‘THE FRESHWATER FLOC: A FUNCTIONAL RELATIONSHIP OF WATER AND ORGANIC AND INORGANIC FLOC CONSTITUENTS AFFECTING SUSPENDED SEDIMENT PROPERTIES 1.G. DROPPO"*, G.G. LEPPARD", D.T. FLANNIGAN" AND 5.N. L1SS* ‘Geography, University of Eager Exctee EXS 48, UK, "Deparonent of Biology, MeMaster University. Hanon, Ontario, Canada, L88 4K1.‘Department of Applied Chemical and Biological Scicnees, Ryerson Polytechnic Ueiversity, Toronto, Ontario, Canad, MSB 2K3 Abstract. Focculsted fine-grained sediment sa complex matrix of microbial communities and organic (tis, ‘significant ecological oleae they can opus the overall water qualty through thelr physica, chemical andlor biological actly. This paper invetgnes the complex sroctaral main of riverine Roce over large range of magnifications using eorelatve microscopic techniques. The significance of fbesteacmal characteristics [G3 ‘eanspot and soting), chemical (oz adbobingluansfeening contaminants and niu), and biological (2, [Hotrastormation an habitat devslopment) behviowe af lo is ivestigned. Rests suggest thats the floes internal sete that has a signfieant pact on contling the above Noe behaviours, This aterm suucte is ‘nya sa en Gunshot yd eases owners hain wt Mls outed Uy Ue actve mirobil community wih he oe. This mate in conjunchon with the organic and biopganie (active and inactive) cnstitens ofa fle, provides an inate poe stuctue that may esl in water being an important ‘bound component ofa fice. These complex interactive tractor and functional poperics ofa Mbe are considered 10 iaence a 19s $ peraMtour som pnysiealy 1 fw ts ansporea of sewed, caemicaly 0 now adsorbstransfoms costuinats and nares, and biologelly in how i develops adverse microhabitst capable ‘of moditying the stuctaal, chemical and biological makeup ofthe Hoc. Key Words, freshwater oe, lcevlation, bacteria, ibis, pores, inorganic parties, setling, siz and structure Flocculation of fine-grained sediment significantly alters the hydrodynamics of the constimient particles and will affect the transport of particle-hound contaminants. Diserete primary particles that typically would not settle in a given flow may settle when incorporated within a floc, Flocs can be viewed as individual microecosystems with autonomous and interactive vhvsical. chemical and biological functions or behaviours ‘operating within the floc matrix. These microzcosystems also continuously interct with their surroundings, a the medium in which they ate transported provides the flocs with building materials, eneray, nutrients and chemicals for biological growth, chemical reactions and morphological development. While flocs can regulats their awn ‘environment they also are known to have the ability to regulate surrounding water quality by their physical, chemical and/or biological activity in both natural and engineered systems (Liss eral, 1996; Decho, 1990; Leppard, 1985). While the existence of flocculated particles within aquatic systems has been recognized for some time, the significance of these on the hydrodynamics of the sediment has only mor recently been explored. The change in the sediment transport behaviour has been attributed to the changes in particle size, density and porosity brought about by flocculation (Li and Ganczarczyk 1986, 1987; Liss er al., 1996; Droppo and Ongley, 1994). While these gross scale floc properties are known fo alfect the physical transport of sediment, there is still a fundamental lack of knowledge an the controlling factors of Water Air and Soil Pollution 99: 43-54, 1997. ©1997 Kluwer Academic Publishers. Printed in the Netherlands.4 1G DROPPO, ETAL. fAvcculation and how floc architecture influences its physical chemical and/or biological behaviour. This lack of knowledge is attributed in part t0 the inadequacy of single ‘observational techniques to observe the whole realm of the floc structure and processes from the fine (oubmieron) to the groce aeale (moron). Studies of floc structure generally require techniques such as conventional optical microscopy (COM) and scanning electron microscopy (SEM). For the observation, rmoacurement and modelling of sediment structure and hehavinur (in. transport and setting), COM is often the technique of choice (Droppo and Ongley, 1992, 1994; Li and Ganczarezyk, 1986), This scale (>1 jm) is used because the outward behaviour of flocs can be observed at this recalution Furthermore, from am engi greater than 1.0 jm will represent the majority of the mass for sediment transport and sedimentation studies, While the outward flac stractin and hehaviowe may ering stand point, flocs he derived. from COM. the internal architecture of the floc cannot be resolved at this scale, This internal structure is believed to influence the outward behaviour of the floc (Liss er al, 1996; Heissenberger et ‘a1, 1998). High resohition techniques such as transmission electron microscopy (TEM) and scanning confocal laser microscopy (SCLM) have revealed a consistent presence of fibrillar extracellular polymeric material that bridges and binds primary particles (organic and inoreanic) together within the floc matrix (Liss et al., 1996). ‘These fibrils, in conjunction with the active biological component of the floc matrix, may influence the chemical, physicochemical and biological processes within the floc itself and in the natural aquatic system as a whole (Leppard, 1995, 1993, 1985; Decko, 1990). The significance of the internal floc structure on the outward gross behaviour of the floc is, however, still poorly understood. To evaluate the internal structure of freshwater flocs and its potential impact on floc behaviour, we have examined numerous flocs with correlative microscopy (Liss et al, 1996; Leppard, 1992), Correlative microscopy is a means of compensating for the artifacts and resolution constraints imposed on results when only one microscopy and related techniques are used, Correlative microscopy is a strategy that uses different microscope typos and their ancillary techniques to derive multiple levels of information from a given specimen. By observing flocculated material over a full range of magnifications (1.0 'mm to <1.0 um, this paper provides a better understanding of how tloc torm an bot its {gross and fine scales can affect the function or behaviour ofa freshwater floc. 2. Experimental 2.1, STUDY SITES Sample sites were chosen to provide information on freshwater floc structure and not for me characlervalion or spatial comparison of specifie sites and weit floc sutucwe Suspended and bed sediments were collected from a variety of freshwater systems including both riverine (Nith River and 14-Mile Creek, Ontario, Canada) and lacustrine environments (rTamimton riarbuur aud FUN Suauley Marbut, Take Onnativ, Outasivs Canada). These sites have been previously studied with their hydrological and ‘geomorphological characteristics documented by Droppo and Jaskot, 1995 and Liss et al. 1990 QMid River), Ongley, 1974 (I4-Iile Creek), Amos and Dreppo, 1996 (lan‘THE FRESHWATER FLOC 45 Harbour), and Skafel and Krishnappan, 1005 (ort Stanley Harbour), 2.2, CORRELATIVE MICROSCOPY 22.1. Conventional Optical Microscopy for Gross Structural Determination Samples were collected following the metiod of Droppo and Ongley (1992). This method allows for the nondestructive dirert sampling. ohservation nd mesennomant of Alocculated material within a settling column (plankton chamber). The flocs that settle ‘onto the plankton chamber’s microscope slide were imaged using a Zeiss Axiovert 100 micrnccope interfaced with an image analysis system (Northern Expose! — Fepiz Imaging, Ine.). Northern Exposure™ allows for the determination of floc morphological characteristics and grain-size distributions down 10 a lower resolution of approximately 2 uum at 100 X magnification (10 X objective). Samples from the 14-Mile Creek were stabilized in agarose following the methods of Droppo et al. (1996) for subsequent use with the higher resolution microscopes. 222. Scanning Confocal Laser Microscopy for Internal Ploc Matrix Determination Samples stabilized im agarose were stained with a solution of wheat germ agglutinin conjugated to Texas Red™ (WGA-TR, Molecular Probes, Inc., USA), This molecular probe is specific to many of the polysaccharides present in the fibrillar material (those containing N-acetyl-D-glucosamine). ‘The flocs were then washed three times with 0.1 M phosphate buffer (pH 7.0). The agarose disk was then divided into multiple sections {sections also used for TEM). One section was placed on a standard microscope slide and covered by a cover slip which was sealed around the edges with nail polish (prior 10 sealing, the area under the cover slip was flooded with buffer solution), “The sample was imaged using a Zeiss Micro System LSM (Model LSM 10 BioMed). The SCLM was equipped with an argon laser (emission lines at 418 and S14 nm) and a 63x (1.4 na) objective with an electronic zoom of 30X. Image slices were collected at 1 um vertical intervals and reassembled to view the whole floc volume specitic to the stained component. 2.2.4. ‘Transmisston blectron Microscopy Jor Ultrastructure Determination More detailed ultrastructural observations of sediment samples (discrete or in agarose) were made by preparing the samples for TEM following the four-fold multi-preparatory Technique (Liss ef ai, 1990), This technique allows for me ennanced observation oF specific components of the floc such as cells and polymeric material. Ultrathin sections ‘were imaged in transmission mode (TEM) at an accelerated voltage of 80 KV using a IDOL 1200 Bx N-TEMSCAN scanning wanisinission elecuun niictoscope, 2.2.4. Stereoscopic Microscopy for Settling Velocity. Density and Porosity Deverminasion Settling experiments were performed similar to that of Li and Ganezarezyk (1987), however with the use of a Nikon SMZ-27 stereoscopic microscope and image analysis ayotoun A diup vf sudinnast wollseied with a wide mouth pipette (3:74 sm) frou a gently fhomogenized sample bottle was introduced into an insulated 2.5 1, capacity setting column, The setting column itself is 5 x 10 x 50 cm with the stereoscopic microscope Focusing of a plane inaide the column at a distance of 38 om from the top. ‘The long46 1. G DROPPO, ET AL. sitting Uiotauwe relative cy dhe sige Uf dhe puis is teyulved qo damp out any turbulence or setling iregularities resulting from the intial introduction of the sediment and to allow the sediment to reach terminal velocity prior to detection, ‘Ao the floes pass through the ficld of view of the miiuuscupe icy ave videu taped Um a SVHS VCR through a CCD camera interface. Using Northern Exposure™ (Empix Imaging Inc.) image analysis software, the setting velocity was derived by digitally overlaying two video frames af @ known time interval apact. Tn this way the san pantie appears on the newly combined image twice and the distance of settling and the particle size van be digitized, The data are automatically sent to a spreadsheet where density and porocity can be mathomatically eotimated, The density of a floc was estimated using Stokes’ Law (Equation 1). As Stokes’ Law is based on the settling of single impermeable spherical particles in a laminar region Geynolds Numhor 7 015), it fe nat ical far the datormination of Aloe dansity besauce of the heterogeneous structure and irregular shape of the floc (Hawley, 1982; Burban er al, 1990), Nevertheless Stokes’ Law or a modification thereof has offen been used to determine the wet density of singulae Nove (Paha and Watanabe, 1070, Gikke, 1085; Li and Ganezarczyk, 1987; Fennessy et al, 1994), and does provide an indication of how aggregate settling velocity, density, and porosity are related to aggregate size Equation | O= 15D (pr—pt Where: @ = soltling velocity, D = diameter, py = wet density of the floc, py = density of the water, and) = dynamie wieensity (Hinemetia wieensity © py) ‘As @ and D ate derived from the image analysis system and p, and 1 are constants for 4 given watne tomparstiee, tho sit dancity af Us fae (p) can be ealeslated. Dencitiae are expressed as excess density (I-p9). Following the method of Li and Ganczarezyk (1987), the floc porosity can be expreseed hy a mace halance aquation (Equation 7) ascuming 2 typical density of dried oilt and clay of 1.65 gmvem Rquation 2 p= PP Dim Do where: @= floc porosity, and p, = density of the dried solid material 3. Results and Discussion Lacustrine and riverine flocs have a complex composition and may be structurally and functionally dynamic due to the multiple interactive processes operating between the various physical, chemical and biological factors (Figure 1). The common thread for most flocculated natural aquatic sediments, however, is that they all have a living and active‘THE FRESHWATER FLOC a biological component in conjunction with inorganio and won Tiving biological particles Pe aes aaa turbulence Tae | & fibrils Brownian motion “S I y, ifforontial cottl o fibril bundles differential cottling a colloids pony | primary particle size temperature Pom : bl TSS ‘MN | detritus surface Properties / | fecal material ae | \ \ ‘oni concentration” ye discharge Heed ™ PH redox potential nutrients and 7 \ gerseicogei composition a eed Pes ee CHEMICAL Fig. |. Interactive physical, cecal and biological ators inlluencing flac development ‘The gross structural characteristics of two riverine flocs (Nith River) can be observed in Figure 2a and b. Bach appears heterogencous in terms of spatial density across the flocs ‘with the presence of macro-pores. All flocs observed were composed of organic and inorganic particles mixed together. Inorganic grains were silts and clays with generally no sand particles present. The majority of organic material can not be differentiated or speciated at this gross scale (only a difference in shading), however diatoms and other large living organisms were occasionally observed within the floc matrix. ‘Using COM in conjunction with image analysis, floc size distributions were ‘determined {an example from the Nith River is given in Figure 3a). While the descriptive Statistics may vary between samples and sites, the same trends were observed for all Pervent by number distributions were positively skewed while percent by volume istributions wete negatively skewed, ‘This suggests that while the majority of the mass is, in the larger size classes the majority of the particles is in the smaller size classes. “This8 1. G DROPPO, ETAL. aes a for tie modeiting ot contaminant movement as the larger flocs will likely settle out much faster than the finer flocs that will travel with their associated contaminant much further within the system. An understanding of the floc distributions (by nwovtns aud volunte) is dius important ror me development ot sediment and contaminant transport models. eae ingplica 4100um Fig, 2aandb, COM observations of flocculated sediment fram the Nith River Many other floc morphological characteristics also are important when studying or ‘modelling floc behaviour, Figures 3b to 3d illustrate three such additional parameters of longest dimension, width, and shape factor which are known to affect settling (Li and ‘Ganczarczyk, 1Y8/). Ihe Hocs observed generally were irregular in shape but tended towards @ circular shape with median values ranging from 0.6 to 0.7 (1.0 = a perfect circle). Longest dimensions and widths yielded similar distributions to the equivalent sphiericat atamerers wath sight variations reilective of the non-spherical shape of flocs Knowledge of floc settling velocity. density and porosity also is useful for the characterization of sediment environments and for the study and modelling of sediment sand Cunuaminan wansport, igure 4a tllustrates the settling velocities of eroded ‘aggregated particles from the bed sediments of Hamilton Harbour, Lake Ontario (Amos and Droppo, 1996). In the observed positive relation between floc size and setting relovicy, a siguificain annount of varlantity (K° = 0.4) 18 evident but not surprising since the majority of the sediment is present in a flocculated form, Factors that can affect the settling rate of a floc and impose variability im the results are floc composition (proportion organic amt invigenis), shape, purusicy amd water content. While selling velocity increases with floc size, Figure 3b demonstrates thatthe density decreases and the porosity increases with floc size. This relationship also has been observed by Li and Ganczarczyk (1987), Logan and Hunt (1900) aud Taunby ail Watanabe (1979). AS a TOC FOS, HS hhumber of linkages to additional particles or flocs increases creating additional pores. This increased porosity leads to an increase in water content that forces the density of the floc towarde the dansity of water and tends to reduc dhe seilling velucigy UF te ME, Logan and Hunt (1987, 1988) found thet the development of porous flocs was advantageous to the microbial community as substrate removal was up to 60% greater than for disposed bactoria. This wae attributed to edvective flow (calvulatud ws Le up i 100 im s*) through the floc pores rather than to traditional molecular diffusion theory. Li and Ganezarczyk (1988) proposed that, if there is significant flow through the floc's pores, then the floc's setting velewity may. be ineroaced rather than reduced. Sherman (1939) un the other hand calculated that flow through flocs studied from Lake Mead was likely‘THE FRESHWATER FLOC 49 erat Decor alert Share! ante ay at get 457m Nise cian oxen Fig. 3. Example of COM and image analysis floc moypholoical characterization forthe 14-Mile Creek. (3) [Equivalent spherical dhamete distributions by volume and rab, (D particle longest mension distribution, () patie wid dstibuton, and (B) parce shape factor distnboion,0 1G DROPPO, ETAL, gible cand hat die bound! water was thus an integral part ot the Moe structure. if water does nove through a floc, it may act as a food stream or supply for the biological component and also will bring additional floc building material of organic and inorganic ds ow amautia, Ollierwise a Hee May assimilate nultients and contaminants primarily through diffusional gradients. composition Seting Viety (mie) xcees Oey (oi la a ge ages aay te elu slo "odo ido a abo ae who ato aba 0 aarone Se oss Sze wm) Fig. 4 The relationship of floc sie to (a) seing velocity, and (&) porosity and excess density for eroded ‘enlace yn Hanon Habe Ge Sete een ANDO ANd WIOppes 19H). ‘The porosity of flocculated sediment usually is calculated from the density as inferred rod floc rotiling velocity (ao above abd by Li and Gasweaiveyhy 1987) Taub and Watanabe, 1979). Porosity is expressed as a percentage which is used mathematically for the prediction or inference of floc hydrodynamics (Li and Ganezarezyk, 1987) or ukstrate uptake by natural or wastewater flogs through adveetive flow aust diffusion ‘gradients (Logan and Hunt, 1987 and 1988), Porosity is a complicated issue as the definition or identification of pores is highly dependent on the resolving power used for their nhservation Potee ahcorved swith COM such ac thoce in Figure 2a und b appear to be devoid of materials and appear to be significant open channels for the movement of water through the floc as it is transported or settled, While we cannot observe the same pores as those viewed with COM, TEM ohservarione aften ravsal that many af thece macro pores are in fact filled with a complex network of cells and fibrils (Figure 5). As such, calculations of pore water movement and implications to floc seuiling and contaminant transport that do not consider the internal structure of the pwere may he srroneous. ‘ng >. ES micrograph ot eroded oc trom Port Stanley showing extensive fibril network among clay pts (Moc stabilined ngloaaldehyéefrahenium rel fixative).‘THE FRESHWATER FLOC st The Guile itluseaed in Figure J aie che Uouinane physical bidging mectanisin between the organic and inorganic constituents of the natural flocs observed. ‘These polymeric fibrils produced by the bacteria serve as a means of attachment and nutrient assimilation for the many vells thot colvmire thy flucculatal material (Custestont €¢ a, 1987), ‘The flocs exhibit « pseudoplastic nature that is likely to be related to the flexible nature of the fibrillar material (Droppo and Ongley, 1992). We believe this polymeric raterial to be the dominant material for the development and stabiliration of flocculated ‘material (Droppo and Ongley, 1994). This does not exclude electrochemical flocculation completely (inorganic particle-particle contact is observed in Figure 5 - inorganic particles, svere composed of Fe, AUSi or i clemonts ao derived from energy dispersive spectroscopy), but rather that it appears to be less significant than the biological flocculation within the natural systems we have examined. In-no case was a floc observed to be aggrogstad colaly by olectrochamical flocculation. “A cignificant quantity of fibrile was always present Fig 6. SCLM images of polymeric material of ¢ floc from 14-Mile Creek. (a) single optical section, (b) SCLM helps to visualize in three-dimensions the significance of fibrillar materiaf to floc building and behavioural (transport and settling) characteristics. The use of the molecular probe wheat germ agglutinin in conjunction with SCLM allows for the observation and estimation of the polymeric fibrillar material. Figure 6a illustrates a Single optical section from a 14-Mile Creek floc. while Figure 6b represents the total floc volume as derived from the computerized stacking of optical sections obtained at | ym intervals. It is evident that the fibrillar material of natural flocs is a significant constituent component of the floc. In association with all other components this extracellular polymeric substances will influence floc size, floc density (increases water content), contaminant and nutrient adsorption andfor modification (large surface areas) and assist in maintaining diffusional and electrochemical gradients 4. Conclusion Flocculation, because it alters the hydrodynamic properties of particles in transport, significantly influences the fate and effect of sediment and associated contaminants. Tt va found that the complex structure and composition of a floc wall have a significant2 1. @ DROPPO, ETAL. influence over its physical, chemical and biotogical Dehaviour. An tmporam. observation was the apparent structural dominance of the fibrillar extracellular polymeric material within freshwater flocs. These fibrils are believed to be the dominant material for the development and siabiizadion uf Moccutated macrlal. Bac geucral vompoueut Uf & Mus (organic and inorganic particles plus water and pores) is diverse in itself and can possess a specific function within a floc, The interactions of these constituents and their functional processes cai weaull i dy snalifivation of a fion's Uhavioue, physically in how i is transported and settled, chemically in how it adsorbs and transforms contaminants and nutrients, and biologically in how it develops a diverse microhabitat capable of modifying fie structural chemical and biological makeup of a floc, Theoe interactions and Function are summarized in Figure 7, re FlocConstiuents a i rantaminant Fe i \,00 Ory Vqutiiont and metabolic j! | transformation & m5 end product transport! | microbial growth | ,7 floc building \\ |) electrochemical and i | ut / Afusional gradients gad 1 hydrodynamic, | anaerobiclaercbic m1 chemical and} processes |" 8) ological a" | \\ behaviour 7 7 | Tay colonization sites cawon progng [yf | contaminant acscron sorption [See eee et tenet Fig, 7. Conceptual model of oe form and function“THE FRESHWATER FLOC 3 Achuumledgenents ‘This work was supported by Environment Canada and by a National Science and Enginccring Research Council of Canada Strategic Grant, STROI67324, awarded to SNL, GGL. and LG.D. We thank M.M. West and C. Jaskot for their analytical assistance, Dr. M. Skafel for the provision of Port Stanley sediment and Dr. K.N, Irvine fond throe anonymous roviowere For their eritioal raviow of tho manacoript References ‘Amos C.L, an Droppo, IG. 1996, "The stability of remediated lake bed sediment, Hamiton Harbour, Lake ‘Ontarie, Canada’, Geological Suevey of Canad Opoa File Report # 2276. Burban, PY, Xu, ¥-J, MeNei, J and Lik, W.: 1990, J Geophys, Res 98(C10), 18213-18220, Rev, Mierblel. 41, 435-468, echo, A.W. 1990, Oceanogr. Mar Bol. Annu. Rev.28, 73: Droppe, LG. and Ongley, E.D. 1992, Wat Res.26, 65-72 Dopo. 1G. snd Ungiey, E10: 1995 Wat Kes. 288) 198-180. Dopp. 1G. and Jskot, C1995, Bnviter, $e. Technol29, 161-170. 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