Toxic potential of paracetamol to freshwater organisms: A headache to environmental regulators?

Ecotoxicology and Environmental Safety 107 (2014) 178–185

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Toxic potential of paracetamol to freshwater organisms: A headache to environmental regulators? Bruno Nunes a,b, Sara C. Antunes a,d,n, Joana Santos b, Liliana Martins c, Bruno B. Castro a,b a

Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal Departamento de Biologia da Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal c Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP), Porto, Portugal d Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal b

art ic l e i nf o

a b s t r a c t

Article history: Received 8 January 2014 Received in revised form 21 May 2014 Accepted 22 May 2014

Paracetamol is one of the most prescribed drugs globally, due to its antipyretic and analgesic properties. However, it is highly toxic at elevated doses, with involvement of an already described oxidative stress pathway. Despite this, the number of ecotoxicological studies on potential effects of paracetamol in wild organisms is still scarce. The present article presents a comprehensive series of standardized assays for the assessment of paracetamol effects in freshwater organisms. The results show that paracetamol toxicity is widely variable among species, even when these species are phylogenetically related. Furthermore, comparisons between data from the literature and our results reinforce this conclusion, providing evidence of the inadequacy of standardized toxicity testing guidelines for pharmaceutical compounds in wild organisms. Paracetamol toxicity can be modulated by unpredictable physiological conditions that might compromise extrapolations and comparisons of responsiveness among species. The ecological relevance of data obtained from classical tests for this compound is further discussed. & 2014 Elsevier Inc. All rights reserved.

Keywords: Aquatic organisms Pharmaceutical drugs Acetaminophen Standard toxicity tests Effects assessment Species sensitivity distribution curve

1. Introduction The presence of pharmaceuticals drugs and residues in the aquatic compartment has been a common issue in environmental chemistry (Winker et al., 2008). These substances reach water bodies as a result of their use by humans in sewage treatment plants – STPs (Ferrari et al., 2003; Jones et al., 2002; Metcalfe et al., 2003; Roberts and Thomas, 2006; Ternes, 1998), but also as residues from other activities that include livestock or poultry production and pet medication (Thomas et al., 2007), aquaculture facilities (Thomas et al., 2007), and industrial discharges (Li and Randak, 2009). As a result, the occurrence of pharmaceutical drugs is consensually widespread, being frequent their detection in wastewater (Ternes, 1998), drinking water (Benotti et al., 2009; Jones et al., 2002), surface water (Jones et al., 2002; Roberts and Thomas, 2006; Ternes, 1998), and even ocean water (Fang et al., 2012), despite the increasing dilution. Levels in which these substances have been detected range from the ng l
1 to the mg l
1 (Schrap et al., 2003), but in specific cases, can reach higher amounts, in the range of mg l
1; the overall tendency points to an increase in the concentrations of drugs in the environment (Oggier et al., 2010). Despite this variation, pharmaceutical drugs are ubiquitously dispersed in the aquatic

n Corresponding author at: Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal. E-mail address: [email protected] (S.C. Antunes).

http://dx.doi.org/10.1016/j.ecoenv.2014.05.027 0147-6513/& 2014 Elsevier Inc. All rights reserved.

environment. Thus, the extrapolation of results from studies with single substances to real scenario of contamination is difficult (Fent et al., 2006). Additionally, these substances possess a combination of exclusive features: drugs are biologically active among a varied number of species, and resist metabolic degradation; pharmaceuticals are lipophilic to allow the entrance into cells; pharmaceutical degradation is responsible for the liberation of new molecules, with distinct pharmacological activities; drugs are resistant to common water treatment processes; drugs are pseudopersistent, because their rate of removal is compensated by the daily input of new molecules into the environment; drugs can coexist in the environment, consequently interacting with each other (Daughton and Ternes, 1999; HallingSö rensen et al., 1998; Jones et al., 2002; Miao et al., 2002). All the referred characteristics turn pharmaceutical drugs into environmentally concerning substances due to the occurrence of potentially deleterious effects in non-target organisms, mainly in the aquatic environment. Among pharmaceuticals that have been reported in aquatic matrices, one assumes an important role due to its intrinsic toxicity – paracetamol (N-acetyl-p-aminophenol, also known as acetaminophen). This is one of the most used drugs worldwide (Lourenção et al., 2009; Solé et al., 2010; Yang et al., 2008), used as an antipyretic and analgesic drug in human therapeutics (Xu et al., 2008). The environmental concern surrounding paracetamol stems from its common presence in the aquatic environment: it has been found in concentrations of up to 6 mg l
1 in European STP

B. Nunes et al. / Ecotoxicology and Environmental Safety 107 (2014) 178–185

effluents (Ternes, 1998), up to 10 mg l
1 in natural waters in USA (Kolpin et al., 2002), and above 65 mg l
1 in the Tyne River, UK (Roberts and Thomas, 2006). Paracetamol has been considered a priority pharmaceutical in the aquatic environment by de Voogt et al. (2009), based on literature review and a series of criteria (including toxicity, persistence, environmental fate, etc.). The concerns regarding paracetamol derive from its toxicity, although not exclusively (see de Voogt et al., 2009). Paracetamol can be hepatotoxic in over dosage, a condition 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 administration of paracetamol, in normal dosages, results in its conjugation with co-factors (namely with sulphate and glucuronic acid, and later with glutathione), forming non-toxic conjugated metabolites (Jaeschke and Bajt, 2006; Klaassen, 2001; Patel et al., 1992; Xu et al., 2008) that are promptly excreted. A different scenario can be observed for higher amounts of paracetamol and/or unavailability of intracellular glutathione. In such cases, the highly reactive and electrophilic metabolite of paracetamol (N-acetyl-p-benzoquinoemine, NAPQI) can accumulate and 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 (Hinson et al., 2004; Jaeschke and Bajt, 2006; Jaeschke et al., 2003; Prescott, 1980). Thus, the toxicity of paracetamol is modulated by a detoxification mechanism likely to suffer saturation by exhaustion of co-factors; this influences the occurrence of a threshold limit that very sharply separates the safe and the toxic levels of paracetamol. It is thus expectable that similar detoxification mechanisms might exist in non-target organisms that are environmentally exposed to this substance. Bearing this in mind, it is of paramount importance to diagnose exposure of distinct representative organisms to paracetamol. However, one must consider that the number and typology of standard ecotoxicological tests (and recommended organisms) are still limited. Additionally, and taking into consideration the need to assess the ecotoxicological effects of drugs at varied levels, it is mandatory to suggest and validate new analytical tools. The commonly adopted tiered approach, implemented by regulation institutions such as Food and Drug Administration (USA FDA) and European Medicine Agency (UE EMA) is still the standard procedure to assist the introduction of new drugs into the market. However, strong criticism has been recently raised about this issue, since new testing methodologies are required to quantify the effects of particular substances such as pharmaceutical drugs, whose environmental effects are not likely to be followed using classical standardized tests (Bound and Voulvoulis, 2004). This is particularly important for drugs already in the market, whose ecotoxicological characterization was not performed under any regulation. However, and given the vast array of drugs in use at this moment, values of toxicity thresholds (calculated by standard test guidelines) are still not available for substances widely employed, which is absolutely critical to ascertain the potential ecotoxicity of such substances. This is the case of paracetamol. In order to build a substantial database of toxicity values concerning paracetamol, this study intended to perform a thorough diagnostic of its aquatic ecotoxicity, using a comprehensive battery of wellestablished test organisms and literature review.

2. Material and methods 2.1. Organism provenance and culture The bioluminescent bacteria Vibrio fischeri (Beijerinck, 1889) was purchased from Azur Environmental (Microtoxs assay), USA, as a lyophilized material, and

179

kept at
20 1C. Prior to testing, the bacteria were reconstituted with a saline solution. The microalga Pseudokirchneriella subcapitata ((Korschikov) Hindák, 1990) is currently recommended as a standard species for algal toxicity tests (Environment Canada, 1992; OECD, 2006a; USEPA, 1994). It was maintained in nonaxenic batch cultures with Woods Hole MBL medium, at 20 7 2 1C, and with a 16 hL:8 hD photoperiod (  6000 lx). Algae were cyclically harvested while still in the exponential growth phase (5–7 days old) and inoculated in fresh medium. The cyanobacterium Cylindrospermopsis raciborskii (Woloszynska, 1972) was obtained from a local eutrophic lake and kept in the laboratory for several months, under the same conditions described for P. subcapitata, but under lower light intensity (  3000 lx, which were adjusted to its physiological requirements). Monoclonal cultures of Daphnia magna (clone A, sensu Baird et al., 1989) and Daphnia longispina (clone EM7, sensu Antunes et al., 2003) were reared in singlecohort group cultures under a 16 hL:8 hD cycle and a temperature of 20 72 1C. ASTM (1980) synthetic hard water medium was used as culture medium, which was supplemented with a standard organic additive to provide essential microelements to daphnids. Culture medium was renewed on Mondays, Wednesdays and Fridays; every two weeks, cultures were renewed with o 24-h-old neonates, and mothers were discarded. Animals were fed with P. subcapitata, which was cultured as described above. Algal ration was determined spectrophotometrically and daily supplied to the cladocerans (3.0  105 cells ml
1 day
1 for D. magna and 1.5  105 cells ml
1 day
1 for D. longispina). More detailed rearing procedures can be found in Antunes et al. (2003, 2007), Castro et al. (2007), or Loureiro et al. (2011). All experiments were initiated with neonates ( o24 h old), born between the 3rd and 5th broods, which were obtained from group cultures. Lemna minor (L, 1753) and Lemna gibba (L, 1753) were isolated from a local pond, and washed with a 0.5 per cent (v/v) sodium hypochlorite solution to remove field contamination (pathogens, microalgae, associated fauna). Healthy fronds were then used to start a culture under laboratory controlled conditions (temperature 207 2 1C; photoperiod 16 hL:8 hD; light intensity:  4000 lx) in Steinberg medium, following the recommendations of OECD (2006b). Cultures were cyclically renewed by transferring a few (  10) individuals to fresh medium every week, under aseptic conditions. For all organisms, range-finding bioassays were carried out prior to definite tests (see below), in order to set appropriate paracetamol concentrations that allow obtaining EC50 values with the best confidence interval. Thus, instead of a general approach, we used species-specific concentration ranges and dilution factors, because of their differential sensitivity to paracetamol (see below).

2.2. Microtoxs procedures The toxicity of paracetamol was assessed following the Basic Test protocol using a Microtox Model 500 Analyser (AZUR Environmental, 1998). The endpoint measured by Microtox assay is the decrease in the intensity of light emitted by the bioluminescent marine bacteria – V. fischeri – after 5 min of exposure to several dilutions of the paracetamol stock solution (1000 mg l
1), after previous osmotic adjustment of the samples. This resulted in a geometric series of seven concentrations, ranging from 819 mg l
1 to 12.8 mg l
1 (using a dilution factor of 2). Bioluminescence inhibition EC50 values and respective 95 per cent confidence intervals were estimated by non-linear regression using the least-squares method to fit the logistic equation to the data.

2.3. Alga and cyanobacterium growth inhibition test Growth inhibition of the microalga P. subcapitata was assessed following USEPA (1994) and OECD (2006a) guidelines, adapted to 24-well microplates (Blaise and Jean-Francois, 2005). Algae were exposed for 72 h to a range of seven paracetamol concentrations (87.8–1000 mg l
1, in a geometric series using a dilution factor of 1.5). A replicated design (three replicates per treatment) was applied, using 900 ml of test solution plus 100 ml of algal inoculum per well. Initial algal density was set to 104 cells ml
1 (after counting in a haemocytometer), following OECD (2006a). Clean MBL medium was used as negative control (optimal growth). The microplates were incubated in an orbital shaker at 23 1C and continuous light (  7000 lx). Similar procedures were used for assessing growth inhibition with C. raciborski, with some modifications (eight paracetamol concentrations, ranging from 48.4 mg l
1 to 510.2 mg l
1, in a geometric series using a dilution factor of 1.4). First, experiments with the cyanobacterium were run in 100 ml Erlenmeyer flasks covered with gauze stoppers, containing 50 ml of test solution. Second, assay duration (14 d) and lighting conditions (  3000 lx) were adjusted to its specific requirements (slow growth and susceptibility to high light intensities). At end of the assays, we measured optical density of microalgal or cyanobacterial suspensions at 440 nm as a surrogate of biomass, since optical density is proportional to cell density (linear relationships can be found at optical densities between 0.010 and 1.000). Optical density data were fitted with a non-linear dose–response toxicity model (Hill's model; Vindimian et al., 1983), following the spreadsheet and macro provided by Éric Vindimian (REGTOX software, version EV7.0.6, accessed at: http://www.normalesup.org/  vindimian/en_index.html in November 2013); this

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B. Nunes et al. / Ecotoxicology and Environmental Safety 107 (2014) 178–185

spreadsheet also allows the estimation of the confidence intervals using a nonparametric bootstrap method (Garric et al., 1990).

and fecundity estimates were used to compute the per capita intrinsic rate of population increase (r), which was iterated from the Euler–Lotka equation: n

1 ¼ ∑ e
rx lx mx x¼0

2.4. Daphnia acute immobilization and reproduction tests Independent experiments were used to assess the acute toxicity of paracetamol to both D. magna and D. longispina. Tests were performed in accordance with standard protocols (ASTM, 1997; ISO, 1996; OECD, 2000), under the same temperature and photoperiod regimes described for rearing procedures. A static design was employed, using twenty animals (randomly divided into four groups of five animals) per treatment concentration. Test vessels consisted of glass tubes containing 10 ml of test solution (i.e. 2 ml per individual Daphnia). For each species, a geometric range of concentrations was obtained by diluting paracetamol with culture medium. Clean ASTM medium was used as negative control. Final concentrations for D. longispina and D. magna were 48.6–85 mg l
1 (five concentrations separated by a factor of 1.15) and 4.0–8.9 mg l
1 (eight concentrations separated by a factor of 1.12), respectively. After 48 h, the proportion of immobilized/mobile individuals was determined; EC50 values were obtained using probit analysis (Finney, 1971). Reproduction tests were conducted for 21 d in accordance with standard protocols (ASTM, 1997; ISO, 2000; OECD, 1998), under the same temperature and photoperiod regimes described for rearing procedures. A semi-static design was employed, using ten individualized animals randomly assigned to the control (clean ASTM medium) and to six paracetamol concentrations (using a dilution factor of 1.5): 7.9, 11.8, 17.8, 26.7, 40.0, and 60.0 mg l
1 for D. longispina; 0.53, 0.79, 1.2, 1.7, 2.7, and 4.0 mg l
1 for D. magna. Test vessels consisted of glass beakers containing 50 ml of test solution. Daphniids were transferred to newly-prepared paracetamol concentrations every other day, and were daily fed with their respective P. subcapitata ration (see above). Animals were checked daily for mortality and reproductive state and, if neonates had been released, they were counted and immediately discarded. The following parameters were registered: mortality, age at first reproduction (AFR), and total number of offspring. Survival Table 1 Summary of EC50 values and corresponding 95 per cent confidence intervals (CI95) obtained with the different species exposed to paracetamol. Species

Lowest CI95% o EC50 (mg l
1) ohighest CI95%

V. fischeri P. subcapitata C. raciborskii D. longispina D. magna L. minor L. gibba

71.83 o 92.2 o 112.58 292.6 o 317.4 o 341.4 171.0 o 192.9 o 213.5 63.2 o 65.9 o68.7 4.2 o 4.7 o5.0 352.2 o429.9 o 551.3 41000

where r is the intrinsic rate of increase (d–1), x is the age class in days (0…n), lx is the probability of surviving to age x, and mx, is the fecundity at age x. Standard errors for r were estimated using the jack-knifing technique described by Meyer et al. (1986). Data from each endpoint were analysed using a one-way analysis of variance (ANOVA), followed by a Dunnett test (if applicable), in order to determine significant effects induced by paracetamol exposure on the life history of each Daphnia species. Additionally, EC50 values for fecundity data were calculated using non-linear regression (REGTOX spreadsheet and macro, see above), similarly to microalgae and cyanobacteria data. 2.5. Lemna sp. growth inhibition test Independent experiments were used to assess the acute toxicity of paracetamol to both Lemna species. Tests were performed in accordance with standard protocols (OECD, 2006b), under the same temperature and photoperiod regimes described for rearing procedures. A range of five paracetamol concentrations (ranging from 62.5 mg l
1 to 1000 mg l
1, using a dilution factor of 2) was used for both species, which were exposed in a final volume of 100 ml of test solution, in triplicate. Clean Steinberg medium was used as negative control (optimal growth). Initial inocula for all test vessels consisted of 9–10 fronds of each species, and experiments lasted for 7 days. At the end of the assays, the number of fronds of each test vessel was recorded. Respective EC50 values were calculated using non-linear regression (REGTOX spreadsheet and macro, see above). 2.6. Species sensitivity distribution (SSD) Species sensitivity distribution curve was obtained using acute toxicity data from this study and from the literature (when possible). A log-probit distribution was used to model the data and to estimate 95 per cent CIs, using the spreadsheet provided by US Environmental Protection Agency (available at: http://www.epa. gov/caddis/da_software_ssdmacro.html, accessed on 22 December 2013). Estimated hazardous concentrations for 5 per cent of species (HC5) were calculated using graphical interpolation (Maltby et al., 2005).

3. Results Paracetamol was toxic to all test organisms in the tested concentrations, except for L. gibba (Table 1). No effects were found for this species at concentrations up to 1000 mg l
1. High among-species variability was found, with the following ranking

Table 2 Summary of paracetamol toxicity in aquatic organisms. Species

Endpoint

Concentration (mg l
1)

Reference

Vibrio fischeri Vibrio fischeri Tetrahymena pyriformis Scenedesmus subspicatus Streptocephalus proboscideus Streptocephalus proboscideus Brachionus calyciflorus Thamnocephalus platyurus Hydra vulgaris Artemia salina Daphnia magna Daphnia magna Daphnia magna Daphnia magna Daphnia magna Daphnia magna Daphnia magna Lemna gibba Oryzias latipes Oryzias latipes Brachydanio rerio Pimephales promelas

EC50 (15 min) EC50 (30 min) EC50 (48 h) EC50 (72 h) EC50 (24 h) LC50 (24 h) LC50 (24 h) LC50 (24 h)

567.5 650 112 134 9.2 29.6 5306 63.8 410 577 30.1 26.6 50 9.2 13 55.5 293 41.0 4160 4160 378 814

Kim et al., 2007. Environ. Int. 33, 275–370 Henschel et al., 1997. Regul. Toxicol. Pharm. 25, 220–225 Henschel et al., 1997. Regul. Toxicol. Pharm. 25, 220–225 Henschel et al., 1997. Regul. Toxicol. Pharm. 25, 220–225 Calleja et al., 1994. Arch. Environ. Contam. Toxicol. 26, 69–78 Kuhn et al., 1989. Water Res. 23(4), 495–499 Webb, 2001. Pharm. Environ. 175–201 Nałęcz-Jawecki and Persoone, 2006. Environ. Sci. Pollut. Res. 13(1), 22–27 Pascoe et al., 2003. Chemosphere 51, 521–528 Webb, 2001. Pharm. Environ. 175–201 Kim et al., 2007. Environ. Int. 33:370–375 Kim et al., 2007. Environ. Int. 33:370–375 Henschel et al., 1997. Regul. Toxicol. Pharm. 25, 220–225 Kuhn et al., 1989. Water Res. 23(4), 495–499 Webb, 2001. Pharm. Environ. 175–201 Calleja et al., 1994. Arch. Environ. Contam. Toxicol. 26, 69–78 Webb, 2001. Pharm. Environ. 175–201 Brain et al., 2004. Environ. Toxicol. Chem. 23, 371–82 Kim et al., 2007. Environ. Int. 33, 370–375 Kim et al., 2007. Environ. Int. 33, 275–370 Henschel et al., 1997. Regul. Toxicol. Pharm. 25, 220–225 U.S. Environmental Protection Agency, 2005. ECOTOX User Guide: ECOTOXicology Database System. Version 3.0. Available: http://www.epa.gov/ecotox/

LC50 (24 h) EC50 (48 h) EC50 (96 h) EC50 (48 h) EC50 (48 h) EC50 (24 h) EC50 (24 h) EC50 (24 h) EC10 LC50 (48 h) LC50 (96 h) LC50 (48 h) LC50 (96 h)

B. Nunes et al. / Ecotoxicology and Environmental Safety 107 (2014) 178–185

B. calyciflorus

Proportion of species affected

1 0.9

P. promelas A. salina L. minor D. rerio P. subcapitata C. raciborskii S. subspicatus T. pyriformis V fischeri D. longispina T. platyurus

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1.00

Central Tendency

S. proboscideus D. magna 10.00

95% Prediction Interval

100.00

1000.00

10000.00

Paracetamol concentrations (mg l-1)

Fig. 1. Species sensitivity distribution (SSD) plot, showing the distribution of EC50s for organisms acutely exposed to paracetamol, with 95 per cent confidence intervals (dotted lines). Data were obtained from the literature and from this study (highlighted in bold and underlined).

of toxicity (in terms of EC50 values, from most sensitive to most tolerant): D. magna oD. longispina o V. fisheri oC. raciborskii oP. subcapitata oL. minor oL. gibba. Daphnids were the most sensitive ecoreceptors of this pharmaceutical, justifying the assessment of potential effects of chronic exposure to paracetamol in these species (see next paragraph). EC50 values from the literature were very variable (Table 2). The relative sensitivity of aquatic species to paracetamol, from literature data and the present study, is shown in Fig. 1. An HC5 value (concentration which is hazardous to 5 per cent of species) of 7.6 mg l
1 (95 per cent CI: 3.4–17.3 mg l
1) was observed. In chronic exposures, paracetamol caused mortality in the highest concentration(s). This was so abrupt in D. magna (between 1.2 mg l
1 and 1.7 mg l
1) that no organisms survived the whole duration of the experiment in the last three concentrations, although they did reproduce (see differences between fecundity at day 10 and day 21). Mortality occurred from day 7 onwards, but mostly after day 10, when reproduction had already occurred (approximately two clutches per female). No significant reproductive impairment was observed in the remaining concentrations (Fig. 2), either considering the full length of the assay (one-way ANOVAs for age at first reproduction and total offspring, p 40.05) or the first 10 days of the test – when most individuals were still alive (one-way ANOVA for fecundity at day 10, p 40.05). For this reason, no reproductive EC50 could be calculated. A smoother transition was found for D. longispina, which experienced a significant delay in the first reproductive event (one way ANOVA on age at first reproduction: MS ¼15.1; d.f. ¼ 5, 45; F¼5.7; p o0.001) and in fecundity (one way ANOVA on number of offspring: MS¼ 6352; d.f. ¼5, 47; F¼ 271; p o0.001) prior to high mortality (Fig. 2). A reproductive EC50 of 12.2 mg l
1 was found for this species. Mortality and fecundity were integrated in the intrinsic rate of increase, which showed a significant population decrease in D. magna and D. longispina from 4.0 mg l
1 and 17.8 mg l
1 onwards, respectively.

4. Discussion Table 1 shows the effectiveness of paracetamol to elicit toxic effects in all tested organisms, with the exception of L. gibba. From a preliminary analysis of the obtained data, it is possible to state that paracetamol was more toxic against the two crustacean species (D. magna and D. longispina), than against primary producers (the algal species and the two selected macrophytes). Intermediate values were obtained for bacteria V. fischeri and C. raciborskii. This set of results is intrinsically interesting, because it implies that the most likely group to be impacted by exposure to

181

paracetamol corresponds to the primary consumers (freshwater crustaceans), which occupy an intermediate position in the food web. No significant effects on producers can be attributed to paracetamol, since neither the macrophytes nor the unicellular algal species were responsive to low levels of this drug. However, and despite the occurrence of effects for almost all tested species, it must be emphasized that the levels of paracetamol required to elicit such effects surpass in several orders of magnitude the concentrations already documented in the aquatic compartment. This is an important consideration in ecological terms, since it implies that exposure to paracetamol alone will not, most probably, result in acute deleterious effects. However, one must also take into account the typology of effects that are quantified when using standardized guidelines of toxicity testing, which often rely in rough estimates of toxic effects, with results being quantified in terms of mortality or growth impairment. Other types of effects can occur at concentrations that are well below those required to cause mortality or growth impairment, but can compromise key functions of the test organisms. Water pollution can lead to different adaptation changes, ranging from biochemical alterations in single cells up to modifications in whole populations (Vasseur and Cossu-Leguille, 2006). In previous studies with freshwater (Brandão et al., 2014) and marine clams (Antunes et al., 2013), we have already shown that oxidative stress (confirmed by GSTs activity and lipid peroxidation alterations) can occur at concentrations in the mg l
1 range, prior to mortality scenarios. Considering that oxidative stress can occur well before mortality, and that the most common outcomes of oxidative stress include oxidation of DNA, proteins and lipids, it is not difficult to establish a connection between low levels of organic pollution (including by the pharmaceutical drug paracetamol), and the onset of long term degenerative processes, such as cancer, that can ultimately lead to death, with obvious consequences at the population level. Another aspect to consider is the extent of the effects; lower levels of contamination might cause slight reductions in population terms, which are indisputably of ecological relevance (Forbes and Calow, 2002). Indeed, as observed in our study, low concentrations of paracetamol caused significant impairments of fecundity and population growth potential in daphnids. Still, the concentrations required to elicit such effects are still fairly high (41.7 mg l
1), if compared to those reported in the wild. However, these data are paradigmatic, as D. magna was consistently (both in acute and chronic exposures) more sensitive than D. longispina, which is not the most common scenario (see Antunes et al., 2007; Gonçalves et al., 2007). An interesting issue can though be raised; considering the bibliographic data concerning results obtained only with D. magna as test species (Table 2), one can observe a large variability of results, of more than one order of magnitude in some cases. This makes the comparison between both species hard to sustain, and difficult to extrapolate to real life conditions. Rather than being of absolute meaning, the comparisons between both Daphnia species are more important to establish rankings of sensitivity. Additional studies should be conducted with crustacean zooplankton species; they are clearly the most sensitive ecorreceptors of this drug, and a literature review on acute toxicity data of paracetamol confirms this trend (Table 2 and Fig. 1). The most sensitive species, based on the hereobtained results, were crustaceans. This may be of significant ecological meaning if one considers that paracetamol is capable of causing oxidative stress (see above), which may interfere with moulting (see Fanjul-Moles and Gonsebatt, 2012; Rodríguez et al., 2007), thus configuring a case of endocrine disruption. The alkylphenolic structure of paracetamol may be responsible for this effect, since this compound shares structural similarities with compounds that belong to a well-known family of endocrine disruptors in crustaceans (Rodríguez et al., 2007), which display

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B. Nunes et al. / Ecotoxicology and Environmental Safety 107 (2014) 178–185

Mortality

Mortality 100

80

80

60

60

40

40

20

20

%

100

0

0

Days

Age at first reproduction

Age at first reproduction

14

14

12

12

10

10

8

8

6

6

4

4

2

2

0

*

0

number of neonates

Total offspring 130 120 110 100 90 80 70 60 50 40 30 20 10 0

Total offspring

Fecundity at day 21 Fecundity at day 10

x

x

x

130 120 110 100 90 80 70 60 50 40 30 20 10 0

EC50= 12.2 mg l-1 (IC95%:11.7-12.8)

* *

Rate of population increase

intrinsic rate of increase (day -1)

*

*

Rate of population increase

0.5

0.5

0.4

0.4

*

0.3

*

0.3

* 0.2

0.2

0.1

0.1

0.0

0.0

-0.1

* *

-0.1 0

0.53

0.79

1.2

1.7

Daphnia magna

2.7

4

0

7.9

11.8

17.8

26.7

40

60

Daphnia longispina

Fig. 2. Life history responses of Daphnia magna and Daphnia longispina exposed for 21 days to a range of paracetamol concentration (x-axis, in mg l
1). Error bars correspond to standard error and * represents statistically significant differences (Dunnett test, p r 0.05) between the different paracetamol concentrations and the negative control. Fecundity (total offspring) is shown at day 10 and day 21 for D. magna for comparative purposes; because all test organisms (mothers) died before the end of the assay, in the last three concentrations (represented by the X), no fecundity estimates could be calculated at day 21.

B. Nunes et al. / Ecotoxicology and Environmental Safety 107 (2014) 178–185

anti-ecdysteroidal activity (LeBlanc, 2007). Further research is needed to clarify whether paracetamol does in fact impair moulting in arthropods (namely crustaceans), thus eliciting endocrine disruption. Table 2 depicts results from the literature regarding acute toxicity of paracetamol for several aquatic species, including bacteria, unicellular algae, cnidarians, crustaceans (both freshwater and saltwater) and fish. The most striking evidence is the multiplicity of the reported toxicity values (Fig. 1); values can range from 9.2 mg l
1 to 5306 mg l
1. It is thus difficult to systemize the assessment of paracetamol toxicity by only referring to the literature data. Paracetamol toxicity also differs among organisms with some phylogenetic similarity (see Table 2 and Fig. 1). In our study, marked differences (approximately 14-fold) were observed when comparing acute EC50 values for D. longispina (65.9 mg l
1) and for D. magna (4.7 mg l
1). Additionally, large variations can be observed for studies that used the same test organism, such as those that assessed toxicity of paracetamol on D. magna (see Table 2). Although being considered a standard test species, D. magna-based bioassays (48 h EC50) resulted in evidently distinct results, which ranged from 9.2 to 50 mg l
1. Although having been only reported for humans, increased toxic effects of paracetamol can be related to nutrient deficits (Berling et al., 2012; Vale, 2012), because nutrition can affect glutathione metabolism, which is the main metabolic pathway explaining paracetamol biotransformation and subsequent excretion (Geenen et al., 2013). Differences in the nutritional status of test organisms can thus justify toxicity differences. This is a possibility that cannot be discarded when discussing variations in toxicity values such as those referred in the literature, given distinct rearing procedures among laboratories. However, this biological variation in response can be of extreme importance, since it is extremely difficult to establish benchmark toxicity values for a specific compound when its toxicity is inherently variable, even for experiments conducted with the same organism in a controlled environment. The described biotransformation of paracetamol in most mammals can occur by two main routes, which depend upon the level of exposure. Low levels of exposure usually do not result in any cellular damage, since paracetamol may undergo conjugation with intracellular co-factors, forming the non-toxic conjugated metabolites paracetamol glucuronide and paracetamol sulphate in reactions catalysed by sulfotransferase and UDP-glucuronyltransferase, respectively (Letelier et al., 2011; Xu et al., 2008). Exposure to low levels of paracetamol thus results in its biotransformation (by more than 90 per cent) by the sulphation and glucuronidation conjugation pathways; the formed conjugates are ultimately excreted in the urine. A smaller portion of paracetamol is excreted without conjugation, and a small amount suffers oxidation by the cytochrome P450 isoenzymes (primarily CYP 2E1, 1A2, and 3A4), leading to the formation of the toxic metabolic NAPQI (Kavitha et al., 2011; Letelier et al., 2011; Xu et al., 2008). However, low doses of paracetamol result in the synthesis of small amounts of Nacetyl-p-benzoquinoneimine (NAPQI), which is its main reactive metabolite, and largely responsible for its toxicity (Davern et al., 2006). In this scenario of low level of exposure, NAPQI is promptly conjugated with intracellular glutathione for detoxification (Xu et al., 2008), with the intervention of GSTs. This step is responsible for the production of a less reactive and more soluble compound, which is readily excreted in urine. The excretion of NAPQI prevents the occurrence of oxidative stress and cellular damage. However, large doses of paracetamol can cause the exhaustion of the required co-factors (glucuronic acid, sulphate and glutathione), leading to a sudden increase in the formation in NAPQI and in its consequent toxic effects, which include covalent modifications of thiol groups of cellular proteins (Xu et al., 2008), DNA and RNA damage, and oxidation of membrane lipids, resulting in necrosis

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and cellular death (Hinson et al., 2004). Despite not being entirely described for non-mammalian species, the metabolism of paracetamol seems to be also mediated by the formation and release of reactive oxygen species, in other organisms, such as fish (Ramos et al., 2014), mollusks and crustaceans (Antunes et al., 2013; Brandão et al., 2014; Parolini et al., 2009; Touliabah et al., 2008). The overproduction of reactive oxygen species by paracetamol metabolism was connected to the establishment of a scenario of oxidative stress, which was even capable of altering population traits in crustacean species, as evidenced by Sarma et al. (2013). It is thus possible to conclude that the nutritional status of a given organism can alter the amounts of the intracellular reserves of cofactors required for paracetamol biotransformation. Considering that test organisms reared for toxicity testing are fed with variable diets and nutrient supplies, it is important to normalize the intake of critical nutrients, which in turn, are fundamental to elicit a standard, straightforward toxicological response. The variation in the observed data among species, also confirmed by the here-obtained results, can also be explained by species-specific biotransformation differences. As shown by previously published data, distinct species do not equally metabolize paracetamol, with important differences in terms of toxicological outcomes; rodent species, for instance, are extremely variable in their capacity to metabolize paracetamol by the oxidative pathway, and exhibit marked toxicological differences (Boobis et al., 1986). Additionally, differences in phase II enzymes can also be accounted for the already referred multiple toxic responses among species, as evidenced by Bessems and Vermeulen (2001). Given the already described all-or-nothing nature of toxicological response (as a function of exposure levels), and the variables that can ultimately condition paracetamol toxicity, from species to species, and even for the same species tested under diverse conditions, it is extremely difficult to produce consistent ecotoxicological data of paracetamol. Therefore, this leads to a large degree of uncertainty in predictions based on benchmark values, and the ability to assess its potential effects at the ecosystem level. However, considering the implications of paracetamol exposure and consequent toxicity that may derive from it, it is of paramount importance to design experiments that include multispecies assessments, combining low/high levels and chronic vs acute conditions, which cover the entire spectra of effects that are expected to occur. The use of a biomarker approach has also proven useful to enlighten the antioxidant and oxidative stress state of the exposed individuals (Antunes et al., 2013; Brandão et al., 2014). The evidences presented here, and supported by specialized literature, show that the evaluation of noxious effects of pharmaceutical compounds – and paracetamol in particular – in the biota has “more than the eye can see”. These substances are designed to be biologically active and their toxicity seems to be intimately linked to the physiological condition of the organisms, namely their detoxification pathways. As already evidenced by previous approaches, a suitable test methodology to be used in environmental assessment of toxic effects posed by specific substances must consider the underlying mechanisms of toxicity (Breitholtz et al., 2006). Most commonly referred paracetamol toxic mechanisms result from the onset of oxidative stress, which occurs not only in common mammalian test species, but also for aquatic organisms (please see references above); however, endocrine effects have not been extensively investigated. Given this fact, it is possible to conclude that paracetamol ecotoxicity testing has relied on sub-optimal testing protocols and inadequate endpoints, which are, consensually, blunt tools that only assess mortality or rough population estimates. Also, there is a gap of knowledge on paracetamol toxicity in bacteria, plants, and invertebrates (which we tried to address here), and there is a

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pronounced bias towards literature with vertebrate data. As such, the resulting uncertainty caused by the variable spectra of benchmark toxicity values, and the underlying mechanistic effects, should indeed be a headache to environmental scientists and regulators – despite paracetamol's analgesic properties.

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