Constructed wetlands for the removal of metals from livestock wastewater - Can the presence of veterinary antibiotics affect removals?

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 (0.4m×0.3m×0.3m), simulating CWs, were constructed with Phragmites australis to treat livestock wastewater spiked or not with 100µg/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.

Interferences of surfactants during determination of chlorophenols by acetylation coupled to headspace-solid phase microextraction-gas chromatography with an electron capture detector.

The micellar extraction (ME) of chlorophenols (CPs) from solid samples is rapid and avoids the use of organic solvents. The cationic surfactant cetyltrimethylammonium bromide (CTAB) is a powerful agent for ME of CPs. However, CTAB will be an important interference when the extracts are subsequently analyzed by direct solid phase microextraction (SPME) without a previous derivatization step. Therefore, CTAB is usually replaced by the nonionic surfactant polyoxyethylene-10-lauryl ether (POLE), which causes less interference but is less efficient in extracting CPs. In order to assess to what extent the derivatization of CPs with headspace (HS) sampling would be able to reduce surfactant interferences, CPs were measured in the presence of CTAB or POLE using acetylation and HS-SPME coupled to GC with an electron capture detector. Experiments were carried out both in water and in a 0.1 g/mL solution of NaCl (salting out agent). Acetylation and HS-SPME strongly decreased the interference of CTAB for CP determination. Therefore, the application of CTAB followed by SPME determination for an efficient ME of CPs from solid samples should be reconsidered since using CTAB with HS-SPME after acetylation proved to be a potential tool for CP determination in those matrixes after method optimization.

Antioxidant response of Phragmites australis to Cu and Cd contamination.

Metals are known to induce oxidative stress in plant cells. Antioxidant thiolic compounds are known to play an important role in plants׳ defence mechanisms against metal toxicity but, regarding salt marsh plants, their role is still very poorly understood. In this work, the involvement of non-protein thiols (NPT), such as cysteine (Cys), reduced glutathione (GSH), oxidised glutathione (GSSG) and total acid-soluble SH compounds (total thiols), in the tolerance mechanisms of the marsh plant Phragmites australis against Cu and Cd toxicity was assessed. Specimens of this plant, freshly harvested in an estuarine salt marsh, were exposed, for 7 days, to rhizosediment soaked with the respective elutriate contaminated with Cu (0, 10 and 100 mg/L) or Cd (0, 1, 10 mg/L). In terms of NPT production, Cu and Cd contamination induced different responses in P. australis. The content of Cys increased in plant tissue after plant exposure to Cu, whereas Cd contamination led to a decrease in GSSG levels. In general, metal contamination did not cause a significant variation on GSH levels. Both metals influenced, to some extent, the production of other thiolic compounds. Despite the accumulation of considerable amounts of Cu and Cd in belowground tissues, no visible toxicity signs were observed. So, antioxidant thiolic compounds were probably involved in the mechanisms used by P. australis to alleviate metal toxicity. As P. australis is considered suitable for phytostabilising metal-contaminated sediments, understanding its tolerance mechanisms to toxic metals is important to optimise the conditions for applying this plant in phytoremediation procedures.

Stability of chlorophenols and their acetylated derivatives in water: sample storage procedures.

The determination of chlorophenols (CPs) in water samples is a subject of increasing interest. Reduction of sample storage space and the stability of CPs when present at very low levels are still problems that deserve research. The stability of CPs at ng/L levels at different temperatures and in the presence or absence of sodium carbonate and acetic anhydride was studied for up to 39 days. Stable and reproducible CP concentrations for about a month of storage in both river and wastewater were achieved in two storage conditions as follows: at -18 degrees C with addition of 10% sodium chloride; and at 4 degrees C with addition of both 10% sodium chloride and 10 mglmL sodium carbonate. These sample treatments are good alternatives to the immobilization of CPs on SPE cartridges in terms of both analyte stability and saving of storage space.

Determination of gaseous polycyclic aromatic hydrocarbons by a simple direct method using thermal desorption-gas chromatography-mass spectrometry.

In the last decade, the development of novel analytical methodologies enabled the identification of several environmental pollutants responsible for health problems associated with indoor exposure. Polycyclic aromatic hydrocarbons (PAHs) are among the potential hazardous chemicals present in ambient air. Due to their bioaccumulation potential and carcinogenic/mutagenic effects, 16 PAHs are currently listed as priority air pollutants. The main goal of this work was to implement a new and simple method for sampling and determination of PAHs in air by using a thermal desorption (TD) technique followed by gas chromatography coupled with mass spectrometry analysis. A detailed study was carried out to optimise the experimental method in each of its phases, including (active) sampling, TD and chromatographic analysis. The results demonstrate that this approach allowed the detection and quantification of the six more volatile PAHs, namely, naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene. Acceptable precision and good linearity over the explored range were obtained. No carry-over was observed during experimental tests and the method provided a reproducible answer. The applicability of the novel methodology was tested in real environment, namely, on the roof of a building in an urban area, in a domestic kitchen and in a collective car garage. The method enabled the identification of two PAHs in the field samples, specifically, naphthalene (two rings) and phenanthrene (three rings). With regard to PAHs sample composition, the most abundant PAH found, in the three different locations, was naphthalene, accounting for about 84-100 % of the total PAH mass detected.

Simultaneous determination of several veterinary pharmaceuticals in effluents from urban, livestock and slaughterhouse wastewater treatment plants using a simple chromatographic method.

Minocycline, oxytetracycline, tetracycline, enrofloxacin and ceftiofur, commonly used veterinary pharmaceuticals, were searched in four urban, two livestock and two slaughterhouse effluents from wastewater treatment plants (WWTPs) in the north of Portugal. A simple method that includes solid-phase extraction followed with analysis by high-performance liquid chromatography with diode array detector was established and applied to the simultaneous determination of the five pharmaceuticals in WWTP effluents. This method, which is expeditious, inexpensive and available in most laboratories, showed to be useful for screening for problematic levels of drugs in WWTP effluents. It is known that several livestock and slaughterhouse effluents (pre-treated or treated) are discharged to the urban network before discharge into the environment. The presence of these drugs in such effluents can constitute a significant environmental problem that should be addressed, by the monitoring of these drugs and by implementation of methodologies that contribute to their decrease/elimination from wastewaters. Minocycline (≤6 µg L(-1)), oxytetracycline (≤7 µg L(-1)), tetracycline (≤6 µg L(-1)) and enrofloxacin (<2 µg L(-1)) could be detected and/or quantified in three urban effluents. Detectable levels of enrofloxacin (<2 µg L(-1)) and quantifiable levels of tetracycline (≤15 µg L(-1)) were found in the slaughterhouse effluents.

Role of natural attenuation, phytoremediation and hybrid technologies in the remediation of a refinery soil with old/recent petroleum hydrocarbons contamination.

Within a search for a biological remediation technology to remove petroleum hydrocarbons (PHC) from a contaminated soil from a refinery, the potential of monitored natural attenuation (MNA) was compared with the use of transplants of Cortaderia selloana both in the absence and in the presence of soil amendments. After 31 months of experiments, MNA was effective in removing most of the recent PHC contamination (50% of the initial total contamination) at 5-20 cm depth. The presence of weathered contamination explains the existence of an established community of PHC degraders, as can be inferred by the most probable number technique. C. selloana, in its turn, showed capacity to mobilize the most recalcitrant fraction of PHC to its roots, nevertheless masking its remediation capacity. The use of a hybrid technology (C. selloana together with treatments with a surfactant and a bioaugmentation product) improved the removal of PHC at 15-20 cm depth, the presence of C. selloana facilitating the migration of additives into the deeper layers of soil, which can be considered a secondary but positive role of the plant. In the surface soil layer, which was exposed to both microorganisms and the atmosphere, a further 20% of weathered PHC contamination disappeared (70% total removal) as a result of photo- and chemical degradation. Periodic revolving of the soil, like tillage, to expose all the contaminated soil to the atmosphere will therefore be a reliable option for reducing the contamination of the refinery soil if conditions (space and equipment) permit this operation.

A headspace SPME-GC-ECD method suitable for determination of chlorophenols in water samples.

A headspace solid phase microextraction coupled to gas chromatography with electron capture detector (HS-SPME-GC-ECD) method was optimized for the determination of seven chlorophenols (CPs) with different levels of chlorination. This is the first time that HS-SPME-GC-ECD with acetylation of the analytes is used for the simultaneous determination of CPs in water samples. The influence of fibre type, derivatization conditions, salt addition, temperature and time of extraction and temperature of desorption was checked. Possible sources of contamination and analyte losses were considered. The best results were obtained with the polydimethylsiloxane/divinylbenzene fibre, derivatization by acetylation using 100 µL of acetic anhydride and 0.1 g of anhydrous sodium carbonate per 10 mL of sample, salt addition of 100 g L(-1) sodium chloride, extraction at 70 °C for 60 min and desorption in the GC injector at 260 °C for 6 min. The limits of detection (LOD) for monochlorophenols were 12 and 122 ng L(-1) for 2-chlorophenol and 4-chlorophenol, respectively. For polychlorinated CPs, the LODs were lower than 6 ng L(-1), values similar to the existing methods that use SPME with derivatization for CPs determination in water samples. The method is suitable for the determination of CPs in most environmental aqueous samples. Repeatability and reproducibility were less than 16.8% and 11.7%, respectively. The optimized method was successfully applied for the analysis of waters with complex matrices such as river and estuarine water samples.

Effects of minocycline and its degradation products on the growth of Microcystis aeruginosa.

This work aimed to investigate the influence of Microcystis aeruginosa on the rate of minocycline (MNC) removal (abiotic degradation, physical binding or chemical transformation by cells) and the toxicity of MNC and its degradation products to the cyanobacterium. Most of the work was carried out in culture media in the presence or in the absence of M. aeruginosa. The rate of the MNC abiotic degradation in culture media strongly decreased with the increase of the MNC initial concentration. The exudates did not influence the rate of MNC degradation in the media. For concentrations ≥0.48 µM, the rate of the MNC removal from the media was faster in the presence of M. aeruginosa. Biotic MNC removal occurs by both physical binding by the cells (10%) and chemical transformations. EC(50) and EC(20) for MNC on the growth of M. aeruginosa were 0.92 and 0.13 µM, respectively. Interestingly, MNC degradation products might promote M. aeruginosa growth.

Butyltin levels in several Portuguese coastal areas.

This work aimed to report present levels (2007-2008 sampling) of tri- (TBT), di- (DBT), and monobutyltin (MBT) in surface sediments from 11 Portuguese coastal sites and discuss the evolution of BTs contamination in the last two decades. All the samples revealed quantifiable values of TBT, DBT, and MBT with total butyltin concentrations between 1 and 565 ng/g (of Sn in dry sediment). Maximum level of TBT, 66 ng/g, was observed in Sado estuary, at Lisnave site, in the proximity of a big shipyard. MBT decreased site by site by the same order as DBT and TBT did, but its concentrations were much higher in many cases, denoting that TBT contamination was much higher in the past. A comparison with the available previous data confirmed a marked decrease of TBT contamination all over the last years, indicating that the main sources of TBT in Portuguese coastwise stopped effectively.

Application of SPME to the determination of alkylphenols and bisphenol A in cyanobacteria culture media.

In order to survey the influence of estrogenic compounds on cyanobacteria, solid-phase microextraction (SPME) with a carbowax-divinylbenzene fibre was used for the determination of tert-octylphenol (tert-OP), n-nonylphenol (n-NP), technical nonylphenol (tech-NP) and bisphenol A (BPA) in cyanobacteria culture media by gas chromatography with flame ionization detection. Determinations were carried out without derivatization in deionized water and filtered culture media. A comparison between f2 and Fraquil culture media was performed, which showed that only f2 allowed quantitative recoveries. Headspace SPME with salting out, requiring only 10 mL of sample, was suitable for tert-OP, n-NP, and tech-NP determination with limits of detection (LOD) of <0.05 microg L(-1). For BPA, direct immersion SPME could provide a LOD of 1 microg L(-1). Automated sampling allowed reproducible extraction. No exudate substances overlapped with the studied compounds during the chromatographic separation and no matrix effects were observed. Ecotoxicity tests can be performed by single spiking of tert-OP and tech-NP and multiple spiking of n-NP due to its lower stability.

Response of a salt marsh microbial community to antibiotic contamination.

Salt marsh plants and associated microorganisms can have an important role in contaminant removal from estuaries, through bioremediation processes. Nevertheless, the interaction between emerging contaminants, namely antibiotics, and plant-microorganism associations in estuarine environment are still scarcely known. In this vein, the aim of the present study was to evaluate, in controlled conditions, the response of a salt marsh plant-microorganism association to a contamination with a veterinary antibiotic. For that a salt marsh plant (Phragmites australis) and its respective rhizosediment were collected in a temperate estuary (Lima estuary, NW Portugal) and exposed for 7 days to enrofloxacin (ENR) under different nutritional conditions in sediment elutriates. Response was evaluated in terms of ENR removal and changes in microbial community structure (evaluated by ARISA) and abundance (estimated by DAPI). In general, no significant changes were observed in microbial abundance. Changes in bacterial richness and diversity were observed but only in unplanted systems. However, multivariate analysis of ARISA profiles showed significant effect of both the presence of plant and type of treatment on the microbial community structure, with significant differences among all treatment groups. In addition, plants and associated microorganisms presented a potential for antibiotic removal that, although highly dependent on their nutritional status, can be a valuable asset to recover impacted areas such as estuarine ones.

Microbial community dynamics associated with veterinary antibiotics removal in constructed wetlands microcosms.

This study aimed to evaluate the response of the microbial community from CWs microcosms tested for the removal of two veterinary antibiotics, enrofloxacin (ENR) and tetracycline (TET), from livestock industry wastewater. Three treatments were tested (control, ENR or TET (100 µg L(-1))) over 12 weeks in microcosms unplanted and planted with Phragmites australis. CWs removal efficiency was relatively stable along time, with removals higher than 98% for ENR and 94% for TET. In addition, CWs were able to reduce wastewater toxicity, independently of antibiotics presence. Despite no significant differences were observed in terms of microbial abundance, bacterial richness or diversity, analysis of similarities (two-way crossed ANOSIM) showed a significant effect of both time and treatments in bacterial community structure. This study points to CWs applicability for veterinary antibiotics removal from livestock wastewaters, showing that CWs microbial communities were able to adapt without significant changes in their diversity or depuration capacity.

A review of plant-pharmaceutical interactions: from uptake and effects in crop plants to phytoremediation in constructed wetlands.

Pharmaceuticals are commonly found both in the aquatic and the agricultural environments as a consequence of the human activities and associated discharge of wastewater effluents to the environment. The utilization of treated effluent for crop irrigation, along with land application of manure and biosolids, accelerates the introduction of these compounds into arable lands and crops. Despite the low concentrations of pharmaceuticals usually found, the continuous introduction into the environment from different pathways makes them 'pseudo-persistent'. Several reviews have been published regarding the potential impact of veterinary and human pharmaceuticals on arable land. However, plant uptake as well as phytotoxicity data are scarcely studied. Simultaneously, phytoremediation as a tool for pharmaceutical removal from soils, sediments and water is starting to be researched, with promising results. This review gives an in-depth overview of the phytotoxicity of pharmaceuticals, their uptake and their removal by plants. The aim of the current work was to map the present knowledge concerning pharmaceutical interactions with plants in terms of uptake and the use of plant-based systems for phytoremediation purposes.

Bioremediation potential of microorganisms from a sandy beach affected by a major oil spill.

The aim of this work was to evaluate the bioremediation potential of microorganisms from intertidal sediments of a sandy beach affected by a major oil spill 7 years before and subject to chronic petroleum contamination since then. For that, the response of microorganisms to a new oil contamination was assessed in terms of community structure, abundance, and capacity to degrade hydrocarbons. Experiments were carried out under laboratory-controlled conditions by mixing sediment with crude oil with three different nitrogen supplementations in 50 ml serum bottles under constant shake for 15 days. Autochthonous microorganisms were able to respond to the new oil contamination by increasing their abundance (quantified by DAPI) and changing the community structure (evaluated by DGGE). This response was particularly clear for some specific bacterial groups such as Pseudomonas, Actinomycetales, and Betaproteobacteria. These communities presented an important potential for hydrocarbon degradation (up to 85 % for TPHs and 70 % for total PAHs), being the biodegradation stimulated by addition of an appropriate amount of nitrogen.

Cyanobacterium Microcystis aeruginosa response to pentachlorophenol and comparison with that of the microalga Chlorella vulgaris.

Pentachlorophenol (PCP) effects on a strain of the cyanobacterium Microcystis aeruginosa were investigated at laboratory scale. This is the first systematic ecotoxicity study of the effects of PCP on an aquatic cyanobacterium. The microalga Chlorella vulgaris was studied in the same conditions as the cyanobacterium, in order to compare the PCP toxicity and its removal by the species. The cells were exposed to environmental levels of PCP during 10 days, in Fraquil culture medium, at nominal concentrations from 0.01 to 1000 µg L(-1), to the cyanobacterium, and 0.01 to 5000 µg L(-1), to the microalga. Growth was assessed by area under growth curve (AUC, optical density vs time) and chlorophyll a content (chla). The toxicity profiles of the two species were very different. The calculated effective concentrations EC20 and EC50 were much lower to M. aeruginosa, and its growth inhibition expressed by chla was concentration-dependent while by AUC was not concentration-dependent. The cells might continue to divide even with lower levels of chla. The number of C. vulgaris cells decreased with the PCP concentration without major impact on the chla. The effect of PCP on M. aeruginosa is hormetic: every concentration studied was toxic except 1 µg L(-1), which promoted its growth. The legal limit of PCP set by the European Union for surface waters (1 µg L(-1)) should be reconsidered since a toxic cyanobacteria bloom might occur. The study of the removal of PCP from the culture medium by the two species is an additional novelty of this work. M. aeruginosa could remove part of the PCP from the medium, at concentrations where toxic effects were observed, while C. vulgaris stabilized it.

Pentachlorophenol toxicity to a mixture of Microcystis aeruginosa and Chlorella vulgaris cultures.

Pentachlorophenol (PCP) is a priority pollutant due to its persistence and high toxicity. For the first time, PCP effects were investigated at laboratory scale on co-cultures of two ubiquitous freshwater phytoplankton species: the cyanobacterium Microcystis aeruginosa and the microalgae Chlorella vulgaris. The cells were exposed to environmental levels of PCP for 10 days in Fraquil culture medium, at nominal concentrations from 0.1 to 10,000 µg L(-1). Growth was assessed by area under growth curve (cell count vs. time). The phytoplankton community structure can be changed as a consequence of a PCP contamination. Low µg L(-1) levels of PCP are advantageous to M. aeruginosa. This is the first report of the promoting effect of PCP on the growth of aquatic cyanobacteria, using mixtures with microalgae. As a result of the direct toxic effects of high PCP concentrations on M. aeruginosa, C. vulgaris cell count increased given that in biological controls M. aeruginosa inhibited the C. vulgaris growth. At 16.7 mg L(-1), PCP already had direct toxic effects also on the microalga. The pH of culture medium tended to decrease with increasing PCP concentrations, which was mostly related to the growth inhibition of cyanobacterium caused by PCP. The PCP concentration was stable in the co-cultures, which differed from what has been observed in monocultures of the same two species. Short-term laboratory assays with two phytoplankton species gives important information on the species interactions, namely possible direct and indirect effects of a toxicant, and must be considered in ecotoxicity studies regarding environmental extrapolations.

Activated sludge systems removal efficiency of veterinary pharmaceuticals from slaughterhouse wastewater.

The knowledge on the efficiency of wastewater treatment plants (WWTPs) from animal food production industry for the removal of both hormones and antibiotics of veterinary application is still very limited. These compounds have already been reported in different environmental compartments at levels that could have potential impacts on the ecosystems. This work aimed to evaluate the role of activated sludge in the removal of commonly used veterinary drugs, enrofloxacin (ENR), tetracycline (TET), and ceftiofur, from wastewater during a conventional treatment process. For that, a series of laboratory-controlled experiments using activated sludge were carried out in batch reactors. Sludge reactors with 100 µg/L initial drug charge presented removal rates of 68 % for ENR and 77 % for TET from the aqueous phase. Results indicated that sorption to sludge and to the wastewater organic matter was responsible for a significant percentage of drugs removal. Nevertheless, these removal rates still result in considerable concentrations in the aqueous phase that will pass through the WWTP to the receiving environment. Measuring only the dissolved fraction of pharmaceuticals in the WWTP effluents may underestimate the loading and risks to the aquatic environment.

Potential of constructed wetlands microcosms for the removal of veterinary pharmaceuticals from livestock wastewater.

The aim of the present work was to evaluate, at microcosm level, the capacity of constructed wetlands (CWs) to remove veterinary pharmaceutical compounds, from wastewater. Results indicated that CWs have potential to mitigate the release of veterinary drugs, namely enrofloxacin (ENR, a fluoroquinolone) and tetracycline (TET, tetracyclines family). Removal efficiencies of 94% and 98% where achieved for TET and ENR, respectively, when treating pigfarm wastewater effluent doped at 100 µg L(-1) drug level, along twelve weeks. Occurrence of adsorption of the drugs to CWs substrate may be the predominant mechanism for ENR, although for TET there are signs that degradation is also occurring.

Extraction and preconcentration techniques for chromatographic determination of chlorophenols in environmental and food samples.

Methods for chlorophenols (CPs) determination (with low limits of detection) that can be applied to real environmental samples (waters, sediments, soils, biological tissues) and food are reviewed. Special emphasis is given to sampling, storage conditions and the application of preconcentration techniques for the determination of CPs using chromatographic methods. Solid phase extraction, solid phase microextraction, stir bar sorptive extraction, liquid phase microextraction, dispersive liquid-liquid microextraction, liquid-liquid-liquid microextraction and purge and trap methods are considered. Methods for microwave and ultrasonic extraction of CPs from solid matrices are also focused.