Rikans and Moore 1988; Maciejewska-Paszeka et al. 2007), but also to human patients (Larson 2007; Mitchell et al. 2011), and these findings raised concerns regarding its environmental relevance (see de Voogt et al. 2009). Paracetamol is a model hepatotoxicant, especially at greater doses, which was already fully documented in both experimental animals and humans (Brind 2007; Hinson et al. 2004; Jaeschke and Bajt 2006; Jaeschke et al. 2003; Prescott 1980; Xu et al. 2008). The metabolism of paracetamol in normal therapeutic doses requires its conjugation with polar intracellular cofactors (sulphate and glucuronic acid, and thereafter glutathione), forming non-toxic metabolites (Jaeschke and Bajt 2006; Klaassen 2001; Patel et al. 1992; Xu et al. 2008) that are quickly excreted with no harm to the organism. However, overdosage leads to a completely distinct scenario, with the most common outcome being the exhaustion of polar cofactors. As a consequence, paracetamol is oxidized with the intervention of cytochrome p450 isoenzymes, forming a highly reactive and electrophilic metabolite of paracetamol, N-acetylp-benzoquinone imine (NAPQI), which can accumulate in tissues and exert multiple toxic effects, including 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 (Hinson et al. 2004; Jaeschke and Bajt 2006; Jaeschke et al. 2003; Prescott 1980). Thus, at greater concentrations, paracetamol can be toxic, depending on the occurrence of cofactor exhaustion. Given the large number of studies describing similar outcomes in many distinct organisms (please see references above), it is believed that this mechanism of paracetamol toxicity can be widely conserved and is likely to also occur in aquatic organisms. In fact, previous studies aiming to assess the effects of paracetamol in aquatic invertebrate species revealed the effects of this drug to bivalves (Corbicula fluminea, Brandão et al. 2011; Ruditapes decussatus and Ruditapes philippinarum, Antunes et al. 2013; Dreissena polymorpha, Parolini et al. 2010; Parolini and Binelli 2012; Parolini et al. 2013) and to crustaceans (Daphnia magna, Nunes et al. 2014a). Additionally, similar effects were also reported for vertebrates, such as fish (Oncorhynchus mykiss, Ramos et al. 2014; Anguilla anguilla, Nunes et al. 2015), and for plants (Lemna gibba and Lemna minor, Nunes et al. 2014b). These authors showed significant increases in enzymatic biomarkers involved in the redox homeostasis (namely, the activities of

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the enzymes glutathione-S-transferases and glutathione reductase, and also lipid peroxidation) evidencing the metabolism of paracetamol that triggered the onset of oxidative alterations and effects. Similarly, the data obtained by Gómez-Oliván (2012) confirmed that paracetamol induced oxidative stress in the amphipod species Hyalella azteca. In addition, a multi-biomarker approach for the evaluation of the cytogenotoxicity of paracetamol on the zebra mussel (Dreissena polymorpha) revealed the capacity of this drug to induce moderate genotoxicity in bivalves exposed to environmentally relevant concentrations (0.75 and 1.51 μg/L; Parolini et al. 2010). These authors further demonstrated a significant destabilization of the lysosomal membrane from baseline levels at the end of the exposure and a high induction of catalase and glutathione-S-transferase enzymes activities. Generally, the majority of studies assessing the effects of anthropogenic substances on benthic invertebrates has been performed with bivalves (among others, Viarengo et al. 1993; Bergayou et al. 2009; Bebianno and Barreira 2009; Coughlan et al. 2009; Ramos-Gómez et al. 2011c; Freitas et al. 2012; Figueira et al. 2012). However, polychaetes are frequently the most abundant group in benthic communities (e.g., Rodrigues et al. 2011). Polychaetes species are suitable and highly pertinent to be used in toxicological studies as they are usually available all the year and frequently occur in high densities. This group of organisms can also be found in a large range of grain size sediments and salinities and has a wide geographic range making them easily available for environmental monitoring tests (Lewis and Watson 2012) and appropriate for a large range of tests with sediments (e.g., Scaps 2002; Méndez et al. 2013; Gomes et al. 2014). Furthermore, polychaetes are key elements in the estuarine and coastal food webs, being the primary food source for many commercial fish and crustaceans, among others, making them important vectors for the transfer of contaminants to higher trophic levels (Serrano et al. 2003; Lewis and Watson 2012). Due to these characteristics, polychaetes have been increasingly used for assessing the toxicity of organic and inorganic contaminants (e.g., Catalano et al. 2012; Durou et al. 2007a, b; Gomes et al. 2013; Kalman et al. 2009; Pérez et al. 2004; Moreira et al. 2006; Solé et al. 2009; Freitas et al. 2012; Tankoua et al. 2012; Carregosa et al. 2014), both under field and laboratory conditions. Concerning pharmaceuticals, to the best of our knowledge, the number of studies with polychaetes is scarce compared to that of other invertebrates (e.g., Canesi et al. 2007; Yang et al. 2008; Li et al. 2011; McEneff et al. 2014; Almeida et al. 2014). In polychaetes, physiological and biochemical markers have been considered a suitable approach to detect early warning signals of organism-level effects (Ayoola, et al. 2011; Sizmur et al. 2013). However, few studies assessed the impacts of anthropogenic or natural stressors on regenerative ability of these organisms (Carregosa et al. 2014; Freitas et al. 2015; Nusetti et al. 2005; Pires et al.

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2015). The ability to regenerate varies widely within polychaetes, and while some species do not have this capacity, others are able to regenerate an entire individual from a single mid-body segment (Bely 2006). Among polychaetes, some Diopatra species are capable to regenerate anterior segments and prostomial structures (including D. sugokai, D. tuberculantennata, D. cuprea, and D. micrura), posterior ends (D. aciculata) and the anterior and posterior regions (D. dexiognatha, D. neapolitana, D. marocensis) (see for review Pires et al. 2012a). Studies conducted by Pires et al. (2012a) further revealed that, under laboratory conditions (simulating environmental conditions), (i) specimens of Diopatra neapolitana amputated at the 20th chaetiger (starting from the prostomium) were not able to regenerate and did not survive; (ii) D. neapolitana specimens amputated up to the 15th chaetiger (e.g., 3rd, 10th, or 15th chaetigers) were able to regenerate a new prostomium at the posterior body part, but the anterior end was unable to survive. Individuals regenerating at these levels presented a survival capacity of 50, 75 and 87.5 %, respectively; (iii) D. neapolitana individuals amputated between the 25th chaetiger and 40th chaetiger were able to regenerate the body after the amputated region, but the posterior ends were not able to regenerate a new anterior part (prostomium). At these amputation levels, organisms presented a survival capacity ranging from 50 %, when amputated at the 25th chaetiger, to 81.3 %, when amputated at the 40th chaetiger; (iv) organisms amputated after the branchial region presented a survival rate of 100 % and were able to regenerate the posterior end. However, the posterior end is not able to regenerate a new prostomium. Thus, the present study aimed to investigate if the regenerative ability of D. neapolitana (Delle Chiaje, 1841) can be used as a sensitive marker to assess environmental exposure to pharmaceuticals, namely paracetamol. Furthermore, since to date mostly the acute toxicity of pharmaceuticals has been tested on organisms, the present work evaluated the chronic effect of paracetamol. Additionally, most toxicity studies with polychaetes have been conducted using the species Hediste diversicolor (e.g., Moreira et al. 2006; Durou et al. 2007a, b; Kalman et al. 2009; Solé et al. 2009; Gomes et al. 2013) and Arenicola marina (Casado-Martinez et al. 2012; RamosGómez et al. 2011a, b). However, considering the importance of the polychaete species D. neapolitana in the Mediterranean and Southeast Atlantic areas, the present study intended to validate the use of this species as a sentinel organism for paracetamol exposure. This species represents a wide spatial distribution, being reported in intertidal and shallow subtidal habitats, namely in the Red Sea and Indian Ocean (Wehe and Fiege 2002), the Mediterranean Sea (Arvanitides 2000; Dagli et al. 2005; Gambi and Giangrande 1986), and the Atlantic Ocean (Fauvel 1923; Lourido et al. 2008; Moreira et al. 2006; Pires et al. 2012b). D. neapolitana is collected to be sold as

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fish bait, and this activity can be locally intense and economically important (Gambi et al. 1994; Conti and Massa 1998; Cunha et al. 2005). Additionally, Diopatra species plays an important ecological role, since its tubes stabilize the sediment, increasing its structural complexity and therefore its biodiversity, by providing refugia from disturbance and predation (Bailey-Brock 1984) while facilitating the settlement and the attachment of some algal species (Thomsen and McGlathery 2005).

Methodology Sampling To assess the toxicity of paracetamol, laboratory experiments were conducted with D. neapolitana collected from the Mira channel, a low contaminated channel at the Ria de Aveiro lagoon (Portugal). The sediment grain size at the sampling site varied from silty very fine sand to mud, with an organic matter content of less than 4 % (Carregosa et al. 2014). D. neapolitana individuals inhabit a membranous tube buried in sediments with mud or a mixture of mud and sand, and might grow up to a total length of 70 cm (Dagli et al. 2005; Pires et al. 2012b), being manually collected with a shovel. Sampling was done in an intertidal area, during the low tide period. Specimens were collected inside their tubes and transported to the laboratory in plastic containers. Laboratory conditions In the laboratory, organisms were removed from their tubes and the ones already regenerating were discarded and not used in the study. Organisms for exposure assays were acclimated in the laboratory in artificial seawater (salinity at 28±1 g/L) for 10 days under continuous aeration, and a temperature regime of 24±1 °C. During this period, organisms were fed ad libitum with frozen cockles every 2–3 days. Salinity and temperature values used were selected taken into account previous works performed with D. neapolitana (Carregosa et al. 2014; Conti and Massa 1998; Freitas et al. 2015; Pires et al. 2012a, 2012b, 2015). After acclimatization, specimens were removed from their tubes and washed with artificial seawater. Then, organisms were anaesthetized with a solution of 4 % MgCl2·6H2O, and, to ensure that individuals with similar size were selected for the experiment, the width of the 10th chaetiger was registered for each individual. Under a stereomicroscope, individuals were amputated between the 60th chaetiger and the 61st (Fig. 1). Amputation at the 60th chaetiger was selected because previous studies conducted by Pires et al. (2012a) revealed higher regenerative capacity of D. neapolitana when amputated after the branchial end (i.e., after chaetigers 45 to

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frozen cockles every 2–3 days. In general, one cockle of medium size was enough for six polychaetes. Data analysis

Fig. 1 Diopatra neapolitana anterior end: P prostomium, chaetigers 3, 10, 45 and 60

56) compared to organisms amputated before this region (cf. Fig. 1). Exposures to paracetamol were conducted following an adapted version of the Ecological Effects Test Guidelines: OPPTS 850.1710. Oyster BCF (EPA 1996a, b). Organisms were exposed to a concentration range of paracetamol (Ctl0.00; 5; 25; 125; 625; 3125 μg/L). The lower concentrations reflected low to moderately contaminated areas (Ternes 1998; Roberts and Thomas 2006). The three higher tested concentrations were up to two orders of magnitude greater than concentrations measured in the environment and were selected to test the responsiveness of the test organism to paracetamol. Test concentrations were obtained by preparing a stock solution of 500 mg/L in water; this paracetamol concentration was confirmed through colorimetric quantification following an adapted version of the procedure no. 430 by Sigma Diagnostics (St. Louis, MO, USA) (Brandão et al. 2011). To assess the effects of paracetamol on the regenerative capacity of D. neapolitana, 36 organisms, distributed by six concentrations (six individuals per concentration), were used. For each concentration, individuals were placed in different glass aquaria, filled with sediment (2 L from a reference site at the Mira channel, Carregosa et al. 2014) and artificial seawater (4 L, salinity 28±1 g/L). The experiment was carried out until regeneration was completed in all the organisms, i.e., when, in all organisms, no differences could be noticed between the width of the older and the new regenerated segments. Every other day, the solution was renewed to maintain the paracetamol levels during the experiment. During this period, oxygen concentrations were monitored every 24 h, and animals were checked for mortality. During the regeneration period, every week, the percentage of the body width regenerated was determined for each specimen, corresponding to the comparison of the width of the new regenerated segments (placed after the 60th chaetiger) and the width of the old body part (60th chaetiger). The number of new segments was also counted weekly. To perform these measurements, organisms were anaesthetized with a solution of 4 % MgCl 2 ·6H 2 O. Regenerated segments were identified by the lighter colour and/or their narrower width when compared to the rest of the body. The regenerative capacity was assessed considering the number of days needed to achieve full regeneration. During the regeneration period, specimens were fed ad libitum with

The regenerative capacity was submitted to hypothesis testing using permutation multivariate analysis of variance, using PERMANOVA+ add-on in PRIMER v6 (PRIMER-E Ltd, Plymouth Marine Laboratory, Plymouth, UK; Anderson et al. 2008). This data was analysed following a one-way hierarchical design, with exposure concentration as the main fixed factor. The null hypothesis tested was as follows: no significant differences existed among concentrations. The pseudo F values in the PERMANOVA main tests were evaluated in terms of significance among different concentrations. When the main test revealed statistical significant differences (p≤0.05), pairwise comparisons between the concentrations were performed. Significance levels (p≤0.05) among concentrations were presented with different letters.

Results The results showed that at the 4th day after amputation, individuals in all treatment groups were healing the cut (cf. Table 1). Eleven days after amputation, specimens from control and specimens exposed to paracetamol had a small reddish differentiated portion with rudimentary anal cirri, which were about 32.5±2.5 and 31.5±2.2 % as wide as the older chaetigers in the control and specimens exposed to the lowest concentration (5 μg/L), respectively; 30.0±3.5 and 28.0± 2.7 % in individuals exposed to 25 and 125 μg/L, respectively; and 16.7±2.0 and 17.5±1.8 % in individuals exposed to 625 and 3125 μg/L, respectively (cf. Table 1, Fig. 2). Chaetiger width was significantly smaller in animals exposed to concentrations greater or equal to 125 μg/L after 11 days of regeneration. Eighteen days after amputation, significant differences in width and in the number of the new chaetigers were observed between organisms exposed to the lower (0, 5, 25 and 125 μg/L) and higher (625 and 3125 μg/L) concentrations: specimens exposed to the highest concentrations regenerated between 15 and 17 chaetigers when exposed to 625 μg/L and between 11 and 15 segments when exposed to 3125 μg/L; with the width of the new segments ranging between 20 and 24 % of the width of the older body, specimens exposed to the control and the remaining concentrations (5, 25 and 125 μg/L) presented between 25 and 35 new chaetigers, and the width of new segments corresponded to 40–43 % of the old body portion (cf. Table 1). At days 31 and 39, it could be noticed that individuals exposed to lower concentrations tend to significantly regenerate faster (cf. Table 1, Fig. 2). Furthermore, with increasing exposure concentrations, specimens tended to significantly decrease the number of segments

Regenerative capacity

91.2±2.6a 54–72a 45–59a,b 68–78a

39 days % body width # chaetigers # days to complete regeneration

45–52a # chaetigers after complete regeneration

52–59b 69–74b

87.1±9.9a,b 52–69a

76.6±9.5a,b 40–56a

65–71b,c

52–59b

78.5±5.5b 48–63a

71.0±8.1b 39–51a

64.2±8.4a 26–42a

40.5±3.7a 25–32a

30.0±3.5a,b

Healing

6.7±0.8a

[25]

66–73c 64–71c

78.5±11.1b,d 51–60a

64.6±13.7b,d 35–49a

59.4±13.0a 29–38a

39.5±5.7a,b 24–31a

28.0±2.7b

Healing

6.6±0.6a

[125]

66–73c 53–59 d

56.5±10.2c 33–44b

46.6±8.7c 25–39b

37.2±5.3b 12–25b

24.0±11.9b 15–17b

16.7±2.0c

Healing

7.0±0.8a

[625]

47–49e

71.25±4.4d 26–32c

59.4±5.1d 23–28b

41.3±0.9b 16–21b

20.0±2.0b 11–15b

17.5±1.8c

Healing

7.0±0.7a

[3125]

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Width are measurements obtained at the 10th chaetiger, after amputation (day 0)

81.3±3.9a 42–57a

% body width # chaetigers

68.8±6.7a 27–47a

41.0±2.2a 26–33a

43.0±2.7a 27–35a 71.5±4.9a 33–46a

31.5±2.2a,b

32.5±2.5a

6.6±0.7a Healing

6.9±0.9a

[5]

Healing

% body width # chaetigers 31 days

24 days

Width (mm)

4 days 11 days % body width 18 days % body width # chaetigers

Biometric data

CTL

Exposure concentration

Table 1 Biometric and regeneration data for Diopatra neapolitana exposed to paracetamol (0, 5, 25, 125, 625, 3125 μg/L). Regeneration was evaluated considering the percentage of regenerated body width (mean±STDEV) and the number of new chaetigers at 11, 18, 24, 31, 39 days after amputation. The minimum and maximum number of days needed to completely regenerate and the minimum and maximum number of new chaetigers at the end of the regeneration is also presented. Significant differences between concentrations are represented by letters (a–e)

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Author's personal copy Environ Sci Pollut Res (2015) 22:13382–13392 Fig. 2 Diopatra neapolitana posterior regeneration. Photographic record of the regenerating process at 11 and 31 days after the amputation, for individuals exposed to a concentration range of paracetamol (Ctl-0.00, 5, 25, 125, 625, 3125 μg/L)

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regenerated: at day 31, organisms presented 35 to 57 new segments when exposed to the lowest concentrations, and 23 to 39 new segments when at the two highest concentrations (cf. Table 1 and Fig. 2); at day 39, organisms exposed to the lowest concentrations regenerated 48 to 72 segments, while organisms exposed to the highest concentrations regenerated 26 to 44 new segments (cf. Table 1). After 31 and 39 days of exposure, chaetiger widths were significantly smaller in animals exposed to 125, 625 and 3125 μ/L paracetamol when compared to those in the controls. Full regeneration (when the new regenerated chaetigers had the same width as the old ones) was observed between days 48 and 76 for organisms exposed to the control and the highest paracetamol concentration, respectively (cf. Table 1). Worms in all but the lowest treatment group took significantly longer to complete regeneration than control animals (Table 1). Similarly, all treatment groups had significantly lesser segments at completion of regeneration compared to the control. At the end of the regeneration, organisms under control conditions regenerated between 68 and 78 segments while specimens exposed to the highest paracetamol concentration regenerated between 47 and 49 chaetigers (cf. Table 1).

Discussion From the obtained results, it was observed that individuals exposed to all but the least exposure concentrations needed a significantly longer period (between 10 and 21 days more, at concentrations 25 and 3125 μg/L) to completely regenerate the amputated body region when compared to the control. Therefore, it is hypothesized that paracetamol negatively affected the regenerative ability of D. neapolitana. This pattern was comparable to the results obtained by Pires et al. (2012a), who demonstrated that under laboratory controlled conditions, simulating environmental conditions, D. neapolitana amputated after the branchial region presented full regeneration within 50 days after amputation, with 40 to 90 new chaetigers. Although no studies have, to date, investigated the effects of paracetamol on polychaetes, in previous studies, different authors demonstrated the impacts of this pharmaceutical drug to different aquatic invertebrates, namely bivalves (Antunes et al. 2013; Brandão et al. 2011; Parolini et al. 2010) and crustaceans (Nunes et al. 2014a). Most of toxicity studies evaluating the impacts of contaminants (other than pharmaceutical drugs) in polychaetes were devoted to analysis of biochemical alterations, and very few studies assessed the effects on their regenerative capacity. However, considering the ecological importance of regenerative capacity and the potential individual and population consequences of its impairment by chemical exposure, this marker represents an important endpoint. In fact, previous studies showed the sensitivity of this parameter, measured in different

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polychaetes (including D. neapolitana), to the exposure to contaminants and natural environmental changes. Carregosa et al. (2014) demonstrated that the regenerative capacity of D. neapolitana was negatively affected by the increase of organic matter in the environment. Nusetti et al. (2005) demonstrated that wound healing of body regions in the polychaete species Eurythoe complanata was retarded when exposed to hydrocarbons resulting from the water-soluble fraction of used crankcase oil. The same authors also noticed that, besides the mere delay on the regeneration of the new region, the number of new regenerated segments after body repair was diminished. Reish et al. (1989) reported, for the same species, a reduced ability of these worms to regenerate the anterior and posterior ends when exposed to cadmium and the organochlorine pesticide DDT. Recently, Freitas et al. (2015) demonstrated that D. neapolitana exposed to extreme salinity conditions (14, 21 and 42 g/L) needed more days to completely regenerate the missing body region and also regenerated less chaetigers, when compared to organisms exposed to salinities 28 and 35 g/L. Pires et al. (2015) further demonstrated that D. neapolitana individuals exposed to lower pH exhibited a significantly lower capacity to regenerate their body, while with the increase of temperature, individuals showed a higher capacity to regenerate their tissues. From these studies, it is possible to conclude that growth and/or regeneration (basically regulated by the same physiological mechanisms) represent a sensitive unspecific response to environmental alterations, including contaminants and climatic changes. Consequently, it is possible to suggest that this is a suitable, ecological, relevant endpoint whose use in environmental monitoring has already been demonstrated for a significant number of species. Considering the already described toxicity mechanism of paracetamol in invertebrate species, which evidenced the involvement of oxidative stress in the toxicity of paracetamol (Antunes et al. 2013; Brandão et al. 2011; Ramos et al. 2014), it is possible to hypothesize the involvement of this toxicity mechanism in the impairment of tissue regenerating from a physical aggression (mechanical cut) in D. neapolitana. Oxidative stress is a condition that is required for the onset of tissue regeneration, but the excess of this controlled prooxidative pathway can compromise the efficacy of the repair process. Being a complex set of events, in which pre-existent tissues give rise to newly formed and fully differentiated cells (Hill 1970), the attainment of a fully regenerated body in annelids is controlled by multiple mechanisms. However, the role of oxidative processes is not fully documented in these organisms, and some assumptions must be based on data for other alternative animal models. In fact, oxidative alterations are important for the onset of physiological processes of recovery in many organisms, since reactive oxygen species (ROS) and nitric oxide (NO)are vital regulators for cell proliferation and tissue differentiation and, in the case of ROS,

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can also defend wounds from pathogen development (Schäfer and Werner 2008). Given the occurrence of inflammatory processes and the involvement of numerous immune cells, it is not surprising that the condition of oxidative stress occurs after the initial physical aggression, when the animal was amputated. However, an excess of ROS during wound repair can trigger a series of alterations, including the production and release of additional amounts of reactive intermediates, for which the cell does not have a compensatory mechanism or response, resulting in cellular and tissue damage (Soneja et al. 2005). A similar mechanism seems also to act in annelids subjected to physical aggression; previous studies show that pro-oxidative conditions favour a longer healing period (Nusetti et al. 2005) and that exposure to antioxidant compounds is related to increased healing efficacy (Petno 2014). Our data showed that organisms exposed to higher doses of paracetamol took considerably longer to heal from the aggression when compared to animals exposed to lower doses. This observation suggests the occurrence of a dose-response relationship that, to the best of our knowledge, was never described. Previous studies using human tissue models showed that pro-oxidative conditions (such as exposure to UV radiation) involved in the enhancement of ROS production and release could be related to increased damage and healing impairment of skin tissue (Marionnet et al. 2010) by increasing inflammation. Considering that the inflammatory process is required for the onset of regeneration, it is thus possible to suggest that paracetamol acts by potentiating the extent of inflammation through the hyperproduction of reactive oxidative metabolites, to which the polychaetes cannot satisfactorily respond. Another factor to consider when interpreting these data is that disturbances of the regeneration process occurred for concentrations of paracetamol that are ecologically relevant, i.e., levels of exposure that caused significant regeneration impairment are comparable to those reported to occur in the wild. As previously stated, the here-selected lower levels of exposure are representative of real contamination scenarios, based on results from the literature. If one assumes that the parameter Bdays to complete regeneration^ was significantly affected in organisms exposed to the second lowest concentration (25 μg/L), it is possible to ascertain about the ecotoxicological potential posed by paracetamol to D. neapolitana. However, it is questionable if this level of 25 μg/L of paracetamol is easily and frequently attained in the wild; on the contrary, it seems to be close to a worst-case scenario, which, despite being possible, is not completely likely. Nonetheless, paracetamol is just one chemical including a larger group of environmental contaminants with redox activity frequently found in the aquatic ecosystem, to which wild organisms will be simultaneously exposed. Consequently, the regeneration process may be significantly delayed, with potential consequences to D. neapolitana specimens. If one takes into account the

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here-obtained data, it is possible to hypothesize that the regeneration process may be impaired by anthropogenic contamination, even under field conditions. As previously observed, D. neapolitana is a natural resource intensively exploited in the Ria de Aveiro area, namely for its use for fishing/aquaculture. Consequently, any interference with physiological traits of this particular species (including body regeneration after physical aggression) may consequently endanger the autochthonous population, causing serious imbalances to the ecology of the Ria de Aveiro or the ecology of other aquatic systems where this species inhabits.

Conclusions The present study demonstrated that the regeneration of D. neapolitana can be a sensitive biomarker for the exposure to anthropogenic contaminants. Overall, D. neapolitana showed different regenerative capacity when exposed to increasing paracetamol concentrations, evidencing a close doseresponse relationship that was not previously reported in the literature. This parameter is of undisputable ecological relevance and can be integrated into future environmental quality assessment studies of impacts posed by pharmaceutical drugs to polychaetes species. Thus, in agreement with previous studies, the results of the present work validate the use of the polychaete species D. neapolitana as a sentinel species to signal the presence of contamination, including pharmaceuticals, being an excellent tool to be integrated into a battery of test organisms for estuarine environmental studies. Nevertheless, further studies are necessary to confirm the use of D. neapolitana as a sentinel species to different contamination sources.

Acknowledgments The authors acknowledge Aldiro Pereira for the invaluable help in collecting the Diopatra neapolitana specimens. This work was supported by the European Funds through COMPETE and by the National Funds through the Portuguese Science Foundation (FCT) within the project PEst-C/MAR/LA0017/2013. Rosa Freitas benefited from a post doc grant (SFRH/BPD/92258/2013) by the Portuguese FCT (Fundação para a Ciência e a Tecnologia). Adília Pires benefitted from a post-doctoral grant (BPD/CESAM/RP/BENTONICAS/2013), funded by CESAM research own funds. Bruno Nunes was hired within project BSustainable Use of Marine Resources - MARES^ (CENTRO-07ST24-FEDER-002033), cofinanced by BMais Centro^ Regional Operational Programme (Centro Region) and European Regional Development Fund (ERDF).

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