Optimization of quadrupole ion storage mass spectrometric conditions for the analysis of selected polybrominated diphenyl ethers. Comparative approach with negative chemical ionization and electron impact mass spectrometry

Gas chromatography coupled to quadrupole ion storage mass spectrometry (QISTMS) operating in the non-resonant mode is presented as an innovative approach for the analysis of selected polybrominated diphenyl ethers (PBDEs). Although reductions in complexity and time needed for optimization are achieved in comparison with the resonant option, precise adjustment of the mass spectrometric conditions is required. Differences in isolation and fragmentation patterns of target species with degree of bromination were observed. The reliability of the method was confirmed by using standard solutions through the evaluation of certain quality parameters such as accuracy (92-108%), injection repeatability and reproducibility (coefficient of variation below 10% and 15%, respectively). Detection limits ranged from 62 to 621 fg, providing sensitivity similar to that of negative chemical ionisation (NCIMS) and greater than that of electron ionization mass spectrometry. The applicability of QISTMS method to real samples and matrix effects were evaluated through the analysis of some PBDE congeners in a sewage sludge sample from a Spanish waste-water treatment plant. Comparable results were obtained using QISTMS and NCIMS. According to these observations, QISTMS performed in the non-resonant mode may constitute a low-cost, rapid and reliable alternative to high-resolution devices for the analysis of selected PBDEs in environmental samples.     

Determination of polybrominated diphenyl ethers in milk samples. Development of green extraction coupled techniques for sample preparation

Ultrasound‐assisted extraction (UAE), cloud point extraction (CPE), and ultrasound back‐extraction (UABE) techniques have been coupled for lixiviation, preconcentration, and cleanup of polybrominated diphenyl ethers (PBDEs) from milk samples for determination by gas chromatography‐electron capture detection (GC‐ECD). Physicochemical parameters that affect the efficiency of the extraction system were investigated using a design of experiments based on multivariate statistical tools, and considering the sample matrix along the development. The coupling of the leaching step, UAE, enhanced ca. 3.5 times the extraction efficiency of the former sample preparation methodology (CPE‐UABE) leading to cleaner sample extracts suitable for GC analysis. Under optimum conditions, the proposed methodology exhibits successful performance in terms of linearity and precision, with recoveries in the range of 68–70% and LODs within the range 0.05–0.5 ng/g dry weight (d.w.). The proposed sample preparation methodology coupled three green analytical techniques. It expands the application frontiers of CPE for the analysis of biological samples by GC. The optimized methodology was used for determination of PBDEs in powder milk samples, from both commercial and human sources.     

Headspace solid-phase microextraction gas chromatography tandem mass spectrometry for the determination of brominated flame retardants in environmental solid samples

A headspace solid-phase microextraction gas chromatography coupled with tandem mass spectrometry (HSSPME-GC-MS-MS) methodology for determination of brominated flame retardants in sediment and soil samples is presented. To the best of our knowledge, this is the first time that SPME has been applied to analyze polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs) in environmental solid samples. Analyses were performed using 0.5-g solid samples moisturized with 2 mL water, employing a polydimethylsiloxane (PDMS) fiber coating, exposed to the headspace at 100 °C for 60 min. Several types of environmental solid samples were included in this study and the extraction efficiency was related to the organic matter content of the sample. Calibration was performed using real samples, and the method showed good linearity over a wide concentration range, precision, and afforded quantitative recoveries. The obtained detection limits were in the sub-ng g⁻¹ for all the target analytes in both samples. The proposed procedure was applied to several marine and river sediments and soils, some of which were found to contain PBDEs at concentrations in the ng g⁻¹ level; BDE-47, BDE-100, and BDE-99 were the major congeners detected. The proposed method constitutes a rapid and low-cost alternative for the analysis of the target brominated flame retardants in environmental solid samples, since the clean-up steps, fractionation, and preconcentration of extracts inherent to the classical multi-step solvent extraction procedures are avoided.      

A Novel Sample Pretreatment Method for the Analysis of Polybrominated Diphenyl Ethers in Polymers of Waste Electrical and Electronic Equipment(WEEE)

A fast sample pretreatment method was developed to determine polybrominated diphenyl ethers (PBDEs) in polymers from waste electrical and electronic equipment (WEEE). Polymers were firstly prepared into "polymer film" to facilitate a complete Soxtec extraction. Then, three extraction parameters (including hot plate temperature, boiling time, and rinsing time) were optimized based on the recovery of target in reference polymer samples. The proposed method was successfully applied to the analysis of PBDEs in standard plastic materials from WEEE and the extraction yields of PBDEs ranged from 78.9% to 91.3%. The detection limits of PBDEs were 0.05–0.10 µg/g, indicating this method can be used as a quality assurance and quality control procedure of PBDEs in the production of electrical and electronic equipment.     

Analysis of polybrominated diphenyl ethers in sewage sludge by matrix solid‐phase dispersion and isotope dilution GC–MS

A method for the determination of 14 polybrominated diphenyl ethers (PBDEs) in sludge from wastewater treatment plants is presented. PBDEs were extracted by matrix solid‐phase dispersion assisted by sonication and determined by isotope dilution gas chromatography with electron impact mass spectrometric detection in the selected ion monitoring mode, using labelled ¹³C‐PBDEs as internal standards. The limits of detection and quantification for the tri‐ to hepta‐BDEs were in the range of 0.05 to 0.5 ng/g dry weight and 0.15 to 1.8 ng/g dry weight, respectively, and 1.6 ng/g dry weight and 5.6 ng/g dry weight for deca‐BDE‐209. The proposed analytical method was applied to determine PBDE levels in sewage sludge samples collected from 19 water treatment plants located in the province of Madrid (Spain). In all of the examined samples, BDE‐100 and BDE‐154 were the main compounds found with a mean concentration of 3.9 and 2.0 ng/g, respectively. PBDEs were detected in all of the samples, and their total concentrations not considering BDE‐209 were between 3.9 and 23.0 ng/g dry weight. The dominant PBDE congener in sewage sludge was BDE‐209, which constituted 38.7 to 97.3% of the total, and showed concentration levels ranging from 8.1 to 717.2 ng/g dry weight.     

Determination of polybrominated diphenyl ethers in domestic dust by microwave-assisted solvent extraction and gas chromatography-tandem mass spectrometry

In this paper, a rapid and simple method for the analysis of polybrominated diphenyl ethers (PBDEs) in house dust samples based on microwave-assisted solvent extraction (MASE) and gas chromatography-tandem mass spectrometry (GC-MS/MS) is presented. Extraction conditions were optimized using a multifactorial experimental design approach. The use of an aqueous NaOH phase in combination with a non-polar organic phase (hexane) to extract the target analytes from dust allowed an efficient extraction and reduced chromatographic background. The final hexane extracts could be analyzed after a simple one-step cleanup procedure using Florisil. The validation of the method was performed in terms of accuracy, linearity, and repeatability. The limits of detection (LODs) ranged from 0.29 to 0.55 ng/g for all compounds. The target PBDEs were found in several real dust samples collected in urban and rural houses of Northwestern Spain.     

Analysis of PBDEs in soil,dust, spiked lake water,and human serum samples by hollow fiber-liquid phase microextraction combined with GC-ICP-MS

A novel method for the analysis of four polybrominated diphenyl ethers (PBDEs) in environmental and human serum samples based on hollow fiber-liquid phase microextraction (HF-LPME) followed by gas chromatography-inductively coupled plasma mass spectrometric (GC-ICP-MS) detection has been developed. The organic solvent in the porous hollow fiber was first dipped into the sample for extraction at a given time, and the retracted organic phase was introduced into the GC-ICP-MS for analysis. The addition of methanol has a strong effect on the HF-LPME extraction efficiency. Other significant parameters affecting the extraction efficiency of HF-LPME were also studied. HF-LPME was effective to isolate the analytes from the complex matrix. Under the optimized conditions, the detection limits of the proposed method varied from 15.2 to 40.5 ng/L. In general, the relative standard deviations (RSDs) were less than 10%. Good linearity was obtained with the correlation coefficients all better than 0.999. The proposed method is simple, quick, few microliters of organic solvent required, and is especially suitable for the analysis of the real sample with small amount available. The overall process of HF-LPME with GC-ICP-MS was applied successfully for the determination of polybrominated diphenyl ethers (PBDEs) in environmental and spiked human serum samples, and the results were satisfactory.     

Microwave-assisted extraction (MAE) for the determination of polybrominated diphenylethers (PBDEs) in sewage sludge

An efficient microwave-assisted extraction (MAE) method has been developed and evaluated for the quantification of eight major polybrominated diphenylethers (PBDEs) in sewage sludge. The PBDEs were extracted from wet and dry sludge in a microwave extraction unit using a hexane/acetone mixture for 35 min at a controlled temperature of 130 °C. The extract was concentrated, cleaned up on a silica gel column, and analyzed by gas chromatography/mass spectrometry (GC/MS) in the negative chemical ionization (NCI) mode. The MAE procedure exhibited higher extraction efficiency, specifically for BDE (brominated diphenylether) 209, than the conventional Soxhlet extraction. The test congeners were clearly separated under specific instrumental operating conditions, at a source temperature of 230 °C and a column length of 20 m. The present analytical method showed recovery efficiencies ranging from 80 to 110% when applied to the PBDE-free sludge spiked with eight PBDE congeners. The efficiency of the MAE method was confirmed using sludge obtained from four sewage treatment plants (STPs). The results indicate that BDE 47, 99, and 209 are the most abundant congeners present in these sewage sludges, which is consistent with previous reports.     

Imprinted sol-gel electrochemical sensor for the determination of benzylpenicillin based on Fe3O4@SiO2/multi-walled carbon nanotubes-chitosans nanocomposite film modified carbon electrode

In this work, we developed a novel graphene-assisted matrix solid-phase dispersion (GA-MSPD) method for extraction of polybrominated diphenyl ethers (PBDEs) and their methoxylated (MeO-) and hydroxylated (OH-) analogs from environmental samples. We found that grinding the solid sample with chemically converted graphene (CCG) powder yielded a tight contact and sufficient dispersion of the sample matrix due to the large surface area and flexible nanosheet morphology of CCG. The resultant blend was eluted using a two-step elution strategy: PBDEs and MeO-PBDEs were eluted firstly by hexane/dichloromethane and analyzed by GC-ECD, and then OH-PBDEs were eluted by acetone and determined by LC-ESI-MS/MS. The GA-MSPD conditions were optimized in detail. Better recoveries were obtained with GA-MSPD than with other sorbents (C18 silica, Florisil and carbon nanotubes) and other extraction techniques (Soxhlet and accelerated solvent extraction). Other advantages of GA-MSPD, including reduced consumption of sorbent and solvent, good selectivity and short extraction time, were also demonstrated. In analysis of soil samples, the method detection limits of five PBDEs, ten MeO-PBDEs and ten OH-PBDEs were in the range of 5.9-28.7, 14.3-46.6, and 5.3-212.6 pg g(-1) dry weight, respectively. The proposed method was successfully applied to the extraction of PBDEs, MeO-PBDEs and OH-PBDEs from different kinds of spiked environmental samples, including soil, tree bark and fish.     

State of the art of environmentally friendly sample preparation approaches for determination of PBDEs and metabolites in environmental and biological samples: A critical review

Green chemistry principles for developing methodologies have gained attention in analytical chemistry in recent decades. A growing number of analytical techniques have been proposed for determination of organic persistent pollutants in environmental and biological samples. In this light, the current review aims to present state-of-the-art sample preparation approaches based on green analytical principles proposed for the determination of polybrominated diphenyl ethers (PBDEs) and metabolites (OH-PBDEs and MeO-PBDEs) in environmental and biological samples. Approaches to lower the solvent consumption and accelerate the extraction, such as pressurized liquid extraction, microwave-assisted extraction, and ultrasound-assisted extraction, are discussed in this review. Special attention is paid to miniaturized sample preparation methodologies and strategies proposed to reduce organic solvent consumption. Additionally, extraction techniques based on alternative solvents (surfactants, supercritical fluids, or ionic liquids) are also commented in this work, even though these are scarcely used for determination of PBDEs. In addition to liquid-based extraction techniques, solid-based analytical techniques are also addressed. The development of greener, faster and simpler sample preparation approaches has increased in recent years (2003–2013). Among green extraction techniques, those based on the liquid phase predominate over those based on the solid phase (71% vs. 29%, respectively). For solid samples, solvent assisted extraction techniques are preferred for leaching of PBDEs, and liquid phase microextraction techniques are mostly used for liquid samples. Likewise, green characteristics of the instrumental analysis used after the extraction and clean-up steps are briefly discussed.     

Development of an analytical method for the determination of polybrominated diphenyl ethers in sewage sludge by the use of gas chromatography coupled to inductively coupled plasma mass spectrometry

Polybrominated diphenyl ethers (PBDEs) are flame retardants. As a consequence of their widespread use, they have been released into the environment. PBDEs are lipophilic organic contaminants that enter wastewater treatment plants (WWTPs) from urban, agricultural and industrial discharges. Because of their low aqueous solubility and resistance to biodegradation, up to 90% of the PBDEs are accumulated in the sewage sludge during the wastewater treatment. To assess the possibilities for sludge re-use, a reliable determination of the concentrations of these PBDEs is of crucial importance. Six PBDE congeners (BDE 28, BDE 47, BDE 99, BDE 100, BDE 153 and BDE 154) are listed as priority substances under the EU Water Framework Directive. In the present work a simple analytical method with minimal sample-preparation steps was developed for a sensitive and reliable determination of the six PBDEs in sewage sludge by the use of gas chromatography coupled to inductively coupled plasma mass spectrometry (GC-ICP-MS). For this purpose an extraction procedure was optimised. Different extracting agents (methanol (MeOH), acetic acid (AcOH)/MeOH mixture (3:1) and 0.1 mol L⁻¹ hydrochloric acid (HCl) in MeOH) followed by the addition of a Tris-citrate buffer (co-extracting agent) and iso-octane were applied under different modes of extraction (mechanical shaking, microwave- and ultrasound-assisted extraction). Mechanical shaking or the microwave-assisted extraction of sewage sludge with 0.1 mol L⁻¹ HCl in MeOH and the subsequent addition of the Tris-citrate buffer and the iso-octane extracted the PBDEs from the complex sludge matrix most effectively. However, due to easier sample manipulation during the extraction step, mechanical shaking was used. The PBDEs in the organic phase were quantified with GC-ICP-MS by applying a standard addition calibration method. The spike recovery test (recoveries between 95 and 104%) and comparative analyses with the species-specific isotope-dilution (ID) GC-ICP-MS confirmed the accuracy of the developed analytical procedure. The procedure is sensitive (limits of detection (LODs) for PBDEs congeners between 0.2 and 0.3 ng g⁻¹), repeatable and reproducible (RSDs 2.2–5.7%) and was applied for the determination of PBDEs in sewage sludge samples collected three times at the municipal WWTP over a period of 16 years.     

高分辨气相色谱-高分辨质谱测定活性污泥中的多溴联苯醚

建立了利用高分辨气相色谱-高分辨质谱定量测定多溴联苯醚(PBDEs)的方法,参与了测定PBDEs的国际比对实验,实验结果表明该法是可行的。对不同地区（北京、山东临沂、上海）的3个污水处理厂的活性污泥进行了索氏提取、多层复合硅胶柱分离,然后用所建立的方法测定了19种多溴联苯醚单体的含量。结果表明,北京某污水处理厂的活性污泥中PBDEs的总量高于其他两者。     

Multiwalled carbon nanotubes coated fibers for solid-phase microextraction of polybrominated diphenyl ethers in water and milk samples before gas chromatography with electron-capture detection

Determination of polybrominated diphenyl ethers (PBDEs) in environmental samples has raised great concerns due to the widespread use of PBDEs and their potential risk to humans. Solid-phase microextraction (SPME) is a fast, simple, cost-effective, and green sample preparation technique and is widely used for environmental analysis, but reports on the application of SPME for determination of PBDEs are very limited, and only a few publications dealing with commercial SPME fibers are available for extraction of PBDEs. Herein, we report a novel SPME method using multiwalled carbon nanotubes (MWCNTs) as the SPME fiber coating for gas chromatography with electron-capture detection (GC-ECD) of PBDEs in environmental samples. The MWCNTs coating gave much higher enhancement factors (616-1756) than poly (5% dibenzene-95% dimethylsiloxane) coating (139-384) and activated carbon coating (193-423). Thirty-minute extraction of 10 mL of sample solution using the MWCNTs coated fiber for GC-ECD determination yielded the limits of detection of 3.6-8.6 ng L(-1) and exhibited good linearity of the calibration functions (r(2)>0.995). The precision (RSD%, n=4) for peak area and retention time at the 500 ng L(-1) level was 6.9-8.8% and 0.6-0.9%, respectively. The developed method was successfully applied for the analysis of real samples including local river water, wastewater, and milk samples. The recovery of the PBDEs at 500 ng L(-1) spiked in these samples ranged from 90 to 119%. No PBDEs were detected in the river water and skimmed milk samples, whereas in the wastewater sample, 134-215 ng L(-1) of PBDEs were found. The PBDEs were detected in all whole fat milk samples, ranging from 13 to 484 ng L(-1). In a semiskimmed milk sample, only BDE-47 was found at 21 ng L(-1).     

Analysis of polybrominated diphenyl ethers in atmospheric deposition and snow samples by solid-phase disk extraction

An extraction method for the quantitative analysis of polybrominated diphenyl ethers (PBDEs) in aqueous samples has been evaluated. The analytical methodology includes the sample filtration through glass fiber filter and subsequent extraction of dissolved phase compounds by C18 solid-phase disk extraction. Dependence of extraction efficiency on factors such as pollutant concentrations, sample volume, and stability during storage has been investigated. Mean extraction efficiencies of 97% for total PBDEs (13 tri- to heptabrominated congeners at spiking levels in the range of 15-90pg) with a RSD between 9 and 20% were achieved. Higher recoveries were observed for the more volatile PBDEs (112%) in relation to more brominated congeners (88%). The developed methodology was successfully applied to the analysis of these compounds in atmospheric deposition and snow samples from remote sites in Europe with method detection and quantitation limits in the range of 2.1-10pgL(-1) for almost all congeners, which allow the determination of PBDEs in remote areas with levels in the range of low to medium pgL(-1) for SigmaPBDEs.     

Simplified and rapid determination of polychlorinated biphenyls, polybrominated diphenyl ethers, and polycyclic aromatic hydrocarbons in fish and shrimps integrated into a single method

In this study, a new rapid and flexible method for the simultaneous determination of 18 key representatives of polychlorinated biphenyls (PCBs), 7 polybrominated diphenyl ethers (PBDEs), and 32 polycyclic aromatic hydrocarbons (PAHs) in fish and shrimps by gas chromatography coupled to mass spectrometry (GC-MS) was developed and validated. A substantial simplification of sample processing prior to quantification step was achieved: after addition of water to homogenized sample, transfer of hydrophobic analytes into ethyl acetate was supported by added inorganic salts. Bulk fat, contained in crude organic extract obtained by partition, was subsequently removed on a silica minicolumn. This approach enabled to process six samples in less than 1h; moreover, the volume of an extraction solvent and consumption of other chemicals can be significantly reduced compared to, e.g., traditional Soxhlet extraction followed by gel permeation chromatography. The recoveries of target analytes were in the range of 73-120% even at the lowest spiking level (1 μg kg(-1)), repeatabilities (relative standard deviations, RSDs) ranged from 1 to 20%. Under optimized GC-MS conditions (time-of-flight mass analyzer, TOF), the limits of quantification (LOQs) were as follows: PCBs 0.1-0.5 μg kg(-1), PBDEs 0.5 μg kg(-1), and PAHs 0.05-0.25 μg kg(-1). Ambient mass spectrometry employing a direct analysis in real time (DART) ion source was shown as an effective tool for fat control in extract, which is needed during the method development and examination of unknown samples prior to the analysis. Further extension of a method scope by other similar analytes is easily possible.     

Simultaneous determination of the endocrine disrupting compounds nonylphenol, nonylphenol ethoxylates, triclosan and bisphenol A in wastewater and sewage sludge by gas chromatography-mass spectrometry

An integrated analytical method for the simultaneous determination of 4-n-nonylphenol (4-n-NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA) and triclosan (TCS) in wastewater (dissolved and particulate phase) and sewage sludge was developed based on gas chromatography-mass spectrometry. Chromatographic analysis was achieved after derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA). Extraction from water samples was performed by solid-phase extraction (SPE). The optimization of SPE procedure included the type of sorbent and the type of the organic solvent used for the elution. Referred to solid samples, the target compounds were extracted by sonication. In this case the optimization of the extraction procedure included the variation of the amount of the extracted biomass, the duration and the temperature of sonication and the type of the extraction organic solvent. The developed extraction procedures resulted in good repeatability and reproducibility with relative standard deviations (RSDs) less than 13% for all the tested compounds for both types of samples. Satisfactory recoveries were obtained (>60%) for all the compounds in both liquid and solid samples, except for 4-n-NP, which gave recoveries up to 35% in wastewater samples and up to 63% in sludge samples. The limits of detection (LODs) of the target compounds varied from 0.03 (4-n-NP) to 0.41 microg l(-1) (NP2EO) and from 0.04 (4-n-NP) to 0.96 microg kg(-1) (NP2EO) for liquid and solid samples, respectively. The developed methods were successfully applied to the analysis of the target compounds in real samples.     

New perspective on the determination of flame retardants in sewage sludge by using ultrahigh pressure liquid chromatography–tandem mass spectrometry with different ion sources

Analysis of 11 polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A bis 2,3-dibromopropylether (TBBPA-bis), tetrachlorobisphenol A (TCBPA), tetrabromobisphenol A (TBBPA) and hexabromocyclododecanes (HBCDs) was optimized by ultrahigh pressure liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) operating in negative ion (NI) mode. Electrospray ionization (ESI), atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were tested and for PBDEs APCI gave higher sensitivity than APPI while for TBBPA-bis APCI and APPI showed similar performance. ESI was the best option for TCBPA, TBBPA and HBCDs. Detection limits were between 20 and 59 fg for the compounds analyzed by ESI, 0.10 and 0.72 pg for PBDEs and 6 pg for TBBPA-bis. The matrix effect of sewage sludge extract was also tested showing negligible ion suppression for APCI and an increase of the background level of all investigated pollutants leading to a worsening of the limits of quantification by a factor between 1.2 and 3.3. The UPLC-APCI/MS/MS method for PBDEs, after pressurized liquid extraction (PLE), was validated by comparison with the concentration values from the NIST 1944 standard reference material. The advantages of the methods include low detection limits, PBDE congeners specificity using selected multiple reaction monitoring (MRM) transitions, and the absence of thermal degradation of higher PBDE congeners, especially BDE-209. The methods were applied for the determination of the above reported flame retardants in sewage sludge in order to get more information about the degradation on PBDEs (in particular BDE-209) during municipal wastewater treatments.     

Determination of polybrominated diphenyl ethers in soil from e-waste recycling site

Soil samples collected from an electronic waste recycling site were prepared by using Soxhlet extraction and multiple-step column chromatographic clean-up. Gas chromatography/ion trap mass spectrometry method was developed to determine polybrominated diphenyl ethers (PBDEs) in the sample extracts. The method performance was evaluated by the recovery of ¹³C-labeled internal standards and by analyzing quality assurance and quality control samples. Relative error and relative standard deviation obtained from the analysis of duplicated samples and spiked matrix were better than 10%. PBDEs were detected in the field soil samples collected from an e-wastes disposal site at levels from low parts-per-billions to 600 parts-per-billions.     

A review of the analysis of novel brominated flame retardants

This review provides a summary of various analytical methodologies applied to the determination of “novel” brominated flame retardants (NBFRs) in various environmental compartments, as reported in peer reviewed literature, either in print or online, until the end of 2010. NBFRs are defined here as those brominated flame retardants (BFRs) which are either new to the market or newly/recently observed in the environment. The preparation and extraction of sediment, water, sewage sludge, soil, air and marine biota samples, the extract clean-up/fractionation and subsequent instrumental analysis of NBFRs are described and critically examined. Generally, while the instrumental analysis step mainly relies on mass-spectrometric detection specifically developed for NBFRs, and hyphenated to liquid or gas chromatography, preceding steps tend to replicate methodologies applied to the determination of traditional BFRs such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD). Shortcomings and gaps are discussed and recommendations for future development are given.