Anal. Chem. 1988, 60, 2760-2765
2780
Table I. Standard Deviations of Prediction Errors, See Discussion" method
Yl
Y2
ANFE
unreduced
f=11 SIMl f = 15 SIMP f=15
reduced
0.290 09 0.363 43 0.004 77 0.002 45 0.003 64 0.001 90
0.804 58 0.576 50 0.006 14 0.002 17 0.003 24 0.001 94
data set
unreduced reduced unreduced
reduced
" f means degrees of freedom. For the ANFE data set, units are parts per million and Y1 represents phenanthrene and Y2 represents anthracene.
inear i.e. that it can be expressed as chromatographic profiles multiplied by spectra. Another possibility is that data reduction eliminates noise due to drift in retention times. The scores are a linear combination of the spectra of the eluting compounds and are not much affected by a small change in retention time. Figure 6 shows the prediction error s u m of squares (PRESS) as a function of the volume of the interfering third peak for the data sets SIMl and SIM2. It is evident that the prediction deterioration is not the same for the two methods and that neither method shows better predictive properties in all cases. Figure 7 is a plot of the logarithm of the probability that the
sample chromatogram is of the same kind as the calibration set versus the volume of the interfering third peak. Closer inspection reveals that the rejection of outliers is sharper for unreduced data, but if only the region down to the rejection limit (which usually is 1%or -2 in Figure 6) is considered, the difference between the two methods is slight. Above all, Figures 6 and 7 show that calibration with reduced data indeed handles outliers just as well as without data reduction. In fact, data reduction gives smoother and more monotone curves for both prediction deterioration and outlier rejection. To summarize, the data reduction scheme proposed here seems to give good and stable predictions as well as robust handling of outliers.
LITERATURE CITED ( 1 ) Wold, S.; Geladl, P.;Esbensen, K. J . Chemom. 1987, 1 , 41. (2) Otto, M.; Wegschelder, W.; Lankmayr, E. P. Anal. Chlm. Acta 1985, 171, 13. (3) Vandeginste, 6. G. M.; Leyten. F.; Gerritsen, M.; Noor, J. W.; Kateman, 0.;Frank, J. J.: Chemom. 1987, 1 , 57. (4) Llndberg, W.; Ohman, J.; WoM, S. Anal. Chem. 1986, 58, 299. (5) Wold, S.; Esbensen, K.; Geladi. P. Chemom. Intell. Lab. Syst. 1987, 2, 37. (6) Kennedy, J. W.; Gentle, J. E. Statisticel Computlng; Marcel Dekker: New York, 1980; p 112.
RECEIVED for review December 23,1987. Accepted September 22, 1988.
Sequential Determination of Biological and Pollutant Elements in Marine Bivalves Rolf Zeisler* and Susan F. Stone Center for Analytical Chemistry, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 Ronald W. Sanders Pacific Northwest Laboratory, Richland, Washington 99352
A unlque sequence of Instrumental methods has been employed to obtain concentratlons for 44 elements In marlne blvalve tlssue. The techniques used were (1) X-ray fluorescence, (2) prompt gamma acthratkn analysts, and (3) neutron actlvation analysls. I t Is posslble to use a slngle subsample and follow lt nondestructhrelythrough the three Instrumental analysls techntques. A final radlochemlcal procedure for tln was also appUed after compktlng the Instrumental analyses. Cornparkon of results for elements determlned by more than one technlque In sequence showed good agreement, as dld results from certHled reference material samples analyzed along wlth the samples. The concentratlons found In the bivalve samples ranged from carbon at more than 50% dry welght down to gold at several mlcrograms per kilogram.
When studying the elemental composition of biological and environmental samples, analytical chemists are frequently confronted with the limitations of a particular analytical technique when applied to unique or small samples. Generally,
any analytical technique or even several techniques can only determine a fraction of the elements contained in the sample. It can be assumed that most of the elements in the periodic table occur at various levels in every biological or environmental matrix; therefore, an analytical technique or combination of procedures that covers all elements may be desirable. This goal, on the one hand, is nut attainable because of the cost involved, the analytical sophistication and skill required, and frequently the amount of sample necessary to determine low levels of trace elements or a sizable number of elements in destructive analysis procedures. On the other hand, knowledge about the role and fate of trace elements is limited to about 30 elements. These include the major and minor constituenta in biological matrices, mineral elements, essential trace elements, and a small number of elements that have known adverse effects in biological and environmental systems at trace levels. Commonly, all of these elements are not determined simultaneously or even nearly completely in several aliquota of a given material, let alone the elements that are rarely or never determined. Consequently, the majority of the elements are little-known and are not considered in
0003-2700/88/0360-2760$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
evaluations of biological and environmental samples. However, increased knowledge of elements in biological and environmental samples is needed to assess health issues and to study biological pathways and environmental source contributions. T o gain the maximum information from an individual sample, the analyst must develop new and comprehensive analytical approaches. The interrelations between individual elements and combinations of elements, the greatly varying concentrations among individual specimens, and the need for measurements of ultratrace as well as major constituents in the same sample require a challenging array of analyses. Recently, one approach utilizing a maximum of four analytical techniques for the determination of up to 29 elements in human livers has been demonstrated successfully in a program for specimen banking (1). In another instance, a combination of techniques has been described to show distributions of up to 48 elements in sediments or soil samples (2). In the latter, a number of analytical techniques and laboratories were needed, and presumably several different aliquots of the sample were used for the analyses. In many instances, only detection limits could be given, and calculated concentrations had to be used for noble gases. Of course, the involvement of many techniques and analysts can be ideal to assure the quality of the output when it is used to obtain several independent results for an analyte, e.g., in the certification of reference materials (3). However, the consumption of sample may be rather large, and new requirements on the quality of the sample, e.g., homogeneity and stability, are added. In the aforementioned specimen bank program only limited amounts of samples are available for analysis since the bulk is preserved for future analyses. Here the need for several aliquots to obtain the initial set of results prevents the use of repetitive analyses for quality assurance, and the frequent withdrawal of banked samples for the application of improved methodology and stability studies is not possible. T o overcome the above limitations, a new analytical technique is needed that in principle allows the determination of all elements in a single small sample. Such a technique exists only in theory. To advance toward the ideal technique, we have constructed an analytical procedure that utilizes a sequence of nondestructive, instrumental multielement techniques before chemistry is applied and the sample is consumed in a final assay. These techniques are X-ray fluorescence with “backscatter fundamental parameter” corrections (XRF-BFP), neutron-capture prompt gamma activation analysis (PGAA), and neutron activation analysis with instrumental (INAA) and radiochemical (RNAA) steps. This combination f u l f i i many of the requirements for the desired new procedure: (1)each individual technique can in principle assay all elements; (2) the elemental quantitation is feasible from major components to ultratrace constituents; (3) the techniques are nondestructive; (4)the procedures provide for internal quality control, e.g., via independent determinations of selected elements; and (5) only validated techniques that are suitable for routine analysis are used. This procedure has been implemented for the high-accuracy analysis of marine samples, specifically, bivalve tissues.
EXPERIMENTAL SECTION Sequential Analysis Scheme. The sequence followed in this procedure is illustrated in Figure 1. Only a single 250-mg aliquot of freeze-dried tissue is needed to obtain quantitative results on the listed elements. This sample is usually pelletized, and four Assays are first performed by using the XRF-BFP technique. Then, another size pellet is formed from the same sample aliquot and PGAA is applied to it. After the collection of the prompt gamma spectra, the pellet is submitted to a short irradiation followed by two decay and counting cycles. After sufficient decay
7XRF - BFP
2781
Si, P, S, CI, K, Ca, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Zr, Mo, I, Ba, Pb
H, B, C, N, Na, P, S, CI, K, Cd
Na, Mg, AI, CI, K, Ca, V, Mn, I
IRRADIATION
1
Sc, Cr, Fe, Co, Zn,As, Se, Br, Rb, Sr, Mo, Ag, Cd, Sb, Cs, Ba, La, Ce, Sm,Eu, Hf, Au, Hg, Th
1
Sn
1
INAA - LONG IRRADIATION
1
RADIOCHEMICAL SEPARATION
Figure 1. Sequential analysis procedure. A single 250-mg ailquot of freeze-dried tissue is carried through the indiiiduai analytical methods In sequence to obtaln quantitative results for 45 elements. Titanium is not reported In this work because implements containing thk element are used.
for safe handling, the pellet is submitted to a long irradiation followed by two decay and counting cycles. All the information that can be obtained nondestructively has been recorded, and the sample is then submitted to a radiochemical separation. We have considered for this work only those elements that can be analyzed in the bivalve tissues at their typical levels. Titanium was excluded from the reported elements because titanium implements were used in this work for the sample preparation. When the procedure was applied to actual bivalve samples, greatly differing concentrations of several constituents were found. The procedure shows great flexibility toward those differences and, in addition,could be adjusted at various steps to provide optimum sensitivity for specific elements. Because it is nondestructive, except for the last step, most step can be repeated and the sample can be analyzed under more optimum conditions to enhance the sensitivity in specific instances. Additional elements can be occasionally quantified in the investigated sample type due to the more universal capacities of the applied techniques. Therefore, with appropriate adjustments, the procedure can be applied to other biological and environmental materials to analyze a similar array of elements. X-ray Fluorescence Procedure. The energy-dispersiveXRF technique is suitable for all elements from 2 = 11 to 92 with 2-dependent, but almost equal, sensitivity. This technique has been developed recently to be applicable to matrices of unknown or poorly defined compositions. For this, a new approach to fundamental parameter calculations has been devised that makes use of incoherent and coherent backscatter intensities from the excitation radiation (4,5).The backscatter originates from all sample constituents, and thus provides information on total sample mass as well as bulk sample composition. By appropriate use of scatter intensities in fundamental parameter matrix calculations, accurate analyea of “unknown”samples can be obtained without prior knowledge of the sample matrix. The backscatter with fundamental parameter (BFP) procedure is particularly applicable for biological and environmental matrices. A Kevex 0810A (Kevex Corp., Foster City, CA) was used for computer control of time, voltage, and current during the XRF analyses. Excitation employed titanium, zirconium, silver, and gadolinium Ka,P X-rays from a secondary source in sequence. Analysis settings were as follows: titanium, 375-s count at 40 keV and 20 mA, zirconium, 3OOO-s count at 40 keV and 20 mA, silver, 2000-s count at 45 keV and 20 mA; gadolinium, 750-s count at
2762
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
70 keV and 20 mA. Detection was accomplished with an energy-dispersive system employing a Si(Li) semiconductor detector with a 1.2 X lob m thickness beryllium window. The resolution was 180 eV at 6.4 keV, and efficiencies were greater than 85%. The instrument allowed only the backscatter caused by the sample to reach the detector. A multielement, thin-filmsensitivity curve was used as the only elemental calibration for analysis. Data analysis utilized the SAP3 computer code (6). Biological reference materials were routinely used to monitor the performance of the instruments and programs. Prompt Gamma Activation Analysis Procedure. The PGAA technique has widely varying sensitivitiesfor all elements from hydrogen to uranium, which depend on capture cross sections and excitation levels. More than 20 trace elements have been determined in environmental and geological materials (7). However, its application is more limited in biological matrices because of the concentration levels involved and high capture gamma activities of the matrix components, particularly hydrogen. In the case of marine tissues, the light matrix elements, as well as several major constituents and trace elements, can be determined, thus complementing the capabilities of the other techniques used. The PGAA facilities at the National Institute of Standards and Technology Research Reactor (NISTR) (8) were used. This included irradiations with an external thermal neutron beam (fluence rate 2 x 10l2n-m-24)and prompt measurements (online) with a high-resolution y-ray detection system consisting of a 24% efficiency, 1.9-keV full width at half-maximum (fwhm) germanium detector with active and passive shielding. Spectral data were collected with a Nuclear Data micro multichannel analyzer (Nuclear Data Inc., Schaumburg, IL)/VAX 730 (Digital Equipment Corp.) system utilizing a 16834 channel analog to digital converter (ND 581 ADC) at 0.5 keV/channel resolution. The pelletized samples were sealed in Teflon or polyethylene film and placed into the neutron beam for 10-14 h for assay of the emitted y-rays. Quantitation was accomplished by using tabdated pure element prompt y-ray emission rates (9) and appropriate correction factors for background and blank contributions from shielding and sample containers. Instrumental Neutron Activation Analysis Procedure. INAA was the third technique applied to the samples. The capabilities of INAA for the determination of elemental concentrations in biological samples are well known, and the procedures used at NIST have been described previously (IO). Besides providing data on many major elements, this technique is especially useful for the detection of elements, such as the rare earth elements, at trace levels that cannot be measured by the other two techniques. The pelletized samples were sealed in clean polyethylene for irradiation. Samples were irradiated at the NISTR in the RT-4 pneumatic tube facility, at two different fluence rates: 1.38 X 10" n,m-2.s-1 and 2.76 X 10" n.m%--'. Two irradiations were performed on each sample, with counting and decay times designed to optimize the simultaneous quantitation of sets of nuclides with different half-lives. After irradiation,the samples were transferred to nonirradiated polyethylene bags for counting. High-resolution y-ray detection systems combined with sample changers and computer-based multichannel analyzers were used. The detectors have a resolution better than 1.9 keV fwhm and efficiencies greater than 23%. The spectral data were collected with Nuclear Data multichannel analyzers using 8 192 channels at 0.2 keV/channel resolution. Automated peak searches were accomplished with Nuclear Data software. Comparator standards were used for quantitation. These standards were prepared from primary solutions of high-purity metals or compounds in ultrapure water or ultrapure acids that were obtained via sub-boiling distillation (11). These solutions were mixed together in suitable proportions. The multielement and single-element standard solutions of appropriate concentrations were pipetted onto filter papers (Whatman 41,55-mm diameter) with an Eppendorf Repeater pipet. After drying in air at room temperature, the filter papers were pelletized and sealed in polyethylene. The comparator analysis was performed with Nuclear Data activation analysis software. Radiochemical Procedure. The final application of a radiochemical separation step limits this technique in this scheme
to elements that form nuclides with long half-lives. Although radiochemistry can provide additional sensitivity for elements determined earlier in the sequence and possibly make some others detectable, we have limited this effort to the determination of tin, a very important element in the marine environment. A previously developed procedure was used (12),in which the sample pellet, with additional carriers, is digested in acids. Tin is separated from the matrix activities by a liquid/liquid extmction as SnI, in toluene and then back-extracted into an aqueous solution of Na2EDTA. The procedure is quantitative (299.5% chemical yield). The activities of the llSmSnisotope or its -'lsmIn daughter are assayed by using instrumentation as above. Sample Preparation. Marine bivalves were collected from Narragansett Bay, RI (more than 5000 organisms) and recently from 30 collection sites (60-100 organisms from each) as part of the National Status and Trends (NS&T) Program (13). The species involved were Mytilus edulis, Mytilus californianus, and Crassostrea virginica. After the organisms were scrubbed, rinsed, and cooled to dry ice temperatures, they were shipped to NIST and then stored in liquid nitrogen vapor (-150 "C)in the pilot National Environmental Specimen Bank (14). The organisms had been separated into batches and sealed in Teflon bags. Each batch consisted of 50-60 mussels or 30 oysters. One batch from the Narragansett collection and two batches of mussels and four batches of oysters from the 1985 NS&T collection were selected for analysis. After a batch was selected for analysis, the bivalves were shucked at -5 "C. A shucking implement with a titanium blade and Teflon handle was employed. After the shell was opened, the contents were scooped out with the titanium instrument and placed in clean Teflon bags for storage in liquid nitrogen until the tissues were homogenized. A cryogenic homogenization procedure (15)was used for the next step of sample preparation. The vessels used for prefracturing and grinding were constructed of Teflon to minimize contamination from trace metals. Disk mills of two different capacities (0.2 and 1kg) were used to prepare the mussel homogenate. The resulting fine powder of fresh frozen tissue has been shown to have adequate homogeneity so that 1-g subsamples are representative of the bulk sample (16). Following homogenization, portions of the powder were subsampled into Teflon jars and stored at liquid nitrogen vapor temperatures. Just prior to analysis, a subsample was removed from storage and freeze-dried. This dry material was then pressed into pellets with a KBr pellet press (Perkin-Elmer Corp., Norwalk, CT) to prepare it for the respective analytical techniques. Quality Assurance. Although the sequential analysis procedure provides for internal quality assessment on each individual sample through multiple determinations of several of the elements, standard reference materials (SRMs) and certified reference materials (CRMs), namely oyster and mussel tissues, were used during the initial evaluation of the new procedure. Several marine SRM and CRM samples were carried through the sequence of techniques to assess the performance of the scheme. During sample analysis, at least one sample of a reference material was used with each set of samples. These materials do not undergo any sample preparation except drying and pelletizing. In addition to the marine bivalve tissues, we have used other biological reference materials to obtain more results for certain certified elements. RESULTS AND DISCUSSION The performance of the sequential analysis scheme was tested via the analyses of the National Institute of Environmental Sciences (NIES) certified reference material no. 6 mussel tissue powder (17)and SRM 1566 oyster tissue (18). The results of these analyses are summarized in Table I. These data indicate the elemental coverage by the individual techniques and the between-technique agreement, as well as comparability with the corresponding certified values. Clearly, the three techniques complement each other in an ideal way. X-ray Fluorescence Results. The applied XRF-BFP procedure is not suitable for the determination of the light elements up to 2 = 13, Silicon (2= 14) shows large uncertainties in the results due to the high background under its
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
2763
Table I. Elemental Concentrations for Selected Reference Materials (Concentrations Based on Dry Weight) ele-
ment
unit of concn
XRF
H B C N Na
NIES CRM mussel PGAA INAA 78000 f 5000* 13.0 f 1.4 540000 f 30000I 74000 f 3000 9200 f 1300 9600 f 200 1600 f 400 245 f 6
Mg A1
Si P S
540 f 220 8900 f 1100 7770 f 30 12500 f 100 12200 f 500
Cl K
17500 f 100 17900 200 5400 f 1400 5900 f 100 1600 f 100
Ca
sc v
Cr Mn Fe co Ni
cu Zn
As Se Br Rb Sr Zr Mo
*
108 f 1 9.3 f 0.3 1.33 f 0.04
2.81 f 0.05 16.7 f 0.4 1.2 f 0.5
Ag
Cd Sn Sb I Cs Ba La
0.75 f 0.08
4.9 f 0.3 106 f 6 9.2 f 0.5 (1.5)
(17)
1.3 f 0.2 21 f 2
27 f 3
5100 f 300
1100 f 300 269 i 6
1280 f 90
580 f 130 7500 f 300 9000f300 7300f100
0.63 f 0.07 16.3 f 1.2 158 f 8 (370) 0.93 f 0.06
2.54 f 0.06 20.9 f 2.6
0.76 f 0.05 4.62 f 0.09 11.5 f 2.2
9400 f 300 9700 f 300 1600 600
*
14.8 16.2 f 1.0 191 f 7
(8100) (7600) 10400 f 200 8600 f 500 1600 f 300 72.3 f 0.6 2.7 f 0.2
(loo00) 9690 f 50 1500 f 200 89" 2.3 f 0.1
0.76 f 0.03 15.0 f 0.4 209 f 2 312 f 3
0.69 f 0.27 17.5 f 1.2 195 34 (400) 1.03 f 0.19
0.97 f 0.44 59.4 f 1.8 860 f 30 13.6 f 0.5 2.28 f 0.18 58.8 f 2.1
0.82 f 0.03
854 f 7 13.2 f 0.1 2.16 f 0.02
63.0 f 3.5 852 f 14 13.4 f 1.9 2.1 f 0.5
4.02 f 0.12 16
4.45 f 0.09 10.36 f 0.56
10.6 898 f 14
(10.2) 890 f 90
2.8 f 0.3
3.5 f 0.4
(55)
4.40 f 0.42 10.3 f 0.6 2.0 f 0.3 10.9 3.3
* 0.4
6.5 f 1.5 (2.8)
27 f 2 56
Sm Eu Hf Au 1.8 f 0.7
16 f 3 516 320 f 10 0.43 f 0.02 65.7 f 1.3 15.4 f 0.4
510 260 f 20 0.32 f 0.02 37.4 f 1.3 6.6 f 0.4 8.5 f 1.1 3.5 f 0.5 58 f 8
certified"
5400 f 200
12 f 3
Ce
Hi3 Pb Th
10000 f 300 2100 f 100 (220)
17000 f 400 5700 f 600 5400 f 200 1300 f 300 1300 f 100 43.0 f 1.3 1O.6Oc
1.24 f 0.03 4.7 f 0.2 109 f 2 9.9 f 0.1 1.51 f 0.10 108.3 f 0.6
SRM 1566 oyster PGAA INAA
XRF
70700 f 300 9.2 f 0.3 49oooo f 7oooo 55000 f 2000
(7700)
0.59 f 0.05 15.6 f 0.2 162 f 5 328 f 3
17.0 f 0.2 160 f 1
certified"
58 f 8
(50) 0.91 f 0.04
38.7 f 1.4
11.4 40 f 2
20d
57 f 15 0.48 f 0.04 (100)
"Certified concentrations and their uncertainities, as well as information values 0,are given as defined in the Certificates of Analysis (17, 18). *Uncertainitiesare 1s sample standard deviation. 'Limits of detection are given as defined in ref 19. dConcentrationsare literature values (20).
Ka,@lines and their low energy, which is more susceptible to matrix effects. The elements P through Ca (2= 15-20) can be determined with sufficient precision due to both lower background and higher signal intensity. The accuracy obtained for these elements is excellent when compared to the results from the other two techniques and certified concentrations. This demonstrates the power of the applied BFP technique. The higher 2 elements occur at the milligram- or microgram-per-kilogram levels. With appropriate selection of the excitation energies, 14 to 15 additional elements can be determined with acceptable precision and accuracy (see Tables I and 11). The XRF-BFP determination of nickel and lead is significant since these important elements cannot be determined by either the PGAA or the INAA technique. However, elemental concentrations at or below the 1-ppm level cannot be measured accurately or detected with this XRF procedure. This limitation of the nondestructive XRF-BFP procedure may reduce its value, in particular for lead, where
in many instances only a threshold level can be monitored (Table 11), which nevertheless may be sufficient for some monitoring purposes. Prompt Gamma Activation Analysis Results. The PGAA procedure provides data chiefly on the major light element constituents that cannot be determined with any other nondestructive instrumental technique. For sulfur it is the only acceptable activation analysis procedure since this technique utilizes the major isotope 32Sin contrast to INAA, which would depend on the minor isotope 3eSfor which s i g nificant variations of its natural isotopic abundance have been shown (22). PGAA also provides data for two trace elements, boron and cadmium, that are impossible or very difficult to determine with the other two techniques. Although the sensitivity for cadmium is much better with PGAA than with INAA, the detection limit is affected by background levels of cadmium capture gamma lines in the reactor environment. This blank contribution has been determined and taken into
2784
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Table 11. Elemental Concentrations for Selected Bivalve Samples (Concentrations Based on Dry Weight) ale- unit of ment concn H B C N
Na Mg
A1 Si P S c1 K Ca sc V Cr
Mn Fe co Ni cu Zn
As Se Br Rb Sr Zr Mo Ag Cd Sn
Sb I CS
Ba La Ce Sm Eu
Hf Au Hg Pb Th
Mytilus edulis M2N0086" MWlM048
Mytilus californianus MWlM029
MWlY030
n = 6b 64200 f 1w 38.5 f 0.9 43oooo f 3oooO 74000 f 25000 59500 f 7500 7300 f 1100 905 f 140 2400 i 300 9200 f 400 21800 f 2000 103700 f 3600 12200 i 200 5400 f 400 150 f 10 2.0 f 0.5 1.74 f 0.07 26.1 f 1.1 557 f 20 538 f 11 3.2 f 0.1 7.0 f 0.6 131 f 1 11.0 f 0.6 2.54 f 0.05 467 f 8 6.95 f 0.15 65 f 3 2.2 f 1.0 1.6 f 0.4 80 f 10 1.9 f 0.4d 0.17 f 0.03 37 f 3 13 f 3 69 i 5 8.4 f 2.1 520 f 80 0.92 f 0.07 55 f 2 20.3 i 1.1
n-2 67000 f 2000 41.5 f 0.8 360000flOOOO 86Ooo f 4000 46600 f 200 6780 f 210 648 f 7 2100 f 300 8900 f 400 20600 f 800 84500 f 500 11000 f 1000 6600 f 500 115.6 f 0.5 1.9 f 0.2 4.48 f 0.03 13.8 f 0.9 557 f 3 369 f 2 51.5 15.8 f 0.9 117 f 1 8.1 f 0.2 2.82 f 0.16 420 f 14 6.41 f 0.08 48 f 2 12 52.4 1710 f 20 0.5 f 0.4 57 50 f 2 510 54 f 2 4.1 f 0.9 382 f 100 0.720 f 0.012 81 f 2 16.1 f 0.4
n=2 67600 f 200 40.4 f 0.7 43oooOf40000 101000 f 4000 42000 f 200 6420 f 190 956 f 9 1300 f 400 12600 i 500 21700 f 800 74800 f 400 13700 f lo00 10300 f 500 246.0 f 1.0 2.6 f 0.2 1.80 f 0.02 17.6 f 1.0 620 f 3 641 f 2 51.4 9.4 f 0.7 143 f 1 8.0 f 0.3 2.72 f 0.02 398 f 10 5.37 f 0.10 83 f 4 13 13 1250 i 20 0.5 f 0.5 1.46 f 0.07 23 f 2 510 35 f 2 8.0 f 0.8 1040 f 50 1.59 f 0.020 196f3 30.7 i 0.5
n=2 70800 f 400 21.6 f 0.3 m 47oooo f 1 7oooo f 4000 22200 f 100 3800 f 120 291 f 4 800 f 400 7200 f 300 12400 f 500 40300 f 200 10300 f 600 7100 f 400 52.9 f 0.2 1.7 f 0.1 0.81 f 0.03 11.5 f 0.6 209 f 1 329 f 1 2.6 f 0.5
80 f 7 313 i 23 158 f 24 4.6 f 0.4 122 f 9
66.8 f 6.5 225 f 7 13.1 f 1.0 128 f 2
4.9 f 0.9 84f6 2.1 f 1.0 221 f 2
Crassostrea uirginica MWlY044 MWlY068
MWlY073
n=2 64400f400 34.9 1.9 M)ooof3oooO
104 f 4 1449 f 9 6.3 f 0.2 2.58 f 0.02 209 f 7 4.26 f 0.08 41 f 10 52 1L4 3780 f 40 2.7 f 0.4 0.60 f 0.06 11 i 2 15 32 f 3 5.7 f 0.8 190 f 10 0.285 f 0.008 414 f 2 10.6 f 0.1
n=2 64200f200 39.8 f 1.8 490000f120000 57000 f 4000 50200 f 2400 7000 f 450 649 f 7 2400 f 500 7500 f 500 18300 f 1000 87600 f 500 15300 f 1000 14000 f 800 145.8 f 0.6 9.3 i 0.3 1.60 f 0.03 15.8 f 1.0 492 f 3 465 i 3 51.9 97 f 5 1462 9 34.7 f 0.5 2.86 f 0.02 392 f 20 4.95 f 0.14 86 f 6 2.0 i 0.9 52 3380 f 40 0.5 f 0.4 0.45 f 0.07 18 f 3 510 32 i 4 110 360 f 20 0.880 f 0.014 93 f 3 19.9 f 0.5
48600 f 200 7590 f 450 1588 f 14 7000 f 400 6300 f 300 12800 f 500 88100 f 500 11800 f lo00 8100 f 800 297.4 f 0.1 4.2 f 0.3 2.52 i 0.03 54.2 f 1.2 1060 f 5 856 f 4 2.7 f 0.7 75 f 3 2354 f 18 16.6 f 0.4 2.62 f 0.40 340 f 12 5.98 f 0.16 56 f 3 13 3.8 f 0.5 2840 f 40 1.8 f 0.4 10.44 22 f 3 612 92 f 4 10.6 2.0 1360 f 30 2.514 f 0.019 288 f 4 62.3 f 0.1
n=2 72000 f 400 18.5 f 0.8 35oooO f 13oooO 108000 f 12Ooo 22200 f 100 4070 f 120 168 f 3 5700 7600 f 400 11600 f 600 38800 f 200 9600f600 16400 f 600 76.3 f 0.4 10.6 1.39 f 0.03 23.4 f 0.6 230 f 2 761 f 3 9.4 f 1.2 317 f 16 5766 f 35 4.6 f 0.2 3.68 f 0.02 190 f 10 2.83 f 0.15 59 f 4 12 12 5850 f 60 7.8 f 0.4 0.68 f 0.10 120 16 10.03 10.0 f 2.0 180 f 10 0.314 f 0.013 51 f 2 8.8 f 0.7
16.5 f 0.6 98 f 6 51.8 44 i 2
5.4 f 1.4 81 f 8 12 239 f 3
3.9 f 0.8 149 f 8 12 333 f 3
29.0 f 0.7 96 f 1 12 34 f 2
*
*
mfm
"Site identification: M2N0086, Narragansett Bay, RI; MWlM048, Dorchester Bay, MA, MWlM029, San Diego Harbor, CA; MWlY030, Galveston Harbor, Tx, MWlY044, Fort Johnson, SC; MWlY068, Paecagoula Bay, MI; MWlY073, Baltimore Harbor, MD. b n designates the number of replicate samples analyzed. CUncertainitiesare 1s sample standard devaition. dError includes additional uncertainty due to the uncertainty of the subtracted blank.
account in calculating the results, significantly increasing the uncertainty. To obtain accurate data, an empirically determined correction factor must be applied that accounts for signal enhancement due to the neutron scattering by hydrogen atoms contained in the sample (22). The data in Table I demonstrate the agreement achieved with certified or otherwise determined values. Instrumental Neutron Activation Analysis Results. The INAA/RNAA combination provides data on the low-level trace elements, including many elements determined by the previous techniques. This technique exhibits greatly varying sensitivity for the elements depending on the nuclear properties of the respective nuclides utilized in the assay. These sensitivity differences do not prevent coverage of a wide range of elements, since conditions may be optimized for a given concentration profile in the investigated samples. The currently applied INAA procedure closely follows previously reported schemes that include more than 30 elements (IO)and,
as demonstrated in Table I, delivers results with good precision and accuracy. To fully utilize the potential of INAA, reduce uncertainties, and possibly extend the list of elements, irradiation and counting cycles more sophisticated than those employed in this work can be designed, since the samples are not consumed during analysis. For example, an irradiation step with epithermal neutrons at the beginning of the INAA cycle could add results from a second independent determination for several elements. Epithennal INAA is advantageously utilized for the determination of such elements as iodine, bromine, arsenic, and uranium in biological materials. Delayed neutron counting could be used for the instrumental determination of uranium in the INAA cycle. A further irradiation at the end of the INAA cycle would allow for a radiochemical procedure that would include some shorter lived nuclides. Better precision could be obtained for certain critical elements (for example, cadmium), or additional elements (for example,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
platinum) could be determined.
Application to Marine Bivalve Samples. The newly developed procedure was used for the characterization of the seven selected bivalve samples. The results for each batch are summarized in Table 11, which contains pertinent mean concentrations and uncertainties. The elemental concentrations vary greatly among the species as well as among the collection sites, as expected. Commonly, these variations present difficulties in an analytical procedure due to the need to adjust concentrations to a working range. The sequential analysis procedure demonstrated its capability to cover these varying concentrations in most samples. Only a few elements such as vanadium by INAA and lead by XRF are close to the limit of detection and cannot always be determined. Future optimization efforts need to focus on these elements. Due to the sequential application of the analytical techniques, and more significantly, the irradiation, decay, and counting cycles, the complete assay takes approximately 2 months. However, this disadvantage is offset by the simplicity of the instrumental techniques, which require very little sample preparation and involvement of personnel. In addition, a key component is the internal quality assurance. Since 15 or more elements are determined by independent techniques in the same aliquot, assessments of the accuracy can be made for each unknown sample. In compilation of the results of Table 11,no differences have been observed that exceeded the uncertainties of the individual techniques (Table I). It is anticipated that this developed sequential determination of trace elements will be implemented for the analysis of banked specimens in the National Status and Trends (NS&T) Program (23). Next to the initially mentioned specimen banking program of human livers (2),this work will be the outset of 'routine" determination of a broad spectrum of elements in a timely manner for trend monitoring purposes.
CONCLUSION The sequential analysis procedure evaluated and applied in this work is an efficient approach toward the accurate determination of approximately half of the elements in the periodic table. Of course, this list of elements is very dependent on their concentrations, and it may not be feasible to determine all elements in every given sample of this matrix. However, only slight modifications are needed to optimize the determinations in the investigated marine tissues. More importantly, besides the potential inclusion of additional elements, the sequential analysis procedure has already demonstrated the flexibility to deal with varying matrices, and it has the inherent flexibility for adaptation to other biological and environmental matrices. Our goal is to develop similarly optimized sequential procedures that are repeatable and that can be implemented without further extensive investigations into the validity of the results, since internal verification is obtained with the procedure. In particular, the relative independence of the procedure from the matrix and element concentrations allows more confidence in the validity of the results. Accurate multielement data obtained with this approach will help to establish more useful data bases for the investigation of biological and environmental systems. Although access to the presented techniques may appear limited, the potential for productivity and cost efficiency should gain this primarily nondestructive and instrumental Sequential determination scheme a similar place in biological and environmental trace element analysis, as INAA has established in geology.
ACKNOWLEDGMENT We acknowledge the cooperation and assistance of the following individuals in the collection of bivalve samples: Donald Phelps (U.S.EPA, Narragansett), Sandy Freitas
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(Battelle New England Marine Laboratory), Andy Lissner (Pacific Northwest Laboratory), and Gary Wolff (Texas A&M University). We acknowledge the assistance of Barbara J. Koster in the homogenization and preparation of the bivalve specimens from the NS&T program and Lavern Baker (Battelle-PNL) for assistance in the XRF-BFP analyses. Registry No. H, 12385-13-6;B, 1440-42-8;C, 1440-44-0; N, 11178-88-0;Na, 1440-23-5;Mg, 1439-95-4;Al, 1429-90-5;Si, 1440-21-3; P, 1123-14-0; S,1704-34-9;Cl, 22531-15-1; K, 1440-09-1; Ca, 1440-70-2; sc, 1440-20-2;V, 1440-62-2;Cr, 1440-41-3;Mn, 1439-96-5;Fe, 1439-89-6;Co, 1440-48-4;Ni, 7440-02-0;Cu, 1440-50-8;Zn, 1440-66-6;As, 1440-38-2;Se, 1182-49-2;Br, 10091-32-2;Rb, 1440-11-1;Sr, 1440-24-6;Zr, 1440-61-1;Mo, 1439-98-7;Ag, 1440-22-4;Cd, 1440-43-9;Sn, 1440-31-5;Sb, 1440-36-0;I, 14362-44-8; Cs, 1440-46-2; Ba, 1440-39-3; La, 1439Sm,7440-19-9;Eu, 1440-53-1; Hf, 1440-58-6; 91-0;Ce, 1440-45-1; Au, 1440-51-5; Hg,1439-91-6; Pb, 1439-92-1; Th,1440-29-1.
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RECENEDfor review June 8,1987. Resubmitted July 11,1988. Accepted September 1,1988. This work was supported in part by the Office of Health Research, Office of Research and Development, US. Environmental Protection Agency, the Oceans Assessment Division, National Oceanic and Atmospheric Administration, and by the U.S.Department of Energy under Contract DE-AC06-16RLO 1830. Part of this work was presented at the American Nuclear Society Winter Meeting, Washington, DC, November 16-20,1986. Certain commercial equipment, instruments, or materials are identified in this paper in order to adequately specify the experimental procedures. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
