Two fish bile reference materials certified for PAH metabolites

Environmental pollution with polycyclic aromatic hydrocarbons (PAHs) from pyrogenic or petrochemical sources is a matter of concern in many aquatic ecosystems. For field monitoring purposes, PAH metabolite levels in fish bile samples can be used as biomarkers of exposure, but previously there were no reference materials for analytical method validation or quality control. In this paper we describe the preparation and certification of two fish bile materials. BCR 720 is a pure bile material from sediment-exposed flounder (Platichthys flesus) and BCR 721 is a pure bile material from oil-exposed plaice (Pleuronectes platessa). Homogeneity and stability tests were carried out and a group of 12 expert laboratories was assembled for the certification measurements. The methods applied were liquid chromatography with fluorescence detection or gas chromatography with mass spectrometric detection. In BCR 720 three PAH metabolites were certified: 1-hydroxyphenanthrene, 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene. In BCR 721 1-hydroxypyrene was certified. The fourth target compound, 2-hydroxynaphthalene, could not be certified, but the results obtained are listed. The major findings from earlier feasibility studies and recommendations for the optimal use of the reference materials are also included.     

A new mussel certified reference material (CRM 477) for the quality control of butyltin determination in the marine environment

The analytical techniques used for the determination of butyltin compounds in biological tissues are generally based on a succession of steps (e.g., extraction, derivatization, separation, detection) which are all prone to systematic errors. An interlaboratory programme performed at the EU level and based on a stepwise approach has enabled one to identify and remove most of these errors and to improve considerably the state of the art so that certification of a mussel reference material has been made possible. This paper describes the preparation of this mussel reference material containing monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT), the homogeneity and stability studies and the analytical work performed for the certification of the contents of MBT [Sn(C4H9)3+], DBT [Sn(C4H9)2(2+)] and TBT [Sn(C4H9)3+]. The results obtained by a group of expert EU laboratories are discussed along with the methods used to certify the mass fractions (based on dry mass) of monobutyltin (1.50 +/- 0.28 mg kg-1 as MBT cations), dibutyltin (1.54 +/- 0.12 mg kg-1 as DBT cations) and tributyltin (2.20 +/- 0.19 mg kg-1 as TBT cations). The paper also describes an attempt to certify the content of triphenyltin in this material which, although reasonable agreement was found among the participants' results, failed owing to the instability of this compound. This new material will be of great support to ensure the quality control of butyltin determination in shellfish, as carried out in environmental monitoring programmes.     

SI-traceable certification of Cu, Cr, Cd and Pb in sediment and fly ash candidate reference materials

Many fields in environmental analytical chemistry deal with very low limits and thresholds as set by governmental legislations or transnational regulations. The need for the accuracy, comparability and traceability of analytical measurements in environmental analytical chemistry has significantly increased and total uncertainties are even asked for by accreditation bodies of environmental laboratories. This paper addresses achieving these goals to guarantee accuracy, quality control, quality assurance or validation of a method by means of certified reference materials. The assessment of analytical results in certified reference materials must be as accurate as possible and every single step has to be fully evaluated. This paper presents the SI-traceable certification of Cu, Cr, Cd and Pb contents in geological and environmentally relevant matrices (three sediments and one fly ash sample). Certification was achieved using isotope dilution (ID) ICPMS as a primary method of measurement. In order to reduce significantly the number of analytical steps and intermediate samples a multiple spiking approach was developed. The full methodology is documented and total uncertainty budgets are calculated for all certified values. A non-element specific sample digestion process was optimised. All wet chemical digestion methods examined resulted in a more or less pronounced amount of precipitate. It is demonstrated that these precipitates originate mainly from secondary formation of fluorides (essentially CaF2) and that their formation takes place after isotopic equilibration. The contribution to the total uncertainty of the final values resulting from the formation of such precipitates was in general < 0.1% for all investigated elements. Other sources of uncertainty scrutinised included the moisture content determination, procedural blank determination, cross-contamination from the different spike materials, correction for spectral interferences, instrumental background and deadtime effects, as well as the use of either certified values or IUPAC data in the IDMS equation. The average elemental content in the sediment samples was 30-130 micrograms g-1 for Pb, 0.5-3 micrograms g-1 for Cd and 50-70 micrograms g-1 for Cu. Cr was measured in one sample and was about 60 micrograms g-1. The concentrations in the fly ash sample were up to 2 orders of magnitude higher. Expanded uncertainty for the investigated elements was about 3% (coverae factor k = 2) except for Cr, (measured by high resolution ICPMS), for which the expanded uncertainty was about 7% (k = 2).     

The application of HPLC-F and GC-MS to the analysis of selected hydroxy polycyclic hydrocarbons in two certified fish bile reference materials

Four selected hydroxy polycyclic aromatic hydrocarbons (OH-PAHs), 2-hydroxy-naphthalene (2-OH-NPH), 1-hydroxy-phenanthrene (1-OH-PHE), 1-hydroxy-pyrene (1-OH-PYR) and 3-hydroxy-benzo[a]pyrene (3-OH-BaP) have been analysed in two certified fish bile reference materials (CRMs) for exposure monitoring of PAHs in the aquatic environment. The two materials, BCR 720 and BCR 721, consist of bile from fish exposed to contaminated sediment and dispersed crude oil, respectively. Both bile samples have been analysed by two different analytical techniques, gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-fluorescence detection (HPLC-F), and the separation performance, detection limits, recoveries and reproducibility for the four target compounds were evaluated. HPLC-F requires a simple sample preparation and the separation capacity is adequate for quantification of 1-OH-PYR and 3-OH-BaP. Detection limits are excellent for 1-OH-PYR (6 pg injected) and 3-OH-BaP (3 pg injected) and generally improved with increasing molecular size. Recoveries ranged from 48 to 99% for the four selected compounds, depending on compound and concentration. Sample preparation prior to GC-MS analysis was more demanding, as reflected by the obtained recoveries for 2-OH-NPH, 1-OH-PHE and 1-OH-PYR (35 to 61%). The sensitivity improved with decreasing molecular size, 2-OH-NPH (1.2 pg injected), 1-OH-PHE (2.4 pg injected) and 1-OH-PYR (6 pg injected). Because of the superior separation power of GC and the extra selectivity of MS detection, GC-MS was the method of choice for the determination of 2-OH-NPH and 1-OH-PHE in both CRMs. In fish bile samples these two compounds are more likely to suffer from chromatographic overlap, and HPLC-F was not sufficiently selective. Determination of 1-OH-PYR was performed with success by both methods, but HPLC-F would be preferred because of the simpler and less time-consuming sample preparation. Detectable concentrations of 3-OH-BaP were present in BCR 720 and could only be determined by HPLC-F. The present work aims to present HPLC-F and GC-MS as complementary methods for the quantitative analysis of OH-PAHs in fish bile.     

Homogeneity and stability study of the candidate reference material Adamussium colbecki for trace elements

The preparation of a new candidate certified reference material (CRM) for trace elements based on the antarctic bivalve Adamussium colbecki(IRMM 813) was carried out by the Istituto Superiore di Sanità(ISS, Rome, Italy) in cooperation with the Institute for Reference Materials and Measurements, Joint Research Centre of the European Commission (EC-JRC-IRMM), in the frame of the Italian National Programme of Research in Antarctica (Programma Nazionale di Ricerca in Antartide, PNRA). Samples were collected at Terra Nova Bay (Ross Sea) during the 2000-2001 expedition in Antarctica. The preparation of a material suitable for certification was performed by the EC-JRC-IRMM. Measurements for homogeneity and short-term stability tests were carried out by ISS. The elements selected for the certification project were As, Cd, Cu, Cr, Fe, Mn, Ni and Zn. For the above-mentioned tests, the determination of the elements of interest was performed on samples mineralised by microwave-assisted acid digestion. The analytical techniques employed to this end were inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma dynamic reaction cell quadrupole mass spectrometry (ICP-DRC-Q-MS). The candidate material was shown to be fit for purpose with regard to homogeneity and short-term stability, thus allowing the following phases of the certification project to be undertaken, in the first place the selection of expert laboratories for the accomplishment of the certification campaign.     

Feasibility study of preparation and certification of reference materials for nitrogen dioxide and sulfur dioxide in diffusive samplers

This paper presents the results of a feasability study for the preparation and certification of reference materials (RMs) for nitrogen dioxide (NO(2)) and sulfur dioxide (SO(2)) in diffusive samplers. RMs for NO(2) were prepared by exposure to gas mixtures in a chamber while the RMs for SO(2) were prepared by liquid spiking. Certification of RMs for NO(2) was found feasible with a certified uncertainty of 5.8% and a proposed shelf life of 5 years. The uncertainty was calculated with contribution from the homogeneity of preparation, stability and transport of the CRMs and from an external verification of the certified value. To reach 5.8% of uncertainty, the contribution of the differences between the results of analysis by ion chromatography and colorimetry must be eliminated. It is proposed to solve this by pre-extracting the samplers with water before analysis. The results of this study indicate that the samplers are stable for at least two years before and after exposure when stored in a refrigerator. By contrast, the certification of RMs for SO(2) was found to not be feasible due to instability problems. This instability was attributed to reaction of sulfate on the walls of the samplers. Alternatively, the preparation of RMs by simultaneous exposure to SO(2) and NO(2) has been tested. Satisfying homogeneities has been reached both for NO(2) and SO(2).     

Natural background concentrations of nutrients in the German Bight area (North Sea)

Natural background concentrations of nutrients are needed for the assessments of eutrophication processes and their status. Natural background concentrations of total nitrogen (TN) and total phosphorus (TP) were modelled for the rivers discharging into the German Bight and the Rhine considering individual catchment sizes, freshwater flows and soil types. These data were validated by comparison with data from unpolluted rivers. The consistency of modelled and some compiled nutrient concentrations was confirmed by their area-specific load dependency on freshwater discharges. Pristine inorganic nutrient concentrations were deduced from modelled relations to TN and TP in unpolluted rivers. Pristine nutrient gradients between rivers and offshore waters were estimated by linear mixing until a salinity of 32, continued by hyperbolic fits towards recent mean offshore values (salinity 34.5?C35). Based on these gradients and recent mean salinities, maps of pristine surface gradients were plotted for the whole German Bight. Variability was transferred from recent conditions as percentage of standard deviation. Reported historical nutrient data and concentrations from unpolluted rivers, coastal and offshore North Sea waters are discussed concerning their relations to natural background conditions.     

Study of heavy metal pollution in seawater of Kepez harbor of Canakkale (Turkey)

In this work, the heavy metals cadmium and lead in Kepez harbor were studied with regard to the determination of the environmental pollution levels. Seawater samples of the Dardanelles (Canakkale Strait) were collected from the nearest station to Kepez harbor in July 2007. Then, the concentrations of these metals were determined after simple pretreatment of samples by the proposed inductively coupled plasma?Catomic emission spectrometry (ICP-AES) method. The analysis of a given sample is completed in about 15 min by the ICP-AES method, and the concentrations of Cd and Pb were found to be 73.80 mg L^???1 (relative standard deviation [RSD], 1.07%) and 9.39 mg L^???1 (RSD, 0.70%), respectively. The accuracy of the applied ICP technique was checked by recovery studies, and good recoveries were obtained. The pollution levels of Cd and Pb recorded in this study were compared with those in other studies to analyze the water sample in Kepez harbor.     

Environmental monitoring of linear alkylbenzene sulfonates and physicochemical characteristics of seawater in El-Mex Bay (Alexandria, Egypt)

In the present work, the influence of different physicochemical characteristics on the distribution of anionic detergents, linear alkylbenzene sulfonates (LAS), was studied. Surface and bottom water samples were collected from eight different sites from a small bay near the main sewage discharge of Alexandria City (El-Max Bay). The results showed great variations in the concentrations, as a function of the regional and seasonal variations. The study revealed that the pH values lie in the normal side, with a range of 8.0–8.5 inside the bay and 7.5–7.7 at El-Umum Drain effluent. Wide variations, observed between the surface and the bottom water of the bay, salinity, dissolved oxygen, oxidizable organic matter, total hardness, and total alkalinity, were scattered in the ranges (3.33–42.73 practical salinity unit), (0.42–8.27 mg O₂/l), (0.12–10.49 mg/l), (1.39–8.99 mg/l), and (0.23–0.48 mg/l), respectively. The regional variations of LAS concentrations in the bay waters showed that the concentration decreased as the distance from the source of drainage water (El-Umum Drain). The seasonal average variations of LAS cleared out that summer and spring periods had the highest concentrations at surface (0.13?±?0.04 mg LAS/l) and bottom (0.12?±?0.10 mg LAS/l) layer, which is attributed to increase in population density and human activities. The inverse relationships between total LAS concentration and salinity, dissolved oxygen, and calcium ions concentration are r?=??0.78, 0.50, and 0.67, respectively. This is related to the occurrence of the untreated wastewater containing detergents, the biodegradation rate of surfactants, and strong precipitation of LAS as Ca.     

Feasibility study prior to the certification of trace elements in urban and industrial wastewater reference materials

Reliable results for the determination of trace elements in urban and industrial wastewaters are of paramount importance for both checking the performance of sewage treatment and for detecting possible urban or industrial contamination sources. The quality control of measurements should in principle rely on external tools such as certified reference materials (CRM), which should represent, as closely as possible, the matrix of samples currently analysed, e.g., in the frame of environmental monitoring. To date, however, no CRM representatives of wastewater composition are available, which limits the possibility for control laboratories to check their QC externally. To fill this gap, the European Commission's Standards, Measurements and Testing Programme (formerly BCR) has started a European collaborative project of which the aim is to test the feasibility of preparation of wastewater reference materials and the analytical state-of-the-art to enable the certification of trace elements in such matrices. This paper presents the results of the first phase of this project, namely the feasibility study and the results of an interlaboratory trial.     

Formaldehyde determination in seawater. Preliminary application to coastal samples at Terra Nova Bay (Antarctica)

A sensitive spectrofluorimetric-FIA (flow injection analysis) method for formaldehyde (HCHO) determination was improved with the aim of analysing seawater samples.The fluorescence emission versus HCHO concentration shows a linear pattern from sub microg L(-1) to about 1000 microg L(-1). The reproducibility at 15 ppb level is about 2%. Interferences from other aldehydes were checked; only glyoxal shows a significative interference, but only when its concentration is about 6000 times higher than that of formaldehyde. Superficial (microlayer, just sub-pack or sea-ice free sea surface) and deep (along the water column, sub-pack or in sea-ice free areas) seawater samples were collected near the coast at Terra Nova Bay (Ross Sea, Antarctica) during the 1998/1999 and 2001/2002 Italian Antarctic Expedition. We report here the preliminary results of the spectrofluorimetric-FIA determination of the HCHO content. The mean seawater superficial formaldehyde concentration was 15 microg L(-1); the concentration along the water column ranged between 4.5 to over 40 microg L(-1)(20 microg L(-1) mean concentration), usually with a maximum value for the 30 m depth, corresponding to a fluorescence maximum. The sampling was repeated 7 times in the austral summer in order to evaluate seasonal changes in the formaldehyde concentration/seawater depth profiles. The results show changes in the formaldehyde concentration at different depths.     

Intra-laboratory evaluation of Microbial Assay for Risk Assessment (MARA) for potential application in the implementation of the Water Framework Directive (WFD)

The Microbial Assay for Risk Assessment (MARA) is an innovative system based on an array of 11 different microbial species freeze-dried in a 96-well micro-titre plate format. Developed for testing the toxicity of chemicals, mixtures and environmental samples, the assay employs species of a taxonomically diverse range. In addition to ten prokaryotic species, a eukaryote (yeast) is included in the range. The MARA's innate scope of a multi-dimensional test allows determination of toxicity based on a unique assay fingerprint or index, numerically expressed as the mean Microbial Toxic Concentration (MTC). The most significant potential of the test is in the additional inference that can be conveyed to the toxicity evaluation because of the presence of each of the constituent species. In view of the fact that conventional aquatic bioassays, like fish or cladoceran tests, are expensive and impractical, the MARA could provide a cost-effective solution for routine ecotoxicological testing. The performance of the MARA was evaluated to ascertain its capability and potential scope. Sensitivity to toxicants and different environmental samples was assessed. Evaluation included comparison with other tests: namely Microtox, invertebrate (Daphnia magna and Thamnocephalus platyurus) microbiotests, and respiration-inhibition and nitrification-inhibition tests. The most sensitive invertebrate test was found to be the T. platyurus microbiotest for three of the four metals tested. The LC(50) values for this test for Cd(ii), Cr(vi) and As(iii) were 0.2, 0.018 and 0.3 mg l(-1), respectively; and the corresponding most sensitive MARA species MTC values were 4.4, 2.8 and 17 mg l(-1), respectively.     

Optical fiber based methodology for assessment of thiocyanate in seawater

A new methodology for the assessment of thiocyanate (SCN(-)) is proposed based on optical fiber (OF) detection coupled to a liquid chromatography system (LC). The developed methodology showed an adequate performance for the analysis of SCN(-) comparable to a high performance liquid chromatography with UV detector (HPLC-UV) methodology: a detection limit of 3 μg L(-1), a linear range from 4 to 400 μg L(-1), and an analytical time of less than 6 min. The OF based methodology was of compact design and easy operation. This simple system has the potential to be used as a sensing approach for SCN(-) in seawater.     

Disturbance of Dabao highway construction on plant species and soil nutrients in Longitudinal Range Gorge Region (LRGR) of Southwestern China

The disturbance of highway construction upon surrounding vulnerable ecosystems is a common threat in the Longitudinal Range Gorge Region of southwestern China. We evaluated the disturbance of highway on plant species richness and diversity and soil nutrients from adjacent to the highway to 300 m upslope and 100 m downslope in forests and grasslands by setting 12 belt transects in forests and grasslands (six belt transects and six control belt transects, respectively). The results showed that there were some significant variances in belt transects with respective control belt transects for species richness and diversity in both forests and grasslands. Species richness and diversity of trees were lower within a 50-m distance from the highway and more noticeable on the downslope portion. Species richness and diversity of shrubs and herbs appeared higher near highway edge. Both species richness and diversity of herbs were similar in forests. In addition, exotic species, such as Eupatorium adenophorum, were further from the road and more widely dispersed in grasslands. Soil nutrients except total potassium (TK) were lower in the downslope area adjacent to highway edge and showed a significant increase with increasing distance from the highway in both forests and grasslands. This indicates that grasslands acted as microhabitats for exotic species and are more easily to be invaded than forests, especially if disturbed. Once destroyed, plant species and soil nutrients will require a significant amount of time to be restored to control levels. This work illustrates that the effects extend considerably to distances upslope and downslope from the construction site. Given that these changes occurred relatively quickly, the study suggests that the environmental "footprint" grows far beyond the road and adjacent zone of disruption.     

Development of an ombrotrophic peat bog (low ash) reference material for the determination of elemental concentrations

Given the increasing interest in using peat bogs as archives of atmospheric metal deposition, the lack of validated sample preparation methods and suitable certified reference materials has hindered not only the quality assurance of the generated analytical data but also the interpretation and comparison of peat core metal profiles from different laboratories in the international community. Reference materials play an important role in the evaluation of the accuracy of analytical results and are essential parts of good laboratory practice. An ombrotrophic peat bog reference material has been developed by 14 laboratories from nine countries in an inter-laboratory comparison between February and October 2002. The material has been characterised for both acid-extractable and total concentrations of a range of elements, including Al, As, Ca, Cd, Cr, Cu, Fe, Hg, Mg, Mn, Na, Ni, P, Pb, Ti, V and Zn. The steps involved in the production of the reference material (i.e. collection and preparation, homogeneity and stability studies, and certification) are described in detail.     

The use of a natural coagulant (Opuntia ficus-indica) in the removal for organic materials of textile effluents

The goal of this study was to investigate the activity of the coagulant extracted from the cactus Opuntia ficus-indica (OFI) in the process of coagulation/flocculation of textile effluents. Preliminary tests of a kaolinite suspension achieved maximum turbidity removal of 95 % using an NaCl extraction solution. Optimization assays were conducted with actual effluents using the response surface methodology (RSM) based on the Box–Behnken experimental design. The responses of the variables FeCl₃, dosage, cactus dosage, and pH in the removal of COD and turbidity from both effluents were investigated. The optimum conditions determined for jeans washing laundry effluent were the following: FeCl₃ 160 mg L^?1, cactus dosage 2.60 mg L^?1, and pH 5.0. For the fabric dyeing effluent, the optimum conditions were the following: FeCl₃ 640 mg L^?1, cactus dosage 160 mg L^?1, and pH 6.0. Investigation of the effects of the storage time and temperature of the cactus O. ficus-indica showed that coagulation efficiency was not significantly affected for storage at room temperature for up to 4 days.     

Speciation of inorganic arsenic in coastal seawater from Ionian and Tyrrhenian Seas (Sicily, Italy) using derivative anodic stripping chronopotentiometry

The purpose of this paper was to use derivative anodic stripping chronopotentiometry (dASCP) as a sensitive and accurate technique, to determine the concentrations of dissolved As (III) and As (V) in coastal seawater samples from the Straits of Messina, the Ionian and the Tyrrhenian seas, and to investigate the relationship between the anthropogenic activities on the coastal areas and the concentration of dissolved inorganic arsenic in seawaters. The obtained data indicated that As (V) was the most abundant species, with concentration ranging from 26.7 to 307 nM, whereas As (III) levels were lower than 48 nM in all the samples. In particular, As (III) and As (V) levels significantly decreased from high to low anthropogenic activities zones (p < 0.00001, ANOVA), with the reference samples, from a wildlife reserve, showing the lowest values. Furthermore it was observed that human activities influenced inorganic arsenic speciation, since the zones that received high human input presented the highest As(V)/As (III) ratio.     

Retention capacities of several bryophytes for Hg(II) with special reference to the elevation and morphology of moss growth

Hg(II) Retention capacities of nine bryophyte species, collected from Jinfo Mountains (JFM) in Chongqing, China, had been investigated with special reference to the effect of morphology and elevation of moss growth. Results indicated that adsorption capacities of bryophytes for Hg(II) became stronger with the increase of multi-branches and leafy-shoots, as well as the elevation of moss growth, which was observed both in adsorption isotherm and adsorption kinetics experiments. Contrarily, the desorption kinetics showed a decrease tendency with the increase of multi-branches and leafy-shoots and the elevation of moss growth. The results demonstrated that bryophytes with higher multi-branches and leafy-shoots and higher growth elevation had a stronger adsorption capacity and a weaker desorption tendency, and therefore had a stronger retention capacity to Hg(II). The results disclosed the different relative sensitivity and retention capacity of mosses to pollution resulting from heavy metals, due to the differences in growth elevation and morphology. These should be considered when bryophytes were chosen as a tool for biomonitoring materials to environmental pollution, especially caused by Hg(II).     

Landscape as a predictor of wetland condition: an evaluation of the Landscape Development Index (LDI) with a large reference wetland dataset from Ohio

RecentAbstract. Recent approaches to wetland assessment have advocated a multilevel approach which incorporates assessments based on landscape (remote sensing) data, on-site but “rapid” methods, and intensive methods where quantitative data is collected. Brown and Vivas (2004) recently pro- posed an assessment method that uses remote sensing information (Landscape Development Index or LDI) and propose that it may also be usable as a quantified human disturbance gradient. The LDI was evaluated using a large reference wetland data set from Ohio using land use percentages within a 1 km radius circle of the wetlands. The LDI had interpretable and significant relationships with another human disturbance gradient (the Ohio Rapid Assessment Method for Wetlands or ORAM) and with most metrics and scores from the Vegetation Index of Biotic Integrity (VIBI) developed for use in the State of Ohio. Metrics from emergent wetlands had the most significant correlations with the LDI (10 of 10 metrics), followed by forested wetlands (8 of 10 metrics) and shrub wetlands (4 of 10). Poor correlation for VIBI scores and metrics of shrub wetlands was due to differences in attainable LDI scores based on ecoregion and natural buffers shielding the wetland from otherwise intensive land uses. The ORAM and VIBI were developed for use in wetlands in Ohio completely independent of the LDI. It is an important test of the LDI concept that so many interpretable and significant relationships occurred between the VIBI scores, VIBI metric values, and the ORAM scores. For the purposes of VIBI development, the LDI is an independent, quantified disturbance gradient that has provided an additional test of the VIBI. Given its theoretical underpinnings and the fact that it uses quantified land use percentages, the LDI has many advantages over more qualita- tive human disturbance gradients. Using land use percentages from increasingly smaller distances from the wetland edge (100-200 m) may improve the resolution of the LDI to detect on-site dis-turbances to a wetland which degrade its ecological condition. The LDI should be evaluated with other large reference data sets in other regions to evaluate its validity and usefulness as an assessment tool.