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Constructed wetlands for the removal of metals from livestock wastewater – Can the presence of veterinary antibiotics affect removals?
Ecotoxicology and Environmental Safety 137 (2017) 143–148
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Constructed wetlands for the removal of metals from livestock wastewater – Can the presence of veterinary antibiotics affect removals?
MARK
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C. Marisa R. Almeidaa, , Filipa Santosa, A. Catarina F. Ferreirab, Carlos Rocha Gomesb, M. Clara P. Bastob, Ana P. Muchaa a Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR / CIMAR), Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal b CIIMAR/CIMAR e Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Alegre, s/n, 4169-007 Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Keywords: Wastewaters Green technology Remediation Enrofloxacin
The presence of emergent antibiotics, in livestock wastewater may affect constructed wetlands (CWs) performance in the removal of other pollutants. The main objective of this study was to evaluate the influence of two antibiotics commonly used in livestock industry, enrofloxacin and ceftiofur, on metal removal by CWs. Microcosms (0.4 m×0.3 m×0.3 m), simulating CWs, were constructed with Phragmites australis to treat livestock wastewater spiked or not with 100 µg/L of enrofloxacin or ceftiofur (individually or in mixture). Wastewater was treated during 20 one-week cycles. After one-week cycle wastewater was removed and replaced by new wastewater (with or without spiking). At weeks 1, 2, 4, 8, 14, 18 and 20, treated wastewater was analysed to determine the removal rates of metals (Zn, Cu, Fe and Mn) and of each antibiotic. At weeks 1, 8 and 20 portions of the plant root substrate were collected and metals determined. At the end of the experiment metal levels were also determined in plant tissues. Removal rate of Fe from wastewater was 99%. Removal rates of Cu and Zn were higher than 85% and 89%, respectively, whereas for Mn removal rates up to 75% were obtained. In general, no significant differences were observed through time in the removals of the different metals, indicating that the systems maintained their functionality during the experimental period. Antibiotics did not interfere with the system depuration capacity, in terms of metals removals from wastewater, and ceftiofur even promoted metal uptake by P. australis. Therefore, CWs seem to be a valuable alternative to remove pollutants, including antibiotics and metals, from livestock wastewaters, reducing the risk the release of these wastewaters might pose into the environment, although more research should be conducted with other antibiotics in CWs.
1. Introduction Constructed wetlands (CWs) are engineered systems designed and built to mimic the biological, chemical and physical processes that occur in natural wetlands (Zhang et al., 2014). These processes include sorption, sedimentation, photolysis, hydrolysis, volatilization, plant uptake and accumulation, plant exudation, microbial degradation, filtration, precipitation and adsorption, removing pollutants from contaminated waters in a more controlled environment (GarciaRodríguez et al., 2014; Wu et al., 2014). CWs can be used to reduce the load of pollutants, such as nutrients, metals or organic matter, present in different types of wastewaters, including livestock wastewaters (e.g. Meers et al., 2005, Meers et al., 2008). In fact, CWs have successfully treated wastewaters contaminated with metals from acid mine drainage, metallurgy, tannery, swine ⁎
production, landfill leachates and domestic effluents (Arivoli et al., 2015) and references therein). Although the efficiency for metal removal varied with the plant species (Vymazal and Březinová 2015) and with the substrate composition (Allende et al., 2011), as well as with the metal itself (Marchand et al., 2010), removal rates as high as 90% have been reported (e.g. Vymazal, 2005; Morari et al., 2015). CWs systems have also recently been used to remove the so-called pollutants of emerging concern, including pharmaceutical compounds. In fact, several review articles on removal of pharmaceuticals from wastewaters by CWs have recently been published (e.g. Verlicchi et al., 2013; Li et al., 2014). Although most of the work has been for urban wastewaters, some studies have already shown CWs potentialities for the removal of pharmaceuticals, including antibiotics, from livestock wastewaters (e.g. Hussain et al., 2012; Carvalho et al., 2013; Hsieh et al., 2015). The more complex matrix of livestock wastewaters when
Corresponding author. E-mail address: [email protected] (C.M.R. Almeida).
http://dx.doi.org/10.1016/j.ecoenv.2016.11.021 Received 18 March 2016; Received in revised form 24 November 2016; Accepted 25 November 2016 0147-6513/ © 2016 Elsevier Inc. All rights reserved.
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ones (Carvalho et al., 2013). All microcosms had a tap at the bottom and worked in batch mode. The wastewater was poured on top, to percolate through the different layers of the solid matrix (matrix water saturation of ca 100%, with water just below the surface, ca. 1 L of wastewater), and drained out through the tap when necessary, simulating a sub-surface vertical flow CW. In this type of CWs wastewater flows under the surface of the planted bed, with the water percolating through the substrate.
compared with that of urban wastewaters makes these wastewaters more difficult to treat. These wastewaters can have high loads of organic matter, solids and nutrients, as well as pharmaceutical compounds such as antibiotics. However, pollutants of emerging concern, including antibiotics, can be harmful for both microorganisms and plants, which are key players in CWs removal processes. For example, there is a growing body of evidence documenting a reduction of microbial diversity in soils contaminated with antibiotics (Jechalke et al., 2014). In addition, exposure of plants to pharmaceuticals, including antibiotics, may influence, for example, plant development, due to phytotoxicity (Carvalho et al., 2014). Thus, the presence of pollutants of emerging concern, namely antibiotics, in livestock wastewaters may affect CWs performance for the elimination of other pollutants, such as metals. This subject needs to be investigated. Therefore, the aim of this work was to evaluate the influence of two antibiotics commonly used in livestock industry, enrofloxacin and ceftiofur, alone or in a mixture, on the removal of metals from livestock wastewaters by CWs.
2.1.3. Microcosms operation The livestock wastewater was treated during 20 one-week cycles. At the beginning of each week, new wastewater (with or without spiking with veterinary antibiotics) was added. Wastewater was daily recirculated to prevent the formation of anaerobic areas. After each one-week cycle, treated wastewater was removed and replaced by new wastewater (with or without spiking with the selected antibiotics), allowing to simulate a hydraulic retention time commonly used in CWs. This continuous addition of wastewater into the CWs systems also allowed simulating the cumulative effect of adding continuously a new load of pollutants to the system, which is the situation in full scale real CWs systems. This continuous load of pollutants can lead in the long term to accumulation of pollutants in the systems, which might affect CWs performance along time. At the end of weeks 1, 2, 4, 14, 18 and 20 the wastewater treated by the CWs was collected for analysis of metals (naturally present in the livestock wastewater) and to estimate their removal rates in the presence and absence of antibiotics. At the same time aliquots (ca. 5 g) of plants roots bed substrate were also collected from each CW microcosm. Microcosms were kept under greenhouse conditions, subjected to environmental temperature variations (21–25 °C) and to natural light exposure, along 20 weeks (May to October 2014), after which they were dismantled. After dismantling, plants were separated from the plants roots substrate, washed and put to dry until constant weight. Then plants were separated into roots, rhizomes, leaves and stems. All substrate samples were lyophilized.
2. Methodologies 2.1. Microcosms experiments Experiments were carried out in controlled conditions in microcosms simulating CWs. Small-scale process experiments allow to fully control the experimental conditions when intending to study the influence of specific variables, such as, in the present case, the presence of antibiotics in wastewater. Microcosms were planted with Phragmites australis, one of the plants more frequently used in CWs (Stottmeister et al., 2003). Two antibiotics commonly used in Portuguese livestock industry for therapeutic purposes were chosen: enrofloxacin (Enr) and ceftiofur (Cef). These antibiotics belong to different families (fluoroquinolone (Enr) and cephalosporin (Cef) families) and present different physical-chemical properties. Considering that more than one antibiotic can be present in wastewaters and that either antagonistic or synergetic effects can occur, the influence of a mixture of the two antibiotics on CWs performance was also assessed. 2.1.1. Sampling Livestock wastewater (after being treated in two lagoons (one anaerobic and another aerobic)) was collected every week in a pig farm. The wastewater was used as collected or spiked with one or both antibiotics. This wastewater already contained significant amounts of different metals (see Section 3). P. australis plants were collected in Lima River (NW Portugal) with the sediment attached to their roots to preserve plants’ rhizosphere. In the laboratory, sediment was removed and mixed with river sand (in a 1:2 proportion) to prepare the plants roots bed substrate into which plants were transplanted (each microcosms had ca. 80 plants). This mixing was carried out to increase the substrate porosity. A few plants and roots bed substrate were set aside to determine initial metal levels. These samples were called field plants and field substrate.
2.2. Samples analysis All reagents were pro analysis or equivalent. All material was washed with deionised water (conductivity < 0.1 µS cm−1), immersed in nitric acid solution (20% v/v) for 24 h, washed again with deionised water and dried in a clean oven. Samples of initial and treated wastewater were filtered (nitrate cellulose filters, 0.45 µm porosity) and acidified (with 1% nitric acid) before direct analysis. Plants roots substrate and plant tissues (dry samples) were digested with concentrated nitric acid (HNO3) and 30% hydrogen peroxide (H2O2) solution (only for plant tissues). Digestions were carried out in a high-pressure microwave system (Ethos, Milestone) in closed Teflon vessels. Metals (Cd, Cu, Fe, Mn, Ni, Pb, Zn) concentrations were measured in an atomic absorption spectrophotometer with flame atomization (AASF-PU 9200X, Philips), as described before (Almeida et al., 2004). A calibration curve obtained with aqueous standard solutions of different metal concentrations (0–3 mg/L) was used. These standard solutions were prepared from 1000 mg/L stock standard solutions of each metal. Antibiotics concentrations in treated wastewaters and plants roots bed substrate were determined by high-performance liquid chromatography (HPLC), after an ultrasonic extraction (only for solid matrix) and a pre-treatment by solid phase extraction (SPE) as described before (Carvalho et al., 2013).
2.1.2. Microcosms’ assemblage Microcosms (0.4 m×0.3 m×0.3 m), simulating CWs, were constructed to treat livestock wastewater not spiked (Control) or spiked with 100 µg/L of Enr or with 100 µg/L of Cef. Another set of microcosms was prepared in which the wastewater was spiked with a mixture of Enr and Cef, each antibiotic with a concentration of 100 µg/ L (Mix). This concentration, although relatively high, has already been found in wastewaters effluents (Babić et al. 2010). Three replicates per variable were constructed. All CWs microcosms had three layers: the first one with gravel (4 cm), the second one with lava rock (2 cm) and the third layer with the plants roots bed substrate (11 cm height) described above. These CWs systems were based on previously used 144
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control (significant differences (p < 0.05) until W8). For Mn and Fe removal rates were very variable (Fig. 1). For Mn, removal rates in wastewater spiked with Enr were more or less constant over time, whereas for remaining treatments removal rates increased over time. For Fe, removal rates in control CWs microcosms (treating wastewater not spiked with antibiotics) decreased over time, whereas in the presence of Enr removal rates were constant until W8, decreasing afterwards. In the presence of Cef and of Cef+Enr (Mix) Fe removal rates varied significantly along time. Both antibiotics were also significantly removed from the wastewater, being below analytical detection limit. In fact, removal rates from wastewater were higher than 90% (considering antibiotics concentration in soluble phase) in all CWs microcosms used to treat wastewater spiked with veterinary antibiotics. Antibiotics were also not detected in plants roots bed substrate (concentrations were always below detection limit ( < 0.09 µg/g for Enr and 0.2 µg/g for Cef)). In plants roots bed substrate, Cd concentrations were always below detection limit ( < 3 µg/g). For Ni and Pb, levels were between 5– 10 µg/g and 10–20 µg/g, respectively without significant differences (p > 0.05) between field substrate (initial substrate used to construct the CWs microcosms and not exposed to wastewater) and plants roots bed substrate collected in the different CWs microcosms along time. For Cu, metal concentrations in substrate increased over time, without significant (p < 0.05) differences among the different CWs microcosms in each week (Fig. 2). Comparatively to field substrate, a significant (p < 0.05) decrease in Cu concentration at W1 was observed, but then Cu concentration increased over time. In W20 Cu levels were significantly (p < 0.05) higher than those initially present, with the exception of Cef treatment for which differences were not significant (p > 0.05). A similar behaviour was observed for Zn, but at W20 metal levels were identical to initial levels in field substrate. The only exception was Cef treatment for which significantly (p < 0.05) higher Cu levels were observed. For Mn, a similar tendency to decrease in W1 was observed, but differences were not significant (p > 0.05). After W1, Mn concentrations were identical throughout time and among treatments. Regarding Fe, in general, concentrations were identical along time and identical to levels in field substrate. The only exception was the substrate exposed to wastewater spiked with Cef which showed an increase in Fe concentrations over time. In plant tissues Cd concentrations were always below detection limit (1.5 µg/g). For Ni and Pb, levels were only above detection limits (which were 2 µg/g and 5 µg/g for Ni and Pb, respectively) in plant roots (ca. 5 µg/g for Ni and ca. 16 µg/g for Pb), without significant differences (p > 0.05) between field plants (plants collected in the field similar to the ones used in CWs microcosms but not exposed to wastewater) and control plants (plants in CWs microcosms exposed to wastewater not spiked with antibiotics) or among plants exposed to different treatments. For the other metals (Cu, Fe, Mn and Zn), in general, concentrations in the different plant tissues increased relatively to concentrations observed in not exposed plants (field plants) (Fig. 3). For Cu, comparatively to field plants, in general, a significant (p < 0.05) accumulation of the metal after the 20-week cycles was observed for all tissues of the plant (not only in belowground but also aboveground tissues). For this metal, in general, no significant (p > 0.05) differences were observed between plants exposed and not exposed to the veterinary antibiotics (control microcosm). The only exception was for the systems exposed to Cef. In this microcosm Cu levels in plants stems were significantly higher than those in plants stems not exposed to the antibiotics. For Mn and Zn, results were very similar to those of Cu, with both stems and leaves of plants exposed to wastewater spiked with Cef presenting higher metal concentrations than plants of the other treatments. However, for Zn, differences in metal levels in belowground tissues, between the field plants and those collected after 20-week cycles of treatment, were only significant (p < 0.05) for wastewater spiked with Cef (alone or in a mixture).
Fig. 1. Iron (A), manganese (B) and zinc (C) removal rates from livestock wastewater along 20 one-week cycles treatment of livestock wastewater (Wi: week i). Wastewater not spiked (Cont) or spiked with enrofloxacin (Enr) or with ceftiofur (Cef) or with a mixture of Enr and Cef (Mix). WP1 in Cont for Mn not determined.
2.3. Data analysis For wastewater and substrate samples, mean and respective errors of metal concentrations were calculated for each treatment (i.e., for each set of three replicates, one from each microcosm). For metals concentrations in plant tissues, due to the high plant natural variability, three replicates were analysed for each microcosm and then mean and respective errors of metal concentrations were calculated for each treatment. To evaluate statistically significant differences between treatments, students’ t-test (p < 0.05) in GraphPad Prism 6 software was used.
3. Results Metal levels (Cd, Cu, Fe, Mn, Ni, Pb and Zn) were measured in livestock wastewater before and after treatment in the different CWs microcosms. Levels of Cd, Ni and Pb were always below detection limit ( < 0.10 mg/L). For Cu, initial levels varied between 0.26 and 2.2 mg/L and final levels were always below detection limit ( < 0.050 mg/L), showing removal rates higher than 85%. Removal rates of Zn were higher than 89% (Fig. 1), whereas for Mn removal rates were lower, ca. 75%. For Fe, removal rates from wastewater were up to 99%. In general, for Zn no significant differences (p > 0.05) were observed among treatments or among weeks, except for wastewater spiked with Cef which had in general lower Zn removal rates than 145
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Fig. 2. Copper (A), iron (B), manganese (C) and zinc (D) in plants roots substrate at the beginning of the experiment (field) and after exposure to 20 one-week cycles of treatment of livestock wastewater (Wi: week i). Wastewater not spiked (Cont) or spiked with enrofloxacin (Enr) or with ceftiofur (Cef) or with a mixture of Enr and Cef (Mix).
lower than those reported in the Portuguese legislation for wastewater discharge (Decreto-Lei no 236/98). These metal concentrations were also identical or lower than those defined in international legislation regarding water quality for irrigation (Norton-Brandão et al., 2013). Removal of metals in CWs occurs mainly by binding to CWs substrate, precipitation as insoluble salts and uptake by bacteria, algae and plants. However, binding to substrate within wetlands is considered the major process for removal of metals (Bhatia and Goyal, 2014). In the present study, removal of metals from wastewater also occurred by sorption to plants roots bed substrate because metal (Cu, Mn and Zn) levels in substrate increased over time. In fact, despite a decrease in the levels of metals after the first week of experiment, probably due to substrate lixiviation, metals levels in substrate increased showing the adsorption capacity of this matrix. Other studies also have shown this feature (e.g. Yadav et al., 2012; Arivoli et al., 2015; Travaini-Lima et al., 2015). The only exception was Fe, probably due to the already high concentration of this metal in the substrate which prevented the detection of Fe inputs from the wastewater. Most CWs employ crushed stones, sand and gravel as substrates not only to support the plant growth but also to act as a filter; however, other substrates such as clay soil, zeolites, shells and industrial wastes (furnace slag, steel slag, sludge from waste treatment plants) had also been found as efficient filter materials (Arivoli et al., 2015). In the present study, lava rock (a very porous material) was also part of the CWs microcosms and as so, metals might have been also adsorbed to this fraction. Nevertheless, metals were not determined in lava rock layer because by the end of the experiments the rock was mostly smash and was not possible to fully separate it from the other components of the microcosms. Removal of metals from wastewaters in CWs may also occurred by plant uptake. In fact, some wetland plants, including those used in CWs, such as P. australis, are known by their ability to uptake metals without showing metal toxicity (Bhatia and Goyal, 2014). Studies involving metal uptake by P. australis in the environment have shown the potential of this plant to phytoremediate metal contaminated soil and
Regarding Fe, although an increase in the metal levels in plant roots from the CWs microcosms relatively to not exposed plants (field plants) was observed, the increase was only significant (p < 0.05) for plants exposed to wastewater spiked with the antibiotics. On the other hand, Fe concentrations in plants rhizomes and leaves were identical among all plants. For plant stems the scenario was different. In fact, Fe levels were statistically identical (p > 0.05) between field plants and those of control and Enr treatments, but Fe levels in stems of plants exposed to wastewater spiked with Cef or with Cef+Enr were significantly (p < 0.05) higher than in plants exposed to wastewater not spiked with antibiotics (control plants). It should be mentioned that pH values in initial water were ca. 7.8, whereas in treated wastewater varied between 7.3 and 8.0. 4. Discussion Livestock wastewaters have different pollutants, including metals (e.g. Meers et al., 2005), that need to be removed before their disposal in the environment. CWs can be a suitable alternative as they have been used to treat livestock wastewaters not only for the removal of conventional pollutants (e.g. Lee et al., 2004; Meers et al., 2005, 2008), but also for the removal of emergent compounds, such as pharmaceuticals (e.g. Hussain et al., 2012; Carvalho et al., 2013; Hsieh et al., 2015). Nevertheless, there is a need to understand if the presence of antibiotics can interfere with removal of metals is this type of effluents. Removal rates of metals present in the used livestock wastewater observed in the present study were similar to those reported before, including for CWs applied for treatment of livestock wastewaters (Meers et al., 2005, 2008). In general, no significant differences were observed through time in the concentrations of the different metals analysed (Fe was the only exception, with a slight decrease in its removal rate over time), indicating that the systems maintained its functionality during the experimental period. It should be mentioned that metals concentrations in CWs treated wastewater were always 146
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the medium. For instance, the ability of Enr to form complexes with metal cations is well known (e.g. (Graouer-Bacart et al., 2013), and Cef has also the capacity to bound Cu (unpublished results). Moreover, in CWs the growth of macrophytes and their depurative performance may be influenced by interactions among mixed pollutants (GuittonnyPhilippe et al., 2015). These features can interfere with the removal of metals and their distribution in CWs. In the present study, in general, metal sorption to plants roots bed substrate was identical among all CWs microcosms, indicating that the amount of antibiotics, either alone or in a mixture, did not affect the retention of metals over time. One should be aware that antibiotics were also retained in the CWs. In CWs, antibiotics can be adsorbed to the solid matrix, biologically degraded or accumulated by plants. In fact, previous studies carried out by the authors (Carvalho et al., 2012, 2013) have shown that all these processes can contribute for Enr and Cef removal from contaminated wastewater in CWs. But, as none of the antibiotics were detected in the solid matrix, either in previous studies (Carvalho et al., 2013) or in the present one, biological degradation was probably the main antibiotics removal process. In that sense a high influence of the antibiotics on metal adsorption was not expected because these compounds were probably being degraded over time and not accumulating in the substrate. However, metal uptake by P. australis was affected by the presence of Cef. In fact, Cef potentiate Cu translocation to plants stems and Mn and Zn translocation to plants stems and leaves, a feature not observed for Enr. In the presence of Enr metal concentrations in plant tissues were identical to those of control (plants in CWs microcosms treating wastewater not spiked with antibiotics). This behaviour was observed also, in general, for the treatment with the mixture of the two antibiotics indicating Enr probably interfered with Cef. Moreover, Cef also potentiated Zn accumulation in plants roots relatively to control. In the case of Fe, Cef potentiated metal translocation to stems, an effect also observed in the systems where both antibiotics were present. Therefore, Cef affected the ability of plants for the absorption of metals, particularly the translocation of the metals, increasing it. Metal uptake by plants depends on the form in which the metal is present in the medium and the simultaneous presence of pollutants of different nature can modify the mechanisms of absorption of metals by the plants. For example, a greater accumulation of Cu by the wetland plant Haliminone portulacoides in the presence of hydrocarbons has been reported, suggesting hydrocarbons can control to some extent Cu sorption by plants or how the plant controls the solubility of Cu (Almeida et al., 2008). In the present case, Cef could have formed complexes with the metals, or potentiated the complexation of the metal by organic ligands exuded by the plant, which could have potentiated the accumulation of Cu, Fe, Mn and Zn. Enhanced plant uptake in the presence of metal complexes have been found (Degryse et al., 2006) and references therein). On the other hand, Cef may passively penetrate root cells membranes without any carrier which can, for example, facilitated the penetration of metals (or a metal complexes) into the cell, a phenomenon already reported for hydrocarbons (Alkio et al., 2005). The effect of Cef on the absorption of metals by plants is a very pertinent subject, with important implications for phytoremediation processes, meriting further investigation.
Fig. 3. Copper (A), iron (B), manganese (C) and zinc (D) in plant tissues (roots, rhizomes, stems and leaves) at the beginning of the experiment (field) and after exposure to 20 oneweek cycles of treatment of livestock wastewater (Wi: week i). Wastewater not spiked (Cont) or spiked with enrofloxacin (Enr) or ceftiofur (Cef) or with a mixture of Enr and Cef, (Mix).
sediment (e.g. Almeida et al., 2011). In the present study, results showed, in general, metal (particularly, Cu and Mn) accumulation in the different plants tissues, indicating that part of the metals present in the wastewater were accumulated by plants. Although in some cases, metal translocation to the aboveground tissues, namely stems, occurred, most of the metals were accumulated in the belowground parts of the plants. These studies corroborate that roots and rhizomes are the primary plant tissues involved in metal accumulation followed by aboveground tissues, like leaves and stems. In fact, even in the plants used in CWs the highest metals concentrations are mostly found in roots, followed by rhizomes, leaves and stems (Morari et al., 2015; Vymazal and Březinová 2015). For P. australis this feature has been also reported (e.g. Almeida et al., 2011). Therefore, in the present study plants seemed to contribute for the removal of Cu, Fe, Mn and Zn present in the livestock wastewater used. In general, antibiotics did not interfere with the depuration capacity of the CWs systems, in terms of removals of metals from wastewater. In fact, Mn and Zn removal rates were identical in the absence and in the presence of the antibiotics and Fe removal rates were even slightly higher in the presence of the antibiotics, alone or in a mixture. Moreover, metal removal rates were identical over time. However, antibiotics can affect the distribution of metals within the CWs microcosms systems. In fact, physical-chemical processes of pollutants retention can be affected by the presence of antibiotics in
5. Conclusions Present results indicate that, under the used laboratory conditions, the presence of veterinary drugs, namely enrofloxacin and ceftiofur, did not influence significantly the removal of metals from livestock wastewater by CWs. Moreover, metal concentrations in wastewater treated by the CWs microcosms were below values established in Portuguese legislation for wastewater discharge and identical or lower than those defined in international legislation regarding water quality for irrigation. Therefore, CWs seem to be a valuable alternative to remove 147
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pollutants, including antibiotics, from livestock wastewaters, reducing the risk the release of these effluents might pose to the environment. Funding This research was partially supported by the Strategic Funding UID/ Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and by the structured Programme of R & D & I INNOVMAR Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, namely within the Research Line ECOSERVICES (Assessing the environmental quality, vulnerability and risks for the sustainable management of the NW coast natural resources and ecosystem services in a changing world) within the R & D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), supported by the Northern Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF). Acknowledgements to Antonio Francisco Cebrian Talaya for his help in antibiotic determination in plant root substrate, to Iolanda Ribeiro for her help in assembling and keeping the experiments, and to Ricardo Emanuel Barbosa Abreu, Ana Cláudia Mendes da Costa and João Simão for their help in metal determination in plant tissues and plant root substrate. References Alkio, M., Tabuchi, T.M., Wang, X., Colón-Carmona, A., 2005. Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms. J. Exp. Bot. 56, 2983–2994. Allende, K.L., Fletcher, T.D., Sun, G., 2011. Enhancing the removal of arsenic, boron and heavy metals in subsurface flow constructed wetlands using different supporting media. Water Sci. Technol. 63, 2612–2618. Almeida, C.M.R., Mucha, A.P., Vasconcelos, M.T.S., 2004. Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant. Environ. Sci. Technol. 38 (11), 3112–3118. Almeida, C.M.R., Mucha, A.P., Bordalo, A.A., Vasconcelos, M.T.S.D., 2008. Influence of a salt marsh plant (Halimione portulacoides) on the concentrations and potential mobility of metals in sediments. Sci. Total Environ. 403, 188–195. Almeida, C.M.R., Mucha, A.P., Teresa Vasconcelos, M., 2011. Role of different salt marsh plants on metal retention in an urban estuary (Lima estuary, NW Portugal). Estuar. Coast. Shelf Sci. 91, 243–249. Arivoli, A., Mohanraj, R., Seenivasan, R., 2015. Application of vertical flow constructed wetland in treatment of heavy metals from pulp and paper industry wastewater. Environ. Sci. Pollut. Res. 22, 13336–13343. Babić, S., Mutavdžić Pavlović, D., Ašperger, D., Periša, M., Zrnčić, M., Horvat, A.M., Kaštelan-Macan, M., 2010. Determination of multi-class pharmaceuticals in wastewater by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Anal. Bioanal. Chem. 398, 1185–1194. Bhatia, M., Goyal, D., 2014. Analyzing remediation potential of wastewater through wetland plants: a review. Environ. Prog. Sustain. Energy 33, 9–27. Carvalho, P., Basto, M.C., Almeida, C.M., Brix, H., 2014. A review of plant– pharmaceutical interactions: from uptake and effects in crop plants to phytoremediation in constructed wetlands. Environ. Sci. Pollut. Res., 1–35. Carvalho, P.N., Araújo, J.L., Mucha, A.P., Basto, M.C.P., Almeida, C.M.R., 2013. Potential of constructed wetlands microcosms for the removal of veterinary pharmaceuticals
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Constructed wetlands for the removal of metals from livestock wastewater – Can the presence of veterinary antibiotics affect removals?
MARK
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C. Marisa R. Almeidaa, , Filipa Santosa, A. Catarina F. Ferreirab, Carlos Rocha Gomesb, M. Clara P. Bastob, Ana P. Muchaa a Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR / CIMAR), Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal b CIIMAR/CIMAR e Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Alegre, s/n, 4169-007 Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Keywords: Wastewaters Green technology Remediation Enrofloxacin
The presence of emergent antibiotics, in livestock wastewater may affect constructed wetlands (CWs) performance in the removal of other pollutants. The main objective of this study was to evaluate the influence of two antibiotics commonly used in livestock industry, enrofloxacin and ceftiofur, on metal removal by CWs. Microcosms (0.4 m×0.3 m×0.3 m), simulating CWs, were constructed with Phragmites australis to treat livestock wastewater spiked or not with 100 µg/L of enrofloxacin or ceftiofur (individually or in mixture). Wastewater was treated during 20 one-week cycles. After one-week cycle wastewater was removed and replaced by new wastewater (with or without spiking). At weeks 1, 2, 4, 8, 14, 18 and 20, treated wastewater was analysed to determine the removal rates of metals (Zn, Cu, Fe and Mn) and of each antibiotic. At weeks 1, 8 and 20 portions of the plant root substrate were collected and metals determined. At the end of the experiment metal levels were also determined in plant tissues. Removal rate of Fe from wastewater was 99%. Removal rates of Cu and Zn were higher than 85% and 89%, respectively, whereas for Mn removal rates up to 75% were obtained. In general, no significant differences were observed through time in the removals of the different metals, indicating that the systems maintained their functionality during the experimental period. Antibiotics did not interfere with the system depuration capacity, in terms of metals removals from wastewater, and ceftiofur even promoted metal uptake by P. australis. Therefore, CWs seem to be a valuable alternative to remove pollutants, including antibiotics and metals, from livestock wastewaters, reducing the risk the release of these wastewaters might pose into the environment, although more research should be conducted with other antibiotics in CWs.
1. Introduction Constructed wetlands (CWs) are engineered systems designed and built to mimic the biological, chemical and physical processes that occur in natural wetlands (Zhang et al., 2014). These processes include sorption, sedimentation, photolysis, hydrolysis, volatilization, plant uptake and accumulation, plant exudation, microbial degradation, filtration, precipitation and adsorption, removing pollutants from contaminated waters in a more controlled environment (GarciaRodríguez et al., 2014; Wu et al., 2014). CWs can be used to reduce the load of pollutants, such as nutrients, metals or organic matter, present in different types of wastewaters, including livestock wastewaters (e.g. Meers et al., 2005, Meers et al., 2008). In fact, CWs have successfully treated wastewaters contaminated with metals from acid mine drainage, metallurgy, tannery, swine ⁎
production, landfill leachates and domestic effluents (Arivoli et al., 2015) and references therein). Although the efficiency for metal removal varied with the plant species (Vymazal and Březinová 2015) and with the substrate composition (Allende et al., 2011), as well as with the metal itself (Marchand et al., 2010), removal rates as high as 90% have been reported (e.g. Vymazal, 2005; Morari et al., 2015). CWs systems have also recently been used to remove the so-called pollutants of emerging concern, including pharmaceutical compounds. In fact, several review articles on removal of pharmaceuticals from wastewaters by CWs have recently been published (e.g. Verlicchi et al., 2013; Li et al., 2014). Although most of the work has been for urban wastewaters, some studies have already shown CWs potentialities for the removal of pharmaceuticals, including antibiotics, from livestock wastewaters (e.g. Hussain et al., 2012; Carvalho et al., 2013; Hsieh et al., 2015). The more complex matrix of livestock wastewaters when
Corresponding author. E-mail address: [email protected] (C.M.R. Almeida).
http://dx.doi.org/10.1016/j.ecoenv.2016.11.021 Received 18 March 2016; Received in revised form 24 November 2016; Accepted 25 November 2016 0147-6513/ © 2016 Elsevier Inc. All rights reserved.
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ones (Carvalho et al., 2013). All microcosms had a tap at the bottom and worked in batch mode. The wastewater was poured on top, to percolate through the different layers of the solid matrix (matrix water saturation of ca 100%, with water just below the surface, ca. 1 L of wastewater), and drained out through the tap when necessary, simulating a sub-surface vertical flow CW. In this type of CWs wastewater flows under the surface of the planted bed, with the water percolating through the substrate.
compared with that of urban wastewaters makes these wastewaters more difficult to treat. These wastewaters can have high loads of organic matter, solids and nutrients, as well as pharmaceutical compounds such as antibiotics. However, pollutants of emerging concern, including antibiotics, can be harmful for both microorganisms and plants, which are key players in CWs removal processes. For example, there is a growing body of evidence documenting a reduction of microbial diversity in soils contaminated with antibiotics (Jechalke et al., 2014). In addition, exposure of plants to pharmaceuticals, including antibiotics, may influence, for example, plant development, due to phytotoxicity (Carvalho et al., 2014). Thus, the presence of pollutants of emerging concern, namely antibiotics, in livestock wastewaters may affect CWs performance for the elimination of other pollutants, such as metals. This subject needs to be investigated. Therefore, the aim of this work was to evaluate the influence of two antibiotics commonly used in livestock industry, enrofloxacin and ceftiofur, alone or in a mixture, on the removal of metals from livestock wastewaters by CWs.
2.1.3. Microcosms operation The livestock wastewater was treated during 20 one-week cycles. At the beginning of each week, new wastewater (with or without spiking with veterinary antibiotics) was added. Wastewater was daily recirculated to prevent the formation of anaerobic areas. After each one-week cycle, treated wastewater was removed and replaced by new wastewater (with or without spiking with the selected antibiotics), allowing to simulate a hydraulic retention time commonly used in CWs. This continuous addition of wastewater into the CWs systems also allowed simulating the cumulative effect of adding continuously a new load of pollutants to the system, which is the situation in full scale real CWs systems. This continuous load of pollutants can lead in the long term to accumulation of pollutants in the systems, which might affect CWs performance along time. At the end of weeks 1, 2, 4, 14, 18 and 20 the wastewater treated by the CWs was collected for analysis of metals (naturally present in the livestock wastewater) and to estimate their removal rates in the presence and absence of antibiotics. At the same time aliquots (ca. 5 g) of plants roots bed substrate were also collected from each CW microcosm. Microcosms were kept under greenhouse conditions, subjected to environmental temperature variations (21–25 °C) and to natural light exposure, along 20 weeks (May to October 2014), after which they were dismantled. After dismantling, plants were separated from the plants roots substrate, washed and put to dry until constant weight. Then plants were separated into roots, rhizomes, leaves and stems. All substrate samples were lyophilized.
2. Methodologies 2.1. Microcosms experiments Experiments were carried out in controlled conditions in microcosms simulating CWs. Small-scale process experiments allow to fully control the experimental conditions when intending to study the influence of specific variables, such as, in the present case, the presence of antibiotics in wastewater. Microcosms were planted with Phragmites australis, one of the plants more frequently used in CWs (Stottmeister et al., 2003). Two antibiotics commonly used in Portuguese livestock industry for therapeutic purposes were chosen: enrofloxacin (Enr) and ceftiofur (Cef). These antibiotics belong to different families (fluoroquinolone (Enr) and cephalosporin (Cef) families) and present different physical-chemical properties. Considering that more than one antibiotic can be present in wastewaters and that either antagonistic or synergetic effects can occur, the influence of a mixture of the two antibiotics on CWs performance was also assessed. 2.1.1. Sampling Livestock wastewater (after being treated in two lagoons (one anaerobic and another aerobic)) was collected every week in a pig farm. The wastewater was used as collected or spiked with one or both antibiotics. This wastewater already contained significant amounts of different metals (see Section 3). P. australis plants were collected in Lima River (NW Portugal) with the sediment attached to their roots to preserve plants’ rhizosphere. In the laboratory, sediment was removed and mixed with river sand (in a 1:2 proportion) to prepare the plants roots bed substrate into which plants were transplanted (each microcosms had ca. 80 plants). This mixing was carried out to increase the substrate porosity. A few plants and roots bed substrate were set aside to determine initial metal levels. These samples were called field plants and field substrate.
2.2. Samples analysis All reagents were pro analysis or equivalent. All material was washed with deionised water (conductivity < 0.1 µS cm−1), immersed in nitric acid solution (20% v/v) for 24 h, washed again with deionised water and dried in a clean oven. Samples of initial and treated wastewater were filtered (nitrate cellulose filters, 0.45 µm porosity) and acidified (with 1% nitric acid) before direct analysis. Plants roots substrate and plant tissues (dry samples) were digested with concentrated nitric acid (HNO3) and 30% hydrogen peroxide (H2O2) solution (only for plant tissues). Digestions were carried out in a high-pressure microwave system (Ethos, Milestone) in closed Teflon vessels. Metals (Cd, Cu, Fe, Mn, Ni, Pb, Zn) concentrations were measured in an atomic absorption spectrophotometer with flame atomization (AASF-PU 9200X, Philips), as described before (Almeida et al., 2004). A calibration curve obtained with aqueous standard solutions of different metal concentrations (0–3 mg/L) was used. These standard solutions were prepared from 1000 mg/L stock standard solutions of each metal. Antibiotics concentrations in treated wastewaters and plants roots bed substrate were determined by high-performance liquid chromatography (HPLC), after an ultrasonic extraction (only for solid matrix) and a pre-treatment by solid phase extraction (SPE) as described before (Carvalho et al., 2013).
2.1.2. Microcosms’ assemblage Microcosms (0.4 m×0.3 m×0.3 m), simulating CWs, were constructed to treat livestock wastewater not spiked (Control) or spiked with 100 µg/L of Enr or with 100 µg/L of Cef. Another set of microcosms was prepared in which the wastewater was spiked with a mixture of Enr and Cef, each antibiotic with a concentration of 100 µg/ L (Mix). This concentration, although relatively high, has already been found in wastewaters effluents (Babić et al. 2010). Three replicates per variable were constructed. All CWs microcosms had three layers: the first one with gravel (4 cm), the second one with lava rock (2 cm) and the third layer with the plants roots bed substrate (11 cm height) described above. These CWs systems were based on previously used 144
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control (significant differences (p < 0.05) until W8). For Mn and Fe removal rates were very variable (Fig. 1). For Mn, removal rates in wastewater spiked with Enr were more or less constant over time, whereas for remaining treatments removal rates increased over time. For Fe, removal rates in control CWs microcosms (treating wastewater not spiked with antibiotics) decreased over time, whereas in the presence of Enr removal rates were constant until W8, decreasing afterwards. In the presence of Cef and of Cef+Enr (Mix) Fe removal rates varied significantly along time. Both antibiotics were also significantly removed from the wastewater, being below analytical detection limit. In fact, removal rates from wastewater were higher than 90% (considering antibiotics concentration in soluble phase) in all CWs microcosms used to treat wastewater spiked with veterinary antibiotics. Antibiotics were also not detected in plants roots bed substrate (concentrations were always below detection limit ( < 0.09 µg/g for Enr and 0.2 µg/g for Cef)). In plants roots bed substrate, Cd concentrations were always below detection limit ( < 3 µg/g). For Ni and Pb, levels were between 5– 10 µg/g and 10–20 µg/g, respectively without significant differences (p > 0.05) between field substrate (initial substrate used to construct the CWs microcosms and not exposed to wastewater) and plants roots bed substrate collected in the different CWs microcosms along time. For Cu, metal concentrations in substrate increased over time, without significant (p < 0.05) differences among the different CWs microcosms in each week (Fig. 2). Comparatively to field substrate, a significant (p < 0.05) decrease in Cu concentration at W1 was observed, but then Cu concentration increased over time. In W20 Cu levels were significantly (p < 0.05) higher than those initially present, with the exception of Cef treatment for which differences were not significant (p > 0.05). A similar behaviour was observed for Zn, but at W20 metal levels were identical to initial levels in field substrate. The only exception was Cef treatment for which significantly (p < 0.05) higher Cu levels were observed. For Mn, a similar tendency to decrease in W1 was observed, but differences were not significant (p > 0.05). After W1, Mn concentrations were identical throughout time and among treatments. Regarding Fe, in general, concentrations were identical along time and identical to levels in field substrate. The only exception was the substrate exposed to wastewater spiked with Cef which showed an increase in Fe concentrations over time. In plant tissues Cd concentrations were always below detection limit (1.5 µg/g). For Ni and Pb, levels were only above detection limits (which were 2 µg/g and 5 µg/g for Ni and Pb, respectively) in plant roots (ca. 5 µg/g for Ni and ca. 16 µg/g for Pb), without significant differences (p > 0.05) between field plants (plants collected in the field similar to the ones used in CWs microcosms but not exposed to wastewater) and control plants (plants in CWs microcosms exposed to wastewater not spiked with antibiotics) or among plants exposed to different treatments. For the other metals (Cu, Fe, Mn and Zn), in general, concentrations in the different plant tissues increased relatively to concentrations observed in not exposed plants (field plants) (Fig. 3). For Cu, comparatively to field plants, in general, a significant (p < 0.05) accumulation of the metal after the 20-week cycles was observed for all tissues of the plant (not only in belowground but also aboveground tissues). For this metal, in general, no significant (p > 0.05) differences were observed between plants exposed and not exposed to the veterinary antibiotics (control microcosm). The only exception was for the systems exposed to Cef. In this microcosm Cu levels in plants stems were significantly higher than those in plants stems not exposed to the antibiotics. For Mn and Zn, results were very similar to those of Cu, with both stems and leaves of plants exposed to wastewater spiked with Cef presenting higher metal concentrations than plants of the other treatments. However, for Zn, differences in metal levels in belowground tissues, between the field plants and those collected after 20-week cycles of treatment, were only significant (p < 0.05) for wastewater spiked with Cef (alone or in a mixture).
Fig. 1. Iron (A), manganese (B) and zinc (C) removal rates from livestock wastewater along 20 one-week cycles treatment of livestock wastewater (Wi: week i). Wastewater not spiked (Cont) or spiked with enrofloxacin (Enr) or with ceftiofur (Cef) or with a mixture of Enr and Cef (Mix). WP1 in Cont for Mn not determined.
2.3. Data analysis For wastewater and substrate samples, mean and respective errors of metal concentrations were calculated for each treatment (i.e., for each set of three replicates, one from each microcosm). For metals concentrations in plant tissues, due to the high plant natural variability, three replicates were analysed for each microcosm and then mean and respective errors of metal concentrations were calculated for each treatment. To evaluate statistically significant differences between treatments, students’ t-test (p < 0.05) in GraphPad Prism 6 software was used.
3. Results Metal levels (Cd, Cu, Fe, Mn, Ni, Pb and Zn) were measured in livestock wastewater before and after treatment in the different CWs microcosms. Levels of Cd, Ni and Pb were always below detection limit ( < 0.10 mg/L). For Cu, initial levels varied between 0.26 and 2.2 mg/L and final levels were always below detection limit ( < 0.050 mg/L), showing removal rates higher than 85%. Removal rates of Zn were higher than 89% (Fig. 1), whereas for Mn removal rates were lower, ca. 75%. For Fe, removal rates from wastewater were up to 99%. In general, for Zn no significant differences (p > 0.05) were observed among treatments or among weeks, except for wastewater spiked with Cef which had in general lower Zn removal rates than 145
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Fig. 2. Copper (A), iron (B), manganese (C) and zinc (D) in plants roots substrate at the beginning of the experiment (field) and after exposure to 20 one-week cycles of treatment of livestock wastewater (Wi: week i). Wastewater not spiked (Cont) or spiked with enrofloxacin (Enr) or with ceftiofur (Cef) or with a mixture of Enr and Cef (Mix).
lower than those reported in the Portuguese legislation for wastewater discharge (Decreto-Lei no 236/98). These metal concentrations were also identical or lower than those defined in international legislation regarding water quality for irrigation (Norton-Brandão et al., 2013). Removal of metals in CWs occurs mainly by binding to CWs substrate, precipitation as insoluble salts and uptake by bacteria, algae and plants. However, binding to substrate within wetlands is considered the major process for removal of metals (Bhatia and Goyal, 2014). In the present study, removal of metals from wastewater also occurred by sorption to plants roots bed substrate because metal (Cu, Mn and Zn) levels in substrate increased over time. In fact, despite a decrease in the levels of metals after the first week of experiment, probably due to substrate lixiviation, metals levels in substrate increased showing the adsorption capacity of this matrix. Other studies also have shown this feature (e.g. Yadav et al., 2012; Arivoli et al., 2015; Travaini-Lima et al., 2015). The only exception was Fe, probably due to the already high concentration of this metal in the substrate which prevented the detection of Fe inputs from the wastewater. Most CWs employ crushed stones, sand and gravel as substrates not only to support the plant growth but also to act as a filter; however, other substrates such as clay soil, zeolites, shells and industrial wastes (furnace slag, steel slag, sludge from waste treatment plants) had also been found as efficient filter materials (Arivoli et al., 2015). In the present study, lava rock (a very porous material) was also part of the CWs microcosms and as so, metals might have been also adsorbed to this fraction. Nevertheless, metals were not determined in lava rock layer because by the end of the experiments the rock was mostly smash and was not possible to fully separate it from the other components of the microcosms. Removal of metals from wastewaters in CWs may also occurred by plant uptake. In fact, some wetland plants, including those used in CWs, such as P. australis, are known by their ability to uptake metals without showing metal toxicity (Bhatia and Goyal, 2014). Studies involving metal uptake by P. australis in the environment have shown the potential of this plant to phytoremediate metal contaminated soil and
Regarding Fe, although an increase in the metal levels in plant roots from the CWs microcosms relatively to not exposed plants (field plants) was observed, the increase was only significant (p < 0.05) for plants exposed to wastewater spiked with the antibiotics. On the other hand, Fe concentrations in plants rhizomes and leaves were identical among all plants. For plant stems the scenario was different. In fact, Fe levels were statistically identical (p > 0.05) between field plants and those of control and Enr treatments, but Fe levels in stems of plants exposed to wastewater spiked with Cef or with Cef+Enr were significantly (p < 0.05) higher than in plants exposed to wastewater not spiked with antibiotics (control plants). It should be mentioned that pH values in initial water were ca. 7.8, whereas in treated wastewater varied between 7.3 and 8.0. 4. Discussion Livestock wastewaters have different pollutants, including metals (e.g. Meers et al., 2005), that need to be removed before their disposal in the environment. CWs can be a suitable alternative as they have been used to treat livestock wastewaters not only for the removal of conventional pollutants (e.g. Lee et al., 2004; Meers et al., 2005, 2008), but also for the removal of emergent compounds, such as pharmaceuticals (e.g. Hussain et al., 2012; Carvalho et al., 2013; Hsieh et al., 2015). Nevertheless, there is a need to understand if the presence of antibiotics can interfere with removal of metals is this type of effluents. Removal rates of metals present in the used livestock wastewater observed in the present study were similar to those reported before, including for CWs applied for treatment of livestock wastewaters (Meers et al., 2005, 2008). In general, no significant differences were observed through time in the concentrations of the different metals analysed (Fe was the only exception, with a slight decrease in its removal rate over time), indicating that the systems maintained its functionality during the experimental period. It should be mentioned that metals concentrations in CWs treated wastewater were always 146
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the medium. For instance, the ability of Enr to form complexes with metal cations is well known (e.g. (Graouer-Bacart et al., 2013), and Cef has also the capacity to bound Cu (unpublished results). Moreover, in CWs the growth of macrophytes and their depurative performance may be influenced by interactions among mixed pollutants (GuittonnyPhilippe et al., 2015). These features can interfere with the removal of metals and their distribution in CWs. In the present study, in general, metal sorption to plants roots bed substrate was identical among all CWs microcosms, indicating that the amount of antibiotics, either alone or in a mixture, did not affect the retention of metals over time. One should be aware that antibiotics were also retained in the CWs. In CWs, antibiotics can be adsorbed to the solid matrix, biologically degraded or accumulated by plants. In fact, previous studies carried out by the authors (Carvalho et al., 2012, 2013) have shown that all these processes can contribute for Enr and Cef removal from contaminated wastewater in CWs. But, as none of the antibiotics were detected in the solid matrix, either in previous studies (Carvalho et al., 2013) or in the present one, biological degradation was probably the main antibiotics removal process. In that sense a high influence of the antibiotics on metal adsorption was not expected because these compounds were probably being degraded over time and not accumulating in the substrate. However, metal uptake by P. australis was affected by the presence of Cef. In fact, Cef potentiate Cu translocation to plants stems and Mn and Zn translocation to plants stems and leaves, a feature not observed for Enr. In the presence of Enr metal concentrations in plant tissues were identical to those of control (plants in CWs microcosms treating wastewater not spiked with antibiotics). This behaviour was observed also, in general, for the treatment with the mixture of the two antibiotics indicating Enr probably interfered with Cef. Moreover, Cef also potentiated Zn accumulation in plants roots relatively to control. In the case of Fe, Cef potentiated metal translocation to stems, an effect also observed in the systems where both antibiotics were present. Therefore, Cef affected the ability of plants for the absorption of metals, particularly the translocation of the metals, increasing it. Metal uptake by plants depends on the form in which the metal is present in the medium and the simultaneous presence of pollutants of different nature can modify the mechanisms of absorption of metals by the plants. For example, a greater accumulation of Cu by the wetland plant Haliminone portulacoides in the presence of hydrocarbons has been reported, suggesting hydrocarbons can control to some extent Cu sorption by plants or how the plant controls the solubility of Cu (Almeida et al., 2008). In the present case, Cef could have formed complexes with the metals, or potentiated the complexation of the metal by organic ligands exuded by the plant, which could have potentiated the accumulation of Cu, Fe, Mn and Zn. Enhanced plant uptake in the presence of metal complexes have been found (Degryse et al., 2006) and references therein). On the other hand, Cef may passively penetrate root cells membranes without any carrier which can, for example, facilitated the penetration of metals (or a metal complexes) into the cell, a phenomenon already reported for hydrocarbons (Alkio et al., 2005). The effect of Cef on the absorption of metals by plants is a very pertinent subject, with important implications for phytoremediation processes, meriting further investigation.
Fig. 3. Copper (A), iron (B), manganese (C) and zinc (D) in plant tissues (roots, rhizomes, stems and leaves) at the beginning of the experiment (field) and after exposure to 20 oneweek cycles of treatment of livestock wastewater (Wi: week i). Wastewater not spiked (Cont) or spiked with enrofloxacin (Enr) or ceftiofur (Cef) or with a mixture of Enr and Cef, (Mix).
sediment (e.g. Almeida et al., 2011). In the present study, results showed, in general, metal (particularly, Cu and Mn) accumulation in the different plants tissues, indicating that part of the metals present in the wastewater were accumulated by plants. Although in some cases, metal translocation to the aboveground tissues, namely stems, occurred, most of the metals were accumulated in the belowground parts of the plants. These studies corroborate that roots and rhizomes are the primary plant tissues involved in metal accumulation followed by aboveground tissues, like leaves and stems. In fact, even in the plants used in CWs the highest metals concentrations are mostly found in roots, followed by rhizomes, leaves and stems (Morari et al., 2015; Vymazal and Březinová 2015). For P. australis this feature has been also reported (e.g. Almeida et al., 2011). Therefore, in the present study plants seemed to contribute for the removal of Cu, Fe, Mn and Zn present in the livestock wastewater used. In general, antibiotics did not interfere with the depuration capacity of the CWs systems, in terms of removals of metals from wastewater. In fact, Mn and Zn removal rates were identical in the absence and in the presence of the antibiotics and Fe removal rates were even slightly higher in the presence of the antibiotics, alone or in a mixture. Moreover, metal removal rates were identical over time. However, antibiotics can affect the distribution of metals within the CWs microcosms systems. In fact, physical-chemical processes of pollutants retention can be affected by the presence of antibiotics in
5. Conclusions Present results indicate that, under the used laboratory conditions, the presence of veterinary drugs, namely enrofloxacin and ceftiofur, did not influence significantly the removal of metals from livestock wastewater by CWs. Moreover, metal concentrations in wastewater treated by the CWs microcosms were below values established in Portuguese legislation for wastewater discharge and identical or lower than those defined in international legislation regarding water quality for irrigation. Therefore, CWs seem to be a valuable alternative to remove 147
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pollutants, including antibiotics, from livestock wastewaters, reducing the risk the release of these effluents might pose to the environment. Funding This research was partially supported by the Strategic Funding UID/ Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and by the structured Programme of R & D & I INNOVMAR Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, namely within the Research Line ECOSERVICES (Assessing the environmental quality, vulnerability and risks for the sustainable management of the NW coast natural resources and ecosystem services in a changing world) within the R & D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), supported by the Northern Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF). Acknowledgements to Antonio Francisco Cebrian Talaya for his help in antibiotic determination in plant root substrate, to Iolanda Ribeiro for her help in assembling and keeping the experiments, and to Ricardo Emanuel Barbosa Abreu, Ana Cláudia Mendes da Costa and João Simão for their help in metal determination in plant tissues and plant root substrate. References Alkio, M., Tabuchi, T.M., Wang, X., Colón-Carmona, A., 2005. Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms. J. Exp. Bot. 56, 2983–2994. Allende, K.L., Fletcher, T.D., Sun, G., 2011. Enhancing the removal of arsenic, boron and heavy metals in subsurface flow constructed wetlands using different supporting media. Water Sci. Technol. 63, 2612–2618. Almeida, C.M.R., Mucha, A.P., Vasconcelos, M.T.S., 2004. Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant. Environ. Sci. Technol. 38 (11), 3112–3118. Almeida, C.M.R., Mucha, A.P., Bordalo, A.A., Vasconcelos, M.T.S.D., 2008. Influence of a salt marsh plant (Halimione portulacoides) on the concentrations and potential mobility of metals in sediments. Sci. Total Environ. 403, 188–195. Almeida, C.M.R., Mucha, A.P., Teresa Vasconcelos, M., 2011. Role of different salt marsh plants on metal retention in an urban estuary (Lima estuary, NW Portugal). Estuar. Coast. Shelf Sci. 91, 243–249. Arivoli, A., Mohanraj, R., Seenivasan, R., 2015. Application of vertical flow constructed wetland in treatment of heavy metals from pulp and paper industry wastewater. Environ. Sci. Pollut. Res. 22, 13336–13343. Babić, S., Mutavdžić Pavlović, D., Ašperger, D., Periša, M., Zrnčić, M., Horvat, A.M., Kaštelan-Macan, M., 2010. Determination of multi-class pharmaceuticals in wastewater by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Anal. Bioanal. Chem. 398, 1185–1194. Bhatia, M., Goyal, D., 2014. Analyzing remediation potential of wastewater through wetland plants: a review. Environ. Prog. Sustain. Energy 33, 9–27. Carvalho, P., Basto, M.C., Almeida, C.M., Brix, H., 2014. A review of plant– pharmaceutical interactions: from uptake and effects in crop plants to phytoremediation in constructed wetlands. Environ. Sci. Pollut. Res., 1–35. Carvalho, P.N., Araújo, J.L., Mucha, A.P., Basto, M.C.P., Almeida, C.M.R., 2013. Potential of constructed wetlands microcosms for the removal of veterinary pharmaceuticals
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