Management of a Toxic Cyanobacterium Bloom - ACS Publications

Nov 20, 2012 - Emanuele Ferretti,. ‡ ... Institute of Health, Department of Environment and Primary Prevention, Viale Regina Elena 299, 00161 Roma, ...
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Management of a Toxic Cyanobacterium Bloom (Planktothrix rubescens) Affecting an Italian Drinking Water Basin: A Case Study Sara Bogialli,† Federica Nigro di Gregorio,‡,§ Luca Lucentini,*,‡ Emanuele Ferretti,‡ Massimo Ottaviani,‡ Nicola Ungaro,∥ Pier Paolo Abis,⊥ and Matteo Cannarozzi de Grazia# †

University of Padua, Department of Chemistry, Via Marzolo 1, 35131, Padova, Italy Italian National Institute of Health, Department of Environment and Primary Prevention, Viale Regina Elena 299, 00161 Roma, Italy § University of Rome “Sapienza”, Department of Chemistry and Drug Technologies, Piazzale Aldo Moro 5, 00185 Roma, Italy ∥ Regional Agency for the Prevention and Environmental Protection, Corso Trieste 27,70126 Bari, Italy ⊥ Aqueduct of Apulia (AQP), Viale Emanuele Orlando 1, 70123 Bari, Italy # ASL Foggia, Department of Prevention, Piazza Pavoncelli 11, 71100 Foggia, Italy ‡

S Supporting Information *

ABSTRACT: An extraordinary bloom of Planktothrix rubescens, which can produce microcystins (MCs), was observed in early 2009 in the Occhito basin, used even as a source of drinking water in Southern Italy. Several activities, coordinated by a task force, were implemented to assess and manage the risk associated to drinking water contaminated by cyanobacteria. Main actions were: evaluation of analytical protocols for screening and confirmatory purpose, monitoring the drinking water supply chain, training of operators, a dedicated web site for risk communication. ELISA assay was considered suitable for health authorities as screening method for MCs and to optimize frequency of sampling according to alert levels, and as internal control for the water supplier. A liquid chromatography-tandem mass spectrometric method able to quantify 9 MCs was optimized with the aim of supporting health authorities in a comprehensive risk evaluation based on the relative toxicity of different congeners. Short, medium, and long-term corrective actions were implemented to mitigate the health risk. Preoxidation with chlorine dioxide followed by flocculation and settling have been shown to be effective in removing MCs in the water treatment plant. Over two years, despite the high levels of cyanobacteria (up to 160 × 106 cells/L) and MCs (28.4 μg/L) initially reached in surface waters, the drinking water distribution was never limited.

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INTRODUCTION Cyanobacteria are a group of prokaryotic organisms that occur in the environment under very different climatic conditions worldwide. About 40 of the 150 known genera of cyanobacteria can produce toxins1 that cause adverse effects on animal2,3 and human health. 4 Planktothrix rubescens, Microcystis spp., Aphanizomenon spp., and Anabaena spp. are quite common cyanobacteria in Europe with microcystins (MCs) and cylindrospermopsin (CYN) being most frequent algal toxins in freshwaters.5 MCs are hepatotoxic compounds6 suspected to be tumor promoters,7,8 their toxicity being due to a severe inhibition of protein phosphatase 1 (PP-1) and 2A (PP-2A) attributed to the presence of the uncommon amino acid Adda. CYN has been described as hepatotoxic, cytotoxic, genotoxic, and a potential carcinogen9 and was indicated as the causative agent in some severe adverse effects on humans.4,10 Anatoxin-a (ANA-a) is a neurotoxin produced by Anabaena f los-aquae, Aphanizomenon f los-aquae, Oscillatoria spp., and Phormidium favosum11 and has been associated with several animal fatalities including cattle and dogs.3,12,13 © 2012 American Chemical Society

Humans could be chronically exposed to cyanotoxins via contaminated drinking water. In order to protect consumers, the World Health Organization (WHO) has recommended a provisional guideline value of 1 μg/L for MC-LR equivalents in drinking water,14 whereas no values are currently set for CYN and ANA-a.15 Currently, the most diffuse monitoring analysis used to check cyanobacteria-affecting water bodies, is based on initial cells counting and on high-throughput screening methods.23 ELISA assays are widely used for analyzing MCs24,25 providing results generally related to the presence of Adda and expressed as MC-LR equivalents. Although reliable toxicological data for MC variants other than MC-LR were not available at that time for the WHO evaluation, nevertheless an effective consumer protection requires detection of the whole spectrum of cyanobacterial toxins and MC Received: Revised: Accepted: Published: 574

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congeners, with particular regard to toxicity of single congeners.26 Moreover, toxin production by certain species, when it occurs, is qualitatively and quantitatively unpredictable.27 Thus, confirmatory analysis based on chemicophysical methods are necessary when cyanobacteria population densities and screening assays results reach concentrations near the critical WHO value and/or an alert level.2 The best performing confirmatory methods for analyzing cyanotoxins in water samples generally involve a solid phase extraction followed by liquid chromatography (LC) coupled to mass spectrometric (MS) detectors.23,28−34 The WHO provisional guideline refers to distributed drinking water, so the management and mitigation of the risk related to the presence of cyanobacteria is mainly handled by a multiple barrier approach according the alert levels2 and water safety plan approach.28 Drinking water treatments to remove cyanobacteria and cyanotoxins have been extensively reviewed29 and more recently data about cyanotoxin byproducts have been published.30 However, data are scarce on the fate of cyanobacteria cells and their toxins from real bloom events in water treatment plants (WTPs), and the majority of information derives from experiments conducted at laboratory or pilot scale, whereas few articles have been supported by full scale tests under typical plant operating conditions.31−34 The science and regulations for dealing with unexpected blooms are inadequate and management must be adaptive, so case studies of bloom occurrence and fate within WTPs are valuable. In Italy, more than 60 water bodies have been reported to be affected by cyanobacterial blooms,35,36 with consequent risks due to the presence of cyanotoxins in basins used for agricultural purposes, bathing, fishing, and drinking water production. This study reports the response actions implemented by a task force involving mainly the water supplier and the health and environmental authorities to manage the risk related to the presence of cyanotoxins in the drinking water supply chain of the Finocchito WTP, after an extraordinary bloom of the cyanobacterium Planktothrix rubescens observed in early 2009 in the Occhito basin,37 the artificial reservoir used for drinking water supply.

area is about 13 km2 and its depth reaches 70 m. It has a storage capacity of over 330 million m3 of water, 250 million m3 of which are used for human consumption. About 60% of this water is used for irrigation, whereas about 20% is supplied as drinking water to surrounding municipalities (about 800 000 inhabitants). The Finocchito WTP intake is sited at a depth of 20 m. Sample Collection. Over 28 months of monitoring (February 2009 to June 2011), water samples were taken at least every 15 days at the WTP and over the piped distribution system, at the following five stations: (1) raw water, at the WTP inlet (2) drinking water, exiting the WTP at the head of the distribution system (3−5) three sampling points on the distribution system, sited in different municipalities, namely Castelnuovo della Daunia, San Severo and Foggia (Figure S1 of the Supporting Information) Additional investigations were conducted from January 2010 to June 2011 on samples collected within the WTP, to evaluate the efficiency of the main treatment process, according to the following scheme: (a) raw water, collected at the WTP inlet (b) treated water after a preoxidation process with chlorine dioxide (c) treated water after flocculation and settling (d) treated water after the filtration system (e) treated water after disinfection with hypochlorite Samples were taken in 0.5 L Ruttner bottles. Temperature and free chlorine content were measured for each sample (data not shown). For LC/MS analysis, water samples were transported in ice chests to the laboratories, where they were stored at −18 °C to determine the total cyanotoxins content after one cycle of freezing-thawing.21 After thawing, water samples were spiked with IS and filtered. Cyanobacteria Cell Density and Analysis of MCs with ELISA. The cell density and ELISA analysis were conducted by ARPA-Foggia.37 Cell densities were estimated according to the Utermöhl method,38 whereas total MCs analysis was performed with an ELISA assay by means of polyclonal antibodies antiMC-LR. The commercial kit ENVIROLOGIX QuantiPlateKit was used for quantifying MCs (0.16 μg/L of Limit of Detection, LOD). LC/MS/MS Analysis of Cyanotoxins. To analyze CYN and ANA-a toxins, 100 μL of the filtered water sample spiked with IS were directly injected into the LC/MS apparatus according to experimental conditions reported in ref 22 for CYN and in ref 25 for ANA-a, except for using nodularin as IS and a calibration range of 0.5−50 μg/L. For the analysis of MCs, an analytical method using a Graphitized Carbon Black as solid phase extraction sorbent,22 was modified and implemented as follows: (a) The number of target MCs was enlarged, including [D-Asp3]MC-RR, [D-Asp3]-MC-LR, MC-LF and MC-LY; (b) nodularin was selected as IS, always at 1 μg/L concentration; (c) 0.25 L of water sample was loaded onto extraction cartridges instead of 0.5 L; (d) 6 mL of eluent phase in frontal mode was adopted for the re-extraction of analytes instead of 4 mL in backflushing mode;

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EXPERIMENTAL SECTION Reagents and Chemicals. MC-RR, MC-YR, MC-LR, MCLA, MC-LW, MC-LF, MC-LY, [D-Asp3]-MC-RR, [D-Asp3]MC-LR, ANA-a, CYN, and nodularin, used as internal standard (IS), were purchased from Alexis Biochemicals, La Jolla, CA, USA. ANA-a was from ICN Biomedical, Aurora, OH, USA. Stock solutions of the nine MCs, IS, CYN, and ANA-a were prepared by dissolving each compound with at least 2 mL of methanol. These solutions were stored at −18 °C in the dark to minimize analyte degradation. Working standard solutions of analytes and IS were obtained by suitable diluting stock solutions with mobile phases, at a final concentration of 1 and 5 μg/mL, respectively. When unused, all working standard solutions were stored at 4 °C in the dark and renewed after two working weeks. All solvents and chemicals were of analytical grade (Carlo Erba, Milan, Italy). Extraction cartridges filled with 0.5 g of Carbograph 4 were supplied by LARA, Rome, Italy. A 125 mm diameter Black Ribbon 589 paper filters were purchased from Schleicher & Schuell, Legnano, Italy. Site Description. Occhito Lake (41°34′48″N 14°56′42″E) is an artificial reservoir sited in the province of Foggia (Southern Italy, Apulia Region) and it is fed from a river and two streams, namely Tappino, Cigno, and La Catola. The lake 575

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(e) 0.5 g/L of Na2S2O3 was added to treated water samples to avoid oxidation of the IS, when concentration of residue free chlorine was higher than 0.3 mg/L. Finally, extract was evaporated at 50 °C to dryness, reconstituted with mobile phases, and 50 μL of this solution was injected into the LC/MS system. Deionized water was used as blank samples after spiking with IS. Analysis of extracts of deionized water samples spiked with analytes at 0.1 μg/L and IS after evaporation were used as reference for the evaluation of the extraction efficiency. The liquid chromatograph consisted of an Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA, USA) equipped with an Alltima C18 (2.1 × 250 mm, ID 5 μm) (Alltech, Sedriano, Italy) column thermostated at 40 °C and interfaced by a Turbo Ion Spray source to a triple-quadrupole mass spectrometer API 3000 (Applied Biosystems, Darmstadt, Germany). Different chromatographic gradients, slightly modified with respect to the original protocols, were employed for determining MCs, CYN, and ANA-a, using acetonitrile and water as mobile phases, both containing 10 mM formic acid. All MS parameters were optimized for each analyte (data are reported in Table S1 of the Supporting Information). Two precursor ion > daughter ion transitions for each analyte were selected for the multi reaction-monitoring mode. The transition with the higher signal-to-noise ratio, S/N was chosen for the quantification. The analyte area/IS area ratio was used to construct a calibration curve by spiking and extracting blank samples with analytes at 0.1, 0.5, 1.0, and 2.5 μg/L and the IS at 1 μg/L. The LODs were assessed for each analyte from the lowest calibration level, considering the transition with the worst S/N (qualifier transition), as reported elsewhere.22 LODs ranged from 0.002 to 0.013 μg/L for MCs, whereas LODs of CYN and ANA-a, evaluated from spiked lake water samples, were 0.08 and 0.2 μg/L respectively (Table 1). When amounts of MCs injected from water sample extracts exceeded the upper limit of the linear dynamic range of the detector response, extracts were suitably diluted and reinjected.

Table 1. Multi-Reaction Monitoring (MRM) transitions, Limit of Detection (LODs), and Accuracy Obtained for Determining Selected Cyanotoxins in Water by Tandem MS; Accuracy, Expressed As Sum of Trueness and withinLaboratory Reproducibility, Was Obtained Analyzing on Three Different Days and by Different Operators N = 9 Water Samples Spiked with 0.1 μg/L of Each Microcystin and IS at 1 μg/L MRM transition, m/z

compound anatoxin-a cylindrospermopsin [D-Asp3]-MC-RR MC-RR nodularin (ISd) MC-YR [D-Asp3]-MC-LR MC-LR MC-LA MC-LF MC-LW MC-LY

166 > 149 166 > 131b 416 > 194 416 > 336 513c > 135 513c > 127 520c > 135 520c > 127 825 > 135 1045 > 135 1045 > 70 981 > 135 981 > 70 995 > 135 995 > 213 910 > 135 910 > 776 986 > 478 986 > 852 1025 > 446 1025 > 891 1002 > 135 1002 > 868

LOD, μg/L truenessa, % RSD, % 0.2 0.08 0.004

95%

11%

0.004

107%

10%

0.013

121%

15%

0.004

118%

11%

0.006

109%

11%

0.002

108%

16%

0.002

119%

13%

0.002

117%

15%

0.002

109%

11%

a Trueness is expressed as percentage recovery of fortified water samples at 0.1 μg/L with respect to a reference solution obtained by spiking extracts of water at the same concentration after evaporation. b Transitions with the worst S/N are reported in bold. cDouble charged ion. dIS = internal standard.

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RESULTS AND DISCUSSION Emergency Phase and Creation of the Task Force. When the first observation of the P. rubescens bloom was reported in February 2009 by the local environmental authorities in this previously unaffected water body, a task force involving all the different stakeholders was readily created to tackle the consequences of the contamination. These included the health (ASL Foggia), environmental (ARPA Puglia), and basin authorities, National Institute of Health (ISS), National Research Council (CNR-IRSA), water supplier (AQP), mayors, civil protection, epidemiological observatory, under the coordination of the Apulia Region (Regione Puglia) to cope with the emergency. The first actions focused on reducing risk related to the human consumption of potentially contaminated drinking water and fisheries products. A monitoring campaign started immediately on the lake, the WTP and the entire drinking water chain, with rapid screening methods and more reliable confirmatory ones. Short-, medium-, and long-term actions were planned to reduce concentrations of cells and toxins in distributed waters. Early actions coordinated by the task force included: (a) 1−2 weekly monitoring of water within the reservoir and the whole water supply chain: algal cells counting and

(b) (c) (d) (e) (f) (g)

(h)

(i)

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toxin analysis with ELISA assay performed by the environmental authorities; analytical sampling and LC/MS confirmatory analysis conducted by the ISS; individuation of possible alternative water supply and additional water treatments; alert to civil protection for emergency water supply; protection measures for workers within the basin; communication to consumers; investigation of factors inducing the algal bloom: eutrophication caused by irregular waste disposal/role of sediments/ sequence of droughts and overflowing; risk assessment for water uses other than human consumption: agriculture, fishing, recreational uses, food production:  banning of fishing in the lake (April 2009);  banning of mussel harvesting (April 2009, exceptional circumstance due to overflowing phenomena from lake to the sea); protocol between National Research Council/irrigation water manager (Consorzio di Bonifica − Capitanata): study on the Occhito Lake: limnology, nutrient flows, dx.doi.org/10.1021/es302260p | Environ. Sci. Technol. 2013, 47, 574−583

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ability of several MC isomers.19 Indeed, the quantification of nine MCs variants, comprising two demethylated variants, obtained at such a low concentration with this LC/MS technique, could be used to critically evaluate different toxicological contributions to support the decisional process. MCs in the Drinking Water Chain. During the massive cyanobacteria bloom that occurred in the Occhito basin in the winter of 2008−2009, ARPA identified P. rubescens as major component reaching the maximum value of 140−160 × 106 cells/L in March 2009.37 Figure 1 shows data of cell density and MCs concentration, obtained from LC/MS analysis (expressed as sum of selected congeners), and from ELISA assay (expressed as MC-LR equivalents). Two species of cyanobacteria were identified, both generally producing MCs: P. rubescens, which was always the predominant strain, and Microcystis aeruginosa, which was found and identified a few times starting in November 2009. No samples positive for ANA-a and CYN were encountered coherently with the absence of cyanobacteria conventionally described as potential producers of these algal toxins. The maximum cell density registered in the initial phase of sanitary emergency (February−March 2009) was consistent with typical winter blooming of P. rubescens, and it was never again reached. However, as the water catchment of the WTP is situated at 20 m depth,37 the density profile has to consider the water column temperature. The hydrologic extreme events with the rapid succession of exceptional drought and flooding in winter 2008−2009 may account for the uncommon blooming until summer of the same year and MCs production in the Occhito lake in which algal growth had not been recorded for many decades. Other important blooms (20−40 × 106 cells/L) were detected in July 2010 and March 2011. As is evident from Figure 1, total content of MCs arising from ELISA assay fits very well with cyanobacteria population trend. This series of ELISA data has generally shown positive results (> 0.16 μg/L) when cells density exceeded one million cells per liter. This fact could be remarkable for choosing analytical tools to adopt for routine surveillance and for identifying a threshold for alert levels. Instead, a comparison between cell density and total MCs determined with the LC/MS system showed some peculiar cases. MCs total levels quantified with the LC/MS system in the raw water ranged from 0.006 μg/L (November 2009) to 28.4 μg/L (April 2009). However, several blooms with high cell density (up to 45 × 106 cells/L), occurring in July−August 2009, did not yield a significant production of toxins (total concentration below 4 μg/L). Analogously, the increasing blooming experienced during the period January−March 2011, did not appreciably produce MCs. The opposite was observed in the same period of 2010, when a significant concentration of MCs was produced (higher than 1.1 μg/L) during a decreasing blooming of P. rubescens (712 000−2 million cells/L). Although a bloom of M. aeruginosa has been detected in the meanwhile, its contribution to toxins production was not supported by the presence of its typical MC-pattern (i.e., MC-LR, below and Figure 2). However, this may be attributed to changes in the genes expression involved in toxin production,36,40 so that a toxin quota was not easily inferable. Environmental parameters like temperature, light, and nutrients contents may have affected the behavior observed in the toxinogenesis.40 Unfortunately, measurements of physicochemical parameters at the water source were impractical in this study due to the technical difficulties in accessing the water intake within the basin. As a conclusion, the

cyanobacteria, and toxins within the irrigation channels (data not reported); (j) extension of monitoring to other basins. Analytical Issues: Optimization of the Solid Phase Extraction-LC/MS/MS Analysis of MCs. Simultaneous determinations of total MCs content were performed with an ELISA test a LC/MS method. Several new MCs congeners are recently available as certified standards, so a previous method 22 was upgraded optimizing it in terms of instrumental response and extraction efficiency for these analytes. In particular, the correct quantification of [D-Asp3]-MC-RR and [D-Asp3]-MC-LR variants is important when a bloom of P. rubescens occurs, these MCs being the most abundant ones produced by this cyanobacterium.35,36 Performance, reliability and feasibility of the method were improved to optimize resources of the health authority’s laboratories for cyanotoxins sampling and analysis in raw, treated, and drinking waters. The entire method was validated in terms of sensitivity, selectivity, repeatability, reproducibility, robustness, and LODs in compliance with the Italian implementation of the Drinking Water Directive 98/83/EC.39 Deionized water, surface water from a cyanotoxins-free lake (Bracciano, Italy), and tap water were chosen as matrices for the evaluation of the matrix effect. Results, reported in Table S2 of the Supporting Information indicated that a weak matrix effect was present for several compounds in all types of water considered. Anyway, the matrix effect is not significantly dependent on the specific matrix selected, as resulted from oneway ANOVA test at the P = 0.05 significance level. Thus, to propose an friendly to-use method for routine analysis, deionized water was chosen as a representative matrix. The validation experiments were planned as follows: (a) a calibration curve, spiking N = 8 (n = 2 replicates for each level) water samples at 0.1, 0.5, 1.0, and 2.5 μg/L concentration levels covering the range of the WHO guideline value (1 μg/L as MC-LR equivalents) for the study of sensitivity and linearity of the method; (b) analysis of N = 3 blank samples for evaluating method selectivity; (c) analysis of N = 9 water samples spiked at 0.1 μg/L, assayed in triplicate over three days by different operators, for the assessment of accuracy, as sum of trueness (recoveries) and intralaboratory reproducibility. Results of these studies are reported in Table 1 and Table S1 of the Supporting Information, whereas representative chromatograms of a standard solution and a blank sample are depicted in Figures S2 and S3 of the Supporting Information. Internal standard accuracy has been used as quality control for all measurements during the monitoring showing that this protocol is robust and not significantly dependent by different matrices processed (79% ± 21% on N = 20 samples). The extraction efficiency of not previously tested MC congeners proved to be satisfactory, with relative recoveries not lower than 95%, and an intralab reproducibility not higher than 16%. A good linearity was achieved over the selected range of interest (Table S2 of the Supporting Information). Finally, adding the antioxidant to the treated waters seems to be effective in the analytes and IS protection enlarging the feasibility of the proposed method to the analysis of drinking water. Including as many MC congeners as possible in the monitoring program is an important issue in risk management, also taking into account recent information on PP-inhibitory 577

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Figure 1. Temporal trend of cell densities [cells/L] of cyanobacteria identified in raw waters of the Finocchito water treatment plant and microcystins level, determined with both LC/MS analysis (expressed as total concentration of the sum of selected congeners) and ELISA assay (expressed as MC-LR equivalents).

Figure 2. Temporal trend of total microcystins levels obtained with LC/MS analysis of all selected congeners and with ELISA assays, both recorded in raw waters of the Finocchito water treatment plant. 578

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sustained without additional data. Anyway, the ELISA test proved to be adequate for a screening purpose, even if caution has to be taken with cyanobacterial blooms that produce MC congeners different from MC-LR. Risk Mitigation: Corrective Actions within the WTP of Finocchito. A comprehensive approach to risk mitigation related to the cyanotoxins occurrence suggests a preventive management of the basin affected by algal blooms followed by the attempt to remove cyanobacterial cells, before they reach the WTP or they can be lysed by physical or chemical treatments within the WTP.29 Anyway, during similar health emergencies, water suppliers often have to tackle high toxin levels in waters and have to carefully choose the best sequence of treatments. For these reasons, the analyses of total content of MCs were conducted as the worst case of the risk assessment given that water treatments could contribute simultaneously to removing algal cells and in releasing free toxins in treated waters.31 Mitigation measures to cope with unexpected, massive contamination with toxic cyanobacteria, mostly rely upon modification of the intake depth, the dilution of the inlet water or the adoption of an alternative water supply.28,29 However, such measures were not practicable in the Finocchito WTP due to its structure, the large volume of water (the biggest WTP in Europe with 543 million m3/year), and size of the served population. For similar reasons, distribution with tankers was considered unfeasible by the civil protection. In such circumstances, without a contingency operational plan utilizing specific treatment barriers, starting from the emergency in 2009 ancillary practices29 were adopted by the water supplier. The basic scheme of the WTP was initially as follows: preoxidation with ClO2 > 3 tanks for flocculation and settling > 10 beds of rapid sand filters > final disinfection with NaClO. The optimization of pre-existing water treatment processes for the oxidation of MCs, such as the use of chlorine dioxide in the early steps of water treatments, was an example of a short-term corrective action. This option has been preferred to a superchlorination with hypochlorite, to narrow the concentration of byproducts, mainly trihalomethanes.29 The conversion of pre-existenting sand filters to mixed sand/GAC beds, and the construction of a new battery of GAC filters could be considered medium- and long-term corrective actions. In detail, during the emergency phase, 658 t of GAC were used for the conversion and reloading of sand filters and their running was completed by early May 2010. After this period, an ordinary maintenance of the filtration system was performed. Data reported in Figure 3 suggest that not systematic MCs contaminations occurring in treated waters in May 2009, June 2009, and January 2010 could be ascribed to a partial failure in removing toxins, probably due to the saturation of the sand and/or mixed bed filters, when relatively high levels of cyanobacteria and cyanotoxins were present in raw waters. In the meanwhile, some significant MCs break through in distributed waters (summer−autumn 2009, November 2010, and June 2011), always lower than WHO guideline value, were detected at Castelnuovo, Foggia, and San Severo. The last two sampling sites are quite far from the WTP (San Severo ca. 2−3 days and Foggia ca. 4−5 days), so that the contamination scenario is a snapshot not in-time correlated with treated waters sampled on the same day due to the long residence time of water in the distribution network (Figure S1 of the Supporting Information). Thus, it was very difficult to trace the origin of these contaminations without the in-time related sampling of raw and treated waters.

concentration of MCs measured with the LC/MS system was not constant or predictable based only on cell density, that is generally, not necessarily, associated to MCs production. According to this finding, for risk management purposes, cell density measurements could be used as a marker for the cyanotoxins class that has to be analyzed, and to optimize the frequency of sampling on the basis of density variation.31 Alternatively, ELISA data seem to match very well with the temporal variations of cyanobacterial population, so this test could be used for routine analysis both to monitor algal blooms producing MCs and to define the alert levels.28 Figure 2 reports the temporal trend of MCs concentration, as obtained from ELISA and LC/MS analysis of all selected variants in raw water. A pattern of toxins production typical of P. rubescens previously occurred in Italy35,36 was experienced, with [D-Asp3]-MC-RR, MC-RR, and [D-Asp3]-MC-LR as congeners most frequently detected with maximum concentration of 28.4, 0.75, and 0.53 μg/L, respectively. Conversely, the presence of MC-LR in raw water at 1 μg/L in August 2009 and other toxins (MC-LA, MC-LY and MC-LF) at much lower concentration, found sporadically in June 2010, was unusual for P. rubescens blooms. The occurrence of MC-LR in 2009 has prompted a further investigation of cyanobacteria potential producers with a typical summer blooming, like Microcystis aeruginosa, which was in fact identified in November 2009. To our knowledge, MC-LY and MC-LF have been never previously identified in Italian freshwaters, but these toxins have been reported to be produced by Microcystis spp.41,42 Taking advantage of the selective determination of MC congeners, the LC/MS method is able to suggest the possible presence of coblooming, which is not easy to identify with phycological analysis. Such information on raw waters, together with algal counts, could be useful for planning mitigation measures in WTPs, optimized for specific cyanobacteria or cyanotoxins, that is with clariflocculation or oxidation systems.17,19 With regard to a correlation between ELISA and LC/MS data, except for a generally similar temporal trend, this was not evident. Taking into consideration that this screening test gives results proportional to the amount of Adda residue (free and/or conjugated) and expressed as MC-LR equivalents, false positive results or an overestimation due to the cross-response of conjugated forms have quite frequently been reported at low concentrations.16,18,43 Instead, this behavior was detected only in March 2009 during the early emergency phase, in July− August 2009, when MC-LR was effectively present, and several times in the period November 2010 to June 2011. Conversely, an unexpected underestimation of the data obtained from ELISA test with respect to LC/MS ones was often observed during monitoring (again, Figure 2). This could be due to the different response of the ELISA test to congeners others than MC-LR, like [D-Asp3]-MC-RR, which are predominant in the P. rubescens strain found in the Occhito basin, as appears from MS analysis. We should point out that analytical standards of [D-Asp3]MC-RR are only recently commercially available. Thus, a correct quantification by LC/MS technique of its contribute on toxins production is now possible and a comparison with ELISA results is more reliable. One could speculate that previous LC/MSbased quantitation of demethylated forms of MC-RR or MC-LR, made on the basis of their fully methylated congeners,22 was underestimated. This is the first time that such a long series of data has been obtained with standards of demethylated congeners. Other assumptions related to the toxinogenesis or the presence of other Adda-conjugated compounds cannot be 579

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Figure 3. Temporal trend of total microcystins levels determined with LC/MS analysis, expressed as sum of selected congeners, sampled in raw and treated waters of the Finocchito Water Treatment Plant, and in distributed waters of the three selected municipalities (Castelnuovo della Daunia, San Severo, and Foggia). Principal corrective actions implemented by the AQP stakeholder to improve efficiency of the water treatment plant are indicated in the graph with numbers: 1 = conversion of 5/10 sand filters to mixed sand/GAC beds; 2 = conversion of all sand filters to mixed sand/ GAC beds; 3 = reloading of carbon; 4 = running of 4/10 GAC filters; 5 = running of 8/10 GAC filters; 6 = running of all GAC filters.

Further corrective actions, consisting of reloading carbon and/or conversion of filtration beds, have led to a definitive cutback of the MCs level in drinking waters ( 10 beds of mixed sand/GAC filters > 10 beds of GAC filters > final disinfection with NaClO. Once fully operative, this system has been able to reduce even a level of 2.8 μg/L in raw water to a final concentration of 0.006 μg/L (May 2011). Thus, the implementation of this scheme avoided any interruption in the distribution of drinking water to about one million consumers served by the Finocchito WTP. Risk Communications. Some main ancillary actions included the risk communication to the public with press release and a dedicated Web site within the Apulia Region portal (http://www.regione.puglia.it/index.php?page=prg&id=18) and a free-call phone number by the regional epidemiologic observatory available for reports from the public. The Web site has reported information about decisions of the task force, press releases, technical reports on cyanobacteria, and real-time data performed by ARPA. Finally, several training courses were organized by the ISS for technical operators of ARPA, ASL, and AQP. In conclusion, the monitoring and corrective actions implemented through an intensive cooperation among the regulatory bodies, water supply company, and the public have been effective in successfully managing the health risk for the affected population without requiring any limitation of water uses. This case study was a solid basis for the production of the Italian guidelines for risk management of drinking water contaminated by cyanobacteria (http://www.iss.it/publ/rapp/ cont.php?id=2557&lang=1&tipo=5&anno=2011).

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AUTHOR INFORMATION

Corresponding Author

*Fax: +39-06-49903115, e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to “Regione Puglia” for the financial assistance and Dr. Fulvio Longo for management of the project “Emergency alga Planktothrix rubescens in the Occhito basin and in the distribution system of Foggia”. The assistance of Antonio Argentile, Antonio Salvatore, Fernando Testa of ASL Foggia is also gratefully acknowledged. Finally, the authors are grateful to all referees who have significantly improved this article.

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GLOSSARY ANA-a anatoxin-a ARPA environmental authority AQP local water supplier ASL health authority CNR National Research Council CYN cylindrospermopsin ELISA enzym-linked immunosorbent assay GAC Granular Activated Carbon ISS Italian National Institute of Health LC liquid chromatography LOD limit of detection IS internal standard MC microcystin MS mass spectrometry PP protein phosphatase WTP water treatment plant WHO World Health Organization

ASSOCIATED CONTENT

S Supporting Information *

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