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Published in J. Environ. Qual. 33:2132-2140 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Organic Compounds in the Environment

Refined Tunable Methodology for Characterization of Contaminant–Particle Relationships in Surface Water

Christopher H. Marvina,*, Gary G. Lepparda,b, M. Marcia Westb,c, Gary A. Sternd, Adrienne R. Bodene and Brian E. McCarryf

a National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, ON, Canada L7R 4A6
b Department of Biology, McMaster University, Hamilton, ON, Canada L8S 4K1
c Electron Microscopy Facility, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada L8N 3Z5
d Freshwater Institute, Department of Fisheries and Oceans, 501 University Crescent, Winnipeg, MB, Canada R3T 2N6
e Ontario Ministry of the Environment, 125 Resources Road, Toronto, ON, Canada M9P 3V6
f Department of Chemistry, McMaster University, Hamilton, ON, Canada L8S 3Z5

* Corresponding author (chris.marvin{at}ec.gc.ca)

Received for publication May 22, 2003.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
To understand contaminant transport in aquatic systems, it is essential to define the physical characteristics of the primary particulate carriers. The distribution of organic pollutants with particle-size class and particle morphology in a freshwater embayment (Hamilton Harbor, western Lake Ontario) was studied using a sequence of novel sample preparation and characterization techniques. Water samples (24 L) were fractionated according to particle-size distribution using differential cascade sedimentation and centrifugation methods. These size fractions were subsequently subjected to a physicochemical characterization using scanning transmission electron microscopy and energy-dispersive spectroscopy to identify flocs and individual colloidal particles in the size range of 1 nm to 1 mm in diameter. Analytical chemical analyses were performed to identify organic contaminants in extracts prepared from particle-size classes, including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). The contaminant distribution trends were very similar for all compound classes studied; contaminants were primarily associated with fractions containing particles less than 2 µm in diameter. Morphological characterization of these fractions showed the majority of the particulates to be humic fractals. The results of this study show that contaminants in aquatic systems can be preferentially associated with specific types of particle carriers, the characteristics of which can be clearly defined in terms of size and morphology.

Abbreviations: EDS, energy-dispersive spectroscopy • EM, electron microscopy • ESEM, environmental scanning electron microscopy • PAH, polycyclic aromatic hydrocarbon • PCB, polychlorinated biphenyl • STEM, scanning transmission electron microscopy • TEM, transmission electron microscopy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
PARTICULATE MATERIAL comprised of both organic and inorganic components is an integral part of complex aquatic environmental systems and can be an important vector for transport, fate, and bioavailability of contaminants and nutrients (Kavanaugh and Leckie, 1980; Buffle, 1988; Sigleo and Means, 1990; Weilenmann et al., 1989; O'Melia et al., 1985). Decho (1990) and Leppard (1993) have published reviews that document the importance of suspended organic-rich particles in accumulation and transport of organic contaminants and metals. These particles can have a range of gross chemical compositions and physical properties, and exhibit diverse microbial and chemical activities. Included in these particle classes are flocs (Leppard, 1993; Liss et al., 1996; Droppo et al., 1997) comprised of aggregated particles including microbiota (both active and moribund), minerals, debris, and colloidal organic matrix fibrils. The mechanisms of transport and dispersion of contaminants by particulate carriers in surface waters remain relatively poorly understood. As a result, contaminant transport by particulate carriers is usually modeled without considering specific properties. Therefore, the carrier becomes an abstract entity that is awarded generalized or averaged properties, whereas specific characteristics 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. It is essential for public health and environmental restoration purposes that transport mechanisms be better understood, and that modeling of contaminant and nutrient dispersion reflects the behavior of the particulate carriers.

Our previous work (Leppard, 1993; Liss et al., 1996; Droppo et al., 1997) focused on the development of tunable methodology to assist in elucidation of the role of resuspended contaminated sediment in influencing the contaminant burden in Hamilton Harbor, an embayment of western Lake Ontario (Leppard et al., 1998). This work also resulted in identification of particle-size classes and morphologies primarily responsible for contaminant transport, and enabled characterization of a number of predominant particle types, including flocs and particles in the colloidal size range. The fractionation methodology was based on sedimentation and centrifugation to isolate particle classes based on size ranges, with confirmation by electron microscopy (EM) (Leppard et al., 1998; Perret et al., 1994; Leppard, 1992a). This multistage approach yielded fractions with little perturbation of unsettled particles (Perret et al., 1994). Analysis of fractions using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM), in combination with energy-dispersive spectroscopy (EDS) (Liss et al., 1996; Leppard, 1992a, 1992b), enabled characterization of individual particles and subsequent classification based on morphology and elemental composition (Leppard et al., 1998). Chemical analyses enabled quantitation of contaminants in relation to particle fractions, while correlative use of electron-optical techniques (Leppard, 1992b) permitted identification of colloidal and/or particulate components. In addition, the methodology produced representative and reproducible particle fractions from water samples that yielded good-quality contaminant data.

Our sample preparation and fractionation scheme was originally developed to assist in the investigation of the role resuspended contaminated sediments play in distributing contaminants over broader areas of aquatic systems. Hamilton Harbor has been designated as an Area of Concern by the International Joint Commission (Canada–USA). The harbor has a relatively small surface area of 40 km2, but receives discharges from a watershed of approximately 900 km2. Sources of contaminants in the harbor include industrial effluents, roadway runoff, and treated municipal sewage. In addition, sediments grossly contaminated by coal tar characterize an area on the south of the harbor. High levels of contaminants including PAHs and metals have been determined in sediments in this area of the harbor (Murphy et al., 1990; Mayer and Nagy, 1992; Marvin et al., 1993). Total PAH content in these coal tar–contaminated sediments can exceed 1000 µg/g (Murphy et al., 1990). The resuspension and transport of coal tar–contaminated sediment in Hamilton Harbor is potentially a major source of PAHs and other contaminants to the rest of the harbor (Murphy et al., 1990). As a result, contaminated sediments in some inshore areas of the harbor have been targeted for remediation.

The objectives of this work were to further refine the tunable methodology based on timed sedimentation followed by centrifugation for the isolation and preparation of specific particle-size classes from surface waters, and to expand the suite of analyses to include a broader range of persistent organic pollutants, including PAHs and PCBs. Particulate material in Hamilton Harbor surface water was characterized using STEM and EDS. Results are presented within the context of both particle size and morphology, to assess the physical characteristics of particles predominantly associated with contaminant transport.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sampling and Initial Sample Preparation
A subsurface water sample (depth = 1 m) from the mouth of the Windermere Arm Hamilton Harbor was collected in late fall (November 2000) in a 50-L polyethylene carboy and transported immediately to the laboratory for processing (Fig. 1) . The carboy was prepared for sampling by washing with soap and water, followed by rinsing with tap water and distilled water, respectively. The carboy was then rinsed with Harbor water immediately before sample collection. Small portions of water and isolated-water fractions were subsampled (<5 mL), with no storage, for high-resolution electron microscope analysis of individual colloids; these subsamples were immediately stabilized in a select variety of fixatives (Liss et al., 1996) as detailed below. In addition, subsampling (<5 mL) was conducted for correlative EM analyses (Leppard, 1992b) of unfixed particles and colloids during the course of fractionation to verify particle size fractionation. Fractions for contaminant analyses that were isolated from water by sedimentation and centrifugation were frozen at –40°C and then freeze-dried.



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  		Fig. 1. Protocol for the sequential sedimentation and centrifugation scheme used to fractionate surface water. EM, electron microscopy; ESEM, environmental scanning electron microscopy; TEM, transmission electron microscopy.

 
Fractionation for Particle Characterization and Microscopic Verification of Size Fractions
In preliminary work, we noticed that the quality of fractionation was a function of the nature of the suspension (e.g., mucilage-rich water vs. water rich in humic substances). Consequently, fractionation protocols for Hamilton Harbor water had to be "tuned" (Leppard et al., 1998) to suit the specific site and time, with the fractionation being monitored and verified for integrity by correlative microscopy (Leppard, 1992a, 1992b). Environmental scanning electron microscopy (ESEM), TEM, and chemical analyses were used to examine the size range of hydrated particles in fractions covering the selected ranges from 10 to >80 µm (Fig. 1). Whereas TEM and STEM–EDS were ultimately used for the characterization of all fractions, TEM was also used initially in size-fraction verification for fractions of 10 to 20 µm and smaller (Fig. 1). This correlative use of ESEM and TEM allowed us to overcome the fact that ESEM has increasingly limited use as one looks at smaller and smaller particles, while TEM becomes impractical for use in examinations of the larger particles. For a high-resolution EM overview of whole mounts of suspended particles, 34 mL of water (an appropriate amount for an optimal display of individual small particles and/or colloids) was deposited onto a Formvar film (Marviac Ltd., St. Laurent, QC, Canada) mounted on a TEM grid (Lienemann et al., 1998). The combination of starting water volume and suspended particle population density was such that particle capture could be arranged to give partial coverage of the grid film surface by essentially a monolayer of particles, with little overlap (Lienemann et al., 1998).

Parameters of the sequential sedimentation and fraction verification by EM are shown in Fig. 1. Water was poured into a 1570-mL settling tube (80 x 5 cm) and the particulate fractions were isolated by settling time and collected as described earlier (F1 to F4; Leppard et al., 1998). The 5-min fractionation by centrifugation (F5) was performed using a Baxter Cryofuge 6000 (Heraeus Sepatech, Osterode, Germany) with a Heraeus-Crist swing-out rotor. The final separation (PF) was performed by ultracentrifugation (of the supernatant from the 2- to 10-µm fraction) using a Beckman L8-70M (Beckman Coulter, Fullerton, CA) with an SW28 rotor. To collect particles directly onto Formvar-covered TEM grids (Perret et al., 1991), customized plugs were installed in the bottom of the centrifugation tubes to provide a flat surface for supporting the grids. Supernatant above the grid was removed with a peristaltic pump set at low speed (approximately 30 mL/min).

Fractionation for Contaminant Analyses
Processing of substantially greater volumes of water was required to collect enough particulate material from each size class for extraction and chemical analyses. For naturally sedimenting particles (e.g., flocs, minerals), a sedimentation separation resulting in a series of subfractions for suspended conventional particles was performed using four settling tubes in a parallel configuration (each tube 10 cm in diameter by 100 cm in length), and the parameters are shown in Fig. 1. The volume of water processed in each settling tube was 6 L, yielding a total capacity of 24 L. For each step of sedimentation, the lowest fraction, containing the largest particles, was drained from the bottom of each tube (300 mL/fraction). To conserve the height of the water column, the tube was then topped up with deionized distilled water, which allowed collection of the desired size-class particles within an acceptable error (Mavrocordatos, 1997). Fractionation by centrifugation was patterned after the combined electron-optical and chemistry approach of Perret et al. (1994), and performed using a Baxter Cryofuge 6000 and a swing-out rotor (Heraeus-Crist), which held six tubes of 250 mL each.

Contaminant Analyses
At each stage of the sedimentation and centrifugation procedures (Fig. 1), subsamples of supernatant water were collected using a peristaltic pump and subsequently freeze-dried to isolate particulate material associated with the supernatant. Particulate material yielded by freeze-drying of isolated fractions and supernatants was extracted using a mini-Soxhlet (PAHs) or accelerated solvent extraction (PCBs and organic contaminants; ASE; Dionex, Sunnyvale, CA) in dichloromethane. All solvents used were of glass-distilled analytical grade. Sulfur was removed from the extracts using mercury (PAHs) or activated copper powder (PCBs and organic contaminants).

Analyses for PAHs were conducted on crude extracts without cleanup. Gas chromatography (GC)–mass spectrometry (MS) analysis was performed on a Hewlett-Packard (Palo Alto, CA) Model 5890 Series II gas chromatograph with an on-column injector (1 µ L) and a Hewlett-Packard Model 5971A mass selective detector operated in selected ion monitoring mode. The following temperature program was used: 130 to 300°C at 1.6°C/min; final time at 300°C, 30 min. The column was a 60-m x 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]anthracene-d12. Typical between-run precision ranged from 4% for benzo[a]pyrene to 11% for chrysene. Based on a signal-to-noise ratio of 2.5:1, the estimated detection limits were 60 pg for 178 amu and 202 amu PAH; 100 pg for 228 amu and 252 amu PAH; and 130 pg for 276 amu PAH. Recoveries of PAH were measured using a standard reference material (SRM 1649; National Institute of Standards and Technology, Gaithersburg, MD); the levels of PAH varied by no more than 15% from the certified values.

Extracts for analyses of PCBs and organochlorine pesticides were evaporated, exchanged into hexane, and separated into three fractions of increasing polarity on Florisil (8 g; 1.2% v/w water deactivated; Fisher Scientific, Hampton, NH). Elution with hexane yielded a PCB fraction. The eluate was treated with activated copper powder to remove elemental sulfur and, after addition of aldrin as a volume corrector, was analyzed by GC with 63Ni electron capture detection (ECD) using an automated Varian 3600 GC (Varian Instruments, Palo Alto, CA). Samples were injected on a 60-m x 0.25-mm-i.d. DB-5 column (film thickness = 0.25 µm). Hydrogen was used as the carrier gas (initial flow set at 1 mL/min) and N2 as the make-up gas (40 mL/min). A total of 103 PCB congeners (including co-eluting congeners) were quantified using external standard mixtures (Ultra Scientific, North Kingstown, RI) that were run after every six samples. Total PCBs ({Sigma}PCBs) corresponds to the sum of all congeners. Recovery standards PCB 30 and octachloronaphthalene (OCN) were added to all sediments before extraction. Recoveries of the surrogates were uniformly greater than 90% and no corrections were made for recoveries. Procedural blanks were run with every 10 sediment samples.

Transmission Electron Microscopy of Sections
To stabilize and prepare samples for the production of ultrathin sections of particles for transmission electron microscopy (TEM), we selected correlative methodology (Liss et al., 1996; Leppard et al., 1996, 1997) that couples sampling with immediate 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 matrices. Four 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 fourfold multimethod protocol of Liss et al. (1996). We used their two treatments involving Nanoplast embedding (Rolf Bachhuber, Ulm, Germany) and their two treatments involving epoxy embedding. For aggregated heterogeneous colloid systems, such as flocs, the image interpretation was performed according to Leppard et al. (1997); for humic substances, image interpretation was performed with reference to Leppard et al. (1986) and Wilkinson et al. (1999). As was the case for Liss et al. (1996), images from the "glutaraldehyde + RR" method could be used as proxy images for the descriptions derived by correlating information from complementary TEM preparations. The Nanoplast (Frosch and Westphal, 1989) melamine resin treatment allows a spatial resolution of 1 nm, while embedding in Spurr's epoxy resin (Spurr, 1969) permits a resolution as fine as 3 nm.

All resin-embedded samples were sectioned with a diamond knife on a RMC MT-7 Ultramicrotome (Boeckeler Instruments, Tucson, AZ), and ultrathin sections (approximately 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 methods outlined by Lewis and Knight (1977). All ultrathin sections (and also all whole mounts) were examined with a JEM 1200 EX-II TEMSCAN scanning transmission electron microscope (STEM) (JEOL, Peabody, MA) operating at 80 kV.

Scanning Transmission Electron Microscope– Energy-Dispersive Spectroscopy of Sections
For microanalysis of individual particles for element composition, sections of approximately 100-nm thickness were used for an optimal compromise of section thickness with image quality. Sections were placed on grids that had been both Formvar- and carbon-coated to improve section stabilization during interaction with the electron beam. These thicker stabilized sections were coated again on their upper surface and then used for the determination of typical element compositions of selected particles, and discrete subcomponents of aggregated particles, by energy-dispersive X-ray microanalysis (Chandler, 1977). For the purpose of this research, absolute quantification of elements was not necessary. Instead, we determined the abundances (Jackson et al., 1999) of elements present in measurable amounts, and then examined element associations in precisely located particles. The scanning mode of the STEM was used to generate a microprobe beam for EDS using the same STEM instrument (above) equipped with an IMIX multichannel analyzer (Princeton-Gamma Tech, Rocky Hill, NJ).

Semiquantitative "fingerprint" analyses (Jackson et al., 1999) of composition for elements of Z > 10 were made over 60-s counting periods adjusted to minimize sample decomposition. The minimum detectable mass fraction of elements of limnological interest was in the range of 0.1 to 0.5%. A constraint on the interpretation of relative abundances of elements in ultrathin sections had to be taken into account, using the "P" value as defined by Russ (1972); this value is essentially the relative intensity of X-ray emission of each element from the same concentration. For each element of limnological interest, considering the K{alpha} line at 80 kV, the "P" values are as follows: Al (58%), Ca (94%), Fe (98%), Mn (100%), P (75%), and Si (67%).

Environmental Scanning Electron Microscopy of Hydrated Samples
Electron microscopy was performed on particle fractions prepared for two different electron optical modes: (i) by topographical imaging (using ESEM) to survey particles measured in tens of micrometers and (ii) through the imaging of internal details of smaller particles by transmitted electrons (as outlined above for sections documented by TEM). The ESEM topographical approach results in "whole volume" information from fresh hydrated particles, including dimensional information on particles that had not been exposed to preparatory fluids. To start an ESEM examination, 5-mL water fractions (containing suspended natural particles) were placed in a funnel/support assembly, in which a 2-µm pore-size polycarbonate Nuclepore membrane filter (Whatman, Maidstone, UK) was mounted on a rigid porous support, below which was applied a low vacuum. After filtration, the moist filters were affixed to stainless steel stubs and in turn fitted into a Peltier stage, cooled to 1°C, and viewed in a Model 2020 environmental scanning electron microscope (Electroscan Corporation, Wilmington, MA) operating at 20 kV. The chamber pressure was carefully decreased to slowly lower relative humidity within the chamber to remove enough surface water from the sample to allow observation of topographical views of particles on a stub.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
The fractionation scheme for colloids and larger conventional-size particles is shown in Fig. 1. These methods were based on an equation derived from Stokes law to separate different classes of aquatic particles according to a combination of size and density (Perret et al., 1994; Lienemann et al., 1998). The equation enabled calculation of specific g forces and times resulting in particle-size fractionations that can be accurate within ±10%, under conditions of low polydispersivity and similarity of density for the predominant particles. In this study, based on Hamilton Harbor surface water collected in late autumn (November 2000), the smaller size fractions were dominated by small colloids whose images showed low polydispersivity, and whose element analyses by EDS showed mineral colloids to be minor contributors to particle number. Our protocol, using settling time plus centrifugation, was originally designed to yield fractions from Hamilton Harbor water having an autumn particle composition. Our previous work showed an extreme predominance of flocs in fractions >20 µm in harbor water sampled in September (Leppard et al., 1998). In addition, total suspended particulate (including colloids) was polydisperse, and the fractions of major interest for PAH analysis contained essentially one particle type in terms of total particle volume (flocs), which was verified by EM. However, this methodology is amenable to modification for application to water samples exhibiting spring-like characteristics (e.g., with a significant contribution of runoff water carrying pedogenic humic substances), or summer-like characteristics (e.g., with an in situ production of microbial mucilage). Consequently, a comparison of earlier findings with water fractions of dissimilar particle composition was merited.

Each of the individual particle-size fractions prepared using sedimentation or centrifugation, and the corresponding individual supernatant fractions, were analyzed for PAHs and PCBs. For both compound classes, there was a distinct trend toward greater contaminant association with decreasing particle size, with a very high predominance of contaminants being associated with particles <2 µm in diameter (Fig. 2) . In the case of PAHs, typically 60 to 80% of the particulate-bound contaminant was associated with particles less than 2 µm. There appeared to be little variation in the percentages of individual PAHs associated with the individual particle-size classes, despite the broad range of molecular weights represented by PAHs. Of the two finer particle-size classes isolated by centrifugation, particles <2 µm generally accounted for roughly 50% of the total PAH binding while particles in the 2- to 10-µm range generally accounted for approximately 20%.



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  		Fig. 2. Relative percentages of selected polycyclic aromatic compounds in particle size diameter subfractions in a surface water sample from Hamilton Harbor processed using the described fractionation methodology. The sixth size range denotes particle-free fractions prepared using ultracentrifugation. PCBs, polychlorinated biphenyls.

 
The predominant association of contaminant with the finer particle-size classes stood in dramatic contrast to results from our previous characterizations of particle–contaminant associations in Hamilton Harbor water (Leppard et al., 1998). In the previous work, almost all PAHs (94%) were associated with particles greater than 0.15 µm; 15% of PAHs were associated with the 0.15- to 0.45-µm size fraction and 79% of the PAHs were detected in the fraction containing particles >0.45 µm. Lower molecular mass PAHs such as phenanthrene, fluoranthene, and pyrene were primarily associated with coarse fractions ranging in size from 20 to >80 µm (98, 89, and 85%, respectively).

The particle fractions ranging in size from 10 µm to fractional mm were shown by EM assessments to be dominated by heterogeneous flocs possessing an extensive internal colloid structure, with consequent extended surfaces available for contaminant binding. These colloidal particles are typically abundant in surface waters and can represent a substantial portion of the total particulate loading relevant to contaminant binding (Leppard, 1993; Liss et al., 1996; Leshniowsky et al., 1970; Wijayaratne and Means, 1984a). Sorption of PAH on colloids is known to occur in aquatic systems, and these contaminant-laden colloids can readily aggregate to form flocs (Wijayaratne and Means, 1984b); aggregated particles in the floc-size range are known from riverine ecosystems to contain PAHs (Evans et al., 1990).

At each stage of the sedimentation and centrifugation scheme, subsamples of supernatant were collected, freeze-dried, and subsequently analyzed to track the relative contributions of the two phases to the overall contaminant burden in the whole water sample. The breakdown of total PCBs in both the particle and supernatant phases is shown in Table 1. Figure 3 illustrates the relative contributions of both phases to the total PCB burden in the Hamilton Harbor whole water sample. In the case of the four individual sedimented fractions containing particles ranging in size from 2 to >80 µm, more than 95% of the PCBs remained in the supernatant fractions. The supernatants for the 2- to 10- and <2-µm particle fractions isolated by centrifugation accounted for 72 and 53%, respectively, of the total PCBs in the sample.


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  		Table 1. Quantities of polychlorinated biphenyl (PCB) homologs and concentrations associated with particle-size classes and corresponding supernatants in Hamilton Harbor surface water.

 


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  		Fig. 3. Mean relative percentage contributions of polychlorinated biphenyl (PCB) homologs in >10- and <10-µm particles prepared from Hamilton Harbor surface water. Mean percentages for >10-µm particles were calculated from values for the two particle fractions isolated by centrifugation, while mean percentages for <10-µm particles were calculated using values from the four fractions isolated by sedimentation.

 
The association of physical properties of contaminants with particle size was also investigated for PCBs. Table 1 also shows the concentrations of PCBs associated with the particle and supernatant phases on a homolog basis. There were no definitive trends in the individual homolog patterns. However, on a percentage basis the lower molecular weight homologs (mono and di, tri, and tetra) appeared to be more preferentially associated with the finer particle classes, while the heavier homologs (hexa and hepta) appeared to be more preferentially associated with coarser particles (Fig. 3). This trend was not unexpected given the generally greater solubility of the lower molecular weight PCBs, while congeners in higher homologs generally have lower solubilities and would correspondingly partition more quickly onto particulate material in the water column.

Electron-optical views of aggregated and colloidal particles sampled from Hamilton Harbor water are presented in Fig. 4 . They reveal the irregular morphology and porous aspect of aggregated particles and indicate that the fractionation proceeded as intended. The sections displayed in Fig. 4 show that the interior of the most abundant aggregates has a colloidal substructure. Overall, several individual colloid "species" were evident (e.g., humic substances, microbes, fibrils, cell parts, minerals) as classified by a combination of size, shape, electron-opacity, and internal differentiation. The ESEM images were selected from many to illustrate the irregularity of particle morphologies and to show that the sizes of particles matched the fractions to which they were attributed. Considering the TEM whole mount image, note the bridging of individual colloids within the heterogeneous aggregate by fine curved fibrils that cannot be resolved by ESEM. Such heterogeneous particles were uncommon relative to the high frequency observed in an earlier study on Hamilton Harbor (Leppard et al., 1998), and to the most abundant aggregates reported in this comparative study.



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  		Fig. 4. Electron-optical views (a–c) of entire aquatic particles (whole mounts) in surface water collected from Hamilton Harbor and separated into size fractions. (a) An environmental scanning electron microscopy (ESEM) image, which indicates the size and morphology of a typical aggregated particle (a multicomponent floc) from Fraction 3 (F3 of Fig. 1) in topographical view. (b) An ESEM image of a smaller floc from Fraction 4. (c) A high-resolution transmission electron microscopy (TEM) image of a small heterogeneous aggregated particle captured, from raw water, on top of a Formvar film (mounted on a TEM grid). Electron-optical images of ultrathin sections of aggregated particles collected from Hamilton Harbor water fractions (d–f). (d) A TEM image showing individual colloids within a porous large aggregate collected from raw water. (e) A TEM image showing individual colloids in an aggregate collected from Fraction 4 (10- to 20-µm range). (f) A TEM image showing individual colloids in an aggregate collected from Fraction 5 (2- to 10-µm range). The colloidal particles shown in all TEM images (of ultrathin sections above) represent the dominant particles by number. Note that for all three images, the protocol used for fixation and subsequent TEM preparation was the proxy protocol beginning with the step "glutaraldehyde + RR."

 
In TEM examinations proceeding through the smaller fractions (Fig. 4c–f), there was an increasing dominance of colloids and small clusters of colloids having nanoscale dimensions. Colloids imaged as apparent individuals (near 3 nm) as small clusters, and as larger aggregates yielded images consistent with those of humic substances described in the TEM literature (Leppard et al., 1986; Wilkinson et al., 1999; Stevenson and Schnitzer, 1982). "Per cluster" EDS analyses of the element composition (Leppard, 1992a; Jackson et al., 1999) indicated a dominance (in terms of particle number) of the water by organic-rich particles whose mineral component was typically less than one part per thousand by mass (as judged by the typical absence of peaks for Al, Ca, Fe, Mn, P, and Si). With regard to these dominant organic colloids, their abundant fractal aggregates revealed sizes at the near-micrometer level, a feature that has been detected by photon correlation spectroscopy for lake humic acids (Caceci and Billon, 1990). The presence of our moderately large aggregates is consistent with recent experimental work on humic acids showing 2.5-nm colloids giving rise to 50-nm fractal aggregates (Senesi, 1999), and earlier TEM-based aggregation studies on pedogenic fulvic acids (Leppard et al., 1986) showing 3-nm colloids producing aggregates of near-micrometer size. Comments on the mechanism of formation of the humic substance aggregates and an analysis of their fractal morphotypes are topics beyond the scope of this paper.

The multimethod correlative EM analyses indicate that the particle composition of the November samples of Hamilton Harbor water in this study was substantially different from that of the water of the earlier Hamilton Harbor study (Leppard et al., 1998). There are a number of potentially influencing factors that may have contributed to the observed differences, including climatic factors and the characteristics of water masses entering the harbor via tributaries, sewage treatment plants, and runoff. In lakes, the origin of natural organic matter has a large effect on the nature of particles present and their relative proportions (Buffle, 1988). Depending on the speciation of the biota at a given time, a lake can have a variable in situ production of mucilage and a variable release of modified organic matter as a result of biological transformations.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The described particle-size fractionation and particle characterization methodology is a significant improvement over our previous work, and represents further incremental progress toward accurate characterization of contaminant–particle carrier relationships. Our earlier work (Leppard, 1992a, 1992b; Leppard, 1993) revealed the feasibility and utility of characterizing colloidal carriers of contaminants by electron-optical means. Liss et al. (1996) used electron-optical characterization in a multimethod, multidisciplinary context to analyze the nanoscale architecture of riverine and wastewater flocs. Droppo et al. (1997) further developed this research by improving the quantification of floc properties, while providing additional confirmation of the importance of individual nanoscale floc components. This tunable methodology has evolved further in the current study through comparative analysis of contaminant binding by colloids and flocs, featuring differential cascade sedimentation and centrifugation to separate different sizes of suspended particles. In comparison with our most recent work (Leppard et al., 1998), the substantial scaling up of the particle-size class sedimentation procedure yielded greater particle mass to enable more complete physical and chemical characterizations.

A proper statistical analysis is required to ensure the validity of any conclusions regarding particle carriers and their role in contaminant transport. Our work to this point has been developmental in nature, and future studies will incorporate replication into the experimental design for statistical purposes and for evaluation of potential spatial variations in carrier characteristics. The major contribution of this preliminary work is the clear demonstration of the association of contaminants with particle carriers represented by specific size classes and distinct morphologies. When considered within the context of our previous work, we have also shown the potential for significant spatial and temporal variations in the physical characteristics of primary carriers within aquatic systems. This entirely adaptable methodology represents a significant step toward incorporating specific particle characteristics into aquatic system models, rather than incorporating particle characterizations based on averaged properties.

Future research will also focus on additional tuning of the methodology that, through EM monitoring, may lead to isolation of fractions based on specific particle morphotypes within the gross categories of colloids and small particles. This in turn could lead to the quantitative isolation of specific major individual colloids. The identification of humic fractals as contaminant concentrators and carriers in Hamilton Harbor, and a capacity to isolate them quantitatively, may enable modeling based on particles with these specific physical properties. Using our methodology, carrier particles can be isolated and characterized in their native state; electron optics, used in conjunction with selected chemical measures, may reveal variations in particle structure–activity relationships as particles undergo successional changes over time. A well-characterized, isolated contaminant carrier can be subjected to focused experiments. Thus, important environmental variables affecting contaminant transport could be investigated directly. For particles isolated and characterized in this study, important properties for future analyses include rate of contaminant uptake, settling rates, aggregation phenomena that alter settling rates, dispersion of particles in terms of distance traveled, factors inducing changes in the nature of contaminant binding, and effects of burial or resuspension on contaminant release. Our methodology provides the potential to assess individual particle morphotypes, their roles in contaminant uptake and release, and the potential to determine factors facilitating or frustrating contaminant containment procedures after burial.


   ACKNOWLEDGMENTS
 
The authors thank the Government of Canada Green Plan and the Great Lakes Action Plan.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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