RECOMMENDED ANALYTICAL TECHNIQUES AND QUALITY ASSURANCE/QUALITY CONTROL GUIDELINES FOR THE MEASUREMENT OF ORGANIC AND INORGANIC ANALYTES IN MARINE SEDIMENT AND TISSUE SAMPLES DRAFT Prepared by U.S. Environmental Protection Agency Environmental Research Laboratory Narragansett, Rhode Island March 1993 ------- TABLE OF CONTENTS I. INTRODUCTION 1 II. CHEMICAL ANALYSIS OF MARINE SEDIMENT AND TISSUE SAMPLES . 1 III. QUALITY ASSURANCE/QUALITY CONTROL GUIDELINES 7 1.0 General QA/QC Requirements 8 1.1 Initial Demonstration of Capability 9 Initial Calibration 9 Calculation of Method of Detection Limits 13 Blind Analysis of Accuracy-Based Material 15 1.2 On-going Demonstration of Capability 15 Laboratory Participation in Intercomparison Exercises 16 Continuing Calibration Checks 17 Routine Analysis of Reference Materials 18 Laboratory Reagent Blank 20 Laboratory Fortified Sample Matrix 21 Duplicates 22 Internal Standards 23 Internal Injection Standards 24 IV. REFERERENCES 25 APPENDIX 1 ANALYTICAL METHODS 27 1.0 Organic Analyses 27 Tissue Extraction 29 Sediment Extraction 35 Extract Cleanup 41 GC Analysis of Extracts for PCBs and Chlorinated Pesticides . 46 GC/MS Analysis of Extracts for PAHs 52 2.0 Inorganic Analyses 57 Tissue Digestion 58 Total Digestion of Sediments 66 Ultrasonic Digestion of Sediments 73 Instrumental Analysis of Metals 79 This document may not reflect the official views and policies of the U.S. Environmental Protection Agency, and mention of trade names or commercial products does not constitute endorsement or recommendation for use. ------- INTRODUCTION This document is intended to provide guidance on the analysis of selected organic and inorganic analytes in marine sediments and tissues. Its purpose is to suggest analytical methods for measuring contaminants in the low parts-per-billion concentration range. The analytical techniques contained herein are those employed by the U.S. EPA Environmental Research Laboratory in Narragansett, R.I. for the analysis of marine environmental samples. They are intended, however, to serve only as examples and are not being suggested as EPA standard methods. These methods have been successfully employed on marine samples to achieve these detection limits. Included with the analytical methods are quality assurance/quality control (QA/QC) guidelines. The overall objective of the document is therefore to ensure that data produced under these guidelines will be of the highest quality, have detection limits necessary for trace level marine samples, and be comparable to data produced by other laboratories employing similar methods. CHEMICAL ANALYSIS OF MARINE SEDIMENT AND TISSUE SAMPLES No procedures have been officially approved by the regulatory agencies for low-level (i.e., low parts-per-billion) analysis of organic and inorganic contaminants in estuarine sediments and tissue samples. This document includes methods and guidance that have been used at ERLN in work related to the Environmental Monitoring and Assessment Program [EMAP] (Valente and Schoenherr 1991) and are similar to those that have been used for NOAA's National Status and Trends Program (Lauenstein in prep., Kralin c.i til. 1988). The EMAP and NS&T programs have I ------- chosen not to specifically require that particular analytical methods always be followed, but rather that participating laboratories demonstrate proficiency through the regular analysis of Standard or Certified Reference Materials' (SRMs or CRMs) or similar types of accuracy-based materials. We support this approach, and have therefore written this document to reflect this type of "performance based" program. Table 1 provides a list of analytes which have been successfully analyzed using the methods provided in Appendix 1. Additional analytes may also be successfully analyzed using these methods, although these methods have been optimized to enhance recovery of these specific analytes. Other analytes, such as heptachlor and the two- and three-ringed. PAHs, are not included in Table 1 because the protocols provided are not suitable for the analysis of these compounds. (Treatment of sediment extracts with copper to remove sulfur may reduce the recovery of heptachlor, and the lower molecular weight PAHs may elute in both fractions from the silica gel column and complicate quantitation.) More polar organic compounds may not be effectively extracted from the sample matrix or may not elute from the silica gel column using the solvent schemes provided. Similarly, additional major or trace elements may be analyzed using these methods, although the methods have been employed only for the elements presented in Table 1. Recoveries of other elements may be lower if a total digestion method is not used. 1 Certified Reference Materials are samples containing precise concentrations of chemicals, accurately determined by a variety of technically valid procedures and accompanied by a certificate or other documentation issued by a certifying body (e.g., agencies such as the National Research Council of Canada (NRC), U.S. EPA, U.S. Geological Survey, etc.). Standard Reference Materials (SRMs) are CRMs issued by the National Institute of Standards and Technology (NIST), formerly the National Bureau of Standards (NBS). A useful catalogue of marine science reference materials has been compiled by Cantillo (1990). 2 ------- TABLE I. ANALYTES MEASURED IN MARINE SAMPLES BY ERLN PAHs Anthracene Benz(a)antliracene Sum of benzofluoranthenes Benzo(g,h,i)perylene Benzo(a)pyrene Benzo(e)pyrene Chrysene Dibenz(a,h)anthracene Fluoranthene Fluorene Indeno(l,2,3-cd)pyrene Phenanthrene C, alkyl phenanthrenes + anthracenes Q alkyl phenanthrenes + anthracenes Cj alkyl phenanthrenes + anthracenes C4 alkyl phenanthrenes + anthracenes Perylene Pyrene PCB Congeners 8 18 28 52 44 66 101 118 153 105 138 187 128 180 170 195 206 209 DDT and its metabolites p.p'-DDD p.p'-DDE p,p'-DDT Chlorinated pesticides other than DDT Gamma-chlordane Alpha-chlordane Trans-nonachlor Heptachlor (tissues only) Heptachlor epoxide Hexachlorobenzene Lindane (gamma-BHC) Mirex Major Elements Aluminum Iron Manganese Trace Elements Antimony Arsenic Cadmium Chromium Copper Lead Mercury Nickel Silver '/,i nc ------- Several general issues are discussed in the following paragraphs, including, but not limited to, the separation of PCBs from chlorinated hydrocarbon pesticides, second column confirmation of PCBs and pesticides, GC column selection, and degradation of analytes in the GC injection port. Calculation of Total PCBs We recommend that PCBs be quantitated as discrete congeners. The methods detailed in Appendix 1 are specifically written to allow for optimal quantitation of the eighteen congeners listed in Table 1. It is possible, although not recommended, to derive an estimate of total PCBs from the sum of the individual congeners. Regressions were reported in two NOAA National Status and Trends Program documents (NOAA 1989, 1991) which convert the sum of congeners to a total PCB concentration. The conversions were based on regressions of over 100 measurements of PCB chlorination levels and PCB congeners conducted by two separate laboratories. Use of the equations would provide a total PCB concentration equivalent to that based on summing the concentrations at each level of chlorination. As the two equations are not the same, we stress the point that the use of them will provide only an estimate of total PCB concentration. An attempt was also made to relate total PCBs measured as Aroclor formulations (following EPA Test Method 608) to total PCBs measured as the sum of individual congeners (Battelle 1990). This attempt resulted in PCB levels that varied by up to a factor of three between the two methods of measurement. Separation of PCBs from chlorinated hydrocarbon pesticides We recommend that the more polar pesticides such as the chlordancs, trans-nonachlor and 1 ------- heptachlor epoxide, as well as DDI) and DDT be separated from the PCBs and analyzed separately. This recommendation is based on our experiences with samples heavily impacted with PCBs, which can overwhelm the pesticides if they have not been separated into different fractions. An overestimation of the pesticide concentrations, or the reporting of false positives could result from this situation. Several methods have been successfully employed to separate PCBs from the more polar pesticides, including HPLC, GPC and silica gel adsorption chromatography. NIST uses liquid chromatography to separate the PCBs from the polar pesticides in their sediment and mussel reference materials (Wise et al. 1991, Schantz et al. 1990) and analyzes each fraction separately. The protocols included as part of this document include a method utilizing silica gel adsorption chromatography to separate PCBs and pesticides. GC column selection The industry standard for the analysis of the semivolatile organic analytes included in Table 1 is a fused silica capillary column with a 95% Dimethyl-5% diphenyl polysiloxane coating (DB-5 or equivalent). Available in several combinations of length, diameter and film thicknesses, this nonpolar column offers good resolution, low bleed and excellent thermal stability. Considerable data, such as retention indices, exist for these semivolatile compounds as analyzed on columns with this phase which can be used for comparison purposes. NIST uses 60 m DB-5 columns in their GCs to obtain the concentrations of these analytes in their standard reference materials, although the use of a 30 m DB-5 column is common as well. A 30 m DB-5 column is recommended for use in quantitating all the semivolatile organic analytes in Table 1. S ------- Analyte confirmation Although the use of a single DB-5 (or equivalent) column for the quantitation of PCBs and chlorinated pesticides provides reliable and comparable results, Durell and Sauer (1990) and others have suggested that the lack of confirmation may result in the incorrect identification of these compounds. Vargo and Mosesman (1992) suggest that the use of a confirmation technique can improve the reliability of pesticide analysis. To avoid the reporting of false positives, confirmation may be made using a second, more polar phase column or, if the concentrations are sufficiently high, by GC/MS. A commonly used confirmation column is coated with 50% Methyl-50% phenyl polysiloxane (DB-17 or equivalent). Due to the complexity of PCB congener analysis (up to 209 congeners may be present in environmental samples), and the availabilty of supporting information such as relative retention indices for many of the congeners on commonly used columns, dual column confirmation of PCBs is not recommended. Degradation of analvtes in the GC injection port As a result of injecting extracts containing these analytes into a hot GC injection port, some of the thermally labile compounds may be degraded. These compounds include the pesticides p,p'-DDT and endrin. Lowering the injection port temperature on the GC may eliminate or minimize the degradation of these compounds, but may also result in poor response of other compounds. In order to monitor the degradation of these compounds, it is necessary'to track their response using the calibration standards or a solution prepared expressly for this purpose. If response is seen to dramatically decrease, corrective action in the form of injection port cleaning, replacement of the injection port liner, packing or changing the glass- or fused silica wool packing in the liner may be required to restore response. 6 ------- QUALITY ASSURANCE/QUALITY CONTROL GUIDELINES The following sections detail the Quality Assurance/Quality Control (QA/QQ procedures recommended to ensure that only the highest-quality data are produced. The QA/QC guidelines presented draw on components from several programs, including, but not limited to, the U.S. EPA Environmental Monitoring and Assessment Program, the National Status and Trends Program and the Puget Sound Estuary Program. As specified earlier, the guidelines presented here reflect a "performance based" approach. The first phase of this "performance based" program is an Initial Demonstration of Capability or Performance Evaluation. Prior to the analysis of samples, the laboratory must demonstrate proficiency in several ways: the lab must provide written protocols for the analytical methods to be employed for sample analysis; method detection limits for each analyte must be calculated; an initial calibration curve must be established for all analytes; and generally, acceptable performance on a known or Blind accuracy-based material must be shown. Following a successful first phase, the laboratory must demonstrate its continued capabilities in several ways: participation in refereed intercomparison exercises; repeated analysis of certified reference materials; calibration checks; and analysis of laboratory reagent blanks and fortified samples. These steps are detailed in the following sections and are summarized in Table 2. The sections are arranged to mirror the elements in Table 2 to allow the user to more easily cross-reference the specific details with the general topics in the table. 7 ------- 1.0 General OA/OC Requirements The guidance provided in the following sections is based largely on the protocols developed for the Puget Sound Estuary Program (U.S. EPA 1989) and the EMAP Program; it is applicable to low parts-per-billion analyses of both sediment and tissue samples unless otherwise noted. The QA/QC requirements are intended to provide a common foundation for each laboratory's protocols; the resultant QA/QC data will enable an assessment of the comparability of results generated by different laboratories and different analytical procedures. It should be noted that the QA/QC requirements specified in this plan represent the minimum requirements for any given analytical method. Additional requirements which are method-specific should always be followed, as long as the minimum requirements presented in this document have been met. It is imperative that the results for the various QA/QC samples be reviewed by laboratory personnel immediately following the analysis of each sample batch. These results then should be used to determine when warning and control limits have been exceeded and corrective actions must be taken, before processing a subsequent sample batch. Warning limits are numerical criteria that serve as flags to data reviewers and data users. When a warning limit is exceeded, the laboratory is not obligated to halt analyses, but the reported data may be qualified during subsequent QA/QC review. Control limits are numerical data criteria that, when exceeded, require specific corrective action by the laboratory before the analyses may proceed. Warning and control limits and recommended frequency of analysis for each QA/QC element or sample type arc summarized in Table 2. Descriptions of the use, frequency of analysis, type of X ------- information obtained, and corrective actions for eacli of these QA/QC sample types or elements are provided in the following sections. l.l Initial Demonstration of Capability A laboratory's initial demonstration of capability should include the following: written protocols that will be followed for sample analysis; the calculation of method detection limits for each analyte; the establishment of an initial calibration curve for each analyte; and if possible, documentation of acceptable performance on a "performance evaluation" sample. These components are described in the following paragraphs. Initial Calibration Equipment must be calibrated before any samples are analyzed, after each major equipment disruption, and whenever on-going calibration checks do not meet recommended control limit criteria (Table 2). All calibration standards should be traceable to a recognized organization for the preparation of QA/QC materials (e.g., National Institute of Standards and Technology, U.S. Environmental Protection Agency, etc.). Calibration curves must be established for each element and batch analysis from a calibration blank and a minimum of three analytical standards of increasing concentration, covering the range of expected sample concentrations. The calibration curve must be established prior to the analysis of samples. Only data within the demonstrated working calibration range may be reported by the laboratory; samples outside this range should be diluted or concentrated, as appropriate, and reanalyzed. ------- TABLE 2. KEY ELEMENTS FOR QUALITY CONTROL OF CHEMICAL ANALYSES (SEE TEXT FOR DETAILED EXPLANATIONS). Element or Sample Type Warning Limit Criteria Control Limit Criteria Frequency 1. Initial Demonstration of Capability (Prior to Analysis of Samples): - Initial Calibration Calculation of Method Detection Limits Blind Analysis of Accuracy-Based Material NA NA Must be equal to or less than target values (see Table 3) NA NA Initial and then prior to analyzing each batch of samples At least once each project Initial 2. On-going Demonstration of Capability: - Blind Analysis of Laboratory Inter- comparison Exercise Samples NA NA Regular intervals throughout the project - Continuing Calibration Checks using Calibration Standard Solutions NA should be within ±15% of initial calibration on average for all analytes, not to exceed ±25% for any one analyte At a minimum, middle and end of each sample batch Continued on following page 10 ------- ^^LE 2 (Continued) Element or Warning Limit ' Control Limit Sample Type Criteria Criteria Frequency ' Analysis of Certified Reference Material (CRM) or Laboratory Control Material (LCM): One with each batch of samples Precision (see NOTE 1): NA Value obtained for each analyte should be within 3s control chart limits Value plotted on control chart after each analysis of the CRM Relative Accuracy (see NOTE 2): PAHs Lab's value should be within ±25% of true value on average for all analytes; not to exceed ±30% of true value for *" more than 30% of individual analytes Lab's value should be within ±30% of true value on average for all analytes; not to exceed ±35% of true value for more than 30% of individual analytes PCBs/pesticides inorganic elements same as above Lab should be within ±15% of true value for each analyte same as above Lab should be within ±20% of true value for each analyte PTE 1: The use of control charts to monitor precision for each analyte of interest should follow generally accepted ractices (e.g., Taylor 1987). Upper and lower control limits, based on three standard deviations (3s) of the mean, lould be updated at regular intervals. PTE 2: "True" values in CRMs may be either "certified" or "non-certified" (it is recognized that absolute accuracy in only be assessed using certified values, hence the term relative accuracy). Relative accuracy is computed by ^mparing the laboratory's value for each analyte against either end of the range of values (i.e., 95% confidence mits) reported by the certifying agency. The laboratory's value must be within ±35% of either the upper or lower 5% confidence interval value. Accuracy control limit criteria only apply for analytes having CRM concentrations 10 times the laboratory's MDL. Continued on following page I I ------- 3LE 2 (Continued) Element or Sample Type Warning Limit Criteria Control Limit Criteria Frequency Laboratory Reagent Blank Analysts should use best professional judgement if analytes are detected at <3 times the MDL No analyte should be detected at >3 times the MDL . One with each batch of samples Laboratory Fortified Sample Matrix (Matrix Spike) NA Recovery should be within the range 50% to 120% for at least 80% of the analytes At least 5% of total number of samples NOTE: Samples to be spiked should be chosen at random; matrix spike solutions should contain all the analytes of interest. The final spiked concentration of each analyte in the sample should be at least 10 times the calculated MDL. Laboratory Duplicate or Sample Matrix Duplicate (Matrix Spike Duplicate) NA RPD1 must be £ 30 for each Same as analyte matrix spike Internal Standards (Surrogates) NA Recovery must be within the range 30% to 150% Each sample Internal Injection Standards Lab develops its own Each sample ' . . ' • RPD = Relative percent difference between matrix spike and matrix spike duplicate results (see section 1.2 Laboratory Duplicates for equation) 12 ------- Calculation of Method Detection Limits Analytical chemists have coined a variety of terms to define "limits" of detectability; definitions for some of the more commonly-used terms are provided in Keith et al. (1983) and in Keith (1991). In this document, the Method Detection Limit (MDL) will be used to define the analytical limit of detectability. The MDL represents a quantitative estimate of low-level response detected at the maximum sensitivity of a method. The Code of Federal Regulations (40 CFR Part 136) gives the following rigorous definition: "the MDL is the minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero and is determined from analysis of a sample in a given matrix containing the analyte." Confidence in the apparent analyte concentration increases as the analyte signal increases above the MDL. Each analytical laboratory should calculate and report an MDL for each analyte of interest in each matrix of interest (sediment or tissue) prior to the analysis of samples. Each laboratory should follow the procedure specified in 40 CFR Part 136 (Federal Register, Oct. 28, 1984) to calculate MDLs for each analytical method employed. The matrix and the amount of sample (i.e., dry weight of sediment or tissue) used in calculating the MDL should match as closely Us possible the matrix of the actual samples and the amount of sample typically used. In order to ensure comparability of results among different laboratories, MDL target values have been recommended (Table 3). The initial MDLs reported by each laboratory should be equal to or less than these specified target values before the analysis of samples may proceed. It is n ------- TABLE 3. TARGET METHOD DETECTION LIMITS INORGANICS (NOTE: concentrations in jug/g (ppm), dry weight) Tissue Sediments Aluminum 10.0 1500 Antimony a 0.2 Arsenic 2.0 1.5 Cadmium 0.2 0.05 Chromium 0.1 1.0 Copper 5.0 1.0 Iron 50.0 500.0 Lead 0.1 1.0 Manganese a 1.0 Mercury 0.01 0.01 Nickel 0.5 1.0 Silver 0.01 0.01 Tin 0.05 0.1 Zinc 50.0 2.0 ORGANICS (NOTE: concentrations in ng/g (ppb), dry weight) Tissue Sediments PAHs 50.0 10.0 PCB congeners 2.0 1.0 Pesticides 2.0 1.0 a = not measured in tissue 14 ------- recognized that the initial MDL is a statistically-derived, empirical value that subsequently may vary in actual samples as a function of sample matrix, volume, percent moisture, etc. Eachlaboratory must periodically (i.e., at least once each year) re-evaluate its MDLs for the analytical methods used and the sample matrices typically encountered. Blind Analysis of Accuracy-Based Material Whenever possible, a representative sample matrix which is uncompromised, homogeneous and contains the analytes of interest should be analyzed blind by each laboratory performing the analyses. The purpose of analyzing the sample(s) "blind" (where the laboratory does not know the concentrations of the analytes) is to assess the accuracy of the laboratory's performance prior to the analysis of actual field samples. Typically, an SRM or CRM is used for this "blind" sample. A laboratory's performance is then determined by comparing the analytical results to the certified concentrations. Acceptable performance is generally indicated by concentrations that are ±30% for organic analytes and ±20% for inorganic analytes of the known concentration of the analytes in the sample. These criteria are recommended only for analyte concentrations that are equal to or greater than 10 times the MDL as established by the laboratory. Failure of the laboratory to meet these criteria should result in reanalysis of the sample until acceptable performance is obtained. 1.2 On-going Demonstration of Capability In order to ensure that the laboratory is consistently producing comparable high-quality 15 ------- data during a project, the following Quality Control elements are suggested: participation in intercomparison exercises; continuing calibration checks; analysis of reference materials, reagent blanks, matrix spikes and duplicate samples; monitoring internal and injection internal standard performance. Criteria for warning and control limits are presented in Table 2, and discussion related to each element is presented in the following sections. Laboratory Participation in Intercomparison Exercises The laboratory intercomparison exercises previously referred to are sponsored jointly by the NOAA National Status and Trends Program and the EMAP-Near Coastal Program to evaluate both the individual and collective performance of their participating analytical laboratories. Participation by other laboratories in these exercises is limited at present, however, plans are being formulated to allow additional laboratories to join in the future. Inquiries should be addressed to the NS&T Program QA Manager (Dr. Adriana Cantillo, NS&T (N/ORCA-21), 6001 Executive Blvd., WSC-1, Rm 312, Rockville, MD 20852). It is highly recommended that each laboratory include some type of intercomparison exercise in their QA/QC program. Typically, three or four different NS&T/EMAP-NC exercises are conducted over the course of a year; each exercise involves the blind analysis of different representative matrices (e.g., standard solutions, sediment or tissue samples) distributed to all laboratories in common by either NIST or NRGC (under contract to NS&T/EMAP-NC). Following the initial demonstration of capability, each laboratory is required to participate in these on-going intercomparison exercises as a continuing check on performance and intercomparability. Laboratories which fail to achieve acceptable performance in any iiuercomparison exercise must provide an explanation and may be required 16 ------- to undertake corrective actions, as appropriate. Continuing Calibration Checks The initial instrument calibration is checked through the analysis of a calibration standard. If possible, the calibration standard solution used for the calibration check should be obtained from a different source than the initial calibration standards, so that it can provide an independent check both on the calibration and the accuracy of the standard solutions. Analysis of the calibration standard should occur at the beginning of a sample set (i.e., batch), at least once every 10 samples, and after the last sample in the batch. If the control limit for analysis of the calibration standard is not met (Table 2), the initial calibration will have to be repeated. If possible, the samples analyzed before the calibration check that failed the control limit criteria should be reanalyzed following the re-calibration. The laboratory should begin by reanalyzing the last sample analyzed before the calibration standard which failed. If the relative percent difference (RPD) between the results of this reanalysis and the original analysis exceeds 30 percent, the instrument is assumed to have been out of control during the original analysis. If possible, reanalysis of samples should progress in reverse order until it is determined that there is less than 30 RPD between initial and reanalysis results. If It is not possible or feasible to perform reanalysis of samples, all earlier data (i.e., since the last successful calibration control check) is considered suspect. It is also possible that the majority of analyies will meet the calibration criteria, while only a few may not. In this case, the best professional judgement of the analyst may he required lo assess the acceptabilty of the 17 ------- calibration. In this case, the laboratory should include a written narrative with the data describing the situation. Routine Analysis of Reference Materials Reference Materials (SRMs or CRMs) generally are considered the most useful QC samples for assessing the accuracy of a given analysis (i.e., the closeness of a measurement to the "true" value). Because Certified Reference Materials have "certified" concentrations of the analytes of interest, as determined through replicate analyses using two independent measurement techniques, these materials can be used to assess accuracy. Thus, routine analysis of reference materials represents a particularly vital aspect of the "performance-based" QA philosophy. A Laboratory Control Material (LCM) is similar to a Certified Reference Material in that it is a homogeneous matrix which closely matches the samples being analyzed. A "true" LCM is one which is prepared (i.e., collected, homogenized and stored in a stable condition) strictly for use in-house by a single laboratory. Alternately, the material may be prepared by a central laboratory and distributed to others (so-called regional or program control materials). Unlike CRMs, concentrations of the analytes of interest in LCMs are not certified but are based upon a statistically-valid number of replicate analyses by one or several laboratories. In practice, this material can be used to assess the precision (i.e., consistency) of a single laboratory, as well as to determine the degree of comparability among different laboratories. If available, LCMs may be preferred for routine (i.e., day-to-day) analysis because CRMs are relatively expensive. However, CRMs still must be analyzed at regular intervals (e.g., monthly or quarterly) to provide a check on accuracy. IX ------- One SRM, CRM or LCM should be analyzed along with each batch of 20 or fewer samples (Table 2). The SRM, CRM or LCM concentrations of the target analytes should be known to the analyst(s) and should be used to provide an immediate check on accuracy for each batch of samples before proceeding with a subsequent batch. If values are outside the control limits (Table 2), the data for the entire batch of samples is considered suspect. Calculations and instruments should be checked; the control material may have to be reanalyzed (i.e., reinjected) to confirm the results. If the control limits are still exceeded in the repeat analysis, the laboratory is required to determine the source(s) of the problem and repeat the analysis of that batch of samples until control limits are met, before continuing with further sample processing. The results of the CRM or LCM analysis should not be used by the laboratory to "correct" the data for a given sample batch. Results of control material analyses also should be recorded on control charts to monitor laboratory precision from batch to batch. This is particularly important in situations where certified concentrations are not available for all the analytes of interest in a particular SRM or CRM. In such instances, each laboratory should be able to demonstrate an acceptable level of batch-to-batch consistency for a given reference material, in accordance with commonly- employed control charting techniques (i.e., wildly fluctuating results are not acceptable). The "absolute" accuracy of an analytical method can be assessed using CRMs only when certified values are provided for the analytes of interest. However, the concentrations of many analytes of interest are provided only as non-certified values in some of the more commonly-used CRMs. Therefore, control limit criteria are based on "relative accuracy", which is evaluated for 19 ------- each analysis of the CRM or LCM by comparison of a given laboratory's values relative to the "true" or "accepted" values in the LCM or CRM. In the case of CRMs, this includes both certified and noncertified values and encompasses the 95% confidence interval for each value as described in Table 2. Accuracy control limit criteria have been established both for individual compounds and combined groups of compounds (Table 2). There are two combined groups of compounds for the purpose of evaluating relative accuracy for organic analyses: PAHs and PCBs/pesticides. The laboratory's value should be within ±30% of the true value on average for each combined group of organic compounds, and the laboratory's value should be within ±35% of either the upper or lower 95% confidence limit for at least 70% of the compounds in each group. For inorganic analyses, the laboratory's value should be within ±20% of either the upper or lower 95% confidence limit for each analyte of interest in the CRM. Due to the inherent variability in analyses near the method detection limit, control limit criteria for relative accuracy only apply to analytes having CRM true values which are >10 times the MDL established by the laboratory. Laboratory Reagent Blank Laboratory reagent blanks (commonly called method blanks) are used to assess contamination during all stages of sample preparation and analysis. For both organic and inorganic analyses, one reagent blank should be run in every sample batch (minimum frequency of one per 20 samples). Warning and control limits for blanks (Table 2) are based on the laboratory's method detection limits as documented prior to the analysis of samples (see Section ------- 1.1). A reagent blank concentration between the MDL and 3 times the MDL should serve as a warning limit requiring further investigation based on the best professional judgement of the analyst(s). A reagent blank concentration equal to or greater than 3 times the MDL requires definitive corrective action to identify and eliminate the source(s) of contamination. Laboratory Fortified Sample Matrix A laboratory fortified sample matrix (commonly called a matrix spike) should be used to evaluate the effect of the sample matrix on the recovery of the compound(s) of interest. A minimum of 5% of the total number of samples submitted to the laboratory in a given project should be selected at random for analysis as laboratory fortified samples. The compounds used to fortify samples should include all the analyte of interest These compounds should be added at 5 to 10 times their MDLs as previously calculated by the laboratory (see Section 1.1). The recovery data for each fortified compound, which should be reported along with the rest of the data for each sample, ultimately will provide additional information on the performance of the methods. If the percent recovery for any analyte is less than the recommended warning limit of 50 percent, the chromatograms and/or raw data quantitation reports should be reviewed. Corrective actions taken and verification of acceptable instrument response should be included. The laboratory should document the recoveries in a control chart as a long-term assessment of method, and laboratory performance. 21 ------- Laboratory Duplicates One sample per batch should be split in the laboratory and analyzed in duplicate to provide an estimate of analytical precision. Duplicate analyses also are useful in assessing potential sample heterogeneity and matrix effects. An alternative to a sample duplicate is a matrix spike duplicate. If results fall outside the control limit (Table 2), calculations and instrument should be checked. A replicate analysis may be required to confirm the results. If results continue to exceed the control limit, subsequent corrective action is at the discretion of the program manager or QA officer, because matrix effects or incomplete homogenization (either in the field or laboratory) may be contributing factors. The relative percent difference (RPD) between the analytical results for the duplicate samples (or matrix spike and matrix spike duplicate) should be less than 30 for each analyte of interest (Table 2). The RPD is calculated as follows: RPD = (C1-C2) x 100% (CI + C2)/2 where: CI is the larger of the duplicate concentrations for a given analyte C2 is the smaller of the duplicate concentrations for a given analyte If results for any analytes do not meet the recommended RPD < 30% control limit criteria, calculations and instrumentation should be checked. It may be necessary to repeat the analysis to confirm the results. Results which repeatedly fail to meet the control limit criteria may indicate poor laboratroy precision. If this is the case, the laboratory should halt analysis of samples and eliminate the source of the imprecision before proceeding with sample analysis. ------- Internal Standards Internal standards (commonly referred to as surrogate spikes or surrogate analytes) are compounds chosen to simulate the analytes of interest in organic analyses. Ideally, the internal standard(s) is an isotopically-labeled analog of an analyte, although this type of standard is suitable for GC/MS analysis only. Alternatively, a compound similar to the analytes to be quantitated should be used as the internal standard(s). PCB congeners 198 and 103 have been successfully utilized as internal standards for PCB quantitation due to their relative retention indices and extremely low concentrations in environmental samples. Similarly, gamma-chlordene has been used to quantitate the more polar pesticides. Wherever possible, the use of multiple internal standards which elute at dramatically different times (e.g. congeners 103 and 198 which elute toward the beginning and end of a GC run, respectively) is highly recommended. The use of multiple internal standards may provide better quantitations by minimizing response differences caused by such factors as automatic injections. The internal standard represents a reference against which the signal from the analytes of interest is compared directly for the purpose of quantitation. Internal standards must be added to each sample, including QA/QC samples, prior to extraction. The internal standard recovery data therefore should be carefully monitored; each laboratory should report the absolute amounts and the percent recovery of the internal standards along with the target analyte data for each sample. Using this approach, the analytes of interest are assumed to behave identically to the 2.1 ------- appropriate internal standard(s). The internal standard is assumed to be fully recovered in an internal standard type calibration. Even if this assumption is not valid, as based on the use of an external standard, as described below, it is still assumed that the analytes of interest behave (i.e., are not fully recovered) in the same manner as the internal standard(s). Therefore, the ratio of the concentration of the analytes to the concentration of the internal standard is constant. Recovery of the internal standard(s) is determined through the use of an external or internal injection standard as described below. Acceptable internal standard recoveries are listed in Table 2. These limits are asymmetrical because low recoveries are provided for as described above, while recoveries greater than 100% for the internal standaxd(s) may indicate an interference with the internal standard(s) which could affect data quality. Internal Injection Standards For gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analysis, internal injection standards are added to each sample just prior to injection. Internal injection standards are used to monitor the actual recovery of the internal standards. The analyst(s) should monitor internal injection standard retention times and response to determine if instrument maintenance or repair is needed. Instrument problems that may have affected the data or resulted in the reanalysis of the sample should be documented properly in logbooks and/or internal data reports and used by the laboratory personnel to take appropriate corrective action. 24 ------- REFERENCES Ballschmiter, K., W. Schafer and H. Buchert. 1987. Fresenius Z. Anal. Chem. 326:253-257. Battelle Memorial Institute. 1990. Phase 4 Final Report on National Status and Trends Mussel Watch Program. Collection of Bivalves and Surficial Sediments from Coastal US Atlantic and Pacific Locations and Analyses for Organic and Trace Elements. Prepared for U.S. Dept. of Commerce, NOAA Ocean Assessments Division, Rockville, MD. Cantillo, A.Y. 1990. Standard and Reference Materials for Marine Sciences. Intergovernmental Oceanographic Commission Manuals and Guides 21. Durell, G. S. and T. C. Sauer. 1990. Simultaneous Dual-Column, Dual-Detector Gas Chromatographic Determination of Chlorinated Pesticides and Polychlorinated Biphenyls in Environmental Samples. Anal. Chem. 62:1867-1871. Keith, L. H., W. Crumett, J. Deegan, Jr., R. A. Libby, J. K. Taylor, and G. Wender. 1983. Principles of environmental analysis. Anal. Chem. 55:2210-2218. Keith, L. H. 1991. Environmental Sampling and Analysis: A Practical Guide. Lewis Publishers, Chelsea, MI, 143 pp. Krahn, M. M., C. A. Wigren, R. W. Pearce, L. K. Moore, R. G. Bogar, W. D. MacLeod, S. L. Chan, and D. W. Brown. 1988. Standard Analytical Procedures of the NOAA National Analytical Facility, 1988, New HPLC Cleanup and Revised Extraction Procedures for Organic Contaminants. NOAA Technical Memo. NMFS F/NWC-153. U.S. Dept. of Commerce, NOAA National Marine Fisheries Service, Seattle, WA. Lauenstein, G. L. in preparation. A Compendium of Methods Used in the NOAA National Status and Trends Program. NOAA. 1989. A Summary of Data on Tissue Contamination from the First Three Years (1986- 1988) of the Mussel Watch Program. NOAA Technical Memo. NOS OMA 49. U.S. Dept. of Commerce, NOAA National Ocean Service, Rockville, MD. NOAA. 1991. Second Summary of Data on Chemical Contaminants in Sediments from the National Status and Trends Program. NOAA Technical Memo. NOS OMA 59. U.S. Dept. of Commerce, NOAA National Ocean Service, Rockville, MD. Schantz, M. M., B. A. Benner, S. N. Chesler, B. J. Koster, K. E. Hehn, S. F. Stone, W. R. Kelly, R. Zeisler, and S. A. Wise. 1990. Preparation and Analysis of a Marine Sediment Reference Material for the Determination of Trace Organic Constituents. Fresenius J. Anal. Chem. 338:501-514. 25 ------- Taylor, J. K. 1987. Quality Assurance of Chemical Measurements. Lewis Publishers, Inc., Chelsea, Michigan. 328 pp. U.S. Environmental Protection Agency, in preparation. EMAP Laboratory Methods Manual: Estuaries. U. S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Office of Research and Development, Cincinnati, Ohio. U.S. Environmental Protection Agency. 1979a. Methods for chemical analysis of water and wastes. EPA-600/4-79/020. U. S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Office of Research and Development, Cincinnati, Ohio (revised March 1983). U.S. Environmental Protection Agency. 1979b. Handbook for analytical quality control in water and wastewater laboratories. U. S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio, EPA/600/4-79/019. U.S. Environmental Protection Agency. 1989. Recommended Protocols for Measuring Selected Environmental Variables in Puget Sound. U.S. Environmental Protection Agency, Puget Sound Estuary Program, Office of Puget Sound, Seattle, Washington. Valente, R. M. and J. R. Schoenherr. 1991. EMAP Near Coastal 1991 Virginian Province Quality Assurance Project Plan. U.S. Environmental Protection Agency, Office of Research and Development, Environmental Research Laboratory, Narragansett, RI. Vargo, C. and N. Mosesman. 1992. Simultaneous Confirmational Analysis of Pesticides and Herbicides. Amer. Environ. Lab. 2/92:25-30. Wise, S. A., B. A. Benner, R. C. Christensen, B. J. Koster, J. Kurz, M. M. Schantz, and R. Zeisler. 1991. Preparation and Analysis of a Frozen Mussel Tissue Refernce Material for the Determination of Trace Organic Constituents. Environ. Sci. Technol. 25(10): 1695- 1704. 26 ------- APPENDIX 1 ANALYTICAL METHODS The following sections include analytical methods which have been utilized by the ERLN Chemistry Group for the trace level analysis of organic and metal contaminants in marine sediment and tissue samples. As previously stated, mention of trade names or commercial products does not constitute endorsement for their use. The analytical techniques detailed in the following sections were developed for a unique suite of analytes, and may not be applicable to other analytes. Table 1 lists the specific target analytes which have been successfully analyzed for from marine sediments and tissue samples by chemists at ERLN. 1.0 ORGANIC ANALYSES As previously mentioned, the procedures outlined in the next section have been employed by ERLN for the analysis of a specific set of semivolatile organic analytes. These analytes are listed in Table 1 of the main text, but generally include selected PCB congeners, chlorinated pesticides and PAHs. The methods may not be suitable for other analytes such as the acid extractable priority pollutants or volatile organics. Procedures included in the following section arc accionitriic/pentanc extraction of tissue samples, methylene chloride/acctone extraction of 27 ------- sediments, silica gel chromatography cleanup and chemical class separations of tissue and sediment extracts, analysis of PCBs and chlorinated pesticides by gas chromatography with electron capture detection (GC-ECD), and analysis of PAHs by gas chromatography/mass selective detection (GC/MSD). The silica gel chromatography, PCB/pesticide and PAH instrumental procedures are applicable to both sediment and tissue sample extracts. The sample weights given in the following methods are not absolute. They are based on our experience with marine samples and are optimized to achieve low detection limits. It may be necessary to adjust the amount of sample used based on such factors as detection limits required and moisture content. While a lower detection limit may be achieved by using more sample, it is possible to overload the silica gel column with an extract containing too much organic material. This could result in poor cleanup and/or separation of the chemical classes. It is important to adjust the sample amount for moisture content so that the dry weight of the samples is consistent with the dry weight of the samples used to calculate the MDLs. 2X ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR TISSUE EXTRACTION OF SEMIVOLATILE ORGANIC ANALYTES (REVISED FEBRUARY 1993) 1.0 OBJECTIVES The objective of this document is to define the standard operating procedure for the extraction of semi-volatile organic compounds from marine tissue samples. The extracts will be further cleaned up by silica gel chromatography procedures prior to analysis by gas chromatography (GC) or gas chromatography/mass spectrometry (GC/MS). 2.0 MATERIALS AND EQUIPMENT Apparatus for homogenizing tissue Brinkman Polytron 100- or 150-ml glass centrifuge tubes Apparatus for determining weight and dry weight Top-loading balance capable of weighing to 0.01 g Aluminum weighing pans 29 ------- Stainless steel spatula Drying oven maintained at 105-120°C Turbo-Vap (Zymark) apparatus, with heated water bath maintained at 25-35° C Nitrogen gas, compressed, 99.9% pure Glass Turbo-vap flasks, 200 ml Glass graduated cylinders, 100- and 500-ml Glass separatory funnels, 1 L. Glass erlenmeyer flasks, 250 and 500 ml. Borosilicate glass vials with Teflon-lined screw caps, 2-ml Microliter syringes or micropipets, solvent rinsed Reagents Pentane, pesticide grade or equivalent Acetonitrile, pesticide grade or equivalent Deionized water, pentane-extracted Sodium sulfate-anhydrous, reagent grade. Heated to 400°C for at least 4 hours, then cooled and stored in a tightly-sealed glass container at room temperature. Internal Standards, to be added to each sample prior u> extraction. 30 ------- 3.0 METHODS 3.1 Weigh approximately 10.0 g of sample into a solvent rinsed centrifuge tube. Weigh approximately 1.0 gram into a preweighed aluminum pan for dry/wet determination. 3.2 Add Internal Standards as required: CB198 for PCB analysis, 2,5-dichloro-m- terphenyl for pesticides, and dl2 Benzo(a)Anthracene and dlO Phenanthrene mix for PAHs. The amount of IS added is dependent on the expected contaminant concentrations and should be equivalent to those concentrations. 3.3 Add 50 ml acetonitrile. 3.4 Polytron the samples for 20 seconds, at a speed setting of ~ 5. Centrifuge for 10 minutes at 1750 rpm and pour off the supernatant into a separatory funnel containing 500 ml pentane extracted deionized water (DI). Repeat this step two more times. 3.5 Back extract the DI/ACETONITRDLE phase in the separatory funnel with 3 X 50 ml pentane. After each addition of pentane has been shaken, draw off the bottom layer irito a 500 ml erlenmeyer flask. Decant the Pentane layer into a 250 ml erlenmeyer flask by pouring it out the top of the separatory funnel. This way the transfer of water into the pentane extract will be avoided. 31 ------- 3.6 Transfer the water layer from the 500 ml erlenmeyer flask back into the separatory funnel for every addition of pentane. Rinse the 500 ml flask 3 x with Pentane and add the rinses to the separatory funnel. 3.7 Combine the pentane extracts and dry over Sodium Sulfate. 3.8 Transfer the sample to a 200 ml Turbo-Vap flask. Rinse the flask 3 x with pentane and add the rinses to the flask. Place the flask into the Turbo-Vap apparatus, and turn on the unit. Open the valve on the nitrogen tank and set the regulator to ensure a pressure of 15 psig is reaching the Turbo-Vap unit. Reduce the volume of sample to approximately 1 ml. 3.9 Adjust the volume to 1.0 ml with pentane. Remove 0.1 ml of sample into a preweighed aluminum pan for lipid weight determination. Allow it to dry at room temperature for at least 24 hours. Record the weight of the pan plus the sample. 3.10 Fractionate the sample following the Column Chromatography SOP. 4.0 QUALITY ASSURANCE/QUALITY CONTROL 4.1 Standard Reference Materials 4.1.1 A certified SRM is prepared with each batcli of samples to validate 32 ------- analytical recovery. Analytical results should then be compared to the certified concentrations. Corrective action is required if the required accuracy goals are not met. 4.1.2 SRMs should be prepared in the exact same manner as the unknowns. 4.2 Analytical Reproducibility 4.2.1 Replicate samples should be prepared to assess the reproducibility of the extraction procedure. 4.2.2 For every batch of samples, one sample should be chosen to extract and analyze in triplicate. Deviation between replicate samples should be <30%. 4.3 Procedural Blanks 4.3.1 Procedural blanks should be carried throughout the entire extraction procedure to verify the absence of contamination of the method. 4.3.2 Trace amounts of analytes in the blanks (less than three times the method detection limit) may be ignored and have no effect on the subsequent sample analyses, but samples should be rejected if significant concentrations (greater than 33 ------- five times the MDL) are present in procedural blanks. 4.3.3 One blank should be prepared for each batch of samples (minimum frequency 5%). 34 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR SEDIMENT EXTRACTION OF SEMIVOLATILE ORGANIC ANALYTES (REVISED FEBRUARY 1993) 1.0 OBJECTIVES The objective of this document is to define the standard operating procedure for the extraction of semi-volatile organic compounds from marine sediment samples. The extracts will be further cleaned up by silica gel chromatography procedures prior to analysis by gas chromatography (GC) or gas chromatography/mass spectrometry (GC/MS). 2.0 MATERIALS AND EQUIPMENT Apparatus for homogenizing sediment Wrist-action shaker 100 ml glass centrifuge tubes Apparatus for determining weight and dry weight Top-loading balance capable of weighing to 0.01 g Aluminum weighing pans 35 ------- Stainless steel spatula Drying oven maintained at 105-120°C Turbo-Vap (Zymark) apparatus, with heated water maintained at 25-35°C Nitrogen gas, compressed, 99.9% pure Glass Turbo-Vap flasks, 200 ml Glass graduated cylinders, 100- and 500-ml Erlenmeyer flasks, 250 ml Microliter syringes or micropipets, solvent rinsed Borosilicate glass vials with Teflon-lined screw caps, 2-ml Reagents Methylene chloride, pesticide grade or equivalent Deionized water, pentane-extracted Acetone, pesdcide grade or equivalent Sodium sulfate-anhydrous, reagent grade. Heated to 400°C for at least 4 hours, then cooled and stored in a tightly sealed glass container at room temperature. Internal Standards, to be added to each sample prior to extraction. 3.0 METHODS 3.1 Find the correct caps for each centrifuge tube to be used by filling them with approximately 25 mis of methylene chloride, putting the caps on and rolling the tube on the lab bench on a paper towel and look for leaks. Once the correct tubes and caps have 36 ------- been matched, weigh approximately 10.0 g of homogenized sample into a solvent rinsed centrifuge tube. Homogenization is accomplished by physical mixing of the sediment with stainless steel or Teflon coated utensils, or by a polyethylene propeller attached to an electric drill. The amount of sample may be adjusted based on expected contaminant concentrations or detection limits required. Weigh approximately 2.0 grams into a preweighed aluminum pan for dry/wet determination. 3.2 Add Internal Standards as required: CB198 for PCB analysis, 2,5-dichloro-m- terphenyl for pesticides, and dl2 Benzo(a)anthracene/ dlO Phenanthrene mix for PAHs. The amount of IS added is dependent on the expected contaminant concentrations and should be equivalent to those concentrations. 3.3 Add 30 g Sodium sulfate and mix with a teflon coated spatula very well. Then add 50 ml 20:80 acetone:methylene chloride. 3.4 Seal the centrifuge tubes with teflon tape and caps, and shake -15 hrs. (overnight). Shake tubes at approximately a 60° angle, at an intensity setting of "5". Centrifuge for 20 minutes at 1750 rpm and pour off the supernatant into an erlenmeyer flask. 3.5 Add 50 ml of 20:80 acetone:methylene chloride, seal and shake as above for ~6 hrs. Centrifuge for 20 minutes at 1750 rpm and add the supernatant to the erlenmeyer flask. Add some additional sodium sulfate to the combined extracts to ensure all water is excluded. 37 ------- 3.6 Gravity filter the extract through a pre-rinsed (methylene chloride) glass fiber filter. Rinse the erlenmeyer 2 x with methylene chloride, and the filter itself once. Collect the filtrate in a clean rinsed 200 ml Turbo-Vap tube. Place the flask into the Turbo-Vap apparatus, and turn on the unit. Open the valve on the nitrogen tank and adjust the regulator to ensure a pressure of 15 psi. Reduce the sample volume to approximately 1 ml, with solvent exchange to pentane. 3.9 Adjust the volume to 1 ml with hexane. 3.10 Fractionate the sample following the Column Chromatography SOP. 4.0 OPTIONAL CLEANUP PROCEDURES Activated copper powder (activated by the addition of 8 M hydrochloric acid and rinsed with the following solvents in succession: deionized water, methanol, methylene chloride, and hexane) may be added to the extract to remove any free elemental sulfur. The copper is added until the formation of black copper sulfide no longer occurs. 5.0 QUALITY ASSURANCE/QUALITY CONTROL 5.1 Standard Reference Materials 5.1.1 A certified SRM is prepared with each batch of samples to validate 38 ------- analytical recovery. Results arc compared to certified concentrations and corrective action is required if the accuracy is outside of the required specifications. 5.1.2 SRMs should be prepared in the exact same manner as the unknowns. 5.2 Analytical Reproducibility 5.2.1 Replicate samples should be prepared to assess the reproducibility of the extraction procedure. 5.2.2 For every batch of samples, one sample should be chosen to extract and analyze in triplicate. Deviation between replicate samples should be <30%. 5.3 Procedural Blanks 5.3.1 Procedural blanks should be carried throughout the entire extraction procedure to verify the absence of contamination of the method. 5.3.2 Trace amounts of analytes in the blanks (less than three times the method detection limit) may be ignored and have no effect on the subsequent sample analyses, but samples should be rejected if significant concentrations (greater than five times the MDL) are present in procedural blanks. 39 ------- 5.3.3 One blank should be prepared for each batch of samples (minimum frequency of 5%). 40 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR COLUMN CHROMATOGRAPHY OF SEMIVOLATILE ORGANIC ANALYTES IN SEDIMENT AND TISSUE EXTRACTS (REVISED FEBRUARY 1993) 1.0 OBJECTIVES The objective of this document is to define the standard operating procedure for the preparation of columns for the cleanup and chemical class separation of semi-volatile organic compounds from marine samples. The extract fractions will be analyzed by gas chromatography (GC) or gas chromatography/mass spectrometry (GC/MS). 2.0 MATERIALS AND EQUIPMENT 9.5-mm ID X 45-cm glass chromatography column with 200 ml reservoir Apparatus for determining weight Top-loading balance capable of weighing to 0.01 g Turbo-Vap (Zymark) apparatus, with heated water bath maintained at 25-35° C Glass Turbo-Vap flasks, 200 ml 41 ------- Nitrogen gas, compressed, 99.9% pure Tumbler, ball-mill Glass graduated cylinders, 100- and 500-ml Glass beakers, 50-ml Borosilicate glass vials with Teflon-lined screw caps, 2-ml Micropipets, solvent rinsed or muffled at 400°C Reagents Pentane, pesticide grade or equivalent Methylene Chloride (CH^Cy, pesticide grade or equivalent Hexane, pesticide grade or equivalent Heptane, pesticide grade or equivalent Deionized water, pentane-extracted BioSil A silicic acid, 100-200 mesh Glass wool, silanized 3.0 METHODS 3.1 Silica gel preparation 3.1.1 Approximately 150 grams of fully activated silica gel is accurately weighed and transferred to a glass jar. 42 ------- 3.1.2 The silica gel is deactivated by adding 7.5% (weight basis) of pentane- extracted deionized water. The water is weighed accurately and an appropriate amount is added dropwise, ~ 1 ml at a time, to the silica gel. After each water addition, the jar is hand-shaken vigorously. 3.1.3 The glass jar is then placed on a ball-mill tumbler and allowed to tumble overnight 3.1.4 After tumbling, the jar is removed from the tumbler. The silica gel is stored tightly sealed in the jar at room temperature until use. 3.2 Column preparation 3.2.1 The glass columns are set up in ring stands in a fume hood. 3.2.2 Glass wool, sufficient to create a 1 cm thick plug in the column is placed into the reservoir of the column. A glass rod is used to push the glass wool to the bottom of the column. 3.2.3 11.5 g of the 7.5% deactivated silica gel is weighed out in a beaker. Approximately 30 ml of CH2Cl2 is added to the beaker to form a slurry. The slurry is then carefully poured into the column. The beaker is rinsed with additional CH2C12, as are the inner walls of the reservoir to ensure all silica is 43 ------- introduced to the column. The total volume of CH2Cl2 should be approximately 50 ml. 3.2.4 The column is allowed to drip, and the eluate is collected and discarded. When the level of the CHjClj just reaches the top of the silica gel, 50 ml of pentane is slowly added to the column. This eluate is also collected and discarded. 3.3 Chemical class separations 3.3.1 The sample extract is introduced to the column just as the pentane rinse level reaches the silica gel. The vial is then rinsed with an additional 1 ml of pentane which is also introduced to the column just before the silica gel is exposed. The eluate is collected in a clean round bottom flask. 3.3.2 As the sample rinse level reaches the silica gel, 55 ml of pentane is added to the column. The eluate is collected as the F-l fraction in a clean Turbo-Vap flask. 3.3.3 As the pentane level reaches the top of the silica, 36 ml of 70:30 pentanermethylene chloride is introduced to the column. The F-2 fraction is collected in a separate Turbo-Vap flask from the F-l fraction. After collection, the flasks are kept tightly capped with aluminum foil. At no time should the column 44 ------- flow rate exceed 6 ml/min. 3.3.4 After the F-2 fraction has been collected from the column, the flasks are placed in the Turbo-Vap. The apparatus is turned on and Nitrogen gas is introduced to the flasks. The solvent is reduced to approximately 1 ml. The samples are then solvent-exchanged to heptane and concentrated to about 1 ml. 3.3.5 The fractions are then transferred to borosilicate glass vials fitted with Teflon-lined screw caps for storage until analysis. 4.0 QUALITY ASSURANCE/QUALITY CONTROL 4.1 Silica Gel Testing 4.1.1 Silica Gel is verified to separate compound classes using the silica gel testing SOP. 4.2 Method Blanks 4.2.1 Method (procedural) blanks are included in each sample set to provide an estimate of contamination from the reagents. 4.3 Internal Standard Recovery 4.3.1 PCB103 is added to final column fractions to calculate recovery of the internal standard. 45 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR GAS CHROMATOGRAPHIC ANALYSIS OF PCBs AND CHLORINATED PESTICIDES (REVISED FEBRUARY 1993) 1.0 OBJECTIVES The objective of this document is to define the standard procedure for analyzing marine environmental samples for polychlorinated biphenyls (PCBs) and chlorinated hydrocarbon pesticides using gas chromatography and electron capture detectors. 2.0 EQUIPMENT USED Hewlett Packard 5890 Gas Chromatographs equipped with electron capture detectors (Ni 63), automatic samplers, 30 m DB-5 fused silica capillary columns (0.25 p film thickness, 0.25 mm i.d.). Perkin-Elmer/Nelson software (ACCESS*CHROM) provides for collection and storage of raw chromatographic data, and for selection and quantitation of analyte peaks. Ultra high purity helium and 95/5% Argon/Methane gases are used as the carrier and auxiliary gas respectively. 3.0 OPERATION 46 ------- 3.1 Instrument checks made prior to data collection 3.1.1 Gas supply 3.1.1.1 Check gas cylinder pressures. Replace tank if pressure is less than 100 psig. 3.1.1.2 Check head pressure gauge on front panel of instrument. Gauge should read 18 psig; adjust to correct setting if reading is high; check for leaks if pressure is low. This setting provides for a carrier gas flow of approximately 1.5 ml/min. 3.1.1.3 Replace injection port septum. Check septum nut and column fittings for leaks with leak detector and tighten as necessary. 3.1.1.4 Check the auxiliary gas flow. A flow of 35 ml/min is required. 3.1.1.5 Check septum purge and split flows. Adjust to 1 and 35 ml/min, respectively, as necessary. 3.1.2 Instrument output signal 3.1.2.1 Display the analog output signal from the detector on the LED panel of the GC. Record the value in the instrument log book, and check for 47 ------- consistency with previous readings. On instruments with dual detectors, ensure the signal is correctly assigned to the detector selected for the analysis. 3.1.3 Instrument operating parameters 3.1.3.1 Temperature programs and run times are stored as workfiles in each GC's integrator. The following conditions are required for the analysis of PCBs and pesticides: Injection port temperature 275°C Detector temperature 325°C Initial column temperature 100°C Initial hold time 1 min Rate 1 5°C/min Ramp 1 final temperature 140°C Ramp 1 hold time 1 min Rate 2 1.5°C/min Ramp 2 final temperature 230°C Ramp 2 hold time 20 min Rate 3 10°C/min Final column temperature 300°C Final hold time 5 min Stop time 100 min Injection port purge open time 1 min 3.1.3.2 Load an appropriate workfile into the integrator. 3.1.3.3 Enter the autosampler parameters into the integrator via Option 11. Indicate which injection port is being used, the number and positions of the samples in the autosampler tray, the number of injections per bottle, and the amount injected (I ul). 48 ------- 3.1.3.4 Check the signal assignments and levels again. If they are correct, store the workfile in the integrator. Data system setup 3.2.1 Scheduling of standards and samples 3.2.1.1 Setting up the instrument queue is accomplished by following instructions laid out in the Perkin-Elmer Nelson manual. 3.2.1.2 Order the samples, standards, and rinses according to the following guidelines: -place hexane rinses before and after standards -bracket groups of no more than five (5) samples with standards. -arrange multiple level standards so that a high and a low standard precede as well as follow samples -procedural and field blanks should be run prior to samples to minimize risk of carryover contamination.- 3.2.1.3 Type in sample weight and internal standard amounts for each sample to be used in final concentration calculations. Double check all manually entered values for accuracy. 49 ------- 3.3 Instrument startup and data collection 3.3.1 After the instrument has been scheduled, arrange the samples and standards to be run in the autosampler trays. Check the order for accuracy against a copy of the queue. Load the trays into the autosampler. 3.3.2 Visually recheck tank regulator gauges and instrument settings to ensure proper settings. 3.3.3 Start GC operation and data collection by pressing 'start' on the integrator. 3.4 Peak identification and quantitation 3.4.1 Peak identification is accomplished by automated routines. Identifications are based on comparison of retention times of actual standards to unknown peaks. Multilevel standards are calibrated to generate a linear regression curve of response according to the manufacturer's instructions. After a calibration curve has been generated, the samples are analyzed. Analytes are quantitated based on the peak areas for the analytes and internal standard, the amount of the internal standard, and the response factors generated from the calibration curve. Chromatograms and data reports are generated for each sample and standard. 50 ------- 4.0 QUALITY ASSURANCE 4.1 Chromatograms of standards are compared to posted references. Peak identifications, resolution and shapes are inspected. Calculated standard amounts are checked for accuracy and documented. Other abnormalities, such as spurious or extra peaks, rising or falling baselines, and negative spiking are examined. Response factors and overall instrument response are compared to previous runs and documented. Blanks are checked for the presence of interferences or analytes of interest Unknown samples are compared to standards to verify peak identifications. 5.0 TROUBLESHOOTING 5.1 Refer to the ERLN GC Troubleshooting notebook, the manufacturer's manuals, or to experienced personnel for guidance in troubleshooting the GCs. 51 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR ANALYSIS OF PAHs BY GC/MS (REVISED FEBRUARY 1993) 1.0 OBJECTIVES The objective of this document is to define the standard procedure for analyzing marine environmental samples for PAHs using GC/MS in electron impact/positive ion mode. 2.0 EQUIPMENT HP Model 5890 Series II Gas Chnomatograph HP Model 5971A Mass Selective Detector HP Model 7673 Autosampler HP MS Chemstation (DOS Series) Software IBM Compatible Personal Computer 3.0 OPERATION A. Instrument Parameters 52 ------- Column: 60 m x 0.25 mm ID x 0.25 um DB-5 (J&W Scientific) Carrier: Helium at 25 psi; 0.8-1.0 ml/min Injector: 270 degrees C; splitless mode, purge on at 0.8 min Interface: 300 degrees C; direct, source 200 degrees C Temperature Program: 1 min, 40 deg; 20 deg/min to 120 deg; 10 deg/min to 310 deg and hold 16 min. This is suitable for Polycyclic Aromatic Hydrocarbons. MS Parameters: Set by Autotune using PFTBA as the calibration compound; Manual Tune is then used to force the 131 and 219 abundances to 20 to 40 percent of the 69 base peak; the electron multiplier is then set to meet the requirements of the particular method. This procedure is done in a series of loops, as new parameter settings for a specific lens will affect the behavior of the others. B. Daily Performance Checks 1) Adequate DFTPP spectrum (see attached criteria), based on a 50 ng injection. 2) Calibration Check - results for a mid-level standard must be within 25 percent of the true value for a single target compound; the average error for all compounds in the method must be less than 15 percent. C. Calibration The calibration method is a 5 point, internal standard, least squares fit, forced through the origin. The levels are chosen to cover a range from 4 to 10 times the instrument detection 53 ------- limit for the lowest point, up to the point at which saturation and/or non-linear behavior is observed. For PAHs in marine sediment or tissue, the current levels are 1.0, 5.0, 10.0, 15.0, and 20.0 ng/ul. Acceptance criteria for each level are the same as listed for the daily check. D. Sample Analysis A 250 uL aliquot of the sample extract is blown down to 20-25 uL with nitrogen or helium. If required, an internal injection standard is added (4-chloro-p-terphenyl). Once the daily performance checks are satisfied, the extracts are queued up on the autosampler. Periodic solvent blanks, standards, etc. are inserted at the judgement of the analyst. E. Identification Compounds are identified by monitoring a characteristic ion within a 12 second retention time window. Additional ions may be monitored at the discretion of the analyst. Confirmation is obtained by inspection of the full mass spectrum. 4.0 QUALITY ASSURANCE A. Standard Reference Materials, Blanks, Calibration Checks 54 ------- Standard reference materials are prepared along with each batch of samples. Calibration standards are verified with independently prepared control standards. B. Method Detection Limits Method detection limits are determined independently for a given sample matrix. Instrument detection limits are generally in the 6-10 pg per injection range, which usually corresponds to a 3-5 ng/g (ppb) method detection limit range in samples. 5.0 TROUBLESHOOTING AND MAINTENANCE On a daily basis, the injection port and liner are cleaned; the septum and glass wool in the liner are changed. It is periodically necessary to break off the first few inches of the column (this is done daily for heavy workloads of dirty samples; compounds most affected are the high molecular weight compounds). 55 ------- Mass 51 68 70 127 197 198 199 275 365 441 442 443 DFTPP ACCEPTANCE CRITERIA (by CLP 3/90) Abundance 30-60% of mass 198 Less than 2% of mass 69 Less than 2% of mass 69 40-60% of mass 198 Less than 1% of mass 198 Base peak, 100% relative abundance 5-9% of mass 198 10-30% of mass 198 Greater than 1% of mass 198 Less than mass 443 40-60% of mass 198 17-23% of mass 442 56 ------- 2.0 INORGANIC ANALYSES The methods outlined in the following section have been utilized by ERLN for the analysis of major and trace elements in sediment and tissue samples. Two sediment digestion procedures are included: a total digestion using concentrated nitric/hydrofluoric acids and microwave heating, and an ultrasonic extraction with 2M nitric acid A tissue digestion method involving concentrated nitric acid, hydrogen peroxide and microwave heating is oudinecL A method for instrumental analysis of sediment and tissue digests is also provided. The sample weights given in the following methods are not absolute. They are based on our experience with marine samples and are optimized to achieve low detection limits. It may be necessary to adjust the amount of sample used based on such factors as detecdon limits required and moisture content. While a lower detection limit may be achieved by using more sample, it is possible that too much sample would produce excess foaming or pressure and lead to a poor digestion and low recoveries. It is important to adjust the sample amount for moisture content so that the dry weight of the samples is consistent with the dry weight of the samples used to calculate the MDLs. 57 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR DIGESTION OF MARINE ORGANISM SAMPLES FOR METALS ANALYSIS 1.0 OBJECTIVES The objective of this document is to establish the standard operating procedure for the total digestion of marine tissue samples. Sample extracts are routinely analyzed by Flame Atomic Absorption Spectrometry (FAA), Graphite Furnace Atomic Absorption Spectrometry (GFAAS) or Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). 2.0 MATERIALS AND EQUIPMENT Top-loading balance (0.01 gram precision) Vacuum Freeze Dryer CEM Microwave Digestion System (Including 100 ml. Teflon vessel liners and pressure control capability) 50 ml. class A volumetric flasks 60 ml. polyethylene screw-cap bottles Instra-Analyzed grade concentrated HNOj for trace metal analysis (70-71 %) 58 ------- Hydrogen Peroxide - H202 (30%) Vacuum filtering apparatus with Whatman 42 filter paper 3.0 METHODS 3.1 Sample Preparation 3.1.1 Organism samples should be thawed, and handled only with plastic or stainless steel utensils. Where necessary, organism tissues should be homogenized. If chromium or nickel is to be analyzed in the samples, the homogenizer dp should be constructed of titanium to avoid contamination of sample tissues. 3.1.2 Obtain the tare weight of labeled, acid-washed 100 ml. Teflon microwave digestion vessel liners. 3.1.3 Weigh approximately 3-5 grams wet tissue into each vessel (-0.5 grams dry). Obtain the wet gross weight of each tube. 3.1.4 Freeze dry samples and obtain the dry gross weight for each sample. Subtract the tare weight and record the weight of dry tissue in each tube. 3.2 Closed Vessel Microwave Digestion (1st Stage) 59 ------- 3.2.1 Add 10 ml. of concentrated HN03 (70-71 %) to each digestion vessel. 3.2.2 Make sure the tissue sample is fully saturated and allow to sit for a minimum of 1 hour, or until all foaming subsides. 3.2.3 Place each liner into a microwave vessel. 3.2.4 Insert a pressure relief membrane into each cap assembly and place on top of the vessels (use the modified cap assembly for the vessel to be used for pressure monitoring). 3.2.5 Place a top on each vessel and hand tighten. 3.2.6 Place the vessels into the carousel. 3.2.7 Insert a vent tube into each vessel, place the free end in the center trap, then place the carousel into the oven. 3.2.8 Connect the pressure sensing line to the modified cap assembly (make suite the valve on the side of the oven is in the "neutral" position). 3.2.9 Program the oven following the parameters below: STAGE 12 3 4 5 60 ------- %POWER 85 85 85 85 85 PSI 20 40 85 150 190 TIME 15:00 15:00 15:00 15:00 15:00 TAP 5:00 5:00 5:00 5:00 5:00 FAN SPEED 100 100 100 100 100 ** Note - Power settings arc for 12 vessels. If a different # of vessels is desired, subtract or add 5% power per vessel. 3.2.10 After completion of the program, allow the pressure in the control vessel to drop below 20 PSI, then manually vent the control vessel, remove the pressure sensing line and place the carousel into the fume hood. Closed Vessel Microwave Digestion (2nd Stage) 3.3.1 Manually vent each vessel, remove the caps and add 2 ml. of 30% H^. 3.3.2 Allow the reaction to subside, then reassemble the vessels as described in sections 3.2.4-3.2.6. 3.3.3 Place the carousel into the oven and reconnect the pressure sensing line to the control vessel. Check to ensure the exhaust fan is operating. 3.3.5 Program the oven following the parameters below: STAGE 1 2 %POWER 85 100 PSI 100 100 61 ------- TIME TAP FAN SPEED 15:00 5:00 100 15:00 5:00 100 ** Note - Power settings are for 12 vessels. If a different # of vessels is desired, subtract or add 5% power per vessel. 3.3.6 Although the oven is automated, individual tissue samples will react differently, so all steps should be monitored in case venting should occur. If venting does occur, remove the vented, vessels and lower the power accordingly. 3.3.7 After completion of the program, allow the vessels to cool in the oven until the pressure in the control vessel is below 20 PSL 3.3.8 Manually vent the control vessel, then remove the carousel and place in a fume hood until the liquid reaches room temperature. 3.3.9 Remove the vent tubes and manually vent the remaining vessels. 3.4 Sample Filtration 3.4.1 Remove the tops and rinse the lids with deionized water, catching the rinse in the vessel liner. 3.4.2 Add -15 ml. of deionized water to each vessel. 62 ------- 3.4.3 Using plastic tweezers, place a sheet of Whatman 42 filter paper in a vacuum filtration funnel and wet the paper with 2M HN03. 3.4.4 Place a 60 ml. acid-cleaned polyethylene bottle and vacuum gasket under the filter funnel and apply vacuum. 3.4.5 Filter the digested sample through the paper and collect the filtrate in the bottle. 3.4.6 Rinse the digestion vessel with deionized water, filter and collect the filtrate in the botde. 3.4.7 Pour the combined filtrates into a 50 ml. acid-cleaned volumetric flask, and dilute to the mark with deionized water. 3.4.8 Shake the solution thoroughly and transfer back to the acid-cleaned 60 ml. polyethylene bottle. Label the bottle appropriately. 4.0 QUALITY ASSURANCE 4.1 Standard Reference Materials (SRM) 4.1.1 A certified SRM should be prepared with every batch of samples to validate 63 ------- analytical accuracy and recovery. 4.1.2 SRMs should be prepared in the exact manner as the unknown samples, including drying, even if the material is already dry. 4.1.3 The frequency of SRM preparation should be approximately 1 for every 20 unknown samples prepared. 4.1.4 The oudined extraction technique should yield close to 100% recoveries for organism SRMs, as oudined in the ERLN QA/QC guidelines. 4.2 Analytical Reproducibility 4.2.1 Replicate samples should be prepared to assess the reproducibility of the digestion procedure. 4.2.2 For every 20 samples prepared, one sample should be chosen to digest and analyze in triplicate. The relative standard deviation between replicate analyses should be <20%. 4.3 Procedural Blanks 4.3.1 Procedural blanks should be carried throughout the entire extraction 64 ------- procedure to verify that contaminants arc not present in the reagents and that no contamination has occurred throughout the procedure. 4.3.2 Trace amounts of metals in the blanks can be subtracted from subsequent sample analyses (blank subtraction), but a sample batch should be rejected if concentrations in the blank are >10% of "average" sample concentrations. 4.3.3 One procedural blank should be prepared for every 20 samples extracted 65 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR TOTAL DIGESTION OF SEDIMENT SAMPLES 1.0 OBJECTIVES The objective of this document is to establish the standard operating procedure for the total dieestion of bulk sediments. Sample digests are routinely analyzed by Flame Atomic Absorption Spectrometry (FAA), Graphite Furnace Atomic Absorption Spectrometry (GFAAS) or Inductively Coupled Plasma Atomic Emission Spectrometry (ICP). 2.0 MATERIALS AND EQUIPMENT Top-loading balance (0.01 gram precision) Vacuum Freeze Dryer CEM Microwave Digestion System (Including 100 ml. Teflon digestion vessel liners with pressure control capability) Protective Clothing (Polyethylene apron, Neoprene gloves, Safety goggles, Face shield) 100 ml. class A volumetric flasks 125 ml. polyethylene screw-cap bottles Instra-Analyzed grade concentrated HN03 for trace metal analysis (70-71 %) Reagent grade concentrated HF (49%) Reagent grade concentrated HCL (36.5-38%) 66 ------- Boric Acid (5%) prepared from H3B03 crystals Deionized water 3.0 METHODS 3.1 Sample Preparation 3.1.1 Sediment samples should be thawed and homogenized with plastic or stainless steel utensils. 3.1.2 Obtain the tare weight of labeled, acid-washed 100 ml. Teflon microwave digestion vessels liners. 3.1.3 Weigh approximately 1.5 grams wet sediment into each vessel (~0.5 grams dry). Obtain the wet gross weight of each liner. 3.1.4 Freeze dry samples and obtain the dry gross weight for each sample. Subtract the tare weight and record the weight of dry sediment in each liner. 3.2 Microwave digestion ** NOTE- Be sure to wear proper safety clothing when working with the concentrated HF. 67 ------- 3.2.1 Add 5 ml. of concentrated HN03 (70-71 %), 4 ml. of concentrated HF (49%) and 1 ml. concentrated HC1 (36.5-38%) to the vessel liners. 3.2.2 Make sure the sediment is fully saturated and allow to sit for a minimum of 1 hour. 3.2.3 Place the liners into their corresponding vessels. 3.2.4 Insert a rupture membrane into each lid and secure into place with a cap. Do not overtighten. 3.2.5 Place the vessels into the carousel. 3.2.6 Insert a vent tube into each vessel and place the free end into the center trap. 3.2.7 Attach the pressure sensing line to thhe control vessel, making sure the lever on the side of the oven is in the "neutral" position. 3.2.8 Program the oven following the parameters below: STAGE % POWER 100 120 30:00 20:00 100 1 2 100 150 15:00 10:00 100 PSI TIME TAP FAN SPEED 68 ------- **Note - Power settings are for 12 vessels. If a different # of vessels is desired, subtract or add 5% power per vessel. 3.2.9 Although the oven is automated, individual sediments will react differently, so all steps should be monitored in case venting should occur. If venting does occur, remove the vented vessels and lower the power accordingly. 3.2.10 When the program is finished, allow the pressure in the control vessel to drop below 20 PSI. 3.2.11 Manually vent the control vessel, detach the pressure sensing line and place the carousel in a fume hood. 3.2.12 Remove the vent tubes and vent the remaining vessels manually. 3.2.13 In a fiime hood, remove the caps and rinse the lids with deionized water, catching the rinse in the vessel liner. 3.2.14 Add 30 ml. of 5% Boric acid to each sample. 3.3 Sample Filtration (This step may not be necessary) 3.3.1 Add -15 ml. of deionized water to each vessel. 69 ------- 3.3.2 Using plastic tweezers, place a sheet of Whatman 42 filter paper in a vacuum filtration funnel and wet the paper with 2M HN03. 3.3.3 Place a 120 ml. acid-cleaned polyethylene bottle and vacuum gasket under the filter funnel and apply vacuum. 3.3.4 Filter the digested sample through the paper and collect the filtrate in the bottle. 3.3.5 Rinse the digestion vessel with deionized water, filter and collect the filtrate in the bottle. 3.3.6 Pour the combined filtrates into a 100 ml. acid-cleaned volumetric flask, and dilute to the mark with deionized water. 3.3.7 Shake the solution thoroughly and transfer back to the acid-cleaned 120 ml. polyethylene bottle. Label the bottle appropriately. 3.4 Sample Dilution (Required only if filtration step was omitted) 3.4.1 Transfer the contents of the vessel liner to a clean 100 ml. volumetric flask and rinse the vessel with deionized water, also adding the rinse to the flask. 70 ------- 3.4.2 Dilute to the volume mark with deionized water. 3.4.3 Shake the extracts thoroughly and transfer into acid-cleaned 125 ml. polyethylene screw-cap bottles. 3.4.4 Label the botdes appropriately and store at room temperature until analysis. 4.0 QUALITY ASSURANCE 4.1 Standard Reference Materials (SRMs) 4.1.1 A certified SRM should be prepared with every batch of samples to validate analytical accuracy and recovery. 4.1.2 SRMs should be prepared in the exact manner as the unknown samples, including drying, even if the material is already dry. 4.1.3 The frequency of SRM preparation should be approximately 1 for every 20 unknown samples prepared. 4.1.4 The outlined extraction technique should yield close to 100% recoveries for sediment SRMs. 71 ------- 4.2 Analytical Reproducibility 4.2.1 Replicate samples should be prepared to assess the reproducibility of the digestion procedure. 4.2.2 For every 20 samples prepared, one sample should be chosen to digest and analyze in triplicate. The relative standard deviation between replicate analyses should be <20%. 4.3 Procedural Blanks 4.3.1 Procedural blanks should be carried throughout the entire digestion procedure to verify that contaminants are not present in the reagents and that contamination has not occurred throughout the procedure. 4.3.2 Trace amounts of metals in the blanks can be subtracted from subsequent sample analyses (blank subtraction), but a sample batch should be rejected if concentrations in the blank are >10% of "average" sample concentrations. 4.3.3 One procedural blank should be prepared for every 20 samples digested. 72 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR ULTRASONIC EXTRACTION OF METALS FROM SEDIMENT SAMPLES 1.0 OBJECTIVES The objective of this document is to establish the standard operating procedure for the ultrasonic extraction of particulate and organically-bound metals from marine sediments. This procedure will not dissolve metals present within sediment mineral phases. Sample extracts are routinely analyzed by Flame Atomic Absorption Spectrometry (FAA), Graphite Furnace Atomic Absorption Spectrometry (GFAAS) or Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). 2.0 MATERIALS AND EQUIPMENT Top-loading balance (0.01 gram precision) Vacuum Freeze Dryer Ultrasonic water bath Centrifuge with 30 ml. tube and 10,000 RPM capabilities 30 ml. polycarbonate screw-cap centrifuge tubes 50 ml. class A volumetric flasks 73 ------- 60 ml. HDPE screw-cap bottles Instra-Analyzed grade concentrated HN03 for trace metal analysis (diluted to 2M concentration) Deionized water 3.0 METHODS 3.1 Sample Preparation 3.1.1 Sediment samples should be thawed and homogenized with plastic or stainless steel utensils. 3.1.2 Obtain the tare weight of labeled, acid-washed 30 ml. polycarbonate centrifuge tubes. 3.1.3 Weigh approximately 5 grams wet sediment into each centrifuge tube. Obtain the wet gross weight of each tube. 3.1.4 Freeze dry samples and obtain the dry gross weight for each sample. Subtract the tare weight and record the weight of dry sediment in each tube. 3.2 Ultrasonic Metal Extraction 74 ------- 3.2.1 Add 23-25 ml. of 2M HN03 (prepared from instra-analyzed grade concentrated HN03 for trace metal analysis) and replace caps hand tight. **NOTE** Acidification may result in dramatic foaming for some samples. Add the acid slowly to prevent loss of sample. 3.2.2 Shake the samples vigorously, then loosen the caps to release any gases evolved. Repeat this process until no further gas is evolved. 3.2.3 Tighten all caps hand tight and place the samples in the ultrasonic water bath. Water level should be at, or above, the acid level in the tubes. 3.2.4 Cover the sonicator bath to prevent excessive water evaporation. 3.2.5 Sonicate for a minimum of 16 hours. The water temperature will rise, but maximum temperature obtained should not exceed 75 degrees centigrade. 3.2.6 Remove the samples from the water bath and allow to cool to room temperature. 3.2.7 Weigh the sample tubes and pair samples accordingly for centrifugation. Centrifuge at 10,000 RPM for 15 minutes. 3.2.8 Decant the supernatant into individually labeled 50 ml volumetric flasks. 75 ------- 3.2.9 Repeat steps 3.2.1-3.2.7 for the second extraction. 3.2.10 Combine the supernatant with that obtained from the first extraction. 3.3 Sample Dilution 3.3.1 Dilute the samples to 50 ml with 2M HN03 3.3.2 Shake the extracts thoroughly and transfer into acid-cleaned 60 ml. polyethylene screw-cap bottles. 3.3.3 Label the bottles appropriately and store at room temperature until analysis. 4.0 QUALITY ASSURANCE 4.1 Standard Reference Materials (SRMs) 4.1.1 A certified SRM should be prepared with every batch of samples to validate analytical accuracy and recovery. 4.1.2 SRMs should be prepared in the exact manner as the unknown samples, including drying, even if the material is already dry. 76 ------- 4.1.3 The frequency of SRM preparation should be approximately 1 for every 20 unknown samples prepared. 4.1.4 The outlined extraction technique will not yield 100% recoveries for sediment SRMs. The recoveries, however, should be relatively consistent between sample batches. 4.2 Analytical Reproducibility 4.2.1 Replicate samples should be prepared to assess the reproducibility of the extraction procedure. 4.2.2 For every 20 samples prepared, one sample should be chosen to extract and analyze in triplicate. Deviation between replicate analyses should be <10%. 4.3 Procedural Blanks 4.3.1 Procedural blanks should be carried throughout the entire extraction procedure to verify that contaminants are not present in the reagents and that no contamination has occurred throughout the procedure. 4.3.2 Trace amounts of metals in the blanks can be subtracted from subsequent sample analyses (blank subtraction), but a sample batch should be rejected if 77 ------- concentrations in the blank are >10% of "average" sample concentrations. 4.3.3 One procedural blank should be prepared for every 20 samples extracted. 78 ------- ERLN CHEMISTRY GROUP STANDARD OPERATING PROCEDURE FOR INSTRUMENTAL ANALYSIS OF METALS IN SEDIMENT AND TISSUE EXTRACTS 1.0 OBJECTIVES The objective of this document is to outline the proper sample preparation and instrumental parameters for the analysis of trace metals in marine sediment or tissue acid digests. 2.0 MATERIALS AND EQUIPMENT Atomic Absorption Spectrometer or Inductively Coupled Plasma Atomic Emission Spectrometer Reagent grade Instra-Analyzed concentrated HN03 for trace metal analysis (diluted to 2M concentration) 3.0 METHODS 3.1 Standard Calibration 79 ------- 3.1.1 Estimate or determine the range of concentrations that exist within the sample analytes. This may require scanning several samples prior to standard calibration in order to approximate the range of absorbances (AA) or emission intensities (ICP) produced from the samples. 3.1.2 Prepare multiple calibration standards that bracket the expected range of sample analyte concentrations. The composition of the standard matrices (Le. acid strength and salt content) should match that in the samples as closely as possible. 3.1.3 Analyze the standards and calculate calibration equations by regression (linear or polynomial) of standard concentrations against measured standard absorbances or intensities. 3.2 Sample Dilutions 3.2.1 In section 3.1 the expected range of sample concentrations is determined. If sample concentrations exceed the upper limit of the chosen analytical technique, then the sample analytes will need to be diluted to fall within the range of standard concentrations. Sample diluent should be of the same acid composition and strength present in the sample analytes (Keep close record of the sample dilutions so that raw analytical concentrations can be dilution-corrected). 80 ------- 4.0 ANALYSIS 4.1 Sample Analysis (Unknown Concentrations) 4.1.1 Analyze the samples and record the absorbances (AA) or emission intensities (ICP). 4.1.2 Triplicate readings should be made for every element. 4.1.3 After approximately 10 (AA) or 20 (ICP) samples, several calibration standards should be re-analyzed to determine instrumental drift 4.2 Concentration Calculation 4.2.1 Calculate sample concentrations by applying the calibration equation obtained from the standard curve to the measured sample signals (absorbances or intensities). Calculate the mean and standard deviation of the individually calculated sample concentrations. 4.3 Dilution Correction 4.3.1 Calculated analyte concentrations must be dilution-corrected to obtain the true metal concentration present in the sample. The analyte concentration, in 81 ------- ug/ml, is converted to ug/g dry sample by inputing the sample prep, information into the following equation: Analyte conc.(ug/ml) X Acid volume (ml.) Sed. Cone, (ug/g dry sed.) = dry sed. wt. (g) 5.0 QUALITY CONTROL 5.1 Determination of Analytical Accuracy (Calibration check) 5.1.1 Analyze several standards as unknown samples to check the accuracy of the standard curve regression. Recoveries should be within 10% of the standard concentration. 5.1.2 Analyze a solution of known and/or certified concentration, prepared independently from the calibration standards, to determine the daily analytical fluctuation. Recoveries should be within 10% of the certified concentration. 5.2 Standard Additions (Spike Additions) 5.2.1 Standard additions are required to investigate instrumental interferences arising from differing sample solution matrices. 82 ------- 5.2.2 Select a sample whose concentrations can be matched fairly closely with a dilution of a calibration standard. 5.2.3 Prepare an acid spike (a dilution of a calibration standard) in the same acid matrix as the samples. Try to match spike concentrations as closely as possible with the sample chosen. 5.2.4 Prepare a sample spike by removing a second sample aliquot and adding the same amount of calibration standard as was used in the acid spike. Hie total volume of sample spike should also be equal to the total volume of acid used in the acid spike. 5.2.5 Analyze the sample, acid spike and sample spike as unknown samples. 5.2.6 Calculate the spike recovery using the following equation: QaMPLE SPIKE " ^SAMPLE R(%) = Qvcid spike 5.2.7 Acceptable spike recoveries fall between 80-120%. 5.2.8 One out of every 20 samples should be chosen for a standard addition. 6.0 DETECTION LIMITS 6.1 Instrument Detection Limits 6.1.1 Instrument detection limits are determined as the concentration equivalent to a signal three times the standard deviation of a blank. The limits should either be determined previously for given instrumental conditions or as part of the instrumental data analysis, and should be comparable to those listed below: 83 ------- ICP GFAA (ug/ml) (ug/L) Cu .020 1.0 Zn .005 0.1 Cr .020 1.0 Pb .050 3.0 Ni .050 2.0 Mn .010 0.5 Fe .020 2.0 Cd .005 0.5 A1 .075 Sn .050 2.0 Sb .100 2.0 As .100 2.0 Ag .020 0.5 6.1.2 Sample Detection Limits, assuming a dry weight of 2 grams and a total volume of 50 mis. (ie. sediment ultrasonic extraction method), are 25 times higher than the instrument D.L.S. Method detection limits should be calculated following the rigorous statistical procedure detailed in 40 CFR Part 136. 84 ------- |