Scaling and Further Tests of Heavy Meson Decay Constant Determinations from Nonrelativistic

Determination of Pesticide Minimum Residue Limits in Essential OilsReport No 3A report for the Rural Industries Research andDevelopment CorporationBy Professor R. C. Menary & Ms S. M. GarlandJune 2004RIRDC Publication No 04/023RIRDC Project No UT-23A© 2004 Rural Industries Research and Development Corporation.All rights reserved.ISBN 0642 58733 7ISSN 1440-6845‘Determination of pesticide minimum residue limits in essential oils’, Report No 3Publication No 04/023Project no.UT-23AThe views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report.This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.Researcher Contact DetailsProfessor R. C. Menary & Ms S. M. GarlandSchool of Agricultural ScienceUniversity of TasmaniaGPO Box 252-54HobartTasmania 7001AustraliaPhone: (03) 6226 2723Fax: (03) 6226 7609Email: r.menary@.auIn submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.RIRDC Contact DetailsRural Industries Research and Development CorporationLevel 1, AMA House42 Macquarie StreetBARTON ACT 2600PO Box 4776KINGSTON ACT 2604Phone: 02 6272 4819Fax: 02 6272 5877Email: rirdc@.auWebsite: .auPublished in June 2004Printed on environmentally friendly paper by Canprint.FOREWORDInternational regulatory authorities are standardising the levels of pesticide residues present in products on the world market which are considered acceptable. The analytical methods to be used to confirm residue levels are also being standardised. To constructively participate in these processes, Australia must have a research base capable of constructively contributing to the establishment of methodologies and must be in a position to assess the levels of contamination within our own products.Methods for the analysis for pesticide residues rarely deal with their detection in the matrix of essential oils. This project is designed to develop and validate analytical methods and apply that methodology to monitor pesticide levels in oils produced from commercial harvests. This will provide an overview of the levels of pesticide residues we can expect in our produce when normal pesticide management programs are adhered to.The proposal to produce a manual which deals with the specific problems associated with detection of pesticide residues in essential oils is intended to benefit the essential oil industry throughout Australia and may prove useful to other horticultural products.This report is the third in a series of four project reports presented to RIRDC on this subject. It is accompanied by a technical manual detailing methodologies appropriate to the analysis for pesticide residues in essential oils.This project was part funded from RIRDC Core Funds which are provided by the Australian Government. Funding was also provided by Essential Oils of Tasmania and Natural Plant Extracts Cooperative Society Ltd.This report, an addition to RIRDC’s diverse range of over 1000 research publications, forms part of our Essential Oils and Plant Extracts R&D program, which aims for an Australian essential oils and plant extracts industry that has established international leadership in production, value adding and marketing.Most of our publications are available for viewing, downloading or purchasing online through our website:•downloads at .au/fullreports/index.html•purchases at .au/eshopSimon HearnManaging DirectorRural Industries Research and Development CorporationAcknowledgementsOur gratitude and recognition is extended to Dr. Noel Davies (Central Science Laboratories, University of Tasmania) who provided considerable expertise in establishing procedures for chromatography mass spectrometry.The contribution to extraction methodologies and experimental work-up of Mr Garth Oliver, Research Assistant, cannot be underestimated and we gratefully acknowledge his enthusiasm and novel approaches.Financial and ‘in kind’ support was provided by Essential Oils Industry of Tasmania, (EOT).AbbreviationsADI Average Daily IntakeAGAL Australian Government Analytical Laboratoriesingredientai activeAPCI Atmospheric Pressure Chemical IonisationBAP Best Agricultural PracticesenergyCE collisionDETA DiethylenetriamineECD Electron Capture DetectorionisationESI ElectrosprayFPD Flame Photometric DetectionChromatographyGC GasResolutionHR HighChromatographyLC LiquidLC MSMS Liquid Chromatography with detection monitoring the fragments of Mass Selected ionsMRL Maximum Residue LimitSpectrometryMS MassNRA National Registration AuthorityR.S.D. Relative Standard DeviationSFE Supercritical Fluid ExtractionSIM Single Ion MonitoringSPE Solid Phase ExtractionTIC Total Ion ChromatogramContents FOREWORD (III)ACKNOWLEDGEMENTS (IV)ABBREVIATIONS (V)CONTENTS (VI)EXECUTIVE SUMMARY (VII)1. INTRODUCTION (1)1.1B ACKGROUND TO THE P ROJECT (1)1.2O BJECTIVES (2)1.3M ETHODOLOGY (2)2. EXPERIMENTAL PROTOCOLS & DETAILED RESULTS (3)2.1M ETHOD D EVELOPMENT (3)2.2M ONITORING OF H ARVESTS (42)2.3P RODUCTION OF M ANUAL (46)3. CONCLUSIONS (47)IMPLICATIONS & RECOMMENDATIONS (50)BIBLIOGRAPHY (50)Executive SummaryThe main objective of this project was to continue method development for the detection of pesticide residues in essential oils, to apply those methodologies to screen oils produced by major growers in the industry and to produce a manual to consolidate and coordinate the results of the research. Method development focussed on the effectiveness of clean-up techniques, validation of existing techniques, the assessment of the application of gas chromatography (GC) with detection using electron capture detectors (ECD), flame photometric detectors (FPD) and high pressure liquid chromatography (HPLC) with ion trap mass selective (MS) detection.The capacity of disposable C18 cartridges to separate components of boronia oil was found to be limited with the majority of boronia components being eluted on the solvent front, with little to no separation achieved. The cartridges were useful, however, in establishing the likely interaction of reverse phases (RP) C18 columns with components of essential oils, using polar mobile phases . The loading of large amounts of oil onto RP HPLC columns presents the risk of permanently contaminating the bonded phases. The lack of retention of components on disposable SPE C18 cartridges, despite the highly polar mobile phase, presented a good indication that essential oils would not accumulate on HPLC RP columns.The removal of non-polar essential oil components by solvent partitioning of distilled oils was minimal, with the recovery of pesticides equivalent to that recorded for the essential oil components. However application of this technique was of advantage in the analysis of solvent extracted essential oils such as those produced from boronia and blackcurrant.ECD was found to be successful in the detection of terbacil, bromacil, haloxyfop ester, propiconazole, tebuconazole and difenaconzole. However, analysis of pesticide residues in essential oils by application of GC ECD is not sufficiently sensitive to allow for a definitive identification of any contaminant. As a screen, ECD will only be effective in establishing that, in the absence of a peak eluting with the correct retention time, no gross contamination of pesticide residues in an essential oil has occurred . In the situation where a peak is recorded with the correct elution characteristics, and which is enhanced when the sample is fortified with the target analyte, a second means of contaminant identification would be required. ECD, then, can only be used to rule out significant contamination and could not in itself be adequate for a positive identification of pesticide contamination.Benchtop GC daughter, daughter mass spectrometry (MSMS) was assessed and was not considered practical for the detection of pesticide residues within the matrix of essential oils without comprehensive clean-up methodologies. The elution of all components into the mass spectrometer would quickly lead to detector contamination.Method validation for the detection of 6 common pesticides in boronia oil using GC high resolution mass spectrometry was completed. An analytical technique for the detection of monocrotophos in essential oils was developed using LC with detection by MSMS. The methodology included an aqueous extraction step which removed many essential oil components from the sample.Further method development of LC MSMS included the assessment of electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI. For the chemicals trialed, ESI has limited application. No response was recorded for some of the most commonly used pesticides in the essential oil industry, such as linuron, oxyflurofen, and bromacil. Overall, there was very little difference between the sensitivity for ESI and APCI. However, APCI was slightly more sensitive for the commonly used pesticides, tebuconazole and propiconazole, and showed a response, though poor, to linuron and oxyflurofen. In addition, APCI was the preferred ionisation method for the following reasons,♦APCI uses less nitrogen gas compared to ESI, making overnight runs less costly;♦APCI does not have the high back pressure associated with ionisation by ESI such that APCI can be run in conjunction with UV-VIS without risk of fracturing the cell, which is pressure sensitive. Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil, terbacil, and the esters of the fluazifop and haloxyfop acids. Further work using APCI in the positive mode formed the basis for the inclusion of monocrotophos, pirimicarb, propazine and difenaconazole into the standard screen already established. Acephate, carbaryl, dimethoate, ethofumesate and pendimethalin all required further work for enhanced ionisation and / or improved elution profiles. Negative ionisation mode for APCI gave improved characteristics for dicamba, procymidone, MCPA and mecoprop.The thirteen pesticides included in this general screen were monocrotophos, simazine, cyanazine, pirimicarb, propazine, sethoxydim, prometryb, tebuconazole, propiconazole, , difenoconazole and the esters of fluroxypyr, fluazifop and haloxyfop.. Bromacil and terbacil were not included as both require negative ionisation and elute within the same time window as simazine, which requires positive ionisation. Cycling the MS between the two modes was not practical.The method validation was tested against three oils, peppermint, parsley and fennel.Detection limits ranged from 0.1 to 0.5 mgkg-1 within the matrix of the essential oils, with a linear relationship established between pesticide concentration and peak height (r2 greater than 0.997) and repeatabilities, as described by the relative standard deviation (r.s.d), ranging from 3 to 19%. The type of oil analysed had minimal effect on the response function as expressed by slope of the standard curve.The pesticides which have an carboxylic acid moiety such as fluazifop, haloxyfop and fluroxypyr, present several complications in any analytical method development. The commercial preparations usually have the carboxylic acid in the ester form, which is hydrolysed to the active acidic form on contact with soil and vegetation. In addition, the esters may be present in several forms, such as the ethoxy ethyl or butyl esters. Detection using ESI was tested. Preliminary results indicate that ESI is unsuitable for haloxyfop and fluroxypyr ester. Fluazifop possessed good ionisation characteristics using ESI, with responses approximately thirty times that recorded for haxloyfop. Poor chromatography and response necessitated improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. The inclusion of acetic acid improved peak resolution.The LC MSMS method for the detection of dicamba, fluroxypyr, MCPA, mecoprop and haloxyfop in peppermint and fennel distilled oils underwent the validation process. Detection limits ranged from 0.01 to 0.1 mgkg-1Extraction protocols and LC MSMS methods for the detection of paraquat and diquat were developed. ESI produced excellent responses for both paraquat and diquat, after some modifications of the mobile phase. Extraction methodology using aqueous phases were developed. Extraction with carbonate buffer proved to be the most effective in terms of recovery and robustness. A total ion chromatogram of the LC run of an aqueous extract of essential oil was recorded and detection using a photodiode array detector confirmed that very little essential oil matrix was co-extracted. The low background noise indicated that samples could be introduced directly into the MS. This presented a most efficient and rapid way for analysis of paraquat and diquat, avoiding the need for specialised columns or modifiers to be included in the mobile phase to instigate ion exchange.The adsorbtion of paraquat and diquat onto glass and other surfaces was reduced by the inclusion of diethylenetriamine (DETA). DETA preferentially accumulates on the surfaces of sample containers, competitively binding to the adsorption sites. All glassware used in the paraquat diquat analysis were washed in a 5% solution of 0.1M DETA, DETA was included in all standard curve preparations, oils were extracted with aqueous DETA and the mobile phase was changed to 50:50 DETA / methanol. The stainless steel tubing on the switching valve was replaced with teflon, further improvingreproducibility. Method validation was undertaken of the analysis of paraquat and diquat using the protocols established. The relationship between analyte concentration and peak area was not linear at low concentrations, with adsorption more pronounced for paraquat, such that the response for this analyte was half that seen for diquat and the 0.1 mgkg-1 level.The development of a method for the detection of the dithiocarbamate, mancozeb was commenced. Disodium N, N'-ethylenebis(dithiocarbamate) was synthesised as a standard for the derivatised final analytical product. An LC method, with detection using MSMS, was successfully completed. The inclusion of a phase transfer reagent, tetrabutylammonium hyrdrogen sulfate, required in the derivatisation step, contaminated the LC MSMS system, such that any signal from the target analyte was masked. Alternatives to the phase transfer reagent are now being investigated.Monitoring of harvests were undertaken for the years spanning 1998 to 2001. Screens were conducted covering a range of solvent extracted and distilled oils. Residues tested for included tebuconazole, simazine, terbacil, bromacil, sethoxydim, prometryn, oxyflurofen, pirimicarb, difenaconazole, the herbicides with acidic moieties and paraquat and diquat. Problems continued for residues of propiconazole in boronia in the 1998 / 1999 year with levels to 1 mgkg-1 still being detected. Prometryn residues were detected in a large number of samples of parsley oil.Finally the information gleaned over years of research was collated into a manual designed to allow intending analysts to determine methodologies and equipment most suited to the type of the pesticide of interest and the applicability of analytical equipment generally available.1. Introduction1.1 Background to the ProjectResearch undertaken by the Horticultural Research Group at the University of Tasmania, into pesticide residues in essential oils has been ongoing for several years and has dealt with the problems specific to the analysis of residues within the matrix of essential oils. Analytical methods for pesticides have been developed exploiting the high degree of specificity and selectivity afforded by high resolution gas chromatography mass spectrometry. Standard curves, reproducibility and detection limits were established for each. Chemicals, otherwise not amenable to gas chromatography, were derivatised and incorporated into a separate screen to cover pesticides with acidic moieties.Research has been conducted into low resolution GC mass selective detectors (MSD and GC ECD. Low resolution GC MSD achieved detection to levels of 1 mgkg-1 in boronia oil, whilst analysis using GC ECD require a clean-up step to effectively detect halogenated chemicals below 1mgkg-1.Dithane (mancozeb) residues were digested using acidified stannous chloride and the carbon disulphide generated from this reaction analysed by GC coupled to FPD in the sulphur mode.Field trials in peppermint crops were established in accordance with the guidelines published by the National Registration Authority (NRA), monitoring the dissipation of Tilt and Folicur residues in peppermint leaves and the co-distillation of these residues with hydro-distilled peppermint oils were assessed.Development of extraction protocols, analytical methods, harvest monitoring and field trials were continued and were detailed in a subsequent report. Solvent-based extractions and supercritical fluid extraction (SFE) was found to have limited application in the clean-up of essential oilsIn conjunction with Essential Oils of Tasmania (EOT), the contamination risk, associated with the introduction of a range of herbicides, was assessed through a series of field trials. This required analytical method development to detect residues in boronia flowers, leaf and oil. The methodology for a further nine pesticides was successful applied. Detection limits for these chemicals ranged from 0.002 mgkg-1 to 0.1 mgkg-1. In addition, methods were developed to analyse for herbicides with active ingredients (ai) whose structure contained acidic functional groups. Two methods of pesticide application were trialed. Directed sprays refer to those directed on the stems and leaves of weeds at the base of boronia trees throughout the trial plot. Cover sprays were applied over the entire canopy. For all herbicides for which significant residues were detected, it was evident that cover sprays resulted in contamination levels ten times those occurring as a result of directed spraying in some instances. Chloropropham, terbacil and simazine presented potentially serious residue problems, with translocation of the chemical from vegetative material to the flower clearly evident.Directed spray applications of diuron and dimethenamid presented only low residue levels in extracted flowers with adequate control of weeds. Oxyflurofen and the mixture of bromacil and diuron (Krovar) presented only low levels of residues when used as a directed spray and were effective as both post and pre-emergent herbicides. Only very low levels of residues of both sethoxydim and norflurazon were detected in boronia oil produced in crops treated with directed spray applications. Sethoxydim was effective as a cover spray for grasses whilst norflurazon showed potential as herbicide to be used in combination with other chemicals such as diuron, paraquat and diquat. Little contamination of boronia oils by herbicides with acidic moieties was found. This advantage, however, appears to be offset by the relatively poor weed control. Both pendimethalin and haloxyfop showed good weed control. Both, however, present problems with chemical residues in boronia oil and should only be used as a directed sprayThe stability of tebuconazole, monocrotophos and propiconazole in boronia under standard storage conditions was investigated. Field trials of tebuconazole and propiconazole were established in commercial boronia crops and the dissipation of both were monitored over time. The amount of pesticide detected in the oils was related to that originally present in the flowers from which the oils were produced.Experiments were conducted to determine whether the accumulation of terbacil residues in peppermint was retarding plant vigour. The level recorded in the peppermint leaves were comparatively low. Itis unlikely that terbacil carry over is the cause for the lack of vigour in young peppermint plants.Boronia oils produced in 1996, 1997 and 1998 were screened for pesticides using the analytical methods developed. High levels of residues of propiconazole were shown to persist in crops harvested up until 1998. Field trials have shown that propiconazole residues should not present problems if the fungicide is used as recommended by the manufacturers.1.2 Objectives♦Provide the industry, including the Standards Association of Australia Committee CH21, with a concise practical reference, immediately relevant to the Australian essential oil industry♦Facilitate the transfer of technology from a research base to practical application in routine monitoring programs♦Continue the development of analytical methods for the detection of metabolites of the active ingredients of pesticide in essential oils.♦Validate the methods developed.♦Provide industry with data supporting assurances of quality for all exported products.♦Provide a benchmark from which Australia may negotiate the setting of a realistic maximum residue limit (MRL)♦Determine whether the rate of uptake is relative to the concentration of active ingredient on the leaf surface may establish the minimum application rates for effective pest control.1.3 MethodologyThree approaches were used to achieve the objectives set out above.♦Continue the development and validation of analytical methods for the detection of pesticide residues in essential oils. Analytical methods were developed using gas chromatography high resolution mass spectrometry (GC HR MS), GC ECD, GC FPD and high pressure liquid chromatography with detection using MSMS.♦Provide industry with data supporting assurances of quality for all exported products.♦Coordinate research results into a comprehensive manual outlining practical approaches to the development of analytical proceduresOne aspect of the commissioning of this project was to provide a cost effective analytical resource to assess the degree of the pesticide contamination already occurring in the essential oils industry using standard pesticide regimens. Oil samples from annual harvests were analysed for the presence of pesticide residues. Data from preceding years were collated to determine the progress or otherwise, in the application of best agricultural practice (BAP).2. Experimental Protocols & Detailed ResultsThe experimental conditions and results are presented under the following headings:♦Method Development♦Monitoring of Commercial Harvests♦Production of a Manual2.1 Method DevelopmentMethod development focussed on the effectiveness of clean-up techniques, validation of existing techniques, the assessment of the application of GC ECD and FPD and high pressure liquid chromatography with ion trap MS, MS detection.2.1.1 Clean-up Methodologies2.1.1.i. Application of Disposable SPE cartridges in the clean-up of pesticide residues in essentialoilsLiterature reviews provided limited information with regards to the separation of contaminants within essential oils. The retention characteristics of disposable C18 cartridges were trialed.Experiment 1;Aim : To assess the capacity of disposable C18 cartridges to the separation of boronia oil components. Experimental : Boronia concrete (49.8 mg) was dissolved in 0.5 mL of acetone and 0.4 mL of chloroform was added. 1mg of octadecane was added as an internal standard. A C18 Sep-Pak Classic cartridge (short body) was pre- conditioned with 1.25 mL of methanol, which was passed through the column at 7.5 mLmin-1, followed by 1.25 mL of acetone, at the same flow rate. The boronia samplewas then applied to the column at 2 mLmin-1 flow and eluted with 1.25 mL of acetone / chloroform (5/ 4) and then eluted with a further 2.5 mL of chloroform. 5 fractions of 25 drops each were collected. The fractions were analysed by GC FID using the following parametersAnalytical parameters6890PackardHewlettGCcolumn: Hewlett Packard 5MS 30m, i.d 0.32µmcarrier gas instrument grade nitrogeninjection volume: 1µL (split)injector temp: 250°Cdetector temp: 280°Cinital temp: 50°C (3 min), 10°Cmin-1 to 270°C (7 mins)head pressure : 10psi.Results : Table 1 record the percentage volatiles detected in the fractions collectedFraction 1 2 3 4 5 % components eluting 18 67 13 2636%monoterpenes 15%sesquiquiterpenes 33 65 2%high M.W components 1 43 47 9Table 1. Percentage volatiles eluting from SPE C18 cartridgesDiscussion : The majority of boronia components eluted on the solvent front, effecting minimal separation. This area of SPE clean-up of essential oils requires a wide ranging investigation, varying parameters such as cartridge type and polarity of mobile phase.Experiment 2.Aim : For the development of methods using LC MSMS without clean-up steps, the potential for oil components to accumulate on the reverse phase (RP) column must be assessed. The retention of essential oil components on SPE C18 cartridges, using the same mobile phase as that to be used in theLC system, would provide a good indication as to the risk of contamination of the LC columns withoil components.Experimental: Parsley oil (20-30 mg) was weighed into a GC vial. 200 µL of a 10 µgmL-1 solution (equivalent to 100mgkg-1 in oil) of each of sethoxydim, simazine, terbacil, prometryn, tebuconazoleand propiconazole were used to spike the oil, which was then dissolved in 1.0 mL of acetonitrile. The solution was then slowly introduced to the C18 cartridge (Waters Sep Pac 'classic' C18 #51910) using a disposable luer lock, 10 mL syringe, under constant manual pressure, and eluted with 9 mLs of acetonitrile. Ten, 1 mL fractions were collected and transferred to GC vials. 1mg of octadecane was added to each vial and the samples were analysed by GC FID under the conditions described in experiment 1.The experiment was repeated using C18 cartridges which had been pre-conditioned with distilled waterfor 15 mins. Again, parsley oil, spiked with pesticides was eluted with acetonitrile and 5 x 1 mL fractions collected.Results: The majority of oil components and pesticides were eluted from the C18 cartridge in the firsttwo fractions. Little to no separation of the target pesticides from the oil matrix was achieved. Table2 lists the distribution of essential oil components in the fractions collected.Fraction 1 2 3 4 5 % components eluting 18 67 13 2663%monoterpenes 15%sesquiquiterpenes 33 65 2%high M.W components 1 43 47 9water conditioned% components eluting 35 56 8 12%monoterpenes 3068%sesquiquiterpenes 60 39 1 0%high M.W components 0 50 42 7Table 2. Percentage volatiles eluting for SPE C18 cartridgesFigure 1 shows a histogram of the percentage distribution of components from the oil in each of the four fractions.Figure 1. Histogram of the percentage of volatiles of distilled oils in each of four fraction elutedon SPE C18 cartridges (non-preconditioned)Figure 2. Histogram of the percentage of volatiles of distilled oils in each of four fraction elutedon SPE C18 cartridges (preconditioned)Discussion : The chemical properties of many of the target pesticides, including polarity, solubility in organic solvents and chromatographic behaviour, are similar to the majority of essential oil components. This precludes the effective separation of analytes from such matrices through the use of standard techniques, where the major focus is pre-concentration of pesticide residues from water or water based vegetative material. However, this experiment served to provide a good indication that under HPLC conditions, where a reverse phase C18 column is used in conjunction with acetonitrile / water based mobile phases, essential oil components do not remain on the column.。

Sustainable desalination using a microbial capacitive desalination cell †Casey Forrestal,a Pei Xu b and Zhiyong Ren *aReceived 15th January 2012,Accepted 13th March 2012DOI:10.1039/c2ee21121aMicrobial desalination cells (MDCs)use the electrical current generated by microbes to simultaneously treat wastewater,desalinate water,and produce bioenergy.However,current MDC systems transfer salts to the treated wastewater and affect wastewater’s beneficial use.A microbial capacitivedesalination cell (MCDC)was developed to address the salt migration and pH fluctuation problems facing current MDCs and improve the efficiency of capacitive deionization.The anode and cathode chambers of the MCDC were separated from the middle desalination chamber by two speciallydesigned membrane assemblies,which consisted of cation exchange membranes and layers of activated carbon cloth (ACC).Taking advantage of the potential generated across the microbial anode and the air-cathode,the MCDC was capable of removing 72.7mg total dissolved solids (TDS)per gram of ACC without using any external energy.The MCDC desalination efficiency was 7to 25times higher than traditional capacitive deionization pared to MDC systems,where the volume of concentrate can be substantial,all of the removed ions in the MCDC were adsorbed in the ACC assembly double layer capacitors without migrating to the anolyte or catholyte,and the electrically adsorbed ions could be recovered during assembly regeneration.The two cation exchange membrane based assemblies allowed the free transfer of protons across the system and thus prevented significant pH changes observed in traditional MDCs.IntroductionThe increasing awareness of the water-energy nexus is compelling the development of technologies that reduce energy requirements during water treatment as well as water demands for energy production.1,2Microbial desalination cells (MDCs)recentlyemerged as a promising technology to simultaneously treat wastewater,desalinate saline water,and produce renewable energy such as electricity or hydrogen gas.3–10MDCs share the same principle of bioelectrochemical reactions with microbial fuel cells (MFCs):electrochemically active bacteria in the anode chamber oxidize biodegradable substrates and generate electron flow (i.e.current)to reduce the electron acceptors in the cathode chamber.The additional desalination function can be achieved in an MDC by adding a middle chamber containing saline water and utilizing the anode–cathode potential difference to drive the migration of anions (e.g.,Cl À)to the anode chamber and cations (e.g.,Na +)to the cathode chamber for charge neutrality.3The MDC process carries great potential in desalination systems,aDepartment of Civil Engineering,University of Colorado Denver,Denver,CO 80004,USA.E-mail:zhiyong.ren@;Tel:+1(303)556-5287bCivil and Environmental Engineering,Colorado School of Mines,Golden,CO 80401,USA†Electronic supplementary information (ESI)available:Two additional figures are included.See DOI:10.1039/c2ee21121aDynamic Article Links CEnergy &Environmental ScienceCite this:Energy Environ.Sci.,2012,5,/eesPAPERP u b l i s h e d o n 13 M a r c h 2012. D o w n l o a d e d b y D a l i a n U n i v e r s i t y o f T e c h n o l o g y o n 13/03/2014 10:23:29.View Article Online / Journal Homepage / Table of Contents for this issuebecause it can either be used as a stand-alone process or serve as a pretreatment for conventional desalination processes such as reverse osmosis (RO)to reduce the salt concentration of RO feed and minimize energy consumption and the membrane fouling potential.Current desalination technologies,such as RO and electrodialysis (ED),are energy and capital intensive.Even the most advanced large scale seawater RO units require 3–7kW h m À3for water desalination,while conventional multi-stage flash evaporation requires 68kW h m À3.11In contrast,the MDC system is considered to be an energy gaining process because it converts the biochemical energy stored in wastewater to elec-tricity or hydrogen b scale MDC studies showed that 180–231%more energy can be recovered as H 2than the reactor energy input when desalinating 5–20g L À1NaCl solutions,4,6and a recent study calculated that a litre-scale MDC can produce up to 58%of the electrical energy required by downstream RO systems.8Current MDC systems use an anion exchange membrane (AEM)to separate the anode and middle chamber,and a cation exchange membrane (CEM)to separate the cathode and middle chamber.Similar to electrodialysis,desalination in MDC is achieved by direct transport of salts from the middle chamber to the anode and cathode chamber.This system faces two main problems.While salts get removed from the middle chamber,they become concentrated in the anode and cathode chambers,resulting in an increase of the volume of saline water.This concern becomes more imperative when wastewater is treated as the anolyte.Although the addition of ions (or total dissolved solids,TDS)increases wastewater conductivity and benefits electricity generation,the increased salinity may affect effluent water quality and prevent subsequent beneficial use of treated wastewater.12,13The high salinity may also affect wastewater treatment efficiency in MDCs because studies showed that high chloride concentration is inhibitory to biological treatment,especially for nutrient removal.14In addition,the AEM between the anode and middle chamber inhibits the free transfer of H +accumulated in the anolyte to other chambers,which causes a significant pH drop in the anode chamber and pH increase in the cathode chamber.15,16A previous study showed that the pH of the wastewater anolyte dropped to 4.2in one batch cycle if no buffer was added to the anolyte.6Additionally,the catholyte pH could increase to 11–13due to the loss of H +.6,16This pH fluc-tuation significantly inhibits bioelectrochemical reaction effi-ciency and reduces system performance.In order to modulate the movement of salts to the anode and cathode chambers,the concept of capacitive deionization (CDI)17was incorporated in this study to develop a sustainable desalination system called a microbial capacitive desalination cell (MCDC).In the proof-of-concept MCDC,salt water can be deionized through electrochemical ion adsorption driven by the electrical field generated by microorganisms.Two activated carbon cloth (ACC)membrane assemblies were designed to connect with the anode and cathode and adsorb ions from water.During desalination,the ions are stored in the electrical double layer capacitors between the solution and the ACC assembly interfaces,thus preventing the salinity increase in treated wastewater.After the ACC is saturated with adsorbed ions,the assembly can be regenerated by removing the electrical potential and the retained salts can be fully recovered in situ for disposal orfurther salt recovery.Another innovative aspect of the MCDC,as compared to conventional MDC,is the use of a second CEM in lieu of AEM between the anode and desalination chamber (Fig.1).This configuration allows cations and protons to move freely from the anode chamber throughout the reactor and therefore maintains electrochemical neutrality and prevents pH fluctuation.In this study,the proof-of-concept MCDC devel-opment and operation are demonstrated,and its advantages over current systems and application potentials are discussed.Materials and methodsMCDC reactor designThe MCDC reactors consisted of three polycarbonate cube-shaped blocks with 3cm diameter holes forming an internal anode,cathode,and desalination chamber volume of 23mL,27mL,and 10mL respectively.The anode and cathode cham-bers had a length of 4cm,while the desalination chamber had a length of 1.5cm.The anode electrode was a graphite brush (Golden Brush,CA)and was pretreated by washing in acetone and heating to 350 C for 30minutes.18Traditional air-cathodes were made by applying 10%Pt/C (0.5mg cm À2)and four PTFE diffusion layers on 30%wet-proofed carbon cloth as previously described.19The desalination chamber was separated from the anode and cathode chamber by two assemblies.Each assembly was constructed by placing a cation exchange membrane (CMX-SB,Astom Corporation,Japan),a Ni/Cu mesh current collector (McMaster Carr,IL),and 3layers of Zorflex Òactivated carbon cloth (ACC,Chemviron Carbon,UK)together.Additionally,the CEM faced the anode/cathode chamber to prevent microbial growth on the assembly.The total weight of the ACC was 1gram with the specific surface area of 1019.8m 2g À1,determined by the Brunauer–Emmet–Teller (BET)method (ASAP 2020,Micro-meritics,Norcross,GA).20The ACC assemblies were connected to the anode/cathode by titanium wires (Fig.1).MCDC operating conditionsTwo reactors were inoculated with anaerobic sludge from the Englewood-Littleton Wastewater Treatment Plant (Englewood,CO)and operated in fed-batch MFC mode.When arepeatableFig.1Diagram of MCDC reactor configuration and operation.Two CEM-ACC assemblies were used to separate the three chambers and capture the removed salts,as well as allow the free transfer of protons.P u b l i s h e d o n 13 M a r c h 2012. D o w n l o a d e d b y D a l i a n U n i v e r s i t y o f T e c h n o l o g y o n 13/03/2014 10:23:29.voltage profile was obtained for consecutive batch cycles,the reactors were shifted to fed-batch MCDC mode by inserting a pair of assemblies and adding one middle chamber as described previously.The anolyte growth media contained per litre:1.6g NaCH 3COO,0.62g NH 4Cl, 4.9g NaH 2PO 4$H 2O,9.2g Na 2HPO 4,0.26g KCl,and 10mL trace metals and 10mL vitamin solution.21The catholyte contained per litre:10g KCl,0.68g KH 2PO 4,0.87g K 2HPO 4.Potassium chloride was used in the cathode chamber to differentiate with sodium transport and monitor the movement of cations from the cathode to the desalination chamber.The salt solution in the desalination chamber contained per litre:10g NaCl,0.49g NaH 2PO 4$H 2O,0.92g Na 2HPO 4.A small amount of buffer was added to the salt solution to some extent mimic the 300to 700m mol kg À1natural buffering capacity of seawater and prevent potential scaling at high pH values.22Two experimental procedures and two controls were per-formed to investigate the desalination performance of the MCDC system.The first experiment investigated simultaneous physical and electrical adsorption capacity by directly adding salt solution into the desalination chamber equipped with ACC assemblies free of adsorbed ion.When the anode and cathode electrodes were connected to the ACC assemblies,physical and electrical adsorption on the ACC assemblies could occur concurrently.The second experimental procedure investigated only electrical adsorption capacity.Electrical adsorption capacity of the ACC assemblies was determined by first adding salt solution to the desalination chamber to allow complete physical adsorption.Electrical adsorption was then characterized by replacing the desalted solution with fresh solution,and con-necting the two assemblies to the anode and cathode,respec-tively.Any residual water from previous experimental washing would have been removed when the salt solution was replaced.Abiotic control experiments were performed by using new brush anodes without bacterial acclimation.The first control experiment measured the physical adsorption capacity by short circuiting the assemblies to ensure no charge was formed across the electrodes.The adsorption capacity of the assemblies was defined as the change in initial and final salt concentration.The second control investigated the electrical adsorption capacity by first allowing complete physical adsorption to occur then by connecting the assemblies to an external power supply at a voltage of 0.53V to simulate the voltage generated by a microbial fuel cell.The MDC control experiment used an anion exchange membrane next to the anode chamber (Astom Corporation,Japan)and a CEM next to the cathode chamber without ACC assemblies in the desalination chamber.An external resistor of 1000Ohms was used between the anode and cathode electrodes,and all other experimental procedures were identical to the MCDC experiments.To regenerate the ACC assemblies in situ for all experiments,the assemblies were either allowed to naturally regenerate or were regenerated by applying an external voltage to increase the rate of regeneration.The natural regeneration was performed by disconnecting the anode and cathode from the assemblies and creating a short circuit between the assemblies with an external wire.Alternatively,an external voltage of 1V in reverse polarity was applied to the assemblies by a programmable power source.The external voltage was applied for 5–10minutes and followedby short circuiting the ACC assemblies,as mentioned above,for 20–30minutes.When the potential difference reached Æ0.5mV,the ACCs were assumed to be regenerated,meaning that any electrically adsorbed ions should have been removed from the electrodes.After regeneration,all electrolytes were emptied and washed with deionized (DI)water to remove any residual salt remaining in the chambers before starting a new batch cycle.Analysis and calculationsConductivity and pH were measured for all three chambers using a conductivity meter and pH meter (HACH Co.,CO).The change in the reactor’s internal resistance was determined through electrochemical impedance spectroscopy (EIS)tests using a Potentiostat.EIS measurements were performed by using the anode as the working electrode,the cathode as the counter electrode,and a saturated Ag/AgCl reference electrode placed in the anode chamber.Results were fitted into equivalent circuit models developed in our previous EIS studies and plotted using Nyquist plots where the ohmic resistance is defined as the intercept of the Zreal axis.21Samples of all three chambers were collected before and after desalination,and after regeneration.Ion concentrations were measured using the Optima 3000Inductive Coupled Plasma (ICP)Spectrometer (Perkin Elmer,CT)and Dionex DC80ion chromatography system (IC)(Dio-nex,CA).Using the data from the IC and ICP,the mass balance of the major ions was determined by summing the concentrations of the ions in each chamber initially,after desalination,and after recovery of the salts.Internal power used was calculated using the following equations:P ¼ðV 2Rd t (1)R ¼r L A(2)where P is power in terms of watt hours,V is the voltage,R is the resistance,r is resistivity,L is length of the resistance,and A is the cross-sectional parisons between the MCDC and CDI were made based off either presented data or estimations from figures in published parison to membrane capacitive deionization (MCDI)was not conducted due to the incompatibility in methodology to the MCDC.Results and discussionReactor desalination performanceDuring MCDC operation,an electrical potential was generated across the microbial anode and air-cathode and applied to the two ACC assemblies to form a double layer capacitor 23–30(Fig.1).The formation of the double layer capacitor has been fully modeled using the Gouy Chapman–Stern theory.29The potential drives the ions to move from the salt solution and adsorb on the activated carbon cloths.The ion adsorption can be observed proportional to the charge formed between the ACC assemblies (Fig.2).Fig.2shows that,in repeated batch cycles,when the potential on the assemblies increases from 0to more than 530mV in each cycle,the solution conductivity in the desalination chamber decreased by 12–18%,from 18mS cm À1toP u b l i s h e d o n 13 M a r c h 2012. D o w n l o a d e d b y D a l i a n U n i v e r s i t y o f T e c h n o l o g y o n 13/03/2014 10:23:29.below 16mS cm À1.The desalination rate was the greatest at the beginning of each cycle and then decreased gradually,suggesting the adsorption capacity of the ACC assemblies decreased along with the increased amount of salt that had been adsorbed in the assemblies.Salt removal was characterized by both conductivity,measured using a conductivity meter,and total dissolved solids (TDS)concentration,measured by IC and ICP (Table 1).Through simultaneous physical and electrical adsorption,the MCDC removed 26.9%of the conductivity or 25.5%of TDS from the desalination chamber in one batch cycle.In addition,a small percentage of salt was removed from the anolyte (4.4%)and catholyte (10.4%)as well.This is likely due to the ions that were driven across the membranes by the electrical potential of the ACC assemblies from the anode and cathode chamber and then adsorbed onto the ACC.Further experiments showed that electrical adsorption alone removed 22.3%TDS from the desa-lination chamber,which contributed up to 88%of the TDS removal compared to the combined physical and electrical adsorption experiments.Table 2compares the normalized TDS removal between the MCDC and CDI studies.The results showed that for the same amount of adsorptive material (ACC),the MCDC improved TDS adsorption by 7–25times.Both MCDC and CDI use an electric field between two electrodes that electrochemically adsorb ions,but the high adsorption from the MCDC may be attributed to the unique feature of the MCDC that uses the internal potential generated by microorganisms.This in situapproach avoided the use of an external power supply and circuit and reduced transportation energy loss,so it demonstrated higher efficiency than traditional CDI processes.The salt adsorption rate in MCDC,however,is lower than published CDI studies,and that is mainly due to the low kinetics of the fed-batch operation and the limited amount of ACC available for ion adsorption.In this study,the MCDC configuration was modified from traditional cubic type MDCs,which only allowed for a total of 1g activated carbon cloth being used in the assembly.This may explain why the amount of salt removed in the desa-lination chamber was relatively small.It was calculated that the amount of salt added in the desalination chamber (114mg TDS)was drastically beyond the control electrical adsorption capacity of the ACC (8.5mg TDS for the 1g ACC applied).Moreover,compared to CDI systems that consume 0.21–1.78Watt hour external energy to generate the potential to remove 1g TDS,the MCDC system does not use any external energy but instead utilized the in situ potential difference between the ACC assem-blies generated during microbial activities.It was calculated that the MCDC reactor saved 2.18Watt hour for 1g of TDS removed.That is why in Table 2the net energy used for the MCDC is negative,indicating that 2.18W h g À1TDS removed was not required,while for the CDI systems an external energy of 0.2–1.78W h is required for removal of 1g TDS.While the MCDC reactor directly uses generated current for desalination,it is possible for electricity to be generated by applying an external load across the ACC assemblies during regeneration.Reactor configuration optimization is underway to increase the ACC loading and further improve desalination efficiency.Sodium,chloride,potassium,and phosphate accounted for greater than 85%of the TDS,and their specific concentration changes in the three chambers are shown in Fig.3.In addition to direct capacitive electrical adsorption that caused concentration decreases in the desalination chamber,a small amount of charged ions migrated from the anode and cathode chamber to the desalination chamber due to the electrical potential or concen-tration gradient.However,the desalination efficiency for the anode and cathode chambers is low compared to the salt removal in the desalination chamber due to the lack of electrical double layer adsorption and the inhibited anion transfer across cation exchange membranes.Results in Table 1showed that saline water can also be used as the catholyte and partially desalinated.Further desalination can be achieved by feeding the treated catholyte to the subsequent reactor’s desalination chamber.The reactor’s internal resistance as measured by EIS at the beginning of the desalination cycle was on average 8.5Ohms.After desalination,the internal resistance increased to anaverageFig.2The correlation between the charge potential across the ACC assemblies and the conductivity changes in the desalination chamber due to electrical adsorption.Arrows indicate changes in electrolyte solution in batch cycles.Table 1Salt removal in terms of conductivity and total dissolved solids in the MCDCPhysical/electrical adsorption Electrical adsorption Desalination chamberAnode chamber Cathode chamber Desalination chamber Anode chamber Cathode chamber %Removal in conductivity 26.9Æ5.113.1Æ3.8 5.6Æ4.410.0Æ0.210.6Æ3.5À2.0Æ2.7%Removal in TDS 25.2Æ3.64.4Æ3.610.4Æ3.622.3Æ3.67.6Æ3.62Æ3.6Total TDS adsorption (mg TDS per g ACC)72.750.7P u b l i s h e d o n 13 M a r c h 2012. D o w n l o a d e d b y D a l i a n U n i v e r s i t y o f T e c h n o l o g y o n 13/03/2014 10:23:29.of 13Ohms (ESI†).The change in conductivity in the desalination chamber correlated closely with the change in internal resistance for the reactor over the course of desalination.The MCDC reactor’s ability to transfer electrons was not inhibited as occurs over the course of desalination in standard MDCs.It is theorized that this is due to the MCDC’s ability to maintain charge neutrality better than MDC reactors.In standard MDC reactors,charge neutrality is reached by ion migrating out of the desali-nation chamber,while in the MCDC reactor charge neutrality is performed by ion migrating through the entire reactor.Assembly regeneration and salt recoveryThe ion saturated ACC assemblies were regenerated using two approaches.The natural regeneration was accomplished by directly connecting the two assemblies in short circuit.The electrical potential across the assemblies was dissipated with the adsorbed salts being released back into solution.When the potential difference across the ACC assemblies reached Æ0.5mV,it was assumed that the ACC assemblies were regen-erated with complete electrical salt desorption.The regeneration rate can be significantly increased by connecting the assemblies to an external power supply of 1V with reverse polarity to facilitate ion desorption (ESI†).Fig.4shows that among the four major ion species,almost all of the electrical adsorbed salts wererecovered during assembly regeneration,shown as a direct correlation between the initial and recovered salt concentrations.The capability of in situ regeneration of the ACC assemblies is another advantage of the MCDC,because the assemblies can be reused many times without investing significantly in materials.Almost all of the adsorbed salts can be recovered in concentrates during regeneration,and the recovered salts can be dewatered or extracted for beneficial uses.Furthermore,MCDC stacks can be developed and integrated with reverse electrodialysis (RED)to capture the energy generated due to the salinity gradient across the concentrate and freshwater.31,32The current MCDC is operated in batch mode,and the desalination and regenerated processes were conducted sequentially.More efficient operation can be achieved by connecting multiple reactors in series or in parallel and operating them in complementary sequential batch reactor (SBR)modes.While some of the units perform desali-nation,others conduct assembly regeneration at the same time.This operation not only provides a continuous flow of produced freshwater but also allows for the direct usage of the electricity produced from regeneration units for desalination units.Reduced pH fluctuationFig.5shows the change in pH units among the three chambers over one typical batch cycle for both the MCDC and the controlTable 2Desalination efficiencies in the MCDC and CDI reactors a Method Electrode materialsElectrodedistance (mm)Net W h/g TDS removed mg TDS/g adsorptive material Reference #MCDC Activated carbon cloth 15À2.1850.74This paper CDI Carbon aerogel2.3+0.217.0017CDI Activated carbon powder NA +1 1.9524CDI Activated carbon powder 0.1+1.78 2.8825CDI Activated carbon powder0.1+1.68 3.1126CDI Activated carbon powder with mesoporous carbon black0.22NA 3.8227CDI MnO 2/nanoporous carbon composite NA NA 0.1028CDI Activated carbon clothNA +0.52NA 30CDIActivated carbon cloth with titaniaNANA4.3834aNA ¼notavailable.Fig.3The concentration changes of the four major ions (potassium,sodium,chloride,phosphate)before and after one typical batch cycle of MCDCoperation.Fig.4Mass balance of the four major ions (potassium,sodium,chlo-ride,phosphate)in the MCDC reactor before and after regeneration.P u b l i s h e d o n 13 M a r c h 2012. D o w n l o a d e d b y D a l i a n U n i v e r s i t y o f T e c h n o l o g y o n 13/03/2014 10:23:29.MDC.The initial pH values in the chambers were all within 7.0Æ0.2.The change in pH for the anode chamber in both the MCDC and the MDC was relatively small,with a drop in pH of between 0.2and 0.5pH units,which was presumably attributed to the high buffering capacity of the anolyte.However,the catholyte had drastically different results between the MCDC and MDC,with the MCDC increasing in pH on average 1.5pH units and the MDC increasing 4.4pH units.Interestingly the change in pH for the desalination chamber for the MCDC is greater than for the MDC control.Previous capacitive deionization studies showed that water electrolysis may cause slight pH variation at low voltages,which may explain the pH increase in the MCDC desalination chamber.30It is difficult to compare the MCDC results directly with CDI studies,because no known CDI experiments have been conducted at a set potential lower than 0.6V.23–30Further investigations should explore the cause of this phenomenon.Because the average percent change between the cathode and desalination chamber were essentially the same,it is assumed that the proton transfer capability of the reactor was not inhibited.The MCDC employs a CEM to separate the anode and desalination chamber.This is different from the AEM used in current MDCs and releases the pH fluctuations in the reactor.In traditional MDCs,anions (Cl À)migrate from the desalination chamber to the anode chamber to compensate for the accumu-lation of H +,but because the AEM prevents the transfer of H +out of the anode chamber,a decrease in pH is observed.By using a CEM,the accumulated H +not only can transfer to the desa-lination chamber but also can transfer further to the cathode chamber and therefore solves the pH change problem in the entire MCDC reactor.Previous studies show that other ions such as Na +and K +also play important roles in maintaining charge balances across different chambers in microbial fuel cells,33but the majority of such ions are adsorbed in the ACC assemblies,so electrolyte charge balance due to ion transfer is not a concern in the MCDC.OutlookThe integration of capacitive deionization with microbial desa-lination provides a sustainable solution that not only addressesthe salt migration and pH fluctuation problems facing current MDC systems,but also improves salt removal and energy effi-ciency compared to CDI systems.Traditional MDCs remove salts from the desalination chamber,but they also add TDS to the anode and cathode chambers and may increase the volume of saline water significantly,depending on different operation configurations.3–10The MCDC reactor demonstrated that desa-lination can be accomplished in the middle chamber without adding salts to the anolyte and catholyte,and therefore released the concerns on the viability of wastewater treatment and reuse due to increased TDS concentration.This proof-of-concept system also demonstrates a microbial desalination reactor to reduce salinity in all three chambers of the reactor.The MCDC system offers a sustainable desalination,renewable energy production,and wastewater treatment.To maximize the benefits and prevent negative effects of salinity changes on the waste-water anolyte,salt migration from the desalination chamber could be modulated by constructing modular plate-shaped ACC-membrane assemblies.If added salt is desired in wastewater to improve the anolyte conductivity,regular MDC operation could be performed.If salt should be prevented from migrating into the anode chamber,the modular ACC assembly plate can be inserted into the reactor to perform salt adsorption.This system inte-gration and operation will provide microbial desalination systems with great flexibility in salt migration management as well as better pH fluctuation control.Despite the potential benefits offered by the MCDC system,many challenges remain to be addressed based on the informa-tion collected from this proof-of-concept study.In addition to the low-cost material development that is required for all bio-electrochemical systems,the adsorptive material can be improved,such as with silica or titanium modification.34,35The reactor configuration needs to be optimized to provide more ACC loading and improve diffusion rate and adsorption capa-bility.Modular stack reactors and flexible operation strategies need to be developed to maximize the integration of desalination and assembly regeneration in multiple units,optimize water recovery,and enhance salt migration management.Improve-ments in MCDCs will also benefit from the continued advances of other bioelectrochemical systems such as microbial fuel cells and capacitive deionization,with the eventual goal of developing a full scale sustainable system directed toward the integration of multiple functions,such as extracting energy from wastewater and water desalination.AcknowledgementsThis work was supported by the Office of Naval Research (ONR)under Awards N0001410M0232.We thank Dr Peter Jenkins for his suggestions and reviewers for their helpful comments.References1M.M.Pendergast and E.M.V.Hoek,Energy Environ.Sci.,2011,4,1946–1971.2J.L.Schnoor,Environ.Sci.Technol.,2011,12,5065.3X.Cao,X.Huang,P.Liang,K.Xiao,Y.Zhou,X.Zhang and B.E.Logan,Environ.Sci.Technol.,2009,43,7148–7152.4M.Mehanna,T.Saito,J.L.Yan,M.Hickner,X.X.Cao,X.Huang and B.E.Logan,Energy Environ.Sci.,2010,3(8),1114–1120.Fig.5The change in pH for the MCDC anode,cathode,and desali-nation chambers compared to the control MDC in one batch cycle.The initial pH values in the chambers of MDC or MCDC all ranged 7.0Æ0.2.P u b l i s h e d o n 13 M a r c h 2012. D o w n l o a d e d b y D a l i a n U n i v e r s i t y o f T e c h n o l o g y o n 13/03/2014 10:23:29.。

This article appeared in a journal published by Elsevier.The attached copy is furnished to the author for internal non-commercial research and education use,including for instruction at the authors institutionand sharing with colleagues.Other uses,including reproduction and distribution,or selling or licensing copies,or posting to personal,institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle(e.g.in Word or Tex form)to their personal website orinstitutional repository.Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:/copyrightAuthor's personal copyWhat is the risk of European sovereign debt defaults?Fiscal space,CDS spreads and market pricing of risk qJoshua Aizenman a ,*,Michael Hutchison b ,Yothin Jinjarak caRobert R.and Katheryn A.Dockson Chair in Economics and International Relations,USC,Los Angeles,CA 90089,USA bDepartment of Economics,UC Santa Cruz,Santa Cruz,CA 95064,USA cDeFiMS,SOAS,University of London,London WC1H0XG,UKJEL classi fications:E43F30G01H631Keywords:CDS spreads Sovereign risk Fiscal space Default risk Eurozonea b s t r a c tWe estimate the pricing of sovereign risk for fifty countries based on fiscal space (debt/tax;de ficits/tax)and other economic funda-mentals over 2005–10.We focus in particular on five countries in the South-West Eurozone Periphery,Greece,Ireland,Italy,Portugal and Spain.Dynamic panel estimates show that fiscal space and other macroeconomic factors are statistically and economically important determinants of sovereign risk.However,risk-pricing of the Eurozone Periphery countries is not predicted accurately either in-sample or out-of-sample:unpredicted high spreads are evident during global crisis period,especially in 2010when the sovereign debt crisis swept over the periphery area.We match the periphery group with five middle income countries outside Europe that were closest in terms of fiscal space during the European fiscal crisis.Eurozone Periphery default risk is priced much higher than the matched countries in 2010,even allowing for differences in fundamentals.One interpretation is that these economies switched to a “pessimistic ”self-ful filling expectational equilibrium.An alternative interpretation is that the market prices not on currentq We thank seminar participants at the Bank for International Settlements,Danmarks Nationalbank,the Bank of Canada,Columbia –Tsinghua international economics workshop,Chulalongkorn University (Sasin),Association for Public Economic Theory 2011Conference,and the National Institute for Public Finance and Policy 9th Research Meeting (New Delhi,India,2012)for very helpful comments.We also thank participants at the conference “The European Sovereign Debt Crisis:Background and Perspectives ”(Bank of Denmark,Copenhagen,April 3–4,2012),and especially our discussant,Marcus Miller,for helpful comments.*Corresponding author.E-mail address:aizenman@ (J.Aizenman).Contents lists available at SciVerse ScienceDirectJournal of International Moneyand Financejournal homepage :www.else /locate/jimf0261-5606/$–see front matter Ó2012Elsevier Ltd.All rights reserved./10.1016/j.jimon fin.2012.11.011Journal of International Money and Finance 34(2013)37–59Author's personal copybut future fundamentals,expecting adjustment challenges in the Eurozone periphery to be more dif ficult for than the matched group of middle-income countries because of exchange rate and monetary constraints.Ó2012Elsevier Ltd.All rights reserved.1.IntroductionDuring 2000–2006,the OECD and most emerging markets experienced a remarkable decline in macroeconomic volatility and the price of risk.This period turned out to be the tail-end of the Great Moderation ,a precursor of the turbulences leading to the global financial crisis of 2008–09,the consequent increase in risk premia,and the focus on fiscal challenges and the importance of fiscal space in navigating future economic challenges.The latter stages of the crisis,unfolding in 2010,focused attention on the heterogeneity of the Euro block,and the unique challenges facing the five South-West Eurozone Periphery countries,or SWEAP group (Greece,Ireland,Italy,Portugal,and Spain),in adjusting to fiscal fragility in the context of a ten-year old currency union.1This paper investigates the pricing of risk associated with the sovereign debt crisis that escalated during 2010in several European countries,and culminating in the selective default on Greek sovereign debt in early 2012.Our objective is to determine whether the perception of relatively high sovereign default risk of the fiscally distressed Euro area countries,as seen in market pricing of credit default swap (CDS)spreads,may be explained by existing past or current fundamentals of debt and de ficits relative to tax revenues –which we term de facto fiscal space –and other economic determinants.2Our analysis allows us to address several questions.Does fiscal space help systematically explain the evolution of the market pricing of risk beyond that embedded in other macroeconomic indicators?Was risk in some markets (e.g.SWEAP)“overpriced ”in 2010judging by model predictions using the pre-vailing values of fiscal space other macro variables?Our objectives for the empirical work are three-fold.Firstly,we determine whether CDS spreads (in a panel regression setting)are related to fiscal space measures and other economic determinants.Secondly,we address whether there is an identi fiable dynamic pattern to CDS spreads during the crisis period.Thirdly,we investigate pricing differentials of CDS spreads in the Euro area,and the SWEAP in particular,compared to other countries.We seek to answer the question of whether Euro area and SWEAP CDS spreads follow the same pattern as the rest of the world or may they considered “mis-priced ”in some sense,especially during the 2010European debt crisis.To this end,we develop a pricing model of sovereign risk for a large number of countries (50)within and outside of Europe,before and after the global financial crisis,based on fiscal space and other economic fundamentals including the foreign interest rate,external debt,trade openness,nominal depreciation,in flation,GDP/Capita and economic growth.We use this dynamic panel model to explain CDS spreads and determine whether the market pricing of risk is comparable in the affected European countries and elsewhere in the world.By this methodology,and using in-sample and out-of-sample predictions,we can determine whether there are systematically large prediction errors for the CDS spreads during the global financial crisis and in 2010when the sovereign debt crisis in Europe intensi fied.Systematically large prediction errors may be due to mispricing of risk or may be attrib-utable to expectations of a future decline in fundamentals that lead to higher default risk.We also “match,”on the basis of similar fiscal space,each SWEAP country with a corresponding Middle Income country.This provides additional information on the market pricing of risk between SWEAP and countries with similar fiscal conditions but,unlike SWEAP,with histories of debt restructuring.1The SWEAP acronym for these five countries is used in Buiter and Rahbari (2010).2Our measure of fiscal space is from Aizenman and Jinjarak (2010).They propose a stock and flow measure of de facto fiscal space.The stock variable is de fined as the inverse of the tax-years it would take to repay the public debt.In this paper,fiscal space is measured both as outstanding government debt and government de ficits,relative to the de facto tax base.The de ficits measure is the realized tax collection,averaged across several years to smooth for business cycle fluctuations.J.Aizenman et al./Journal of International Money and Finance 34(2013)37–5938Author's personal copyJ.Aizenman et al./Journal of International Money and Finance34(2013)37–5939 Our investigation reveals a complex and time-varying environment in the market for sovereign default risk.Specifically,wefind empirically a key role offiscal space in pricing sovereign risk, controlling for other relevant macro variables.The link is economically and statistically strong,and robust to the time dimension of the CDS premium,sample period,included control variables and estimation technique.Wefind that risk of default in the SWEAP group appeared to be somewhat “underpriced”relative to international norms in the period prior to the globalfinancial crisis and substantially“overpriced”countries during and after the crisis,especially in2010,with actual CDS values much higher than the model would predict given fundamentals.In addition,compared to the matched group,controlling forfiscal space and macroeconomic conditions,all of the SWEAP countries have much higher default risk assessments measured by CDS premiums.One potential explanation for the switch from under-to over-pricing of default risk is that markets were forward looking,not pricing entirely on current fundamentals but on expected further deterioration in future SWEAP fundamentals, especially in the realm offiscal space.Alternatively,the results are consistent with multiple equilib-rium with an abrupt switch from a“good”(optimistic)expectations equilibrium in the Euro Area–with low expected default rates and low interest rates wherefiscal positions are sustainable–to a“bad”(pessimistic)expectations equilibrium in these same countries–with high expected default rates and high interest rates wherefiscal positions are not sustainable.The next section discusses the data.The third section provides a preliminary statistical analysis.The fourth section presents the empirical results.We close the paper with a discussion on possible inter-pretation of the emerging SWEAP risk premia,including the handicapping effect of being a member of a currency union,which reduces the country’s scope of adjustment via exchange rate and monetary policy.2.CDS spreads as a measure of sovereign default risk2.1.CDS spreads on sovereign bondsWe measure the market perception of sovereign default risk by the spreads on sovereign credit default swaps(CDS)at various maturities(tenors).3CDS instruments are mainly transacted in over-the-counter(OTC)derivative markets.The spreads represent the quarterly payments that must be paid by the buyer of CDS to the seller for the contingent claim in the case of a credit event,in this case non-payment(or forced restructuring)of sovereign debt,and is therefore an excellent proxy for market-based default risk pricing.4The total CDS market grew from about10trillion USD in2004,when statistics werefirst systematically reported,to a peak prior to the globalfinancial crisis of almost60 trillion USD in mid-2008,and then fell sharply.The estimated gross(net)notional amount of sovereign CDS outstanding was2447(233)billion USD in2010,compared to about2196billion USD in government-issued international debt securities(BIS,2010).Fig.1shows the outstanding notional amounts of CDS instruments on sovereign bonds across countries in late February2011.Italy has the largest outstanding CDS notional amounts,at almost USD300billion,followed by Brazil,Spain and Turkey with notional amounts outstanding of over USD150billion.Sovereign CDS provide a market-based realtime indicator of sovereign credit quality and default risk.We consider sovereign CDS spreads at several maturities d three,five and ten-year maturities, across industrial countries and emerging markets.Despite the low probability of a credit event in most advanced economies,CDS markets are still active in most markets as buyers can use the sovereign CDS3See Packer and Suthiphongchai(2003)and Fontana and Scheicher(2010)for discussions of sovereign CDS markets.4An alternative proxy for default risk is the interest rate spread of sovereign debt.From an empirical standpoint,there are three main advantages of using CDS spreads rather than interest rate spreads.Firstly,CDS statistics provide timelier market-based pricing with larger coverage of industrial and developing countries than sources for national bond market rates. Secondly,using CDS spreads avoids the difficulty in dealing with time to maturity as in the case of using interest rate spreads(of which the zero yields would be preferred).Recent estimates from the Bank for International Settlements suggest that the average original and the remaining maturities of government debt instruments vary markedly across countries(BIS,2010). Thirdly,interest rate spreads embed inflation expectations and demand/supply for credit conditions as well as default risk.We are only interested in default risk.Author's personal copyas a hedge and for mark-to-market response.5Buyers of the sovereign CDS may or may not own the underlying government bonds.The latter case is termed ‘naked ’sovereign CDS,and frequently labeled as a speculation.CDS prices in our study are taken from CMA Datavision,a platform that is based on quotes collected from a consortium of over thirty independent swap market participants.CMA aggregates the most recent quotes and delivers the data intraday.The quoting convention for CDSs is the annual premium payment as a percentage of the notional amount of the reference obligation.The sovereign CDS spreads are reported in basis points,with a basis point equals to $1000to insure $10million of debt.6CDS spreads are London closing values.While CMA is not the sole provider of CDS prices,Mayordomo et al.(2010)find that,in a comparison of six major providers,CMA quotes are most consistent with a price discovery process.The majority of sovereign CDS in the market are denominated in the US dollar;in our sample,about one-third of the CDS is Euro-denominated.The CMA data set provides a broad coverage of CDS pricing over countries and years.Appendix A provides data sources and Appendix B a list of countries in the entire sample,the subset of countries included in the empirical estimation,and means of 3,5and 10-year CDS spreads (in basis points),fiscal space and other macroeconomic indicators during the sample (2005–10).2.2.Empirical studies on CDS spreadsEmpirical studies of CDS (corporate and sovereign)are relatively new and usually deal with market microstructure issues.Our study,by contrast,is in line with the macro/international finance literature which considers macroeconomic determinants of country risk assessments and financial crises.Several findings are particularly relevant to our analysis.As noted by Packer and Suthiphongchai (2003)and others,the CDS premium should in principle be approximately equal to the credit spread10020030A R G c d s A U S c d s A U T c d sB E L c d s B G R c d s B R A c d sC H L c d s C H N c d s C O L c d s C Z E c d sDE U c d s D N K c d s E S P c d s E S T c d sF I N c d s F R A c d sG B R c d s G R C c d sH R V c d s H U N c d sI D N c d s I R L c d s I S L c d s I S R c d s I T A c d sJ P N c d sK A Z c d s K O R c d sL B N c d s L T U c d s L V A c d sM E X c d s M Y S c d sN L D c d s NO R c d s N Z L c d sP A N c d s P E R c d s P H L c d s P O L c d s P R T c d sQ A T c d sR O M c d s R US c d s S V K c d s S V N c d s S W E c d sT H A c d s TU N c d s T U R c d s U K R c d s U S A c d sV E N c d s V N M c d s Z A F c d sFig.1.Global sovereign CDS positions in early 2011.This figure provides the gross notional amount of outstanding sovereign CDS positions (billion US$)as of February 25,2011.Source:Depository Trust &Clearing Corporation (DTCC).5Sovereign CDS can also be used to supplement corporate CDS to hedge for country risk.6For example,if the spread is 197basis points,meaning 197,000USD to insure against 10,000,000in sovereign debt for 10years;1.97%of notional amount needs to be paid each year,so 0.0197Â10million ¼$197,000per year.J.Aizenman et al./Journal of International Money and Finance 34(2013)37–5940Author's personal copyJ.Aizenman et al./Journal of International Money and Finance34(2013)37–5941 of the reference bond of the same maturity under certain conditions.However,Fontana and Scheicher (2010)demonstrate that the“basis”,i.e.difference between CDS spreads and the spreads on the underlying government bonds,was not zero in Eurozone CDS markets during late2010.They suggest that sizable deviations are attributable to limits to arbitrage and slow moving capital.Secondly,at high frequency(intraday),differences in credit quality(measured by CDS prices)are found to explain sovereign yield spreads of the Euro area governments(Beber et al.,2009).7Fontana and Scheicher (2010)argue that high CDS premium in late2010during the Eurozone debt crisis may be partly attributable to a decline in the appetite for credit-risky instruments and falling market liquidity rather than entirely due concerns about principle losses on outstanding sovereign debt.In addition,empirical researchfinds that daily sovereign interest spreads are more likely to lead sovereign CDS spreads in emerging markets(Ammer and Cai,2007).8Taken together,both studies suggest that sovereign interest rates and CDS spreads have common underlying causes,rather than one driving the other.This is consistent with other work where some studies indicate that price discoveryfirst occurs in the CDS market and follows in the bond market,and vice versa.9There is evidence that CDS spreads may be driven by macroeconomic conditions.Amato(2005) decomposes CDS spreads into risk premium and risk aversion andfinds that both are influenced by macroeconomic conditions.Packer and Zhu(2005)find that contractual terms influence CDS spreads, but that significant“regional effects”(differential pricing across regions)is also evident.Micu et al. (2006)alsofind that credit rating announcements have a large influence on CDS spreads.In explain-ing recent developments,Berndt and Obreja(2010)suggest that“economic catastrophe risk”rose sharply in2007–08pushing up CDS spreads.Cecchetti et al.(2010)plot severalfiscal indicators against CDS spreads for advanced economies.Theyfind correlations across countries with substantial hetero-geneity.10Longstaff et al.(2011)study sovereign CDS of several emerging markets from October2000to January2010.They found that the sovereign spreads are more associated with global factors(US stock, treasury,and high yield markets)than local factors(stock return,exchange rate,and foreign reserve). Dooley and Hutchison(2009)find thatfinancial,economic and regulatory“news”emanating from the U.S.during the globalfinancial crisis quickly impacted sovereign CDS spreads in emerging markets.Concerns about price determination,systemic risk to thefinancial system,and the general opacity of the over-the-counter markets(where CDS is now traded)have led to calls more government regulation and for CDS to be traded on organized exchanges.Presently CDSs are not traded on an exchange and there is no required reporting of transactions to a government agency.One reason for the traditionally limited government oversight is that OTC markets are considered wholesale markets for professional participants,rather than retail investors.However,the recentfinancial turmoil has shown that OTC derivative markets can negatively affect other functioningfinancial markets and can be a serious risk to the health of the banking system.These observations have led to increasing calls for the regulation and oversight CDS markets.For example,in the event that regulatory reforms require that CDS be traded and settled via a central exchange/clearing house,there would no longer be‘counter-party risk’,as the risk of the counterparty will be held with the central exchange/clearing house.113.Statistical contoursMonthly5-year CDS spreads from2005to2011are plotted for the SWEAP and other selected countries(countries which are matched with the SWEAP group,discussed below)in Fig.2.Judging by7Beber et al.study microstructure data of bond quotes and transactions from the interdealer markets,covering Austria, Belgium,Finland,France Germany Greece,Italy,Netherlands,Portugal and Spain.Their sample period is April2003–December 2004.8Ammer and Cai examine daily data from February2001to March2005,covering Brazil,China,Colombia,Mexico, Philippines,Turkey,and Uruguay.On interest rates,CDS spreads,and speculation,see also the discussion offindings from the European Commission(Tait and Oakley,2010),which suggests no strong causality between the two.9See Carboni(2011)for a recent review of the empirical literature on this point.10For example,they plot the forecast level of general government debt/GDP against January2010CDS spreads for21 advanced economies andfind a positive correlation.11Kiff et al.(2009)discuss systemic risk and calls and government regulation policy options in the CDS market.Author's personal copythese spreads,financial stress in global markets emerged in early 2008but became dramatic and widespread in late 2008.The turbulence subsided by mid-2009in most countries,with the notable exception of the SWEAP group:Greece,Ireland,Italy,Portugal,and Spain.For Greece,the unraveling of its fiscal condition in late 2009resulted in a manifold increase in sovereign CDS spreads.For Ireland,the sovereign CDS spreads have widen sharply twice,in early 2009and in late 2010.By 2010,spreads of the SWEAP group were already much higher than those of most emerging market countries (e.g.above the spreads of South Africa,Mexico,Panama,Malaysia and Colombia in Fig.2).On the other hand,sovereign spreads of Germany and the US remained very low throughout the period (not shown).Table 1reports the annual mean values (standard deviation in parentheses)of sovereign CDS spreads (5-year tenor),fiscal space and macroeconomic fundamentals for SWEAP and other country groupings.The table shows average values for the period before crisis (2005–07)and during the crisis (2008–10and individual years).The fall of 2008was the height of the global financial crisis,while the latter part of 2009was a recovery period as financial panic and the liquidity crisis subsided for most countries.However,the SWEAP sovereign debt crisis manifested mainly in 2010and later.Prior to the crisis,SWEAP CDS values were quite low,ranging from 8to 17basis points on average,which are somewhat but not markedly higher than the mean of other Euro countries (11basis points)and lower than the non-Euro OECD group (35basis points).During the early months into the global financial crisis,2008–09,sovereign CDS spreads rose in virtually every country.However,spreads dropped very substantially in all regions by 2010except for the Euro area excluding SWEAP,where it remained roughly unchanged,and SWEAP,where it rose dramatically.SWEAP CDS average values in 2010ranged from 238basis points in Italy to 1027basis points in Greece.By contrast,in 2010,OECD countries (non-Euro members)had an average CDS spread of 118basis points and non-SWEAP Euro members had an average spread of 101basis points.As our main objective is to investigate the link between fiscal space and the pricing of default risk,we also track the adjustment of fiscal capacity across countries.Table 1shows the fiscal balance to tax base ratio and the public debt to tax base ratio.OECD (excluding the Euro countries)and non-SWEAP Euro countries experienced an increase in debt/tax ratios by 0.2percent and 0.3percent,respectively,100200300400ESP100200300400ZAF02000400060008000GRC100200300400PAN0200400600800IRL050100150200250MYS0100200300400500ITA 0100200300400MEX500100015002005q42006q42007q42008q42009q42010q42011q4PRT1002003004002005q42006q42007q42008q42009q42010q42011q4COLFig.2.Evolution of sovereign CDS prices.This figure plots CDS 5-year tenor (basis points)for selected middle-income countries and Eurozone members.J.Aizenman et al./Journal of International Money and Finance 34(2013)37–5942Author's personal copyT a b l e 1S u m m a r y s t a t i s t i c s o f s o v e r e i g n C D S v a l u e s a n d f u n d a m e n t a l s .C o u n t r y S o v e r e i g n CD S 5-y r t e n o r (b a s i s p o i n t s )F i s c a l b a l a n c e /t a x b a s eP u b l i c d e b t /t a x b a s e05–0708091008–1005–0708091008–1005–0708091008–10S p a i n 16.7100.7113.5347.7187.30.05À0.12À0.31À0.27À0.231.11.11.51.71.4G r e e c e 15.0232.1283.41026.5514.0À0.18À0.29À0.48À0.33À0.373.23.54.04.54.0I r e l a n d 8.6181.0158.0619.2319.40.05À0.24À0.47À1.08À0.600.91.52.23.22.3I t a l y 13.4156.9109.2238.0168.0À0.07À0.07À0.13À0.11À0.102.52.52.82.82.7P o r t u g a l 10.496.391.7497.3228.4À0.13À0.11À0.30À0.27À0.221.91.92.22.42.2M i d d l e -i n c o m e g r o u p 124.2(102.8)829.9(1069.4)295.2(331.3)233.2(221.5)452.8(540.7)À0.06(0.2)À0.08(0.2)À0.22(0.2)À0.19(0.1)À0.16(0.2)2.7(2.6)2.2(2.1)2.3(1.9)2.3(1.9)2.3(2.0)H i g h -i n c o m e (n o n O E C D )32.1(13.9)330.7(158.9)164.0(90.5)172.3(118.4)222.3(122.6)0.13(0.4)0.24(0.5)0.10(0.4)0.16(0.5)0.17(0.5)1.2(0.8)1.0(0.6)1.7(0.1)1.4(0.8)1.4(0.5)O E C D (n o n E u r o )34.8(41.5)260.3(238.8)120.3(103.5)117.8(96.4)166.2(146.2)0.04(0.2)0.01(0.2)À0.12(0.1)À0.08(0.1)À0.06(0.1)1.7(1.7)1.7(1.7)1.9(1.8)1.9(1.8)1.8(1.8)E u r o (e x c l u d i n g S W E A P )11.0(13.3)95.1(40.2)54.2(24.6)101.3(55.2)83.5(40.0)À0.03(0.0)À0.02(0.0)À0.15(0.1)À0.14(0.1)À0.10(0.1)1.4(0.5)1.4(0.5)1.6(0.5)1.7(0.4)1.6(0.5)T r a d e /G D P I n fla t i o n (%)E x t e r n a l d e b t /G D P05–0708091008–1005–0708091008–1005–0708091008–10S p a i n 0.60.60.50.50.53.551.400.802.781.661.41.51.71.51.6G r e e c e 0.60.60.50.50.53.322.642.9717.347.651.31.41.81.71.7I r e l a n d 1.51.61.60.91.43.754.05À4.481.300.297.88.910.711.010.2I t a l y 0.60.60.50.60.52.302.261.022.101.791.11.01.21.11.1P o r t u g a l 0.70.80.60.70.72.600.710.002.521.081.91.92.32.42.2M i d d l e -i n c o m e g r o u p 0.9(0.5)0.9(0.5)0.8(0.4)0.9(0.5)0.9(0.4)6.74(4.7)9.73(6.8)5.24(5.9)6.61(5.4)7.19(6.0)0.5(0.3)0.4(0.3)0.4(0.3)0.4(0.2)0.4(0.3)H i g h -i n c o m e (n o n O E C D )0.9(0.0)0.9(0.1)0.8(0.0)0.8(0.0)0.8(0.0)8.20(6.2)8.02(7.3)À0.90(3.9)1.37(0.7)2.83(4.0)0.6(0.2)0.7(0.2)0.9(0.2)0.8(0.2)0.8(0.2)O E C D (n o n E u r o )0.8(0.3)0.9(0.4)0.8(0.3)0.8(0.4)0.8(0.4)3.38(2.3)4.60(3.4)3.02(3.5)3.08(1.4)3.57(2.8)1.0(1.1)1.3(2.0)1.5(2.5)1.5(2.4)1.4(2.3)E u r o (e x c l u d i n g S W E A P )1.2(0.4)1.3(0.4)1.2(0.5)1.2(0.4)1.2(0.4)2.56(1.0)2.13(1.1)0.94(0.5)2.05(0.6)1.71(0.7)1.8(1.0)1.8(0.9)2.0(0.9)1.9(0.9)1.9(0.9)T h i s t a b l e r e p o r t s m e a n a n d s t a n d a r d d e v i a t i o n (i n p a r e n t h e s i s )o f s o v e r e i g n C D S s p r e a d s (b a s i s p o i n t s )a n d m a c r o f u n d a m e n t a l s .T a x b a s e i s a n a v e r a g e t a x /G D P o v e r a p e r i o d o f p r e v i o u s 5y e a r s .A p p e n d i x A p r o v i d e s d a t a s o u r c e s a n d A p p e n d i x B d e t a i l s t h e c o u n t r y g r o u p s .J.Aizenman et al./Journal of International Money and Finance 34(2013)37–5943Author's personal copybetween 2005and 07and 2010.For Ireland and Greece,the deterioration in fiscal circumstances was much more drastic:the government debt of Ireland climbed from 25%of GDP in 2007to almost 100%of GDP in 2010,while the government debt of Greece grew from 95%to over 140%.As a result,the debt/tax ratio of Ireland jumped from 0.9to 3.2and that of Greece from 3.2to 4.5,leaving both countries with a much less room for a flexible policy response on the fiscal side.The large increase of debt/tax ratios in both countries captures a high degree of distress in their economic fundamentals,including the government assumption of banking sector liabilities in the case of Ireland.Similarly large deteriora-tions in fiscal space for these countries are also evident in the fall in fiscal balance to tax ratios.We illustrate further the 2005–07fiscal preconditions,as measured by debt/tax (and debt/gdp)and de ficit/tax (de ficit/gdp),in the two panels of Fig.3by country group.Lower pre-crisis government debt and lower fiscal de ficits relative to the tax base imply greater fiscal capacity.The figure shows that fiscal space was weakest (highest levels of debt and de ficits relative to the tax base)in the middle-incomecountries,although the average debt to gdp ratio was lower than the other groups.This re flectsgenerally lower tax bases in the middle income countries.Generally,the SWEAP had more limited fiscal space during the tranquil period than other high-income groups –higher average debt and de ficits to the relative to the tax base (despite a signi ficant budget surplus in Ireland during this period),and a higher level of debt to GDP.4.Methodology and empirical results 4.1.MethodologyThe dependent variables in our formal empirical work are sovereign CDS spreads of 5year maturity (regressions with 3and 10-year maturities are shown in the appendix table),12estimated in a panelregression,y it ¼a i þl t þq D y it À1þX 0it b þ3it ;where y is the CDS spread,i stands for country and t foryear;X is a vector of controls.The sample covers a panel of 50countries from 2005to 2010.The vector X includes fiscal space (government debt/tax and fiscal de ficit/tax)d the focus of our work d as well as several other variables that frequently employed in the literature to explain country risk.These variables are the TED spread,external debt (total foreign liabilities/GDP),trade openness (trade/GDP)and in flation.As the conditions for consistent estimation in the dynamic panel are known to be demanding,our solution is to work with both a simple fixed-effects model and other estimation speci fications,including clustered standard errors and the Arellano-Bond dynamic panel estimator.Arellano-Bond is a GMM estimator with instruments (exogenous and lagged values),well suited to the problem at hand,with a substantially larger number of cross-section units (50countries),but requires many period of data as the instruments to account for the correlation of lagged dependent variable and the unobserved error terms.We consider fixed effects,clustered standard errors,and GMM in several variants of the model as bounding the causal effects of interest.The sovereign spreads are estimated in level.We also consider for GMM the dependent variable in log multiplied by a hundred,allowing the coef ficients to be interpreted in terms of a percentage change of sovereign default risks (this terminology also aligns with standard practice in the financial sector that discusses the percentage change of CDS spreads).4.2.Dynamics of CDS spreads and Euro/SWEAP pricing differentialsTable 2considers the dynamics and structure of CDS pricing over the 2005–10sample period.Differential pricing for the SWEAP and other Eurozone countries (non-SWEAP)is investigated.To focus on CDS pricing dynamics during the global and European financial turmoil,we include time dummy variables (t 2008–t 2010)for three crisis years:2008is identi fied as the central part of the global financial crisis,2009is identi fied as a partial recovery period,and 2010is identi fied with the SWEAP12Our CDS data set contains 1–10year maturities.We focus on the 5-year maturity since this is the deepest and most actively traded CDS market.While there is no precise international account of government debt maturity,recent statistics suggest that the average original maturity of central government debts is around 10years for both emerging markets and industrial countries (BIS,2010).Hence,we also report results for 10-year maturities in the appendix,as well as 3-year maturities for a robustness test.J.Aizenman et al./Journal of International Money and Finance 34(2013)37–5944。

ORIGINAL ARTICLEBioremediation of heavy metal-contaminated effluent using optimized activated sludge bacteriaEbtesam El.Bestawy •Shacker Helmy •Hany Hussien •Mohamed Fahmy •Ranya AmerReceived:14October 2011/Accepted:3December 2012/Published online:16December 2012ÓThe Author(s)2012.This article is published with open access at Abstract Removal of heavy metals from contaminated domestic-industrial effluent using eight resistant indigenous bacteria isolated from acclimatized activated sludge was investigated.Molecular identification using 16S rDNA amplification revealed that all strains were Gram-negative among which two were resistant to each of copper,cadmium and cobalt while one was resistant to each of chromium and the heavy metal mixture.They were identified as Entero-bacter sp.(Cu 1),Enterobacter sp.(Cu 2),Stenotrophomonas sp.(Cd 1),Providencia sp.(Cd 2),Chryseobacterium sp.(Co 1),Comamonas sp.(Co 2),Ochrobactrum sp.(Cr)and Delftia sp.(M 1)according to their resistance pattern.Strains Cu 1,Cd 1,Co 2and Cr were able to resist 275mg Cu/l,320mg Cd/l,140mg Co/l and 29mg Cr/l respectively.The four resistant strains were used as a mixture to remove heavy metals (elevated concentrations)and reduce the organic load of wastewater effluent.Results revealed that using the pro-posed activated sludge with the resistant bacterial mixture was more efficient for heavy metal removal compared to the activated sludge alone.It is therefore recommended that the proposed activated sludge system augmented with the acclimatized strains is the best choice to ensure high treatment efficiency and performance under metal stresses especially when industrial effluents are involved.Keywords Acclimatization ÁActivated sludge ÁBioremediation ÁDomestic ÁHeavy metals ÁIndustrial wastewaterIntroductionHeavy metals are considered one of the most common and hazardous pollutants in industrial effluents that might cause serious problems to the sewage network pipelines.The deleterious effects of heavy metals on biological processes are complex and generally related to species,solubility and concentration of the metal and the characteristics of the influent,such as pH,as well as presence and concentration of other cations and/or molecules and suspended solids (Gikas 2008).Metal toxicity results from alterations in the conformational structure of nucleic acids,proteins or by interference with oxidative phosphorylation and osmotic balance (Yaoa et al.2008).The most common mechanisms by which metals are eliminated from wastewater treatment processes depend on precipitation,adsorption to suspended solids during primary sedimentation or adsorption to extra-cellular polymers (Qodah 2006).Use of bio-adsorbentsE.El.Bestawy (&)ÁS.HelmyDepartment of Environmental Studies,Instituteof Graduate Studies and Research,Alexandria University,163Horria Ave.El-Shatby,P.O.Box 832,Alexandria,Egypt e-mail:ebtesamelbestawy@ S.Helmye-mail:shackerh@Present Address:E.El.BestawyDepartment of Life Sciences,Faculty of Science,King Abdul Aziz University,P.O.Box 42805,Jeddah 21551,Kingdom of Saudi ArabiaH.Hussien ÁM.Fahmy ÁR.AmerDepartment of Environmental Biotechnology,City for Scientific Research and Technology Application,P.O.Box 21934,Alexandria,Egypt e-mail:hhussein66@ M.Fahmye-mail:mohamedfahmy73@R.Amere-mail:ranyaamer@;r.amer@mucsat.sci.egAppl Water Sci (2013)3:181–192DOI 10.1007/s13201-012-0071-0such as bacteria,fungi,algae and some agricultural wastes that emerged as an eco-friendly,effective and low cost material option could offer potential inexpensive alterna-tives to the conventional adsorbents(Valls and Lorenzo 2002).Different species of Aspergillus,Pseudomonas, Sporophyticus,Bacillus,Phanerochaete,etc.,have been reported as efficient chromium and nickel reducers.The response of microorganisms towards toxic heavy metals is very important for reclamation of polluted sites(Conge-evaram et al.2007).Adsorption of heavy metals on the sludge surface is usually attributed to the formation of complexes between metals and carboxyl,hydroxyl and phenolic surface func-tional groups of the extracellular polymeric substances (EPS)produced by many different species of bacteria isolated from activated sludge(Yuncu et al.2006).EPS are metabolic products of bacteria result from the organic matter of the effluent and from microbial lysis or hydro-lysis.They serve as a protective barrier for cells against the harsh external environment and play a crucial role in sequestration and biosorption of metal ions as well as ions which constitute metabolic elements for bacteria.Metal biosorption performance depends on external factors,such as pH,other ions in bulk solutions(which may be in competition),organic material in bulk solution and tem-perature(Comte et al.2007).Identification of sludge bacteria that are adapted to the new toxic metal environment provide efficient potential candidates for heavy metal bioremoval from contaminated media.Because of its size(*1,500base pairs),the16S rRNA gene is the most widely used sequence in bacterial identification(Weisburg et al.1991;Drancourt et al.2000). Segments from these genes can be easily amplified by PCR using universal primers and sequenced.Sequence com-parison of16S rRNA has been used as a powerful tool for establishing phylogenetic and evolutionary relationships among organisms(Amer2003).Alexandria is the second largest industrial city in Egypt where more than40%of the industrial activity localized.In Alexandria,industrial effluents are mixed with domestic sewage and discharged into the main sewage network (Hussein1999).The aim of the present study was to isolate and identify heavy metal resistant bacteria from acclimatized activated sludge to be used for remediation of heavy metal contaminated wastewater at their highest expected levels. Materials and methodsSampling and pre-treatmentActivated sludge samples were obtained from the aeration tank of the activated sludge unit at Damanhore Wastewater Treatment Plant located in the North West side of Egypt. Prior to use,sludge was pre-treated by allowing the sludge to settle at room temperature followed by decanting the supernatant and replacement with synthetic wastewater with aeration to maintain dissolved oxygen above4mg/l. After24h,aeration was stopped and the mixture was allowed to settle,then the supernatant was decanted and fresh synthetic wastewater was added to provide the same original volume.The procedure of settling,decanting, addition of new effluent and nutrients was repeated at24-h interval until the desired acclimation time(approximately 1month)had been achieved.Heavy metal determinationFour heavy metals(Cu,Cd,Cr and Co)in the wastewater and sludge were determined using Atomic Adsorption Spectrophotometry(Perkin Elmer)after digestion with microwaves digester following the manual instructions with the addition of nitric acid,hydrofluoric acid and hydrogen peroxide for sample digestion(Costley and Wallis2001;Borowski2004).Measurement of dehydrogenase activity(DHA) Dehydrogenase activity was determined using method described by Weddle and Jenkins(1971).Triphenyl tetra-zolium chloride(TCC)and sodium sulfate solutions(5%) were prepared in distilled water.One milliliter of TCC, 10ml liquor or cell suspension and3drops of NaSO3were placed in centrifuge tube and shaken at30°C for1h in dark.This was followed by centrifugation for5min;the color of the supernatant was determined using spectro-photometer at wavelength480nm.Inhibition of the bac-teria and consequently the activated sludge activity were determined according to the following equation: Inhibition%ðÞ¼I0ÀIðÞ=I0½ Â100Where I0represents intensity of the control without treat-ment and I represents intensity of sample after treatment with heavy metal(Weddle and Jenkins1971;Shim et al. 2003).Isolation of heavy metal-tolerant bacteriaTolerant bacterial isolates were collected fromfive differ-ent acclimatized activated sludge batch reactors treated by synthetic wastewater(Table1)with elevated levels of Cu, Cd,Co,Cr(prepared as10%stock solutions of CuSO4, CdCl2ÁH2O,K2Cr2O7,CoCl2Á6H2O salts)and metal mix-ture(El.Bestawy et al.2012)as described by O¨zbelge et al. (2005).Tolerant bacteria were isolated from thefinal stage of the metal enrichment experiment at all reactors.One-milliliter liquor from each reactor was used to inoc-ulate liquid Luria–Bertani(LB)medium containing the investigated heavy metal(sterilized byfiltration)in that reactor.Heavy metal concentrations used during bacterial isolation were15,100,40and40mg/l for the individual Cu,Cr,Cd and Co,respectively.While a mixture of Cu, Cr,Cd and Co at2,10,10and10mg/l respectively was used for isolation of strains with multiple accumulation ability.After24h incubation at30°C,100l l of the grown culture was transferred into10-ml fresh LB medium con-taining the same metal concentration and left for another 24h under the same conditions.The highest metal con-centration reduced the activated sludge activity by50% was amended in Petri dish containing20ml sterile LB agar and mixed well.100l l of the grown culture was spread on the surface of the plate and incubated for24h at30°C. Large identical colonies from each plate were picked out and cultured for purification and identification. Molecular identificationTotal genomic DNA was extracted from5ml overnight LB culture of the purified isolates(Sambrook et al.1989).PCR was performed in a light cycler Eppendorf PCR machine. A1,300-bp fragment was obtained by PCR amplification of the16S rDNA gene(Ausubel et al.1999)using the primers B341F(50-CCTACGGGA GGCAGC)and1392R(50-ACG GGCGGTG TGTRC-30).The PCR mixture was composed of100ng of genomic DNA,30pmol of each primer, 200l M of dNTPs,1U of Taq polymerase and10l l of10X PCR buffer,the reaction volume was adjusted to100l l in 0.5ml eppendorf tube.The PCR amplification conditions were performed by an initial denaturation step at94°C for 10min followed by30denaturation cycles at94°C for 1min,annealing at60°C for1min and an extension at 72°C for1min followed by afinal extension step at72°C for10min.Amplicons of16S rDNA were purified using PCR purification kit(QIAGEN).Each of these purified products was sequenced by the chain terminator method (ABI3130XL system,DNA technology,Denmark)using the two corresponding PCR primers separately.The resulted DNA sequences were phylogenetically analyzed using the BLAST search program.Multiple sequence alignment and molecular phylogeny were performed using BioEdit software(Hall1999).Screening for heavy metal resistance patternEffect of the individual and mixed tested metals on the growth(measured as optical density,OD at600nm)and dehydrogenase activity of each of the resistant strains was investigated at elevated metal concentrations compared to their controls.An overnight culture of each strain was inoculated in20-ml LB medium supplemented with defi-nite concentrations of the corresponding heavy metals and incubated for24h under the previously mentioned condi-tions.Concentrations of the tested metals(not shown)were calculated according to the IC50which is also included in the screening test.Concentrations of heavy metal mixtures were calculated by dividing the growth IC50of each heavy metal by4and two higher and two lower concentrations were applied for screening bioassay.Treatability studiesThe most efficient strains for metal removal were selected based on the resistance screening bioassays and manipu-lated in activated sludge treatment of mixed domestic-industrial wastewater contaminated with heavy metals and organic matter.The acclimatized sludge was prepared by centrifugation to remove the supernatant which was replaced by wastewater.The resistant strain for each investigated metal was seeded individually in LB-medium and incubated at30°C and200rpm for24h.Cultures were centrifuged and the supernatants were replaced with wastewater and used as inocula for batch reactors for the treatability study with moderate concentrations of heavy metals.Three sets of500-ml reactors were used to compare the removal efficiencies of the sludge,bacterial strains and mixture of both.Each set contained four different con-centrations(not shown)of the heavy metals with initial COD of1,400mg/l as following:1.Thefirst set consists of200ml of sludge with differentconcentrations of the investigated heavy metals.2.The second set consists of200ml of bacterial cultureonly with different concentrations of the investigated heavy metals.Table1Composition of the concentrated synthetic wastewater Serial Constituents Concentration(g/l)1Tripton122.12NaCl40.73Na2SO4 4.464K2H2PO4 4.465MgCl2Á6H2O0.376CaCl2Á2H2O0.377MnSO40.578H2MoO40.319NaOH0.0810ZnSO40.4611CoSO40.4912CuSO40.7610ml of concentrated synthetic wastewater were added to each 1,000ml of deionized waster3.The third set consists of100ml of sludge and100mlof bacterial culture with different concentrations of the investigated heavy metals.Heavy metal removal per unit biomass was determined using the following equation:M uptake=g¼½MðInf:ÞÀMðEff:Þ =MðMLSS)where M=uptake/g:heavy metal removed per unit mass of biomass,M(Inf.)=the initial heavy metal concentration in the influent,M(Eff.)thefinal metal concentration after treatment, MLSS:Biomass concentration in gramResultsIsolation and molecular identification of metal resistant-sludge bacteriaA total of eight strains representing morphologically different bacterial colonies were isolated and purified. Two showed resistance to copper(Cu1and Cu2),two for cadmium(Cd1and Cd2),two for cobalt(Co1and Co2), one for chromium(Cr)and the heavy metal mixture resistant strain(M1).PCR products of the chromosomal DNA extracts of the resistant isolates were purified, sequenced and aligned against the16S rDNA sequences of the ribosomal database project http://www.cme.msu. edu/RDP/html/index/html(Maidak et al.1994;Rainey et al.1996).Sequences were deposited in GenBank for obtaining accession numbers.The Phylogenetic relation-ship among the tested isolates and the closely related species(Fig.1)was analyzed using multi-sequence alignment program(BioEdit Sequence Alignment Editor). Accession numbers and similarity of the tested isolates to the related species are presented in Table2.It is well documented that both Gram-positive and Gram-negative bacteria are ubiquitous in nature with highly resistant anionic cell walls.This anionic cell wall canfix metal and provide sites for nucleation and growth of minerals, a property that has been the basis for many heavy metal removals using bacterial biomass(Kelly et al.2004). Heavy metal resistance and/or sensitivity of the sludge bacteriaResistance and/or sensitivity of the acclimatized sludge bacteria towards the investigated metals(individuals or mixture)were determined in terms of growth(OD)and dehydrogenase activity to select the most efficient for treatability study.Effect of individual metalsComparing data of the individual effects of the four tested metals on the acclimatized strains(Fig.2)revealed the following points:1.IC50of all the tested metals that induced50%reduction in the growth of all the investigated strains was higher than that induced50%reduction in their DHA.This indicates that tested metal could inhibit the enzyme activity much higher than its inhibition to the growth because dehydrogenase activity is a direct reflection of microbial activity(Gong1997;Mathew and Obbard2001)and metal toxicity(Aoyama and Nagumo1997;Kelly and Tate1998;Nweke et al.2006).2.IC50of each metal depends mainly on the microbialspecies as well as acclimatization period,culturing or operation conditions,nutrient status and the presence of other inhibitors.Isolated strains Cu1,Cd1,Co2and Cr had the highest ability to grow individually in the presence of Cu,Cd,Co and Cr with IC50of275,320, 140and29mg/l,respectively.3.Although possessing multiple resistances,most of theacclimated strains exhibited their highest resistance with their corresponding metals.For example Enter-obacter sp.(Cu1and Cu2)showed the highest OD(275 and190,respectively)with copper that declined to190 and46,respectively with Cd followed by more toxicity with Co and Cr.Similarly,Stenotrophomonas sp.(Cd1)and Providencia sp.(Cd2)also showed high resistance to Cd(OD320and260,respectively;DHA 50and15,respectively)compared to their resistance pattern for other metals.4.Ochrobactrum sp.(Cr)isolated from activated sludgeamended with chromium was more sensitive to chromium(OD29)compared to copper and cadmium (OD75and150,respectively).On contrary Ochro-bactrum sp.strain CSCR-3isolated from chromium landfills was able to tolerate and reduce Cr VI to Cr III in cultures containing800mg/l Cr(He et al.2009).This is mainly attributed to factors controlling resis-tance and/or toxicity of metals of which microbial species and the form of the metal are key factors (Gikas and Romanos2006).Results confirmed that gradual increase of the applied metals produced generations with high metal resistance range that can be efficiently used in decontamination pur-poses.These results are in agreement and supported by other studies.For example a Cd-resistant Stenotropho-monas sp.showed resistance to Se(Antonioli et al.2007) and Cu(Zaki and Farag2010);a Cd-resistant Providencia sp.showed very high resistance(1,000–1,500mg/l)to Cd,Cr,Ni,Co and Zn(Thacker et al.2006)and Pseudomonas with multi resistance to Cr,Cu,Cd and Ni(Kelly et al. 2004).In addition,Comamonas sp.(Co2)that showed moderate growth and DHA activity(140and82,respec-tively)when cultivated with50mg/l cobalt was found containing plasmid encoding cobalt and nickel resistance (Siunova et al.2009).According to their average DHA(reflecting their resis-tance)against the individual metals,acclimatized bacteria can be arranged in the following order from the highly resistant to the highly sensitive Cd1[Co2[Cr[ Cu2[Cu1[M1[Cd2.Therefore,Stenotrophomonas sp. (Cd1)represents the highest DHA activity indicating the lowest toxic effects upon exposure to individual metals.Effect of mixed metalsThefinal stage in the screening process was to investigate each of the acclimatized strain isolated against mixture of the tested heavy metals(Cu,Cd,Co and Cr)based on its IC50concentrations obtained in the previous stage.The concentration of each heavy metal for each strain was calculated by dividing its growth IC50by4,and then two higher and two lower concentrations were used for the screening process(i.e.,five mixtures with elevated metals concentration).Applying the investigated metals as mix-tures led to completely different responses from the investigated acclimatized strains(Fig.3).Results revealed the followingpoints:1.As with the individual metals,IC50of all testedmetals,those induced50%reduction in the growth,of all the investigated strains were higher than those induced50%reduction in their DHA,indicating that metal inhibits enzyme activity much higher than growth.2.As expected,tolerance of the investigated strains wasreduced dramatically towards the tested metal mixtures compared to their individual application.This was shown by the growth IC50recorded70,81.25,43.75 and12.5mg/l for strains Cu1,Cd2,Co2and Cr, respectively in the metal mixtures compared to275, 320,140and29mg/l,respectively when grown individually in their corresponding metals.3.IC50of DHA of the isolated strains Cu1,Cu2,Cd1,Cd2,Co2,Cr and M against the tested metals(Cu,Cd, Co and Cr)recorded35,20,2and2;37.5,18.75,11.25 and1.5;5,20,7.5and1;5,16.25,1.75and1.75;8,12.5,17.5and2;10,20,3.5and5;andfinally18,18,30and6mg/l respectively(Fig.3).4.According to their average DHA against the mixedmetals,acclimatized bacteria can be arranged in the following order from the highly resistant to the highly sensitive M[Cu2[Cu1[Co2[Cr[Cd.Delftia sp.exhibited the highest resistance to the mixture ofthe investigated metals due to the adaptation acquired during the acclimatization process when grown in the presence of heavy metal mixture.These results are consistent with those of Jackson et al. (2009)who reported a multiple heavy metal resistance of Delftia sp.when used within a consortium for bioremedi-ation of River Plankenburg water in South Africa con-taminated with aluminum,nickel,copper and manganese due to the presence of highly incidence plasmids(Zolg-harnein et al.2007).Treatability bioassayThree parallel strategies were used to evaluate the reme-diation capability of the proposed system towards heavy metal-and organic matter-contaminated wastewater. Treatment technologies included the proposed activated sludge unit amended with the acclimatized bacterial strains (ASBS),the plain activated sludge unit(AS)and the acclimatized bacterial cultures(BS)alone represented by Cu1,Cd1,Co2and Cr.The performance of the three pro-cedures was evaluated to determine the most efficient application.Also a comparison was made between the removal efficiency of the contaminated metals andtheCFig.1continuedorganic matter load (as COD).Four different concentra-tions of each heavy metal were investigated in the three planned treatment procedures and the removal was deter-mined after 24h exposure.Treatment of wastewater effluent under individual metal stressesWastewater collected from the Western Wastewater Treatment Plant (WWTP),Alexandria was subjected to 24h treatment using ASBS,AS and BS technologies after the addition of four elevated concentrations of individual heavy metals (20–200mg/l for each of Cu,Cd and Co and 20–300mg/l for Cr).Results achieved (Figs.4and 5)can be summarized asfollows:Table 2Resistant isolates’accession numbers and their similarity to the related species Isolate Accession no.Similar Strain Similarity (%)Cd 1FJ205387Stenotrophomonas sp.99Cd 2FJ205388Providencia sp.98Cu 1FJ205389Enterobacter sp.98Cu 2FJ205390Enterobacter sp.99Co 1FJ205391Chryseobacterium sp.98Co 2FJ205392Comamonas sp.97Cr FJ205394Ochrobactrum sp.98M 1FJ205393Delftia sp.9950100150200250300350Fig.2IC50for the growth (OD)and dehydrogenase activity (DHA)of the acclimatized strains after 24-h exposure to individual heavy metals:Cu copper,Cd cadmium,Co cobalt,Cr chromiumFig.3IC50for the growth (OD)and dehydrogenase activity (DHA)of the acclimatized strains after 24h exposure to the heavy metals in mixture:Cu copper,Cd cadmium,Co cobalt,Cr chromium1.As a general rule,removal of all the investigated metals was stimulated with increasing metal levels in the three investigated procedures reaching maximum efficiency at the highest applied concentration while COD removal decreased.Moreover,the removal of the investigated metals by the proposed technologies recorded the following order Co [Cd [Cr [Cu.2.The highest Cu removal achieved (at 200mg/l)by the ASBS,BS and AS technologies recorded 15.84,22.29and 31.37Cu/g biomass,respectively.Therefore,the magnitude of increasing Cu removal efficiency (RE %)is AS [BS [ASBS.On the contrary,COD removal under the highest Cu stress recorded 58.10,57.82and 38.16%using the ASBS,BS and AS technologies,respectively.Therefore,the magnitude of increasing COD RE %is ASBS [BS [AS.3.The proposed technologies ASBS,BS and AS could achieve Cd removal (at 200mg Cd/l)of 53.17,69.36and 56.25mg Cd/g sludge biomass,respectively.Therefore,Cd removal magnitude power order is BS [AS [ASBS technology.While COD removalwas recorded as 23.12,16.64and 50.99%using the ASBS,BS and AS technology,respectively.Therefore,the magnitude of increasing COD RE %is AS [ASBS [BS.4.Co removal achieved (at 200mg/l)by the ASBS,BS and AS technologies recorded 60.93,70.72and 67.12Co/g biomass,respectively indicating that Co removal efficiency by the three techniques is in the following order BS [AS [ASBS technology.COD removal under Co stress recorded 64.44,48.44and 55.16%using ASBS,BS and AS technology,respec-tively indicating magnitude of increasing COD RE %as ASBS [AS [BS.5.Cr removal (at 300mg/l)recorded as 55.17,47.89and 44.04mg Cr/g using ASBS,BS and AS technology,respectively indicating an order of ASBS [BS [AS technology in Cr removal efficiency.COD removal under the highest Cr stress recorded 67.51,10.51and 51.05%using the ASBS,BS and AS technology,respectively.Therefore,the magnitude of increasing COD RE %is ASBS [AS [BS.C DFig.4Comparison among the three bioremediation technologies AS,ASBS and BS in the removal efficiency of a Cu,b Cd,c Co and d Cr after 24-h exposureTreatability bioassays of the contaminated wastewater in the presence of the four tested metals applied as individuals achieved the following points:1.Native activated sludge (AS technology)exhibited the maximum efficiency in the removal of Cu.2.For Cd and Co removal,BS technology where acclimatized bacterial cultures were used,exhibited the highest efficiency followed by AS technology and ASBS technology with no significant variations.3.Activated sludge amended with the acclimatized bacteria (ASBS technology)performed the highest efficiency for removing Cr.4.Activated sludge amended with the acclimatized bacteria (ASBS technology)performed almost the highest efficiency for removing COD from wastewater under the stress of all the investigated metals even under their highest levels.Metal biosorption by microbial biomass is a well-known advanced biotechnological tool used for efficient removal of contaminant metals.Optimization tools of metalbiosorption included many factors among which pH is the most important one (Comte et al.2007)as well as the C/N ratio of activated sludge (Yuncu et al.2006)and temper-ature (Zouboulis et al.2004).Acclimatization is a very important process for acquiring new advanced features of microorganisms that can be efficiently applied in metal biosorption.During the present study,using strains previ-ously acclimated by exposing microbial biomass to metal concentration gradients (El.Bestawy et al.2012)proved high efficiency in the enhancement of activated sludge performance for metals and organic matter removal even at high metal stresses.For example,Enterobacter sp.(Cu 1)acclimated to survive under high levels of Cu stress (200mg/l)showed high efficiency for Cu (31.37Cu/g biomass)and organic matter (58.10%)removal compared to 2.93mg Cu/g achieved as the maximum removal by plain activated sludge (Principia et al.2006).Similar results were obtained by manipulating metal binding capacity of Nocardia amarae cells to optimize the overall Ni,Cu and Cd binding capacity of activated sludge (Kim et al.2002).As in the present study the pure cultureofC DFig.5Comparison among the three bioremediation technologies AS,ASBS and BS in the removal efficiency of COD in the presence of elevated levels of a Cu,b Cd,c Co and d Cr after 24-h exposureNocardia exhibited significantly higher metal sorption capacity than the activated sludge biomass attributed to the fact that the Nocardia cells growing at stationary phase have substantially more specific surface area than that of activated sludge.The metal sorption capacity of activated sludge increased proportionally with the amount of Nocardia cells present in the mixed liquor.Sirianuntapi-boon and Ungkaprasatcha(2007)also reported efficient removal of Pb and Ni as well as organic matter by com-paring acclimatized and un-acclimatized biosuldge.Reductions in the COD removal with increasing metal concentrations during the present study are attributed mainly to the toxic effects and inhibition of the biodegra-dative microbes in the proposed treatment systems induced by the investigated metals at their elevated levels(Jefferson et al.2001;Gikas2007).Treatment of wastewater effluent under mixed heavy metal stressesTreatability study was also performed with mixtures of the four investigated metals with elevated concentrations after 24-h exposure.Three technologies were applied here included BS,where mixture(strains Cu1,Cd1,Co2and Cr) of the acclimatized bacteria was used,ASBS,where acti-vated sludge supplied with the acclimatized bacteria was used and plain activated sludge(AS).Heavy metal removalsAs general trends for all the investigated metals and the three proposed treatment technologies,increasing metal concentrations from5to75mg/l enhanced their removal which was in the following order Cu\Cr\Cd\Co. The highest achieved RE(s)%of Cu,Cd,Co and Cr recorded were6.61,16.58,27.58and19.83mg/g sludge, respectively using ASBS technology(Fig.6a);4.39,22.89, 30.38and26.52mg/g biomass,respectively using BStechnology(Fig.6b)andfinally7.09,15.02,21.08and 18.58mg/g biomass,respectively using AS technology (Fig.6c).The efficiency of using activated sludge under the stress of microbial inhibiting agents(i.e.heavy metals)is one of the issues with lots of environmental concern.Many factors affect the efficiency of activated sludge systems used for metal removal from contaminated media.pH considered as the most critical factor for microbial metal biosorption (Ozdemir et al.2003;Sheng-lian et al.2006;Congeevaram et al.2007)which is mainly attributed to organism-specific physiology(Congeevaram et al.2007).Other factors such as initial metal concentrations,activated sludge dosage, temperature,contact time(treatment duration),presence of other ions and organic materials in the bulk solutions (which may be in competition)could affect the efficiency of activated sludge for heavy metal removal.(Sheng-lian et al.2006;Comte et al.2007;Pamukoglu and Kargi 2007).Bieszkiewicz and Hoszowski(1978)reported that acti-vated sludge could tolerate up to0.8,1.15and20mg/l only of Cu,Cr III and Cr(VI),respectively after which the activated sludge experienced inferior purification and reduced intensity of respiration of its microorganisms.In another study,removal efficiency%of Cu,Cd and Cr using activated sludge recorded only80,62and46%, respectively with initial concentrations of0.8,20.9and 0.63mg/l of the same metals(Petrasek and Kugelman 1983).Comparison with the present work indicated remarkable enhancement of the metal tolerance acquired 051015202530355101520253035510152025303502550750255075 0255075 ACBFig.6Comparison among the removal efficiency of the investigated metals mixture of Cu,Cd,Co and Cr using the proposed a ASBS, b BS and c AS technology for24-h exposure。

Accelerated testing for long-term fatigue strength of various FRP laminates for marine useMasayuki Nakada *,Yasushi MiyanoMaterials System Research Laboratory,Kanazawa Institute of Technology,3-1Yatsukaho,Hakusan,Ishikawa 924-0838,Japana r t i c l e i n f o Article history:Received 14September 2007Received in revised form 19February 2008Accepted 25February 2008Available online 8March 2008Keywords:A.Polymer–matrix compositesB.Durability B.FatigueD.Life predictiona b s t r a c tThe prediction of long-term fatigue life of various FRP laminates combined with resins,fibers and fabrics for marine use under temperature and water environments were performed by our developed acceler-ated testing methodology based on the time–temperature superposition principle (TTSP).The five kinds of FRP laminates were prepared under three water absorption conditions of Dry,Wet and Wet +Dry after molding.The three-point bending constant strain rate (CSR)and fatigue tests for these FRP laminates at three conditions of water absorption were carried out at various temperatures and loading rates.As results,the mater curves of fatigue strength as well as CSR strength for these FRP laminates at three water absorption conditions are constructed by using the test data based on TTSP.It is possible to predict the long-term fatigue life for these FRP laminates under an arbitrary temperature and water absorption conditions by using the master curves.The characteristics of time,temperature and water absorption dependencies of flexural CSR and fatigue strengths of these FRP laminates are clarified.Ó2008Elsevier Ltd.All rights reserved.1.IntroductionRecently carbon fiber reinforced plastics (CFRP)has been used for the primary structures of airplanes,ships,spacecrafts and others,in which the high reliability should be kept during the long-term operation.Therefore,it is strongly expected that the accelerated testing methodology for the long-term life prediction of composite structures exposed under the actual environments of temperature,water,and others is established.The mechanical behavior of polymer resins exhibits time and temperature dependence,called viscoelastic behavior,not only above the glass transition temperature T g but also below T g [1–7].Furthermore,the viscoelastic behavior of polymer resins also de-pends on the water absorption [8–11].Thus,it can be presumed that the mechanical behavior of polymer composites significantly depends on the water absorption as well as time and temperature.We have proposed the accelerated testing methodology to pre-dict the long-term fatigue life of polymer composites under tem-perature condition [12].Our accelerated testing methodology shown in Fig.1rests on the three hypotheses,(A)the time–tem-perature superposition principle (TTSP)held for the viscoelastic modulus of matrix resin is applicable to constant strain-rate (CSR),creep and fatigue strengths of polymer composites,(B)linear cumulative damage law for monotonic loading is applicable to pre-dict the creep strength from CSR strength,and (C)linear depen-dence of fatigue strength upon stress ratio is applicable to predict the fatigue strength under an arbitrary stress ratio between zero and unity.When these hypotheses are met,the fatigue strength under an arbitrary combination of frequency,tempera-ture,and stress ratio can be determined based on the following test results:(a)master curve of CSR strength and (b)master curve of fatigue strength at zero stress ratio.The master curve of CSR strength is constructed from the test results at single strain-rate and various temperatures based on the hypothesis (A).The master curve of fatigue strength at zero stress ratio can be constructed from the test results at single frequency for various temperatures based on the hypothesis (A).The loading frequency for fatigue test should be selected as the evolution of heat in specimen does not occur during fatigue test.We applied this prediction method to the fatigue strength of various kinds of FRP combined with fiber/matrix and its structure under various types of loading methods under temperature condi-tion.Concretely,the prediction method was applied to the tensile and flexural fatigue strengths of PAN-based carbon fiber/epoxy,the flexural fatigue strengths of PAN-based carbon fiber/vinylester,PAN-based carbon fiber/PEEK,pitch-based carbon fiber/epoxy,glass fiber/epoxy and glass fiber/vinylester,and the tensile fatigue strengths of GFRP/metal tapered joint,adhesive joint,and bolted joint [13–20].The applicability of prediction method is shown in Table 1.The tensile fatigue strength in the longitudinal direction,the flexural fatigue strengths in the longitudinal and transverse directions of unidirectional PAN-based carbon fiber/epoxy resin can be predicted by using this method,because the hypotheses0266-3538/$-see front matter Ó2008Elsevier Ltd.All rights reserved.doi:10.1016/pscitech.2008.02.030*Corresponding author.E-mail address:nakada@neptune.kanazawa-it.ac.jp (M.Nakada).Composites Science and Technology 69(2009)805–813Contents lists available at ScienceDirectComposites Science and Technologyj o ur na l h o me pa ge :w w w.e ls e v ie r.c o m/lo c a t e/c om p s c it e ch(A)–(C)are met.The flexural fatigue strengths of satin-woven PAN-based carbon fiber/epoxy resin,PAN-based carbon fiber/epoxy resin quasi-isotropic laminates,plain-woven PAN-based carbonfiber/vinylester resin and plain-woven glass fiber/vinylester resin can be also predicted.The flexural fatigue strengths of PAN-based carbon fiber/PEEK resin and pitch-based carbon fiber/epoxy resin can not be predicted,because the TTSP does not hold for the mechanical behavior of PEEK and the mechanical behavior of high modulus pitch-based carbon fibers itself shows time dependent behavior.For satin-woven glass fiber/epoxy resin,in the case of which the fracture of specimen is controlled by the delayed frac-ture of glass fiber,the hypothesis (A)does not met.The tensile fa-tigue strengths of FRP/metal tapered joint,adhesive joint,and bolted joint can be predicted by using thismethod.Fig.2.Constitution of five kinds of FRP laminates combined with different resins and fibers.Table 2Conditions for Dry,Wet,and Wet +Dry specimens Specimen In air In water In airDry As cured WetAs cured +95°C Â120h Wet +DryAs cured+95°C Â120h+150°C Â2hTable 1Applicability of proposed method to FRP and joint structures FiberMatrixTypeFiber/matrixLoading directionHypothesis (A)(B)(C)CarbonPANEpoxyUDT400/828LT T300/2500LB TB SW T400/3601LB QIL T800/3900-2B LB Vinylester PW T300/vinylester LB PEEKUD T300/PEEK LB ÂÂÂTB ÂÂÂPitchEpoxy UD XN40/25C LBÂÂGlassEpoxy SW E-glass/epoxyLB Â ÂVinylesterPWE-glass/vinylesterLT ÂLB ÂNotice:UD:unidirectional;SW:satin woven;PW:plain woven;QIL:quasi-isotropic laminates;LT:longitudinal tension;LB:longitudinal bending;TB:transverse bending FRP joint systemHypothesis (A)(B)(C)Conical shaped joint of GFRP/metal Brittle adhesive joint of GFRP/metal Ductile adhesive joint of GFRP/metal Bolted joint of GFRP/metal Bolted joint of CFRP/metalFig.1.Accelerated testing methodology for polymer composites.806M.Nakada,Y.Miyano /Composites Science and Technology 69(2009)805–813This paper is concerned with the prediction of long-term fatigue life offive kinds of FRP laminates for marine use under water absorption condition as well as temperature condition.These FRP laminates were prepared under three water absorption conditions of Dry,Wet and Wet+Dry after molding.The three-point bending CSR tests forfive kinds of FRP laminates at three conditions of water absorption were carried out at various temperatures and strain rates.Furthermore,the three-point bending fatigue tests at zero stress ratio for these specimens were carried out at various temperatures and frequencies.The characteristics of time,temper-ature,and water absorption dependencies offlexural fatigue strength as well asflexural CSR strength for these FRP laminates are discussed based on theTTSP.Fig.3.Water content versus soakingtime.Fig.4.Water content in resin and FRP for Wet and Wet+Dryconditions.Fig.5.Configuration of three-point bending test.Table3Test conditionsLoading type Deflection rate V(mm/min)Frequencyf(Hz)Stress ratio R(r min/r max)TemperatureT(°C)Creep forneat resin–––25–150CSR for FRP0.02*,2,200*––25–160Fatigue forFRP–0.02*,20.0525–140*Test conditions for confirming of applicability ofTTSP.Fig.6-1.Master curve of creep compliance for neat vinylester resin and shiftfactors.Fig.6-2.Master curve of creep compliance for neat epoxy resin and shift factors.M.Nakada,Y.Miyano/Composites Science and Technology69(2009)805–8138072.Experimental procedures2.1.Preparation of specimensThe base material offive kinds of FRP laminates employed in this study was plain fabric CFRP laminates T300carbonfibers/viny-lester(T300/VE).Thefirst selection of FRP laminate to T300/VE was the combinations of different fabrics,that isflat yarn plain fabric T700carbonfibers/vinylester(T700/VE-F)and multi-axial knitted T700carbonfibers/vinylester(T700/VE-K)for marine use and the second selection of FRP laminates to T300/VE was the combina-tions with differentfibers and matrix resin,that is plain fabric T300carbonfibers/epoxy(T300/EP)and plain fabric E-glass fibers/vinylester(E-glass/VE)shown in Fig.2.These FRP laminates were formed by resin transfer molding(RTM)except T300/EP which was formed by conventional hand lay up.The thickness of the laminates was approximately2mm.These FRP laminates were prepared under three water absorp-tion conditions of Dry,Wet and Wet+Dry after molding.Dry spec-imens by holding the cured specimens at150°C for2h in air,Wet specimens by soaking Dry specimens in hot water of95°C for 120h and Wet+Dry specimens by dehydrating the Wet specimens at150°C for2h in air were respectively prepared as shown on Ta-ble2.Fig.3shows water content versus soaking time at95°C and the soaking condition of95°C and120h was determined to Wet specimens.Fig.4shows water content in resin and FRP for Wet and Wet+Dry specimens.The water absorption of all FRP lami-nates increases with Wet condition of hot water of95°C for 120h.The water absorption of neat vinylester resin and its CFRP laminates returns to0%with Wet+Dry condition by re-drying and that of T300/EP and E-glass/VE does not return to0%.2.2.Experimental proceduresFig.5shows the configuration of three-point bending test and Table3shows the test conditions.To evaluate the viscoelastic behavior of vinylester(VE)and epoxy(EP)as matrix neat resin, the three-point bending creep tests for the neat vinylester and epoxy resins prepared at Dry,Wet and Wet+Dry conditions were carried out under various temperatures using an creep testingma-Fig.7.Master curves offlexural CSR strength at Dry,Wet and Wet+Dry conditions.808M.Nakada,Y.Miyano/Composites Science and Technology69(2009)805–813chine with temperature chamber.The creep compliance D c was calculated from the deflection d at the center of specimen using the following equation:D c¼4bh3dP0L3ð1Þwhere P0is the applied constant load(58.8N),L is the span (50mm),and b and h are the width(25mm)and the thickness (3.0mm)of specimen,respectively.The three-point bending CSR tests forfive kinds of FRP lami-nates at Dry,Wet and Wet+Dry conditions were carried out at various temperatures and strain rates.The span is L=80mm, and the width and thickness are b=15mm and h=2.0mm, respectively.The CSR tests were conducted at three loading-rates V=0.02,2,200mm/min and various constant temperatures T using an universal testing machine with temperature chamber. Theflexural CSR strength r s is calculated from the maximum load P s byr s¼3P s L2bh2:ð2ÞFurthermore,the three-point bending fatigue tests for thesespecimens were carried out at various constant temperatures Tand two loading frequencies f=2Hz and0.02Hz using an electro-hydraulic servo testing machine with temperature chamber.Thestress ratio R(=minimum stress/maximum stress)was0.05.Thelength,width,thickness of specimen,and span are the same tothose for theflexural CSR tests.Theflexural fatigue strength r f isdefined by maximum applied load P max for the number of cyclesto failure N f.r f¼3P max L2bh2:ð3ÞIn order to prevent the dryness of specimens at Wet conditionduring creep,CSR,and fatigue tests,the specimens were wrappedby a vinyl bag with including distilled water in the bag.3.Results and discussion3.1.Creep complianceThe left sides of upper graphs in Fig.6shows the creep compli-ance D c versus testing time t at various temperatures T for Dry,Wet Fig.8.Flexural CSR strength versus creep compliance of matrix resin at Dry,Wet and Wet+Dry conditions.M.Nakada,Y.Miyano/Composites Science and Technology69(2009)805–813809and Wet +Dry specimens of VE and EP resin.The master curves of D c versus the reduced time t 0were constructed by shifting D c at various constant temperatures along the log scale of t and the log scale of D c .Since the smooth master curve of D c for each specimen can be obtained as shown in the right sides of each graph,the time–temperature superposition principle (TTSP)is applicable for each D c .From these master curves,it is cleared that D c increases with water absorption and returns perfectly to that of Dry speci-men by re-drying after water absorption.The horizontal time–temperature shift factor a T 0(T )and the ver-tical temperature shift factor b T 0(T )at a reference temperature T 0plotted in lower graphs of Fig.6are,respectively,defined by log a T 0ðT Þ¼log t Àlog t 0ð4Þlog b T 0ðT Þ¼log D C ðt ;T ÞÀlog D C ðt 0;T 0Þð5Þ3.2.Flexural CSR strengthThe left side of each graph in Fig.7shows the flexural CSR strength r s versus time to failure t s at various temperatures T for Dry,Wet and Wet +Dry specimens of five kinds of FRP laminates,where t s is the time period from initial loading to maximum load during testing.The master curves of r s versus the reduced time to failure t 0s were constructed by shifting r s at various constant temperatures along the log scale of t s and the log scale of r s using the same time–temperature shift factors and a half of the temper-ature shift factors for D c of matrix resin shown in Fig.6.The reason why the shift amount to the log scale of r s is a half of that to the log scale of D c is mentioned after.Since the smooth master curve of r s for each specimen can be obtained as shown in the right side of each graph,the TTSP for D c of matrix resin is also applicable for the r s of corresponding FRP laminates.It is cleared from Fig.7that the r s for all five FRP laminates strongly decreases with increasing time and temperature and that these r s decreases with water absorption and returns to that of Dry specimens by re-drying after water absorption except that of GFRP laminates (E-glass/VE).The r s of Wet +Dry specimens of E-glass/VE does not return to that of Dry specimens.Fig.8shows the flexural CSR strength versus the creep com-pliance of matrix resin for the same conditions of time,temper-ature and water absorption for five FRP laminates.The degradation of flexural CSR strength for all CFRP laminates except GFRP laminates (E-glass/VE)is uniquely determined by the creep compliance of matrix resin.Therefore,the degradation rate of flexural CSR strength of these CFRP laminates is determined only by increasing of time,temperature and water absorption and is independent upon fiber constitutions which are the type,volume fraction and weaves.The slope is approximately 0.5shown in each graph of this figure.This indicates that the trigger of failure is the microbuckling of carbon fibers in the compression side of specimen shown by the following equation based on Dow’s the-ory [21]:Fig.9.Fracture appearances of specimens after flexural CSR test at 25°C at Dry condition.810M.Nakada,Y.Miyano /Composites Science and Technology 69(2009)805–813log r s ¼log K 0À12log D C ð6Þwhere r s is the CSR strength of CFRP laminates,K is the material con-stant and D c is the creep compliance of matrix resin.Actually,the frac-ture appearance indicates that the fracture mode for these CFRP laminates is the compressive fracture of warp carbon fibers in the compression side of specimen for all condition tested as shown in Fig.9.Therefore,this is the reason why the vertical shift amount for r s is a half of that for D c as mentioned above.The fracture mode for T300/EP laminates and E-glass/VE is the tensile fracture in the tension side of specimen at T =25°C.However the fracture mode at high tem-peratures is the compressive fracture in the compression side of spec-imen same to that for T300/VE laminates.3.3.Flexural fatigue strengthTo construct the master curve of flexural fatigue strength r f ,we need the reduced frequency f 0in addition to the reduced time to failure t 0f ,each defined by f 0¼f Áa T 0ðT Þ;t 0f ¼t f a T 0ðT Þ¼N ffð7Þwhere N f is the number of cycles to failure.The r f versus N f at frequency f =2Hz at various temperatureswere measured for Dry,Wet and Wet +Dry specimens of five kinds of FRP laminates.For examples,the r f versus N f curves at various temperatures for Dry specimen are shown in Fig.10.By converting f and N f into f 0and t 0f using Eq.(7),the time–tem-perature shift factors a T 0(T )and temperature shift factors b T 0(T )of the creep compliance of matrix resin for each specimen shown in Fig.6,the r f versus t 0f for each f 0were constructed for Dry,Wet and Wet +Dry specimens of five kinds of FRP laminates shown in Figs.11-1and 11-2.The curves consisted by solid cir-cles in these graphs show the master curves of CSR strengths which can be considered as the fatigue strength at stress ratio R =0and N f =1/2.Each curve consisted by hollow circles in these graphs shows the curve of fatigue strength r f versus re-duced time to failure t 0f at each reduced frequency f 0to diverge from the master curve of CSR strength.In order to confirm the applicability of TTSP for fatigue strength,we predicted the r f –N f curves at f =0.02Hz and com-pared them with the test results.The predicted r f from fatigue master curves for all FRP laminates agree well with experimental ones,therefore,the TTSP for the creep compliance of matrix re-sin also holds for fatigue strength of the corresponding FRPlaminates.Fig.10.r f versus N f curves at frequency 2Hz for Dry specimen.M.Nakada,Y.Miyano /Composites Science and Technology 69(2009)805–813811It is cleared from Figs.11-1and 11-2that the r f of all five FRP laminates strongly decreases with time to failure,temperature and water absorption and that the r f of four kinds of FRP laminates except GFRP laminates (E-glass/VE)decreases scarcely with N f although that of E-glass/VE decreases strongly with N f .And the degradation rate to time and temperature for the fatigue strength r f of these CFRP laminates is very similar to that for CSR strength.The r f of all FRP laminates also decreases with water absorption and that returns to that of Dry specimens by re-drying after water absorption except that of T300/EP in the range of long timeandFig.11-1.Master curves of flexural fatigue strength for T300/VE,T700/VE-F andT700/VE-K.Fig.11-2.Master curves of flexural fatigue strength for T300/VE,T300/EP and E-glass/VE.812M.Nakada,Y.Miyano /Composites Science and Technology 69(2009)805–813that of E-glass/VE in all range of time employed.The r f of Wet+ Dry specimens of T300/EP and E-glass/VE does not return to that of Dry specimens and shows irreversible behavior.4.ConclusionThe prediction of long-term fatigue life offive kinds of FRP lam-inates combined with matrix resin,fiber and fabric for marine use under temperature and water environments were performed by our developed accelerated testing methodology based on the time–temperature superposition principle(TTSP).The three-point bending CSR and fatigue tests forfive kinds of FRP laminates at three conditions of water absorption were carried out at various temperatures and loading rates.As results,theflexural fatigue strength of three kinds of CFRP laminates with vinylester resin as matrix strongly depends on water absorption as well as time and temperature,however scar-cely depends on the number of cycles to failure.The master curves of fatigue strength for these CFRP laminates are constructed by using the test data based on TTSP.The fatigue strength of these CFRP laminates decreases with water absorption and that returns to the initial fatigue strength by re-drying after water absorption. It is possible to predict the long-term fatigue life for these CFRP laminates under an arbitrary temperature and water absorption conditions by using the master curves.Furthermore,it is clear that the degradation rate of fatigue strength of these CFRP laminates is determined only by increasing of time,temperature and water absorption and is independent uponfiber constitutions which are the type,volume fraction and weaves.On the other hand,CFRP laminates with epoxy resin as matrix and GFRP laminates with vinylester resin as matrix chemically change by the process of water absorption and re-drying and the flexural fatigue strength of these FRP laminates decrease with this process.AcknowledgementsThe authors thank the Office of Naval Research for supporting this work through an ONR award(N000140110949)with Dr.Yapa Rajapakse as the program manager of solid mechanics.The authors thank Professor Richard Christensen at Stanford University as the consultant of this project and Toray Industries, Inc.as the supplier of CFRP laminates.All of experimental data were measured by the staffs and graduate students of author’s laboratory,Kanazawa Institute of Technology.The authors thank these staffs and graduate students,Dr.Naoyuki Sekine,Dr.Junji Noda,Ms.Jun Ichimura,Mr.Eiji Hayakawa and Mr.Takahito Uozu.References[1]Aboudi J,Cederbaum pos Struct1989;12:243–56.[2]Sullivan pos Sci Technol1990;39:207–32.[3]Gates T.Experimental Mechanics1992:68–73.[4]Miyano Y,Kanemitsu M,Kunio T,Kuhn H.J Compos Mater1986;20:520–38.[5]Miyano Y,McMurray MK,Enyama J,Nakada M.J Compos Mater1994;28:1250–60.[6]Miyano Y,McMurray MK,Kitade N,Nakada M,Mohri pos Mater1994;4:87–99.[7]Miyano Y,Nakada M,McMurray MK.J Compos Mater1995;29:1808–22.[8]Shen CH,Springer GS.J Compos Mater1976;10:2–20.[9]Kibler KG.In:AGRD conference proceedings;1980.p.8-1.[10]Neumann S,Marom G.Polym Compos1985;6:9–12.[11]Selzer R,Friedrich pos Part A1997;28A:595–604.[12]Miyano Y,Nakada M,McMurray MK,Muki R.J Compos Mater1997;31:619–38.[13]Miyano Y,Nakada M,Kudo H,Muki R.Prediction of tensile fatigue life undertemperature environment for unidirectional CFRP.Adv Compos Mater 1999;8:235–46.[14]Miyano Y,Nakada M,Muki R.Applicability of fatigue life prediction method topolymer composites.Mech Time-Dependent Mater1999;3:141–57.[15]Miyano Y,Nakada M,Muki R.Prediction of fatigue life of a conical shaped jointsystem forfiber reinforced plastics under arbitrary frequency,load ratio and temperature.Mech Time-Dependent Mater1997;1:143–59.[16]Sihn S,Miyano Y,Nakada M,Tsai SW.Time-and temperature-dependentfailures of a metal-to-composites bonded joint with PMMA adhesive material.J Compos Mater2003;37:35–54.[17]Sekine N,Nakada M,Miyano Y,Tsai SW.Time–temperature dependence oftensile fatigue strength for GFRP/metal and CFRP/metal bolted joints.In: Proceedings of13th international conference on composite materials,Beijing;June2001.p.1610.[18]Miyano Y,Nakada M,Sekine N.Accelerated testing for long-term durability ofGFRP laminates for marine pos Part B2004;35:497–502.[19]Miyano Y,Nakada M,Sekine N.Accelerated testing for long-term durability ofFRP laminates for marine use.J Compos Mater2005;39:5–20.[20]Miyano Y,Nakada M,Nishigaki K.Prediction of long-term fatigue life of quasi-isotropic CFRP laminates for aircraft use.Int J Fatigue2006;28:1217–25. [21]Dow NF,Gruntfest IJ.Space Sciences Laboratory,Structures and dynamicsoperation.T.I.S.R60SD389;1960.M.Nakada,Y.Miyano/Composites Science and Technology69(2009)805–813813。

57第15卷 第4期 2013 年 4 月辽宁中医药大学学报JOURNAL OF LIAONING UNIVERSITY OF TCMVol. 15 No. 4 Apr .，2013中药注射剂由于来源于天然药材，在栽培、贮存及生产加工过程中可能受到重金属的污染。

而重金属在人体中累积达到一定程度，会造成慢性中毒。

尤其是注射液不经消化，直接进入体液，风险更大。

目前，国内外将铅、镉、铜、砷、汞作为主要监控指标，但有关中药注射剂中重金属残留的检测鲜有报道[1]。

因此，对中药注射剂中重金属及有害元素进行测定具有重大现实意义。

为测定鸦胆子油乳注射剂中重金属及有害元素的含量，我们分别采用原子吸收法和原子荧光法对样品中铅、镉、铜、砷、汞残留量进行测定，并根据用法用量计算其每日最大摄入量，根据考察结果，结果表明本品的原料由中药材而来，虽然经过精制过程，但其重金属及有害元素仍有所残留，故应对其加以严格控制。

1 仪器与试剂岛津AA-6300型原子吸收分光光度计（日本岛津公司）；AFS-830双道原子荧光光度计（北京吉天仪器有限公司）；Mars 型微波消解仪（CEM 公司）；BHW-097赶酸加热板。

Pb、Cd、Cu、Hg、As 单元素标准溶液（国家有色金属及电子材料分析中心，浓度均为1000μg・mL -1）。

高纯级硝酸、优级纯硫酸和盐酸，其余试剂均为分析纯，实验用水为重蒸水。

鸦胆子油乳注射液来源于市售三家企业。

2 方法与结果 2.1 测定条件[2]Pb、Cd 采用石墨炉法，Cu 采用火焰法，仪器测定条件见表1，石墨炉参数见表2，As 采用氢化物发生法，Hg 采用冷原子法，仪器测定条件见表3。

2.2 溶液的制备2.2.1 供试品溶液的制备精密量取样品0.5 mL 于消解罐内，加入硝酸10原子吸收及原子荧光法测定鸦胆子油乳注射剂中5种重金属及有害元素的残留量李秀贤1，周海莉2，王怡君2（1.辽宁中医药大学附属第二医院，辽宁省中医药研究院，辽宁 沈阳 110034；2.辽宁省食品药品检验所，辽宁 沈阳 110023）摘 要：目的：建立鸦胆子油乳注射剂中重金属及有害元素（铅、镉、铜、砷、汞）的分析检测方法。

Using “Smart Calibration” To Ensure Accurate WeighingBy Steve Watson, Sartorius IntecSmart calibration; or the ability to commission tank load cells without test weights orflow meters, can significantly reduce both the installation cost and improve overallweighing accuracy. Using factory calibration data instead of weights even allowsthe changing of load cells or instrumentation while the tank remains filled withmaterial.Before we dive into the nuances of calibration without weights, let’s go over some of the basics, starting with the load cell.A load cell consists of two components – the load cell housing and the transducer, usually referred to as the “strain gauge.” The strain gauge is an array of tiny resistors bonded to the load cell housing with epoxy glue or etched into the metal surface of the load cell itself. As the load cell compresses the resistance changes. This change in resistance is translated into a weight value by the indicator.In order to amplify the signal from the load cell a constant voltage (commonly referred to as excitation voltage) is transmitted applied to the load cell from the indicator. The greater the excitation voltage the greater the output from the strain gauge, which is measured in millivolts (mV). Typically, a strain gauge will output 2 mV per volt of excitation. Therefore, if an indicator supplies 12 volts of excitation the strain gauge will transmit 24 mV at full loaded capacity and approximately zero mV in an unloaded condition. Any mV output between 0-24 mV represents the weight measured by the load cell and is displayed on the indicator.The transducers that are used to manufacture strain gauges are extremely difficult to duplicate but in order to have the ability to calibrate without weights the load cell has to be perfectly matched, meaning the load cells output identical mV values when loaded with identical mass. Matching of load cells usually involves a post-production selection process to pair up cells that output similar mV values at similar mass loads. Some manufacturers take it one step further and adjust every load cell during production to match all others in the same accuracy class using certified calibration weights. Among other advantages, this allows a single load cell to be replaced without re-calibration of the entire system, an expensive and time consuming job. A reputable manufacturer will even give the calibration data for each load cell so that the exact value can be entered for each load cell in cases where the highest accuracy is needed. This is only possible because each load cell is individually tested and documented, not spot checked like some other manufacturers.Don’t assume that the load cells being used in your system are capable of calibration without weights because chances are they are not. Most load cells are not factory matched or individually certified to meet specific tolerances and so their mV output must be determined using test weights and then compensated for electronically on the indicator. When they are matched, however, no weights are required to determine the mV output since it’s predetermined in the factory. Once installed the user only needs to enter the certified mV output (typically 2.000 mV) directly into the indicator and the “calibration” is complete. More accurately, the “smart calibration” is not a calibration at all but instead it’s a simple data entry function of certified factory calibration values.Although it would be convenient to be able to use smart calibration all the time, there arelimitations. While it’s possible to predict with certainty that load cells will remain accurate without re-calibration it’s impossible to guarantee that the tank installation is flawless and does not change over time. Extraneous forces such as rigid pipe connections, tank settling, in-feed and out-feed conveyormotion, excess vibration or tank damage will change the vertical force introduction on the load cells which has a negative impact on accuracy. A poor tank design will produce a non-repeatable weight reading when standard weights are applied. This condition must be repaired by eliminating or minimizing the external forces. A marginal tank installation may show a reproducible reading but not necessarily an accurate result compared to standard weights. In this case the weight reading must be adjusted using calibration weights to achieve an acceptable accuracy- assuming the marginal installation itself cannot be improved.Many procedures call for routine tank weight testing regardless of the certified load cell accuracy. In these cases it is always best to install the tank using the factory certified mV output values and then re-test using physical calibration weights. It’s sometimes difficult to load more than 5% of the total tank capacity in calibration weights so it’s always better to adjust calibration to factory certified mV values and then verify the tank reading with physical weights.Unfortunately, there are many factors beyond the load cells that determine tank weighing accuracy, particularly in process environments. Accuracy itself is a term relative to individual expectations. Often times the limitations on accuracy are the result of a restrictive tank installation and not the accuracy of the load cells. Rarely is a tank installation so free of external influences that the accuracy of the system comes close to the accuracy rating of the load cells. This is especially true when empty vessel weights are disproportionately larger than the net amount being weighed. In these cases more accurate load cells can help but only to the extent that the external tank connections do not offset the increased load cell accuracy.Whether dealing with a perfect or flawed installation, using pre-calibrated and matched load cells will always save time and provide a much safer installation than any attempt to calibrate with standard weights. A good weight indicator that is capable of accurately dissecting the mV signal into millions of internal steps will combine with smart calibration load cells to provide a powerful tool in controlling process weighing accuracy.。

METHOD STATEMENT FOR ROAD WORKSTABLE OF CONTENTS1.0 SCOPEThe purpose of this Method Statement is to describe the Methodology involved in construction of Road Works in accordance with project technical specifications, international standards and codes, local rules and regulations (QCS-2010). This method statement will cover the following works:❖Earth Works Preparation to Road Formation❖Granular Sub-base Preparation Works❖Asphalt Works2.0 PERMITS AND LICENSESThe Permits and License required for commencement of the work shall be obtained prior starting any site activities, the permits and license shall be obtained from the Following:❖Electricity and Water Authority❖Tel-communication Operator❖Municipality Government❖Ministry of Environment❖Public Work Authority (Roads)3.0 SEQUENCE OF WORKS FOR ROADS3.1 Site preparation❖Prior to commencement of work, the contractor shall obtain the relevant work permits from Client❖The contractor shall under take clearing of site in order to remove any existing vegetation; steel, concrete, unused buildings identified for demolition, piping debris, loose windblown sand and other foreign materials.❖All existing structures, foundations, etc. as indicated above shall be entirely demolished, removed, transported and disposed of by the contractor to a debris yard designated by Client. All soft spots left shall be filled with suitable material and compacted as perspecification.❖Excavated material, if any, and suitable and approved by Client for backfilling shall be transported and placed in fill areas within the limits of the work.3.2 Survey and Setting Out3.2.1 Survey Markers❖Survey Markers shall be durable, appropriate to location and intended use.❖Survey Markers shall be clearly identifiable and protected from construction traffic. For installation of a Survey Marker into reinforced concrete, the JV's will ensure that steel reinforcement bars are to be avoided.❖Either removable anchors or epoxy adhesive shall be used as the methods of fixing Benchmarks are a particular type of Survey Markers used in the control of elevation.❖Benchmarks shall have a domed surface for unambiguous staff placement. Primary Benchmarks shall comprise a stainless steel bolt securely placed vertically into aconcrete slab, or horizontally into a column.❖The protrusion shall not pose a safety hazard. Survey Markers used for horizontal control shall have an unambiguous point above (or below) which a survey instrument can be precisely centered.❖The point shall comprise either a punch mark or the intersection of 2 lines forming a cross. Cross-headed road nail (75mm long) complete with a colored washer shallgenerally be used as a Survey Marker.3.2.2 Control Observation, Adjustment and Presentation❖Homogeneous horizontal and vertical survey control is required prior to any setting out.Survey control shall evolve from the whole to the parts. Where practical, all survey points within a horizontal survey control network shall be occupied and observed from.❖Forced centering techniques shall be used throughout. A round of angular observations shall comprise the mean of observations taken on both faces of the total station. A minimum number of 4 rounds of horizontal and vertical angles shall be observed at each instrument set up.❖For control work, the angular spread of horizontal angles shall not exceed 3’’ of arc.Distances shall be measured in both directions. All raw data pertaining to each set up shall be electronically recorded. Survey control shall include redundant observations.Observation networks shall be processed using the method of least squaresadjustment and the resulting residuals to the observations shall be inspected formagnitude.❖Any large residuals or error ellipses shall be examined and appropriate remedial action shall be taken. Precise leveling shall be double run using equal back and fore sights at each instrument set up. Leveling sights shall never exceed 30 meters. On the ground surface, the Contractor shall establish stable bench marks adjacent to the site so that the distance between adjacent benchmarks does not exceed 250 meters.❖Height datum transfer accuracy shall be better than 2mm in a 30m deep shaft. The transfer of height datum shall be by various independent means. azimuth transfer from surface to underground shall be better than 3“ of arc and point transfer shall beaccurate to within 2mm. Such transfers shall be by various independent means.❖ At 3 month intervals, the Contractor shall conduct a complete survey of all existing survey control. A bound, A4 size survey control report shall be submitted to theEngineer for acceptance within 2 weeks of completing the field work.❖The convention adopted shall comprise Station Name, Easting, Easting, Northing and Elevation reading from left to right report shall contain the following information report shall contain the following information: dates of survey, fixed survey control and values, specification of instrumentation used, calibration status of instrumentation used,observational acceptance criteria, list offinal adjusted co-ordinates findings andconclusion.❖Attached to the report shall be the observations (A4 print out of electronic booking sheet or customized spread sheet), adjustment with residuals and station error ellipses, table of differences in mm from previous co-ordinates and elevations (if applicable),a drawing clearly showing layout of scheme and measured quantities and final co-ordinates in tabular form.3.2.3 General Setting Out❖The Contractor shall carry out a comprehensive level survey of the Contract area before any work commences on the site that may alter original ground levels.❖Pre-computation shall be carried out prior to any setting out. For rail projects, the effects of cant and throw shall be incorporated into pre-computation wherever relevant.❖ All pre-computation shall be readily available in a spread sheet format for use on site.The method of setting out for each particular element of the work shall commensurate with the required accuracy, the method of construction, and shall be appropriate for site conditions.❖In the setting out process, all elevation transfer conducted by leveling shall start on an established benchmark and finish on a different benchmark. If a significant disclosure is detected, the reason shall be determined and the necessary corrective action taken.❖After the erection of the formwork and prior to concreting, a survey check shall be carried out on the formwork to ensure that the setting out has been done correctly.❖ A spreadsheet shall be used in all instances to tabulate the difference (or offset) in mm between the actual set out (or as-built) co-ordinates from the design co-ordinates. In cases where the design is an alignment, offsets to the alignment shall be computed for each surveyed point.3.3Earth Works3.3.1Earthwork General❖The JV will carry out all earth work in such a manner as to prevent erosion or slips and shall limit working faces to safe slopes and height.❖The JV will ensure that all surfaces have at all times sufficient gradients to enable them to shed water without causing erosion.❖Hauling of material from cuttings or the importation of fill material to the embankments or other areas of fill shall proceed only when sufficient compaction plant is operating at the place of deposition to ensure compliance with the requirements of Client.❖Construction traffic other than that required for the excavation and trimming shall not use the surface of the bottom of a cutting unless the cutting is in Rock or the Contractor maintains the level of the bottom surface at least 300 mm above formation level.❖Any damage to the sub-grade arising from such use of the surface shall be made good by the Contractor at his own expense with material having the same characteristics as the material which has been damaged.❖The JV will arrange for the rapid dispersal of water shed on to the earth works or completed formation during construction, or which enters the earth works from any source❖The JV will provide where necessary temporary water courses, ditches, drains, pumping or other means of maintaining the earth works free from water❖The JV will take special care that naturally occurring materials within the site are not rendered unsuitable by his method of working. Areas of cuttings and excavation shall be so worked that rainfall is conducted rapidly away from the exposed material and at times of expected heavy rain that the cutting areas are protected by appropriatemethods of working and drainage provisions.3.3.2Classification of Earth Work MaterialThe following definitions of earth works shall apply to this and other clauses of the Specification in which reference is made to the definitions:❖ Top Soil' shall mean the top layer of soil that can support vegetation.❖ Suitable Material' shall comprise all that is acceptable in accordance with the Contract for use in the works and which is capable of being compacted in the manner specified in project Technical Specifications to form a stable fill having side slopes as indicated on the drawings.❖ Not Suitable Material' shall mean other than suitable material and shall include :•Material from swamps, marshes or bogs;•Peat, logs, stumps or other organic matter and perishable and toxic materials;•Material susceptible to spontaneous combustion.•Clay of liquid limit exceeding 80 and/or plasticity index exceeding 55.❖‘Rock' shall mean hard material found in ledges or masses in its original position,which would normally have to be loosened either by blasting or by pneumatic tools, or if excavated by hand, by wedges and sledge hammers. ‘Rock’ shall also includeall solid boulders or detached pieces of rock exceeding 0.10 m 3 in size in trenchesor exceeding 0.20 m 3 in general excavation.3.3.3 Excavation❖Excavation will be carried out to the dimensions, lines, levels and slopes as indicated on the IFC Drawings.❖If the JV excavates deeper than the depths shown on the Drawings or as instructed by the Client, then he shall fill in such excessive depths with acceptable materials to the satisfaction of the Client.❖The bottom of all excavations shall be free from mud and water, trimmed clean, protected from the effects of weather and thoroughly compacted and consolidated❖The JV will be responsible to excavate to the top of soft or defective soil below the formation level and backfill with Suitable Material. Such backfill will be well compacted in layers of accepted thickness by approved manners and means.3.3.4Cuttings and Cut Slopes❖Unless otherwise specified, no portion of the earth cutting shall vary from the specified or ordered formation level by an amount exceeding 150mm.❖In the case of cut slopes, no portion of the completed slope shall vary by an amount exceeding 100mm (measured at right angles to the slope).In all cuttings, whether in earth or rock, undulations in the general plane of the slope will not be permitted.Unless otherwise specified, excavation in rock shall extend to at least150mm below the specified formation level and backfilled with approved materials.❖Any overhanging, loose or unstable material shall be removed. The excavation shall be so arranged that the working areas are adequately drained throughout the period of construction.❖In cutting where the strata consists of earth overlaying rock which is required to be cut to a slope of 1 (H): 2 (V) or steeper, the face of the rock shall be given its appropriate slope up to the junction of the rock and earth and the latter shall be stripped to form a bench 900mm wide and sloped to the gradient as specified.3.3.5Maintenance of Excavated Area❖ Continuously monitor the dimensions / level of excavation to avoid over Cutting if any accidental over cutting takes place, fill and compact with Suitable material to thedesired level.❖ Remove any undesirable material (vegetation, organic soils etc.)Encountered duringexcavation and fill with approved granular material.❖ Take necessary measures to keep the excavation free from water by De-wateringwherever required.❖ Arrange shoring so that it does not interfere with the progress of work andFunctioning of nearby facilities as work proceeds, remove the shoring carefullywithout causingany danger to the excavation.❖ If any accidental slips or settlement occurs in the excavated area, Properly fill byselected fill or with mass concrete.3.3.6Sloping the Sides of Excavation❖ Wherever there are chances of sliding, properly slope sides of Excavation.❖ Trim the slopes by hand to uniform batters as shown on the IFC Drawings.❖ Trim any rock or boulder appearing in the face of the slope, and remove italtogether, if it seems unstable.❖ If any rock is removed, fill the resulting void with compacted material.❖‘Imported Rock Fill' shall be clean well-graded quarry waste provided by the Contractor from sources outside the Site. It shall be resistant to weathering, to the acceptance of the Client.❖ The maximum size particles in the material shall pass through a rigid 200mm square grid and the largest dimension of any particle shall not exceed 300 mm. The material shall not have more than 10% of its particles passinga0.75 mm BS sieve.❖‘Fill Material' shall mean Suitable Material for backfilling with the exc eption of Rock and Imported Rock Fill❖‘Special Fill Material’ shall mean Suitable Material of which at least 95 per cent shall pass a 125mm BS sieve and at least 90 percent shall pass the 75mm BS sieve. Up to 5 per cent of the material may be made up from isolated boulders of maximum dimensions not exceeding half the thickness of the layer of material being placed.Liquid limit not exceeding 35❖plasticity index not exceeding 12(h) ‘Selected Fill' shall be well graded granular natural sands, gravel, crushed rock, crushed concrete, well burnt shale or othermaterialsaccepted by the Client. The material passing a 425 micron BS sieve, when tested inaccordance with BS 1377, shall have a plasticity index of less than 6%.❖‘Imported Fill Material' shall be Fill Material supplied by the Contractor from a source outside the Site and accepted by the Client's.3.3.7 Compacting and Levelling the Fill❖ During compaction, maintain the M.C of fill material, usually +/- 3 Percent of OMC. If Required, add clean water to the fill material in order To maintain the necessary M.C❖ Achieve minimum density of specified M.D.D. of soil, by using roller / Plate compactor or any other approved equipment.❖ Around new (or existing remaining) concrete foundations or other Substructures do Not compact by rolling. Instead, use mechanical Rammers or plate compactors to Compact the space in vicinity of Concrete structure.❖ After compaction, level the surface free from undulations using suitable Equipment.3.4 Materials forSub-base3.4.1Fine Aggregate for Sub-Base❖Fine aggregate (Passing the 4.75 mm sieve) shall consist of crushed mineral aggregate and/or natural sand.❖The fine aggregate shall be clean and free from organic matter, clay — balks and other extraneous or detrimental materials.❖The ratio of aggregates passing the 0.075 ram sieve shall not exceed 66% of the portion passing the 0.425 mm sieve.❖The material passing the 0.425 mm sieve shallhave a maximum liquid limit of 25% and the plasticity index shall not exceed 6%.Where the source of fine aggregate does not meet the above requirements, with the Engineer's approval, add fine aggregate and filler to correct the gradation or to change the characteristics of the material passing the 0.42 mm sieve so as to meet the requirements. Such additional material shall be added in a manner which ensures a completely homogeneous material3.4.2Coarse Aggregate for Sub-Base❖Coarse aggregate (retained on the 4.75 mm sieve) shall consist of crushed stone crushed gravel with minimum of 50% by weight having at least one fractured face.❖The flakiness index of the aggregate shallnot exceed 35% when tested in accordance with BS 812.❖The Los Angeles Abrasion Loss, as defined by ASTM C131/C535, shallbe 40% maximum.❖The aggregate type for the sub-base shall be crushed limestone.170-7.5.66 ❖The coarse aggregate shall be hard and durable and free from organic matter, clay and other extraneous or detrimental materials.❖Coarse aggregate shall have a maximumloss of 20% by the magnesium sulphate soundness test when performed as per ASTM C88 for 5 cycles.❖The minimum CBR value obtained when preparing samples of aggregate base and aggregate Sub-base at optimum moisture content and at 100% relativecompaction and soaking them for 4 days shall be 80% and 60%; respectively. The maximum permitted swell shall he 0.5% and 1.0%; respectively3.4.3 Mix Design for Sub-base❖ Aggregates sub-base shall consist of crashed mineral aggregates or natural mineral aggregates of the designated gradation and thickness.❖The maximum dry density and the Optimum moisture content of the material shall ascertained as per the test procedure given in CML 12- 97 andthis shall beused to assess the degree of compaction of the mix after rolling❖The pavement layer designated as sub-base, the material shall conform to Class B or Class C❖The particle size shall be determined by the washing and sieving method of BS 812: Part. 103.❖Fine and coarse aggregates for sub-base shall be combined gradation of material that meets the requirements of the specification for density and other properties,❖The aggregate bases shall conform to the class A or class B gradation and the aggregate sub-base shall conform to the class C gradation, as given in the following Table:❖Immediately comply with such instructions without being entitled to any indemnities or extensions as a result of such instructions. Any equipment or plant shall not be used beforeobtainingthe approval of the Client and to engage skilled And trained operators, mechanics and labour to carry out the works.3.5 Plant Generally❖ The machinery and tools used in constructing the various items involved in asphaltworks shall be in good workingcondition and free of oil and fuel leaks This variousitems shall be maintained and preserved for the whole duration of the work. TheClient shall approve the machinery and tools before works begin and supply of such machinery with adequate quantities in order to execute the work with due speed and precision.❖ For the Client if required, the manufacturer's catalogues, specifications and other published data for the equipment and machinery shall be supplied.❖ On first erecting a batching plant and at least once each three months thereafter, the plant shall be calibrated by a calibration service organization approved by theCentral Materials Laboratory. Production shall not be permitted if the weigh batch calibration does not comply with the requirements of BS 5328.❖ The Client shall have the right to stop the use of any equipment or plant which he deems to be inferior to the quality required or detrimental to the permanent works and to instruct the removal of such equipment and to have it replaced by suitable equipment❖Immediately comply with such instructions without being entitled to any indemnities or extensions as a result of such instructions. Any equipment or plant shall not be used beforeobtainingthe approval of the Client and to engage skilled. And .trained operators, mechanics and labor to carry out the works.3.6 Inspection and Testing Control❖For verification of plant weights and measures, character of materials used in the preparation of the mixes, testing and other quality control requirements, the Engineer shall at all times be provided access to all portions of the mixing plant, aggregate plant, storage yards, crushers and other facilities used for producing and processing the materials of construction❖The Engineer shall have authority to take samples and perform tests on any material supplied to the site from any source whatsoever in order to establish complianceandtoaccept or reject as he deems necessary. Samples shall also be taken from completedwork to determine compliance. The frequency of all sampling and testing shall be asdesignated by the Engineer.❖Suitable facilities at the quarry or plant shall be provided to carry out all necessarytests on the raw materials and mixes.❖For obtaining specimens of materials and samples taken from stockpiles shall be arranged, including the provision of any necessary equipment and plant. This work shall be performed in the presence of the Engineer if so directed by the Engineer.❖Materials that are not in compliance shall be rejected and removed immediately from the site of the works times otherwise instructed by the Engineer.❖Where defects in the material or the completed work have been corrected, until approval has been given by the Engineer subsequent work shall not proceed.❖Materials which require drying before performing any of the designated tests shall be dried out at temperatures not greater than 100 OC.3.7Installation Procedure for Sub-base❖Before commencing the construction of the sub-base, written approval for the Engineer must be obtained thatthe sub-grade is in compliance.❖Before starting the sub-base all the structures have to be installed like kerbs, dropped kerbs, barrier’s foundations, median b arriers, channel block andgully's pre-cast concrete part with road gully pot.❖If the naturalmoisture content is less than the optimum moisture content, the necessary amount of water must be added to obtain the optimum content.❖Allowance shall be made for the quantity of moisture which may be lost by evaporation in the process of raking, leveling and compacting, depending on atmospheric temperature❖The compacted layer shall have moisture content within. ±2% of the optimummoisture content.❖The moisture content shall be uniform in all parts of the section where the work is being carried out and in the various depths of the layer thicknessCompaction shall start immediately afterthe material has been laid.❖Work on the sub-base courses shall not continue during rainy weather.❖Material shall be spread as 2 layers to a thickness that would result in layers not more than 150 mm thick after compaction❖The course shall not be rolled when the underlying material is soft or yielding or when the rolling causes a wave like motion in this course.❖When the rolling develops irregularities, the irregular surface shall be loosened, then refilled with the same kind of material as used in constructing the course and again rolled.❖Rolling must continue until a relative density of not less than 100 % of the maximum dry density has been obtained as determined by the moisture density relationship in CML 12-97❖Care shall be taken so that layers already compacted under the layer being executed are not damage, or that the formation is not damaged❖Any such damage resulting in mixing the various layers constituting the different sub-grades and sub- base courses shall be carefully made good by our own expense and to the satisfaction of the Engineer.❖Any material that fails to meet test requirements shall either be reworked or removed and replaced and then retested to check for compliance. Also the corrective actions and protection shall be done accordance with QCS section 6 parts 4.7 and 4.8.❖All testing shall be can-led out in accordance with AASHTO T96, AASHTO T104 and the relevant parts of BS 1377 or CML Standard Methods of test❖The sub-base shall be compacted and tested for acceptance in accordance with SML 12-97 and BS 1377 to attain a minimum density of 100 % of the maximum city density ofthe material. Two tests every 500 m2 shall be carried out unless otherwise directed bythe Engineer.❖Whenever the degree of compaction is found to be less than required, the area of sub-base involved shall be satisfactorily corrected.❖The gradation of the placed material shall be checked by taking samples from an area0.5x0.5 m from the full depth of the layer for every 1000 m3 of laid material. Thegradation shall be tested in accordance with BS 812: Part 101❖Wherever the gradation is found to be outside the designated limits, the area of sub-baseinvolved shall be scarified, removed or reworked to provide a gradation incompliance.❖The final surfaces of the sub-base shallbe tested by means of a 4 meter long straightedge. And no rises or depressions in excess of 10 mm shall appear inthe surface.❖The finished surface shall be checked by dips or spot levels and shall be constructed to the designated grade levels to within ± 10 mm.❖Where these requirements are not met, the full extent of the area which is out of tolerance shall be determined and shall make good the surface of the course by scarifying to a minimum depth of 75 mm or4 times the maximum particle size. Whichever is greater, reshaping by adding or removing material as necessary, adding water if necessary and re-compacting.3.8 Protection of the Surface❖The sub-base course shall be protected sothat it shall be maintained sound during work progress, after its completion and before receiving the bituminous layers orbefore laying the surface overlay thereon.❖Any damage caused to the layer if exposed to traffic or natural conditions resulting in damage to its surface shall be made good according to the satisfaction of theEngineer.❖The Engineer has the right to stop all hauling over completed or partially completed sub-base course when there is any proof such hauling is causingdamage.❖Following the completion of the sub-base course, all maintenance work necessary shall be performed to keep the course in a condition for priming.3.9 Materials for Asphalt Work❖Fine aggregate is that portion of the mineral aggregate passing the 2.36 mmBS Sieve.❖Fine aggregate shall be non-plastic and chemically stable.❖Unless permitted elsewhere in the contract, the aggregate type for the wearing and intermediate courses shall be Gabbro.❖Fine aggregate shall be clean and free from organic matter, clay, cemented particles and other extraneous or detrimental materials.❖Sampling of fine aggregate shall be in accordance with BS 598 part 100.6❖Fine aggregate shall be consist of crushed hard durable rock and shall be of such Gradation that it will meet the required gradation Wadi beach or dune sand won't be Used.❖Individual stockpiles of crushed -fine ag g re g ate shall have a sand equivalent of not less than 30.❖The loss by the magnesium sulphate soundness test, as determined by ASTM C88, shall be a maximum of 18 %❖The maximum acid-soluble chloride content shall be 0.1% and the maximum acid- soluble sulphate content shall be 0.5 %.❖The fine aggregate shall havea maximum plasticity index of 4 % when sampled form stockpiles and shall be non-plastic when sampled from hot bins.3.10 Coarse Aggregate for Asphalt Work❖ Coarse aggregate is that portion of the mineral aggregate retained on the 2.36 mm BSSieve. Coarse aggregate shall consist of crushed natural stones and gravel. Crushedparticles shall be cubic and angular in shape and shall not be thin, flakyor elongated.❖ The source of crushed aggregate is considered to the crushing site from which it is produced. Sampling of coarse aggregate shall be in accordance with BS 598 : part 100.❖Coarse aggregate shall be clean and free from organic matter, clay, cemented particles and other extraneous or detrimental material The degree of crushingshallbe such that a minimum of 99 % by weight of aggregate (each stockpile) having at least one fractured face and 85 % having at least two fracturedfaces. No rounded or sub-rounded particles shall be used_ The flakinessindex (of each stockpile) shall not exceed 25 % for wearing course and 30 % for intermediate and base courses and the elongation index (of each stockpile) shall not exceed 25 % for the wearing course and 30 % for the intermediate and base courses.❖The loss by magnesium sulphate soundness test, as determined by ASTM C88, shall be a maximum of 10 % for aggregate used in wearing and intermediatecourses and a maximum of 15 % for aggregate used in base course.。

地幔中石榴石的残痕元素分布——以Zabargad为例R. Vannncci 1,2, N. Shimizu 3, G.B. Piccardo 4 L. Ottolini 2, and P. Bottazzi 2t Dipartimento di Scienze della Terra, Universitfi di Pavia, via Bassi 4, 1-27100 Pavia, Italy2 CNR-Centro di Studio per la Cristallochimica e la Cristallografia, via Bassi 4, 1-27100 Pavia, Italy3 Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USADipartimento di Scienze della Terra, Universit/t di Genova, Corso Europa 26, 1-16132 Genova, Italy摘要：在Zabargad的橄榄岩体的辉石矿物形成阶段的Al-Di离子探针显示组成铁镁质层的残斑状的辉石记录了重稀土的异常，Zr,Sc的富集并没有记录在尖晶石二辉岩中。

在斜方辉石和尖晶石的晶簇中的斜方辉石在岩体的内部形成，在层状的辉石中自由生长的单斜辉石带显示出强烈的稀土元素分离模式(HREE N/LREE N>1000;Yb>100×ch)以及非常高的Zr,Sc和Y的丰度(分别高达30,672,600 ppm)。

在外部带，富单斜辉石带的残斑状辉石具有非常高的重稀土异常(HREE N/LREE N~29;Yb~269×ch)，并且显示出Sc、Zr非常高的丰度(分别高达819ppm和164ppm)。