Modification of the Zahn-Wellens test: determination of the biodegradability of poorly soluble, adsorbing and volatile substances by measurement of oxygen consumption and carbon dioxide production

Based on the Zahn-Wellens test (OECD 302 B, 1992; DIN EN ISO 9888, 1999), a test system has been developed which enables a continuous and parallel determination of oxygen consumption (pressure measurement) and carbon dioxide production (conductivity measurement). It is a closed test system consisting of a culture flask, a carbon dioxide absorption flask, a pump as well as integrated measuring and control instruments. The air circulating within the test system causes the carbon dioxide present in the test solution to be stripped out completely and directly absorbed by the absorption solution. Avoiding costly thermostatting of the test apparatus, the results of the pressure measurements were temperature-corrected arithmetically. The functional reliability of the measuring apparatus has been demonstrated exemplary in degradation experiments with selected test substances. This new test system also facilitates to test poorly soluble, adsorbing and volatile substances for inherent biodegradability and constitutes an appropriate complement to the standardised Zahn-Wellens test. The Federal Environmental Agency will use it as input to international standardisation activities ongoing within the OECD, as a draft standard.     

Testing biodegradability with standardized methods

Laboratory test methods are used by industry laboratories to determine biodegradability, an important parameter for the evaluation of the ecological behaviour of substances. Biodegradability has a key role due to the simple fact that a degradable substance will cause no long term risk in the environment. The great variety of biodegradation processes in the natural environment and in technical plants for treating waste water and solid wastes gave rise to a rather large number of test methods based on different test principles. To guarantee the acceptance of the test results by authorities and customers internationally standardized methods (ISO, OECD) and established quality criteria (GLP, EN 45000, ISO 9000) are used.     

Anaerobic inhibition and biodegradation of antibiotics in ISO test schemes

Municipal sewage is the main exposure route for antibiotics that are used in human medical care. Antibiotics that adsorb to the primary sludge and/or sur-plus activated sludge will enter the anaerobic digesters of municipal sewage treatment plants. Here anaerobic biodegradation or inhibition of anaerobic bacteria resulting in a disturbance of the process might occur. ISO standards 13641 (2003) and 11734 (1999) were used for assessing the anaerobic inhibition of 16 and the anaerobic biodegradability of 9 antibiotics respectively. Digestion sludge from a municipal sewage treatment plant (1g/l d.s.) was used as inoculum in both tests. In ISO 13641 (2003) most antibiotics showed only moderate inhibition effects after a 7 day incubation period, with EC50 values between 24 mg/l and more than 1000 mg/l (equal to mg/g d.s.). In contrast, metronidazol was decisively toxic to anaerobic bacteria with an EC50 of 0.7 mg/l. In the anaerobic degradation tests according to ISO standard 11734 (1995), only benzylpenicillin showed certain ultimate biodegradation after 60 days and most antibiotics inhibited the digesting sludge in the respective parallel tested inhibition controls. Thus the inhibition of anaerobic bacteria by antibiotics observed in the degradation tests was higher than expected from the results of the inhibition tests. The possible explanations are that distinct substrates are used (yeast extract versus sodium benzoate), that the digestion sludge loses activity during the washing steps performed for the degradation tests and that the exposure time in the degradation tests was 8 times longer than in the inhibition test.     

Application of the headspace CO2 method (ISO 14 593) to the assessment of the ultimate biodegradability of surfactants: results of a calibration exercise

In the recent review of the control of marketing surfactants used in detergents, the EU decided to increase the severity of the testing procedure by using the criterion of ultimate biodegradability (mineralization) rather than primary biodegradation (removal of the parent molecule) to ensure that possible harmful organic metabolites do not reach the environment. The relatively new ISO headspace CO2 test, considered to be an improvement on the OECD 301B (Sturm CO2) test was chosen. The method was subjected to a ring test by 11 laboratories using one of each of four classes of surfactants plus a poorly degradable reference surfactant; all laboratories satisfactorily applied the method. The necessary addition of silica gel to the medium containing the cationic surfactant, known as a class to be more inhibitory than other classes, was confirmed as a technique for avoiding inhibition of the inoculum. The biodegradability of the surfactants was in general agreement with results reported in the literature and the often reported variable values of % inorganic carbon (IC) produced of the theoretical was found. The anionic and cationic surfactants were readily biodegradable (%IC > 60), the non-ionic surfactant was well below the pass value, while the amphoteric was borderline. The IC production by the blank controls, one of the validity criteria, was about 0.3 mg C/100 ml test medium, equivalent to 3 mg C/l, as recommended in the ISO text. Mild conditions of pre-exposure of the inoculum to the test surfactant did not produce consistent worthwhile effects on either the percentage biodegradation or on its variability.     

Hydrogen storage studies on palladium-doped carbon materials (AC,CB, CNMs) @ metal–organic framework-5

Metal organic frameworks (MOFs) are a rapidly growing class of porous materials and are considered as best adsorbents for their high surface area and extraordinary porosity. The MOFs are synthesized by using various chemicals like triethylamine, terepthalic acid, zinc acetate dihydrate, chloroform, and dimethylformamide (DMF). Synthesized MOFs are intercalated with palladium/activated carbon, carbon black, and carbon nanomaterials by chemical reduction method for the purpose of enhancing the hydrogen adsorption capacities. We have observed that the palladium doped activated carbon on MOF-5 showed high hydrogen storage capacity. This may be due to the affinity of the palladium toward hydrogen molecule. The samples are characterized by X-ray diffraction, scanning electron microscopy (SEM), and Brunauer–Emmett–Teller (BET) surface area analysis. We have observed a clear decrease in the BET surface area and pore volume. The obtained results show a better performance for the synthesized sample. To our best knowledge, no one has reported the work on palladium-doped carbon materials (activated carbon, carbon black, carbon nanomaterials) impregnated to the metal–organic framework-5. We have attempted to synthesize carbon nanomaterials using indigenously fabricated chemical vapor deposition (CVD) unit as a support. We have observed an increase in the hydrogen storage capacities.     

Harmonisation of ready biodegradability tests

Seven ready biodegradability tests (AFNOR, OECD, ISO, RDA, MITI(I), Sturm, Closed Bottle Test) have been assessed, with particular consideration of those aspects which are, at least in part, responsible for the variability in results. Proposals for harmonising certain test conditions, together with recommendations for minimising the effect of nitrification, are made.     

Total and partial digestion of sediments for the evaluation of trace element environmental pollution

Four different sample treatment methods for the determination of trace elements have been compared: a total digestion with HNO3-H2O2-HF using microwave, and three different standardized methods of fractionation: BCR three-steps sequential extraction, USEPA standard 3050B and ISO standard 11466. The four treatment methods were applied to the determination of Cu and Ni in four samples collected in different areas of Cienfuegos Bay (Cuba). The location of samples and the analytes were selected on the basis of results obtained by previous studies. Analyses following total digestion and BCR three-steps procedure were performed by inductively coupled plasma mass spectroscopy whereas analyses following EPA and ISO procedures were performed by flame atomic absorption spectroscopy. The results obtained have been compared with an estimated anthropic fraction evaluated in each sampling point as the difference between the total concentration and an estimated background concentration level. The BCR three-steps provided the best approximation of the estimated anthropic fraction and was therefore applied also in the determination of Pb and Cd for further consideration.     

Ecotoxic Effect of Photocatalytic Active Nanoparticles (TiO2) on Algae and Daphnids (8 pp)

Background, Aim and Scope Due to their large potential for manifold applications, the use of nanoparticles is of increasing importance. As large amounts of nanoparticles may reach the environment voluntarily or by accident, attention should be paid on the potential impacts on the environment. First studies on potential environmental effects of photocatalytic TiO2 nanoparticles have been performed on the basis of widely accepted, standardized test systems which originally had been developed for the characterization of chemicals. The methods were adapted to the special requirements of testing photocatalytic nanoparticles. Materials and Methods: Suspensions of two different nanoparticles were illuminated to induce their photocatalytic activity. For testing, the growth inhibition test with the green alga Desmodesmus subspicatus and the immobilization test with the daphnid Daphnia magna were selected and performed following the relevant guidelines (algae: ISO 8692, OECD 201, DIN 38412-33; daphnids: ISO 6341, OECD 202, DIN 38412-30). The guidelines were adapted to meet the special requirements for testing photocatalytic nanoparticles. Results: The results indicate that it is principally possible to determine the ecotoxicity of nanoparticles. It was shown that nanoparticles may have ecotoxicological effects which depend on the nature of the particles. Both products tested differ in their toxicity. Product 1 shows a clear concentration-effect curve in the test with algae (EC50: 44 mg/L). It could be proven that the observed toxicity was not caused by accompanying contaminants, since the toxic effect was comparable for the cleaned and the commercially available product. For product 2, no toxic effects were determined (maximum concentration: 50 mg/L). In the tests with daphnids, toxicity was observed for both products, although the concentration effect-curves were less pronounced. The two products differed in their toxicity; moreover, there was a difference in the toxicity of illuminated and non-illuminated products. Discussion: Both products differ in size and crystalline form, so that these parameters are assumed to contribute to the different toxicities. The concentration-effect curves for daphnids, which are less-pronounced than the curves obtained for algae, may be due to the different test organisms and/or the differing test designs. The increased toxicity of pre-illuminated particles in the tests with daphnids demonstrates that the photocatalytic activity of nanoparticles lasts for a period of time. Conclusions: The following conclusions can be drawn from the test results: (I) It is principally possible to determine the ecotoxicity of (photocatalytic) nanoparticles. Therefore, they can be assessed using methods comparable to the procedures applied for assessing soluble chemicals. - (II) Nanoparticles may exert ecotoxicological effects, which depend on the specific nanoparticle. - (III) Comparable to traditional chemicals, the ecotoxicity depends on the test organisms and their physiology. - (IV) The photocatalytic activity of nanoparticles lasts for a relevant period of time. Therefore, pre-illumination may be sufficient to detect a photocatalytic activity even by using test organisms which are not suitable for application in the pre-illumination-phase. Recommendations and Perspectives: First results are presented which indicate that the topic 'ecotoxicity and environmental effects of nanoparticles' should not be neglected. In testing photocatalytic nanoparticles, there are still many topics that need clarification or improvement, such as the cause for an observed toxicity, the improvement of the test design, the elaboration of a test battery and an assessment strategy. On the basis of optimized test systems, it will be possible to test nanoparticles systematically. If a potential risk by specific photocatalytic particles is known, a risk-benefit analysis can be performed and, if required, risk reducing measures can be taken.     

Comparison of calculation models for wind-driven rain deposition on building facades

Wind-driven rain (WDR) is an important factor in the dry and wet deposition of atmospheric pollutants on building facades. In the past, different calculation models for WDR deposition on building facades have been developed and progressively improved. Today, the models that are most advanced and most frequently used are the semi-empirical model in the ISO Standard for WDR assessment (ISO), the semi-empirical model by Straube and Burnett (SB) and the CFD model by Choi. This paper compares the three models by applying them to four idealised buildings under steady-state conditions of wind and rain. In each case, the reference wind direction is perpendicular to the windward facade. For the CFD model, validation of wind-flow patterns and WDR deposition fluxes was performed in earlier studies. The CFD results are therefore considered as the reference case and the performance of the two semi-empirical models is evaluated by comparison with the CFD results based on two criteria: (1) ability to model the wind-blocking effect on the WDR coefficient; and (2) ability to model the variation of the WDR coefficient with horizontal rainfall intensity R_h. It is shown that both the ISO and SB model, as opposed to the CFD model, cannot reproduce the wind-blocking effect. The ISO model incorrectly provides WDR coefficients that are independent of R_h, while the SB model shows a dependency that is opposite to that by CFD. In addition, the SB model can provide very large overestimations of the WDR deposition fluxes at the top and side edges of buildings (up to more than a factor 5). The capabilities and deficiencies of the ISO and SB model, as identified in this paper, should be considered when applying these models for WDR deposition calculations. The results in this paper will be used for improvement and further development of these models.     

Mixtures of quaternary ammonium compounds and anionic organic compounds in the aquatic environment: Elimination and biodegradability in the closed bottle test monitored by LC-MS/MS

Quaternary ammonium compounds (QACs) are widely used as disinfectants, detergents and fabric softeners. Anionic detergents are one of the most widely used chemical substances. QACs and anionic surfactants can form ionic pairs. In the present study we investigated the biodegradability of QACs in the presence of different anionic surfactants. The biodegradability of three QACs, namely benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDMAC) and ethacridine lactate (EL), when applied as single substances and in combination with anionic surfactants such as benzene sulfonic acid (BSA), LAS, naphthalene sulfonic acid (NSA) and sodium dodecylsulfonate (SDS) was studied applying the closed bottle test (CBT) [OECD 301D, 1992. Guidelines for Testing of Chemicals. Closed bottle test. Organisation of Economic Cooperation and Development, Paris] at a ratio of 1:1 (mol:mol). Biodegradation was monitored by measuring oxygen concentration in the test vessels with an oxygen electrode in accordance with international standard methods [ISO 5414, 1990. Water quality - determination of dissolved oxygen. In: German Standard Methods for the Examination of Water, Wastewater and Sludge. VCH Verlagsgesellschaft, Weinheim, New York, Basel Cambridge]. Primary elimination of the QACs and of LAS was monitored by LC-MS/MS. There was little biodegradability of the QACs as either single compounds or in the presence of organic counter ions. The biodegradability of the organic counter ions was lower in the presence of QACs as compared to the single substances. Primary elimination of the QACs by sorption took place.     

Comparative analysis of the structure of carbon materials relevant in combustion

The determination of the structure of carbon materials is an analytical problem that join the research scientific communities involved in the chemical characterization of heavy fuel-derived products (heavy fuel oils, coal-derived fuels, shale oil, etc.) and of carbon materials (polycyclic aromatic compounds, tar, soot) produced in many combustion processes.

The knowledge of the structure of these “difficult” fuels and of the carbon materials produced by incomplete combustion is relevant to research for the best low-environmental impact operation of combustion systems; but an array of many analytical and spectroscopic tools are necessary, and often not sufficient, to attempt the characterization of such complex products and in particular to determine the distribution of molecular masses.

In this paper the size exclusion chromatography using N-methyl-pyrrolidinone as eluent has been applied for the characterization of different carbon materials starting from typical carbon species, commercially available like polyacenaphthylene, carbon black, naphthalene pitch up to combustion products like soot and soot extract collected in fuel-rich combustion systems. Two main fractions were detected, separated and molecular weights (MWs) determined by comparison with polystyrene standards: a first fraction consisted of particles with very large molecular masses (>100 000 u); a second fraction consisted of species in a relatively small MW range (200–600 u). The distribution of these fractions changes in dependence on the carbon sample characteristics.

Fluorescence spectroscopy applied on the fractions separated by size-exclusion chromatography has been used and comparatively interpreted giving indications on the differences and similarities in chemical structure of such different materials.     

Improvement of Vicia-micronucleus test for assessment of soil quality: a proposal for international standardization

The Viciafaba root tip micronucleus test is one of the most employed plant genotoxicity assays, and has been used on various types of contaminated materials. This test has been standardized by AFNOR, the French member organization of ISO. However, this test is usually performed with a water extraction step but soil genotoxicity assessment would be more relevant when performed directly in the soil itself. In order to harmonize these protocols, an ISO standard for the V.faba micronucleus test in both liquid phase (exposure of plants to different liquid matrix, including soil water extracts) and solid phase (direct exposure of plants to the soil) would be very useful. In this context, we compared two exposure durations in the solid phase (48 h and 5 d) for the V.faba micronucleus test with two different well-known genotoxicants, maleic hydrazide and copper sulfate. We concluded that these two durations induced equivalent sensitivity: the micronucleus frequency was significantly increased with 5 μmol maleic hydrazide per kg dry soil and with 2 mmol copper sulfate per kg dry soil with both exposure durations. However, exposing roots to soil during 48 h is more practical. Moreover, organically and conventionally cultured seeds were employed to determine whether the seed provenance influenced the test sensitivity. Organic seeds were less sensitive to copper, possibly because copper-based treatments are permitted, and often applied, in organic farms. Therefore, in the absence of completely non-treated seeds, organically-cultured seeds did not appear to offer any advantages over conventional seeds.     

Contribution of vehicle emissions from an attached garage to residential indoor air pollution levels

The infiltration of vehicle emissions into a house from the attached garage was studied for 16 homes of differing designs using the same extensively characterized vehicle at each home. Before the in-home measurement program, the cold-start and hot-start tailpipe emissions and hot-soak evaporative emissions from a 1993 Buick Regal were measured using standard vehicle emissions measurement methods. The emissions were chemically characterized for methane, nonmethane hydrocarbons (NMHC), and carbonyl compounds. The in-home measurements occurred over two winter seasons (1997-1998 and 1998-1999) in Ottawa, Ontario, Canada. Samples of indoor air and garage atmosphere were characterized for carbon monoxide, carbon dioxide, methane, NMHC, and carbonyl compounds. During the second year, real-time measurements of carbon, carbon dioxide, and total hydrocarbons were made to determine when and for how long the emissions plume infiltrates the house. Chemical mass balance modeling results using 31 NMHC species suggest that between 9 and 71% of the concentrations measured in the house during the hot-soak test and between 13 and 85% of the concentrations measured in the house during the cold-start test could be attributed to vehicle emissions infiltrating from the garage. In contrast, increases in carbonyl compound concentrations caused by the vehicle were difficult to detect above the already significant levels found in the houses.     

Comparative study of extraction methods for the determination of PAHs from contaminated soils and sediments

The following four methods were compared on the extraction efficiency of 16 EPA (US Environmental Protection Agency) polycyclic aromatic hydrocarbons (PAHs): German method of the Verband Deutscher Landwirtschaftlicher Untersuchungs und Forschungsanstalten (VDLUFA), two methods of the International Organization for Standardization using shaking (ISO A) and Soxhlet extraction (ISO B) and an ultrasonic method. Recovery rates of 16 PAHs were determined in two soils. Extraction efficiency was evaluated in five soils and three sediments. Effect of drying soils and sediments on extraction efficiency was tested using the VDLUFA and the ultrasonic methods. Our study shows that the number of aromatic rings, rather than extraction procedures, significantly influenced recovery rates of individual PAHs. No significant differences in extraction efficiency of the four methods were observed for less polluted samples. For highly polluted soils, extraction efficiency decreased in the following order: VDLUFA method > ISO A > ultrasonic method > ISO B. Influence of soil moisture on extraction efficiency depended to some extent on both solvent used and content of PAHs in samples. A mixture of dichloromethane/acetone (5:1) is recommended for PAH extraction from moist samples when the ultrasonic method is used.     

Comparison of two methods for the determination of total organic halogen (TOX) in receiving waters

Two methods for the determination of total organic halogen (TOX), orginating from bleaching of pulp, in receiving waters have been compared. One of the methods (AC/MC) is based on adsorption of the halogenated matter onto an activated carbon sorbent. The halogen is determined by a microcoulometric technique after combustion of the carbon. The other method (XAD/PT) uses an XAD resin as sorbent and the determination of the halogen is carried out by potentiometric titration after a schöniger combustion of the resin eluate.Both methods showed good performance for samples consisting of spent bleach liquors diluted with distilled, fresh and brackish water. The repeatability was very good with a relative standard deviation less than a few per cent. The detection limit of the AC/MC method is about 0.1 μmol/1 and for the XAD/PT method about ten times higher. The AC/MC method gave in most cases 10–20% higher TOX concentrations compared to the XAD/PT method.     

Determination of the aerobic biodegradability of polymeric material in a laboratory controlled composting test

A laboratory method is presented for investigating the biodegradation of an organic test material in an aerobic composting system based on the evolution of carbon dioxide. In addition to carbon conversion, biodegradation can also be monitored through weight loss and physical disintegration. The test method is different from other biodegradation tests, especially aquatic tests, because of the elevated temperature representative for real composting conditions and also because of enhanced fungal degradation activities. A ring test was run using paper and poly-β-hydroxybutyrate/valerate as test materials and cellulose powder as a reference material. The test results and the experience gained by the participants showed that the method is suitable and practicable. Experience with real technical-scale composting facilities confirms that the method provides test results of high predictive value. The test is designed to become a European Standard in connection with determining the compostability of packagings and packaging materials.     

不同改性方式对控释碳源材料脱氮性能的影响

以玉米淀粉(S)为碳源,聚乙烯醇(P)为骨架载体,根据是否用硼酸(B)和硫酸钠(N)做改性处理,制备了GSP、GSP-N、GSP-B和GSP-BN 4种控释碳源材料,研究N、B和BN 3种改性方式对碳源材料脱氮性能的影响。在添加4种碳源的静态实验中,硝酸盐氮去除率均在96.1%以上。碳源材料的改性对硝酸盐氮的降解情况影响不明显,但对脱氮过程中TOC、亚硝酸盐氮、氨氮和TN浓度的变化有不同程度的影响。以TOC、亚硝酸盐氮、氨氮和TN为指标,N、BN和B 3种改性方式综合效益指数分别为5.62、4.6和1.92,硫酸钠(N)改性处理对提高控释碳源材料整体性能具有最佳效果。     

Adaptation of the CAS test system and synthetic sewage for biological nutrient removal. Part II: design and validation of test units

A global increase in biological nutrient removal (BNR) applications in wastewater treatment and concern for potential effects of anthropogenic substances on BNR processes resulted in the adaptation of the Continuous Activated Sludge (CAS) laboratory test system (cf. guideline OECD 303A or ISO 11733). In this paper two novel systems are compared to the standard CAS unit: the Behrotest KLD4 and a University of Cape Town system (CAS-UCT). Both are 'single sludge' systems with an anoxic/aerobic and an anaerobic/anoxic/aerobic configuration, respectively. They both can simulate the essential processes of full-scale BNR installations. The units where fed with a specially designed synthetic sewage, Syntho (cf. Part I of this study), or its precursor BSR3 medium. The performance of the two new units was benchmarked against the standard CAS system in terms of carbon/nitrogen/phosphorus (C/N/P) removal, as well as primary biodegradation of the surfactants linear alkylbenzene sulfonate (LAS) and glucose amide (GA). Both systems allow to easily achieve stable excess N- and P-removal. Experimental C/N/P removal data compared closely with simulations obtained with the IAWQ Activated Sludge Model No. 2 (ASM2), and with full scale BNR plants with a similar configuration. In both units the effluent concentrations of the surfactants tested were significantly reduced in comparison to the standard CAS system (up to 50% less). No adverse effects on BNR were noted for the test surfactants dosed at 400 microg/l together with an overall surfactant background concentration in the feed of ca. 20 mg/l. The proposed systems hold potential to complement the standard CAS system for situations where advanced sewage treatment plants with BNR need to be simulated in the laboratory with minimum effort.     

Improvement of oxygen-containing functional groups on olive stones activated carbon by ozone and nitric acid for heavy metals removal from aqueous phase

Recently, modification of surface structure of activated carbons in order to improve their adsorption performance toward especial pollutants has gained great interest. Oxygen-containing functional groups have been devoted as the main responsible for heavy metal binding on the activated carbon surface; their introduction or enhancement needs specific modification and impregnation methods. In the present work, olive stones activated carbon (COSAC) undergoes surface modifications in gaseous phase using ozone (O₃) and in liquid phase using nitric acid (HNO₃). The activated carbon samples were characterized using N₂ adsorption–desorption isotherm, SEM, pHpzc, FTIR, and Boehm titration. The activated carbon parent (COSAC) has a high surface area of 1194 m²/g and shows a predominantly microporous structure. Oxidation treatments with nitric acid and ozone show a decrease in both specific surface area and micropore volumes, whereas these acidic treatments have led to a fixation of high amount of surface oxygen functional groups, thus making the carbon surface more hydrophilic. Activated carbon samples were used as an adsorbent matrix for the removal of Co(II), Ni(II), and Cu(II) heavy metal ions from aqueous solutions. Adsorption isotherms were obtained at 30 °C, and the data are well fitted to the Redlich–Peterson and Langmuir equation. Results show that oxidized COSACs, especially COSAC(HNO₃), are capable to remove more Co(II), Cu(II), and Ni(II) from aqueous solution. Nitric acid-oxidized olive stones activated carbon was tested in its ability to remove metal ions from binary systems and results show an important maximum adsorbed amount as compared to single systems.     

Measuring the Organic Carbon Content of Source Emissions for Air Pollution Control

Measuring emissions of organic materials from such sources as paint bake ovens, degreas-ing operations, and printing processes is a necessy part of a control program for solvents. Over the intervening years since 1966 when Los Angeles first enacted its solvent Rule 66, a considerable number of tests have been performed and the present test method has gone through a period of experience and improvement. A sample is collected from a stack or vent in a freeze-out trap cooled with dry ice followed by an evacuated 8-liter tank. Analysis is done by a system of gas chromatography and catalytic combustion to yield the total organic carbon content. Representative industrial emission analysis results, which demonstrate the practical applicability of the system, are shown.