CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999

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1 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, RESIDUES AND TRACE ELEMENTS Analyses of Polychlorinated Biphenyls and Chlorinated Pesticides in Biota: Method and Quality Assurance CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 MARIANNE CLEEMANN and GUDRUN B. PAULSEN National Environmental Research Institute, Frederiksborgvej 399, DK-4000 Roskilde, Denmark EVA STORR-HANSEN Emergency Management Agency, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark ARVID FROMBERG Danish Veterinary and Food Administration, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark An analytical method for polychlorinated biphenyls (PCBs) and chlorinated pesticides in mussels, fish liver, bird liver, and seal blubber is described. Different calibration functions were tested to establish the optimal fit to the nonlinear response of all included compounds. Detection limits and within-batch variations were determined from standard deviations of duplicate analyses of real samples. Within-batch, between-batch, and total relative standard deviations were calculated from regular use of control material. Percentage recovery was estimated by spiking samples with 3 selected PCB congeners and by spiking selected samples with all compounds included in the procedure. Quality assurance is described, covering analysis of about 450 samples divided into 38 batches. Because between-batch variation was twice the within-batch variation, the use of in-house control material and control charts is considered to be mandatory for this type of analyses. Use of certified reference material and regular participation in intercomparison exercises have demonstrated the high quality of the method and the adequacy of the quality assurance applied. Appropriate methods for analysis of polychlorinated biphenyls (PCBs) and chlorinated pesticides in environmental samples have been available for several years (1 3). Although extensive recommendations on analytical techniques have been published (4), one of the main problems has been obtaining reliable and comparable results at low levels from different laboratories. The present work describes an analytical method, the documentation of the method, and the quality assurance performed to obtain reproducible results over a long period, documented by participation in international intercomparison exercises. The work involved analyses Received January 11, Accepted by JS April 19, of about 450 samples of mussels, fish, birds, and seals originating from Greenland (5). Experimental The method includes analyses of PCBs (IUPAC No. CB-28, CB-31, CB-52, CB-101, CB-105, CB-118, CB-138, CB-153, CB-156, and CB-180), hexachlorobenzene (HCB), hexachlorocyclohexanes [α-hch, β-hch, and -HCH (Lindane)], trans-nonachlor, and DDTs (p,p -DDE, p,p -DDD, and p,p -DDT). The procedure was developed from previously described methods (3, 6, 7) and includes analyses of mussels, fish and bird liver, and seal blubber. General Precautions To avoid contamination of samples, all glass equipment was heat treated at 450 C for 6 h prior to use. Glass wool, sodium sulfate, silica, aluminum oxide, and Soxhlet inserts were rinsed with dichloromethane by Soxhlet extraction for 24 h. All organic solvents were glass-distilled grade and tested before use for compounds interfering with the analytes. Extraction and Determination of Dry Weight and Extractable Lipid Seal blubber samples were extracted by a wet, cold-blend method. Other matrixes were dried with sodium sulfate prior to Soxhlet extraction. Cleanup procedures were identical for all matrixes. Seal blubber was homogenized (Ultra-Turrax) and divided into 2 subsamples. A1gsubsample was used for determination of dry weight (dried at 105 C until a constant weight was obtained), and a 0.5 g sample was extracted 3 times by cold blending (Ultra-Turrax) with 60 ml hexane acetone (1 + 1). Before the sample was extracted, it was spiked with 500 µl of a solution containing CB-3 (5 µg/ml), CB-40 (0.3 µg/ml), and CB-198 (0.2 µg/ml). The combined extracts were dried with g anhydrous sodium sulfate. Part (10%) of the extract was used for gravimetric determination of extractable

2 1176 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 Table 1. Parameter lipid, and the remainder was concentrated to ca 1 ml in a rotary evaporator with 100 µl isooctane added as keeper. For mussel soft tissue and fish or bird liver,a 1 g subsample of the homogenized tissue was used for dry weight determination as described for seal blubber. Homogenized mussel tissue (12 g), fish liver (2 g), or bird liver (4 5 g) was ground in a mortar with 60, 12, or 40 g anhydrous sodium sulfate, respectively. The sample was spiked with 100 or 200 µl spiking solution. The dried homogenate was transferred to a Soxhlet insert, left for at least 4 h, and then extracted for 6 h with hexane acetone (4 + 1). Part (10%) of the extract was used for gravimetric determination of extractable lipid. The rest was concentrated to ca 1 ml in a rotary evaporator with 100 µl isooctane added as keeper. Cleanup GC conditions Setting Carrier gas (flow) Hydrogen (1.7 ml/min) Injector temperature 270 C Injection volume 2 µl Splitless time 1 min Septum purge 2.7 ml/min Detection 63 Ni ECD Detector temperature 300 C Makeup gas (flow) Nitrogen (46 ml/min) Retention gap Fused silica, methyl deactivated, 2.5 m, 0.53 mm id Columns (in parallel) DB-5 (J&W): 60 m, 0.25 mm id, film 0.25 µm DB-1701 (J&W): 60 m, 0.25 mm id, film 0.25 µm Initial oven temperature (period) 90 C (2 min) Initial programming rate 25 C/min Second isotherm temp. (period) 180 C (2 min) Second programming rate 1.5 C/min Third isotherm temp. (period) 220 C (3 min) Third programming rate 3 C/min Final temperature (period) 275 C (20 min) Cleanup was done through a multilayer column (1 20 cm) packed from the bottom with: 5 g aluminum oxide activated at 300 C for 24 h and deactivated with 10% water, 1 g silica activated at 160 C for 24 h, 5 g silica activated and impregnated with concentrated sulfuric acid (30 g silica + 10 ml 98% sulfuric acid), and 1 cm anhydrous sodium sulfate. Prior to use, the column was eluted with 20 ml hexane, which was discarded. The column can destroy up to 1.5 g lipid, but usually 1 g lipid is considered as the maximum load. The concentrated sample extract was quantitatively transferred to a column that was subsequently eluted with 200 ml hexane. The hexane extract was concentrated to 1 ml in a rotary evaporator with 1 ml isooctane added as keeper. The sample extract was analyzed by gas chromatography (GC) with or without internal standards. GC Conditions GC analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a Hewlett Packard 7673A autosampler. Two columns were installed in parallel through a glass T-split (6). GC conditions are described in Table 1. A chromatogram representing a sculpin liver is depicted in Figure 1. Calculations Calibration solutions were made in isooctane at 5 or 6 different concentrations adjusted to the levels found or expected in samples. For mussels, for example, the concentrations of the calibration solutions were in the range ng/ml, whereas for seal blubber, a range of ng/ml was chosen. Calibration solutions were prepared by diluting a stock solution containing ca 2 µg/ml isooctane, which was stored in ampoules in a freezer. The same stock solution was used for all analyses of the study. Fresh calibration solutions were made every month and stored in a refrigerator. CB-53 and CB-155 are appropriate internal standards in most cases. However, small amounts of impurities in CB-53 and CB-155 required that the concentration of internal standards was of the same order of magnitude as the concentrations of analytes in samples: 10 ng/ml for mussel samples, 40 ng/ml for fish liver samples, and 100 ng/ml for bird liver and seal blubber samples. These impurities interfered with CB-52 and CB-153 on the DB-5 column and, to a smaller extent, with CB-105 on the DB-1701 column. Depending on the matrix being analyzed, unknown compounds could interfere with internal standards. This problem was minimized by analyzing the samples without, as well as with, internal standards to select the internal standard that was less subject to interference. The final concentration of internal standards in the samples always equaled the concentration of internal standards in the calibration solutions. At an early stage of the study, octachloronaphthalene (OCN) was used as a time reference standard eluting late in the chromatogram. The response of OCN was not stable enough for quantitative purposes, and as OCN disclosed a still growing tendency to degrade into compounds interfering with especially CB-198, it was omitted from the standard solution. As a consequence, CB-207 was adopted as a late-eluting internal standard. In the present study, it was used as a time reference standard only, but in later studies, it was used as a quantitative internal standard as well. All calibration solutions were analyzed at least twice within each batch, and mean values were used for a piece-wise calibration at both columns. Only results within the range of the calibration curve were accepted. Sample extracts giving results above the range were diluted, and results from samples

3 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, below the range were not reported. If results from the 2 columns differed by less than 10%, the mean value was calculated; if the difference was greater than 10%, the lowest value was chosen as result in order not to overestimate the concentrations that might be influenced by interfering compounds. For the same reason, all calculations were made on basis of peak height. The possibility of interfering compounds could not be totally excluded. CBs eluting late in the chromatogram and found in low concentrations (like CB-156) were supposed to be most exposed to interference of, for example, toxaphene. However, none of the toxaphene congeners that are of importance to mammals (e.g., Parlars No. 32, 50, and 62) interfered with the analytes in the present study. Further, it was assumed that toxaphene would be present in concentrations significantly lower than those of PCBs. Results and Discussion Linearity Previous studies have shown that PCBs give a nonlinear response in analysis by GC with electron capture detection (ECD) (3, 8, 9). Storr-Hansen (3) tested different functions for the best fit to the response curve of PCBs. The test was repeated in this work to investigate lower concentrations and to include chlorinated pesticides, which appear to have nonlinear response curves as well. Figures 2 and 3 show calibration curves of CB-52 and p,p -DDE, respectively, the compounds being analyzed on a DB-1701 column. The responses were calculated on a peak height basis relative to the internal standard, CB-155. The curvature of the calibration curves is made more visible by depicting the function of the response factors (response/concentration), which ideally should be horizontal straight lines if the calibration curves were straight lines through the origin. As seen from Figures 2 and 3, this is not the case: p,p -DDE produces a calibration curve bending to the opposite site compared with the calibration curve of CB-52. According to our experience, the calibration curves degree of curvature depends not only on the ECD signal but also on the production batch of the columns. To test the calibration functions, 6 calibration solutions (1, 4, 12, 40, 120, and 200 ng/ml) and 6 test solutions (0.8, 2.4, 9.6, 24, 96, and 160 ng/ml) were prepared, and the 12 solutions were analyzed in duplicate. Three different functions were tested for calibration: the second-order function (ax 2 +bx + c), the power function (ax b ), and the piecewise calibration curve. After calibration with the 6 calibration solutions and each of the 3 calibration functions, all 12 solutions were calculated, and the calculated concentrations were compared with the assigned values. CB-28, CB-52, CB-118, -HCH, p,p -DDT, and p,p -DDE were selected as test compounds. The relative fit error was calculated as the difference between the calculated value and the assigned value relative to the assigned value. The relative fit error of the 3 functions is depicted for CB-52 in Figure 4. The relative fit error of a linear calibration curve is also included to illustrate the magnitude of the error obtained by this calibration. Figure 1. Chromatogram of a sculpin liver extract analyzed on a DB-5 column (CBs are named by their numbers only, and trans-nonachlor, by TNC).

4 1178 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 Concentration (ng/ml) Figure 2. Calibration curve and response factor (response/concentration) of CB-52 analyzed on a DB-1701 column. Unacceptable large relative fit errors were determined for all 3 fit functions for a solution of 0.8 ng/ml, the lowest level of calibration being 1 ng/ml. Consequently, quantitation below the lowest calibration level was not reported. The second-order fit function showed relatively large fit errors especially in the low concentrations (up to about 40%), whereas the power and piecewise fit functions were almost equal (Figure 4). As a first choice, the power function was the most appropriate. However, in the case of CB-105 and CB-198, the presence of interfering compounds from the internal standards (maybe from OCN) and the lack of a constant in the power function caused some problems. To avoid those p,p -DDE (DB-1701) Concentration (ng/ml) Figure 3. Calibration curve and response factor (response/concentration) of p,p -DDE analyzed on a DB-1701 column.

5 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, Concentration (ng/ml) Second-order Figure 4. Dependence of relative fit error [100(calculated value assigned value)/assigned value] on concentration levels of CB-52 analyzed on a DB-5 column. Six solutions (1, 4, 12, 40, 120, and 200 ng/ml) were used for calibration according to 4 functions: power (ax b ), piecewise, second-order (ax 2 +bx + c), and linear (ax + b). Concentrations of the 6 calibration solutions and of 6 test solutions (0.8, 2.4, 9.6, 24, 96, and 160 ng/ml) were calculated according to the different calibrations; the relative differences between calculated and assigned values are shown for the 12 solutions (analyzed in duplicate). problems, the piecewise calibration was regarded as the safest choice and has since been used for more than 450 samples without any problems. Detection Limits The detection limit (DL) can be calculated from repeated analyses of the same matrix (10): DL = t (N-1,1-α = 99) s where t is a students t-distribution, N is the number of analyses, and s the standard deviation. The concentrations of the analyzed compounds shall be less than 5 times DL (10). In this study, DL was calculated as: value was chosen as DL. These calculations were conducted for all compounds in the 2 matrixes, mussels and sculpins. About 90% of DLs obtained this way derived from the concentration of the lowest calibration level. A few compounds (HCB, α-hch, p,p -DDE, and trans-nonachlor) did not occur in sculpin liver samples made in duplicate at concentrations less than 5 times the estimated DLs. In these cases, DL was obtained only from the lowest calibration level. In general, bird liver and seal blubber revealed high concentrations of the compounds analyzed, and DLs were determined from the lowest calibration level only. Taking into account the differences in amount of matrix and in calibration range, the different DLs are summarized in Table 2. DL=3s w where s w was the standard deviation typically obtained from analyses of 7 8 duplicates. The number of duplicates with measurable concentrations <5 DL was unfortunately low. For the compounds in question, only 1 5 (often 5) duplicates of the mussel samples and 4 9 (often 8 9) duplicates of sculpin liver fulfilled the demand. Because results below the lowest calibration level could not be accepted, the matrix concentration equal to this level was calculated. This value was compared with the first estimate of DL (based on standard deviations), and the highest Table 2. Detection limits for PCB congeners and chlorinated pesticides Matrix Amount of sample, g Detection limit, µg/kg (w/w) Blue mussels Bird liver Fish liver Seal blubber

6 1180 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 Table 3. Relative standard deviations of real samples and in-house control samples Real samples (duplicates from 8 batches of shorthorn sculpin liver) Control samples (duplicates from 38 batches of sand eel oil) Analyte Concn range, µg/kg No. of db a s w b, % s w b, % s b c, % s t d, % Mean concn, µg/kg CB CB CB CB CB CB CB CB CB CB Sum of CBs e HCB α-hch HCH HCH Sum of HCHs e p,p -DDE p,p -DDD p,p -DDT Sum of DDTs e trans-nonachlor CB-3 recovery, % CB-40 recovery, % CB-198 recovery, % Lipid, % Dry weight, % a b c d e Only values above the detection limits were included. s w = within-batch variation calculated from s 2 w =(s 2 1 +s s 2 n )/n, where s 1,s 2..s n are the estimated standard deviations of the n replicates. s b = between-batch variation calculated from s 2 b = [(m 1 m v ) 2 +(m 2 m v ) 2..+(m n m v ) 2 ]/(n 1) ½s 2 w, where m 1,m 2..m n are the mean values of each duplicates, while m v is the overall mean value of 2n results. s t = total standard deviation includes within- and between-batch variation, calculated from s 2 t =s 2 w +s 2 b ;s t,s w, and s b are all calculated relative to the concentration levels. The sums are based on results above detection limits. Relative Standard Deviations Relative standard deviations (RSDs) within a batch of sculpin liver samples were determined from duplicate analysis of the samples. The standard deviations covering within- and between-batch deviations were calculated on the basis of internal control material. The mean values and the RSDs for sculpin liver and control material (sand eel oil) are given in Table 3. Sculpin liver samples analyzed in duplicate showed a within-batch variation of 2.6 6%, with some few exceptions: CB-105 and CB-156 had RSDs of 8.4 and 11.6%, respectively, mainly caused by very low concentrations and interference from other compounds (CB-105 may interfere with CB-132 and CB-141, while CB-156 interferes with CB-171 and CB-202 [1]). p,p -DDT can degrade in the GC injector, and the degradation may be reflected in a slightly elevated RSD of 7.2% The in-house control material of sand eel oil had concentration levels of times the concentrations found in sculpin liver samples. However, for most compounds, the relative within-batch variations were of the same order of magnitude (3.5 6%) as found in sculpins. The exceptions were CB-105 and CB-156, showing RSDs of 6.6 and 15.4%, while CB-28, CB-31, and β-hch showed slightly elevated values of 8.3,

7 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, Table 4. liver Recoveries of polychlorinated biphenyls and chlorinated pesticides from seal blubber and glaucous gull Seal blubber Glaucous gull liver Analyte Mean (n = 2) recovery, % STD, % a Mean (n = 3) recovery, % STD, % CB CB CB CB CB CB CB CB CB CB HCB α-hch β-hch HCH p,p -DDE p,p -DDD p,p -DDT trans-nonachlor CB CB CB a STD = standard deviation calculated relative to the mean recovery. 9.4, and 7.8%, respectively. The elevated RSDs may be explained by interfering compounds. Although the patterns of pollutants in fish may be quite similar, the extent to which different compounds interfere with each other are different and may explain the differences between the sculpin liver and the sand eel oil. The determination of extractable lipid showed comparable within-batch variations of 2.8 and 3.7% for sculpin liver samples and control samples, respectively. Control samples showed between-batch variations of almost twice the magnitude of the within-batch variations, giving total variations between 6 and 11% for most compounds. CB-28, CB-31, CB-105, CB-156, and -HCH showed total variations between 12 and 21%. Total variations in the present work are the result of the work of 7 different technicians, applying 2 different GCs (both Hewlett Packard 5890), 4 different pairs of GC columns, 2 different internal standards at 3 different concentration levels, and analyzing 38 batches over 30 months. Between-batch variation was considerably higher than within-batch variation (Table 3). This result stresses the importance of keeping the analyses under control by using an internal reference material. Recovery To estimate the efficiency of extraction and cleanup, a solution containing CB-3, CB-40, and CB-198 was added to all samples before extraction. CB-40 and CB-198 were added in concentrations ranging from 2 µg/kg for mussels to about 300 µg/kg for seal blubber. However, because of its low ECD response, CB-3 was added in concentrations 20 times higher. Whether recoveries found in this way were representative for all analyzed compounds was tested by spiking a sample of seal blubber (in duplicate) and a sample of glaucous gull liver (in triplicate) with all compounds. The seal blubber was spiked at about 200 µg/kg seal blubber, making a spike of times the original concentration levels. The glaucous gull liver was spiked at about 15 µg/kg liver, which was from 0.5 to 48 times the original concentrations. Unspiked samples were analyzed in triplicate along with the spiked samples. The results are presented in Table 4. Recoveries of CB-40 and CB-198 ranged from 85 to 104 % and were comparable with the % recoveries found for all other compounds analyzed, except for HCB and the HCHs. Thus, with these exceptions, it was found appropriate to use the recovery of CB-40 and CB-198 spiked to every single

8 1182 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 Figure 5. X-chart of CB-52: mean concentrations of duplicates analyzed in a control material (sand eel oil) over a period of 30 months. Solid, horizontal line indicates the average of 7 batches analyzed with either CB-53 or CB-155 as internal standards. Dotted lines represent average ±2s(warning limits). Bold, solid lines represent average ±3s (limits of acceptance). Batches 9 and 18 were reanalyzed. sample as an estimate of the recovery of the single compounds. Analysis of CB-40 quite often was interfered with by other compounds, and recoveries of CB-198 was accepted as the most reliable estimate of the general recovery. The lower chlorinated PCBs might, however, exhibit slightly lower recoveries. HCB and HCHs gave lower recoveries of 76 97%, which, however, was higher than the 68 80% found for CB-3. These compounds are relatively volatile compared with higher chlorinated PCBs and DDTs, and the low recoveries are probably due to evaporation losses. Recovery of CB-3 was used as a warning of possible low recoveries of HCB and HCHs. All 3 recovery standards were included in the control chart and used for judging whether the single samples were in control. The limits of acceptance were % for CB-3, % for CB-40, and % for CB-198. The recoveries of the 3 standards were not judged to be safe enough for correction of the results but were always reported. Quality Assurance To avoid cross-contamination between high and low polluted samples, one matrix only was analyzed simultaneously. Each batch included an equal number of samples from each sampling area, and all samples from a batch were analyzed in random order. In-house control material was included in all batches. Certified reference materials were regularly analyzed, and the laboratory participated in 1 or 2 intercomparison exercises per year. As a general rule and so as not to make any mistakes, 1 or 2 technicians together analyzed all samples in a batch right from the beginning to the last calculations. Afterwards, another person controlled all calibrations and calculations and approved the results of a batch. Table 5. Certified reference materials Name Matrix Source Certified compounds CRM 365 Isooctane BCR a PCBs, 5 20 mg/l CRM 350 Mackerel liver oil BCR a PCBs, µg/kg SRM 2261 n-hexane NIST b Chlorinated pesticides, 2 mg/l SRM 1588 Cod liver oil NIST b Chlorinated pesticides, µg/kg a b Community Bureau of Reference, Brussels, Belgium. National Institute of Standards and Technology, Gaithersburg, MD.

9 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, In-House Control Material A sand eel oil was used as in-house control material and analyzed in duplicate in all batches: 400 mg of the oil was spiked with recovery standards, mixed with anhydrous sodium sulfate, extracted, and cleaned up like real samples. Each batch further contained a blank sample and 12 samples, one of them analyzed in duplicate. Results from analyses of in-house control material were used for making control charts (11). At the beginning, the first 7 duplicates were used to determine mean concentrations, warning limits (mean ± 2 s), and action limits (mean ±3s). Control charts were produced for all compounds, including the 3 recovery standards, and for the lipid determination. Standard deviations used for the limits of the control charts were a bit smaller than those depicted in Table 3, which includes results from 38 batches. For the results of a given batch to be approved, all compounds analyzed in the in-house control samples had to be within the action limits. However, if a few compounds only were outside the limits, whereas the recovery and the lipid determinations were within the limits, these few compounds were omitted in the report of the results of the specific batch. Two of 38 batches were re-analyzed. A number of single compounds found outside the action limits were not reported. Unfortunately, the sand eel oil contained some unknown compounds that interfered with the internal standards, especially CB-53. This constituted a problem when the concentration of internal standards were lowered in order to follow the concentration levels of the pollutants in the less polluted samples. The internal standard used for calculating the results of a batch was always chosen as the one interfering least with the unknown peaks of the real samples. Thus, in some cases, the control samples interfered with up to 10% of the peak height of the internal standard chosen this way. Obviously, this interference changed the levels of the control charts and resulted in revised action and warning limits calculated separately for samples analyzed with internal standards in concentrations of 10, 40, and 100 ng/ml, respectively. Figure 5 depicts a control chart of CB-52, representing samples analyzed with internal standard concentrations of 40 ng/ml. This experience made it obvious that one in-house control material hardly can cover the wide range of concentration levels found in mussels, fish, birds, and seals. The ideal solution would be to find another in-house control material better suited for mussel samples. Certified Reference Materials For every 5 6 batches, 2 3 certified reference materials were included in triplicate in the batch. The reference materials used are described in Table 5. The mean of the results obtained were tested statistically on the basis of the certified values, the given variation of the certified values, the known variation of the obtained results, and a t-distribution using a 95% confidence interval. Except for a single batch displaying significant deviation for HCB and trans-nonachlor, all compounds were within the calculated limits. Intercomparison Exercises Our laboratory participated in intercomparison exercises arranged by the International Council for Exploration of the Sea, the Intergovernmental Oceanographic Commission, and the Oslo and Paris Commissions (ICES/IOC/OSPARCOM) (9, 12, 13) and by the Quality Assurance of Information for Marine Environmental Monitoring in Europe (QUASIMEME) (14 16). Along with analyses of the 38 batches mentioned above, the laboratory analyzed the QUASIMEME round 5 (November 1995), round 6 (November 1996), and round 8 (May 1997) samples. Of the results obtained in the 3 QUASIMEME rounds, 93, 96, and 100%, respectively, were within the limits defined as satisfactory by QUASIMEME (17). Only a minority of the laboratories participating in the QUASIMEME exercises repeatedly obtained such good results. Conclusions Many precautions were taken to obtain the results described above. The main objectives are reliable calibration solutions, multilevel calibrations, use of internal standards for identification and quantitation, control of compounds interfering with the internal standards, control of recovery for all samples, control of all calculations, use of in-house control material, use of certified reference materials, and regular participation in relevant intercomparison exercises. However, because the between-batch variation was about 2 times the within batch variations, use of an in-house control material is considered to be a mandatory part of the daily work, whereas participation in intercomparison exercises is essential on a regularly basis. The total RSDs were 6 11% for the majority and 12 21% for a minority of the compounds. These results were considered satisfactory. The sand eel oil used as in-house control material disclosed some disadvantages in combination with the internal standards, possibly causing estimates of the RSDs to be somewhat too high. For a wide range of concentration levels like those found in mussels, fish, birds and seals, at least 2 in-house control materials of different concentrations ideally should be used. However, use of control materials in this study and participation in intercomparison exercises have demonstrated that the method combined with the quality assurance is robust and able to produce results with a known and stable uncertainty. Acknowledgments We thank Annegrete Ljunqvist, Dorthe Thil Hansen, Hanne Høgsberg Knudsen, Lars Renvald, Lone Hertz, Inge Merete Worsøe, and Michael Zeeberg Nielsen for their skilled technical assistance and the Department of Arctic Environment, National Environmental Research Institute, for collecting the samples. Furthermore, we thank the Danish Environmental Pollution Agency for funding the great number of

10 1184 CLEEMANN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 analyses that made this assessment of the analytical method possible. References (1) De Boer, J. (1988) Chemosphere 17, (2) Jansson, B., Andersson, R., Asplund, L., Bergman, Å., Litzén, K., Nylund, K., Reutergårdh, L., Sellström, U., Uvemo, U.-B., Wahlenberg, C., & Wiedeqvist, U. (1991) Fresenius J. Anal. 340, (3) Storr-Hansen, E. (1991) J. Chromatogr. 558, (4) Smedes, F., & de Boer, J. (1997) Trends Anal. Chem. 16, (5) Cleemann, M., Fromberg, A., Larsen, H., Paulsen, G.B., Poulsen, M.E., Pritzl, G., Riget, F., & Storr-Hansen, E. (1997) in Arctic Monitoring and Assessment Programme, Greenland , Marine Environment, Chapter 3, Terrestrial and Freshwater Environment, Environmental Project No. 356, Danish Environmental Protection Agency, Copenhagen, Denmark (6) Storr-Hansen, E. (1991) Int. J. Environ. Anal. Chem. 43, (7) Storr-Hansen, E., & Spliid, H. (1993) Arch. Environ. Contam. Toxicol. 24, (8) Wells, D.E., Maier, E.A., & Griepink, B. (1992) Int. J. Environ. Anal. Chem. 46, (9) De Boer, J., Duinker, J.C., Calder, J.A., & van der Meer, J. (1992) J. AOAC Int. 75, (10) Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., & Budde, W.L. (1981) Environ. Sci. Technol. 15, (11) De Boer, J., Stronck, C.J.N., Traag, W.A., & van der Meer, J. (1993) Chemosphere 26, (12) De Boer, J., van der Meer, J., Reutergårdh, L., & Calder, J.A. (1994) J. AOAC Int. 77, (13) De Boer, J., van der Meer, J., & Brinkman, U.A.T. (1996) J. AOAC Int. 79, (14) De Boer, J., & Wells, D.E. (1996) Mar. Pollut. Bull. 32, (15) De Boer, J., & Wells, D.E. (1997) Mar. Pollut. Bull. 35, (16) De Boer, J. (1997) Mar. Pollut. Bull. 35, (17) QUASIMEME Performance Studies, Year 3, June 1998 to May 1999, February 4, 1998, Aberdeen, UK