Total Reflection X-ray Fluorescence Spectrometry Research Articles

Evaluation of Dicentrarchus labrax Meats and the Vegetable Quality of Beta vulgaris var. cicla Farmed in Freshwater and Saltwater Aquaponic Systems

The purpose of this study is to exploit the euryhaline nature of commercially attractive species for their cultivation in freshwater aquaponic systems. This approach may increase the profitability of aquaponic production in coastal countries where the consumption of marine fish is traditional and of commercial relevance. For this purpose, juvenile European sea bass (Dicentrarchus labrax) were reared in an aquaponic freshwater (AFW) system and an aquaponic saltwater (ASW) system (salinity 20 ppt), in combination with chard (Beta vulgaris var. cicla) seedlings, a salt tolerant plant. At the end of the trial, nitrate and phosphate concentration in water significantly increased in the ASW system, suggesting that the ability of B. vulgaris to absorb these substances was limited by salinity. Total reflection X-ray fluorescence spectrometry revealed that the concentration of some oligoelements such as Fe remained lower with respect to the concentration in the freshwater hydroponic solution, in both AFW and ASW. FTIR-Fourier transform infrared spectroscopy on plants showed that growth at high salinity affected their lipid content. In the case of fish, freshwater had no effects on mono- and poly-unsaturated fatty acid profiles, although saturated fatty acids were significantly decreased in D. labrax reared in AFW. Our results demonstrates that it is possible to increase aquaponic profitability by farming D. labrax juveniles in an aquaponic freshwater system together with Beta vulgaris, obtaining good quality products.

Quorum Quenching and Microbial Control through Phenolic Extract of Eugenia Uniflora Fruits.

We describe the characterization of the centesimal composition, mineral and phenolic content of Eugenia uniflora fruit and the determination of the antioxidant, antimicrobial and quorum quenching activities of the pulp phenolic extract. Centesimal composition was determined according to standard methods; trace elements were measured by total reflection X-ray fluorescence spectroscopy. The phenolic compounds were extracted by solid-phase chromatography and quantified by spectrophotometry. Antioxidant activity was determined by using 3 different methods. Antimicrobial activity was evaluated against a panel of foodborne microorganisms and antiquorum sensing activity in Chromobacterium violaceum was performed by measuring inhibition of quorum sensing dependent violacein production. The centesimal composition (per 100 g of pulp) was as follows: protein 3.68 ± 0.21 g, lipids 0.02 ± 0.03 g, carbohydrates 10.31 g and fiber 2.06 g. Trace elements (mg/g of pulp) were determined as: K 0.90, Ca 3.36, Fe 0.60, Zn 0.17, Cl 0.56, Cr 0.06, Ni 0.04, and Cu 0.07. The pulp is a source of phenolic compounds and presents antioxidant activity similar to other berries. The fruit phenolic extract inhibited all tested bacteria. We also found that the fruit phenolic extract at low subinhibitory concentrations inhibited up to 96% of violacein production in C. violaceum, likely due to the fruit's phenolic content. This study shows the contribution of E. uniflora phenolic compounds to the antioxidant, antimicrobial and the newly discovered quorum quenching activity, all of which could be used by the food and pharmaceutical industries to develop new functional products.

Multi-element analysis of vegetal foodstuff by means of low power total reflection X-ray fluorescence (TXRF) spectrometry

In this work, an analytical methodology in combination with a low power benchtop total reflection X-ray fluorescence (TXRF) system has been developed for multi-elemental analysis of vegetal materials. An easy and rapid sample preparation consisting in suspending 20mg of sample in de-ionized water showed to be the most suitable for this kind of samples. However, for comparison purposes, two digestion procedures were also applied.A set of fifteen certified reference materials and three real vegetal samples were employed for the quantitative determination of K, Ca, Mn, Fe, Cu, Zn, Br, Rb, Sr, Pb, As and Sn. All the parameters affecting sample preparation and TXRF measurements conditions were carefully evaluated. Accurate and precise results were obtained for mid-high Z elements (Mn-Sr) using internal standardization as quantification approach, while for light Z elements (K and Ca), due to absorption effects, an improvement of analytical results by external calibration was necessary.

Bromine and bromide content in soils: Analytical approach from total reflection X-ray fluorescence spectrometry

Monitoring total bromine and bromide concentrations in soils is significant in many environmental studies. Thus fast analytical methodologies that entail simple sample preparation and low-cost analyses are desired.In the present work, the possibilities and drawbacks of low-power total reflection X-ray fluorescence spectrometry (TXRF) for the determination of total bromine and bromide contents in soils were evaluated.The direct analysis of a solid suspension using 20 mg of fine ground soil (<63 μm) gave a 3.7 mg kg−1 limit of detection for bromine which, in most cases, was suitable for monitoring total bromine content in soils (Br content range in soils = 5–40 mg kg−1). Information about bromide determination in soils is also possible by analyzing the Br content in water soil extracts. In this case, the TXRF analysis can be directly performed by depositing 10 μL of the internal standardized soil extract sample on a quartz glass reflector in a measuring time of 1500 s. The bromide limit of detection by this approach was 10 μg L−1.Good agreement was obtained between the TXRF results for the total bromine and bromide determinations in soils and those obtained by other popular analytical techniques, e.g. energy dispersive X-ray fluorescence spectrometry (total bromine) and ionic chromatography (bromide).As a study case, the TXRF method was applied to study bromine accumulation in two agricultural soils fumigated with a methyl bromide pesticide and irrigated with regenerated waste water.

Metal impurity-assisted formation of nanocone arrays on Si by low energy ion-beam irradiation

Fabrication of nanocone arrays on Si surfaces was demonstrated using grazing incidence irradiation with 1keV Ar+ ions concurrently sputtering the surface and depositing metal impurity atoms on it. Among three materials compared as co-sputtering targets Si, Cu and stainless steel, only steel was found to assist the growth of dense arrays of nanocones at ion fluences between 1018 and 1019ions/cm2. The structural characterization of samples irradiated with these ion fluences using Scanning Electron Microscopy and Atomic Force Microscopy revealed that regions far away from co-sputtering targets are covered with nanoripples, and that nanocones popped-up out of the rippled surfaces when moving closer to co-sputtering targets, with their density gradually increasing and reaching saturation in the regions close to these targets. The characterization of the samples' chemical composition with Total Reflection X-ray Fluorescence Spectrometry and X-ray Photoelectron Spectroscopy revealed that the concentration of metal impurities originating from stainless steel (Fe, Cr and Ni) was relatively high in the regions with high density of nanocones (Fe reaching a few atomic percent) and much lower (factor of 10 or so) in the region of nanoripples. Total Reflection X-ray Fluorescence Spectrometry measurements showed that higher concentrations of these impurities are accumulated under the surface in both regions. X-ray Photoelectron Spectroscopy experiments showed no direct evidence of metal silicide formation occurring on one region only (nanocones or nanoripples) and thus showed that this process could not be the driver of nanocone array formation. Also, these measurements indicated enhancement in oxide formation on regions covered by nanocones. Overall, the results of this study suggest that the difference in concentration of metal impurities in the thin near-surface layer forming under ion irradiation might be responsible for the differences in surface structures.

Total reflection x-ray fluorescence spectroscopy as a tool for evaluation of iron concentration in ferrofluids and yeast samples

In this study, total reflection x-ray fluorescent (TXRF) spectrometry was applied for the evaluation of iron concentration in ferrofluids and biological samples containing iron oxide magnetic nanoparticles obtained by the laser target evaporation technique. Suspensions of maghemite nanoparticles of different concentrations were used to estimate the limitation of the method for the evaluation of nanoparticle concentration in the range of 1–5000ppm in absence of organic matrix. Samples of single-cell yeasts grown in the nutrient media containing maghemite nanoparticles were used to study the nanoparticle absorption mechanism. The obtained results were analyzed in terms of applicability of TXRF for quantitative analysis in a wide range of iron oxide nanoparticle concentrations for biological samples and ferrofluids with a simple established protocol of specimen preparation.

Quantification of purple non-sulphur phototrophic bacteria and their photosynthetic structures by means of total reflection X-ray fluorescence spectrometry (TXRF)

TXRF spectrometry proves useful in analysing bacterial cells and their substructures as exemplified byRhodobacter sphaeroides. Trace element profiling complemented with correlation analysis under varying growth conditions.

Determination of trace elements in freshwater rotifers and ciliates by total reflection X-ray fluorescence spectrometry

Element determination in plankton is important for the assessment of metal contamination of aquatic environments. Until recently, it has been difficult to determine elemental content in rotifers or ciliates derived from natural plankton samples because of the difficulty in handling and separation of these fragile organisms. The aim of this study was to evaluate methods for separation of rotifers and large ciliates from natural plankton samples (μg range dry weight) and subsequent analysis of their elemental content using total-reflection X-ray fluorescence spectrometry (TXRF). Plankton samples were collected from different aquatic environments (three lakes, one river) in Chile, Argentina and Hungary. From one to eighty specimens of five rotifer species (Brachionus calyciflorus, Brachionus falcatus, Asplanchna sieboldii, Asplanchna sp., Philodina sp.) and four to twelve specimens of one large ciliate (Stentor amethystinus) were prepared according to the dry method originally developed for microcrustaceans, and analysed by TRXF following in situ microdigestion. Our results demonstrated that it possible to process these small and fragile organisms (individual dry mass: 0.17–9.39μg ind-1) via careful washing and preparation procedures. We found species-dependent differences of the element mass fractions for some of the elements studied (Cr, Mn, Fe, Ni, Cu, Zn, As, Pb), especially for Cu, Fe and Mn. One large rotifer species (A. sieboldii) also showed a negative correlation between individual dry weight and the element content for Pb, Ni and Cr. We conclude that our application of the in situ microdigestion-TRXF method is suitable even for rotifers and ciliates, greatly expanding the possibilities for use of plankton in biomonitoring of metal contamination in aquatic environments.

Cr speciation in water samples by dispersive liquid–liquid microextraction combined with total reflection X-ray fluorescence spectrometry

A novel method based on dispersive liquid–liquid microextraction (DLLME) and total-reflection X-ray fluorescence spectrometry (TXRF) is proposed for the determination and preconcentration of inorganic Cr species (Cr(VI) and Cr(III)) in water samples.Parameters affecting the extraction procedure for Cr speciation (pH, sample volume, disperser solvent, complexing agent, organic solvent, temperature) and TXRF conditions (sample volume, drying mode, measurement time, internal standardization) have been carefully evaluated to ensure the highest sensitivity for Cr determination. It was found that the minimum Cr content that can be detected was 0.8μgL−1. This value is significantly lower than the maximum Cr content permissible in drinking waters and it is better or comparable with those obtained in other published works based on the use of solid phase extraction or liquid phase microextraction in combination with atomic absorption spectrometry techniques. A good linearity (R2=0.9937) was obtained in the range of 5 to 4000μgL−1 demonstrating the suitability of the DLLME+TXRF for both the analysis of drinking and waste water samples.Additional advantages of the DLLME-TXRF systems include the use of very small amount of reagents and the simplicity and low cost of operation of the benchtop instrument used (no cooling media and gas consumption are needed).

Study of metal release from stainless steels in simulated food contact by means of total reflection X-ray fluorescence

This study shows a reliable procedure to prescribe the preferential use of a material for food contact. Release tests with optimized parameters were performed on six different stainless steel accepted for the use in food contact: AISI 420, AISI 430, AISI 202, AISI 303, AISI 304, and AISI 316. Total reflection X-ray fluorescence spectroscopy was used to measure the concentration of Cr, Mn and Ni in contact solutions from release tests. Results show that AISI 202 and 430 release the lowest amount of Mn, Cr and Ni. While, AISI 420 is the worst material, exceeding the limit set in the Italian regulation for all the three metals of interest. One sample was selected to test the reproducibility of TXRF measurements performed in three different laboratories around the world. Results show that quantitative analyses by means of TXRF satisfy the requirements of this field of application.

Quantification of silane molecules on oxidized silicon: are there options for a traceable and absolute determination?

Organosilanes are used routinely to functionalize various support materials for further modifications. Nevertheless, reliable quantitative information about surface functional group densities after layer formation is rarely available. Here, we present the analysis of thin organic nanolayers made from nitrogen containing silane molecules on naturally oxidized silicon wafers with reference-free total reflection X-ray fluorescence (TXRF) and X-ray photoelectron spectroscopy (XPS). An areic density of 2-4 silane molecules per nm(2) was calculated from the layer's nitrogen mass deposition per area unit obtained by reference-free TXRF. Complementary energy and angle-resolved XPS (ER/AR-XPS) in the Si 2p core-level region was used to analyze the outermost surface region of the organic (silane layer)-inorganic (silicon wafer) interface. Different coexisting silicon species as silicon, native silicon oxide, and silane were identified and quantified. As a result of the presented proof-of-concept, absolute and traceable values for the areic density of silanes containing nitrogen as intrinsic marker are obtained by calibration of the XPS methods with reference-free TXRF. Furthermore, ER/AR-XPS is shown to facilitate the determination of areic densities in (mono)layers made from silanes having no heteroatomic marker other than silicon. After calibration with reference-free TXRF, these areic densities of silane molecules can be determined when using the XPS component intensity of the silane's silicon atom.

Accelerated solvent extraction (ASE) for purification and extraction of silicone passive samplers used for the monitoring of organic pollutants.

Silicone passive samplers have gained an increasing attention as single-phased, practical and robust samplers for monitoring of organic contaminants in the aquatic environment in recent years. However, analytical challenges arise in routine application during the extraction of analytes as silicone oligomers are co-extracted and interfere severely during chemical analyses (e.g. gas chromatographic techniques). In this study, we present a fast, practical pre-cleaning method for silicone passive samplers applying accelerated solvent extraction (ASE) for the removal of silicone oligomers prior to the water deployment (hexane/dichloromethane, 100 °C, 70 min). ASE was also shown to be a very fast (10 min) and efficient extraction method for non-polar contaminants (non-exposed PRC recoveries 66–101 %) sampled by the silicone membrane. For both applications, temperature, extraction time and the solvent used for ASE have been optimized. Purification of the ASE extract was carried out by silica gel and high-pressure liquid size exclusion chromatography (HPLC-SEC). The silicone oligomer content was checked by total reflection X-ray fluorescence spectroscopy (TXRF) in order to confirm the absence of the silicone oligomers prior to analysis of passive sampler extracts. The established method was applied on real silicone samplers from the North- and Baltic Sea and showed no matrix effects during analysis of organic pollutants. Internal laboratory standard recoveries were in the same range for laboratory, transport and exposed samplers (85–126 %).

Microchemical characterization of biogeochemical samples collected from the Buda Thermal Karst System, Hungary

The Buda Thermal Karst System (BTKS) is an active hypogenic karst area, where different processes such as microbially mediated sulfuric acid speleogenesis and mixing corrosion are involved. Chemical analysis of biogeochemical samples formed on the water-covered part of wall surfaces in three caves belonging to two different discharge areas of BTKS was carried out using microchemical methods. Total carbon (TC), total organic carbon (TOC), total inorganic carbon (TIC) and total nitrogen (TN) content were measured using a TC-TN analyzer. TC content was found to be rather high for all the samples (4.6–14.9m/m%); however, TOC concentration was much lower (0.45–3.3m/m%) despite the proven biological activity of the samples. These findings suggest that the biogeochemical samples contain high amounts of carbonates. The TN content changed between 0.05 and 0.2m/m%. For quantification of further elements, total reflection X-ray fluorescence (TXRF) spectrometry was applied. The dried samples were mineralized by applying microwave-assisted acidic digestion (3:2 mixture of cc. HNO3 and 48% HF). In the case of several elements, significant concentration differences were found in the two discharge areas (e.g. Ca, Fe, S, Cr, Mn). Elemental concentrations of water samples, collected simultaneously with the solid samples, were measured using inductively coupled plasma mass spectrometry (ICP-MS) and the enrichment factors (EFs, EF=concentration in biogeochemical sample/concentration in water) were calculated. According to these investigations, unusually high EF values (107, 108) were found for certain elements (e.g. Fe, Sn), while for the acidic corrosion responsible S had relatively low EF values (101–102) in both discharge areas.

Measurement uncertainty in Total Reflection X-ray Fluorescence

Total Reflection X-ray Fluorescence (TXRF) spectrometry is a multi-elemental technique using micro-volumes of sample. This work assessed the components contributing to the combined uncertainty budget associated with TXRF measurements using Cu and Fe concentrations in different spiked and natural water samples as an example. The results showed that an uncertainty estimation based solely on the count statistics of the analyte is not a realistic estimation of the overall uncertainty, since the depositional repeatability and the relative sensitivity between the analyte and the internal standard are important contributions to the uncertainty budget. The uncertainty on the instrumental repeatability and sensitivity factor could be estimated and as such, potentially relatively straightforward implemented in the TXRF instrument software. However, the depositional repeatability varied significantly from sample to sample and between elemental ratios and the controlling factors are not well understood. By a lack of theoretical prediction of the depositional repeatability, the uncertainty budget can be based on repeat measurements using different reflectors. A simple approach to estimate the uncertainty was presented. The measurement procedure implemented and the uncertainty estimation processes developed were validated from the agreement with results obtained by inductively coupled plasma — optical emission spectrometry (ICP-OES) and/or reference/calculated values.

The quantification of total lead in lipstick specimens by total reflection X-ray fluorescence spectrometry

Lipstick is known to contain lead, and this has been a general area of concern. Methods of quantifying lead in lipstick currently require the use of rather harsh digestion procedures given that lipstick specimens are high in their lipid content and contain many refractory materials. A simple method of performing lead analysis in lipstick specimens based on total reflection X-ray fluorescence spectrometry (TXRF) is presented here. Samples were prepared by melting lipstick specimens along with a non-ionic surfactant and an yttrium internal standard followed by homogenization. Solid prepared samples were then finely streaked directly onto a quartz reflector, and TXRF measurements made for 900-s live time. The method was found to produce a mean limit of detection for lead of 0.04μg/g. Precisions were found to be on the order of 11–38% relative standard deviation (RSD) and apparent recoveries for lead between 92% and 106% (n = 8). Although the spreading technique may result in thickness variations that may contribute to the higher than expected variances about the determined lead concentrations, the method presented in this work does show promise as a means of performing routine lead analysis in lipstick specimens without the need for harsh digestion procedures. Copyright © 2015 John Wiley & Sons, Ltd.

Elemental bioimaging of Cisplatin in Caenorhabditis elegans by LA-ICP-MS.

cis-Diamminedichloroplatinum(II) (Cisplatin) is one of the most important and frequently used cytostatic drugs for the treatment of various solid tumors. Herein, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method incorporating a fast and simple sample preparation protocol was developed for the elemental mapping of Cisplatin in the model organism Caenorhabditis elegans (C. elegans). The method allows imaging of the spatially-resolved elemental distribution of platinum in the whole organism with respect to the anatomic structure in L4 stage worms at a lateral resolution of 5 μm. In addition, a dose- and time-dependent Cisplatin uptake was corroborated quantitatively by a total reflection X-ray fluorescence spectroscopy (TXRF) method, and the elemental mapping indicated that Cisplatin is located in the intestine and in the head of the worms. Better understanding of the distribution of Cisplatin in this well-established model organism will be instrumental in deciphering Cisplatin toxicity and pharmacokinetics. Since the cytostatic effect of Cisplatin is based on binding the DNA by forming intra- and interstrand crosslinks, the response of poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutants to Cisplatin was also examined. Loss of pme-1, which is the C. elegans ortholog of human poly(ADP-ribose) polymerase 1 (PARP-1) led to disturbed DNA damage response. With respect to survival and brood size, pme-1 deletion mutants were more sensitive to Cisplatin as compared to wildtype worms, while Cisplatin uptake was indistinguishable.

Determination of trace elements in Italian wines by means of total reflection X-ray fluorescence spectroscopy

This work was performed to highlight the advantages of total reflection X-ray fluorescence spectroscopy (TXRF) for multi-elemental qualitative and quantitative analysis of wine. Indeed the International Organization of Vine and Wine (OIV) selected some potentially toxic elements and proposed limit values for their concentration in wines.Direct TXRF analysis of nine wine samples from Emilia Romagna region of Italy was performed in two different laboratories: Italy and Japan. Wine dehydration was also evaluated as sample conservation mean.Traces of Fe, Cu, Zn and Pb are present in all the analysed samples, with concentrations lower than the limits established by the OIV. The target hazard quotients (THQs) were also calculated for seven elements (Cl, Mn, Fe, Ni, Cu, Zn and Sr) to determine their potential detrimental effects.The results show that TXRF is a fast, simple and accurate analytical technique for trace element analysis of wine. Moreover, dehydration is an effective way to store wine samples for further elemental analysis.

Determination of trace amounts of hexavalent chromium in drinking waters by dispersive microsolid-phase extraction using modified multiwalled carbon nanotubes combined with total reflection X-ray fluorescence spectrometry

A methodology based on the combination of dispersive microsolid-phase extraction (DMSPE) with total reflection X-ray fluorescence (TXRF) spectrometry is proposed for the determination of hexavalent chromium in drinking waters.Multiwalled carbon nanotubes (MWCNTs) modified with the anionic exchanger tricaprylmethylammonium chloride (Aliquat 336) were used as solid sorbents. After the sorption process of Cr(VI) on the modified MWCNTs, the aqueous sample was separated by centrifugation and the loaded MWCNTs were suspended using a small volume of an internal standard solution and analyzed directly by a benchtop TXRF spectrometer, without any elution step.Parameters affecting the extraction process (pH and volume of the aqueous sample, amount of MWCNTs, extraction time) and TXRF analysis (volume of internal standard, volume of deposited suspension on the reflector, drying mode, and instrumental parameters) have been carefully evaluated to test the real capability of the developed methodology for the determination of Cr(VI) at trace levels.Using the best analytical conditions, it was found that the minimum Cr(VI) content that can be detected in an aqueous solution was 3μgL−1. This value is almost 20 times lower than the maximum hexavalent chromium content permissible in drinking waters, according to the World Health Organization (WHO). Recoveries for spiked tap and mineral water samples were, in most cases, in the range of 101–108% which demonstrates the suitability of the TXRF methodology for monitoring Cr(VI) at trace levels in drinking water samples.

Green approach for ultratrace determination of divalent metal ions and arsenic species using total-reflection X-ray fluorescence spectrometry and mercapto-modified graphene oxide nanosheets as a novel adsorbent.

A new method based on dispersive microsolid phase extraction (DMSPE) and total-reflection X-ray fluorescence spectrometry (TXRF) is proposed for multielemental ultratrace determination of heavy metal ions and arsenic species. In the developed methodology, the crucial issue is a novel adsorbent synthesized by grafting 3-mercaptopropyl trimethoxysilane on a graphene oxide (GO) surface. Mercapto-modified graphene oxide (GO-SH) can be applied in quantitative adsorption of cobalt, nickel, copper, cadmium, and lead ions. Moreover, GO-SH demonstrates selectivity toward arsenite in the presence of arsenate. Due to such features of GO-SH nanosheets as wrinkled structure and excellent dispersibility in water, GO-SH seems to be ideal for fast and simple preconcentration and determination of heavy metal ions using methodology based on DMSPE and TXRF measurement. The suspension of GO-SH was injected into an analyzed water sample; after filtration, the GO-SH nanosheets with adsorbed metal ions were redispersed in a small volume of internal standard solution and deposited onto a quartz reflector. The high enrichment factor of 150 allows obtaining detection limits of 0.11, 0.078, 0.079, 0.064, 0.054, and 0.083 ng mL(-1) for Co(II), Ni(II), Cu(II), As(III), Cd(II), and Pb(II), respectively. Such low detection limits can be obtained using a benchtop TXRF system without cooling media and gas consumption. The method is suitable for the analysis of water, including high salinity samples difficult to analyze using other spectroscopy techniques. Moreover, GO-SH can be applied to the arsenic speciation due to its selectivity toward arsenite.

Seasonal variation in trace and minor elements in Brazilian honey by total reflection X-ray fluorescence.

Honey is used as an alternative medicine and is a constituent of a healthy diet worldwide. Its composition is associated with botanical origin and, to some extent, geographical origin because soil and climate characteristics determine the melliferous flora. Also, the elements content in honey samples could give an indication of environmental pollution or geographical origin. The main purpose of this study was to evaluate seasonal patterns of essential elements of Brazilian honey. Honey was collected during spring, summer, autumn, and winter for 2years to quantify K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br, and Sr using total reflection X-ray fluorescence spectroscopy (TXRF). Our results indicate no seasonal differences in concentration of Cr, Ni, Se, and Ti, although there were significant seasonal patterns in the composition of essential elements in honey, with higher concentrations of minor and trace elements, especially K and Ca of samples collected in spring and summer.