Environ. Sci. Technol. 1997, 31, 2303-2306
Evaluation of Digestion Procedures for Determining Silver in Mussels and Oysters K O S T A S D . D A S K A L A K I S , †,‡ T H O M A S P . O ’ C O N N O R , * ,† A N D ERIC A. CRECELIUS§ NOAA N/ORCA21 National Status and Trends Program, 1305 East West Highway, Silver Spring, Maryland 20910, and Battelle/Marine Sciences Laboratory, 1529 West Sequim Bay Road, Sequim, Washington 98382
Here we report a method using HCl along with HNO3 and microwave digestion in sealed vessels that produces reliable and reproducible Ag determinations. This method produces a digestate that could be analyzed by atomic absorption and emission techniques. The digestion method was used to reanalyze 800 archived mussel and oyster samples from the NOAA MW program. The Ag redeterminations, using HNO3HCl digestion (NHD), proved that original results using either HNO3-HClO4 or plain HNO3 digestion were not accurate and in many cases did not recover all silver. For MW samples, original concentrations (OR) may be compared with NHD results on a relative basis:
C ) 100
Determinations of silver concentrations in mussel and oyster tissue digested with nitric acid were low relative to those measured in undigested tissue analyzed by ultrasonic graphite furnace atomic absorption spectroscopy. Good results were obtained, however, using a mixture of hydrochloric and nitric acids for digestion. Archived NOAA Mussel Watch samples collected in 1986-1993 and originally analyzed following HNO3 or HNO3-HClO4 digestion were reanalyzed with HNO3-HCl digestion in 1995. Results suggest that only 44% of the redeteremined Ag concentrations were within 20% of the original values. Most of the reanalyses yielded higher concentrations, and one-tenth of them were more than 100% higher. Temporal trends in Ag concentrations in the coastal United States have been estimated using the revised data. Statistically significant decreases, mostly in the Northeast, have been observed. This is probably an indication of lower Ag discharged in waste waters.
Introduction Common methods for metal analyses of environmental samples, such as atomic absorption or emission spectroscopy, require digestion of the samples, and many environmental laboratories use HNO3 for this purpose. This method has been found to cause problems with Ag recoveries (1). D’Elia et al. (2) also suggest that common HNO3 acid digestion procedures used in preparation of samples for the determination of metals in marine tissue can cause low recovery of silver. However, the dry ashing method used by D’Elia et al. (2) may result in loss of volatile elements (As, Se, etc.) and is not of general interest to environmental analysis. Metal analysis performed by Battelle/Marine Science Laboratory for the NOAA National Status and Trends Mussel Watch (MW) project from 1986 to 1990 had been done by HNO3-HClO4 digestion, followed by graphite furnace atomic absorption spectroscopy (GFAAS) analysis. Between 1991 and 1993, this method was abandoned in favor of the faster and safer HNO3 microwave digestion, and analysis was performed by inductively coupled plasma mass spectrometry (ICP-MS) (3). Evaluation of the MW data revealed reproducibility problems with both digestion methods. * Corresponding author e-mail:
[email protected]; fax: 301713-4388. † NOAA N/ORCA21 National Status and Trends Program. ‡ Present address: Hops Frog, Inc., 407 Upham Pl., Vienna, VA 22180. § Battelle/Marine Sciences Laboratory.
S0013-936X(96)00895-4 CCC: $14.00
1997 American Chemical Society
NHD - OR OR
where C is the % change, NHD is the silver concentration determined from HNO3-HCl digestion, and OR is the HNO3 or HNO3-HClO4 digestion results for the same sample.
Analytical Methods Details of sample collection and preparation have been described elsewhere (3, 4). Composite samples were prepared by freeze-drying and grinding 20 oysters or 30 mussels. For analysis by ICP-MS or GFAAS, 0.3 g of tissue was hotacid digested by two methods. Either 0.3 g of tissue was digested with 5 mL of concentrated HNO3 or the tissue was digested with the combination of 5 mL of HCl and 3.5 mL of HNO3 in a Teflon digestion vessel for 20 min at 6 atm (90 psi) in a microwave oven. After digestion, the digestate was diluted with deionized water to 20 mL. In preparation for analysis by ICP-MS, the digestate was further diluted 1:10 with 1% HNO3 acid, and indium (In) was added as an internal standard. Additional details of the digestion and analysis methods are described by Crecelius et al. (3). Quantification was done on a Perkin-Elmer Elan-5000 ICP-MS. The detection limit for Ag by ICP-MS was approximately 0.03 µg/g. The isotope dilution mass spectrometry (IDMS) used the HNO3 sample digestion procedure, and a known mass of isotopically enriched 109Ag was added before the digestion step. The 109Ag was used as an internal standard for silver quantification. In the current work, no samples were digested with HNO3-HClO4 because of environmental and safety concerns. We compared results obtained by these digestions with those from two methods that do not require complete dissolution of Ag. For analysis by X-ray fluorescence spectroscopy (XRF), 0.5-1.0 g of tissue powder was pressed into a pellet and then analyzed by the method of Zeisler et al. (5). The detection limit was approximately 7 µg/g dry weight, thus suitable only for samples with high concentrations. The other method, ultrasonic slurry sampling graphite furnace atomic absorption spectroscopy (USS-GFAAS), has been used for various solid matrixes without any previous digestion of the sample (6, 7). Usually 10-50 mg of tissue powder was weighed into a 1-mL Teflon autosampler cup. A slurry solution was prepared with 1 mL of 5% HNO3 solution containing 0.005% (V/V) Triton X-100 (Sigma Chemicals). A Model USS-100 ultrasonic slurry sampler (Perkin-Elmer) was used to automatically mix the slurry. The slurry was pipetted into the HGA-600 furnace of a Zeeman 5100PC Perkin-Elmer spectrophotometer equipped with an AS-60 autosampler. The detection limit was, generally, lower than 0.02 µg/g.
Results Tissue Analysis. Initial comparison between HNO3 digestion (ND) and HNO3 + HCl digestion (NHD) of oyster and mussel tissue samples indicated that in many cases the ND procedure
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TABLE 1. Initial Comparison between Analytical Methods for Ag in Mussel and Oyster Tissuea
TABLE 4. Results from Analysis of Ag in New York Harbor Sediments
ICP-MS HNO3
HCI + HNO3
XRF
2.4 1.8 1.8 2.4 2.6 2.4-4.7 1.6 4-3
15 19 6.7 7.8 16 26 5.8 11 9.6
15 20 11 9.7 14 36 7.2 13 8.6
sample ID 2-06 733-10 733-11 733-12 570-7 570-8 YR6-168 YR7-168 YR6-34 a
Concentrations in µg g-1 dry weight.
TABLE 2. Comparison between Analytical Methods for Ag (µg g-1 Dry Weight) sample
HCI + HNO3 ICP-MS
HNO3 IDMS
USS GFAAS
1-05 2-06 3-11 4-12 5-15 6-18
14 15 0.91 0.62 0.28 0.27
12 9.8 0.70 0.89 0.21 0.19
13 12 0.77 0.47 0.22 0.24
TABLE 3. Percent Recovery of Ag in Standard Reference Material Oyster Tissue (SRM 1566a) by ICP-MS by Different Heating Methods
Ag Cd Cu Pb Sn Zn Al a
microwave heat 20 min
conventional oven 16 h
certified (µg g-1)
104 98 113 116 70 98 34
99 98 107 101 112 104 95
1.68 4.15 65 0.371 (3)a 830 203
Value not certified but provided on the certificate as information.
did not completely dissolve the Ag (Table 1). Presumably during the ND procedure, reaction with low concentrations of Cl- present in the tissue precipitated insoluble AgCl. Alternatively, some Ag was stored in the animals in a form that was insoluble in HNO3. However, the NHD contained high levels of Cl-, forming AgCln1-n complexes that promote the dissolution of Ag and its stabilization in solution. Several samples were analyzed using three methods: NHD followed by ICP-MS; USS-GFAAS; and XRF analysis. The agreement between the concentrations measured by all three methods lends credence to the NHD (Tables 1 and 2). In addition, results from standard reference material (SRM 1566a) and matrix spike recoveries were also excellent for the NHD procedure. Results from NHD compare very well with those obtained using instrumental neutron activation analyses (INAA) (8). A limited number of sediment analyses showed good results for Ag using the same HNO3-HCl digestion method. The NHD procedure also provides very good results for Cd, Cu, Pb, and Zn in the National Institute for Standards and Technology (NIST) standard reference material oyster tissue SRM 1566a (Table 3). Recoveries for Sn and Al using NHD were not good. Better recoveries were found using longer conventional heating (16 h) instead of microwave heating (20 min). Prolonged contact of the acid mixture with
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digestion method sample 1
HNO3 + HClO4 + HF EPA Method 7760 HNO3 + HCl HNO3 + HCl sample 2 HNO3 + HClO4 + HF EPA Method 7760 HNO3 + HCl HNO3 + HCl sample 3 HNO3 + HClO4 + HF EPA Method 7760 HNO3 + HCl HNO3 + HCl sample 39 EPA Method 200.2 HNO3 + HCl sample 49 EPA Method 200.2 HNO3 + HCl
sample spike recovery (µg g-1 Ag) (µg g-1 Ag) (%) 2.06 1.74 2.20 2.30 3.89 5.20 2.06 9.65 14.1 2.91 3.38 6.22 7.02
5.00 5.00 5.00 10.00 5.00 5.00 5.00 10.00 5.00 5.00 5.00 10.00 20.00 20.00 20.00 20.00
6 81 106 108 8 22 92 99 27 20 104 101 10 93 93 93
the tissue may aid in the dissolution of insoluble phases. The IDMS method (with HNO3 digestion) was used to quantify Ag in several samples already analyzed by USS-GFAAS and by NHD. The IDMS results (Table 2) were about 20% lower than the other methods for tissue samples containing about 10 µg g-1 Ag. These results indicate that the enriched 109Ag isotope was not at equilibrium with the tissue Ag. Silver is known to form very insoluble sulfide granules (9, 10), which could account for lower recoveries. Sediment Analysis. Analysis of sediments for the MW program has traditionally been done after HNO3 + HClO4 + HF digestion. Using this method, recoveries of 5 µg g-1 Ag spike from Oakland Harbor sediments were low. To investigate it further, several New York Harbor sediment samples were analyzed using two U.S. EPA methods: (a) Method 7760 (11) starts with digestion (for sediments with HNO3 + HClO4 + HF). The solution pH is adjusted to above 7 with NH4OH, and then a mixture of KCN + I2 + KI in ammonia is added. This procedure is used to avoid plating of AgCl onto the walls of the vessel. (b) Method 200.2 (12) for total recoverable elements is based on heating 100 mL of a dilute slurry sample (no more than 0.25% total solids) with 2 mL of 1:1 HNO3 and 1 mL of 1:1 HCl. After volume reduction to 20 mL, the beaker is covered and refluxed for 30 min. Solids are centrifuged out of the solution before analysis. The same samples subjected to the EPA methods were also analyzed by HNO3 + HClO4 + HF and by HNO3 + HCl digestion (NHD). Results were consistently higher for the NHD, and spike recoveries were in the range of 92-108% (Table 4). The other three methods gave lower Ag concentrations, and with one exception, spike recoveries were below 27% (Table 4). It appears that the NHD method is appropriate for Ag analysis in both tissue and sediment matrix. The influence of HF seems to be detrimental. Presumably, the more complete dissolution of the silicate matrix releases ion moieties that, in the absence of excess Cl-, can potentially precipitate Ag. In any case, most of the Ag in these sediments should be found on the surface, and complete dissolution of the them is not necessary. Revised Silver Data. Since these results indicate that there could be many MW tissue samples with incorrectly determined silver concentrations, it was decided to reanalyze archived mussel and oyster tissue samples collected between 1986 and 1993. The MW protocol called for the collection of three composite samples per site for 1986-1991 and one composite thereafter. To decrease the number of analyses, Ag was redetermined in one composite sample per year per site.
a
b
FIGURE 2. Plot of reanalyzed Ag concentration in mussels and oysters as a function of original concentrations. Over half of the new values are outside the (20% limit. Very large increases after re-analysis are distributed across the concentration spectrum.
TABLE 6. Statistical Results of Silver Concentrations in Mollusks FIGURE 1. Histogram of NOAA Mussel Watch samples: original Ag concentrations (b) and after reanalysis (a). The number of samples with concentrations greater than 5 µg g-1 has increased significantly with HNO3-HCl digestion.
TABLE 5. Selected Results from Re-analysis of Ag in Mollusks with Ag Significantly Higher Than Original Value Ag (µg g-1) site
speciesa
collection year
original
NHD
CBBO CBJB CBJR CBMP DBAP DBAP DBAP DBAP DBAP DBFE DBHC DBKI DBKI DBWB QIUB QIUB QIUB PLLH PLLH PLLH TBSM BHHB
CV CV CV CV CV CV CV CV CV CV CV CV CV CV CV CV CV MC MC MC MC ME
90 90 89 90 86 87 90 91 92 86 89 86 87 89 87 91 91 90 91 92 92 87
1.73 0.56 2.30 2.20 2.40 3.30 1.50 6.40 2.32 2.40 1.70 9.10 6.10 1.30 1.10 4.40 4.50 3.19 13.00 2.49 0.09 1.40
13.81 17.88 10.01 9.28 13.08 8.86 11.93 16.15 9.45 9.85 21.78 15.44 8.03 11.11 9.08 8.17 9.63 27.18 33.76 8.73 9.24 5.16
a CV, Crassostrea virginica; MC, Mytilus californianus; ME, Mytilus edulis.
The reanalysis showed that there were many more high concentrations of Ag in mollusks than originally found. Over 60 samples had above 5 µg g-1 Ag dry weight, using NHD, as compared to only 10 originally (Figure 1). While the greatest concentration with HNO3 or HNO3-HClO4 digestion was 13 µg g-1, NHD results were as high as 33.7 µg g-1 (Table 5). No pattern could be found for the low recoveries of Ag observed during the original digestion. For example, there were concentrations of Ag < 2 µg g-1 that did not change significantly after NHD. However, in other samples original concentrations < 2 µg g-1 increased by as much as an order of magnitude (Figure 2 and Table 5).
Crassostrea virginica Mytilus californianus Mytilus edulis a
na
geometric mean (µg g-1 dry)
maximum (µg g-1 dry wt)
222 109 409
2.13 0.24 0.13
21.78 33.76 5.16
n, number of samples.
Approximately 44% of the NHD reanalyzed samples resulted in concentrations within 20% of the original values (Figure 2). While most of the changes of more than 20% were increases, some were some decreases. These were limited to samples with low initial determinations and may have been the result of increased blank samples or signal suppression due to the HNO3-HCl matrix. No NHD concentration was less than half of the original one. Concentrations above 0.5 µg g-1 showed mainly increases after re-analysis, presumably due to the improved Ag recoveries using the NHD method (Figure 2). One-quarter of the samples had more than a 50% increase over the original concentrations, and one-tenth of the samples after NHD had at least two times (100% difference) the concentration measured originally (Figure 2 and Table 5). Oysters accumulate Ag to a much greater extent than mussels, and more increases in Ag concentrations after reanalysis were found for oysters than for mussels. At some sites in California, even though the sampled species is a mussel, the redetermined Ag concentrations were much greater than the original. However, in general, concentrations in mussels (Mytilus edulis) after re-analysis were much closer to the original levels. The geometric mean of Ag concentrations by species were 2.13, 0.24, and 0.13 µg g-1 for Crassostrea virginica, Mytilus californianus, and M. edulis, respectively (Table 6).
Temporal Trends in Silver Concentrations With the revisions, it is possible to examine the silver data for temporal trends in concentrations. O’Connor (4) did this for As, Cd, Cu, Hg, Ni, Pb, Se, Zn, and organic compounds by searching for correlations between concentrations and year at individual stations and among annual geometric means. There were enough samples available for Ag re-analysis to provide at least 6 years of data between 1986 and 1993 for 62 East and West Coast sites. Spearman rank correlation coefficients were calculated for each site. For 90% confidence level, the absolute coefficients have to be above 0.829, 0.714,
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in Figure 3, it could be claimed that Ag concentration in the coastal environment is probably lowered between 1986 and 1993. This may be a reflection of better recovery of Ag from photographic solutions, since over 50% of its use is in photography. The difficulty in detecting trends may be due to their large Ag variability (13, 14).
Conclusions
FIGURE 3. Histogram of Spearman correlation coefficients of Ag concentration vs year, for 62 sites with 6 or more years of data. Negative coefficients predominate, and several are statistically significant, suggesting a decreasing Ag trend in mollusks (see text).
TABLE 7. Significant Silver Trends in Mollusks state
speciesa
mean Ag (µg g-1)
sampling (years)
spearman r
MA RI CT NY NY NY DE OR WA WA
ME ME ME ME ME ME CV ME ME ME
0.93 0.18 0.10 0.19 0.21 0.07 8.07 0.05 0.23 0.08
8 6 7 8 8 7 7 8 7 7
-0.81b -0.94b -0.86b -0.88b -0.81b -0.72 -0.82b -0.69 -0.86b -0.86b
a CV: Crassostrea virginica; ME, Mytilus edulis. Total of 62 sites with 6 or more years of data.
b
Significant at 95%.
and 0.643 for 6, 7, and 8 years. For the 95% confidence level, these values increase to 0.886, 0.786, and 0.738 for 6, 7, and 8 years of data, respectively. The results reveal decreasing trends in Ag concentrations (Figure 3). Specifically, out of 62 sites with 6 or more years of data, there are 10 trends that are significant at the 90% confidence level; all of these trends were in the decreasing direction (Table 7). This is more than the 6 (10%) one would expect from chance. One decreasing trend was found among oysters; all other trends were at sites where mussels were sampled. Among the remaining 9 decreasing trends, 6 were found in the Northeast, one in Oregon, and 2 in Washington state. At the 95% confidence level there are 8 decreasing trends nationwide (randomly one would expect 3, i.e., 5% of 62). Because there is so large a difference between Ag concentrations in oysters and in mussels, it is necessary to calculate descriptive statistics separately. After doing so, only about 30 samples remain per species for each year, and the sample concentrations were not lognormally distributed in all cases. This precludes us from using the geometric mean as a measure of the overall trend. However, given the results
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Comparison of Ag concentrations determined by two different digestion procedures and two methods not needing digestion, i.e., USS-GFAAS and XRF, indicates that the HCI + HNO3 digestion procedure is effective in solubilizing very high tissue Ag concentration. Digestion procedures using only HNO3 produce erroneous results and should not be used for Ag analyses. Based on re-analyzed samples from the NOAA MW program, it appears that there is a decrease in tissue Ag concentrations. Noise in the Ag concentrations that obscures trend detection will be reduced with subsequent sampling and analyses already under way.
Acknowledgments The authors, particularly K.D.D., would like to thank Dr. Nancy Miller-Ihli and Mrs. Ella Greene (Beltsville Agricultural Research Center, USDA) for providing the analytical facilities for UGFAAS slurry analysis.
Literature Cited (1) Crecelius, E. A.; Daskalakis, K. D. In Proceedings of Agrentum 2nd International Conference; University of WisconsinsMadison: Madison, WI, 1994. (2) D’Elia, C. F.; Sanders, J. G.; Capone, D. G. Environ. Sci. Technol. 1989, 23, 768. (3) Crecelius, E. A.; Apts, C.; Bingler, L.; Cotter, O.; Kiesser, S.; Sanders, R. In Sampling and Analytical Methods of the National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects, Volume III Comprehensive Descriptions of Elemental Analytical Methods Lauenstein, G. G., Cantillo, A. Y., Eds.; NOAA Technical Memorandum NOS ORCA 71; NOAA: Silver Spring, MD, 1993; pp 187-212. (4) O’Connor T. P. Mar. Environ. Res. 1996, 41, 183. (5) Zeisler, R.; Stone, S. F.; Sanders, R. W. Anal. Chem. 1988, 60, 2760. (6) Miller-Ihli, N. J.; Fresen. J. Anal. Chem. 1993, 345, 482. (7) Miller-Ihli, N. J.; Greene, F. E. J. AOAC Int. 1992, 75, 354. (8) Personal communication. B. J. Presley, Dept. of Oceanography, Texas A&M University, College Station, TX. (9) Martoja, R.; Ballan-Dufrancais, C.; Jeantet, A. Y.; Gouzerh, P.; Amiard, J. C.; Amiard-Triquet, C.; Berthet, B.; Baud, J. P. Can. J. Fish Aquat. Sci. 1988, 45, 1827. (10) Berthet, B.; Amiard-Triquet, C.; Martoja, R. Water Air Soil Pollut. 1990, 50, 355. (11) Test Methods for Evaluating Solid Waste: Physical/Chemical Methods. SW-846; U.S. Document 955-001-00000; U.S. Environmental Protection Agency: Washington, DC, 1986. (12) Methods for the Determination of Metals in Environmental Samples; EPA-600/4-91-010; EPA, Environmental Services Division, Monitoring Management Branch: Washington, DC, 1991. (13) Daskalakis, K. D. Mar. Pollut. Bull. 1996, 32, 794. (14) Wright, D. A.; Mihursky, J. A. Mar. Environ. Res. 1985, 16, 181.
Received for review October 18, 1996. Revised manuscript received April 3, 1997. Accepted April 9, 1997.X ES9608959 X
Abstract published in Advance ACS Abstracts, June 15, 1997.
