Handbook of Residue Analytical Methods for Agrochemicals

Handbook_of_Residue_Analytical_Methods_for_Agrochemicals/Handbook_of_Residue_Analytical_Methods_for_Agrochemicals/91942_01a.pdf

Regulatory guidance and scientificconsideration for residue analyticalmethod development andvalidation

Assessment of residue analyticalmethods for crops, food, feed, andenvironmental samples: the approachof the European Union

Johannes Siebers and Ralf HanelFederal Biological Research Centre for Agriculture and Forestry (BBA),Braunschweig, Germany

1 Introduction

Plant protection products are widely used throughout the world to reduce the lossin crop production caused by harmful organisms and weeds. However, their usageposes potential risks to humans, animals and the environment, especially if usedwithout having been evaluated for safety and without having been authorized. Inorder to minimize the risks and to facilitate the trade of plant protection products andagricultural produces within the common market, the European Community (EC)has adopted Council Directive 91/414/EEC of 15 July 1991 concerning the placingof plant protection products on the market.1 As a result, the evaluation of the safety ofactive ingredients (a.i.) contained in plant protection products is now carried out onthe basis of data requirements which are harmonized throughout the EC. For rea-sons of preventive health protection and protection of the environment, the use ofplant protection products has to be limited to the minimum level compatible witheffective crop protection. Maximum residue limits (MRLs) are established for cropsand food. Member States are responsible for monitoring the compliance of food-stuffs with these MRL levels on a regular basis to ensure that no misuse of productshas taken place. In view of the importance of the quality of water intended for humanconsumption, a general limit for crop protection products and toxicologicallyrelevant metabolites/degradation products is also established for drinking water.For surface water, soil, and air, there are no harmonized limits; however, pesticideresidue levels in these environmental compartments are regulated at the nationallevel.

Residue analytical methods are needed to enforce these legally based limits or guid-ance values and to perform monitoring projects. For existing a.i., validated analyticalprocedures for only a few selected compounds have been published in journals or

Handbook of Residue Analytical Methods for Agrochemicals.C 2003 John Wiley & Sons Ltd.

14 Regulatory and scientific consideration for residue analytical methods

handbooks. But for many compounds in use and especially for new a.i., there are nosufficiently validated residue analytical methods available in open literature. There-fore, legal provisions are created to supply laboratories involved in post-registrationcontrol and monitoring with residue analytical methods for plant protection prod-ucts. Analytical methods are required, as part of the registration data package, to beevaluated at national and/or at Community level.

The purpose of this article is to clarify the assessment of residue analytical methodsin the context of Directive 91/414/EEC. After discussing the legal and historical back-ground, requirements for enforcement methods as well as data generation methodsare reviewed. Finally, an outlook over further developments in the assessment andvalidation of analytical methods is provided.

2 Legal background

2.1 General

Since the foundation of the European Communities was laid in 1952 with the Euro-pean Coal and Steel Community (ECSC), the importance of the European Commu-nities within their own borders and for the global economic system has increased.Starting with six European countries in 1952, the EC now comprises of 15 MemberStates, and enlargement negotiations are in progress. The European Communitieshave continued to develop, becoming the European Union (EU), an umbrella for thethree extant European Communities, ECSC, European Atomic Energy Community(EURATOM), and European Community [EC, formerly European Economic Com-munity (EEC)]. Institutions involved in the legislative process are the Council of theEuropean Union, usually known as the Council of Ministers (of the Member States),the European Commission (the administration of the EC) and, with limited powers,the European Parliament. The Court of Justice ensures that the law is observed inall Community and Member State activities. Community law may take the followingforms: regulations are applied directly in all Member States without the need fornational measures to implement them.2 Directives bind Member States to achieve theobjectives while leaving the national authorities the power to choose the form andthe means for implementing the Directives. Decisions are binding in all their aspectsfor those to whom they are addressed.2 A decision may be addressed to any or allMember States, to undertakings or to individuals. Recommendations are not legallybinding. Community legislation is published in the Official Journal of the EuropeanCommunities in all official languages of the EC. Guidance documents do not intendto produce legally binding effects and by their nature do not prejudice any measuretaken by a Member State within its implementation of Directives. Details of the legalbackground are described, for example, by Wirsing et al.2

2.2 Council Directive 91/414/EEC

Until 1991, all Member States of the EC applied their own registration regime forplant protection products and operated independently with very little collaboration

Assessment of residue analytical methods for crops, food, feed, and environmental samples 15

between the countries in most cases. These individual regimes were considered toconstitute a barrier to trade in plant protection products and agricultural producewithin the internal market of the EC.

In order to set up a harmonized framework for the regulation of plant protectionproducts in the EC, Council Directive 91/414/EEC of 15 July 1991 concerning theplacing of plant protection products on the market was adopted and implementedin all Member States. Six annexes were established within this Directive, providingthe basis for the harmonization of registration procedures and regulatory decisions(Table 1).

Through the adoption of Directive 91/414/EEC, a decision-making regime fordetermining the acceptability of a.i., which are denoted as active substances (a.s.)in the EUs legislation, was established. Authorization of plant protection prod-ucts was still to be undertaken at national level by the individual Member States.A national authorization may be granted providing that the a.i. has been included inthe positive Community list of a.i. (Annex I to the Directive), and the uniformprinciples for evaluation are applied, as defined in Annex VI to the Directive.Annex I inclusion of an a.i. is the result of a harmonized evaluation and decision-making procedure, performed on the basis of harmonized data requirements, as de-tailed in Annexes II and III to the Directive.

These annexes set out the requirements for the dossier to be submitted by applicantseither for inclusion of an a.i. in Annex I or for authorization of a plant protectionproduct. Active ingredients are listed in Annex I if their use and their residues, resultingfrom applications consistent with good plant protection practice [or Good AgriculturalPractice (GAP)] do not have harmful effects on human and animal health, or on groundwater or any unacceptable influence on the environment (Article 5 of the Directive).In order to take account of developments in science and technology, the inclusionof an a.i. in Annex I is limited to a period not exceeding 10 years to ensure thatthe inclusion is regularly reviewed to meet modern safety standards. Furthermore,Annex I listing is the prerequisite for the mutual recognition of authorizations betweenMember States, whereby one Member State is obliged to accept the evaluation andauthorization prepared by another Member State in situations where the agricultural,plant health, and environmental (including climatic) conditions relevant to the use ofthe plant protection product are comparable in the regions concerned (Article 10 ofthe Directive).2

2.3 Legislation related to MRLs

Pesticide residue levels in foodstuffs are generally regulated in order to:

minimize the exposure of consumers to the harmful or unnecessary intake of pes-ticides

allow control over the use of plant protection products permit the free circulation of products treated with pesticides as long as they comply

with the established MRL.

The MRL for pesticide residues is the maximum concentration of a pesticide residue(expressed milligrams per kilogram) legally permitted in or on food commodities and


Table 1 Annexes of Council Directive 91/414/EEC of 15 July 1991 concerning the placing ofplant protection products on the market and its implementation (status: published up to February2002)

Annex Content Implementation

Annex I Active substances(a.s.)a autho-rized for incorporationin plant protection products

New asb Existing asc

Acibenzolar-S-methyl AmitrolAzimsulfuron BentazonAzoxystrobin -CyhalothrinCyclanilide 2,4-DFenhexamid DiquatFlupyrsulfuron-methyl FluroxypyrIron(III) phosphate EsfenvaleratKresoxim-methyl GlyphosatePaecilomyces ImazailProhexadion-calcium IsoproturonPymetrozine Metsulfuron-methylPyraflufen-ethyl PyridatSpiroxamine Thiabendazole

TriasulfuronThifensulfuron-methyl

Annex II Requirements for the dossierto be submitted for the inclu-sion of an active substance inAnnex I

Part A: Chemicals as Directive

Efficacy 93/71/EECPhysical-chemical prop-erties

94/37/EC

Part A: Chemical substances Analytical methods 96/46/ECPart B: Microorganisms andviruses

Toxicology and metabo-lism

94/79/EC

Residues 96/86/ECAnnex III Requirements of the dossier to Fate and behavior in the 95/36/EC

be submitted for the authoriza- environmenttion of a plant protection prod- Ecotoxicology 96/12/ECuct Part B: MicroorganismsPart A: Chemical preparations and viruses DirectivePart B: Preparations of micro-organisms or viruses

93/71/EEC2001/36/EC

Annex IV Risk phrases In preparationAnnex V Safety phrases In preparationAnnex VI Uniform principles for the

evaluation of plant protectionproducts

Directive 97/57/EC

a Term for a.i. used in EU legislation.b New a.s. are active substances not on the market of EC in protection products before 25 July 1993.c Noninclusion has been decided for the following as after evaluation: azinphos-ethyl, chlozolinate,chlorfenapyr, cyhalothrin, dinoterb, DNOC, fentin-acetate, fentin-hydroxide, fenvalerate, ferbam,lindane, monolinuron, parathion, permethrin, propham, pyrazophos, quintozen, tecnazen, zineb.


animal feed. MRLs are based on GAP. These should reflect minimum quantities ofpesticide necessary to achieve adequate pest control, applied in such a manner thatthe residues are as low as practicable. MRLs are also established at or about the limitof determination where there are no approved uses or where no residues occur whenthe pesticide is used according to GAP. MRLs are not toxicological limits but mustbe toxicologically acceptable. Exceeding the MRL is a violation of GAP.

Legislation at Community level dates back to November 1976 when Council Direc-tive 76/895/EEC3 established MRLs for 43 active substances in fruits and vegetables.These MRLs were based on the best data available at that time. These MRLs aregradually being reviewed and, where appropriate, replaced with MRLs based onmore current information and higher standards.

Current pesticide MRL legislation is derived from/based on four Council Direc-tives:

Council Directive 76/895/EEC3 establishing MRLs for fruits and vegetables Council Directive 86/362/EEC 4establishing MRLs for cereals and cereal products Council Directive 86/363/EEC5 establishing MRLs for products of animal origin Council Directive 90/642/EEC6 establishing MRLs for products of plant origin,

including fruits and vegetables.

Legislation for pesticide residues, including the setting of MRLs in food commodities,is a shared responsibility between the Commission and the Member States. To date,Community MRLs have been established for about 130 pesticide a.i. For pesticidesand commodities where no Community MRL exists, the situation is not harmonizedand the Member States may set MRLs at national levels to protect the health of itscitizens.

Where nonharmonized national MRLs exist, there is always a possibility of tradedisputes. Until 1997, MRLs were established on raw commodities only. Directive97/41/EC changed three important aspects of the work:

it provided a mechanism to set MRLs in processed products and composite food-stuffs, based on the MRLs fixed for raw agricultural products

it established a conciliation procedure through which cases where national MRLsled to barriers of trade within the Community could be resolved

it transferred the competence for setting MRLs from the Council of the MemberStates to the Commission in Brussels.

Member States monitor the compliance of foodstuffs with these MRLs regularly.Inspections and monitoring should be carried out in accordance with the provisionsof Council Directive 89/397/EEC7 on the official control of foodstuffs, and Coun-cil Directive 93/99/EC8 on additional measures concerning the official control offoodstuffs.

The MRLs are derived from data from supervised residue trials that are generallycarried out in the context of food production. Specific conditions of feed productionare not considered. Therefore, many practical problems for the official control of feedmust be solved in future, e.g., application of transfer factors and the calculation ofMRLs for mixed feed.

Besides national monitoring programs, the participation of each Member State in anEU-coordinated monitoring program is recommended. These monitoring programs


have existed since 1996, and are intended to provide an accurate dietary pesticideexposure throughout the EU and Norway. They will have covered all major pesticidecommodity combinations by the end of 2003. The choice of commodities includes themajor components of the Standard European Diet of the World Health Organization.

In recent years, new legislation (Council Directive 99/39/EC) has placed severerestrictions on the use of pesticides in the production of food for infants and youngchildren.

2.4 Legislation related to residues limits for soil, water, and air

The natural and socio-economic differences within the EU require the most decisionson the monitoring and enforcement of residues in the environment as well as measuresto redress failures at local, regional, and national levels. Therefore, no harmonizedlimits for pesticides in soil and in air exist.

Because of the great importance of drinking water for human health, quality stan-dards for pesticides in water were developed at Community level based on the pre-cautionary principle.9 Toxicological considerations were not taken into account toderive the general limit for pesticides.

Within the EU, many water-related Directives have been established over the pastyears. The most important one for the assessment of analytical methods for plantprotection products is Directive 98/83/EC on the quality of water intended for humanconsumption.10 According to Annex I Part B to the Directive, a general limit of0.1 g L1 applies uniformly to each individual pesticide. The sum of all individualpesticides detected may not exceed 0.5 g L1. Only those pesticides which are likelyto be present in a water supply need to be monitored. As a result, analytical methodsused for water monitoring purposes must be able to determine pesticide residues atthe 0.1 g L1 level. As a contrast to the concept of setting MRLs, the concept ofa general limit excludes specific considerations on the properties of individual a.i.,e.g., toxicity. From an analytical point of view, this concept leads in some cases toinconsistencies regarding naturally occurring insecticides listed by the Commissionsuch as carbon dioxide, rape seed oil, nitrogen, or naturally occurring herbicides likesuch as iron (II) sulfate and iron (III) sulfate. Moreover, additional specific limitsapply to copper compounds (copper: 3 mg L1) and cyanide (50 g L1).

For surface water, there are no legally binding limits except for parathion,HCH, and dieldrin in surface water intended for drinking water preparation(Directive 75/440/EEC). Possibly the establishment of the Water Frame Directiveof 22 December 2000 will lead to harmonized quality standards for selected pesti-cides in surface water. Currently, provisions of Annex VI to Directive 91/414/EECconcerning the acceptable exposure of aquatic nontarget organisms are the basis forcalculating guidance limits for assessing analytical methods for surface water.

2.5 Provisions for residue analytical methods

The first step to define data requirements and criteria for decision making forresidue analytical methods was attempted in Council Directive 94/43/EC, establishing


Annex VI to Directive 91/414/EEC concerning the placing of plant protection prod-ucts on the market. The section concerning residue analytical methods was not fullyfinalized when the Directive was first adopted. There were no provisions for methodsto determine residues from a.i. and relevant metabolites in soil, water, and air. Thecriteria for foodstuffs partly proved to be not helpful for the practice of assessment(e.g., with regard to reproducibility, ISO 5725 requires validation in at least eightindependent laboratories).

Although Directive 94/43/EC was later substituted by Council Directive 97/57/ECof 22 September 1997,11 the provisions for analytical methods remained unchanged.

Commission Directive 96/46/EC of 16 July 1996, amending Annex II to the Di-rective 91/414/EEC, is the basis for the assessment of residue analytical methods forcrops, food, feed, and environmental samples.12 Provisions of this Directive covermethods required for post-registration control and monitoring purposes but not datageneration methods. Because it is necessary to provide applicants as precisely as pos-sible with details on the required information, the guidance document SANCO/825/00rev. 6 dated 20 June 2000 (formerly 8064/VI/97 rev. 4, dated 5 December 1998)13

was elaborated by the Commission Services in cooperation with the Member States.Moreover, this document provides guidance to Member States on the interpretation ofthe provisions of Directive 96/46/EC concerning minimum validation requirementsfor residue analytical methods.

For analytical methods used for generating data required in the field of residue be-havior, environmental fate, and other fields, the guidance document SANCO/3029/99rev. 4 was developed.14

According to guidance document 7109/VI/94 rev. 6, the development and validationof an analytical method for monitoring purposes and post-registration control are notsubject to Good Laboratory Practice (GLP) regulation. However, where the method isused to generate data for registration purposes, for example residue data, these studiesmust be conducted according to GLP.15

Table 2 Relevant legal provisions for residue analysis

Document Year of publication Scope

Directive 85/591/EEC 1985 Analytical methods for food con-trol

Directive 89/397/EEC 1989 General principles of food controlDirective 94/43/EC (Annex VI of91/414/EEC)

1994 Uniform principles for nationalauthorizations

Directive 96/46/EC 1996 Data requirements and principlesfor evaluation

Guidance document 8064/VI/97 1997 Details concerning Directive96/46/EC

Directive 97/57/EC 1997 Substitutes Directive 94/43/ECRecommendation 1999/333/EC(Annex II)

1999 Quality control measures for mon-itoring laboratories

Guidance document SANCO/825/00 2000 Substitutes 8064/VI/97 (LC/MS,LC/MS/MS possible)

Guidance document SANCO/3029/99 2000 Details concerning data genera-tion methods


In addition to data requirements and assessment criteria in the context of Annex Ilisting and the authorization of plant protection products, there are legislative demandsfor analytical methods addressed to food control and monitoring laboratories. CouncilDirective 89/397/EEC lays down general principles to be followed by the official foodcontrol. Additional measures are stipulated by Council Directive 93/99/EEC. Crite-ria which should be tested, as far as possible, are described in Annex I to CouncilDirective 85/591/EEC of 20 December 1985 concerning the introduction of Com-munity methods and analysis for the monitoring of foodstuffs intended for humanconsumption.16 Quality control measures are highlighted in guideline 7826/VI/97,which is published as Annex II to the Commission Recommendation 1999/333/EC.17

Relevant legal provisions for residue analysis are summarized in Table 2.

3 Evaluation of the submitted methods

3.1 Institutional background

The evaluation of a.i. including the evaluation of the analytical methods is jointly car-ried out by competent authorities of the Member States and the European Commission.For each a.i., a designated Rapporteur Member State performs the evaluation of thedossier, which is submitted by the applicant and in which all requirements of AnnexesII and III to Directive 91/414/EEC must be addressed. The Rapporteur evaluates thedata and prepares a draft assessment report (monograph) including a proposal forinclusion or noninclusion in Annex I. The monograph is distributed by the EuropeanCommission. Any comments from the Member States and the applicant as well asdetails of the monograph are discussed in peer review meetings. Issues relating toanalytical methods are discussed together with physico-chemical properties inan expert group consisting of about 57 alternating scientists named by theCommission as private experts. Their task is to identify problems and to confirmopen data requirements. Specific scientific issues may be transferred to the ScientificCommittee on Plants. The conclusions of the evaluation of an a.i. are laid down ina Review Report, prepared by the Commission. After consideration by the StandingCommittee on Plant Health (since January 2002, the Standing Committee on theFood Chain and Animal Health), a final decision on Annex I inclusion is taken bythe European Commission and a Directive is adopted. A detailed description of thewhole procedure is given by Wirsing et al.2

Inclusion in Annex I is the prerequisite for the mutual recognition of authoriza-tions between Member States. At the time Directive 91/414/EEC was adopted in1991, there were over 800 a.i. authorized for use in the Member States. The goalwas to evaluate these at Community level within 12 years. However, the resourcesnecessary to carry out this exercise were not fully recognized when the legislation wasadopted. Moreover, time-consuming decision procedures delay the review process.Up to February 2002, 15 existing a.i. and 13 new a.i. were listed in Annex I, whereas19 a.i. were rejected (see also Table 1). There is clearly a lack of mutual recognitionbetween Member States.

In addition to the evaluation at Community level, Member States have to evaluate thedata submitted by applicants, because the authorization of plant protection products


is the responsibility of the individual Member State. It is not possible to apply forauthorization at Community level. Therefore, every Member State has establisheda Competent Authority which may grant authorization (Table 3). For this reason,there are various procedures of data evaluation at Member State level under nationallegislation and with different institutional backgrounds. Details of the 15 differentprocedures applied in the Member States cannot be discussed in this article.

3.2 Validation parameters

Validation may mean different things to different people, depending on the contextand the application of analytical science. For food control and monitoring purposes,it is generally expected that validation includes the establishment of performancecharacteristics and evidence that the method fits the respective purpose.18

Analytical methods submitted by applicants are evaluated using harmonizedcriteria (see Section 2.5). The following presentation provides a brief overview ofthe validation parameters used in the registration of plant protection products andtheir a.i. These parameters are as follows:

TruenessThere are various approaches to determine the trueness of methods.19 The mostcommon is the performance of recovery experiments. According to the guidancedocument SANCO/825/00,13 the mean recovery should be in the range of 70110%.In justified cases, recoveries outside this range can be acceptable.

RepeatabilityRepeatability is defined as precision under conditions where independent testresults are obtained with the same method on identical test material in the samelaboratory by the same operator using the same equipment within short intervals oftime. The replicate analytical portion for testing can be prepared from a commonfield sample containing incurred residues. This approach is used extremely rarely.Normally, repeatability is estimated by the relative standard deviation of recoveries,which should be lower than 20% per commodity and fortification levels accordingto SANCO/825/00. In justified cases, higher variability can be accepted.

ReproducibilityReproducibility in the context of Directive 96/46/EC is defined as a validation ofthe repeatability of recovery, from representative matrices at representative levels,by at least one laboratory, which is independent of the laboratory which initiallyvalidated the study. This independent laboratory may be within the same company,but may not be involved in the development of the method. This concept of inde-pendent laboratory validation (ILV) substitutes the conduct of interlaboratory trials(e.g., according to ISO 5725) because the resources are not available taking intoconsideration the high number of a.i., matrix types and concentration levels whichmust be validated in the registration procedure.

SpecificitySpecificity is defined in Directive 96/46/EC as the ability of a method to dis-tinguish between the analyte being measured and other substances. According toSANCO/825/00, blank values must be reported using representative matrices. They


Table 3 Competent authorities for the authorization of plant protection products (status: August2001)

Authority Address

Bundesamt und Forschungszentrum fur Spargelfeldstrae 191,Landwirtschaft 1226 Vienna,Institut fur Pflanzenschutzmittelprufung Austria

Ministere des Classes Moyennes et de lAgriculture WTC 3, 8e etage,Inspection Generale des Matieres Premieres et Boulevard Simon Bolivar 30,Produits Transformes 1000 Brussels,

Belgium

Biologische Bundesanstalt fur Land- und Messeweg 11/12,Forstwirtschaft 38104 Braunschweig,Abteilung fur Pflanzenschutzmittel und GermanyAnwendungstechnik (BBA)

Miljoestyrelsen Strandgade 29,1401 Copenhagen,Denmark

Ministerio de Agricultura Pesca y Alimentacion Velazuez 147,Subdireccion General de Medios de Produccion 28002 Madrid,Agricola Spain

Ministere de lAgriculture 251 rue de Vaugirard,Protection des Vegetaux 75732 Paris Cedex 15,

France

Plant Production Inspection Centre Vilhonvuorenkatu 11 C, V Floor,Pesticide Division 00500 Helsinki,

Finland

Ministry of Agriculture Hippokratus Str. 35,Directorate of Plant Produce Protection 10164 Athens,Department of Pesticides Greece

Ministero della Sanita Piazza Marconi 25,Dipartimento per lIgiene degli Alimenti 00144 Rome,e della Sanita Pubblica Veterinaria Italy

Pesticide Control Service Abbotstown, Castleknock,Abbotstown Laboratory Complex Dublin 15,

Ireland

Administration des Services Techniques de 16 route dEsch,lAgriculture BP 1904,

1019 Luxembourg,Luxembourg

College voor de Toelating van de Bestrijdingsmiddelen Stadsbrink 5,6700 AA Wageningen,The Netherlands

Centro Nacional de Proteccao Quinta do Marques,da Producao Agricola 2780 Oeiras,

Portugal

Kemikalie Inspektionen PO Box 13 84,17127 Solna,Sweden

Pesticides Safety Directorate 3 Peasholme Green,Mallard House, Kings Pool York Y01 7PX,

UK


should not be higher than 30% of the limit of determination. Moreover, confirma-tion techniques must be presented in order to avoid false positive results.

Limits of determinationThe limit of determination [or limit of quantitation (LOQ)] is defined in Directive96/46/EC as the lowest concentration tested at which an acceptable mean recovery(normally 70110%) and acceptable relative standard deviation (normally 100 areused for identification/quantitation. The rationale for the selection of the ions mon-itored should also be provided. When a confirmatory method/technique is requiredto demonstrate specificity, the properties of the analyte should be considered whendeciding on an appropriate method/technique. In SANCO/825/00 acceptable confir-matory techniques are specified as follows:

HPLC/DAD, if the UV spectrum is characteristic; in this case a UV spectrumobtained under the conditions of determination must be submitted

alternative chromatographic principle (e.g., substitution of HPLC by GC) from theoriginal method

alternative detection method derivatization, if it was not the first-choice method different stationary and/or mobile phase of different selectivities.

In addition, variation of partitioning and/or cleanup steps can be useful for confirma-tion in special cases.

The extent of validation of confirmatory techniques is currently under consider-ation. One approach is that the extent of validation may be smaller than for theenforcement method. In principle, validation in triplicate at the relevant concentra-tion level (LOQ or MRL) is sufficient. In the case where an MRL is set for multiplecrops, a single validation in all representative crop groups is sufficient. A confirmatorymethod for residues in air is not required if a corresponding method was submittedfor the other sample matrices. This approach is realized in Germany.30

4.2 Specific requirements

4.2.1 Food of plant and animal origin

The enforcement method must be suitable for the determination of all compoundsincluded in the residue definition in order to enable Member States to determinecompliance with MRLs. It is not feasible to validate a method for all commodities ifa wide range of MRLs are set. Therefore, a concept of crop groups was developed inSANCO/825/00. The following crop groups with representative crops are presented:

cereals and other dry crops (e.g., barley, wheat, rye) commodities with high water content (e.g., lettuce, cucumber)


commodities with high fat content (e.g., rape seed, linseed, olives) fruits with high acid content (e.g., lemons, grapefruits).

For each group, one representative sample matrix has to be used for method validation.If the intended use is restricted to one of the crop groups, the method must be validatedonly for this group. On the other hand, the method has to be validated for all groups ifthe use is intended for a variety of crops that belong to two or more different groups.In addition, specific crops which are difficult to analyze due to matrix interferencerequire individual method validation (e.g., hops, brassica varieties, bulb vegetables,herbs, tea).

There is some discussion within the Member States aimed at method validation forall crop groups in every case in order to support the enforcement of MRLs establishedfor other crops. Additionally, detailed lists of the crop groups are under development.For example, it seems to be that almost all fruits can be classified as fruits with highacid content (exception: e.g., bananas and certain varieties of apples). Depending onthe variation of the analytical method necessary to obtain acceptable results, it maybe possible to cover more than one group by validation using one crop. For example,if the validation is performed with lemons and the pH value has no influence on therecovery of the a.i., it may be acceptable to waive the validation using a representativecommodity with a higher water content.

Validation of the analytical methods for food of animal origin has to be performedwith milk, egg, meat, and fat. The latter is required only if log PO/W is >3 andmetabolism studies indicate significant residues in fat, because in this case it is likelythat an MRL will be set. Other tissues such as kidney or liver must be validated onlyif an MRL is set or proposed for these tissues. The issue of the general necessity ofanalytical methods for food of animal origin is not addressed in Directive 96/46/EC orSANCO/825/00. At this moment, the Working Group Pesticide Residues proposesan MRL on a case-by-case basis. However, a pragmatic approach is presented inSANCO/825/00.

According to Directive 96/68/EC,31 an analytical method for the determination ofresidues in food of animal origin is not required when metabolism study in animalsis not required. On the other hand, according to Point 6.4 of the Directive, where afeeding study is required, an analytical method for the determination of residues inproducts of animal origin must be submitted. In other cases, the requirement for ananalytical method depends on the establishment of an MRL for food commodities ofanimal origin.

Two additional requirements are specific to the analysis of residues in food. Thefirst requirement depends on the LOQ to be achieved (see Table 5).

Table 5 Relation between the maximum residue limit (MRL) and the limit of quantitation (LOQ)

MRL (mg kg1) LOQ (mg kg1)

>0.1 0.10.1 0.050.05 0.02

3.5 m

40.5

m>

20

m10

.5 m

Tre

ated

plo

tsB

uff

er z

one

Un

trea

ted

plo

ts

> 71

m

3 m

2 - 2.5 m

1.5

m1.

5 m

0.5m buffer

Sampling area

Sprayed area

Figure 4 Randomized block design using four replications having 20 sub-plots each

856 Best practices in the generation and analyses of residues in environmental samples

The width of an individual treated replicate should not be wider than 3 m to enabletest substance application using a single pass of a conventional plot sprayer. Theapplication is made in the same direction as the layout of the plot. If multiple boomwidths are used in treating a single plot, it is critical that areas of potential over-sprayand under-spray are avoided during soil sampling. Study designs requiring multipleapplication passes within a single treated area are not recommended owing to potentialissues arising from areas of over- or under-spray.

Five soil cores are typically collected from a predetermined subplot within eachreplication at each sampling period. As mentioned previously, the number of soil corescollected increases with increasing residue variability. The order of subplot samplingis determined using a randomization procedure38 or by random-number subroutinescommon to many computer spreadsheet programs. The areas between the treatedreplicates serve as buffer zones and provide access lanes for study personnel andvehicles. Within each row, the subplots are separated by a buffer zone of 0.5 m.An important advantage of the completely randomized block design is that samplecollection is distributed across the entire test plot, helping to capture effects of soilspatial variability on agrochemical dissipation. The design presented in Figure 4 isreadily adapted to bare-soil and cropped studies.

Additional planning and sample numbers are often required when agrochemicalsare applied as banded rather than broadcast applications. The soil sampling techniquesdevised for banded fertilizer applications provide a good basis for the sampling ofagrochemical residues.39,40 For example, the recommended approach for samplingfields receiving banded nitrogen fertilizer applications involves the collection of 1530 composite cores taken between the banded rows and inter-rows of the field.39

Sampling at multiple positions perpendicular to the application band provides a mea-sure of agrochemical distribution throughout the surface soil. Similarly, determiningrepresentative soil sampling locations for agrochemicals applied by chemigation is nota trivial undertaking and requires increased sample numbers to account for increasedresidue variability.18

2.4.5 Plot markers

A field soil dissipation study usually lasts between 1 and 2 years; long-term soilaccumulation studies may last for up to 6 years. Hence, it is essential that test plotsare clearly marked to ensure accurate sampling for the duration of the study. Durable,highly visible markers (stakes) made of plastic, metal, or wood should be locatedat the main corners of the treated and control plots. Additional markers indicatingreplication and subplot number or line number, as appropriate, must also be installed.Weather-proof signs must be installed that clearly indicate the Study Director andcontact information, study number, test substance and application rate, and studyinitiation and termination dates. This information helps to prevent application andsampling errors. Plot markers and signs should be checked regularly to ensure thatthey are legible and in good physical condition.

Permanent markers outside the study area should also be located and used in theevent that one or more plot markers are inadvertently moved or lost. One option isto locate a minimum of two permanent reference points outside of the study area

Sampling and analysis of soil 857

50to80cm

Stake markingindividualsub-plot

Post markingindividualreplicate

Permanentmarker

50 to80cm

Sub-soil marker

Figure 5 Techniques used to mark test plots in field soil dissipation trials

that can be used to re-survey the test area by triangulation (Figure 5). The distancesto prominent points such as the ends of sampling plots should be recorded in thestudy records and indicated on corresponding plot maps. Another option is the useof sub-soil markers that are detected by induction (Figure 5). Because these markersare placed 6080 cm directly below prominent points in the study area, it is un-likely that they will be moved during the study. The sub-soil markers are especiallyuseful in long-term accumulation studies that involve seasonal plowing or cultiva-tion and when permanent landmarks are not conveniently located near the studyarea.


2.5 Additional considerations

2.5.1 Study documentation

Overall study success depends upon the careful documentation of key aspects ofstudy conduct. As mentioned previously, a formal, written study plan (protocol) isrequired for GLP studies and is highly recommended for non-GLP studies. Other keyinformation to document in the study records includes example calculations involvingthe application rate, anticipated zero-time concentration, and those associated with theanalysis of soil. Additional documentation should include the source, purity, test sitelocation(s), soil textural class, diagrams of test site layout, type and inner diameters ofsoil corers, sampling depths, pertinent weather parameters, amount and timing of allsupplemental irrigation, and the names of all personnel involved with study conduct.The date and time of each application, sample collection, freezer storage, sampleextraction, and analysis should all be carefully recorded. Any events that result indeviations from the written protocol must be carefully recorded in the study recordsand, in the case of GLP studies, the Study Director notified of these events within 24 hof their occurrence. Photographs taken during test substance application and samplingand of the equipment related to these activities are useful in reconstructing key aspectsof study conduct. Thorough documentation is as vital for non-GLP research as it isfor studies conducted for regulatory purposes.

2.5.2 Safety

Equipment used to apply agrochemicals and to collect and process soil is inherentlydangerous. The appropriate personal protective equipment must be worn and mini-mally includes protective eyewear and gloves. Additional protective equipment mayinclude spray suits, respirators, steel-toed boots, and hearing protection, dependingon the particular materials being investigated and equipment being used. Large phys-ical force is required to insert a soil probe into the ground; this same force can crushor amputate human limbs. Hence, workers must be well trained in the operation ofsampling equipment. Fieldwork also requires physical exertion so caution should beobserved when working in high temperature and humidity conditions. Studies involv-ing the application of radiolabeled materials require prior written permission from theappropriate regulatory authorities as well as special provisions for the proper removaland disposal of treated soils and sub-soils.

3 Phase II: field study conduct

Each of the five main steps in field conduct (site selection, test plot layout, testsubstance application, sample collection, and sample storage/handling) is addressedbelow.

3.1 Test site selection

Once the targeted study regions, soil textures, space requirements, and other keyaspects of study design have been determined, the search for suitable test sites


begins. Test site selection is critical to the success of a field soil dissipation studyas field-related factors have a major influence on the overall outcome of the study.Even for bare-soil studies, an agriculturally viable soil that would be capableof growing a healthy crop is usually desired. Hence it is important to ascertainthe soils recent cropping and management history before choosing a particularsite.

Table 2 lists basic criteria that can be used during field site selection for bare-soil and cropped studies. Priority among the selection criteria depends upon theparticular goals of the study but certain factors (e.g., slope >1%, excessive rocks,flood prone, potential plot disturbance by wildlife) usually serve to exclude cer-tain sites automatically. If the region of interest is far away, it is best to seek theassistance of university investigators, extension agents, and consultants who arefamiliar with the regional agricultural practices and local soil and climatic con-ditions.

Table 2 Site-selection criteria for field soil dissipation studies

Selectioncriterion Prioritya Basis for selection Comments

Region A or B Site must match the climatic, soil, andagricultural conditions typical of thetarget crop

Some crops are grown only in certain regions(e.g., rice) while others are common tomany regions (e.g., maize). Thus, selectionof a test region may be restrictive orrelatively flexible

Soil properties A Soil texture (sand, silt, clay), organicmatter/carbon content, and pH

Stones, roots, and hardpans must belargely absent to allow representativesampling of soil profile

Soil properties should appear uniformover test site

Soil texture data should be available at timeof site selection. Soil properties mustmatch study purpose. This can be realisticuse conditions, realistic worst-case orworst-case in terms of agrochemicalmobility and persistence

Must ensure that the majority of samples canbe taken from the deepest samplinghorizon. Information about sub-soils can beobtained from soil maps, test coring andon-site interviews

Site topography Exclusion Must have slope 1%Site must not be susceptible to floodingShallow water table or tile drains must not

interfere with sampling

These are exclusion criteria that have to becarefully determined during on-siteinspection

Site must be level to prevent losses ofagrochemical due to surface run-off andsoil erosion

Site must not be susceptible to runoff fromother areas higher than test site

Size of test site B Depends on study design. The minimumarea required for a typical large-plotdesign is about 0.25 ha

Test site must allow for test design plussufficient buffer zone around perimeter offield to protect against external disturbance

For bare-soil studies, shady sites should beavoided

(Continued overleaf )


Table 2 Continued

Selectioncriterion Prioritya Basis for selection Comments

Cropping historyand previouspesticide use

Exclusion The cropping and pesticide history for theprevious 3 years must be welldocumented

The test substance must not have beenapplied to site within the past 3 years

This information is crucial and evidence ofcareful record keeping reflects favorablyupon the future reliability of a fieldcooperator

Prior application of agrochemical formingidentical/similar degradation products astest substance should be considered aspotential analytical interferences

Previous management practices (e.g., soilamendments, tillage, crop type) shouldhave been uniformly applied across test site

Irrigation Exclusion Site must be equipped with sprinklerirrigation

Irrigation is necessary to ensure 110% ofhistorical rainfall for dryland settings or tofollow regional irrigation practices inirrigated cropping settings

Test site security A Access of unauthorized persons, livestock,etc., must be restricted

Potential impact of any nearby construction,utility lines, rights-of-way, etc., must alsobe assessed

Plot maintenance B Expertise must be available to maintainthe test site and, if cropped, to take careof the crop

For bare-soil studies, the soil surface must becarefully prepared prior to test substanceapplication and kept weed-free withoutdisturbing the test areas. If the test iscropped, the crop should be treatedaccording to Good Agricultural Practice. Incase of a soil accumulation study, the fieldmay be cultivated and cropped each seasonfor up to 6 years

Ownership A Access to test site must be guaranteed forthe duration of study

Owner must agree to grant access to the sitefor duration of study plus possible timeextensions. As a result, sub-leasing of thetest site is not preferred. This criterion isextremely important for long-term studiessuch as field soil accumulation studies

Weather station/weather datarequirements

A On-site weather station is preferred andmay be mandatory for certain studies.Minimally, a station must be locatedwithin 10 km of test site

In certain cases, a weather station locatedwithin 10 km of the test site may besufficient. If water balances are to bedetermined, an on-site weather station isnecessary to measure, at a minimum,precipitation, solar radiation, wind speed,relative humidity, and air temperature

a Exclusion implies that criteria must be fulfilled without compromise since the study may be jeopardized if the criteria are notmet; Priority A implies some flexibility after careful consideration; Priority B factors offer the greatest flexibility in terms ofsite selection.

3.1.1 Collection of control soil

Once test sites have been identified, control soil should be collected and returned tothe laboratory. This soil is used to (1) verify soil texture and related properties, (2)ensure adequate analytical recovery of target analytes, and (3) determine the presenceof potential background interferences in the soil.


3.1.2 Soil surface preparation

Preparation of the soil surface is critical to achieving acceptable results with minimalvariability. Surface roughness due to the presence of crop debris or soil clods makesrepresentative sampling nearly impossible. This same material also interferes withsample homogenization. As a result, the importance of proper soil surface prepara-tion for bare-soil studies cannot be overstated. If vegetation exists on the selectedsite, it must be removed for bare-soil study designs. Vegetation can be removed byapplication of a nonselective herbicide such as glyphosate, paraquat, or glufosinatefollowed by mowing, raking, and harrowing once the vegetation has died.

A combination of techniques is normally required to smooth the soil properly. Forexample, disking is usually followed by multiple passes of a rolling-cage cultivator. Ifnecessary, individual subplots can be hand-raked. Sandy soils are the easiest to prepareand dry quickly after rainfall. Silt loam to clay loam soils form clods when workedtoo wet. Hence timing field preparation around rainfall and soil moisture content isalways a factor in preparing test plots. Heavy clay soils containing >40% clay posereal challenges in terms of surface preparation owing to excessive clod formationand surface cracking and should be avoided. When clayey soils are investigated,increased numbers of soil samples should be collected to compensate for the additionalvariability typically associated with these soils.

In addition to being smooth, it is preferable that the soil surface be firmly packed.This is because loose soil is not always retained in large-diameter sampling probes.Firming of the soil surface may be accomplished using a turf roller or equivalent.Alternatively, the soil surface may be prepared in advance of study initiation to allowrainfall or irrigation to settle and firm the soil. This latter approach also allows soilsurface depressions to be observed and avoided when laying out the test plots.

3.2 Test substance application

Accurate and even application of test substance is absolutely critical to study success.If the application is highly variable or deviates significantly from the target applicationrate, the study results may be technically unusable and/or unacceptable to regulatoryauthorities. Accurate agrochemical application begins with careful calibration of thespray equipment. Hence Study Directors should be familiar with sprayer calibrationtechniques,41,42 even if they will not be personally making the applications.

Braverman et al.43 found that factors responsible for inaccurate pesticide applica-tions made for crop residue trials (i.e., application rates applied at >10% or 5 cm with good results under a variety of fieldconditions.

3.3.2 Minimizing plot disturbance and cross-contamination

Great care should be taken while moving in and around the plots so that the samplingareas are not disturbed. The importance of minimizing soil surface disturbance anddrag down during sampling is critical as one tries to assess the potential mobilityof an agrochemical. This is particularly an issue when one attempts to collect manysamples from a relatively small area. In general, the risk of sub-surface contaminationis greatly minimized by using zero contamination sampling techniques.

To avoid cross-contamination of control samples, untreated controls are collectedbefore the treated samples. Preferably, personnel who handle the upper cores shouldbe different from those handling the lower depth cores. This further reduces poten-tial cross-contamination of lower depth cores. Sampler handlers should change theirgloves each time a new subplot is sampled. The use of disposable shoe covers alsolessens the possibility of cross-contamination.

Once the soil cores have been collected, all boreholes must be backfilled withuntreated soil (with frequent tamping) to prevent bypass flow that could transportresidues into the lower soil profile. After backfilling, flags or stakes should be placedat the boreholes. This serves as an additional check to ensure that sub-plots are notsampled more than one time during the study. (Note that these boreholes should


0 cm

10 cm

20 cm

40 cm

Top Soil Layer 0 - 5 cm

Sub-soil Layer5 - 20 cm

Stainless steel retainer sleeve

12-cm (ID)

Step 1 Step 2

5 - 20 cm

Step 3

Disassembling of thecorer

and replacing the cartridge

0 cm

Step 5

10 cm

20 cm

40 cm

Step 4

Reassembling of the corerand drilling down to 40 cm

60 cm

80 cm

Step 6

Reassembling of the corerand drilling further down

5 - 20 cm

20 - 40 cm

Figure 7 Alternative zero-contamination sampling method for soil


be periodically checked for subsidence over time and backfilled with soil again, ifnecessary, to prevent water infiltration.)

3.3.3 Cleaning procedure for soil sampling equipment

All sampling equipment coming in contact with treated soil (e.g., sample probes andsectioning equipment) must be thoroughly cleaned between compounds and collec-tion periods. Cleaning is best accomplished by first brushing off any soil adhering toequipment. The next step is washing with pressurized water or soap and water, andfinally rinsing with a solvent such as acetone or isopropyl alcohol, alone or in combi-nation with clean water. The use of a solvent will facilitate faster drying of equipment.

3.3.4 Protection of sample integrity

All application verification and soil samples must be individually labeled with uniquesample identification (ID) and other identifying information such as study ID, testsubstance name, sample depth, replicate, subplot and date of collection, as appropriate.Proper study documentation requires that sample lists and labels be created priorto work commencing in the field. Water- and tear-resistant labels should be usedsince standard paper labels may become water-soaked and easily torn during samplehandling. Sample lists should have the same information on them as the labels andare a convenient place to record plot randomization, initials of the individual whocollected the sample, and date of collection. As such, the sample list is important inestablishing chain of custody from the point of sample collection until its arrival atthe laboratory.

As soon as the sample has been properly labeled and recorded, it should be placedin a generator-powered chest freezer located directly in the field. A flat-bed trailer canbe used to transport freezers to and from the field site. Insulated boxes filled with dry-ice can be used as a substitute for freezers. However, chest freezers typically workbetter than dry-ice since they allow more cold air circulation around the samples,facilitating more rapid freezing.

After the samples have been placed in the freezer, it is critical that they remain frozenuntil analysis. Electronic temperature data-loggers can be used to monitor conditionsduring storage. Simpler techniques, such as inverting plastic tubes partially filled withice or placing plastic bags containing ice cubes, can also be used in combination witha mercury thermometer (any movement of the ice in the inverted tube or melting ofthe ice cubes indicates that the soil samples may have been subjected to temperatures>0 C and, hence, sample integrity potentially compromised). Since electronic data-loggers are fairly inexpensive, however, continuous monitoring of freezer storageconditions is strongly recommended.

3.3.5 Zero-time recovery and importance of the soil micro-layer

Proper sample collection and handling are the key to acceptable agrochemical re-covery at zero time. The zero-time sample interval is defined as the first samplecollected after application. Zero-time soil samples should be collected within 3 hafter application. Zero-time soil core concentrations, such as those given in Table 3,

868B

estpracticesin

thegeneration

andanalyses

ofresiduesin

environmentalsam

ples

Table 3 Summary of zero-time soil concentration and application verification (AV) monitor results for Pyraclostrobin applied at two field sites

Site Nominal soil Calculated soil concentration Maximum observed Day maximum Recovery (%) basedlocation concentration based on average concentration concentration on application rate(state) (mg kg1) pass time (mg kg1) on (mg kg1) observed (DALA)a (0.28 kg a.i. ha1)

(A) Zero-time soil recovery results

CA bare soil 0.25 0.281 0.003 0.236 1 94 (104)bFL bare soil 0.25 0.282 0.003 0.123 0 49 (53)ba Days after last application.b The number in parentheses denotes procedural correction using a 90% recovery for the CA site and a 93% recovery for the FL site.

AV fortified samples: AV spray samples:mean concentration (g) total a.i. recovered (g)

Expected fortification Observed Expected ObservedSite/application no. (nominal/assessed) fortification AV spray AV spray Recovery (%)

(B) Application verification (AV) monitor results

CA App. 1 420.0/423.8 419.3 535 529.3 99CA App. 2 403.4 535 483.2 90CA App. 3 387.0 535 480.3 90CA App. 4 413.1 535 507.4 95FL App. 1 420.0/423.8 365.4 535 476.2 89FL App. 2 349.1 535 482.4 90FL App. 3 385.0 535 501.2 94FL App. 3 372.3 535 482.2 90


are calculated by first subtracting any parent residue present in the core before lastapplication (e.g., T4) from the parent residue measured immediately after the lastapplication (e.g., T4). For example, at the CA site, the soil concentration of BAS 500 Fone day after last application (DALA) was 0.769 mg kg1. Prior to application, the soilconcentration was 0.533 mg kg1. By subtraction, a concentration of 0.236 mg kg1

was determined for BAS 500 F in the 08-cm section. This results in a zero-time soilrecovery of 94% [(0.236 mg kg1)/(0.25 mg kg1) 100]. The parent residue con-centration used to calculate recovery was the maximum concentration reached at anytime during sampling after the last application. Zero-time core recoveries (corrected)ranged from 53 to 104% for the FL and CA sites (Table 3). These data show that evenwhen considerable effort has been expended on proper test substance application (asevident by the excellent pass-time and AV recovery results) and sampling, zero-timerecoveries are frequently lower and more variable than desired.

Discrepancies between AV monitor and pass-time (or catch-back) results and actualzero-time soil concentrations are most likely due to residue losses occurring duringsample handling. Similar discrepancies may also arise for very labile compoundsowing to rapid abiotic and/or biotic losses in soil; the presence of degradates in zero-time samples would indicate that low zero-time recovery was due to degradationlosses. Immediately after application, all residues, with the exception of those com-pounds that are soil incorporated, are located in the uppermost layer of the soil core.This thin layer of surface soil is called the soil micro-layer. Loss of soil micro-layerresidues is believed to be the main reason for low and/or highly variable zero-timerecoveries from soil cores. Initial loss of the soil micro-layer is also believed to be thereason why maximum residue concentrations commonly occur days to weeks afterapplication rather than at time-zero.46 Until these surface residues are redistributedinto the core by capillary action, precipitation, or irrigation, they remain subject toloss. Careful handling of the soil samples in the field and laboratory remains especiallycritical until surface residue redistribution has occurred.

Empirical evidence supporting the role of soil micro-layer losses in zero-time issuesis given by the often-seen rise in post zero-time residue recoveries. The improvedrecoveries likely result from the micro-layer residue redistribution that reduces lossesof the highly concentrated surface residues. There has been some speculation that zero-time core recoveries may be due to volatilization losses not measured by standardlaboratory studies. If this were the case, however, increases in residue concentrationswould not occur over time since volatilized residues would be lost to the atmosphere.46

3.3.6 Sectioning of soil cores

The upper soil core can be further sectioned into 2.5-cm lengths according to studyneeds and purposes. Sectioning of the upper core can be done in the laboratory but ismost efficiently performed immediately after the core has been removed from the soilprofile. In-field sectioning begins by using a metal or plastic punch having a widecircular surface on one end to push the lower portion (i.e., the end furthest from soilsurface) of the core out of the liner to the desired length. Next, a metal cutting tool(e.g., knife or spatula) is used to slice the soil core at the correct length. As the soilis being sliced, it is directed into a pre-labeled sample bag. This process is repeated,working from the lower to upper portion of the core, until all the appropriate sections


have been sliced away. The sample bags should be rotated in and out of the on-sitefreezer until all the sectioning depths have been collected from each core within asubplot. This technique works well for all soil textures.

Once the lower 15120-cm cores are completely frozen, they can be further sec-tioned into 1015-cm lengths using a hacksaw or band saw. As before, red and blackcaps are placed on the tops and bottoms of each newly created core section. Eachnew section also receives a unique sample ID number and new label containing allpertinent sample information. Care must be used when cutting frozen cores to preventdamage to original sample labels. An advantage of the sampling approach shown inFigure 7 is that the soil cores generally require no additional sectioning.

3.3.7 Field-fortification samples

In order to determine the dissipation rate and assess the potential mobility of anagrochemical in soil, it is crucial that the residue level measured in a particular samplereflects the actual concentration present in the soil profile at the time of sampling. If thisbasic assumption cannot be assured, the validity of resulting data may be questioned.Regulatory concerns have arisen over past improper sample-handling practices thatmight have artificially accelerated agrochemical dissipation in the soil samples. Thiscould occur, for example, whenever samples are exposed to elevated temperaturesand/or direct sunlight for extended periods of time prior to freezer storage. As aresult, regulatory authorities have requested that a set of fortified samples having aknown amount of active ingredient be prepared in the field. These field fortificationsamples are intended to indicate how well the integrity of the actual field samples waspreserved during sample collection, transportation, and storage. If the field-fortifiedresidues are found to be stable, the sample handling conditions are deemed sufficientalso to have protected the integrity of the actual field samples. In contrast, if therecovery from the field fortification samples is low, this implies that sample integritywas compromised at some point during study conduct.

Although theoretically sound, field fortification samples often generate as manyquestions as they answer. This is because accurate and precise fortification of soil isdifficult to accomplish under field conditions except when the field site is very nearthe supporting laboratory. For a distant field site, the fortification solution is typicallyprepared and assayed in the laboratory prior to overnight shipment. If agrochemicalrecovery from the resulting field fortification samples is low, this may be due toaccelerated dissipation, problems associated with the fortification solution itself orimproper technique used by field personnel. Shipping fortifying solutions to the fieldis further complicated by the fact that many active ingredients make only suspensions,not true solutions. Once frozen or left without agitation for extended periods, theseformulations are difficult to re-suspend, as is required for proper soil fortification. Asa result, acceptable recovery from field spikes helps to address the issue of sampleintegrity, but poor recovery only results in more questions as to its cause.

A solution to this dilemma is to place soil samples immediately in a freezer lo-cated in the field, the temperature of which is continuously monitored, as describedpreviously. Laboratory-prepared storage study samples can then be used to determinetest substance stability under freezer storage conditions that match those used in thefield and during transportation and final storage. If a valid laboratory storage stability


study indicates that residues are stable, any observed decline in soil residues can thenbe assumed to have occurred in situ. Details on the conduct of a freezer storage studyare given in Section 4.

3.3.8 Test plot maintenance

The guiding principles in test plot maintenance are to (1) minimize soil surface distur-bance at all times, (2) ensure that control and treated plots are similarly maintained,(3) avoid applying other agrochemicals that may interfere with sample analysis orthat are otherwise contrary to the purpose of the study, (4) follow the prescribed ir-rigation policy determined for the study site, and (5) keep bare-soil test plots free ofvegetation, as follows.

For bare-soil studies, vegetation is controlled on an as-needed basis by applicationof nonselective herbicides (e.g., glyphosate, paraquat, glufosinate) or by careful handweeding. Vegetation control may be required on a weekly basis during the growingseason. The use of glyphosate or paraquat is a widely accepted means of controllingunwanted vegetation in and around test plots, and has the added advantage of control-ling weeds without physically disturbing soil surfaces. Because physical disturbanceof the soil surface is to be avoided, hoeing or other forms of mechanical removalshould not be used in the actual test plots. Vegetation that is pulled by hand shouldremain on the test plots to avoid inadvertent removal of agrochemical residues.

3.3.9 Irrigation

Because soil moisture plays such a critical role in determining agrochemical dissipa-tion rate and mobility, it is important to devise carefully an irrigation plan that clearlyspecifies the timing and amount of irrigation that is to be added at each study site. Onemust be able to justify all irrigation applications based upon the relevant agriculturalpractices in the study region and actual use pattern of the agrochemical.

For studies conducted in regions of irrigated agriculture, the plots must be irrigatedaccording to the soil-water budget method. This is determined by calculating theevapotranspiration rate for the target crop (ETc) and adjusting irrigation amounts to110% of the ETc:

ETc = ET0 Kc (4)Irrigation to apply = ETc 110% (5)

where ET0 is the actual daily evapotranspiration rate and Kc is the specific cropcoefficient based on the targeted crop and appropriate growth stage. Deficienciesshould be reconciled about every 10 days, as required.

In regions of rain-fed agriculture, the test plots must receive 110% of the monthlyhistorical rainfall. Differences in this total should be reconciled every 10 days. If theplots do not receive 110% of historical monthly rainfall, the study may be severelycompromised.

Apply the supplemental water inputs via sprinkler irrigation. Do not flood or furrowirrigate since these practices may disturb soil surface residues. Be aware that even


sprinkler irrigation can cause uneven application of water and, if leaks occur, severeerosion of the soil surface. Therefore, regularly inspect irrigation equipment andfunction. The control and treated plots must be irrigated in a similar manner. Recordthe volume and date of all irrigations, the source of irrigation water, and the typeof irrigation system used. If water begins to pool or run off of the soil surface, stopirrigating immediately. Resume irrigation only after the risk of runoff is over. To avoidrunoff, carefully match the application rate to the soil infiltration rate. Note that, incold climates, irrigation equipment is winterized to prevent damage from freezingand is generally not available for use during the winter months.

4 Phase III: sample processing and analysis

Once soil samples have been received and properly logged in by the laboratory, there isa multi-step process required to isolate agrochemical residues from the sample matrixso that sensitive, reproducible analysis can occur. Residue methods for agrochemicalsin soil involve the basic steps shown in Figure 8.

Cleanup

Derivatization

AnalyteQuantitation*

Cleanup

Extraction

Homogenization

*HPLC-UV, GC-ECD, GC-MS, LC-MS

Figure 8 Schematic of general analytical method for soil analysis


A general overview of each of these steps is given below. This is followed by aspecific example involving an increasingly powerful quantitation technique, liquidchromatography/tandem mass spectrometry (LC/MS/MS).

4.1 Sample homogenization

Soil homogenization is the critical first step in the analysis of soil samples. Improperhomogenization can lead to variable results that seriously confound the interpretationof soil residue data. Samples are commonly homogenized using equipment calledsize-reducing mills. Size-reducing mills can be further categorized as being grinder,rotary blade, or hammer type mills. Each of these has advantages and disadvantagesbut the ability to mix uniformly the anticipated volume of soil and the ease with whichthe mill can be cleaned are key considerations when choosing a particular mill. Thedesign of the mill should also prevent the loss of fine soil particles generated during theblending process. Other key aspects of sample homogenization are addressed below.

4.1.1 Protecting sample integrity

When processing samples, they should always be milled using dry-ice in amounts suf-ficient to ensure that the samples remain frozen during homogenization. As discussedpreviously, protecting sample integrity is of utmost concern throughout every aspectof study conduct. The use of adequate dry-ice also helps keep soil from sticking tothe mill. Some mills have been designed to use liquid nitrogen rather than dry-ice forcooling, and also work well with soils.

4.1.2 Minimizing cross-contamination

To minimize cross-contamination, soil cores are processed beginning with the lowestdepth samples and progressing to the surface samples. It is very important that themill be thoroughly cleaned between samples so as to minimize the risk of cross-contamination. The machinery should be thoroughly cleaned with water followed bya watersolvent solution such as acetone. Typically, the machine should be cleanedafter running one replicate set of samples from the lowest depth to the surface. If thesamples have coarse fragments in them, it may be necessary to sieve the samples priorto homogenization. As mentioned previously, soils with a large percentage of clodsor rocks should be excluded during the site selection process since they also interferewith sample collection in the field.

4.1.3 Ensuring thorough sample homogenization

Before processing actual study samples, and periodically during the course of a study,it is important to test the thoroughness of the homogenization procedure using soilshaving a range of textures. This is typically done by measuring the analytical variancebetween sub-samples, and is the only reliable method for determining the effectivenessof a blending technique. Depending on the soil type and sample size, it may benecessary to pass the sample through a mill twice to ensure proper homogenization.For example, experience has shown that when using a rotary-blade type mill, twopasses are normally required for proper homogenization of turf or sod samples. When


Table 4 Tepraloxydim analytical results used to determine efficacy of soil homogenizationprocedure

Description Residue found (mg kg1)

Sample weight = 10 g of soilSample 1 Control Not detectedSample 2 Fortified sample, 0.1 mg kg1 0.101Sample 3 Treated sample, replicate 1 0.120Sample 3, duplicate analysis Treated sample, replicate 1 0.110Sample 4 Treated sample, replicate 2 0.050Sample 4, duplicate analysis Treated sample, replicate 2 0.057



turf samples are being processed, it is also essential that the sod plug be totally frozenso that the plug will break up as it passes through the mill.

An example of adequate sample homogenization is given in Table 4. The exper-iment was conducted with two replicate treated soil samples. Each replicate wasanalyzed in duplicate. Three different sample aliquots (2, 5 and 10 g) were usedfrom each replicate. Analyses of controls and fortified samples were also conductedconcurrently with treated samples to evaluate method performance (i.e., extractionrecoveries). These results show that residue values are the same regardless of samplesize. Thus, thorough homogenization of soil samples coupled with rugged analyticalmethodology provides for satisfactory residue analysis.

4.2 Sample extraction

An efficient and reproducible extraction procedure is mandatory when analyzingagrochemicals in soil. An overview of common soil extraction techniques is givenbelow.

4.2.1 Solvent selection

Soil samples are generally extracted with one or more organic solvents mixed withup to 10% (v/v) water. A wide variety of solvents is used for extraction, the choice


of which depends upon the polarity of the compound to be extracted.47 For example,extraction with methanol and methanolwater usually works well for compoundswith medium to high polarity. Acetonitrile is another common solvent used in soilextractions. Sometimes pH adjustment is also required for compounds that are acidicor basic in nature (e.g., ammonium carbonate is often added to improve the extract-ability of weak organic acids). Starch-encapsulated formulations may benefit froman enzymatic pretreatment prior to extraction from soil.48

Several extraction techniques are used in the analysis of soil. The following arebrief descriptions of some of the most commonly used techniques.

4.2.2 Mechanical shaker

A commonly used extraction technique involves shaking soil with a suitable solvent ona mechanical shaker at about 300 rpm. After extraction, the soil extracts are collectedby centrifugation followed by decantation or filtration. This technique could be usedfor any amount of soil samples (from 10 to >100 g). Soil samples greater than 100 grequire efficient agitation to achieve acceptable recoveries.

4.2.3 Soxhlet extraction

This technique is used to extract effectively analytes that are polar in nature andstrongly bound to soil. Typically, a solvent mixture containing a water-miscible solventand an apolar solvent (e.g. methanoldichloromethane) is used. A small aliquot of soil(1030 g) is dried by mixing with sodium sulfate and refluxed for 816 h to extractthe residues.

4.2.4 Sonication

This technique is used mainly for nonpolar compounds. Typically a small aliquot ofsoil (1030 g) is dried by mixing with sodium sulfate prior to extraction. Next, thesample is extracted with a solvent for 1020 min using a sonicator probe. The choiceof solvent depends on the polarity of the parent compound. The ultrasonic powersupply converts a 50/60-Hz voltage to high-frequency 20-kHz electric energy that isultimately converted into mechanical vibrations. The vibrations are intensified by asonic horn (probe) and thereby disrupt the soil matrix. The residues are released fromsoil and dissolved in the solvent.

4.2.5 Supercritical fluid extraction (SFE)

SFE is used mainly for nonpolar compounds [e.g. polychlorinated biphenyls (PCBs)].Typically, small aliquots of soil (0.510 g) are used for extraction. The extraction sol-vent is a supercritical fluid, most commonly carbon dioxide, which has propertiesof both a liquid and gas. The supercritical fluid easily penetrates the small pores ofsoil and dissolves a variety of nonpolar compounds. Supercritical carbon dioxide ex-tracts compounds from environmental samples at elevated temperature (100200 C)and pressure (500010 000 psi). High-quality carbon dioxide is required to minimize


analytical interferences. Compounds with different chemical natures can be selec-tively extracted by varying the extraction pressure and temperature. The addition of anorganic modifier, such as methanol, may improve the recoveries of polar compounds.

4.2.6 Accelerated solvent extraction (ASE)

This fully automated process developed by Dionex is used for a variety of compoundshaving a wide range of polarities.49 Typically, a small aliquot of soil (0.520 g) isextracted using a variety of solvents. As with other techniques, the solvent choicedepends upon the polarity of the compound to be extracted. The unit extracts soil atelevated temperatures (>60 C) and pressures (>1000 psi). The increased temperatureaccelerates the extraction kinetics while the elevated pressure keeps the solvent(s)below the boiling point, thus allowing safe and rapid extraction. Both time and solventconsumption are dramatically reduced compared with mechanical shaking. There arenow several published United States Environmental Protection Agency (USEPA)methods that use ASE (e.g., USEPA Method 600/4-81-055, Interim Methods for theSampling and Analysis of Priority Pollutants in Sediment and Fish Tissue).

4.2.7 Microwave extraction

This is a relatively new technique that is used for PCBs and other nonpolar, volatileand semi-volatile organic compounds. Typically, a small aliquot of soil sample(0.520 g) is used for the extraction. Soil samples are extracted with one or moreorganic solvents using microwave energy at elevated temperature (100115 C) andpressure (50175 psi). This method uses less solvent and takes significantly less timethan Soxhlet extraction but is limited to thermally stable compounds.

4.3 Sample cleanup

Trace analysis of soil samples often requires post-extraction cleanup to remove co-extracted matrix interferences. There are several difficulties that may arise duringchromatographic analysis due to interferences present in sample extracts. To avoidthese and other issues, one or more of the following cleanup techniques are often used.

4.3.1 Liquidliquid partition

This technique provides a convenient method for separating an agrochemical com-pound from a highly aqueous extraction mixture. The partitioning solvent is usuallya volatile, water-immiscible organic solvent that can be removed by evaporation afterthe desired component has been extracted. This technique is based on the principlethat when a substance is soluble to some extent in two immiscible liquids, it can betransferred from one liquid to another by shaking. The degree of partitioning from onesolvent to the other depends on the agrochemicals distribution coefficient betweenthe immiscible liquids. This technique is particularly useful for the cleanup of ioniz-able compounds, since the pH of the aqueous solution can be adjusted to maximizepartitioning into the organic or water phases, as desired.


4.3.2 Solid-phase extraction (SPE)

This technique is based on the same separation mechanisms as found in liquid chro-matography (LC). In LC, the solubility and the functional group interaction of sample,sorbent, and solvent are optimized to effect separation. In SPE, these interactions areoptimized to effect retention or elution. Polar stationary phases, such as silica gel,Florisil and alumina, retain compounds with polar functional group (e.g., phenols,humic acids, and amines). A nonpolar organic solvent (e.g. hexane, dichloromethane)is used to remove nonpolar inferences where the target analyte is a polar compound.Conversely, the same nonpolar solvent may be used to elute a nonpolar analyte, leavingpolar inferences adsorbed on the column.

The most common technique used for agrochemicals is reversed-phase SPE. Here,the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon(e.g. C4C18) or styrenedivinylbenzene copolymer. This technique operates in thereverse of normal-phase chromatography since the mobile phase is polar in nature(e.g., water or aqueous buffers serve as one of the solvents), while the stationary phasehas nonpolar properties.

Ion-exchange solid-phase extractions are used for ionic compounds. The pH of theextracts is adjusted to ionize the target analytes so that they are preferentially retainedby the stationary bonded phase. Selection of the bonded phase depends on the pKa orpKb of the target analytes. Sample cleanup using ion exchange is highly selective andcan separate polar ionic compounds that are difficult to extract by the liquidliquidpartition technique.

A variety of solid-phase cartridges are available from a number of different manu-facturers (e.g. J.T. Baker, Varian). Most cartridges, however, use a similar extractionprocedure that consists of these basic steps:

1. Conditioning the column. This step prepares the column to absorb the analytes andalso pre-washes the column with the solvents that are used for the cleanup.

2. Sample application. The sample extract is dissolved in the weaker solvent andapplied to the top of the column. The analytes of interest are extracted from thecrude sample extract and are adsorbed on the column.

3. Wash. Solvents, weaker than the elution solvents, are used to remove interferencesselectively.

4. Elution. The compound of interest is selectively eluted with a stronger solvent.

4.4 Derivatization techniques

A derivatization technique is commonly applied to an agrochemical with certain re-active functional groups (e.g., carboxylic acid, amine, phenol) to make the compoundamenable to either gas chromatography (GC) or LC analysis. An in-depth discus-sion of derivatization reactions used in the analysis of agrochemicals is beyond thescope of this article. For more information on this topic, the reader is referred toKnapp.50


4.5 Analytical detection and quantitation techniques

The most common final separation techniques used for agrochemicals are GC and LC.A variety of detection methods are used for GC such as electron capture detection(ECD), nitrogenphosphorus detection (NPD), flame photometric detection (FPD)and mass spectrometry (MS). For LC, typical detection methods are ultraviolet (UV)detection, fluorescence detection or, increasingly, different types of MS. The excellentselectivity and sensitivity of LC/MS/MS instruments results in simplified analyticalmethodology (e.g., less cleanup, smaller sample weight and smaller aliquots of theextract). As a result, this state-of-the-art technique is becoming the detection methodof choice in many residue analytical laboratories.

An example of an LC/MS/MS method with an LOQ of 0.01 mg kg1 is illustratedin Figure 9. This method was used to analyze tepraloxydim and its primary metabolite

Soil (25 g)

- Extract with dichloromethane 3 X 50 mL- Centrifuge

Combined dichloromethane extract Marc

(discard) - Evaporate to dryness

Dissolve in acetonitrile-water (80:20, v/v)

- Dilute with:1

Acetonitrile-water (1:1) + 0.1% formic acid or Methanol-water (1:1) + 0.1% formic acid,

4 mM ammonium formate

LC/MS/MS determinationAnalysis for tepraloxydim (m/z 342 to 250) and DP-6 (m/z 253 to 197) in positive ion mode

1Modifications were used for different soil types.

Tepraloxydim DP-6

O

OH

ON

CIO

O

OH

O O

Figure 9 Method diagram for the determination of tepraloxydim and its degradate, DP-6, in soil(LOQ 0.01 mg kg1)


Table 5 Recoveries of tepraloxyim and degradates from soil dissipation studies conducted in the USA and Canada

Recovery range (%) Mean recovery (%)Compoundfortifieda North Dakota Mississippi California North Dakota Mississippi California

(A) US sites

Tepraloxydim 78119 74106 86113 96 10 86 7 100 9(n = 46) (n = 44) (n = 26)

DP-6 69116 71102 77102 93 11 92 7 89 7(n = 46) (n = 44) (n = 26)

Recovery range (%) Mean recovery (%)Compoundfortifieda Manitoba Saskatchewan Alberta Manitoba Saskatchewan Alberta

(B) Canadian sites

Tepraloxydim 77110 72121 70107 92 9 90 9 88 8(n = 39) (n = 44) (n = 43)

DP-6 71116 74119 72118 90 10 94 11 94 16(n = 39) (n = 44) (n = 43)

a Fortification range for all three sites was 0.010.1 mg kg1.

DP-6 over 3000 soil samples collected from several terrestrial field dissipation studies.The sample procedural recoveries using this method, conducted concurrently with thetreated samples during soil residue analysis, are summarized in Table 5. This methodwas proven to be short, rugged, sensitive, and suitable for measuring residues in soiland sediment at levels down to 0.01 mg kg1. The reproducibility of the methods alsoindicated acceptable method performance and, as a result, thousands of samples wereanalyzed using this methodology.

4.6 Freezer storage stability

Most agrochemicals remain stable in frozen soil for many months. However, it isimportant to verify this stability by conducting a freezer storage stability study. Onetype of study is conducted by fortifying known amounts of test substance and itsmajor transformation products into control soil collected from a participating fieldsite. Fortification normally occurs at two levels: replicate soil samples are fortifiedat the LOQ and at the highest expected residue concentration for each analyte ofinterest. The fortified soil samples are stored under the same conditions as the fieldsamples and analyzed at different time periods that bracket the storage time of theactual field samples. The recoveries of the storage samples are compared with thoseobtained from day zero analyses to obtain the storage stability. In general, the methodof analysis is the same as used for the soil residue analysis.

A second approach to determining freezer storage stability involves the reanalysisof incurred residues found in actual samples that are stored over time. Using thisapproach, soil from an actual field sample containing residues is periodically analyzed


during the course of the analysis phase of the study. A key

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Handbook of Residue Analytical Methods for Agrochemicals

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