Environ. Sci. Technol. 1998, 32, 3633-3639
Binding of Polycyclic Aromatic Hydrocarbons by Size Classes of Particulate in Hamilton Harbor Water G A R Y G . L E P P A R D , * ,†,‡ DERRICK T. FLANNIGAN,‡ D E N I S M A V R O C O R D A T O S , ‡,§ C H R I S H . M A R V I N , |,⊥ DOUGLAS W. BRYANT,4 AND BRIAN E. MCCARRY| Aquatic Ecosystem Protection Branch, National Water Research Institute, Burlington, Ontario L7R 4A6, Canada, Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada, ICMA, University of Lausanne, BCH, 1015 Lausanne, Switzerland, Department of Chemistry, McMaster University, Hamilton, Ontario L8S 4M1, Canada, Pest Management Research Centre, Agriculture and Agri-Food Canada, Vineland Station, Ontario L0R 2E0, Canada, and Department of Biochemistry, McMaster University, Hamilton, Ontario L8S 3Z5, Canada
In aquatic systems there is considerable transport of organic contaminants on suspended particles that act as carriers and influence the redistribution, bioavailability, and ultimate fate of contaminants. Using methodology not previously applied to the analysis of lake water, we demonstrate that polycyclic aromatic hydrocarbons (PAH) in Hamilton Harbor are predominantly sorbed to suspended flocs. Techniques employed were as follows: (i) differential cascade sedimentation and centrifugation to separate suspended particles; (ii) scanning transmission electron microscopy and energy-dispersive spectroscopy to identify flocs and individual particles in the size range of 10-3103 µm; (iii) gas chromatography-mass spectrometry to identify PAH in extracts prepared from size classes. Heterogeneous flocs larger than 20 µm accounted for roughly 98% of phenanthrene binding, 89% of fluoranthene binding, and 85% of pyrene binding.
Introduction Suspended organic and inorganic particles are ubiquitous in surface waters and are important vectors for the transport and distribution of contaminants and nutrients in aquatic ecosystems (1, 2). Suspended particles also play a role in the bioavailability and fate of contaminants (3-5). Included among these particles are flocs (6-8), which are aggregated particles comprised of microbiota (both active and moribund), minerals, debris, and colloidal organic matrix fibrils, which are secretion products of microbiota. The mechanisms * Corresponding author e-mail:
[email protected]; phone: (905)336-4787; fax: (905)336-4420. † National Water Research Institute. ‡ Department of Biology, McMaster University. § University of Lausanne. | Department of Chemistry, McMaster University. ⊥ Agriculture and Agri-Food Canada. 4 Department of Biochemistry, McMaster University. 10.1021/es980055p CCC: $15.00 Published on Web 09/24/1998
1998 American Chemical Society
of transport and dispersion of contaminants by specific particulate carriers in surface waters are not well understood. However, it is essential for public health and environmental restoration purposes that contaminant transport be better understood and that modeling of contaminant dispersion reflect the behavior of the particulate carriers. Contaminant transport by particulate carriers is usually modeled without considering the specific properties of the carrier; the carrier becomes an abstract entity that is awarded generalized or averaged properties. There is evidence, reviewed by Decho (9) and Leppard (6), that suspended organic-rich particles can accumulate and transport organic contaminants and/or metals. Such particles have a range of gross chemical compositions as well as diverse microbial and chemical activities and physical properties. These specifics should be incorporated into models to refine them sufficiently for practical uses. The isolation and subsequent investigation of particulate carriers with their contaminant burdens should, in turn, provide new information on contaminant bioavailability changes, release rates during carrier decomposition, and behavior in burial scenarios. The characterization and isolation of contaminant-carrier complexes are our research goals for a highly contaminated portion of Hamilton Harbor (Ontario, Canada). Hamilton Harbor is an embayment of western Lake Ontario designated as an Area of Concern by the International Joint Commission (Canada-U.S.A.). The harbor has a surface area of 40 km2 and receives discharges from a watershed of some 900 km2. Sources of contaminants in the harbor include industrial effluents, roadway runoff, and treated municipal sewage. High levels of contaminants including polycyclic aromatic hydrocarbons (PAH) have been identified in harbor sediments (10-12). Total PAH content in coal tar-contaminated sediments from the south shore can exceed 1000 µg/g (10). We previously reported that PAH including benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene were primary contributors to mutagenic activity associated with this coal tar-contaminated sediment (12). The resuspension and transport of coal tar-contaminated sediment in Hamilton Harbor is potentially a major source of genotoxic PAH in harbor-wide sediment samples (10-13). As a result, heavily contaminated sediments in some inshore areas of the harbor have been targeted for remediation. It is our intention to develop methods for elucidating the role that resuspension of coal tar-contaminated sediment plays in influencing the contaminant burden in Hamilton Harbor and identifying the primary carriers on which PAH are distributed. A sampling, fractionation, and analysis scheme has been developed and applied to particles in harbor surface water, including flocs and particles in the colloidal size range. The methodology included the following: (i) gravitational sedimentation and differential cascade centrifugation (including ultracentrifugation) to afford particle fractions containing operationally defined size ranges, which could be checked for size overlap by electron microscopy (EM); (ii) simultaneous collection and analysis of the sedimented and centrifuged particulate using scanning and scanning-transmission electron microscopy in combination with energy-dispersive spectroscopy to characterize individual particles in each of the size fractions and to classify them on the basis of their morphology and element composition; and (iii) gas chromatography-mass spectrometry analysis to identify and quantify PAH in organic extracts prepared from these individual sized fractions. This multimethod approach yielded several size fractions with little perturbation of the unsettled sediment particles (14). CorVOL. 32, NO. 22, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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relative use of the electron-optical techniques (7, 15, 16) permitted identification of the colloidal/particulate components of each fraction from which organic extracts were prepared and analyzed. The present work outlines a versatile tunable protocol for the sampling and analysis of suspended sediments for trace organic compounds.
Experimental Section Sampling and Sample Preparation. Subsurface water samples (depth ) -1 m) from Hamilton Harbor were collected in September 1995; each was taken in a 50-L polyethylene carboy and transported immediately to the laboratory for processing. The depth was chosen to minimize the contribution from resuspended particles coming from -9 m. Small portions of raw water and isolated water fractions were taken, with no storage, for EM analysis; subportions were stabilized immediately in a selection (below) of different fixatives. For PAH analysis, fractions isolated from the fresh raw water by sedimentation and centrifugation techniques were frozen at -40 °C and then freeze-dried, prior to analysis. Polycyclic Aromatic Hydrocarbon Analysis. Dried particulate material was extracted from each fraction using a 300-W Fisher Dismembrator model 300 ultrasonicator with a 0.75 in. diameter titanium probe (Fisher Scientific, Fairlawn, NJ). Samples were placed in glass beakers containing 50 mL of dichloromethane. Eight consecutive ultrasonic pulses of 15 s duration each were applied at full power. An interval of 1 min was maintained between pulses, and the beaker was immersed in ice to minimize solvent heating. The suspension was filtered and re-extracted with 50 mL of fresh dichloromethane as described. Solvent extracts were combined to afford a single extract. Extracted material was adsorbed to neutral alumina (Brockman activity 1, 80-200 mesh, 1 g, activated at 170 °C for 48 h) by gentle solvent reduction using a rotary evaporator. The sample, adsorbed to alumina, was applied to the top of fresh activated alumina (2 g) contained in a glass column (1 cm × 20 cm). Polycyclic aromatic compounds (PAC) were eluted by the addition of 25 mL of dichloromethane. Extracts were gently reduced to dryness under nitrogen and reconstituted in toluene for analysis. Gas chromatography-mass spectrometry (GC-MS) analysis was performed on a Hewlett-Packard model 5890 series II gas chromatograph with an on-column injector (1 µL) and a Hewlett-Packard model 5971A mass selective detector operated in selected ion monitoring mode (Hewlett-Packard Co., Mississauga, ON). The following temperature program was used: 130 °C to 300 °C at 1.6 °C/min; final time at 300 °C, 30 min. The column was a 60 m × 0.25 mm i.d. DB-5MS with a 0.25 µm stationary phase film coating (J & W Scientific, Folsom, CA). The internal standard was benz[a]anthracened12. The following PAH were monitored: phenanthrene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b/j]fluoranthene (which coeluted), benzo[e]pyrene, benzo[a]pyrene, perylene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene. Typical between-run precision ranged from 4% for benzo[a]pyrene to 11% for chrysene. Based on a signalto-noise ratio of 2.5:1, the estimated detection limits were 60 pg for 178 and 202 amu PAH; 100 pg for 228 and 252 amu PAH; and 130 pg for 276 amu PAH. Recoveries of PAH were measured using a standard reference material (SRM 1649); the levels of PAH varied by no more than 15% from the certified values. Fractionation for Particle Characterization. Raw water was poured into a 1-L vessel (height ) 123 mm) with two aluminum SEM stubs resting on the bottom. During the 2-h sedimentation, large coarse particles were deposited directly onto the stub by adhesion (particle capture). The suspended 3634
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particle population density was such that particle capture occurred to give partial coverage of the stub with little overlap. For fractions derived later by sequential sedimentation, early runs revealed that flocs dominated every fraction >20 µm, a fact well exploited in protocol development. The supernatant was removed using a peristaltic pump set at low speed (333 mL/min). The adherent particles were dried before being sputter-coated with 5-8 nm of gold for optimal viewing. Each of the subsequent separations (Figure 1A) was carried out with supernatant from ultracentrifugation using a Beckman L8-70M with an SW28 rotor. To collect particles directly onto TEM grids, customized plugs were installed in the bottom of the centrifugation tubes to provide a flat surface for supporting the grids (17). Particles were transported during centrifugation, coming to rest on the Formvar-covered grid. The combination (14) of suspended particle population density and water volume centrifuged yielded essentially a monolayer of particles that partially covered the grid. Supernatant above the grid was removed with the peristaltic pump and used in the next separation step. Fractionation for Polycyclic Aromatic Hydrocarbon Analysis. Using an ultracentrifugation protocol, patterned after the combined electron-optical and chemistry approach of Perret et al. (14) and correlated with the small-volume protocol of the previous section, a larger volume of raw water was processed to collect a sufficient number of particles in each size class for extraction and chemical analysis of PAH. Fractionation was carried out using a Baxter cryofuge 6000 and a swing-out rotor (Heraeus-Crist), which held six tubes of 250 mL each. For naturally sedimenting particles (e.g., flocs), a second separation resulting in a series of subfractions for suspended conventional particles was carried out using a settling tube (80 × 5 cm; 1570 mL) and the parameters shown in Figure 1B. For each step of sedimentation, the lowest fraction (largest particles) was removed with a peristaltic pump (77 mL/fraction). To conserve the height of the water column, the tube was topped with deionized distilled water. This procedure allowed us to collect the desired size-class particles within an acceptable error (18). Electron Microscopy. To stabilize and prepare samples for the production of ultrathin sections of particles for transmission electron microscopy (TEM), we selected correlative methodology (7, 19, 20) that couples sampling with stabilization and allows one to identify and minimize artifacts. The principal artifacts in TEM analysis of colloid-rich water samples and aggregated colloid systems are (i) extraction by processing fluids, leading to selective losses of colloids; (ii) physical perturbation of delicate aggregated structures; and (iii) dehydration leading to shrinkage of floc matrixes. Four different (independent but complementary) preparatory TEM treatments were applied to aliquots of each sample to detect and assess these sources of artifacts. In preliminary work with these treatments, micrographic data favored the application of the protocol of Liss et al. (7). We used their two treatments involving Nanoplast embedding and their two treatments involving epoxy embedding. For aggregated colloid systems (such as flocs), the image interpretation was carried out according to Leppard et al. (20). The Nanoplast (21) melamine resin treatment provided a spatial resolution of 0.001 µm, while embedding in Spurr’s epoxy resin (22) resolved colloid structure as fine as 0.003 µm. All resin-embedded samples were sectioned with a diamond knife on a RMC MT-7 Ultramicrotome, and ultrathin sections (50-70 nm thick) were collected on Formvar-covered copper grids. To maximize specimen contrast in the TEM, Nanoplast sections were counterstained with 1% aqueous uranyl acetate for 3 h while Spurr’s epoxy resin sections were counterstained with uranyl acetate in 50% ethanol for 10 min followed by Reynolds’ lead citrate stain according to
FIGURE 1. (A) Sequential centrifugation scheme used for the fractionation and chemical/structural analyses of an autumn water sample from Hamilton Harbor. Size classes of each colloid fraction were verified by TEM. (B) Sequential sedimentation scheme yielded five size classes of visualized conventionally defined particles for extraction and analysis of PAH species by GC-MS. Fractions 1-4 were collected using an extended settling tube. Size classes were verified by SEM, and ultrastructure was examined by TEM and STEM-EDS. methods outlined by Lewis and Knight (23). All ultrathin sections were examined with a JEOL JEM 1200 EX-II TEMSCAN scanning transmission electron microscope (STEM) operating at 80 kV in transmission mode. For microanalysis of individual particles for element composition, sections with thicknesses up to 100 nm were placed on grids that had been both Formvar- and carboncoated in order to improve section stabilization during interaction with the electron beam. These thicker stabilized sections were coated again on their upper surface and then used to determine typical element compositions of selected particles and discrete subcomponents of aggregated particles by energy-dispersive X-ray (EDS) microanalysis procedures (24). EDS was performed using the same STEM instrument equipped with a Princeton Gamma Tech IMIX multichannel analyzer. Analyses of composition for elements of Z > 10 were made over counting periods adjusted to minimize sample decomposition. For whole volume information on large particles (including aggregated particles) in fractionated samples (whole mounts), the SEM used was a dual-stage scanning electron microscope (I.S.I. DS-130) operated at 30 kV. EM was carried out on particle fractions prepared for two different electron optical modes, topographical imaging, and imaging of internal details by transmitted electrons. The topographical approach resulted in “whole volume” information from a SEM, providing gross information on particle shape and size. The second approach (TEM) yielded diagnostic details on the internal structure of aggregated particles/colloids at higher resolution. For aggregated particles, TEM is applied to ultrathin sections (15); for small
colloid fractions, however, TEM can also be applied to whole mounts (17).
Results and Discussion The tunable fractionation schemes for colloids and conventional-size particles are shown in Figure 1. Each was based on methodology (14, 25) that uses an equation derived from Stokes law to separate different classes of aquatic particles according to a combination of size and density. The equation enables calculation of specific g forces and times for particle size fractionations and can be accurate within (10%, under conditions of low polydispersivity and similarity of density for the predominant particles. Our specific protocol information (times and g forces) were established to yield fractions from harbor water having an autumn particle composition. This choice of sample time minimized complication so as to permit the most simple of approaches to size fractionation by sedimentation. For raw water sampled in the spring (with its contribution of runoff water carrying pedogenic humic substances) or in summer (with an in situ production of microbial mucilage), adjustments to protocol would be necessary. Our few adjustments were guided by a simplifying feature of the water body, the extreme predominance of flocs in fractions >20 µm. While our total suspended particulate (including colloids) was polydisperse, the fractions of major interest for PAH analysis contained essentially one particle type (flocs), as verified by EM. Accessory technology is available (26) to help design protocols for raw water presenting a more complex situation. For more complex environmental situations and for refining our sedimentation protocol further to minimize particle size VOL. 32, NO. 22, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Electron-optical views of entire aquatic particles (whole mounts) collected from Hamilton Harbor and separated into size fractions. (a) A SEM image that indicates the morphology of an aggregated particle (a multicomponent floc) in topographical view; the bar ) 6 µm. (b-d) TEM images of entire small particles captured (according to average dimension) on top of Formvar-coated grids: (b) >0.45 µm, (c) 0.45-0.15 µm, and (d) 0.45 µm. 3636
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Table 2 shows the PAH data from a subsequent analysis of size fractions isolated using the scheme of Figure 1B. There were negligible levels of PAH in the four size fractions containing particles less than 2 µm, including the particlefree fraction. The lower molecular mass PAH such as phenanthrene (98%), fluoranthene (89%), and pyrene (85%) appeared to be preferentially associated with the coarser fractions ranging in size from 20 to >80 µm. When we will be better able to minimize size overlap in our fractions, this relationship will receive intensive examination. Fractions ranging in size from 10 µm to fractional millimeters in size were shown by EM assessments to be dominated by heterogeneous flocs possessing an extensive internal colloid structure and its consequent extended surface available for binding. The high porosity of the flocs, established in part by matrix fibrils of 0.005 µm (least diameter) size, is also consistent with a capacity to concentrate PAH from the bulk water phase. The near absence of PAH among the freely dispersed colloids lent rationale for focusing on analyses of sedimenting particles in the lower range of the conventional particle size range (mm to µm). Such particles are typically abundant in the water column and represent a substantial portion of the total particulate loading relevant to organic contaminant
FIGURE 3. (A) TEM imaging of individual colloids within a large aggregated particle (ultrathin section) from a water sample embedded in Spurr’s epoxy resin. A guide to identification based on morphology and element composition analysis by STEM-EDS is found in Leppard (15). Bar ) 1 µm. Symbols for individual colloids are as follows: star ) bacterium; arrow ) micelle of a clay mineral; 1 ) manganese-rich colloids; 2 ) globular colloid of iron oxyhydroxide; 3 ) iron-coated clay micelle. Surrounding the moribund bacterium (star) is a capsule () 4) of short fibrils impregnated with iron compounds derived from Hamilton Harbor water. Note the variable but high porosity of this portion of the aggregated particle and the scattered fibrils. The extracellular fibrils have a patchy distribution but tend to be woven into a three-dimensional web, most of which is out of the plane of this individual ultrathin section chosen for its variety of colloids. (B) Spectra from STEM-EDS applied to individual colloids above. Note that the copper peak comes from the grid and the osmium peak comes from a stain for organics; these peaks are marked by an asterisk. (1) Manganese oxide colloids associated with small iron oxide colloids. (2) Globular colloid of iron oxyhydroxide. (3) Clay micelle containing potassium and coated with iron. (4) Iron-impregnated bacterial capsule. An asterisk (*) indicates those peaks that are not from the water sample; the * peak centered at approximately 8.91 keV reveals both Cu and Os. binding in the water column (6, 7, 28, 29). Sorption of PAH on colloids is known to occur in aquatic systems; contaminant-laden colloids can readily aggregate to form flocs (30). Aggregated particles in the floc size range are known from riverine ecosystems to contain PAH (31). Colloid systems within flocculated organic-rich materials can include macromolecular gel networks where sorption can be viewed as a dissolution of a contaminant into an organic polymer matrix (32). The matrix fibrils of flocs are an example of such a
macromolecular gel network (33). Future studies will focus on fibril contributions to PAH binding. Whether fibrils interact with PAH directly or by way of mineral colloids and organic macromolecules collected on their surface (34) has yet to be determined. Brief reviews of fibril functions and activities are found in Leppard (6, 15). Additional tuning of the methodology, through EM monitoring, could lead to direct attempts to produce fractions based on specific particle morphotypes within the gross VOL. 32, NO. 22, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Absolute Quantities of Individual PAH (ng) Contained in Material from the Four Size Fractions (Including Three in the Colloid Size Range) from the Sequential Centrifugation Scheme of Figure 1a size fraction compound
>0.45 µm
0.15-0.45 µm
0.02-0.15 µm
80 µm
40-80 µm
20-40 µm
10-20 µm
2-10 µm
phenanthrene fluoranthene pyrene benz[a]anthracene chrysene benzo[b/k]fluoranthene benzo[e]pyrene benzo[a]pyrene perylene indeno[1,2,3-cd]pyrene benzo[ghi]perylene
50 (45%) 69 (63%) 50 (60%) 12 (38%) 10 (32%) 12 (27%) 5 (33%) 3 (8%) 1 (8%) 4 (18%) 6 (16%)
47 (42%) 15 (14%) 10 (12%) 3 (9%) 3 (10%) 7 (16%) 2 (13%) 4 (10%) 1 (8%) 3 (14%) 9 (24%)
12 (11%) 13 (12%) 11 (13%) 6 (19%) 6 (19%) 9 (20%) 3 (20%) 8 (20%) 1 (8%) 4 (18%) 8 (21%)
ND 4 (4%) 6 (7%) 4 (13%) 4 (13%) 5 (11%) 2 (13%) 16 (40%) 8 (67%) 3 (14%) 5 (13%)
3 (3%) 8 (7%) 7 (8%) 7 (22%) 8 (26%) 12 (27%) 3 (20%) 9 (23%) 1 (8%) 8 (36%) 10 (26%)
112 109 84 32 31 45 15 40 12 22 38
225 (41%)
107 (19%)
84 (15%)
58 (10%)
81 (15%)
555 (100%)
total PAH a
total
Percentages of individual PAH in each size class are shown in parentheses. ND denotes not detected.
category of porous flocs. This in turn could lead to the quantitative isolation of specific floc varieties and, via mild floc disaggregation procedures, of the major individual colloids comprising them. The identification of flocs as PAH concentrators and carriers in Hamilton Harbor and a capacity to isolate them quantitatively should enable modeling based on a particle with specific physical properties rather than on a particle with an averaged or potential set of properties. Using our methodology, carrier particles can be isolated and characterized in their native state; electron-optics, used in conjunction with selected chemical measures, is likely to reveal variations in particle structure/activity relationships as particles undergo successional changes over time. A wellcharacterized, isolated, contaminant carrier can be subjected to focused experiments. Thus, important environmental variables impacting on PAH transport could be investigated directly. For the flocs isolated in this study, important properties for future analyses include rate of PAH uptake, settling rate, aggregation phenomena that alter settling rate, dispersion of flocs in terms of distance traveled, stability of the flocs with regard to microbial and geochemical breakdown processes, factors inducing changes in the nature of PAH binding within floc structure, and effects of burial/ resuspension on PAH release from the floc. Our methodology provides the potential to assess individual subcomponents of the floc and their roles in PAH uptake and release and the potential to determine colloidal factors facilitating or frustrating PAH containment procedures applied after burial. 3638
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Acknowledgments We thank the McMaster University Eco-Research Program for Hamilton Harbor, the Government of Canada Green Plan, the Great Lakes Action Plan, the Fonds National Suisse (Berne, Switzerland), and the McMaster University Life Sciences EM Unit.
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(12) Marvin, C. H.; Allan, L.; McCarry, B. E.; Bryant, D. W. Environ. Mol. Mutagen. 1993, 22, 61. (13) Harlow, H. E.; Hodson, P. V. Can. Technol. Rep. Fish. Aquat. Sci. 1988, No. 1603. (14) Perret, D.; Newman, M. E.; Negre, J.-C.; Chen, Y.; Buffle, J. Water Res. 1994, 28, 91. (15) Leppard, G. G. In Environmental Particles, Vol. 1; Buffle, J., van Leeuwen, H. P., Eds.; IUPAC Environmental Chemistry Series; Lewis Publishers: Chelsea, MI, 1992; pp 231-289. (16) Leppard, G. G. Analyst 1992, 117, 595. (17) Perret, D.; Leppard, G. G.; Muller, M.; Belzile, N.; DeVitre, R.; Buffle, J. Water Res. 1991, 25, 1333. (18) Mavrocordatos, D. Ph.D. Dissertation, University of Lausanne, Switzerland, 1997. (19) Leppard, G. G.; Heissenberger, A.; Herndl, G. J. Mar. Ecol. Prog. Ser. 1996, 135, 289. (20) Leppard, G. G.; West, M. M.; Flannigan, D. T.; Carson, J.; Lott, J. N. A. Can. J. Fish. Aquat. Sci. 1997, 54, 2334. (21) Frosch, D.; Westphal, C. Electron Microsc. Rev. 1989, 2, 231. (22) Spurr, A. R. J. Ultrastruct. Res. 1969, 26, 31. (23) Lewis, P. R.; Knight, D. P. Staining Methods for Sectioned Material; North-Holland Publishing Co.: Amsterdam, 1977. (24) Chandler, J. A. X-ray Microanalysis in the Electron Microscope; North-Holland Publishing Co.: Amsterdam, 1977.
(25) Lienemann, C.-P.; Heissenberger, A.; Leppard, G. G.; Perret, D. Aquat. Microb. Ecol. 1998, 14, 205. (26) Buffle, J.; Leppard, G. G. Environ. Sci. Technol. 1995, 29, 2176. (27) Fortin, D.; Leppard, G. G.; Tessier, A. Geochim. Cosmochim. Acta 1993, 57, 4391. (28) Leshniowsky, W. O.; Dugan, P. R.; Pfister, R. M.; Frea, J. I.; Randles, C. I. Science 1970, 169, 993. (29) Wijayaratne, R. D.; Means, J. C. Environ. Sci. Technol. 1984, 18, 121. (30) Wijayaratne, R. D.; Means, J. C. Mar. Environ. Res. 1984, 11, 77. (31) Evans, K. M.; Gill, R. A.; Robotham, P. W. J. Water, Air Soil Pollut. 1990, 51, 13. (32) Freeman, D. H.; Cheung, L. S. Science 1981, 214, 790. (33) Leppard, G. G.; Burnison, B. K.; Buffle, J. Anal. Chim. Acta 1990, 232, 107. (34) Leppard, G. G. Colloids Surf. A 1997, 120, 1.
Received for review January 22, 1998. Revised manuscript received July 9, 1998. Accepted July 30, 1998. ES980055P
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