Document ID: EPA-HQ-OPP-2007-0725-0008
Agency: epa
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Title: 
Posted Date: 2007-11-20T05:00Z

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<h2>Review of Existing Immunoassay Kits for Screening of Acetochlor and Other  Acetanilides in Water</h2>

<p>Acetochlor Registration Partnership<br />
c/o ZENECA Ag Products<br />
1800 Concord Pike<br />
Wilmington, Delaware 19897</p>

<p>March 1995</p>

<h2>TABLE OF CONTENTS</h2>

<ol style="list-style-type:upper-roman;">

<li><h3><a href="#I">SUMMARY</a></h3></li>

<li><h3><a href="#II">INTRODUCTION</a></h3></li>

<li><h3><a href="#III">FACTORS USED IN COMPARISON OF IMMUNOASSAYS</a></h3>
	<ol style="list-style-type:upper-alpha;">    
	<li><h4>Dose-Response Curves</h4></li>
	<li><h4>Analytes of Interest</h4> </li>
	<li><h4>Cross-Reactants</h4> </li>
	<li><h4>Threshold Screening Levels</h4> </li>
	</ol>
</li>
<li><h3><a href="#IV">COMPARISON OF COMMERCIALLY AVAILABLE IMMUNOASSAYS FOR ACETANILIDES</a></h3>
	<ol style="list-style-type:upper-alpha;">    
	<li><h4>Metolachlor (Ohmicron)</h4></li>
	<li><h4>Metolachlor 1.0(Idetek/Quantix)</h4></li>
	<li><h4>Alachlor 1.0 (Idetek/Quantix)</h4></li>
	<li><h4>Alachlor (Millipore)</h4></li>
	<li><h4>Alachlor (Ohmicron)</h4></li>
	<li><h4>Acetanilide (Millipore)</h4></li>
	<li><h4>Summary</h4></li>
	</ol>
</li>
<li><h3><a href="#V">EVALUATION OF ENVIROGARD<sup>TM</sup> ACETANILIDE PLATE KIT</a></h3>
	<ol style="list-style-type:upper-alpha;">     
	<li><h4>Background</h4></li>
	<li><h4>Principles of the Assay</h4></li>
	<li><h4>Reproducibility of the Standard Curve</h4></li>
	<li><h4>Cross-Reactivity</h4></li>
	<li><h4>Accuracy and Precision</h4></li>
	</ol>
</li>
<li><h3><a href="#VI">ENVIRONMENTAL HEALTH LABORATORIES (EHL) ACETANILIDE ASSAY</a></h3>
	<ol style="list-style-type:upper-alpha;">    
	<li><h4>Background</h4></li>
	<li><h4>Principles of the Assay</h4></li>
	<li><h4>Reproducibility of Standard Curve</h4></li>
	<li><h4>Cross-Reactivity</h4></li>
	<li><h4>Accuracy and Precision</h4></li>
	</ol>
</li>
<li><h3><a href="#VII">PERFORMANCE TESTING OF ACETANILIDE IMMUNOASSAYS</a></h3>
	<ol style="list-style-type:upper-alpha;">    
	<li><h4>Purpose</h4></li>
	<li><h4>Experimental Design</h4></li>
	<li><h4>Results and Discussion</h4></li>
	</ol>
</li>
<li><h3><a href="#VIII">CONCLUSIONS</a></h3></li>

<li><h3><a href="#IX">TABLES</a></h3>
	<ol style="list-style-type:upper-alpha;">    
	<li><h4>Tables</h4></li>
	</ol>
</li>
<li><h3><a href="#X">REFERENCES</a></h3></li>
</ol>

<p class="pagetop"><a href="#content">Top of page</a></p>
<hr />

<ol style="list-style-type:upper-roman;">

<li><h3 id="I">SUMMARY</h3>

<p>As part of the conditional registration of acetochlor in March 1994, the Acetochlor Registration Partnership (ARP) agreed to provide immunoassay methods for cost effective  state monitoring of ground and surface water samples for the presence of acetanilide  herbicides.  This report is submitted to the Environmental Protection Agency in  fulfillment of that agreement. </p>

<p>At the time of registration of acetochlor, only specific acetanilide assays, directed toward the detection of a single analyte had been reported.  Whilst engaged in an assessment of the potential utility of these specific immunoassays, the ARP became aware  of a class-specific assay in pre-commercial development by Millipore Corporation.  This  acetanilide immunoassay will be available in a microtiter plate format for use in  experienced laboratories with appropriate equipment and an automated format available as  an analytical service from the Environmental Health Laboratories (EHL) in South Bend,  Indiana. </p>

<p>A summary of the performance and characteristics of the existing analyte specific assays has been compiled and included within this report.  Also experimental data are presented for an ARP sponsored independent evaluation of the Millipore assay at the Water  Quality Laboratory (WQL) of Heidelberg College in Tiffin, Ohio and the results of the  analysis of blind fortified surface and ground water samples at the both the WQL and the  EHL. </p>

<p>Five commercially available analyte specific acetanilide immunoassays were evaluated for cross-reactivity with acetochlor.  Two of the assays were designed for metolachlor and three were directed toward alachlor.  Three of the assays were reported to have some  cross-reactivity towards acetochlor but the sensitivities as measured by I50 (concentration of analyte resulting in a 50% inhibition of control response) were from 6.55 to 70 ppb acetochlor.   Based on the typical characteristics of immunoassay 
dose-response curves, these levels of sensitivity were considered inadequate to provide sufficient precision of measurement at lower concentrations of interest.  </p>

<p>The pre-commercial acetanilide assay of Millipore was reported to have I50's for metolachlor, acetochlor and alachlor of 0.26 ppb, 1.7 ppb and 4.9 ppb, respectively, suggesting that it offers the best prospects for an acetanilide screen including acetochlor.  The independent WQL evaluation of this kit confirmed the sensitivity of the  assay for the reported acetanilides as well as demonstrating that dimethenamid is also  detected with an I50 of 0.5 ppb.  Atrazine and the common soil metabolites of alachlor  and acetochlor were not detected by the assay at concentrations up to 500 ppb.   Calibration of the assay using acetochlor calibrators provided by Millipore was reproducible and an acceptable level of accuracy for acetochlor in reagent water was  demonstrated at 0.1 and 1.0 ppb. The within-assay and between-assay precision varied with  the level of measurement but were typical of this type of immunoassay (% CV 8.3 to  35.2%).  The automated assay available from the EHL gave similar results except with  slightly better precision as would be anticipated for instrumented operation.</p>

<p>The analysis of blind surface and ground water samples fortified with acetochlor at 0.1 or 1.0 ppb provided a direct comparison of the manual operation of the acetanilide  assay at WQL with the automated version available at the EHL using samples with a typical  matrix composition.  In addition, since these samples were analyzed before and after  fortification by GC/MS for corn herbicides, it was possible to establish potential  interferences due to the presence of other acetanilides.  The results of the blind  analyses by the WQL and the EHL were in general agreement but differed significantly from  the GC/MS in their estimation of acetochlor level for the unfortified and the 0.1 ppb  fortifications.  These discrepancies were shown to be due to the presence of an average  of 0.091 ppb metolachlor in the surface water samples.  Improved agreement among all  assays was obtained for the 1.0 ppb fortifications.</p>

<p>The results described in this report indicate that the acetanilide immunoassay in 
pre-commercial development by Millipore when used as a plate kit or in an automated format has sufficient accuracy and precision to serve as a screen for acetochlor in water  at 1.0 ppb.   Use of the assay as a screen at 0.1 ppb was considered to be unadvisable  due to low precision at this level and the fact that the lowest calibrator provided in  the kit was 0.1 ppb acetochlor.  The assay was shown to have improved precision at 0.2  ppb and to be within the linear range of the standard curve at this level.  These  observations suggest that 0.2 ppb would be a reasonable low level threshold for  application of the assay.</p>

<p>The acetanilide immunoassay of Millipore is not spectific and does not differentiate among alachlor, acetochlor, metolachlor and dimethenamid but rather detects their presence in varying degrees.  Therefore, it is imperative that any positive detects obtained using this immunoassay as a plate kit or in an automated format be confirmed  with another analytical method, such as GC/MS, to establish the presence and level of  acetochlor in water. </p>

<p class="pagetop"><a href="#content">Top of page</a></p>
<hr />
</li>
<li><h3 id="II">INTRODUCTION</h3>

<p>As part of the conditional registration of acetochlor in March 1994, the EPA requested  that the Acetochlor Registration Partnership provide immunoassay methods for cost  effective state monitoring of ground and surface water samples for the presence of  acetanilide herbicides.</p>

<p>The ARP has adopted a structured approach towards fulfilling this requirement. In the first instance, this has involved discussions with commercial immunoassay kit manufacturers, in order to gain a clear understanding of the potential issues associated with the development, validation, and use of appropriate methods. Subsequent discussions  with possible end-users have served to define additional criteria for the acceptability  of such methods in routine use.</p>

<p>On the basis of these initial findings, the ARP has reviewed the potential  applicability of currently available immunoassays against the criteria listed below:
</p>

	<ul>

	<li><p>Only commercially available immunoassays are suitable for the proposed  	application.  This reflects the need to ensure a guaranteed, quality-assured supply  	to the end-user, with an appropriate level of after-sales support.</p></li>

	<li><p>The chosen assay must be sufficiently rugged to operate reliably in the 	hands of end-users who may not have extensive experience in the use of this 	technology.</p></li>

	<li><p>The assay must offer the prospect of a rapid, high-throughput screen for  	samples prior to confirmatory analysis of positive detects.</p></li>

	<li><p>The assay should perform with a suitably high degree of reliability at the  	requisite levels.  The incidence of "false negatives" (samples  	incorrectly identified as not containing acetanilides above a specified threshold)  	must be suitably low.  The permissible incidence of "false positives" may  	be relatively higher, but should not be so high that the potential benefits are  	negated by unwarranted repeat analysis.</p></li>

	<li><p>The assay should be able to detect those acetanilides currently used as corn  	herbicides, namely alachlor, acetochlor, dimethenamid and metolachlor. </p></li>

	<li><p>The possible inadvertent detection of interfering compounds 
	("cross-reactants") must be defined. The incidence of such detects must  	not prevent the application of the assay for detection of the specified analytes.	</p></li>

	</ul>

<p>At the time of registration of acetochlor, only specific acetanilide assays, directed towards the detection of a single analyte, had been reported:  this encompassed both commercially available assays and those developed by agrochemical companies1,2.  Consequently, the ARP concentrated attention on commercially available assays (see Section IV) designed for specific acetanilides; the reported levels of cross-reactivity for related compounds in these assays were used to provide an initial assessment of their  potential utility for class-specific determination of acetanilides.  Additionally, whilst  engaged in this process, the ARP became aware of a potential class-specific assay in 
pre-commercial development.  The basic performance characteristics of all these assays  were subsequently compared as a means of prioritizing them for further evaluation.</p>

<p>The factors taken into account in comparing the assays are discussed in Section III.  The assays are described and compared in Section IV.</p>

<p class="pagetop"><a href="#content">Top of page</a></p>
<hr />
</li>
<li><h3 id="III">FACTORS USED IN COMPARISON OF IMMUNOASSAYS</h3>

	<ol style="list-style-type:upper-alpha;">

	<li><h4>Dose-Response Curves</h4>

	<p>Immunoassay dose-response curves are usually defined in terms of the inhibition  	of binding of a labelled ligand to an antibody as a function of concentration of  	the analyte in a test sample.  The concentration of the analyte is related to the  	absorbance (color) produced by the bound, labelled ligand.  As the concentration of  	the test analyte increases, the color produced by the labelled ligand decreases.   	The resulting absorbance versus log [concentration] curves are sigmoidal and  	express the inverse relationship between concentration of the test analyte and  	intensity of color.  </p>

	<p>Two pieces of information are often quoted to define these curves:  I50 and I10  	or I20.  The I50 concentration defines the limiting analyte concentration producing  	50% inhibition and is the mid-point of the dose-response curve.  The I10 or I20 	concentration defines the limiting analyte concentration producing a 10% or 20% 	inhibition of color formation, respectively.  A sigmoidal dose-response curve is 	necessarily steepest at the I50 and shallowest at its upper and lower (I10)  	extremes.  Consequently, the relative precision is higher at the I50 and lower at  	concentrations represented by the lower inhibition values3.  </p>

	</li>
	<li><h4>Analytes of Interest</h4>

	<p>The analytes of interest are acetochlor, alachlor, metolachlor and dimethenamid.   	A suitable class-specific assay must be capable of identifying any sample in which  	any of these analytes occur at or above their respective threshold levels.  It is,  	however, unlikely that any assay will be capable of measuring all four analytes  	with the same degree of sensitivity.  Under these circumstances, it is important to  	evaluate the candidate assays in terms of their performance for all four potential  	analytes.</p>

	</li>
	<li><h4>Cross-Reactants</h4>

	<p>An important criterion of immunoassay suitability is the selectivity or  	specificity of the assay for the analytes of interest.  Interference caused by  	materials of related structure can lead to a high level of "false 	positives" and severely limit the usefulness of the assay for measuring  	acetochlor, alachlor, metolachlor and dimethenamid.  Previous studies with an  	alachlor immunoassay have shown the potential of metabolites such as alachlor  	ethane sulfonic acid to cause false positive results4,5. </p>

	<p>The state ground and surface water programs outlined in the acetochlor  	registration agreement do not include degradates of acetochlor since these  	materials are not of toxicological concern. Therefore, an assessment of the 
	cross- reactivity of degradates or metabolites of the analytes is a key factor in  	determining the suitability of an assay.</p>

	</li>
	<li><h4>Threshold Screening Levels</h4>

	<p>The use of immunoassays to screen for the presence of analytes in environmental 	samples has been successfully tested for a number of analytes including alachlor6  	and atrazine7.  The need for standardized validation of screening procedures has  	been recognized and a number of groups including the Association of Official  	Analytical Chemists (AOAC), Analytical Environmental Immunochemical Consortium  	(AEIC) and International Union of Pure and Applied Chemistry (IUPAC) are reported  	to be developing guidelines for the  evaluation of immunoassay kits8.  The need to  	verify immunoassay results at a specified threshold point by an alternative  	technique such as GC/MS is generally recognized as a requirement of a suitable  	validation program.</p>

	<p>The acetochlor registration agreement has specified the detection of acetochlor  	at 0.10 ppb in ground water in the state monitoring program as a 	"trigger" for  further regulatory action.  In other monitoring programs  	outside of the Prospective Ground Water (PGW) program or the State Monitoring  	Programs, detection of acetochlor at 0.20 ppb requires follow up action.   	Regulatory action for the State Surface Water Monitoring Program is based on  	exceeding an annual time-weighted mean concentration of 2.0 ppb for acetochlor or a  	single peak concentration of 8.0 ppb for this analyte.</p>

	<p>In determining the suitability of an immunoassay for use as a screen for  	threshold levels of acetochlor and other corn herbicides two action levels are  	worthy of consideration:</p>

		<ol>
		<li>0.1 - 0.2 ppb and</li>
		<li>1.0 - 2.0 ppb.</li>
		</ol>

	<p>Sufficient sensitivity at the lower level would provide an assay capable of  	satisfying both thresholds since the more concentrated samples could be easily  	diluted.</p>

	</li>
	</ol>

<p class="pagetop"><a href="#content">Top of page</a></p>
<hr />
</li>
<li><h3 id="IV">COMPARISON OF COMMERCIALLY AVAILABLE IMMUNOASSAYS FOR ACETANILIDES</h3>

<p>Acetanilide immunoassays either currently commercially available, or in 
pre-commercial development, are listed below:</p>

<p>Metolachlor (Ohmicron)<br />
Metolachlor 1.0 (Idetek/Quantix)<br />
Alachlor 1.0 (Idetek/Quantix)<br />
Alachlor (Millipore)<br />
Alachlor (Ohmicron)<br />
Acetanilide (Millipore)</p>

<p>The first five of these are already commercially available, and are promoted as specific assays for the title compound.  The class-specific acetanilide kit is currently in evaluation/development by Millipore.  The properties of these assays are described below.</p>

	<ol style="list-style-type:upper-alpha;">

	<li><h4>Metolachlor (Ohmicron)</h4>

	<p>The Metolachlor Ohmicron RaPID Assay Kit9 is intended for the detection of 	metolachlor in water and soil.  The key assay characteristics are outlined in Table  	1.  The assay shows high sensitivity towards metolachlor (I10 = 0.05 ppb; I50 =  	0.85 ppb), and some cross-reactivity towards acetochlor (I10 = 0.06 ppb; I50 = 6.55 	ppb).  The level of cross-reactivity towards alachlor is, however, comparatively  	low (I10 = 1.30 ppb; I50 = 84.0 ppb), making the assay of limited use for detection  	of the latter.</p>

	<p>Notably, metalaxyl, N-(2,6-dimethyl-phenyl)-N-(methloxyacetyl)alanine methyl  	ester, also shows strong cross-reactivity, comparable to that of acetochlor, and  	might therefore constitute a significant source of false positive detects for  	chloroacetanilides were this kit to be used. </p>

	</li>
	<li><h4>Metolachlor 1.0 (Idetek/Quantix)</h4>

	<p>This assay10 shows high specificity towards metolachlor (I50 = 0.75 ppb). The  	levels of cross-reactivity to both alachlor and acetochlor are very low (I50 = 100  	ppb and 70 ppb, respectively).  I10/20 values are not quoted in the technical data  	sheet, but the I50 values indicate that this assay would be of no use for the  	detection of either alachlor or acetochlor.</p>

	</li>
	<li><h4>Alachlor 1.0 (Idetek/Quantix)</h4>

	<p>This assay11 is specific for alachlor (I50 = 0.48 ppb). The levels of 
	cross-reactivity towards acetochlor (I50 = 23 ppb) and metolachlor (I50 = 80 ppb)  	indicate that the assay would not be of use for the detection of the compounds.   	Limited cross-reactivity was demonstrated for alachlor metabolites as indicated:   	alachlor sodium oxanilate (I50 = 50 ppb),   alachlor sulfonic acid (I50 = 10 ppb)  	and alachlor sulfinyl acetic acid (I50 = 7 ppb).  I10/20 were not reported.</p>

	</li>
	<li><h4>Alachlor (Millipore)</h4>

	<p>The EnviroGard Alachlor Plate Kit12 has a least detectable dose for alachlor of  	0.046 ppb and an I50 of 0.6 ppb.  The least detectable dose of metolachlor is 0.6  	ppb, with an I50 of 40 ppb, again indicating that the assay would be of little  	utility for metolachlor detection.  No data describing the cross-reactivity of  	acetochlor in this assay were reported.</p>

	</li>
	<li><h4>Alachlor (Ohmicron)</h4>

	<p>The characteristics of the alachlor Ohmicron RaPID Assay Kit13 are summarized in 	Table 2.  The assay does not have sufficient cross-reactivity towards metolachlor 	(I10/20) to form the basis of a useful screen for acetanilides.  In addition, there  	is significant inadvertent cross-reactivity to the ethane sulfonic acid metabolite  	of alachlor.</p>

	</li>
	<li><h4>Acetanilide (Millipore)</h4>

	<p>The EnviroGard Acetanilide Plate Kit14 is the only one of the assays described  	which is primarily intended as a class-specific screen for acetanilides including  	acetochlor.  The assay characteristics are summarized in Table 3.  The assay shows  	the highest sensitivity towards metolachlor (I10 = 0.02 ppb; I50 = 0.25 ppb), but  	also has strong cross-reactivity towards acetochlor (I10 = 0.02 ppb; I10 = 1.7 ppb)  	and alachlor (I10 = 0.55 ppb; I50 = 4.9 ppb).</p>

	</li>
	<li><h4>Summary </h4>

	<p>Table 4 shows a direct comparison of the six assays with respect to their  	capabilities to detect metolachlor, acetochlor and alachlor.  Published data for  	dimethenamid were not available for any kit.  The metolachlor (Idetek and Ohmicron)  	and alachlor (Idetek, Millipore and Ohmicron) assays are all clearly deficient in  	their capabilities to detect at least one of the three analytes.  This is most  	readily demonstrated where I10/20 values, corresponding to the least detectable  	doses, are in excess of the concentrations of interest.  In those cases where  	I10/20 values are not quoted, large I50 values indicate that the sigmoidal 
	dose-response curves must be very shallow, and therefore imprecise, if indeed even  	measurable, at the low concentrations of interest.</p>

	<p>The Millipore Acetanilide assay offers the best prospects for a acetanilide  	screen including acetochlor.  The least detectable doses for alachlor, acetochlor  	and metolachlor are relatively low, and the ranges of the respective dose-response  	curves appear sufficiently narrow to enable reasonably precise measurements to be  	made.</p>

	</li>
	</ol>

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<hr />
</li>
<li><h3 id="V">EVALUATION OF ENVIROGARD<sup>TM</sup> ACETANILIDE PLATE KIT</h3>

	<ol style="list-style-type:upper-alpha;">

	<li><h4>Background</h4>

	<p>The pre-commercial development of an acetanilide immunoassay by Millipore 	Corporation was confirmed on December 15, 1994 with Brian A. Skoczenski, Vice 	President of Research and Development of Immunosystems Inc., a division of 	Millipore. Based on this information it was clear that the EnviroGard<sup>TM</sup>  	Acetanilide Plate Kit offered the best prospects for an acetanilide screen and the  	ARP was able to obtain, by special order, twelve kits for independent evaluation. 	</p>

	<p>The ARP selected the Water Quality Laboratory at Heidelberg College in Tiffin,  	Ohio to conduct an evaluation of the acetanilide kit because of their extensive  	experience in water analysis using a variety of methods and their prior work with  	Millipore immunoassays4.  A protocol was written which specified the design of the  	study and was finalized by the signature of the study director on January 4, 1995.   	The study was conducted with clear guidelines for record keeping and documentation  	but not under the specific dictates of Good Laboratory Practices (GLP) as written  	in 40 CFR Part 160. </p>

	<p>The objective of the evaluation was to determine how the kit performed for the 	analysis of acetochlor and other corn herbicides when used by experienced 	investigators.  Included within this evaluation was an analysis of blind coded  	surface and ground water samples that had been fortified with acetochlor at 0.10  	and 1.0 ppb levels. </p>

	</li>
	<li><h4>Principles of the Assay</h4>

	<p>The Millipore assay is a competitive ELISA method that uses a microtiter plate 	format (12 strips of 8 antibody-coated wells each, in a strip holder) supplied by  	the manufacturer with wells precoated using the acetanilide antibody.  Water  	samples of calibration standards are added to individual wells of the microtiter  	plates, followed by an acetochlor-enzyme conjugate for binding to the limiting  	concentration of antibody during a fixed incubation period.  Following a wash step,  	which serves to remove any unbound acetochlor-enzyme conjugate, an enzyme substrate  	is added.  This results in the formation of a blue color in the presence of the  	bound acetochlor-enzyme conjugate.  The intensity of the color is inversely related  	to the concentration of acetanilide in the sample.  The actual concentration is  	measured by reference to a standard curve of log[concentration] versus absorbance  	which is generated concurrently during the analysis using standards of known  	concentration.  Since acetochlor calibrators are used, the concentration is  	expressed as acetochlor equivalents.  However, since the assay has broad  	specificity for a number of acetanilides including alachlor, acetochlor and  	metolachlor, a positive response in the assay does not mean that acetochlor is  	present in the sample; the assay will only detect unspecified cross-reacting  	acetanilides.</p>

	<p>The method performance has been evaluated  in terms of the following assay  	characteristics:</p>

		<ul>   
		<li><p>Reproducibility of the standard curve</p></li>
		<li><p>Cross-reactivity towards related analytes</p></li>
		<li><p>Precision and accuracy of measurement of replicate analyses of 			fortified samples in reagent water</p></li>
		</ul>

	<p>The results of these evaluations are reported and discussed below.</p>

	</li>
	<li><h4>Reproducibility of the Standard Curve</h4>

	<p>The acetanilide kit relies on a three point calibration curve using the  	following acetochlor calibrators in reagent water:  0.0 ppb (negative control or  	blank), 0.1 ppb, 0.5 ppb and 5.0 ppb.  The standard curve is expressed as %Bo  	[(average absorbance of calibrator)/(average absorbance of negative control) x  	100%] versus log [acetochlor].  The reproducibility of the standard curve was  	evaluated by analyzing in triplicate a series of acetochlor standard solutions on  	ten separate days.    The data indicates acceptable reproducibility (%CV of 5.7 to  	14.5%).  </p>

	</li>
	<li><h4>Cross-Reactivity</h4>

	<p>The ARP provided a certified sample of acetochlor as well as the following  	reference materials for testing:</p>

		<ol>
		<li>Alachlor</li>
		<li>Metolachlor</li>
		<li>Dimethenamid</li>
		<li>Atrazine</li>
		<li>Alachlor Sulfonic Acid</li>
		<li>Alachlor Oxanilic Acid</li>
		<li>Alachlor Sulfinyl Acetic Acid</li>
		<li>Acetochlor Oxanilic Acid</li>
		<li>Aceotchlor Sulfonic Acid</li>
		<li>Acetochlor Sulfinyl Acetic Acid</li>
		</ol>

	<p>Compounds 5-10 represent the major soil metabolites of alachlor and acetochlor.   	A stock solution of each standard was prepared in methanol and dilutions in water  	gave a series of solutions between 500 ppb and 0.1 ppb.  Each of the aqueous  	solutions were tested using the acetanilide kit, sigmoidal plots of %Bo versus log 	[concentration] were prepared and the values of I10 (10% inhibition) and I50 (50% 	inhibition) were determined from the graphs. The results are shown in Table 5. </p>

	<p>The sensitivity of the assay for acetochlor, alachlor and metolachlor was  	confirmed as demonstrated by the I50 values (Table 5).  The acetanilide data sheet  	from Millipore for this assay lists a lower I50 for metolachlor than acetochlor  	contrary to the results of the Heidelberg College determination.  The reason for  	this discrepency is not known but one possibility could be the use of linear plots  	of %Bo versus log [concentration] by Millipore.  Since Heidelberg College used  	sigmoidal rather than linear fit of the data different estimates would be obtained.  	Dimethenamid was shown to be a sensitive analyte in the assay with a lower I50 than  	acetochlor.  Atrazine and the soil metabolites of alachlor and acetochlor were  	unreactive even at 500 ppb demonstrating that these materials have a very low  	probability of giving false positives at typical environmental concentrations. </p>

	</li>
	<li><h4>Accuracy and Precision</h4>

	<p>The acetanilide assay was used to measure samples, generally in triplicate, on  	ten different days.  At total of twenty nine individual measurements were made at  	each fortification. </p>

	<p>At 0.1 ppb, individual concentration measurements ranged from 0.09 - 0.29 ppb.  	Mean concentrations from daily measurements ranged from 0.10 - 0.26 ppb and the 	overall mean was 0.16 ppb.</p>

	<p>At 1.0 ppb, individual concentrations ranged from 0.72 - 1.90 ppb.  Mean daily 	concentrations ranged from 0.83 - 1.48 ppb, while the overall mean was 1.06 ppb.	</p>

	<p>The within-assay precision of measurement of samples fortified at 0.1 ppb ranged 	between 10.0 - 23.2% (CV).  The between-assay precision was 32.5% (CV).</p>

	<p>The within-assay precision of measurement of samples fortified at 1.0 ppb ranged 	between 8.3 - 35.2% (CV). The between -assay precision was 17.6%.</p>

	</li>
	</ol>

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</li>
<li><h3 id="VI">ENVIROMENTAL HEALTH LABORATORIES (EHL) ACETANILIDE ASSAY</h3>

	<ol style="list-style-type:upper-alpha;">

	<li><h4>Background</h4>

	<p>A draft Standard Operating Procedure for an automated acetanilide immunoassay  	was received by the Partnership on December 7, 1994 from Jerry Thoma, President of 	Environmental Health Laboratories (EHL) located in South Bend, Indiana.  The EHL 	was founded in 1986 with the goal of providing drinking water analysis to both the 	public and private water suppliers as well as complex analytical testing for the  	commercial sector and state and federal contracting agencies.  The laboratory is 	certified in 33 states and EPA Regions to perform laboratory analyses for regulated 	parameters monitored by Public Water Supplies for compliance with the Safe Drinking  	Water Act.</p>

	<p>The EHL, in an exclusive agreement with Millipore, adapted the EnviroGard 	Acetanilide Plate Kit for an automated assay format.  The goal of the program was  	to provide an automated acetanilide immunoassay service to public and private water 	suppliers.  The first circular advertising the service was distributed January 12,  	1995 and more information is available by contacting the Client Services Department  	of the EHL at (800) 332-4345.</p>

	</li>
	<li><h4>Principle of the Assay</h4>

	<p>EHL assay relies on reagents supplied by Millipore Corporation and has been  	fully automated using a Bio-Tek Els1000 integrated microplate system.  In all other 	respects the assay resembles the typical competitive ELISA method that is the basis 	for the EnviroGard Acetanilide Plate Kit.  The Standard Operation Procedure for the 	EHL method 1294A contains information on the performance on the assay which will be  	summarized below.</p>

	</li>
	<li><h4>Reproducibility of Standard Curve</h4>

	<p>The EHL method relies on a four-point standard curve using the following  	acetochlor calibrators in reagent water: 0.0 ppb (negative control or blank), 0.1  	ppb, 0.25 ppb, 1.0 ppb and 5.0 ppb.  The standard curve is determined in the same  	way as the acetanilide plate kit by expressing %Bo versus log [acetochlor]. </p>

	<p>Ten observations of the standard curve, each in duplicate, were made on separate 	occasions between October 27, 1994 and December 1, 1994.  The reproducibility of 	the automated EHL method is acceptable and somewhat better than that observed for 	the manual plate kit assay (see Table 5.).</p>

	</li>
	<li><h4>Cross-Reactivity</h4>

	<p>The cross-reactivity results obtained with the EHL method are shown in Table 6  	and are in general agreement with the results reported in the acetanilide plate kit  	data insert.  The EHL analysis indicates that metolachlor is a more sensitive  	analyte than acetochlor with I50's of 0.25 and 0.71 ppb, respectively. </p>

	</li>
	<li><h4>Accuracy and Precision</h4>

	<p>The EHL assay was used to measure samples in triplicate on each of seventeen  	days, giving a total of fifty one individual determinations at each concentration.	</p>

	<p>At 0.1 ppb individual concentration measurements ranged from 0.081 - 0.126 ppb.  	Mean concentration measurements from daily determinations ranged from 0.088 - 0.119  	ppb while the overall mean was 0.103 ppb.</p>

	<p>At 1.0 ppb, individual concentrations ranged from 0.845 - 1.309 ppb. Mean 	concentrations from daily measurements ranged from 0.934 - 1.228 ppb, and the 	overall mean was 1.100 ppb.</p>

	<p>The within-assay precision of measurement of samples fortified at 0.1 ppb ranged 	between 0.9 - 13.1% (CV).  The mean between-assay precision was 7.9% (CV, n=17).	</p>

	<p>The within-assay precision of measurement for samples fortified at 1.0 ppb  	ranged from 1.7 - 17.0% (CV).  The mean between-assay precision was 7.0% (CV, 
	n=17).</p>

	</li>
	</ol>

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</li>
<li><h3 id="VII">PERFORMANCE TESTING OF ACETANILIDE IMMUNOASSAYS</h3>

	<ol style="list-style-type:upper-alpha;">

	<li><h4>Purpose</h4>

	<p>This performance test was conducted as part of the evaluation of the 
	EnviroGard<sup>TM</sup> Acetanilide Plate Kit and the automated acetanilide  	immunoassay test.  </p>

	</li>
	<li><h4>Experimental Design</h4>

	<p>Water samples were collected from 14 finished surface water sites in the greater 	metropolitan area surrounding St. Louis, Missouri and from one raw surface water 	site on the Missouri River near Defiance, Missouri.  In addition ground water from 	two Missouri wells, one well in Illinois and two wells in Wisconsin were also 	collected.  These samples were analyzed by GC/MS for the presence of corn 	herbicides and then fortified with acetochlor at either 0.1 or 1.0 ppb.  The  	samples were reanalyzed by GC/MS to confirm the fortification level and identical  	coded sets were sent to the Water Quality Laboratory (WQL) at Heidelberg College in  	Tiffin, Ohio and to the Environmental Health Laboratories (EHL) in South Bend,  	Indiana for analysis using an acetanilide immunoassay.</p>

	</li>
	<li><h4>Results and Discussion</h4>

	<p>No acetochlor or dimethenamid was detected in the surface and ground water  	samples analyzed by GC/MS. Low levels of atrazine and metolachlor which averaged  	0.225 ppb and 0.091 ppb, respectively, were found primarily in the surface water.   	Alachlor was detected at trace levels that were <0.05 ppb in all cases.</p>

	<p>Analysis of the unfortified water samples by immunoassay using the 
	EnviroGard<sup>TM</sup> Acetanilide Plate Kit (WQL, Heidelberg College) and an  	automated acetanilide assay (EHL) gave results which were in general agreement but  	differed significantly from the acetochlor analysis by GC/MS.  No acetochlor was  	present in these samples and yet the WQL analysis indicated an average of 0.19 ppb  	acetanilide with five samples <0.10 ppb and the EHL results gave an average  	level of 0.32 ppb acetanilide with 9 samples <0.10 ppb.  The discrepancy was due  	primarily to the presence of metolachlor  which has been shown to be a strong  	cross-reactant in this immunoassay.</p>

	<p>Following fortification at 0.10 ppb with acetochlor and reanalysis by GC/MS, the 	water samples were shown to contain 0.091 ppb acetochlor in good agreement with the  	theoretical concentration.  Immunoassay at WQL and EHL gave acetanilide results 	that averaged 0.26 and 0.37 ppb, respectively, once again reflecting the presence  	of other chloroacetanilides in addition to acetochlor.  Fortification at 1.0 ppb  	with acetochlor gave immunoassay values that were more precise and accurate and in 	better agreement with the GC/MS.  WQL and EHL measured an average of 1.20 ppb 	acetanilide compared with 1.09 ppb acetochlor obtained by GC/MS.  This agreement 	between ELISA and GC/MS at 1.0 ppb acetochlor was due to the fact that interfering 	acetanilides were minor compared with acetochlor and the values were within the 	range of the immunoassay where good precision for this analyte was obtained (I50).	</p>

	</li>
	</ol>

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<hr />
</li>
<li><h3 id="VIII">CONCLUSIONS</h3>

<p>The sensitive and specific ELISA immunoassays developed for alachlor and metolachlor  have been shown in some cases to cross-react with acetochlor. However, the I50's  (concentration of acetochlor required to achieve 50% inhibition) in these assays were  between 6.55 and 70 ppb (see Table 4) making them unsuitable for measuring acetochlor in  water at 0.1 to 2 ppb.</p>

<p>A new kit,the EnviroGard Acetanilide Plate Kit, in pre-commercial development by Millipore Corporation has been shown to have greater sensitivity for acetochlor (I50 =  0.7 ppb) and broad cross-reactivity for other acetanilides including metolachlor and alachlor with I50's of 0.26 ppb and 4.9 ppb, respectively.  These characteristics of the  new Millipore kit give it the potential for use as a screen for the acetanilide corn herbicides including alachlor, metolachlor, acetochlor and dimethenamid in water.  The  Millipore acetanilide kit has also been adapted for use in an automated format by the  Enviromental Health Laboratories in South Bend, Indiana and offered as a commercial  service to public and private water suppliers by this analytical laboratory.</p>

<p>With the support of the Acetochlor Registration Partnership, an evaluation of the EnviroGard Acetanilide Plate Kit was conducted by the Water Quality Laboratory of Heidelberg College in Tiffin, Ohio.  A similar evaluation of the automated format for the  Millipore acetanilide kit was provided by Enviromental Health Laboratories and presented  in their Standard Operating Procedure for EHL Method 1294A. </p>

<p>For this report four criteria were used to assess the characteristics and performance  of the two assays:</p>

<p>Reproducibility of calibration curve<br />
Cross-reactivity toward related analytes<br />
Accuracy and precision of replicate analyses of acetochlor<br />
fortified reagent water<br />
Accuracy of blind fortified surface and ground water samples</p>

<p>Since both assays are based on the same antibody and reagents from the same company  (Millipore Corporation), similar results would be expected. Differences are likely to  reflect the precision afforded by instrumented transfer and reproducible plate mixing,  washing and incubation provided by the automated assay.</p>

<p>Both assays gave reproducible standard curves.  The acetanilide plate kit utilizes a three point calibration curve with acetochlor calibrators at 0.1 ppb, 0.5 ppb and 5.0 ppb.  The EHL method relies on a four point calibration that includes calibrators of 0.1  ppb, 0.25 ppb, 1.0 ppb and 5.0 ppb.  Somewhat better precision was observed for the  automated EHL assay.</p>

<p>Cross-reactivity results for both methods were consistent with those reported in the acetanilide kit technical data sheet from Millipore although differences in the absolute values for the I50's were observed.  Dimethenamid was found to be a sensitive analyte for  the assay with comparable levels of cross-reactivity to that observed for metolachlor.   The assay was shown to be non responsive to atrazine and the major soil metabolites of  alachlor and metolachlor at levels up to 500 ppb.</p>

<p>Investigation of the accuracy and precision using acetochlor fortified in reagent  water indicated improved accuracy and between-assay precision evident at 1.0 ppb.</p>

<p>A final aspect of the immunoassay evaluation was an analysis of representative blind surface and ground water samples fortified with acetochlor at 0.1 and 1.0 ppb.  The surface and ground water samples were analyzed by GC/MS prior to fortification to measure  the presence of indigenous acetanilides and after fortification to verify the level of  acetochlor.  The results of the two ELISA immunoassay systems were found to be relatively  inaccurate for measuring acetochlor at 0.1 ppb but satisfactory at 1.0 ppb acetochlor.   The inaccuracy at low levels of acetochlor appeared to be due to the presence metolachlor  in the surface water samples. </p>

<p>In summary, the EnviroGard Acetanilide Plate Kit and the EHL automated acetanilide assays have sufficient accuracy and precision to serve as a screen for acetochlor in water at 1.0 ppb.  Use of the assay as a screen at 0.1 ppb was considered to be unadvisable due to low precision at this level and the fact that the lowest calibrator provided in the kit was 0.1 ppb acetochlor.  At 0.2 ppb, values were found to range between 0.13 ppb and 0.34 ppb and improved precision was obtained.  In addition, 0.2 ppb  was shown to be within the linear range of the standard curve.  These observations  suggest that 0.2 ppb would be a reasonable low level threshold for application of the  assay.</p>

<p>The acetanilide immunoassay of Millipore is not specific and does not differentiate among alachlor, acetochlor, metolachlor and dimethenamid but rather detects their presence to varying degrees.  Therefore, it is imperative that any positive detects obtained using this immunoassay as a plate kit or in an automated format be confirmed  with another analytical method, such as GC/MS, to establish the presence and level of  acetochlor in water.   </p>

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<hr />
</li>
<li><h3 id="IX">TABLES/FIGURES</h3>

<table class="table zebra">
<caption>Table 1.<br />
Cross-Reactivity of Metolachlor Ohmicron RaPID Assay Kit for Acetanilides
</caption>
<thead><tr>
<th scope="col">Compound</th>
<th scope="col">Least Detectable Dose<br />(I10 in ppb)</th> 
<th scope="col">Dose-Response Mid-Point<br />(I50 in ppb) </th>
</tr></thead>
<tbody>
<tr><th scope="row">Metolachlor</th>
<td> 0.05 </td> <td> 0.85 </td> </tr>
<tr><th scope="row">Acetochlor</th>
<td> 0.06 </td> <td> 6.55 </td> </tr>
<tr><th scope="row">Metalaxyl</th>
 <td> 0.06 </td> <td> 5.60 </td> </tr>
<tr><th scope="row">Butachlor</th>
<td> 0.26 </td> <td> 52.0 </td> </tr>
<tr><th scope="row">Propachlor</th>
<td> 1.00 </td> <td> 2500 </td> </tr>
<tr><th scope="row">Alachlor</th>
<td> 1.30 </td> <td> 84.0 </td> </tr>
</tbody>
</table>

<table class="table zebra">
<caption>Table 2.<br />
Cross-Reactivity of Alachlor Ohmicron RaPID Assay Kit for Acetanilides</caption>
<thead><tr>
<th scope="col">Compound</th>
<th scope="col">Least Detectable Dose<br />(I10 in ppb)</th>
<th scope="col">Dose-Response Mid-Point<br />(I50 in ppb)</th>
</tr></thead>
<tbody>
<tr><th scope="row">Alachlor</th>
<td> 0.05 </td> <td> ca. 1.0 </td> </tr>
<tr><th scope="row">Acetochlor</th>
<td> n/aa </td> <td> n/a </td> </tr>
<tr><th scope="row">Butachlor</th>
<td> 6.0 </td> <td> ca. 100 </td> </tr>
<tr><th scope="row">Metolachlor</th>
<td> 5.6 </td> <td> ca. 80 </td> </tr>
<tr><th scope="row">Propachlor</th>
<td> 6000 </td> <td> n/a </td> </tr>
<tr><th scope="row">Alachlor sulfonic acid</th>
<td> 0.03 </td> <td> n/a </td> </tr>
</tbody>
</table>

<p>a n/a = not available</p>

<table class="table zebra">
<caption>Table 3.<br />
Cross-Reactivity of EnviroGard Acetanilide Plate Kit for Acetanilides</caption>
<thead><tr>
<th scope="col">Compound</th>
<th scope="col">Least Detectable Dose<br />(I10 in ppb)</th>
<th scope="col">Dose-Response Mid-Point<br />(I50 in ppb)</th>
</tr></thead>
<tbody>
<tr><th scope="row">Metolachlor</th>
<td> 0.02 </td> <td> 0.25 </td> </tr>
<tr><th scope="row">Acetochlor</th>
<td> 0.02 </td> <td> 1.7 </td> </tr>
<tr><th scope="row">Metalaxyl</th>
<td> 0.02 </td> <td> 0.24 </td> </tr>
<tr><th scope="row">Butachlor</th>
<td> 0.13 </td> <td> 4.9 </td> </tr>
<tr><th scope="row">Propachlor</th>
<td> 0.18 </td> <td> 9.2 </td> </tr>
<tr><th scope="row">Alachlor</th>
<td> 0.55 </td> <td> 4.9 </td> </tr>
</tbody>
</table>

<table class="table zebra">
<caption>Table 4.<br />
Comparison of  Cross-Reactivity of Immunoassays for Corn Herbicides</caption>
<thead><tr>
<th rowspan="3" scope="col">Assay<br />(Company)</th>
<th colspan="2" scope="colgroup">Metolachlor</th>
<th colspan="2" scope="colgroup">Acetochlor</th>
<th colspan="2" scope="colgroup">Alachlor</th></tr>
<tr><th scope="col">I10 (ppb)</th><th scope="col">I50 (ppb)</th>
<th scope="col">I10 (ppb)</th><th scope="col">I50 (ppb)</th>
<th scope="col">I10 (ppb)</th><th scope="col">I50 (ppb)</th>
</tr></thead>
<tbody>
<tr><th scope="row">Metolachlor<br />(Ohmicron)</th>
<td>0.05</td><td>0.85</td><td>0.06</td><td>6.55</td><td>1.30</td><td>84.0</td></tr>
<tr><th scope="row">Metolachlor<br />(Idetek)</th>
<td>n/aa</td><td>1.3</td><td>n/a</td><td>70</td><td>n/a</td><td>100</td></tr>
<tr><th scope="row">Alachlor<br />(Idetek)</th>
<td>n/a</td><td>80</td><td>n/a</td><td>23</td><td>n/a</td><td>0.6</td></tr>
<tr><th scope="row">Alachlor<br />(Millipore)</th>
<td>0.6</td><td>40</td><td>n/a</td><td>n/a</td><td>0.15</td><td>5.0</td></tr>
<tr><th scope="row">Alachlor<br />(Ohmicron)</th>
<td>5.6</td><td>ca.80</td><td>n/a</td><td>n/a</td><td>0.05</td><td>1.0</td></tr>
<tr><th scope="row">Acetanilide<br />(Millipore)</th>
<td>0.02</td><td>0.26</td><td>0.02</td><td>1.7</td><td>0.55</td><td>4.9</td></tr>
</tbody>
</table>

<p>a n/a = not available</p>
          
<table class="table zebra">
<caption>Table 5.<br />
Cross-Reactivity of EnviroGard Acetanilide Plate Kit (WQL)</caption>
<thead><tr>
<th scope="col">Compound</th>
<th scope="col">Least Detectable Dose<br />(I10 in ppb)</th>
<th scope="col">Dose-Response Mid-Point<br />(I50 in ppb)</th>
</tr></thead>
<tbody>
<tr><th scope="row">Acetochlor</th><td> ca. 0.1 </td> <td> 1.0 </td> </tr>
<tr><th scope="row">Alachlor</th><td> 2.0 </td> <td> 5.0 </td> </tr>
<tr><th scope="row">Metolachlor</th><td> 0.2 </td> <td> 1.5 </td> </tr>
<tr><th scope="row">Dimethenamid</th><td> 0.1 </td> <td> 0.5 </td> </tr>
<tr><th scope="row">Atrazine</th><td> n/da </td> <td> > 500 </td> </tr>
<tr><th scope="row">Alachlor Sulfonic Acid</th><td> n/d </td> <td> > 500 </td> </tr>
<tr><th scope="row">Alachlor Oxanilic Acid</th><td> n/d </td> <td> > 500 </td> </tr>
<tr><th scope="row">Alachlor Sulfinyl Acetic Acid</th><td>n/d</td><td>> 500</td></tr> <tr><th scope="row">Acetochlor Oxanilic Acid</th><td> n/d </td> <td> > 500 </td> </tr>
<tr><th scope="row">Acetochlor Sulfonic Acid</th><td> n/d </td> <td> > 500 </td> </tr>
<tr><th scope="row">Acetochlor Sulfinyl Acetic Acid</th><td>n/d</td><td>> 500</td>
</tr>
</tbody>
</table>

<p>a n/d = not determined</p>

<table class="table zebra">
<caption>Table 6.<br />Cross-Reactivity of Automated Acetanilide Assay (EHL)</caption>
<thead><tr>
<th scope="col">Compound</th>
<th scope="col">Least Detectable Dose<br />(I10 in ppb)</th>
<th scope="col">Dose-Response Mid-Point<br />(I50 in ppb)</th>
</tr></thead>
<tbody>
<tr><th scope="row">Metolachlor</th><td> 0.04 </td> <td> 0.25 </td> </tr>
<tr><th scope="row">Acetochlor</th><td> 0.08 </td> <td> 0.71 </td> </tr>
<tr><th scope="row">Metalaxyl</th><td> 0.02 </td> <td> 0.13 </td> </tr>
<tr><th scope="row">Butachlor</th><td> 0.81 </td> <td> 8.9 </td> </tr>
<tr><th scope="row">Alachlor</th><td> 0.52 </td> <td> 3.9 </td> </tr>
</tbody>
</table>

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<hr />
</li>
<li><h3 id="X">REFERENCES</h3>

	<ol>

	<li><p> Feng, P. C. C.; Wratten, S. J.; Logusch, E. W.; Horton, S. R.; Sharp, C. 	R., "Immunoassay Detection Methods for Alachlor In Immunochemical Methods for 	Environmental Analysis", Van Emon, J. M. and Mumma, R. O. Eds. American 	Chemical Society Symposium Series 442, Washington, D. C. 1990, pp 180-192.</p></li>

	<li><p> Schlaeppi, J. M.; Moser, H.; Ramsteiner, K., "Determination of  	Metolachlor by Competitive Enzyme Immunoassay Using a Specific Monoclonal  	Antibody", J. Agric. Food Chem. 1991, 39, 1533-1536.</p></li>

	<li><p> Harrison, R. O.; Braun, A. L.; Gee, S. J.; O'Brien, D. J.; Hammock, B. D.,  	"Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Direct 	Analysis of Molinate (Ordam<sup>TM</sup>) in Rice Field Water", Food and  	Agric. Immunology 1989, 1, 37-51.</p></li>

	<li><p> Baker, D. B.; Bushway, R. J.; Adams, S. A.; Macomber, C., "Immunoassay 	Screens for Alachlor in Rural Wells:  False Positives and an Alachlor Soil 	Metabolite", Environ. Sci. Technol. 1993, 27, 562-564.</p></li>

	<li><p> Aga, D. S.; Thurman, E. M.; Pomes, M. L., "Determination of Alachlor  	and Its Sulfonic Acid Metabolite in Water by Solid-Phase Extraction and 
	Enzyme-Linked Immunosorbent Assay", Anal. Chem. 1994, 66, 1495-1499.</p></li>

	<li><p> Feng, P. C. C.; Wratten, S. J.; Horton, S. R.; Sharp, C. R.; Logusch, E. 	W., "Development of an Enzyme-Linked Immunosorbent Assay for Alachlor and Its 	Application to the Analysis of Environmental Water Samples", J. Agric. Food  	Chem. 1990, 38, 159-163.</p></li>

	<li><p> Thurman, E. M.; Meyer, M.; Pomes, M.; Perry, C. A.; Schwab, A. P.,  	"Enzyme-Linked Immunosorbent Assay Compared with Gas Chromatography/Mass 	Spectrometry for the Determination of Triazine Herbicides in Water", Anal.  	Chem. 1990, 62, 2043-2048.</p></li>

	<li><p> Sherry, J. P., "Environmental Chemistry:  The Immunoassay Option. In  	CRC Critical Reviews in Analytical Chemistry"; Coleman, D. M.; Coleman, P. B.,  	Eds.; CRC Press, Inc., Boca Raton, FL; 1992; Vol. 23, p. 276.</p></li>

	<li><p> Lawruk, T. S.; Lachman, C. E.; Jourdan, S. W.; Fleeker, J. R.; Herzog, D. 	P.; Rubio, R. M., "Determination of Metolachlor in Water and Soil by a Rapid 	Magnetic Particle-Based ELISA", J. Agric. Food Chem. 1993, 41, 1426-1431.</p>	</li>

	<li><p> Metolachlor 1.0 Immunoassay Kit, Specification Sheet, Idetek/Quantix 	Systems, 1995, and Supporting Data from Idetek.</p></li>

	<li><p> Alachlor 1.0 Immunoassay Kit, Specification Sheet, Idetek/Quantix Systems, 	1995, and Supporting Data from Idetek.  </p></li>

	<li><p> EnviroGard Alachlor Plate Kit (Catalogue No. ENVR P00 04), Technical Data 	Kit Insert, Millipore Corporation, 1994. </p></li>

	<li><p> Lawruk, T. S.; Hottenstein, C. S.; Herzog, D. P.; Rubio, F. M.; 	"Quantification of Alachlor in Water by a Novel Magnetic Particle-Based  	ELISA", Bull. Environ. Contam. Toxicol. 1992, 48, 643-650.</p></li>

	<li><p> EnviroGard Acetanilide Plate Kit (Catalogue No. SD3P228S4), Technical Data 	Kit Insert, Millipore Corporation, 1995 (See Appendix A).</p></li>

	</ol>

</li>
</ol>
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