Environ. Sci. Technol. 1990, 24, 506-513
Settling and Coagulation Characteristics of Fluorescent Particles Determined by Flow Cytometry and Fluorometry Kathleen A. Newman, Frangois M. M. Morel, and Keith D. Stolzenbach' R. M. Parsons Laboratory, Department of Civil Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02 139
A new technique for detecting particles in natural waters relies upon analysis of fluorescent emission by flow cytometry. Fluorescent pigment particles ranging in radius from -0.1 to 5 pm are available in sufficient quantity to be useful as model particles. Laboratory coagulation and settling experiments analyzed by fluorometry demonstrate that the efficiency with which the pigment particles coagulate with sewage particles is very low (less than 5 X lo-*). Hence, in field applications these particles provide the limiting case of low-interaction behavior relative to natural particles. Removal from laboratory columns occurs primarily by noninteractive settling while thermal convection currents maintain nearly uniform particle concentration within the columns. Observed decreases in particle number are exponential (first order) for each size class as predicted for settling from well-mixed suspensions. The decrease in total suspended particle mass is higher order as a result of the difference in settling rates among particles. Thus, a system in which noninteractive settling dominates mimics systems in which coagulation processes are important. Flow cytometric analysis of particles removed by noninteractive settling shows the fluorescent emission from individual particles to be proportional to the particle surface area. These results enable flow cytometry to be used to detect, count, and size large numbers of particles rapidly. Introduction
The dynamics of aquatic particles are important both in the geochemical cycling of natural substances and in the transport and fate of particle-reactive anthropogenic pollutants. Particles in natural water bodies are subjected to a variety of processes: advection, settling, coagulation, and repackaging by biota in the water column, as well as burial, bioturbation, and resuspension after incorporation in the sediment. Most of these processes and their relative importance in the environment are poorly understood. Progress in this area of study is well served by field experiments using well-characterized model particles. Soluble fluorescent dyes have commonly been used to study mixing and transport of water masses ( I ) , but model particles have been used rarely because their analysis was labor intensive and lacking in sensitivity. Fluorescent painted sand grains (2),ZnS crystals with fluorescent inclusions ( 3 ) ,and fluorescent pigment particles (4, all counted by microscopic observation of filtered samples, have been used with success in field studies. However, a major limitation in these studies was the inability to count the large number of particles necessary for good counting statistics. We have developed a new technique using flow cytometry to count and size fluorescent particles, thereby automating the limiting step in particle tracer studies. This article describes the flow cytometric technique and presents the results of systematic laboratory studies characterizing the settling and coagulation behavior of a model particle. Application of this technique to a field study of deposition at a sewage outfall is described elsewhere (5). 506
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Materials and M e t h o d s
Fluorescent Pigment Particles. Fluorescent pigment particles (AX series) were obtained from the Day Glo Color Corp., Cleveland, OH. The particles are nonporous and amorphous, consisting of a solid solution of fluorescent dyes in a sulfonamide-triazine-aldehyde thermoplastic resin matrix. The material is produced in large, brittle slabs, which are pulverized to produce fine particles. The size range of the particles is narrowed by air classification. The specific gravity of the particles is 1.38 (manufacturers specification as measured by picnometer). The fluorescent pigments degrade under intense ultraviolet light but are expected to be very stable in water. Rocket Red (Amm = 600 nm) was chosen from a selection of colors as its fluorescence spectrum (Figure 1)was best resolved from that of natural particles ( I , 5). The hydrophobic particles were supplied predispersed in a 5050 (wt:wt) aqueous suspension stabilized by Tam01 731 (Rohm and Haas Co., Philadelphia, PA), the sodium salt of a carboxylated polyelectrolyte. The size distribution of the particles was determined by filtering a dilute suspension through a 0.02-pm filter, digitizing a major and minor axis for each of 1200 particles viewed on scanning electron micrographs (SEM) of the filter, and fitting an ellipsoid (rotation about the major axis) to calculate volume. The equivalent radius of the pigment particles, defined as the radius of a sphere of the same volume, ranges from 0.1 to 5 pm. The number distribution (Figure 2) is dominated by particles with volumes less than 1 pm3. Despite the large number of particles sized, the distribution tails off to counts of two and three particles in the size classes above 100 pm3 so that the uncertainty in the number of large particles remains high. Stokes fall velocities calculated by using equivalent radii range from 0.0002 to 18 m day-l. For the most eccentric particles this approximation results in a 4 % error in calculated setting velocity (6); the average error is 0.3%. Sewage Particles. Primary sewage effluent was collected in 4-L carboys from the pump house of the Boston Metropolitan District Commission Deer Island Sewage Treatment facility early in the morning and carried by car (20-30 min) to the laboratory. The carboys were shaken to distribute solids evenly through the suspension before withdrawing aliquots for the experimental vessels. Suspended Particle Analysis. Number concentrations and size distributions of pigment particles in suspension were measured by flow cytometry using a modified version of the instrument described by Olson et al. (7) and Shapiro (8). In the flow cytometer, particles are hydrodynamically focused by a sheath stream and surveyed individually as they pass single file beneath the objective of an epifluorescence microscope (Figure 3). The mercury arc lamp source of the microscope excites each fluorescent particle as it passes. The fluorescent emission from the particle is split by a filter; the two resulting beams are further filtered and focused onto photomultiplier tube detectors. For each particle, the fluorescence intensities in the two wavelength bands are amplified (linearly or
0013-936X/90/0924-0506$02.50/0
0 1990 American Chemical Society
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Flgure 1. Fluorescence emission spectrum of Rocket Red fluorescent pigment particles in water suspension. Excitation at 436 nm. Equivalent Radius
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logarithmically) and then recorded on linear scales from channel 1to channel 254 (Figure 4), with the larger, offscale intensities assigned to channel 255. The number concentration of particles of a given intensity is calculated by dividing the number of particles detected in a channel by the volume of sample. Intensity distributions are smoothed by grouping the channels by tens to yield 25 classes. The number concentration of particles in each class is designated as n,. The flow cytometer data presented in this article were collected by using a 580-nm short-pass filter as a beam splitter and filtering the long-wavelength beam with a 600-nm wide-band and a 620-nm long-pass filter. The short-wavelength beam was not filtered. Signal amplification was achieved by use of linear amplifiers so that channel number is proportional to emission intensity. Size calibration of the linear scale was difficult, as monodisperse particles with the same emission characteristics as Rocket Red were not available and repeated settling of the particles did not produce a sufficiently narrow size distribution
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Flgure 4. Output from the flow cytometer in llnear amplification mode for a standard solution of Rocket Red pigment particles. Histograms show relative number of particles in each of 254 llnear channels for the short- and long-wavelength bands. Each point on the scattergram denotes a single particle with short- and long-wavelength band intensities given by the ordinate and abscissa. Brlght, off-scale particles recorded in channel 255 are not shown.
of particles to be of use on the linear scale. Estimates of the range of sizes detected (1.5-4-pm radius) and the dependence of the fluorescence intensity of individual particles, I,, on particle radius were deduced from the laboratory experiments as will be described later. The dayto-day sensitivity of the instrument was maintained constant by calibration to Polysciences 3-pm fluorescent microspheres. The integrated fluorescent emission intensity, I , of a suspension of pigment particles was measured with a Perkin-Elmer LS-5 luminescence spectrometer with an excitation wavelength of 565 nm and fluorescence detected at 600 nm. Sewage concentrations were measured gravimetrically after evaporation at 105 O F . Samples (20-40 mL) were withdrawn from the experimental vessels by syringe and filtered through predried, preweighed 47-mm 0.2-pm Nucleopore filters in a glass-fritted Millipore filter holder. Each filter was rinsed twice with 10-15 mL of distilled deionized water and then dried at 105 O F to constant weight. The mass of sample filtered was determined by Environ. Sci. Technol., Vol. 24, No. 4, 1990 507
Table I. Quiescent Settling Experiment Conditions
column
A B C D E"
water column depth, cm
pigment particle concn, ppm
75 75 75 37.5 37.5 37.5 37.5 15
F*
G
H'
dry-filterable sewage concn, PPm
100 10 10 60 60
60 60
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10
60 20
0 0 0 0 60 0
"Added surfactant (Kodak PhotoFlo 500). *Sargasso Sea water instead of NaCl solution. 'Added surfactant (Kodak PhotoFlo 500); no salt. Large size distribution of presettled pigment particles.
weighing the sample syringe before and after transfer of the sample to the filter. Reported sewage concentrations, C,, are dry mass filterable solids/volume suspension. Quiescent Settling Experiments. Quiescent settling experiments were carried out under a variety of conditions (Table I). Suspensions were mixed from a stock suspension of pigment particles, a stock solution of sodium chloride and sodium azide, and fresh primary sewage effluent immediately before filling the columns. The final suspensions (except column H) were M in sodium azide (to prevent biological growth) and 0.71 M in ionic strength at a pH of 7.5. Columns A-C were Plexiglas columns (7-cm diameter) sampled through ports mounted in the column walls at 5-cm intervals by using 2-in. gauge 12 needles. Time course samples were taken from middepth. Columns C-G were glass 1-L graduated cylinders sampled through the air-water interface with &in. gauge 12 needles. In column H, also sampled through the interface, conditions were arranged to minimize the effect of possible coagulation; hence, a short column was used and extra surfactant (Kodak PhotoFlo 200, p-tert-octylphenoxypolyethyl alcohol) rather than salt was the only additive to the suspension. The tops of all the columns were sealed with Parafilm to prevent evaporation. Samples were taken over time and stored in 10-mL glass vials until the time course was complete (columns A-G, 600 h; column H, 60 h), at which time 0.01 mL of a 0.5% solution of surfactant (Kodak PhotoFlo 200) was added to each vial and the vials were thoroughly agitated before analysis. Samples from columns A-G were analyzed for integrated fluorescence emission. Samples from columns A and H were analyzed by flow cytometry in order to determine the number concentration of pigment particles, n,, remaining in the water column over time as a function of particle size. Stirred Settling Experiments. The stirred experiments were carried out in 2-L beakers mixed with a Phipps & Bird six-paddle stirrer (see Table 11). Average energy
dissipation in the beakers was calculated from an empirical relationship dependent on the beaker and paddle dimensions and the stirring rate (9). Experiments were run with sewage effluent (S2 series) and with effluent amended with salts to simulate seawater major ion chemistry (Sl series). A small particle size fraction of pigment particles (decanted after settling for a day in an 8 c m water column) was added at the beginning of the experiment to yield a concentration of -25 ppm. The beakers were stirred for a time (see Table 11)to promote coagulation between the pigment and sewage particles and then allowed to settle quiescently to remove the settleable sewage particles and their associated pigment particles. Control beakers with pigment particles in sewage filtrate (0.2 l m filtered) were run in parallel for the S2 series. Sewage suspended solids concentrations were measured in parallel beakers to which pigment particles had not been added. All beakers were sampled over time through syringes without needles; 5-mL fluorescence samples were stored for analysis by fluorometer on completion of the experiment; 20-30-mL sewage concentration samples were filtered immediately after withdrawal. Results Quiescent Settling Experiments. Samples taken at multiple depths at a number of sampling times showed that bulk fluorescence intensity was uniform throughout the water column. Thus, the time course samples taken at middepth in the quiescent columns may be taken as representative of the entire column. In all quiescent sedimentation experiments the integrated fluorescence intensity I dropped off rapidly initially, with the rate of loss decreasing over time. When the intensity is normalized to intensity at time zero, and the time scale is normalized by column height, all seven data sets superimpose (Figure 5). In the absence of sewage or added surfactant (C, D, F) the air-water interface became coated with pigment particles, causing scatter in the data from top-sampled columns (D and F) (not shown). For columns A and H the number concentrations n,, of fluorescent pigment particles of different emission intensities, I,, plotted on a log-linear scale as a function of time fall on straight lines (Figure 6a). Data for the higher channel numbers are unavailable for column A because of the scarcity of large particles relative to small particles, but concentration data for the higher channel numbers from the 15-cm column indicate that the loss in each case is exponential in time, and that the rate of loss increases with particle emission intensity (Figure 6b). Stirred Settling Experiments. The filterable sewage concentration in the stirred beakers shows a slight decrease over the 23.5-h stirring period. This we attribute to sampling near the top of the beaker in the presence of an increasing vertical gradient of sewage particles within the beaker. No deposition was observed until stirring was stopped, at which point the settleable fraction of sewage
Table 11. Stirred Experiment Conditions beaker S1E S1A S2A S2B S2C S2D S2E S2F
added approx init init dry-filterable dry-filterable time estimated energy salts pigment part. concn, ppm sewage concn, ppm nonsettleable sewage concn, ppm stirred r, hr dissipation rate, e," cmz yes yes no no noc no no noc
0
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25 25
106 106b 69 6gb
35 35' 24 24b
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26 26b
0
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22
22 23.5 23.5 23.5 6 6 6
0.36 0.36 7.2 7.2 1.2 7.2 7.2 7.2
By use of empirical relationship dependent on beaker and paddle dimensions and stirring rate (9). bDry-filterable sewage concentrations from parallel sewage-only beakers. e Sewage filtrate (0.20 fim) used for control with pigment particles only. a
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settled out completely within 30 min (Figure 7a,c). The integrated fluorescence intensity of pigment particles was constant during the stirring period and decreased very little after stirring was stopped (Figure 7b,d), implying that a small fraction of pigment had coagulated with the settleable sewage. Further decrease in pigment concentration was apparently by settling at about the same rate as in the control beakers.
Discussion Convective Mixing of the Quiescent Columns. In analysis of sedimentation data the assumption is often correctly made that in an unstirred column particles will settle without being mixed vertically (10). However, depending on column geometry and thermal insulation, convection currents within the column may produce significant mixing (11). A temperature difference A T between a vertical surface and a fluid results in a free convection boundary layer with velocity scale V = ufGr1/2L-1and thickness 6 = LGr-'I4 where L is the vertical length scale of the surface and vf is the fluid viscosity cm2 s-l for water) (12). The Grashof number is Gr = /3gATL3vt2,where /3 is the fluid thermal expansion coefficient (-2 X lo4 OC-l for water) and g is gravitational acceleration (980 cm s-l). thus, the induced discharge per unit width of surface is V6 = (/3gATL3uf?)1/4. For a cylindrical volume of length L and radius R the convection current is capable of "turning over" the entire fluid volume in a time (R4L/vf2@gAT)'l4. For the typical dimensions of the quiescent settling columns used in this study ( R = 3.5 cm, L i= 50 cm) and a temperature difference AT = 0.1 "C, the boundary layer scales are 6 = 0.7 cm and V = 1 cm s-l and the mixing time is -250 s. This time is much shorter than the measured rates of particle settling, making it likely that thermal convection is the cause of the observed vertically uniform particle concentration. The convective stirring is appar-
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Figure 6. (a) Number concentration of particles of a given intensity vs timelcolumn height. Key: channels 15-24, 75-cm column A (O), 15-cm column H (W); channels 55-64, 75-cm column A (0),15-cm column H (0).Error bars represent the 1u counting uncertainty limits. Solid lines are weighted nonlinear least-squares fffs of the 15-cm column H data to exponentially decreasing functions. (b) Number concentration of particles of a given intensity for 1-m column H. Key: channels 25-34 (W), channels 65-74 (O), channels 95-104 (A), channels 235-244 (+). Error bars and fits as for Figure 6a.
ently vigorous enough to keep the column well mixed without producing resuspension of particles that have settled to the bottom. The above estimates of convective overturn are based on the assumption that the thickness of the thermal boundary layer generated is small relative to the radius of the column (6 = 0.71 cm
