Original article iq.unesp.br/ecletica | Vol. 45 | n. 2 | 2020 | 23 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 Estrogenic mycotoxins in surface waters of the Rico Stream micro- basin, São Paulo, Brazil: occurrence and potential estrogenic contribution Elissandro Soares Emídio+ , Claudia Pereira da Silva , Mary Rosa Rodrigues de Marchi São Paulo State University (Unesp), Institute of Chemistry, 55 Prof. Francisco Degni St, Araraquara, São Paulo, Brazil +Corresponding author: Elissandro Soares Emídio, Phone: +55 16 3301-9735 email address: elissandro_se@yahoo.com.br ARTICLE INFO Article history: Received: July 21, 2019 Accepted: December 11, 2019 Published: April 1, 2020 Keywords: 1. estrogenic mycotoxins 2. occurrence 3. surface waters 4. estrogenicity 5. southeast Brazil 1. Introduction Among the hundreds of mycotoxin-producing fungi, the genus Fusarium is considered one of the most prevalents in terms of animal health implications and economic damage1. Resorcyclic acid lactones (RALs) are compounds that exhibit endocrine disruptive behavior and are produced by fungi of the genus Fusarium2. The representative mycotoxin of this class of RALs is zearalenone (ZEN), cited among the most common mycotoxins worldwide3. In Brazil, there have been few studies on the incidence of fusariotoxins. For example, the occurrence of ZEN was reported in maize4, rice5 and wheat6 in the south and southeast Brazilian regions. Fusariotoxins are mostly produced under high humidity and at temperatures of approximately 20 to 26 °C. The Brazilian climate offers good conditions for these toxins to be present in grains used in the diets of animals and humans7. The incidence of ZEN and its metabolites has been reviewed extensively in food for humans and animals8. However, little is known about its impact or environmental distribution, with few studies having been carried out concerning these attributes9. Some publications have reported the occurrence of ZEN in surface water, groundwater ABSTRACT: The aim of this study was to assess the occurrence of zearalenone (ZEN) and its metabolites zearalanone (ZAN), α-zearalenol (α- ZEL), β-zearalenol (β-ZEL) α- zearalanol (α-ZAL), and β-zearalanol (β-ZAL) in the surface waters of northern São Paulo state (Brazil) and to evaluate the associated potential estrogenic contribution to the aquatic environment. The determination of the estrogenic mycotoxins in water samples from the Rico Stream microbasin yielded levels of up to 59 ng L-1 and their corresponding calculated estrogenic equivalent (cEEQ) values were between < 0.03 and 1.4 ng L-1, which are associated with negative effects on the reproduction and growth of some fish species. The physicochemical and microbiological parameters were evaluated to determine the water quality in the Rico stream region. This study revealed the first data about the presence of estrogenic mycotoxins in the Brazilian river waters and emphasizes the need to include these substances in future public policies concerning water quality, since these compounds are not yet legally regulated. From an environmental aspect, it is necessary to take into account the continuous introduction into surface water of microcontaminants associated with wastewater effluent, such as estrogenic mycotoxins. http://revista.iq.unesp.br/ojs/index.php/ecletica/index https://doi.org/10.26850/1678-4618eqj.v45.2.2020.p23-32 mailto:elissandro_se@yahoo.com.br https://orcid.org/0000-0003-0679-4123 https://orcid.org/0000-0002-2806-9021 https://orcid.org/0000-0001-5381-249X Original article 24 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 and effluent from sewage treatment plants and industries, with concentrations of ZEN or its metabolites ranging from undetected levels to 4120 ng L-1 10. Zearalanone (ZAN) and β- zearalenol (β-ZEL) have rarely been detected or analyzed in aqueous environmental samples. ZEN and its metabolites can contribute to the overall estrogenic activity in the environment and therefore could pose a risk to wild fish in their natural habitat, as indicated by a zebrafish study in which there was a change in the reproductive capacity of animals exposed to low concentrations of estrogenic compounds (100 to 3200 ng L-1) for 21 days3. Some studies conducted in Brazil have reported the occurrence of endocrine disruptors in surface waters, with monitoring results showing the periodic presence of these substances in natural waters, suggesting that different sources in addition to untreated sewage contribute to the input into water sources11. Possible aqueous environmental contamination routes of estrogenic mycotoxins include: drainage water from fields cultivated with infected plants, runoff from livestock facilities or manure applications to field crops and human excretions in urban wastewaters12. The purpose of this study was to examine for the first time the occurrence of estrogenic mycotoxins in Brazilian water samples, using dispersive liquid- liquid microextraction (DLLME) followed by liquid chromatography–tandem mass spectrometry and to identify possible sources of contamination, seasonal variations and potential contribution to total estrogenic activity in the southeast region of Brazil (São Paulo state). 2. Experimental 2.1 Standards and reagents ZEN, α–ZEL, β–ZEL, α–ZAL, β–ZAL and ZAN standards were all obtained from Sigma Aldrich (St. Louis, MO, USA) with purities of at least 98%. The zearalenone–d6 (surrogate internal standard, ZEN–d6) was supplied from Toronto Research Chemicals (North York, ON, Canada). Acetonitrile (ACN) and methanol (MeOH), both HPLC grade were provided from J. T. Baker (Phillipsburg, NJ, USA). Bromocyclohexane was acquired from Alfa Aesar (MA, USA). Hydrochloric acid (37% m/v) and sodium hydroxide were purchased from J.T. Baker (Phillipsburg, NJ, USA) to adjust the pH of water samples. Deionized water was obtained using a Milli–Q purification system (Bedford, MA, USA). 2.2 Sampling area The Rico Stream microbasin is localized in the northeast region of São Paulo state and consists of the municipalities Jaboticabal, Taquaritinga, Monte Alto, Guariba and Santa Ernestina (Fig. 1), occupying an area of approximately 563 km2 at an altitude between 498 and 754 m. The Rico Stream is the main source of water in the micro-basin, with a total of 59.2 km of watercourse length discharging into the Mogi Guaçu River. Because of its high representativeness in the region, Rico Stream stands out as the main supply of water for the city of Jaboticabal (population ca. 75,000). Anthropogenic impacts on the environment may occur both directly or indirectly, due to effluent discharges from domestic sewage treatment plants of the Monte Alto municipality and animal husbandry (poultry, swine and cattle)13. In accordance with the classification proposed by Köeppen, the region possesses a climate defined as Aw, characterized as tropical with summer rainfall and dry winters. The average temperature is 23 °C, and the total average annual rainfall is 1405 mm occurring mainly during the period from October to March, with the dry season extending from April to September14. https://doi.org/10.26850/1678-4618eqj.v45.2.2020.p23-32 Original article 25 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 Figure 1. Map of the studied area showing the location of four sampling sites (Adapted from Pissarra et al.15 and Takahashi et al.16). 2.3 Sample collection and pretreatment The collection of water samples was performed from October 2014 to July 2015 (ten sampling campaigns) at four different sampling sites located in the Rico Stream, except one that was located in Tijuco Stream (stream tributary) between the municipalities of Monte Alto and Jaboticabal. References sites: P1 situated near the Rico Stream source with minor contamination impacts and upstream of wastewater treatment plant (WWTP) effluent discharge (21º18'37.26"S; 48º27'39.49"W); P2 situated at the downstream discharge point of the WWTP, which also represents the influence of input from agricultural industry and runoff from livestock facilities or fields receiving livestock manure applications (21º18'46.08"S; 48º26'56.33"W); P3 located in the Tijuco Stream, upstream of the public water supply and near the confluence with the Rico Stream (21º17'56.08"S; 48º22'31.69"W) and P4 located freshwater abstraction site for the public water supply to Jaboticabal city (21º18'36.07"S; 48º19'26.11"W). The water samples were collected at 30 cm depth in 1000 mL amber glass bottles, previously rinsed thoroughly with ultrapure water. After collection, samples were transported to the laboratory under refrigeration at 4 °C (ice packs) and processed within 48 h. In the laboratory prior to extraction, the samples were filtered through a glass fiber filter with a pore size of 0.6 µm (Macherey-Nagel GF-3), acidified to pH 4 and spiked to 200 ng L-1 with an internal surrogate (ZEN-d6). Samples were then immediately extracted and analyzed using the previously developed and validated dispersive liquid-liquid microextraction (DLLME) method and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)17. https://doi.org/10.26850/1678-4618eqj.v45.2.2020.p23-32 Original article 26 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 2.4 Determination of physicochemical and microbiological parameters A total of twelve physicochemical and microbiological parameters were used to assess water quality control and pollution. Parameters such as fecal coliforms, total organic carbon, chemical oxygen demand (COD), biochemical oxygen demand (BOD), total phosphorous, total nitrogen, nitrate, nitrite, ammoniacal nitrogen, potassium, turbidity and total dissolved solids were determined using standard procedures described by the American Public Health Association18. 2.5 Analytical Methodology An aliquot of 10 mL of the water sample was added into a 12 mL conical glass centrifuge tube with screw cap. Thereafter, 100 μL of bromocyclohexane (extraction solvent) was added to the sample solution. The mixture was then vigorously shaken using a vortex mixer at 2500 rpm for 120 s. After centrifugation for 10 min at 3500 rpm, the organic sedimented phase was collected in a glass insert (300 μL) using a 100 μL Hamilton microsyringe and was evaporated in a N2 flow. The residue was reconstituted in 80 μL of MeOH/0.1% formic acid in H2O (50:50, v/v) and injected into the LC–ESI–MS/MS for analysis. 2.6 Instrumentation The HPLC analyses were carried out on an Agilent 1200 series LC system coupled with a 3200 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex Instruments) with an electrospray ionization source (ESI). The mass spectrometric parameters selected for experiments of full scan and ion fragmentation were as follow: ion spray voltage of −4 kV, curtain gas (nitrogen) at 15 psi, auxiliary gas at 40 psi, nebulizer gas at 50 psi, source temperature at 600 °C, interface heater and entrance potential of -10 V. Chromatographic separation of the estrogenic mycotoxins was achieved at 40 °C using a Gemini C18 column (150 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) preceded by a Phenomenex C18 column guard (4 × 3 mm). The mobile phase consisted of 0.1% formic acid in water (48%, v/v), acetonitrile (25%, v/v) and methanol (27%, v/v), with a flow rate of 1.0 mL min-1 for 17 min and injection volume was set to 20 μL. Applied Biosystems Analyst 1.5 software was used for data acquisition. 2.7 Quality assurance/quality control The calibration was performed by internal standardization method with matrix-matched blank samples. The calibration curve consists of eight different concentration values, in the range 8 to 680 ng L-1. The analytes were extracted by DLLME prior to chromatographic analysis for each point of the calibration curve. The calibration curve was obtained from the ratio between the peak areas of analytes and the internal standard versus the analyte concentrations (ng L-1). Every experimental point was analyzed in triplicate. The extraction efficiency (recovery percentage) of the analytes was evaluated by spiked with 200 ng L-1 of the internal surrogate (ZEN-d6) to all river water samples. 2.8 Estrogenicity The estrogenic potential of the aqueous samples was calculated in equivalent activity compared to the reference substance (natural hormone 17-β- estradiol)19. The potential contribution of each estrogenic mycotoxin to the total estrogenic activity of the surface waters was calculated according to the Eq. 1: 𝑐𝐸𝐸𝑄 = 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑒𝑠𝑡𝑟𝑜𝑔𝑒𝑛𝑖𝑐 𝑚𝑦𝑐𝑜𝑡𝑜𝑥𝑖𝑛 × 𝑅𝑃 (1) where cEEQ is the calculated estrogenic equivalent (17-β-estradiol) of a specific estrogenic mycotoxin and RP is the relative potency of the mycotoxin in an individual bioassay. 3. Results and Discussion 3.1 Occurrence of estrogenic mycotoxins in environmental samples The results obtained from the collected samples (during the period of October 2014 to July 2015) are shown in Tab. 1. Surrogate recovery values (ZEN-d6) ranged from 71 to 124% from the extraction method. These recovery values are https://doi.org/10.26850/1678-4618eqj.v45.2.2020.p23-32 Original article 27 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 suitable when dealing with complex environmental samples such as the river water analyzed in this study. Table 1. Estrogenic mycotoxin concentrations (ng L-1) at four sampling sites in the Rico Stream micro-basin. aZEN-d6 - Recovery (%), spiked at 200 ng L-1 in triplicate b limit of quantification value c n.d. – not detected Compounds October/2014 November/2014 P1 P2 P3 P4 P1 P2 P3 P4 β-ZAL n.d.c n.d. n.d. n.d. 48 n.d. n.d. n.d. β-ZEL n.d. <40b n.d. n.d. n.d. n.d. n.d. n.d. α-ZAL n.d. n.d. n.d. n.d. <20b <20b 27 <20b α-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZAN n.d. n.d. n.d. n.d. <8b n.d. n.d. n.d. ZEN-d6 a 112 108 107 106 107 96 76 91 ZEN <8b <8b <8b <8b <8b <8b <8b <8b Compounds December/2014 January/2015 P1 P2 P3 P4 P1 P2 P3 P4 β-ZAL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. β-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZAL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZAN n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZEN-d6 a 89 80 91 93 112 81 74 77 ZEN n.d. n.d. n.d. n.d. 22 29 25 27 Compounds February/2015 March/2015 P1 P2 P3 P4 P1 P2 P3 P4 β-ZAL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. β-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZAL n.d. n.d. n.d. n.d. <20b n.d. n.d. n.d. α-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZAN n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZEN-d6 a 118 105 97 97 73 71 115 100 ZEN n.d. n.d. n.d. n.d. 59 46 51 42 Compounds April/2015 May/2015 P1 P2 P3 P4 P1 P2 P3 P4 β-ZAL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. β-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZAL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZEL n.d. n.d. n.d. n.d. <20b n.d. n.d. n.d. ZAN n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZEN-d6 a 91 81 73 78 114 124 101 127 ZEN n.d. n.d. n.d. n.d. 18.8 n.d. n.d. n.d. Compounds June/2015 July/2015 P1 P2 P3 P4 P1 P2 P3 P4 β-ZAL n.d. n.d. <40b n.d. n.d. n.d. n.d. n.d. β-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZAL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. α-ZEL n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ZAN n.d. n.d. 19 n.d. n.d. n.d. n.d. n.d. ZEN-d6 a 117 119 119 88 102 103 98 88 ZEN 14 12 11 <8b n.d. n.d. n.d. n.d. https://doi.org/10.26850/1678-4618eqj.v45.2.2020.p23-32 Original article 28 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 All analytes were detected at least once at each point over the sampling period. ZEN compound had a higher frequency of detection (52.5%), followed by α-ZAL (12.5%), β-ZAL (5.0%), ZAN (5.0%), α-ZEL (5.0%) and β-ZEL (2.5%). A total of 47.5% of the samples had concentrations above the limit of quantification (LOQ), ranging from 11 to 59 ng L-1. Among the six analytes, ZEN was determined at all sampling points in five of the sampling campaigns performed. The highest levels were determined during the period of greatest rainfall. In addition, most ZEN metabolites were detected during the rainy season. The compounds β-ZAL, α-ZAL and ZAN were quantified only once, whereas α-ZEL and β-ZEL did not present in concentrations above the LOQ (Tab. 1). The limits of detection were for β-ZAL and β-ZEL of 20 ng L- 1, for α-ZAL and α-ZEL of 8 ng L-1 and for ZAN and ZEN of 4 ng L-1. The results obtained in this work are comparable to those obtained by other researchers. In previous studies, the detection frequencies for ZEN ranged from 0 to 30% and differed according to the seasons during sampling9,20-22. The highest concentrations of ZEN (up to 96 ng L-1) were detected in small streams or channels near agricultural fields with crops contaminated by Fusarium sp.9,20,23. Low levels (up to 35 ng L-1) were found in river and lake waters18,22-25. The metabolites β-ZAL and α-ZAL were investigated in two rivers contaminated by wastewater effluents24. The maximum concentration of both compounds was 3 ng L-1. The concentrations of β- ZEL and α-ZEL metabolites were investigated in agricultural streams upstream and downstream of the WWTP in New York (USA). In agricultural fields, the concentrations of these compounds were generally below the limit of detection, but the highest detected concentrations reached tens or even hundreds of ng L-1. In summary, the levels of estrogenic mycotoxins detected in rivers, streams and drainage channels occurred generally at levels of dozens of ng L-1. However, much higher concentrations have occasionally been reported (single study), such as the detection of hundreds of ng L-1 for α-ZEL and β-ZEL in streams near agricultural areas or wastewater effluents in New York, USA9. The results obtained for physical, chemical and microbiological parameters at the sampling sites (Tab. 2) showed that variables such as fecal coliforms (December–July), BOD5 (October and January), total phosphorus (October), nitrite concentration (December), and total ammoniacal nitrogen concentration (October) exceeded the values established by CONAMA for class 2 freshwater (Brazilian Environmental Council)26. According to the results of these parameters (Tab. 2), there was increase in the total phosphorus, potassium concentration, total dissolved solids and turbidity during the rainy season (November to March), which was characteristic of rainfall events and the corresponding transport of fine soil particles into the body of water. There was an increase in the concentration total nitrogen during the extensive dry season in October 2014. It is assumed that the high concentration of ammoniacal nitrogen (42 mg L-1 for P2 site) probably originated from runoff waters that carried residues from livestock animals or by the drainage of agricultural soils where animal excrement was used as fertilizer. The fecal contamination indicator (fecal coliforms) exceeded the maximum limit of 1000 CFU 100 mL-1 24 in samples from all months, with the exception of October and November 2014. These results indicated that Rico Stream is subject to a great anthropogenic influence, probably related to the use and soil occupation of this microbasin. The Rico Stream microbasin presents serious problems of environmental degradation, characterized by residential and rural areas with large deforestation events and lack of adequate management of animal husbandry waste, resulting in contamination of the whole drainage network. The P2 site had the highest concentrations of eight of the hydrological variables (fecal coliforms, BOD, total phosphorous, nitrate, nitrite, potassium, total dissolved solids, ammoniacal nitrogen concentrations), including 14 values above CONAMA legislation. This sampling site is characterized by tributaries that drain from the urban area of the Monte Alto municipality and is located downstream of the effluent discharge of the wastewater treatment plant (WWTP) (site that receives the highest contribution among others). Soil occupation and land use in the area of the Rico Stream consists of small farms with diversified production. According to field observations during the sampling period, horticulture, swine and several agricultural crops (especially sugarcane and peanuts) were present in the area. The presence of mechanized irrigation of agricultural crops during both the dry and rainy periods was also observed during the sampling period. It may be inferred that the presence of https://doi.org/10.26850/1678-4618eqj.v45.2.2020.p23-32 Original article 29 Eclética Química Journal, vol. 45, n. 2, 2020, 23-32 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v45.2.2020.p23-32 estrogenic mycotoxins in the Rico Stream was probably due to effluent discharge from the WWTP of Monte Alto municipality (detection in downstream of the WWTP – P2 site), runoff from livestock facilities or fields receiving livestock manure applications (detection in upstream of the WWTP – P1 site). Rico Stream contamination from fungi in agricultural crops is unlikely since maize (temporary tillage) is insignificant in the region. Table 2. Data on physicochemical and microbiological parameters determined in Rico Stream micro-basin over the period of sampling. Month Sampling sites Fecal coliforms (CFU 100 mL-1) Total organic carbon (mg L-1) Chemical oxygen demand (mg L-1) Biological oxygen demand (mg L-1) Total phosphorus (mg L-1) Nitrate (mg L -1) Nitrite (mg L-1) Total nitrogen (mg L-1) Total ammoniacal nitrogen (mg L-1) Total dissolved solids (mg L-1) Potassium (mg L-1) Turbidity (NTU) October 2014 P1 850 10 24 5 0.098 1.0 <0.011 46 0.30 112 <1.109 11 P2 65 72 180 81 1.820 3.4 0.621 46 42 343 <1.109 16 P3 22 16 40 15 0.230 <1.0 <0.011 0.5 0.51 132 <1.109 7 P4 40 21 52 30 0.176 5.5 0.040 6.0 0.50 159 <1.109 10 November 2014 P1 <1.0 9 23 5 0.123 6.3 <0.011 6.5 0.21 210 6.162 13 P2 500 16 40 <5.0 0.420 7.2 <0.011 10 2.90 184 5.217 12 P3 <1.0 22 55 <5.0 0.120 5.7 <0.011 5.9 0.20 168 6.801 12 P4 70 8 20 <5.0 0.181 6.2 <0.011 6.4 0.25 198 5.209 13 December 2014 P1 2300 5 12 <5.0 0.055 0.33 0.024 0.5 0.16 48 2.482 14 P2 2200 5 12 <5.0 0.707 0.78 1.301 2.4 0.26 83 4.091 16 P3 3300 6 17 <5.0 0.059 0.75 0.032 0.9 0.13 48 2.719 15 P4 3900 2 4 <5.0 0.072 2.6 0.026 2.7 0.11 47 3.073 15 January 2015 P1 600 14 37 15 <0.050 <1.0 0.020 0.4 0.12 94 1.593 4 P2 20000 72 187 94 1.157 1.1 0.355 6.4 <0.05 111 3.892 7 P3 5000 16 41 21 <0.050 <1.0 0.036 0.3 <0.05 138 1.946 46 P4 6000 11 29 13 <0.050 1.6 0.032 2.3 0.25 132 2.034 58 February 2015 P1 95 <0.04 18 <5.0 <0.050 <1.0 0.013 0.2 <0.05 53 7.53 12 P2 48 <0.04 18 <5.0 <0.050 <1.0 0.118 0.5 0.06 92 11.07 19 P3 165 <0.04 22 <5.0 <0.050 <1.0 0.029 0.2 <0.05 54 7.699 14 P4 1100 <0.04 11 <5.0 <0.050 3.8 0.071 4.0 0.14 54 9.994 15 March 2015 P1 61 8 19 <5.0 <0.050 <1.0 0.068 0.4 0.15 44 3.237 10 P2 620 22 54 <5.0 0.196 <1.0 0.143 0.6 0.24 62 4.396 11 P3 380 5 12 <5.0 <0.050 <1.0 0.069 0.6 0.36 40 3.886 56 P4 3100 5 12 <5.0 0.037 1.3 0.100 2.0 0.34 40 3.611 37 April 2015 P1 140 <0.04 <4 <5.0 <0.050 1.0 <0.011 1.9 0.54 50 2.165 10 P2 510 22 54 <5.0 0.237 12 0.108 14 1.82 90 4.569 10 P3 1100 19 46 <5.0 <0.050 2.3 0.036 2.8 0.29 50 2.286 92 P4 900 35 84 <5.0 <0.050 4.0 0.016 4.5 0.15 46 2.796 74 May 2015 P1 410 69 186 <5.0 <0.050 0.31 0.015 0.7 0.33 90 2.420 9 P2 2000 136 367 <5.0 <0.050 9.0 0.019 10 0.44 152 3.230 11 P3 4300 2 4 <5.0 <0.050 0.70 0.014 1.3 0.40 84 2.119 21 P4 1200 40 108 <5.0 <0.050 2.3 0.011 2.5 0.09 84 2.276 19 June 2015 P1 1600 16 40 <5.0 <0.050 1.0 0.015 1.2 0.16 47 1.79 8 P2 420 35 88 <5.0 <0.050 1.5 0.232 2.4 0.64 96 3.991 14 P3 5000 22 56 <5.0 <0.050 0.8 0.020 0.9 0.07 42 1.892 14 P4 960 21 52 <5.0 <0.050 3.4 0.047 3.4 <0.05 42 2.232 13 July 2015 P1 200 42 86 <5.0