Experimental Analysis of Sediment Deposition Due to the Effect of an Upstream Reservoir Backwater

Effects of salts and organic matter on Atterberg limits of dredged marine sedimentsR.Zentar ⁎,N.-E.Abriak,V.DuboisCivil and Environmental Engineering Department,Ecole des Mines de Douai.941,rue Charles Bourseul,B.P.838,59508Douai,FranceA B S T R A C TA R T I C L E I N F O Article history:Received 7December 2007Received in revised form 4April 2008Accepted 9April 2008Available online 22April 2008Keywords:Dredged sediment Organic matter Atterberg limits Fall cone Bentonite KaolinThis paper examines the effects of treatments inducing the reduction of salts and the organic content on the Atterberg limits of marine dredged sediments.To de fine the liquid limits of the sediments,the results of the percussion-cup test and the fall cone test are compared for raw sediments and treated sediments.For the determination of the plastic limit,the results of the rolling test method are compared to the prediction of the fall cone test.Finally,the relationship between the water contents and the penetration depths between the liquid limit and the plastic limit is explored.©2008Elsevier B.V.All rights reserved.1.IntroductionThe dredging is a permanent and a necessary operation to maintain navigation in waterways,access to ports and in the coastal engineering.The dredging operation generates a considerable amount of sediments of various geotechnical properties and various levels of pollution.The traditional solutions such as the immersion and the inland deposit of these sediments,due to the environmental constraint,are likely to become unsatisfactory.In the field of civil engineering,public works and manufacture,the increase in the demand of granular materials combined with the environmental constraints for opening new careers impose to enhance the management of natural resources.In this context,dredged sediments could constitute a new source of materials in these various fields (Centre Saint Laurent,1993;Ulbricht,2002;LIFE,2002).In France,the civil engineering work consumes more than 450million tons of granular materials (UNPG,2007).Among various sectors of civil engineering,the field of road construction,with an annual consumption of 50.7%,is the bigger consumer (Michel,1997).In the context of bene ficial uses of dredged sediments,the road construction sector offers several advantages among which the material performances needed vary and depend on the road category,the layer of the road structure and the type and the amount of binders used.However,dredged marine sediments are complex materials by the presence of organic matter,salts and different levels of pollution.For their bene ficial use in the road construction field,in addition to thephysical and the mechanical characteristics,it is important to deter-mine their potential impacts on the environment.In the case of an unsatisfactory result,the treatment of dredged sediments has to be envisaged before a bene ficial use.In the last decade,several types of treatments of dredged sediments were proposed.These treatments range from mechanical separation techniques to biological techniques.These treatments generally induce changes in the physical and the mechanical characteristics in comparison to the untreated sediments.In the field of road construction,for fine materials,the index pro-perties,proposed in 1911by the Swedish agronomist Atterberg (1911)and introduced in the field of soil mechanics by Terzaghi and Peck (1948),with the grain size distribution,constitute the starting point for the geotechnical study.In this study,firstly,the effects of sediment treatments inducing a reduction in salts content in the pore fluid and the organic matter content on the liquid limit are discussed.At this stage,the liquid limits measured,using the fall cone test,are compared to the results of the percussion-cup test,commonly used in practice in several countries.Secondly,the plastic limits measured by the rolling test method are compared to the prediction of the fall cone test results.At this stage,the relationships between the water content and the penetration depth of the cone,between the liquid limits and the plastic limits,are investigated.2.Studied materials and basic characteristicsThe materials used in this study are dredged marine sediments from four different locations in Dunkirk Harbor (north of France).Two samples denoted A1and A2are typical sediments of the east harbor and two samples denoted B1and B2are typical sediments of the westApplied Clay Science 42(2009)391–397⁎Corresponding author.Tel.:+33327712418;fax:+33327712916.E-mail addresses:zentar@ensm-douai.fr (R.Zentar),abriak@ensm-douai.fr (N.-E.Abriak),dubois@ensm-douai.fr (V.Dubois).0169-1317/$–see front matter ©2008Elsevier B.V.All rights reserved.doi:10.1016/j.clay.2008.04.003Contents lists available at ScienceDirectApplied Clay Sciencej o u r n a l ho m e p a g e :w w w.e l se v i e r.c o m /l o c a t e /c l a yharbor.For the comparison of the test methods in de fining the liquid limits and the plastic limits,complementary tests on a bentonite and two types of kaolin (denoted respectively Be,Ka1and Ka2),com-mercially acquired,with signi ficantly different index properties in comparison to dredged sediments,are performed.Test results of the grain size distribution,the initial water content,the organic matter content and the speci fic gravity for each material,following respectively test standards NF 13320-1(2000),NF P94-050(1995),and XP-P94-047(1998),are reported in Fig.1and Table 1.From these results it appears that the grain size distributions,performed by a laser diffraction technique,of dredged sediments from the different locations are identical.The materials are composed mainly of silt;the clay fraction is between 9.9%and 14.0%and the sand fraction is in general less than 6.2%.The two types of kaolin and the bentonite are finer than the dredged sediments with a percentage diameter d 50varying between 4.7µm and 5.3μm.The organic matter content,de fined by the ignition test at 450°C,is higher for samples from the east harbor (A1and A2)than the samples from the west harbor (B1and B2).The speci fic gravity of the materials,measured using a helium pycnometer,decreases as the organic content increases;this is qua-litatively consistent with expectations and previous observations.3.Specimen preparation,experimental program and test equipments for the determination of the liquid limit and the plastic limit The preparation of dredged sediments starts by sieving the material in the 400µm diameter sieve (NF-P94-051,1993).At this stage seawater is used to facilitate sieving.For each type of sediments,after a settling period of 24h,the supernatant saline solution is removed and the sieved material is divided into three parts.The first part is placed in a container and is mixed with distilled water to form a suspension.After a settling period of 24h,the supernatant saline solution is replaced by distilled water and the sediments are mixed again to form a suspension.The replacement of the supernatant salinesolution is renewed three times before performing the Atterberg limits tests.The second part of the sieved material is treated with hydrogen peroxide following a similar procedure as described in the test standard for determining the organic content of materials (NBNFig.1.Grain size distribution of studied materials.Table 1Material characteristics SamplesA1A2B1B2Ka1Ka2Be %b 2μm10.339.9313.0314.0124.1924.6517.762μm b %b 63μm 89.6686.8480.7980.1075.2375.3482.2363μm b %0.01 3.23 6.18 5.890.580.010.01d 50(μm)9.2310.229.128.19 5.29 4.99 4.71Water content (%)122118148150Dry Dry Dry Organic content (%)9.79.37.0 6.7///Speci fic density2.482.492.532.532.622.642.68Fig.2.Percussion-cup test results and fall cone test results of dredged sediments.Table 2Liquid limit values as de fined from the percussion-cup test and the fall cone test for two different penetration depthsCasagrande method Fall cone method w L (%)w L (%)w L (%)N a =25d =20mm d =17mm A1129.4108.392.0A1-W 130.3115.4102.8A1-H 56.058.156.3A2113.9105.790.2A2-W 120.0108.598.9A2-H 50.753.050.5B1101.990.782.8B1-W 95.588.580.9B1-H 40.944.342.7B293.081.673.7B2-W 88.780.073.1B2-H 43.545.944.3Ka137.641.039.0Ka252.053.750.1Be377.7221.4153.3aNumber of blows.392R.Zentar et al./Applied Clay Science 42(2009)391–397589-207-3,1969).The remaining third part is kept to perform the tests on the raw material.For the measurement of the liquid limits on washed samples(A1-W,A2-W,B1-W and B2-W)and treated samples with hydrogen peroxide(A1-H,A2-H,B1-H and B2-H)by the percussion-cup test,at least four percussion numbers,between10and40,required to close the grove at varying moisture content levels,are defined.For each percussion number,the corresponding penetration depth is measured by the fall cone apparatus.At the plastic limit,as defined by the rolling test method,the penetration depth of the cone is also measured.For the two types of kaolin and the bentonite,after sieving the material in the400μm diameter sieve,the samples are prepared with water content greater than the liquid limit and stored overnight before testing.The test program performed on these standard materials,by the same operator,is similar to that performed on dredged sediments. Moreover,to explore the relationships between the water content and the penetration depth,additional measurements are performed for water content between the end of the percussion-cup test and the plastic limits as defined by the rolling test method.The fall cone equipment used in this study conforms to the test standard NF-P94-052-1(1995)and to BS1377(1975).The weight of the cone is80g with a30°cone angle.The cone penetration depth is measured by a dial gage and is estimated to within0.01mm.The percussion-cup test equipment used fulfills the test Standard NF-P94-051(1993).4.Test results and discussion4.1.Determination of the liquid limit by the percussion-cup methodTo explore the effects of the treatments,which induce the reduction of salt and organic content,on the liquid limits of dredge sediments,the experimental results of the percussion-cup test are reported in Fig.2a.From these results,it appears that the liquid limit values of dredged sediments,defined for a blows number of25,are very high and range between93%and129%(Table2).The samples from the east harbor exhibit systematically higher liquid limit values than those measured on samples from the west harbor.For washed samples,the liquid limit values measured are of the same order as the values measured on raw sediments(between89%and130%).The relative difference observed on each type of sediment is in general less than6%.To explore the effectiveness of the washing process to reduce the salts content in the material,an X-rayfluorescence(XRF)analysis is performed on raw sediments and on washed samples to undertake an elemental analysis.The results of this analysis,reported in Tables3and4 for samples from the east harbor,show a significant decrease in the sodium and the chlorides contents in washed material in comparison to the raw material.The magnesium and the potassium contents,to a lesser extent,are also reduced.For samples from the west harbor,the com-parison of the X-rayfluorescence analysis results performed on raw sediments and washed samples shows a similar trend.Table3Results of X-rayfluorescence analyses on raw sediments from the east harborO Si Ca Al Fe Na Cl S Mg 51.2514.713.4 4.73 4.29 2.91 2.75 2.00 1.81K Ti P Zn Cu Mn Pb Zr1.480.2710.1510.1260.09040.04980.03860.01Table4Results of X-rayfluorescence analysis on washed sediments from the east harborO Si Ca Al Fe Na Cl S Mg 52.615.214.5 4.91 4.94 1.330.60 2.16 1.51K Ti P Zn Cu Mn Pb Zr1.440.3030.1380.1440.09890.05480.04760.01Fig.3.Percussion-cup test results and fall cone test results of kaolin andbentonite. parison of test methods in defining the liquid limit of studied materials.393R.Zentar et al./Applied Clay Science42(2009)391–397For treated sediments with hydrogen peroxide,the measured liquid limit values,between41%and56%,decrease significantly in comparison to measured values on raw sediments.In terms of absolute values decrease,the dredged sediments from the east harbor are more affected by the treatment than the sediments from the west harbor.However,in terms of the relative differences,similar values are calculated for both types of sediments.To explore the effectiveness of hydrogen peroxide treatment to reduce the organic matter content,ignition tests at450°on treated sediments with hydrogen peroxide were performed.The loss of weight by ignition is respectively of2.3%for sediments from the east harbor and of1.6%for sediments from the west harbor.These values are significantly lower than the measured values on raw sediments (Table1).parison of fall cone test method with the percussion-cup method in determining the liquid limit of tested materialsIn order to compare the liquid limit values as defined by the fall cone test and the percussion-cup test,for each water content level corresponding to a percussion number,a fall cone test is performed. The test results are reported in Fig.2b.From these results,the liquid limit is calculated for a corresponding penetration depth of20mm,as prescribed in BS1377(1975)and for a penetration depth of17mm,as prescribed in NF-P94-052-1(1995).Qualitatively,in terms of the effects of salts and organic contents,the trend observed from the percussion-cup test results is confirmed(Table2).However,quantita-tively,for raw sediments and washed sediments,with high liquid limits,the water content at a penetration depth of20mm and at a penetration depth of17mm seem to be lower than the liquid limits defined from the percussion-cup test method.For treated sediments with hydrogen peroxide,with low liquid limits,a better agreement between the two test methods is observed.To compare the prediction of the liquid limits with both test methods,for materials with signi-ficantly different liquid limits,a percussion-cup test and a fall cone test are performed on standard materials which consist of two types of kaolin and a bentonite.The test results in terms of water content versus the percussion number and the water contents versus the penetration depths are shown respectively in Fig.3a and b.The calculated liquid limits from the percussion-cup test method and the fall cone test for all the samples are reported in Fig.4.As discussed above,it appears clear that there is an agreement in defining the liquid limit between the two test methods for materials with low liquid limit values(less than60%in this study),whereas for materials with higher liquid limits(more than100%)the fall cone test method predicts systematically lower values in comparison to the percussion-cup method.For the bentonite,with a liquid limit defined by the percussion-cup test method of377%,the relative difference observed in comparison to the measured value with the cone method reaches almost100%.Moreover,for materials with low liquid limit values,the result is less sensitive to the definition adopted for the penetration depth at the liquid limit(d=20mm or d=17mm)in comparison to materials with high liquid limits.For the30°,80g cone,Budhu(1985)observed that at liquid limit values above about50%,the majority of the fall cone liquid limit values,assuming a penetration depth of20mm at the liquid limit,are lower than those from the cup device.This result is in agreement with the reviewed data by Sherwood and Ryley(1970)and in agreement with the result of this paper.However,Leflaive(1972),on the basis of reviewing test results of about77soils,shows that for soils with liquid limits between60%and100%a penetration depth equal to20mm is adequate to define equivalent liquid limit values as measured by the percussion-cup test,whereas for soils with liquid limits less than40%, a penetration depth of16mm seems more adequate.Also,Leroueil and Le Bihan(1996)concluded that the liquid limits obtained from the percussion method are lower than those from the cone method for soils of a lower liquid limit range from the eastern Canada.This later result was confirmed by the data reviewed by Prakash and Sridharan (2006).In this paper,for materials with low liquid limits,a good agreement in defining the liquid limit between the two test methods is observed.Moreover,for materials with low liquid limits,the ob-tained results seem to be insensitive to the penetration depth in the range of17mm to20mm.The increase of the equivalent penetration depth at the liquid limit with an increase of the liquid limit,as defined by the percussion-cup test,or in other words the underestimation of the liquid limits by the cone method for materials with high liquid limits,was also pointed out by several authors using a Swedish type of fall cone(Kumapley and Boakye,1980;Mendoza and Orozco,1999;Koumoto and Houlsby, 2001).parison of the rolling test method with the fall cone test method in determining the plastic limitTo explore the effects of salts and organic contents on the plastic limits of dredge sediments,the experimental results of the rolling test method are reported in Fig.5and in Table5.From these results,it appears that the plastic limits of sediments from the east and the west harbor are parisons of the test results onraw Fig.5.Effects of sediment treatments on plastic limit.Table5Plastic limit values as defined from the rolling thread method and the fall cone test fortwo different penetration depthsRolling thread method Fall cone methodw p(%)w p(%)w p(%)d=2mm d=1.7mmA148.435.032.2A1-W53.238.435.4A1-H25.025.423.9A247.232.129.5A2-W47.432.429.7A2-H25.628.227.0B146.431.829.6B1-W44.428.125.9B1-H33.024.823.8B243.621.920.0B2-W47.835.333.3B2-H30.924.623.5Ka123.719.518.5Ka233.829.928.8Be52.162.455.9394R.Zentar et al./Applied Clay Science42(2009)391–397sediments with test results on washed samples reveal that the plastic limits are not affected by the washing process and the absolute difference observed is in general less than 5%.For treated sediments with hydrogen peroxide,the plastic limit values measured are signi ficantly lower than the values measured on raw and washed sediments.The relative difference observed is higher than 50%and this difference is higher for sediments from the east harbor than for sediments from the west harbor.In order to compare the rolling test method and the fall cone test method in de fining the plastic limits of dredged sediments,the penetration depth at the plastic limit for the fall cone test method is assumed to be in the ratio of 10in comparison to the penetration depth at the liquid limit (Wood and Wroth,1978;Wroth and Wood,1978).Moreover,based on the critical state theory,Wood and Wroth (1978)and Belviso et al.(1985)suggest that the relationship between the logarithm of the penetration depth and the water content is linear between the liquid limit and the plastic limit and the slope of this relationship is equal to one half of the plasticity index.However,for a number of soils,this relationship has been found to be highly non-linear (Karlsson,1961;Wood,1985;Harisson,1988;Feng,2000,2001).To explore the relationship between the water content and the penetration depth of the cone between the liquid limit and the plastic limit,the tests undertaken on the two types of kaolin and the bentonite are performed for additional penetration depths between 20mm and 2mm.The obtained test results are shown in Fig.6respectively in a semi-logarithmic scale (Fig.6a)and in a bi-logarithmic scale (Fig.6b).From these results,on standard materials with signi ficantly different liquid limits,it appears that for materials with high liquid limits the relationship between the water content and the penetration depth is linear in the bi-logarithmic scale whereas it appears highly non-linear in the semi-logarithmic scale.For materials with low liquid limits,both types of relationships seem to be adequate.The ef ficiency of the bi-logarithmic relationship for de fining the plastic limit from the fall cone test is pointed out by several authors in the literature (Kodikara et al.,1986,Feng,2000),Feng,2001,Kodikara et al.,2006).For the tests performed on dredged sediments,the water content at the penetration depth in the ratio of 10in comparison to the penetration depth at the liquid limit is determined on the basis of the bi-logarithmic plot.Also,in addition to the results of the fall cone test shown in Fig.2b,complementary penetration depths of the cone at water contents close to the plastic limits,as de fined by the rolling test method,are measured and taken into account in de fining the regression coef ficient of the bi-logarithmic relationship.Based on this procedure,the water contents de fined at penetration depths of 2mm and 1.7mm,reported in Table 5,are compared in Fig.7to the plastic limit values as de fined by the rolling test methods.From this figure,except for the results on the bentonite,it appears that the plastic limits as de fined by the fall cone test are lower than the values de fined by the rolling test methods for materials with high liquid limits.Moreover,it appears that the results are slightly affected by the penetration depth in the range of 2mm to 1.7mm.For materials with low liquid limits,the test results of the two test methods in de fining the plastic limits are in better agreements.In order to investigate the effect of the ratio assumed between the penetration depth at the liquid limit and the penetration depth at the plastic limit,it is reported in Fig.8the water contents at a penetration depths in the ratio of 8,10and 12in comparison to the penetration depth of 20mm,as assumed at the liquid limit in the BS 1377(1975).From these results it appears that for materials,with low liquid limits,the effect of the ratio assumed between the penetration depths at the liquid limit and the plastic limit seems to be moderate whereas for materials with high liquid limits,the ratio assumed seems to introduce a higher effect.4.4.Effects of treatment on the dredged sediments classi fication Following the observed effects of treatment on Atterberg limits of dredged sediments,the changes in the material characteristics of the materials are highlighted on the basis of the Casagrande chart as shown in Fig.9.As shown above,the effect of the washing process on the Atterberg limits changes is small in spite of the diminution ofchlorides,Fig.6.Investigation of the relationship between the water content and the penetration depth between the liquid limit and the plasticlimit.parison of test methods in de fining the plastic limit of studied materials.395R.Zentar et al./Applied Clay Science 42(2009)391–397sodium,potassium and magnesium observed on the basis of the X-ray fluorescence analysis performed on the washed sediments in compar-ison to the analysis performed on raw sediments.The low effect of the washing process on the Atterberg limits of dredged sediments can be due to several factors among them the presence of salts in the pore fluid after the washing process and the type of clay particles in the dredged sediments.To determine the crystalline phases in the materials studied,X-rays diffraction analysis is performed on dredged sediments from the east and the west harbor.From these analyses,the three principal crystalline phases detected are:calcite,quartz and halite.To specify the nature of the clay minerals in the dredged sediments,speci fic analysis are carried out on particle sizes lower than 2µm.These analyses consist in characterising the raw material,the material heated at 450°C for 2h in order to characterise the kaolinite and the saturated material by vapors of ethylene glycol to highlight smectites.For samples from the east harbor,the results of this analysis are shown in Fig.10and summarised in Table 6.The obtained results show that the clay phases of marine sediments are composed mainly of illite and kaolinite which are less affected by salts concentration in the pore fluid in changing the Atterberg limits.The effect of decreasing the organic content on the Atterberg limits is more signi ficant and both the liquid limits and the plastic limits decrease.As shown in Fig.9,the liquid limits as theplasticity indexes decrease with reducing the organic content in the sediments.However,the liquid limits decrease faster than the plasticity index.Casagrande (1948)suggests that the effect of increasing the organic content of a soil is to increase the liquid limit with no appreciable change in the plasticity index,thus causing the soil to shift below the A A-line.The presented results in this study,suggest a shift of the experimental data following the A-line.This statement is veri fied to some extend by the data from Holtz and Krizek (1970),which suggest an increase of the plasticity index with the increase in the liquid limit for materials with increasing amounts of organic content.5.ConclusionThis paper examines the effects of treatments inducing the reduction of salts and the organic matter content on the Atterberg limits of marine dredged sediments.To reduce the salts,in the pore fluid,and the organic contents,the sediments are washed with distilled water and treated with hydrogen peroxide.To measure the liquid limits of the dredged sediments,two test methods are performed.In terms of the test results,for dredged marine sediments,the process of washing seems to be ef ficient in reducing the salt content in the material;however the liquid limits seem to be unaffected.The alteration of the organic content by the hydrogen peroxide has shown its ef ficiency on the basis of the ignition test.With reducing the organic content of dredged sediments,a signi ficant decrease of the liquid limits is observed in comparison to the values measured on raw materials.In terms of test methods comparisons,in de fining the effects of the treatments on the liquid limits,the general trend observed is con firmed by both methods of determination.However,the fall cone test predicts systematically lower values in comparison to the percussion-cup test for materials with high liquid limits.For materials with low liquid limits,better agreement between the two test methods is observed.Based on the rolling tests method,the plastic limits of the dredged sediments seem to be unaffected by the washing process.By reducing the organic matter content,a signi ficant decrease of plastic limits is observed.In order to compare the test results of the rolling method to the prediction of the plastic limits by the fall cone tests,the relationship between the water content and the penetration depth of the cone is explored.Based on tests on standard materials with signi ficantly different liquid limits and plastic limits it seems that for materials with high liquid limits,the relationship between the watercontentparison of the plastic limit values de fined by the rolling thread method to the plastic limit values de fined by the fall cone test by assuming different penetrationdepths.Fig.9.Dredged sediments classi fication before and aftertreatments.Fig.10.Clay mineral phase of marine sediments from the east harbor.Table 6Results of X-ray diffraction analyses on raw sediments from the east harbor MineralsSmectites Illite Kaolinite Chlorite Marine sediments From the east harbor30%32%30%8%396R.Zentar et al./Applied Clay Science 42(2009)391–397and the penetration depth is linear in the bi-logarithmic scale.For materials with low liquid limits both types of relationships examined seem to be adequate.In terms of test methods,comparisons in defining the effects of treatments on plastic limits,the general trend observed is confirmed by both methods of determination.However, the fall cone test results,on the basis of the penetration depths at the plastic limit assumed,predict generally lower values in comparison to the rolling test method for materials with high liquid limits whereas for materials with low liquid limits a better agreement between the two test methods is observed.In terms of characteristic changes observed on the dredged sedi-ments following the treatments,based on the Casagrande chart,the results in this study suggest a shift of the experimental data following the A-line.AcknowledgementsThe research reported herein was conducted as part of a project to develop alternative solution than immersion for dredged sediments sponsored by a regional program PREDIS(Plan-Régional-d'Elimina-tion-des-Déchets-Industriels-Spéciaux)and industrials.Their support is gratefully acknowledged,as is the help provided by the technicians under the supervision of the authors.ReferencesAtterberg,A.,1911.Lorornas Forhallande Till Vatten,Deras Plasticitets-granser och Plastitesgrader.Kungliga Lantbruksakademiens Handlingar och Tidskrift50(2), 132–158.Belviso,R.,Ciampoli,S.,Cotecchia,V.,Federico,A.,e of the cone penetrometer to determine consistency limits.Ground Engineering18(5),21–22.BS1377,1975.British Standard Methods of test for soils for civil engineering purposes. 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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.。

THIEME239Comparative Analysis of Erythrocyte Sedimentation Rate Measured by Automated and Manual Methods in Anaemic PatientsVikram Narang 1 Sumit Grover 1, Amandeep Kaur Kang 1 Avantika Garg 1 Neena Sood 11Department of Pathology, Dayanand Medical College & Hospital,Ludhiana, Punjab, India Address for correspondence Sumit Grover, MD, Department of Pathology, Dayanand Medical College & Hospital, Tagore Nagar,Ludhiana,Punjab,141001,India(e-mail:*************************).Purpose Erythrocyte sedimentation rate (ESR) is a widely used indicator of inflam-mation and a routinely done hematology investigation to monitor patients of auto-immune and infectious diseases. We aimed to compare the ESR results obtained by Roller 20LC automated instrument and standard reference Westergren method and analyzed the effect of anemia (hematocrit) on ESR measurements through the auto-mated method.Methods We analyzed 1377 random anemic OPD patients (hematocrit [HCT] < 35%) for ESR levels measured by Roller 20LC using EDTA blood and Westergren method using citrated blood for a one and half year period from January 1, 2018 to June 30, 2019. Fabry’s formula was used to correct the Westergren ESR.Results The total number of samples after evaluation were divided into low (n = 232), intermediate (n = 417), high (n = 406), and very high range of ESR (≥100 mm/hr; n = 422). Mean difference between values of corrected and automated ESR for the low, intermediate, high and very high ESR range was 2.33 ± 5.03, 10.95 ± 8.04, 28.22 ± 19.11 and 43.3 ± 19.22 mm/hr, respectively. The 95% limit of agreement calculated by the Bland–Altmann analysis between the two methods for low-ESR range was −7.53 to 12.2 (highest correlation coefficient –0.65), while for very high ESR, range was −5.1 to 81.5 (least coefficient of 0.18) (p < 0.001).Conclusion In laboratories with high-sample load and where manual measurement may be tedious, the automated method of ESR measurement can safely replace the Westergren method for low-ESR values in patients with low hematocrit. While for high-ESR values, validation by the standard Westergren method may be needed.AbstractKeywords►corrected ESR ►Hematocrit ►Fabry’s formula ►AnemiaDOI https:///10.1055/s-0040-1721155 ISSN 0974-2727 .BackgroundThe erythrocyte sedimentation rate (ESR) is widely used in clinical practice as an indicator of inflammation, infection, trauma, or malignant disease.1 Many methods can be used for measuring the ESR such as Westergren method, Wintrobe’s method, Zeta sedimentation ratio, and micro-ESR. The mostsatisfactory method of performing the test was introduced by Westergren in 1921.2 The Westergren method is recom-mended for measuring the ESR by the International Council for Standardization in Hematology (ICSH).3,4 ESR ranges in adults from 2 to 20 mm/hour.5Various factors affect ESR value such as ratio of red blood cells to plasma, and cellular factors like cell size and cellJ Lab Physicians:2020;12:239–243©2020. The Indian Association of Laboratory Physicians.This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https:///licenses/by-nc-nd/4.0/).Thieme Medical and Scientific Publishers Pvt. Ltd. A-12, 2nd Floor, Sector 2, Noida-201301 UP, IndiaOriginal Article240Journal of Laboratory Physicians Vol. 12 No. 4/2020 © 2020. The Indian Association of Laboratory Physicians.Comparative Analysis of Erythrocyte Sedimentation Rate Narang et al.surface area and their intrinsic capability to aggregate and sediment. Zeta potential plays an important role in this. Increase in rouleaux formation is due to increased plasma proteins such as haptoglobin, ceruloplasmin, α1-acid glycoprotein, α1-antitrypsin, and C-reactive protein (CRP), whereas globulin contributes the least. Fibrinogen is the most abundant acute phase protein with the greatest impact on ESR.6,7 ESR is retarded by albumin. ESR measured by the Westergren method is affected by many factors such as room temperature and length and angle of placement of the tube.8Since ESR performed by the manual standard Westergren method is also affected by hematocrit, Fabry’s formula (Westergren ESR X 15/55-HCT) can be used to correct ESR values obtained by the manual method.9 Also, to overcome the confounding factors, recently many new automated techniques for measuring ESR have been developed and introduced in clinical laboratories. They also provide many advantages like safety of operators, reducing biohazards risks, quicker results, speedy processing time, and ease of perfor-mance of other hematological tests (erythrocyte, leukocyte and reticulocyte concentrations) in a single specimen.10 In 2010 and 2011, ICSH and Clinical and Laboratory Standards Institute (CLSI) released new recommendations.11,12 They kept the Westergren method as reference procedure and stated that all new technologies, instruments, or methodol-ogies have to be evaluated against the Westergren reference method before being introduced into clinical use. Also, it was recommended that the “systems which give the results same as the Westergren method with diluted blood at 60 minutes or normalized to 60 minutes are the only ones of clinical value.”10The automated Roller 20 LC method is based on the mea-surement of change in blood impedance after the red cell aggregation-sedimentation phenomenon has occurred. Roller 20 LC works on the principle of photometrical capil-lary stopped flow kinetic analysis.10 Roller 20 LC recreates the physiological body conditions as it is thermostat at 37°C. The inbuilt microcapillary mimics the blood vessel. The blood sample in the capillary is accelerated and immediately stopped in the flow, which is known as stopped flow system. This simulates the blood pressure given by the cardiac mus-cle, which pumps the blood in the body. Roller 20 LC instru-ment can measure ESR of 18 samples in 10 minutes with minimal blood volume of 800 µL, whereas the Westergren method takes 60 minutes to interpret the result.As per the ICSH guidelines,10,11 for comparison of automated method to the Westergren method, correlations and bias should be calculated for the entire analytical range as well as the low, middle, and upper third of the analytical range sepa-rately. Correlation coefficients for the three parts of the analyt-ical range should be compared with each other and to the total correlation coefficient. The statistical methods recommended for validations of alternate ESR methods are the coefficient of correlation, Passing–Bablok regression, and the Bland–Altman method. If these criteria are met, results can be mathematically transformed to the corresponding Westergren values. ICSH has also recommended to perform interference studies foranemia, hemolysis and lipemia, and indicate the level where interfering factors begins to affect the ESR results.10,11According to our knowledge, very few studies have been conducted following these guidelines properly to statisti-cally compare automated ESR with the Westergren method for the entire range of ESR values. We also evaluated the interference by anemia (hematocrit [HCT]) in an auto-mated method. Hence, we compared ESR values in anemic patients using automated method with the corrected manual Westergren ESR.In this study, automated ESR values of patients having HCT < 35% were measured on Roller 20 LC instrument using EDTA anticoagulated blood samples and compared with the cor-rected manual ESR performed on blood samples of the same patients by the Westergren method using citrated blood.Materials and MethodsCollection of DataIn this study conducted over one and half year period from January 1, 2018 to June 30, 2019, in a tertiary referral insti-tute of north India, ESR of random anemic patients measured by automated method was compared with manual method. Permission was obtained from the ethical committee to conduct the study. ESR measurement was done on random blood samples of anemic indoor patients (HCT < 35%) by the standard Westergren method (citrated blood) and auto-mated method (EDTA blood) using Roller 20 LC. The manual Westergren values were corrected using Fabry’s formula (Westergren ESR X 15/55-HCT). During the study period, a total of 1800 samples of anemic patients were tested for ESR using both the methods. The duplication or triplication of tests was avoided using the hospital information system (HIS) and patient unique identification number or medical record department number (MRD). The ESR tests with the same MRD if repeated within a week of admission or during the same visit were excluded by authors (S.G., V.N., and A.K.). The cases were further categorized into four groups, includ-ing ESR up to 20 mm/hour and elevated ESR (> 20 mm/hour), which were further categorized into mildly, moderately and markedly elevated ESR.Exclusion CriteriaPatient samples whose HCT > 35% and sample was sent for re-evaluation of ESR within a week during same admission or same visit were excluded from the studyStatistical AnalysisData were described in terms of range, mean ± standard devia-tion (SD), frequencies (number of cases), and relative frequen-cies (percentages) as appropriate. All the entries were entered in a Microsoft Excel sheet and the duplication or triplication was further rechecked by AG. Evaluation of Roller 20 LC method was done as described by Bland and Altman. The 95% limits of agreement were calculated as d ± 1.96 SD, where d = mean difference between the two measurements and SD = standard deviation of differences. Pearson correlation was used to find241Comparative Analysis of Erythrocyte Sedimentation Rate Narang et al.Journal of Laboratory Physicians Vol. 12 No. 4/2020 © 2020. The Indian Association of Laboratory Physicians.the correlation among various parameters. A probability value (p value) less than 0.05 was considered statistically significant. All statistical calculations were done using SPSS (Statistical Package for the Social Science) 21 version statistical program for Microsoft Windows.ResultsA total of 1800 random samples having HCT value < 35% were evaluated during the study period of one and a half years. Of these, duplicates and triplicates ordered during the same visit within a week were omitted, as described in materials and methods. Finally, 1377 samples were evaluated after excluding repeats. ESR was first measured by the Westergren method and corrected using Fabry’s formula (Westergren ESR X 15/55-HCT), followed by the automated method. On dividing the cases on the basis of corrected ESR and auto-mated ESR values into low range, intermediate, high and very high range, the cases falling in each range were 232 (16.8%), 317 (23.1%), 406 (29.5%) and 422 (30.6%), respectively.The mean difference between the ESR values of subjects measured by corrected manual and automated method was calculated. Mean difference between values of corrected and automated ESR for the low, intermediate, high and very high ESR range was 2.33 ± 5.03, 10.95 ± 8.04, 28.22 ± 19.11 and 43.3 ± 19.22 mm/hr, respectively. Ninety five percent limit of agreement and correlation coefficient was calculated between corrected manual ESR and automated ESR values using Bland and Altmann analysis (►Table 1).We inferred that for low range of ESR, the ESR values mea-sured by automated method for 95% of subjects would be 7.53 mm/hour, below the corrected ESR or 12.2 mm/hour above it (►Fig. 1). For intermediate range, the ESR measured by automated method for 95% of subjects would be 4.81 mm/hour, below the corrected ESR or 26.72 mm/hour above it and so on for high and very high ESR ranges. Thus, maximum vari-ation and least correlation was found in very high ESR ranges, signifying that the ESR measured by two methods showed least correlation for very high ranges. The maximum correla-tion was seen in the low and intermediate ranges with coeffi-cient of 0.65 and 0.69, respectively (►Fig. 1)DiscussionESR is commonly used as an indicator of inflammation and infection, although it is not a specific test. The Westergrenmethod is a recommended method for measuring the ESR by the ICSH. However, many confounding factors, includ-ing decreased RBC concentration and fall in HCT in anemic patients, and plasma proteins like globulins and fibrinogen which affect the plasma viscosity and hence sedimentation of RBCs, may affect the results.5,13-15 This method requires longer time and more specimen.14,15Various automated ESR instruments like Roller 20 LC (Alifax S.p.A, Polverara, Italy), Ves-matic 60 (Menarini Diagnostics S.r.l. Milan, Italy), Sediscan (Becton Dickinson, Meylan Cedex, France), Sedimatic (Technicon international Inc, Tokyo, Japan), and others 10 claim to overcome all the confounding factors involved in manual method. Also, these methods use a very small amount of blood samples with higher throughput in less time.But these instruments need to be validated against the standard Westergren method to enable their routine use in laboratories and hospitals.10,11 Roller 20 LC is one such instru-ment which can measure ESR of 18 samples in 10 minutes with minimal blood volume of 800 µL, whereas the Westergren method takes 60 minutes to interpret the result. The Westergren method uses the principle of sedimentation ESR, while Roller 20 LC is based on capillary photometry and measures ESR by converting aggregation of RBCs by optical density, which is then converted into mm/hr.16In this study, we compared the ESR of anemic patients measured by Roller 20 LC with the reference Westergren Method. While reviewing the literature, we found that our study is the biggest study on comparison of automated meth-ods with standard methods of ESR measurement with maxi-mum number of samples (n = 1377), while other authors like Dhruva et al 17 included just 209 cases, Patil et al 18 included just 162 cases, Subramanian et al 19 studied 200 cases, and Alfadhli et al 20 had 150 cases.While evaluating the influence of HCT levels on ESR measurement by Roller 20 LC, we found that the differ-ence between corrected Westergren ESR and automated ESR increased with increase in ESR values. Thus, the more the ESR value, the more the variability between corrected Westergren ESR value and the automated value.Sonmez et al and Romero et al, similar to our study, eval-uated the effect of low HCT (< 35%) on ESR levels. Sonmez et al 9 used samples of 755 patients and divided the corrected ESR values into low (≤20 mm/hour) and high (> 20 mm/hour) range and according to HCT (≥35%, < 35%) levels. Like our study, Sonmez et al 9 also found that in anemic patients,Table 1 Mean difference between corrected manual ESR and automated ESR value, 95% limit of agreement and correlation coefficientESR range (mm/hr)Mean difference between corrected ESR and automated ESR value (mm/hr)95% limit of agreement Correlationcoefficient p value Low (n = 232) 2.33± 5.03− 7.53 to 12.20.65< 0.001Intermediate (n = 317)10.95 ± 8.04− 4.81 to 26.720.69< 0.001High (n = 406)28.22 ± 19.11− 9.24 to 65.70.63< 0.001Very high (n = 422)43.3 ± 19.22− 5.1 to 81.50.18< 0.001Abbreviation: ESR, erythrocyte sedimentation rate.ESR values measured by the direct Westergren method were higher than automated method. Hence, Fabry’s formula was applied to correct the overestimation. After applying Fabry’s formula, the mean of difference between the Westergren ESR and automated ESR values dropped down in low and inter-mediate range; however, still a significant difference wasobserved for high and very high ESR ranges.In our study, at high and very high ESR values, morevariation was found between the two methods. For highESR values, mean difference was 28.22 ± 19.11 (95% limit of agreement − 9.24 to 65.7; correlation coefficient 0.63; p < 0.001), and for very high ESR values, mean difference was 43.3 ± 19.22 (95% limit of agreement − 5.1 to 81.5 withleast correlation coefficient 0.18; p < 0.001). Sonmez et al also found a poor correlation between the two methods at high ESR values (p > 0.10).9 Dhruva et al also had similar observa-tions in patients with HCT between 30 to 35%. They realized Fig. 1 Bland and Altman analysis of the comparison between auto-mated method erythrocyte sedimentation rate (ESR) and corrected ESR with mean difference 2.3; 95% limits of agreement from −7.5 to 12.2.243Comparative Analysis of Erythrocyte Sedimentation Rate Narang et al.Journal of Laboratory Physicians Vol. 12 No. 4/2020 © 2020. The Indian Association of Laboratory Physicians.10 Jou JM, Lewis SM, Briggs C, Lee SH, De La Salle B,McFadden S; International Council for Standardization in Haematology. ICSH review of the measurement of the erytho-cyte sedimentation rate. Int J Lab Hematol 2011;33(2):125–13211 Clinical Laboratory Standards Institute (CLSI), Procedure forthe Erythrocyte Sedimentation Rate (ESR) Test; Approved Standard (5th ed., H2–A5). Villanova, PA: CLSI; 201112 Kratz A, Plebani M, Peng M, Lee YK, McCafferty R,Machin SJ; International Council for Standardization in Haematology (ICSH). ICSH recommendations for modified and alternate methods measuring the erythrocyte sedimentation rate. Int J Lab Hematol 2017;39(5):448–45713 Hardeman MR, Levitus M, Pelliccia A, Bouman AA. Test1 analyser for determination of ESR. 1. Practical evaluation and comparison with the Westergren technique. Scand J Clin Lab Invest 2010;70(1):21–2514 Romero A, Muñoz M, Ramírez G. Length of sedimentationreaction in blood: a comparison of the test 1 ESR system with the ICSH reference method and the sedisystem 15. Clin Chem Lab Med 2003;41(2):232–23715 Arikan S, Akalin N. Comparison of the erythrocyte sedimen-tation rate measured by the Micro Test 1 sedimentationanalyzer and the conventional Westergren method. Ann Saudi Med 2007;27(5):362–36516 Alifax. Available at: https://. AccessedJuly 10, 202017 Dhruva G, Agravat A, Kakadiya M, Pansuriya H. Automatederythrocyte sedimentation rate analyser v/s the Westergren’s manual method in measurement of erythrocyte sedimenta-tion rate: a comparative study. Med Sci 2014;3:376–37818 Patil A, Deepak NM, Belurkar SV, Verma S. Validation of anerythrocyte sedimentation rate analyzer with modified Westergren method. Journal of Evolution of Medical and Dental Sciences. 2013;2:284–29019 Subramanian A, Rangarajan K, Pandey RM, Gandhi JS,Sharma V, Bhoi SK. Evaluation of an automated erythrocyte sedimentation rate analyzer as compared to the Westergren manual method in measurement of erythrocyte sedimenta-tion rate. Indian J Pathol Microbiol 2011;54(1):70–7420 AlFadhli SM, Al-Awadhi AM. Comparison of erythrocyte sedi-mentation rate measurement by the automated SEDIsystem and conventional Westergren method using the Bland and Altman statistical method. Med Princ Pract 2005;14(4):241–244。

地震沉积学概念、方法及其应用研究王正和1　蒋能春2　吕其彪2(1.中国地质大学(北京)能源学院,北京100083;2.中石化西南分公司研究院德阳分院,德阳618000)摘　要:地震沉积学是继地震地层学和层序地层学之后出现的一门现代地震技术与沉积学相结合的新兴交叉学科。

地震沉积学继承了地震地层学和层序地层学的思想,但又有着更为不同的内涵与外延。

将此地震沉积学思想应用于某地区的勘探研究中效果良好。

关键词:地震沉积学;地震地层学;层序地层学中图分类号:P315 文献标识码:A 文章编号:167321980(2008)0320025203收稿日期:2008202218作者简介:王正和(19762),男,四川大竹人,中国地质大学(北京)在读博士研究生,研究方向:沉积与层序地层。

1998年,Zeng Hongliu 首次提出了地震沉积学(Seismic Sedimento log y )这个名词,并指出随着3-D 地震勘探以及解释技术的不断发展,地震沉积学可以作为新的常规方法用于盆地分析[1]。

2000年Wolf gang Schlager 又指出,为了满足沉积学应用、地质情况预测及地震解释的需要,地震沉积学将作为沉积学的一个新兴的分支学科而发展[2]。

2005年2月,地震沉积学国际会议在美国休斯顿召开,这标志着地震沉积学作为一门新的学科开始受到人们的关注。

地震勘探作为一种油气勘探的技术手段一直与地质应用紧密结合。

地质记录是沉积环境的响应,而从地震资料所获得的信息又是地质记录的响应。

所以,从地震记录可以间接地反映和反演出地质记录的原始沉积环境。

随着现代地震技术的发展,还可以进一步从地震记录中获得沉积单元的岩性、岩相、几何形态以及内部结构等沉积学及沉积岩石学方面的信息。

这是地震勘探技术用于沉积学研究并能与沉积学联姻而形成地震沉积学的理论基础与前提。

1　地震沉积学的概念及内涵地震沉积学是依据现代沉积、地震数据和古老露头等资料综合研究,对沉积过程进行解释的方法[2]。