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The small volume particle microsampler (SVPM): a new approach to particle size distribution and composition
Deep-Sea Research I 48 (2001) 2331–2346
Instruments and methods
The small volume particle microsampler (SVPM): a new approach to particle size distribution and composition Marie-Claude Archambault*, Jon Grant, Annamarie Hatcher Department of Oceanography, Dalhousie University, Halifax, NS, Canada B3H 4J1 Received 12 May 2000; received in revised form 3 January 2001; accepted 22 February 2001
Abstract The characterization of trophically and geochemically important suspended particulate matter (SPM) has traditionally relied on bottle sampling and subsequent analysis with Coulter Multisizers and other instruments, which are not sufficient in preserving the in situ size, shape and composition of aggregated particles. The small volume particle microsampler (SVPM) is a sampling device that captures individual particles on filters with minimal disturbance for microscope image analysis of size distribution and composition. Sand grains, microalga (Dunaliella tertiolecta) and laboratory cultivated flocs were used to test the SVPM’s ability to determine particle size. For statistical analysis of the SVPM’s capabilities, sand grain and algal size distribution, calculated as equivalent spherical diameter (ESD), were compared to Multisizer data while video images provided a comparison for the flocs. Non-aggregated sand particles sampled by the SVPM showed a size distribution that was similar to that of the Multisizer. Aggregated D. tertiolecta flocs were broken up by the Multisizer, and SVPM data indicated a significantly greater mean ESD. The SVPM showed significantly smaller mean ESDs than the video images because of the higher resolution of the sampler for small particles. In terms of particle concentration, the microsampler measured values similar to those of the Multisizer and video camera. The most important feature of the SVPM is its ability to capture aggregates for the analysis of composition, by histological stains or other means. The SVPM is an alternative method of sampling that is more effective in preserving aggregates for laboratory analyses and is less complicated and expensive than in situ optical sampling techniques, especially in documenting the lower end of the particle size spectrum. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Small volume particle microsampler (SVPM); Image analysis; Aggregates; Flocculation; Particle size distribution; Suspended particulate matter (SPM); Coulter Multisizer
*Corresponding author. Tel.: +1-902-494-3675; fax: +1-902-494-3877. E-mail addresses: [email protected] (M.-C. Archambault), [email protected] (J. Grant), [email protected] (A. Hatcher). 0967-0637/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 7 - 0 6 3 7 ( 0 1 ) 0 0 0 1 5 - 2
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1. Introduction Suspended particulate matter (SPM) in the ocean is often present in aggregated states (Syvitski et al., 1995; Eisma et al., 1996), but flocs and aggregated particles are fragile, and do not remain intact during bottle sampling and analysis, e.g. by Coulter counting (Gibbs, 1982; Eisma and Kalf, 1996; Knowles and Wells, 1996; Milligan, 1996; Pfeiffer, 1996; Wotton, 1990). It has long been recognized that particle size analysis requires in situ methods of observation, the most widely used methods being still and video photography (Bale, 1996; Eisma and Kalf, 1996; Eisma et al., 1996; Honjo et al., 1984; Jackson et al., 1997; Katz et al., 1999; Knowles and Wells, 1998; Milligan, 1996; Pfeiffer, 1996; Syvitski and Hutton, 1996; Van Leussen and Cornelisse, 1996). The results of in situ photography and subsequent image analysis are the determination of floc characteristics such as size, shape, settling velocity, porosity and to an extent composition (Cowen and Holloway, 1996; Kilps et al., 1994; Syvitski et al., 1995; Eisma and Kalf, 1996; Ratmeyer and Wefer, 1996). However, the lower size limit of many camera systems (>50 mm) eliminates information on trophically and geochemically important particles (550 mm), e.g. food for benthic suspension feeders. In addition, the lack of physical specimens for observation and further analysis becomes problematic in understanding flocculation and the roles of particles in biological, chemical and geophysical processes. Particle-specific information concerning size and organic quality may provide superior characterization compared to bulk measures of seston. However, current methods of determining nutrient and chemical constituents in marine systems rely on bulk measurements such as total chlorophyll and carbon content. SPM quality and flux are obviously important components of benthic–pelagic coupling (e.g. deposition of phytodetritus), specifically affecting the ecophysiology and behavior of suspension feeders, including trophically and economically important benthic bivalves (Grant, 1996). It has been difficult to relate bivalve growth to food resources, possibly because present assessment of bulk food quality is crude (Grant and Bacher, 1998). Characterization of the components of individual particles in suspension may provide valuable information on food sources and feeding response of filter feeders. An advantage of capturing individual particles and aggregated flocs is that they can be further analyzed in the laboratory for organic quality via proximate content (% carbohydrate, protein, or lipid) using staining procedures. The small volume particle microsampler (SVPM), improved from Schubel and Schiemer’s (1972) sampler, attempts to capture individual particles in a minimally disturbed state for the determination of size and composition. The SVPM works by sampling a small volume of water and trapping SPM on filters, which are then observed microscopically and analyzed from captured images. Fluorescent or other staining procedures may be applied to the filterpreserved aggregates for composition analyses. Before attempting to use the SVPM for composition analyses, it was important to determine its ability to preserve the in situ size distribution of aggregates. The objectives of the present research were to design the sampler, to determine whether the SVPM takes representative water samples in terms of particle size distribution and, to an extent, particle concentration, and to assess the SVPM’s capabilities as a sampling tool in comparison to other widely used sampling methods (Coulter Multisizer and in situ photography).
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2. Material and methods 2.1. The sampler The SVPM is a lightweight device, approximately 20 cm 10 cm in size, used to capture individual particles in a minimally disturbed state for the quantification of size and composition of SPM (Fig. 1A and B). Schubel and Schiemer (1972) developed the original concept for this type of device and used it to capture suspended particles by rapidly freezing a thin layer of water in dry ice/acetone. Using updated materials, design and analytical techniques, the SVPM works by isolating a small volume of water in situ with an electric Burkert 3-way solenoid valve mounted on a frame of Delrin plastic, trapping SPM on Nucleopore or other filters (25 mm diameter, 0.2 mm pore size). The filter is positioned between two solenoid plates and secured by a bezel ring. A battery-activated electrical signal is applied to clamp the plunger vertically a distance of 5.0 mm and subsequently trap the water on the filter. The water trapped between the filter and plunger (max 1.2 ml 0.1 SD) is expelled from the bottom of the instrument by applying gentle suction with a syringe, allowing particles to settle on the filter with minimal disturbance. A 5.0 mm groove in the frame of the SVPM allows suspension in water by hydrowire or other wires/ropes. The approximate cost of building the SVPM is 400 US$. 2.2. Particle types Sampling of small volumes for particles has the potential for a variety of biases, e.g. rarity of particles, and it was anticipated that the SVPM would be more useful to quantify size and
Fig. 1. (A) The small volume particle microsampler (SVPM) showing the plastic frame, and solenoid–plunger assembly which clamps to the filter holder upon firing. Water is removed through the drain. (B) Schematic drawing of the SVPM with dimensions, including distance between plunger and filter holder.
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Fig. 2. Schematic drawing of the Floc-a-Tron experimental tank used to produce clay-microalgal flocs, and compare video imaging to SVPM filter samples.
composition than particle concentration. In order to assess the capabilities of the SVPM with regard to conservation of original particle size and composition, as well as concentration, several types of particles were sampled during testing and calibration. Sterile sand grains of known size (pre-sieved to 63–75 mm) were used since they do not form aggregates during sampling. Aggregates are defined here as particles that are larger in equivalent spherical diameter (ESD) than any of the ESDs of the individual constituents of the solution. Secondly, the cultured microalga Dunaliella tertiolecta was used with expectations that some particles would aggregate and that the Multisizer would likely break up these particles (Gibbs, 1982). The last particle type that was sampled were flocs cultivated in an enclosed upwelling system with a gentle impellordriven water current (Fig. 2) that consisted of the microalgae Phaeodactylum tricornutum and drilling mud, in 208C filtered seawater (Hatcher et al., 2001). The drilling mud was originally collected at the Cohasset/Panuk drilling site on the Scotian Shelf in 1993 (White, 1997). The exact composition is unknown, but it consisted mainly of bentonite and barite, and is well known to readily produce large flocs on its own or with phytoplankton (White, 1997). The purpose of the tank was to create flocs; the data are presented in Hatcher et al. (2001). Flocculation processes were not stable, and over time disaggregation occurred, resulting in smaller flocs over time. 2.3. Sampling technique To determine the size distribution of the sand grains and D. tertiolecta, stock solutions were run through the Multisizer. The tube apertures for particles were 140 mm for the sand grains, and 70 mm for the microalgae. The lower size resolution of particles for the Multisizer is 4% of the
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aperture size. For the SVPM filter sampling, solutions of sand grains were made to concentrations of 5 and 20 mg l1. The microalgae stock was diluted to 1000 and 100 cells ml1. This difference in cell density was used to assess whether the SVPM was sensitive to concentration when determining size distribution and whether particle counts approximated those of the Multisizer. In all cases, at least three microsampler filter samples were obtained and immediately photographed wet. The microalgae SVPM filters were taken on the same day as the Multisizer samples to minimize temporal effects on algal growth. The flocs were sampled on three separate days using the SVPM and video photography in the experimental tank to determine the size distribution. On each day, the flocs were video taped for a minimum of 5 min at the same time that SVPM samples were taken. At least 6–9 randomly captured images from the tape (see below) were analyzed per sampling day. The first sampling date was conducted 37 days after the initial addition of individual particles (P. tricornutum and drilling mud) to the experimental seawater tank. The second sampling occurred on day 58, and the last sample occurred on day 63. 2.4. Concentration calculations To determine whether the SVPM sampled particle load accurately, the Multisizer sand grain concentrations for the 5 and 20 mg l1 solutions were compared to SVPM filter particle concentrations of the same solutions. Each solution was run through the Multisizer three times for a mean number of particles per ml. Three SVPM filters for each sand solution were analyzed for the number of sand grains per three fields of view. The number of particles per filter was extrapolated from the fact that the microscope field of view was 38% of the total filter area. The SVPM filter particle concentrations were calculated with the mean number of particles per filter divided by a mean SVPM volume of 1.2 ml 0.1 SD. The floc concentration for day 37 was calculated by the same method and compared to the concentration of particles derived from the video images (particle number per image in a video sampling field of 2.0 ml). 2.5. Imaging technique A small sampling volume may have few particles, and in a compound microscope, particles per field of view were too rare for effective size characterization of the population. Photographs of SVPM filters (three fields of view, 75% of the filter) were thus taken under a dissecting microscope with a Model XC-711 CCD Vision Camera (Sony), and an ArcSoft Zipshot frame grabber. Microscope resolution at the highest magnification (625 ) was 7.0 mm, at 38% of the filter area in a single field of view. Subsequent work with SVPM filters has been conducted with compound and epifluorescence microscopes and used CytoClear Slides (Osmonics, Inc.), which eliminated background filter noise. Floc images in the upwelling tank were captured with a Pulnix TM-745E high resolution CCD shutter video camera with Edge Enhancer fitted with a Zoom 6000II Nauter lens (29.2 ) through a glass window on the side of the experimental tank (Fig. 2). The video imaging system had no upper limit on particle size; the sampling volume was 2.0 ml per image. All images were converted to 8 bits per pixel and 256 gray scale, and contrast was maximized. Analysis of photographs was completed with SigmaScan Pro version 4.01 (SPSS Inc.). To minimize any observer bias associated with image analysis, e.g. ‘‘blooming’’ of the image and
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differences in illumination, all photographs were enhanced (gray scale conversion, intensity, contrast) equally. Particle area was quantified and converted from measured area to the equivalent spherical diameter (ESD) (mm) for comparison to Multisizer data using Eq. (1). The ESD is the diameter of any particle assuming the area represents a circle: ESD ¼ 2ðArea=pÞ1=2 :
ð1Þ
Size distribution comparisons of various sampling methods were analyzed using the nonparametric Mann–Whitney U-test with significance level a ¼ 0:05. Following convention, the minimum particle size measured was three screen pixels (Russ, 1995); therefore the minimum size recorded for video floc images was approximately 12 mm. Control photographs using filtered seawater indicated that video images and SVPM filter photographs did not require correction for noise or extraneous particles. Particle number (n) refers to the total number of individual particles or flocs counted from the Multisizer, filters or video images. Replicate samples for size distribution were combined to maximize the particle number obtained for the small sampling volumes.
3. Results 3.1. Sand particles The Multisizer size distribution for the sand grains (n ¼ 2569) showed that the majority of the particles were in the 60.0–80.0 mm size range with a mean diameter of 68.4 mm 9.5 SD (Fig. 3A). The presence of smaller grains may be attributed to the inefficiency of the original sieving, or to oblong grains lying across sieve openings and preventing smaller particles from passing through the holes (Knowles and Wells, 1998). Larger sand grains can be accounted for by the presence of long, thin particles that passed through sieve openings vertically. The ESD distribution of the sand particles as determined by the SVPM 5 mg l1 concentration (n ¼ 35) showed a shift towards larger particles and a mean diameter size of 75.5 mm 11.7 SD (Fig. 3B). Statistical analysis indicated that the SVPM ESD distribution at this concentration was significantly larger (p ¼ 0:0002) than the distribution determined by the Multisizer. In the case of the sand grains and D. tertiolecta cells, Multisizer concentrations were purposely large to ensure an accurate description of particle size distribution. The possibility that the size distribution of the small water samples was biased at low concentration was evaluated by using the SVPM at the higher concentration of 20 mg l1. The SVPM ESD distribution for the 20 mg l1 solution (n ¼ 47) indicated that the majority of the particles were in the 60.0–70.0 mm size range, with a greater abundance of particles >80.0 mm than particles within the 70.0–80.0 mm range (Fig. 3C). The mean ESD at this concentration was 66.5 mm 14.8 SD and the size distribution was not significantly different from the Multisizer distribution (p ¼ 0:254). Therefore in the case of sand particles, SVPM samples indicated a comparable size distribution to the Multisizer at 20 mg l1, but may produce less accurate size distributions at relatively low SPM concentrations. The Multisizer indicated a concentration of 5.3 particles ml1 4.2 SD for the 5 mg l1 solution and 25.3 particles ml1 3.1 SD for the 20 mg l1 solution. In comparison the SVPM filter particle concentrations were 5.4 particles ml1 4.1 SD and 22.8 particles ml1 12.6 SD. These
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Fig. 3. Sand grain ESD distributions as determined by (A) Coulter Multisizer, (B) SVPM at 5 mg l1 and (C) SVPM at 20 mg l1 concentrations.
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results suggest that for sand, the SVPM provides a reasonable measure of particle concentration, but that when particle numbers are low, size distributions may be biased. 3.2. Microalgae The Multisizer size distribution (n ¼ 811) for Dunaliella tertiolecta showed that the majority of algae ranged from 6.0 to 8.0 mm with a mean size of 7.4 mm 1.0 SD (Fig. 4A). The Multisizer distribution indicated non-aggregated cells, as confirmed under microscopy and image analysis, which showed a similar mean diameter (51 mm difference) from the Multisizer. It was assumed that any particles larger than 10.0 mm were likely to be aggregates and that there were no algal cells smaller than 5.0 mm. The SVPM size distributions for both the 100 cells ml1 (n ¼ 29) and 1000 cells ml1 (n ¼ 31) indicated that the majority of the algal cells were distributed towards the high end of the size spectrum, with particles as large as 12 mm (Fig. 4B and C). The SVPM mean ESD for D. tertiolecta was 9.6 mm 1.6 SD and 9.5 mm 1.5 SD for the 100 and 1000 cells ml1 SVPM filters, respectively. At the highest magnification, the microscope camera resolution had a lower size limit of approximately 7.0 mm; therefore, analysis of the SVPM filters under-represented the smallest algal cells ranging 57.0 mm. Unlike the difference in sand concentrations, the solutions used for D. tertiolecta were not sufficiently different and in both cases particles were rare. In analyzing size distributions, both concentrations sampled by the SVPM indicated significant differences from the Multisizer size distribution (p50:0001). By deleting the particles 57.0 mm from Multisizer data, the microscope camera resolution bias was eliminated, and the effects of floc disruption by the Multisizer were emphasized. Comparing these truncated data with the SVPM filter concentrations indicated that there were still significant differences (p50:0001) in size distributions, which may be attributed to the breakup of particles by the Multisizer and their conservation by the SVPM. Microalgae trials thus indicated that the SVPM has superior sampling ability to preserve particle size for particles that are likely to aggregate compared to the Multisizer. Particle concentration of algal flocs from filters was not calculated, since comparisons to the relatively disaggregated Multisizer concentration would be biased as a result of the inability to distinguish individual particles within an aggregate on the filters. 3.3. Floc particles The size distribution of the particles (n ¼ 52) as determined by video images of the flocs in situ from day 37 showed no particles 530.0 mm, but larger particles up to 200.0 mm (Fig. 5A). The peaks found in the size distribution at 90.0–110.0 mm are attributed to two video images consisting of an abundance of large particles. The mean ESD of the flocs from all video images was 84.9 mm 33.8 SD. The SVPM size distribution (n ¼ 113) indicated that there were many flocs ranging between 40.0 and 60.0 mm, with an abundance of flocs >150.0 mm, similar to the size distribution from the video images (Fig. 5B). The mean ESD of flocs from the SVPM filters was 74.6 mm 36.7 SD, and was significantly smaller than that of the video images (p ¼ 0:019). The floc concentration for day 37 calculated from SVPM filters was 72.8 particles ml1. The video images for this day indicated that the floc concentration in the experimental tank was 81.7 particles ml1.
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Fig. 4. Dunaliella tertiolecta ESD distributions as determined by (A) Coulter Multisizer, (B) SVPM at 100 cells ml1 and (C) SVPM at 1000 cells ml1 concentrations.
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Fig. 5. ESD distributions of cultivated flocs on day 37 as determined by (A) video image recording and (B) SVPM filter samples.
The video image size distribution of the floc particles (n ¼ 129) on the second day of sampling (day 58) showed a spread in floc size between 20.0 and 60.0 mm (Fig. 6A). The mean ESD of the particles from the video images was 39.8 mm 23.6 SD. The SVPM filter size distribution (n ¼ 252) indicated that most of the particles ranged from 10.0 to 30.0 mm (Fig. 6B). The mean ESD of the flocs calculated from SVPM filters was 29.1 mm 15.1 SD. The distribution is influenced by smaller particles that the microscope can detect as a result of better resolution than the video camera. At this sampling time, there was a significant difference of the distribution (p50:005) between the two sampling methods. On the last day of sampling, day 63, the video image size distribution (n ¼ 115) indicated an abundance of particles ranging from 10.0 to 60.0 mm with a mean ESD of the particles of 43.3 mm 24.0 SD (Fig. 7A). The size distribution as calculated by the SVPM filters (n ¼ 132) indicated that the majority of the particles ranged from 20.0 to 40.0 mm (Fig. 7B) with a mean ESD of 39.0 mm 30.4 SD. There was a significant difference in distribution (p ¼ 0:009) between
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Fig. 6. ESD distributions of cultivated flocs on day 58 as determined by (A) video image recording and (B) SVPM filter samples.
the two methods at this last sampling time. Thus, in every case, the SVPM indicated a significantly smaller mean ESD for flocs than did in situ photography. We emphasize that this is not because the SVPM was biased against large flocs; Figs. 5–7 indicate relatively similar absolute numbers of large flocs (>150 mm, day 37, >100 mm days 58, 63) in the sampling volumes of the SVPM (1.2 ml) and video imagery (2.0 ml). The difference in the two methods is instead attributed to the superior size resolution at the lower end of the size spectrum of the SVPM’s microscope analysis.
4. Discussion The SVPM improves upon the device originated by Schubel and Schiemer (1972) in that the original design captured particles for microscopic analysis by freezing a thin layer of water. The SVPM foregoes the intermediate and potentially disruptive step of freezing the particles in water
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Fig. 7. ESD distributions of cultivated flocs on day 63 as determined by (A) video image recording and (B) SVPM filter samples.
with acetone and dry ice. Particles are settled directly on a filter, observed under a microscope, and photographed immediately. Although any handling of particles is possibly disruptive, the sampling of small volumes and the gentle settlement to filters attempts to minimize these impacts. The availability and access of image capture and analysis have increased tremendously in recent years, allowing an in-depth look into size, shape and composition of particles. With the ready access to electronic components such as solenoids, the SVPM is simple to produce and is likely to be a useful complement to in situ photography. The comparison made with the Multisizer distribution of sand at 20 mg l1 verified that the SVPM could be used to quantify size distribution. It has a concentration threshold for accurate size portrayal, which may be lower than that determined with sand, as intermediate concentrations were not tested. A rarity of particles will not affect the SVPM’s ability to determine particle composition. Under adequate particle loads, the SVPM effectively has no limitations as an in situ sampling instrument for determining size distributions of various particles. The SVPM limits the upper size threshold to 5000 mm in diameter due to the size of the opening between the plunger and filter base. Large aggregates could
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be sampled, but their apparent size would be distorted by this constriction. However the SVPM is meant to identify that part of the particle size spectrum that cannot be sampled by video imaging. Large aggregates have alternate sampling procedures, which includes in situ photography, as well as hand collection (Alldredge and Silver, 1988). The microalgae trials indicated that the SVPM is more effective than the Multisizer in sampling aggregated particles in that the latter showed an abundance of particles of smaller size, while the SVPM tended toward larger particles. This discrepancy can be attributed to the microscope resolution or to the break up of aggregates by the Multisizer. After eliminating the bias of differential resolution at the lower limits of both methods, the results indicated that the difference in size distributions resulted from the preservation of aggregated particles by the SVPM. It has long been recognized that the Multisizer has a tendency to break up aggregates (Gibbs, 1982; Kranck and Milligan, 1988; Wotton, 1990), prompting the development of in situ sampling techniques. Microscope resolution was the limiting factor in determining the smallest particle sampled by the SVPM, and can be enhanced when particles are abundant on the filter by using stronger objective lenses or a compound microscope. In these trials, the dilute suspensions required the large field of view of the dissecting microscope, but there are otherwise no limitations regarding microscope type or magnification. In subsequent work with the SVPM we have used an epifluorescence microscope to examine particle composition. Microscope resolution is a trade-off between particle size and abundance. Eisma et al. (1996) noted that the minimum particle size measured is related to characteristics of the sampling instruments and methods used, and the maximum size is primarily related to the concentration of the largest flocs and the volume of water measured. The limitation in determining the smallest particles using in situ photography is the camera resolution (Wotton, 1990). An example of a high resolution camera is the video plankton recorder (VPR), which can image particles down to 10 mm (Davis et al., 1996). However, such camera systems are complex and expensive. In addition to retaining aggregates, a major advantage of the SVPM is its ability to allow sampling of very small particles that are undetectable by most in situ cameras. The purpose of the SVPM is to provide an alternative to other sampling techniques, i.e. bottle samples, and to deliver particles intact, so that various post-sampling techniques, such as confocal laser scanning microscopy and scanning electron microscopy (Cowen and Holloway, 1996; Norton et al., 1998), can be conducted, and composition analysis can be accomplished with intact aggregates. Large sampling devices, such as cameras and sediment traps, may disturb the natural shape and size of particles as a result of local changes in current speed (Kranck and Milligan, 1988; Kranck, 1993; Maldiney and Mouchel, 1996; Milligan, 1996) or shear stress (Bale, 1996; Eisma and Kalf, 1996; Katz et al., 1999; Knowles and Wells, 1996, 1998; Milligan, 1996). The SVPM is a small device, and is unlikely to disrupt flow significantly as it is lowered into the water, but may affect flow as a result of water passing through the small space between the solenoid plates. However, no flow artifacts are obvious in that the size and numbers of large flocs were similar between the SVPM and video methods (Figs. 5–7), despite sampling during flow from the upweller. Our ongoing flume experiments with clay-algal flocs will help to elucidate this influence further. Flow alteration may be less important if sampling for individual particle composition is the primary interest.
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Unlike large camera systems, the SVPM is small and manageable, and several devices can be installed on a hydrowire for sampling at different depths simultaneously. The devices can be attached to a rosette and triggered at different depths for water column profiles of particle distribution. Resulting images of the filters can be stored on film or digital media to provide longlived records of experimental data, unlike Multisizer samples, which must be analyzed fresh (Billones et al., 1999). The filters can be preserved, but ideally the samples should be observed without delay to minimize the risk of particle alteration due to drying or preservation effects. The coupling of SVPM particle collection and image analysis has the potential to provide valuable information on the nature of particles in suspension. The comparison between Multisizer and SVPM filter particle concentrations indicated that the SVPM accurately sampled particle load composed of sand grains. However, sand grains were not aggregated and were thus evenly distributed on filters. Trials with flocs suggested that despite indicating a wider size spectrum compared to video imaging, particle numbers per volume (ml) were again comparable between the two methods. Replicate sampling with multiple SVPMs or multiple deployments will be important in accurate determination of particle concentration, especially in the open ocean where particle concentration may be 51 mg l1. Caution should be extended in attempting to extrapolate particle concentrations from small sampling volumes. However, the SVPM’s ability to sample at low concentrations cannot be dismissed as it has yet to be tested. The goal of this research was to establish the SVPM more as a tool for sampling aggregate size distribution and as a means to obtain intact aggregates for composition analyses. An interest in flocculated SPM has prompted many studies of its roles in biological, chemical and geological processes in the ocean (Fowler and Knauer, 1986; Kranck and Milligan, 1988; Knowles and Wells, 1996). These aggregates are known to be important carriers for carbon and other elements and may contain rich microbial communities (Alldredge and Silver, 1988; Fowler and Knauer, 1986; Wotton, 1996). As a result, it is important to identify the components of these flocs with respect to distribution of organic matter and microbes (Alldredge and Silver, 1988; Schumann and Rentsch, 1998). Where particles are rare, the SVPM can still be useful by capturing them for the investigation of composition. Our particular area of interest is the use of the SVPM to understand trophic resources of benthic suspension feeders, wherein the definition of food quality is obscured by bulk water sampling, e.g. total chlorophyll a content. By capturing individual particles, the SVPM may provide valuable information on composition and assist in resolving questions about the relationship between food source and bivalve growth. A variety of other applications are apparent including zooplankton feeding, microbial colonization and decomposition of aggregates, and basic questions of floc structure such as organic/inorganic composition in relation to sinking rate and shear strength. In situ photography provides an opportunity to determine size distribution and other particle characteristics such as shape, volume, porosity and settling velocity (Cowen and Holloway, 1996; Syvitski et al., 1995), but fails to identify the composition of the aggregates. The SVPM allows sampling of particles with minimized disruption of the aggregates, and since the filters can be examined in the laboratory, composition analysis on a particle basis may be conducted. Photographs and image analysis may identify components such as microalgae and bacteria, and selective staining has the potential for quantifying the amount of carbohydrates, protein and lipid in the flocs. Schumann and Rentsch (1998) have used DTAF to stain polysaccharides in aggregates from plankton and sediment samples. SVPM investigations of floc compo-
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sition by selective staining and epifluorescence microscopy are currently underway in our laboratory. The SVPM has proved to be a device with enormous potential in the sampling and analysis of SPM and aggregated particles. The sand trials indicated that distributions quantified with the SVPM are comparable to those produced with the Multisizer. Trials with microalgae and cultivated flocs indicated that the SVPM is ideal for sampling aggregated particles, and is more effective in covering a wide size range than the Multisizer and in situ video photography. Camera systems that are available for in situ observation of SPM are limited by their resolution, but the SVPM surpasses that limitation via adjustments to other equipment, such as the microscope and image analysis software. The advantage of sampling particles with the SVPM is that filters with intact SPM specimens can be taken to the laboratory for other analyses such as floc composition using imaging techniques or staining procedures. The SVPM has several limitations specific to sampling small volumes and will not replace the current systems used for the determination of size distribution or concentrations. However the potential of the SVPM for better definition of size distribution of both large flocs and smaller particles, as well as collection of particles for analysis, provides an excellent complement to existing techniques. Moreover, it provides a means to view aggregates from an individual standpoint rather than simply as a bulk quantity. Acknowledgements We wish to thank Bryan Schofield, Paul Macpherson and Geoff MacIntyre for their assistance and advice with this work. This project was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic Grant to Paul Hill and J.G., and from an NSERC Undergraduate Research Award to M.-C.A.
References Alldredge, A.L., Silver, M.W., 1988. Characteristics, dynamics and significance of marine snow. Progress in Oceanography 20, 41–82. Bale, A.J., 1996. In situ laser optical particle sizing. Journal of Sea Research 36 (1/2), 31–36. Billones, R.G., Tackx, M.L.M., Flachier, A.T., Zhu, L., Daro, M.H., 1999. Image analysis as a tool for measuring particulate matter concentrations and gut content, body size, and clearance rates of estuarine copepods: validation and application. Journal of Marine Systems 22 (2/3), 179–194. Cowen, J.P., Holloway, C.F., 1996. Structural and chemical analysis of marine aggregates: in situ macrophotography and laser confocal and electron microscopy. Marine Biology 126 (2), 163–174. Davis, C.S., Gallager, S.M., Marra, M., Stewart, W.K., 1996. Rapid visualization of plankton abundance and taxonomic composition using the Video Plankton Recorder. Deep-Sea Research II 43 (7/8), 1947–1996. Eisma, D., Bale, A.J., Dearnaley, M.P., Fennessy, M.J., Van Leussen, W., Maldiney, M.-A., Pfeiffer, A., Wells, J.T., 1996. Intercomparison on in situ suspended matter (floc) size measurements. Journal of Sea Research 36 (1/2), 3–14. Eisma, D., Kalf, J., 1996. In situ particle (floc) size measurements with the Nioz in situ camera system. Journal of Sea Research 36 (1/2), 49–53. Fowler, S.W., Knauer, G.A., 1986. Role of large particles in the transport of elements and organic compounds through the oceanic water column. Progress in Oceanography 16 (3), 147–194. Gibbs, R.J., 1982. Floc stability during Coulter-counter size analysis. Journal of Sedimentary Petrology 52 (2), 657–659.
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Grant, J., 1996. The relationship of bioenergetics and the environment to the field growth of cultured bivalves. Journal of Experimental Marine Biology and Ecology 200 (1/2), 239–256. Grant, J., Bacher, C., 1998. Comparative models of mussel bioenergetics and their validation at field culture sites. Journal of Experimental Marine Biology and Ecology 219 (1/2), 21–44. Hatcher, A., Hill, P., Grant, J., 2001. Optical backscatter of marine flocs. Journal of Sea Research 46. Honjo, S., Doherty, K.W., Agrawal, Y., Asper, V.L., 1984. Direct optical assessment of large amorphous aggregates (marine snow) in the deep ocean. Deep-Sea Research I 31 (1), 67–76. Jackson, G.A., Maffione, R., Costello, D.K., Alldredge, A.L., Logan, B.E., Dam, H.G., 1997. Particle size spectra between 1 mm and 1 cm at Monterey Bay determined using multiple instruments. Deep-Sea Research I 44 (11), 1739–1767. Katz, J., Donaghay, P.L., Zhang, J., King, S., Russell, K., 1999. Submersible holocamera for detection of particle characteristics and motions in the ocean. Deep-Sea Research I 46 (8), 1455–1481. Kilps, J.R., Logan, B.E., Alldredge, A.L., 1994. Fractal dimensions of marine snow determined from image analysis of in situ photographs. Deep-Sea Research I 41 (8), 1159–1169. Knowles, S.C., Wells, J.T., 1996. Suspended aggregate analysis using ISAAC, Elbe River, 9–10 June 1993. Journal of Sea Research 36 (1/2), 69–75. Knowles, S.C., Wells, J.T., 1998. In situ aggregate analysis camera (ISAAC): a quantitative tool for analyzing finegrained suspended material. Limnology and Oceanography 43 (8), 1954–1962. . Hydrobiologie Supplement 75 (3/4), 299–309. Kranck, K., 1993. Flocculation and sediment particle size. Archiv fur Kranck, K., Milligan, T.G., 1988. Macroflocs from diatoms: in situ photography of particles in Bedford Basin, Nova Scotia. Marine Ecology Progress Series 44, 183–189. Maldiney, M.-A., Mouchel, J.-M., 1996. In situ video recording of suspended flocs. Journal of Sea Research 36 (1/2), 87–91. Milligan, T.G., 1996. In situ particle (floc) size measurements with the Benthos 373 Plankton Silhouette camera. Journal of Sea Research 36 (1/2), 93–100. Norton, T.A., Thompson, R.C., Pope, J., Veltkamp, C.J., Banks, B., Howard, C.V., Hawkins, S.J., 1998. Using confocal laser scanning microscopy, scanning electron microscopy and phase contrast light microscopy to examine marine biofilms. Aquatic Microbial Ecology 16 (2), 199–204. Pfeiffer, A., 1996. In situ measurements of flocculated suspended matter with a video multi sensor system. Journal of Sea Research 36 (1/2), 115–118. Ratmeyer, V., Wefer, G., 1996. A high resolution camera system (ParCa) for imaging particles in the ocean: system design and results from profiles and a three-month deployment. Journal of Marine Research 54 (3), 589–603. Russ, J.C., 1995. The Image Processing Handbook, 2nd Edition. CRC Press, Inc., Boca Raton. Schubel, J.R., Schiemer, E.W., 1972. A device for collecting in-situ samples of suspended sediment for microscopic analysis. Journal of Marine Research 30 (2), 269–273. Schumann, R., Rentsch, D., 1998. Staining particulate organic matter with DTAF–a fluorescence dye for carbohydrates and protein: a new approach and application of a 2D image analysis system. Marine Ecology Progress Series 163 (1), 77–88. Syvitski, J.P.M., Asprey, K.W., LeBlanc, K.W.G., 1995. In-situ characteristics of particles settling within a deep-water estuary. Deep-Sea Research II 42, 223–256. Syvitski, J.P.M., Hutton, E.W.H., 1996. In situ characteristics of suspended particles as determined by the floc camera assembly FCA. Journal of Sea Research 36 (1/2), 131–142. Van Leussen, W., Cornelisse, J.M., 1996. The underwater video system VIS. Journal of Sea Research 36 (1/2), 77–81. White, M.J., 1997. The effect of flocculation on the size-selective feeding capabilities of the sea scallop Placopecten magellanicus. M.Sc. Thesis, Dalhousie University, Nova Scotia. Wotton, R.S., 1990. The Biology of Particles in Aquatic Systems. CRC Press, Inc., Boca Raton. Wotton, R.S., 1996. Colloids, bubbles, and aggregates}a perspective on their role in suspension feeding. Journal of the North American Benthological Society 15 (1), 127–135.
Instruments and methods
The small volume particle microsampler (SVPM): a new approach to particle size distribution and composition Marie-Claude Archambault*, Jon Grant, Annamarie Hatcher Department of Oceanography, Dalhousie University, Halifax, NS, Canada B3H 4J1 Received 12 May 2000; received in revised form 3 January 2001; accepted 22 February 2001
Abstract The characterization of trophically and geochemically important suspended particulate matter (SPM) has traditionally relied on bottle sampling and subsequent analysis with Coulter Multisizers and other instruments, which are not sufficient in preserving the in situ size, shape and composition of aggregated particles. The small volume particle microsampler (SVPM) is a sampling device that captures individual particles on filters with minimal disturbance for microscope image analysis of size distribution and composition. Sand grains, microalga (Dunaliella tertiolecta) and laboratory cultivated flocs were used to test the SVPM’s ability to determine particle size. For statistical analysis of the SVPM’s capabilities, sand grain and algal size distribution, calculated as equivalent spherical diameter (ESD), were compared to Multisizer data while video images provided a comparison for the flocs. Non-aggregated sand particles sampled by the SVPM showed a size distribution that was similar to that of the Multisizer. Aggregated D. tertiolecta flocs were broken up by the Multisizer, and SVPM data indicated a significantly greater mean ESD. The SVPM showed significantly smaller mean ESDs than the video images because of the higher resolution of the sampler for small particles. In terms of particle concentration, the microsampler measured values similar to those of the Multisizer and video camera. The most important feature of the SVPM is its ability to capture aggregates for the analysis of composition, by histological stains or other means. The SVPM is an alternative method of sampling that is more effective in preserving aggregates for laboratory analyses and is less complicated and expensive than in situ optical sampling techniques, especially in documenting the lower end of the particle size spectrum. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Small volume particle microsampler (SVPM); Image analysis; Aggregates; Flocculation; Particle size distribution; Suspended particulate matter (SPM); Coulter Multisizer
*Corresponding author. Tel.: +1-902-494-3675; fax: +1-902-494-3877. E-mail addresses: [email protected] (M.-C. Archambault), [email protected] (J. Grant), [email protected] (A. Hatcher). 0967-0637/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 7 - 0 6 3 7 ( 0 1 ) 0 0 0 1 5 - 2
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1. Introduction Suspended particulate matter (SPM) in the ocean is often present in aggregated states (Syvitski et al., 1995; Eisma et al., 1996), but flocs and aggregated particles are fragile, and do not remain intact during bottle sampling and analysis, e.g. by Coulter counting (Gibbs, 1982; Eisma and Kalf, 1996; Knowles and Wells, 1996; Milligan, 1996; Pfeiffer, 1996; Wotton, 1990). It has long been recognized that particle size analysis requires in situ methods of observation, the most widely used methods being still and video photography (Bale, 1996; Eisma and Kalf, 1996; Eisma et al., 1996; Honjo et al., 1984; Jackson et al., 1997; Katz et al., 1999; Knowles and Wells, 1998; Milligan, 1996; Pfeiffer, 1996; Syvitski and Hutton, 1996; Van Leussen and Cornelisse, 1996). The results of in situ photography and subsequent image analysis are the determination of floc characteristics such as size, shape, settling velocity, porosity and to an extent composition (Cowen and Holloway, 1996; Kilps et al., 1994; Syvitski et al., 1995; Eisma and Kalf, 1996; Ratmeyer and Wefer, 1996). However, the lower size limit of many camera systems (>50 mm) eliminates information on trophically and geochemically important particles (550 mm), e.g. food for benthic suspension feeders. In addition, the lack of physical specimens for observation and further analysis becomes problematic in understanding flocculation and the roles of particles in biological, chemical and geophysical processes. Particle-specific information concerning size and organic quality may provide superior characterization compared to bulk measures of seston. However, current methods of determining nutrient and chemical constituents in marine systems rely on bulk measurements such as total chlorophyll and carbon content. SPM quality and flux are obviously important components of benthic–pelagic coupling (e.g. deposition of phytodetritus), specifically affecting the ecophysiology and behavior of suspension feeders, including trophically and economically important benthic bivalves (Grant, 1996). It has been difficult to relate bivalve growth to food resources, possibly because present assessment of bulk food quality is crude (Grant and Bacher, 1998). Characterization of the components of individual particles in suspension may provide valuable information on food sources and feeding response of filter feeders. An advantage of capturing individual particles and aggregated flocs is that they can be further analyzed in the laboratory for organic quality via proximate content (% carbohydrate, protein, or lipid) using staining procedures. The small volume particle microsampler (SVPM), improved from Schubel and Schiemer’s (1972) sampler, attempts to capture individual particles in a minimally disturbed state for the determination of size and composition. The SVPM works by sampling a small volume of water and trapping SPM on filters, which are then observed microscopically and analyzed from captured images. Fluorescent or other staining procedures may be applied to the filterpreserved aggregates for composition analyses. Before attempting to use the SVPM for composition analyses, it was important to determine its ability to preserve the in situ size distribution of aggregates. The objectives of the present research were to design the sampler, to determine whether the SVPM takes representative water samples in terms of particle size distribution and, to an extent, particle concentration, and to assess the SVPM’s capabilities as a sampling tool in comparison to other widely used sampling methods (Coulter Multisizer and in situ photography).
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2. Material and methods 2.1. The sampler The SVPM is a lightweight device, approximately 20 cm 10 cm in size, used to capture individual particles in a minimally disturbed state for the quantification of size and composition of SPM (Fig. 1A and B). Schubel and Schiemer (1972) developed the original concept for this type of device and used it to capture suspended particles by rapidly freezing a thin layer of water in dry ice/acetone. Using updated materials, design and analytical techniques, the SVPM works by isolating a small volume of water in situ with an electric Burkert 3-way solenoid valve mounted on a frame of Delrin plastic, trapping SPM on Nucleopore or other filters (25 mm diameter, 0.2 mm pore size). The filter is positioned between two solenoid plates and secured by a bezel ring. A battery-activated electrical signal is applied to clamp the plunger vertically a distance of 5.0 mm and subsequently trap the water on the filter. The water trapped between the filter and plunger (max 1.2 ml 0.1 SD) is expelled from the bottom of the instrument by applying gentle suction with a syringe, allowing particles to settle on the filter with minimal disturbance. A 5.0 mm groove in the frame of the SVPM allows suspension in water by hydrowire or other wires/ropes. The approximate cost of building the SVPM is 400 US$. 2.2. Particle types Sampling of small volumes for particles has the potential for a variety of biases, e.g. rarity of particles, and it was anticipated that the SVPM would be more useful to quantify size and
Fig. 1. (A) The small volume particle microsampler (SVPM) showing the plastic frame, and solenoid–plunger assembly which clamps to the filter holder upon firing. Water is removed through the drain. (B) Schematic drawing of the SVPM with dimensions, including distance between plunger and filter holder.
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Fig. 2. Schematic drawing of the Floc-a-Tron experimental tank used to produce clay-microalgal flocs, and compare video imaging to SVPM filter samples.
composition than particle concentration. In order to assess the capabilities of the SVPM with regard to conservation of original particle size and composition, as well as concentration, several types of particles were sampled during testing and calibration. Sterile sand grains of known size (pre-sieved to 63–75 mm) were used since they do not form aggregates during sampling. Aggregates are defined here as particles that are larger in equivalent spherical diameter (ESD) than any of the ESDs of the individual constituents of the solution. Secondly, the cultured microalga Dunaliella tertiolecta was used with expectations that some particles would aggregate and that the Multisizer would likely break up these particles (Gibbs, 1982). The last particle type that was sampled were flocs cultivated in an enclosed upwelling system with a gentle impellordriven water current (Fig. 2) that consisted of the microalgae Phaeodactylum tricornutum and drilling mud, in 208C filtered seawater (Hatcher et al., 2001). The drilling mud was originally collected at the Cohasset/Panuk drilling site on the Scotian Shelf in 1993 (White, 1997). The exact composition is unknown, but it consisted mainly of bentonite and barite, and is well known to readily produce large flocs on its own or with phytoplankton (White, 1997). The purpose of the tank was to create flocs; the data are presented in Hatcher et al. (2001). Flocculation processes were not stable, and over time disaggregation occurred, resulting in smaller flocs over time. 2.3. Sampling technique To determine the size distribution of the sand grains and D. tertiolecta, stock solutions were run through the Multisizer. The tube apertures for particles were 140 mm for the sand grains, and 70 mm for the microalgae. The lower size resolution of particles for the Multisizer is 4% of the
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aperture size. For the SVPM filter sampling, solutions of sand grains were made to concentrations of 5 and 20 mg l1. The microalgae stock was diluted to 1000 and 100 cells ml1. This difference in cell density was used to assess whether the SVPM was sensitive to concentration when determining size distribution and whether particle counts approximated those of the Multisizer. In all cases, at least three microsampler filter samples were obtained and immediately photographed wet. The microalgae SVPM filters were taken on the same day as the Multisizer samples to minimize temporal effects on algal growth. The flocs were sampled on three separate days using the SVPM and video photography in the experimental tank to determine the size distribution. On each day, the flocs were video taped for a minimum of 5 min at the same time that SVPM samples were taken. At least 6–9 randomly captured images from the tape (see below) were analyzed per sampling day. The first sampling date was conducted 37 days after the initial addition of individual particles (P. tricornutum and drilling mud) to the experimental seawater tank. The second sampling occurred on day 58, and the last sample occurred on day 63. 2.4. Concentration calculations To determine whether the SVPM sampled particle load accurately, the Multisizer sand grain concentrations for the 5 and 20 mg l1 solutions were compared to SVPM filter particle concentrations of the same solutions. Each solution was run through the Multisizer three times for a mean number of particles per ml. Three SVPM filters for each sand solution were analyzed for the number of sand grains per three fields of view. The number of particles per filter was extrapolated from the fact that the microscope field of view was 38% of the total filter area. The SVPM filter particle concentrations were calculated with the mean number of particles per filter divided by a mean SVPM volume of 1.2 ml 0.1 SD. The floc concentration for day 37 was calculated by the same method and compared to the concentration of particles derived from the video images (particle number per image in a video sampling field of 2.0 ml). 2.5. Imaging technique A small sampling volume may have few particles, and in a compound microscope, particles per field of view were too rare for effective size characterization of the population. Photographs of SVPM filters (three fields of view, 75% of the filter) were thus taken under a dissecting microscope with a Model XC-711 CCD Vision Camera (Sony), and an ArcSoft Zipshot frame grabber. Microscope resolution at the highest magnification (625 ) was 7.0 mm, at 38% of the filter area in a single field of view. Subsequent work with SVPM filters has been conducted with compound and epifluorescence microscopes and used CytoClear Slides (Osmonics, Inc.), which eliminated background filter noise. Floc images in the upwelling tank were captured with a Pulnix TM-745E high resolution CCD shutter video camera with Edge Enhancer fitted with a Zoom 6000II Nauter lens (29.2 ) through a glass window on the side of the experimental tank (Fig. 2). The video imaging system had no upper limit on particle size; the sampling volume was 2.0 ml per image. All images were converted to 8 bits per pixel and 256 gray scale, and contrast was maximized. Analysis of photographs was completed with SigmaScan Pro version 4.01 (SPSS Inc.). To minimize any observer bias associated with image analysis, e.g. ‘‘blooming’’ of the image and
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differences in illumination, all photographs were enhanced (gray scale conversion, intensity, contrast) equally. Particle area was quantified and converted from measured area to the equivalent spherical diameter (ESD) (mm) for comparison to Multisizer data using Eq. (1). The ESD is the diameter of any particle assuming the area represents a circle: ESD ¼ 2ðArea=pÞ1=2 :
ð1Þ
Size distribution comparisons of various sampling methods were analyzed using the nonparametric Mann–Whitney U-test with significance level a ¼ 0:05. Following convention, the minimum particle size measured was three screen pixels (Russ, 1995); therefore the minimum size recorded for video floc images was approximately 12 mm. Control photographs using filtered seawater indicated that video images and SVPM filter photographs did not require correction for noise or extraneous particles. Particle number (n) refers to the total number of individual particles or flocs counted from the Multisizer, filters or video images. Replicate samples for size distribution were combined to maximize the particle number obtained for the small sampling volumes.
3. Results 3.1. Sand particles The Multisizer size distribution for the sand grains (n ¼ 2569) showed that the majority of the particles were in the 60.0–80.0 mm size range with a mean diameter of 68.4 mm 9.5 SD (Fig. 3A). The presence of smaller grains may be attributed to the inefficiency of the original sieving, or to oblong grains lying across sieve openings and preventing smaller particles from passing through the holes (Knowles and Wells, 1998). Larger sand grains can be accounted for by the presence of long, thin particles that passed through sieve openings vertically. The ESD distribution of the sand particles as determined by the SVPM 5 mg l1 concentration (n ¼ 35) showed a shift towards larger particles and a mean diameter size of 75.5 mm 11.7 SD (Fig. 3B). Statistical analysis indicated that the SVPM ESD distribution at this concentration was significantly larger (p ¼ 0:0002) than the distribution determined by the Multisizer. In the case of the sand grains and D. tertiolecta cells, Multisizer concentrations were purposely large to ensure an accurate description of particle size distribution. The possibility that the size distribution of the small water samples was biased at low concentration was evaluated by using the SVPM at the higher concentration of 20 mg l1. The SVPM ESD distribution for the 20 mg l1 solution (n ¼ 47) indicated that the majority of the particles were in the 60.0–70.0 mm size range, with a greater abundance of particles >80.0 mm than particles within the 70.0–80.0 mm range (Fig. 3C). The mean ESD at this concentration was 66.5 mm 14.8 SD and the size distribution was not significantly different from the Multisizer distribution (p ¼ 0:254). Therefore in the case of sand particles, SVPM samples indicated a comparable size distribution to the Multisizer at 20 mg l1, but may produce less accurate size distributions at relatively low SPM concentrations. The Multisizer indicated a concentration of 5.3 particles ml1 4.2 SD for the 5 mg l1 solution and 25.3 particles ml1 3.1 SD for the 20 mg l1 solution. In comparison the SVPM filter particle concentrations were 5.4 particles ml1 4.1 SD and 22.8 particles ml1 12.6 SD. These
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Fig. 3. Sand grain ESD distributions as determined by (A) Coulter Multisizer, (B) SVPM at 5 mg l1 and (C) SVPM at 20 mg l1 concentrations.
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results suggest that for sand, the SVPM provides a reasonable measure of particle concentration, but that when particle numbers are low, size distributions may be biased. 3.2. Microalgae The Multisizer size distribution (n ¼ 811) for Dunaliella tertiolecta showed that the majority of algae ranged from 6.0 to 8.0 mm with a mean size of 7.4 mm 1.0 SD (Fig. 4A). The Multisizer distribution indicated non-aggregated cells, as confirmed under microscopy and image analysis, which showed a similar mean diameter (51 mm difference) from the Multisizer. It was assumed that any particles larger than 10.0 mm were likely to be aggregates and that there were no algal cells smaller than 5.0 mm. The SVPM size distributions for both the 100 cells ml1 (n ¼ 29) and 1000 cells ml1 (n ¼ 31) indicated that the majority of the algal cells were distributed towards the high end of the size spectrum, with particles as large as 12 mm (Fig. 4B and C). The SVPM mean ESD for D. tertiolecta was 9.6 mm 1.6 SD and 9.5 mm 1.5 SD for the 100 and 1000 cells ml1 SVPM filters, respectively. At the highest magnification, the microscope camera resolution had a lower size limit of approximately 7.0 mm; therefore, analysis of the SVPM filters under-represented the smallest algal cells ranging 57.0 mm. Unlike the difference in sand concentrations, the solutions used for D. tertiolecta were not sufficiently different and in both cases particles were rare. In analyzing size distributions, both concentrations sampled by the SVPM indicated significant differences from the Multisizer size distribution (p50:0001). By deleting the particles 57.0 mm from Multisizer data, the microscope camera resolution bias was eliminated, and the effects of floc disruption by the Multisizer were emphasized. Comparing these truncated data with the SVPM filter concentrations indicated that there were still significant differences (p50:0001) in size distributions, which may be attributed to the breakup of particles by the Multisizer and their conservation by the SVPM. Microalgae trials thus indicated that the SVPM has superior sampling ability to preserve particle size for particles that are likely to aggregate compared to the Multisizer. Particle concentration of algal flocs from filters was not calculated, since comparisons to the relatively disaggregated Multisizer concentration would be biased as a result of the inability to distinguish individual particles within an aggregate on the filters. 3.3. Floc particles The size distribution of the particles (n ¼ 52) as determined by video images of the flocs in situ from day 37 showed no particles 530.0 mm, but larger particles up to 200.0 mm (Fig. 5A). The peaks found in the size distribution at 90.0–110.0 mm are attributed to two video images consisting of an abundance of large particles. The mean ESD of the flocs from all video images was 84.9 mm 33.8 SD. The SVPM size distribution (n ¼ 113) indicated that there were many flocs ranging between 40.0 and 60.0 mm, with an abundance of flocs >150.0 mm, similar to the size distribution from the video images (Fig. 5B). The mean ESD of flocs from the SVPM filters was 74.6 mm 36.7 SD, and was significantly smaller than that of the video images (p ¼ 0:019). The floc concentration for day 37 calculated from SVPM filters was 72.8 particles ml1. The video images for this day indicated that the floc concentration in the experimental tank was 81.7 particles ml1.
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Fig. 4. Dunaliella tertiolecta ESD distributions as determined by (A) Coulter Multisizer, (B) SVPM at 100 cells ml1 and (C) SVPM at 1000 cells ml1 concentrations.
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Fig. 5. ESD distributions of cultivated flocs on day 37 as determined by (A) video image recording and (B) SVPM filter samples.
The video image size distribution of the floc particles (n ¼ 129) on the second day of sampling (day 58) showed a spread in floc size between 20.0 and 60.0 mm (Fig. 6A). The mean ESD of the particles from the video images was 39.8 mm 23.6 SD. The SVPM filter size distribution (n ¼ 252) indicated that most of the particles ranged from 10.0 to 30.0 mm (Fig. 6B). The mean ESD of the flocs calculated from SVPM filters was 29.1 mm 15.1 SD. The distribution is influenced by smaller particles that the microscope can detect as a result of better resolution than the video camera. At this sampling time, there was a significant difference of the distribution (p50:005) between the two sampling methods. On the last day of sampling, day 63, the video image size distribution (n ¼ 115) indicated an abundance of particles ranging from 10.0 to 60.0 mm with a mean ESD of the particles of 43.3 mm 24.0 SD (Fig. 7A). The size distribution as calculated by the SVPM filters (n ¼ 132) indicated that the majority of the particles ranged from 20.0 to 40.0 mm (Fig. 7B) with a mean ESD of 39.0 mm 30.4 SD. There was a significant difference in distribution (p ¼ 0:009) between
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Fig. 6. ESD distributions of cultivated flocs on day 58 as determined by (A) video image recording and (B) SVPM filter samples.
the two methods at this last sampling time. Thus, in every case, the SVPM indicated a significantly smaller mean ESD for flocs than did in situ photography. We emphasize that this is not because the SVPM was biased against large flocs; Figs. 5–7 indicate relatively similar absolute numbers of large flocs (>150 mm, day 37, >100 mm days 58, 63) in the sampling volumes of the SVPM (1.2 ml) and video imagery (2.0 ml). The difference in the two methods is instead attributed to the superior size resolution at the lower end of the size spectrum of the SVPM’s microscope analysis.
4. Discussion The SVPM improves upon the device originated by Schubel and Schiemer (1972) in that the original design captured particles for microscopic analysis by freezing a thin layer of water. The SVPM foregoes the intermediate and potentially disruptive step of freezing the particles in water
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Fig. 7. ESD distributions of cultivated flocs on day 63 as determined by (A) video image recording and (B) SVPM filter samples.
with acetone and dry ice. Particles are settled directly on a filter, observed under a microscope, and photographed immediately. Although any handling of particles is possibly disruptive, the sampling of small volumes and the gentle settlement to filters attempts to minimize these impacts. The availability and access of image capture and analysis have increased tremendously in recent years, allowing an in-depth look into size, shape and composition of particles. With the ready access to electronic components such as solenoids, the SVPM is simple to produce and is likely to be a useful complement to in situ photography. The comparison made with the Multisizer distribution of sand at 20 mg l1 verified that the SVPM could be used to quantify size distribution. It has a concentration threshold for accurate size portrayal, which may be lower than that determined with sand, as intermediate concentrations were not tested. A rarity of particles will not affect the SVPM’s ability to determine particle composition. Under adequate particle loads, the SVPM effectively has no limitations as an in situ sampling instrument for determining size distributions of various particles. The SVPM limits the upper size threshold to 5000 mm in diameter due to the size of the opening between the plunger and filter base. Large aggregates could
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be sampled, but their apparent size would be distorted by this constriction. However the SVPM is meant to identify that part of the particle size spectrum that cannot be sampled by video imaging. Large aggregates have alternate sampling procedures, which includes in situ photography, as well as hand collection (Alldredge and Silver, 1988). The microalgae trials indicated that the SVPM is more effective than the Multisizer in sampling aggregated particles in that the latter showed an abundance of particles of smaller size, while the SVPM tended toward larger particles. This discrepancy can be attributed to the microscope resolution or to the break up of aggregates by the Multisizer. After eliminating the bias of differential resolution at the lower limits of both methods, the results indicated that the difference in size distributions resulted from the preservation of aggregated particles by the SVPM. It has long been recognized that the Multisizer has a tendency to break up aggregates (Gibbs, 1982; Kranck and Milligan, 1988; Wotton, 1990), prompting the development of in situ sampling techniques. Microscope resolution was the limiting factor in determining the smallest particle sampled by the SVPM, and can be enhanced when particles are abundant on the filter by using stronger objective lenses or a compound microscope. In these trials, the dilute suspensions required the large field of view of the dissecting microscope, but there are otherwise no limitations regarding microscope type or magnification. In subsequent work with the SVPM we have used an epifluorescence microscope to examine particle composition. Microscope resolution is a trade-off between particle size and abundance. Eisma et al. (1996) noted that the minimum particle size measured is related to characteristics of the sampling instruments and methods used, and the maximum size is primarily related to the concentration of the largest flocs and the volume of water measured. The limitation in determining the smallest particles using in situ photography is the camera resolution (Wotton, 1990). An example of a high resolution camera is the video plankton recorder (VPR), which can image particles down to 10 mm (Davis et al., 1996). However, such camera systems are complex and expensive. In addition to retaining aggregates, a major advantage of the SVPM is its ability to allow sampling of very small particles that are undetectable by most in situ cameras. The purpose of the SVPM is to provide an alternative to other sampling techniques, i.e. bottle samples, and to deliver particles intact, so that various post-sampling techniques, such as confocal laser scanning microscopy and scanning electron microscopy (Cowen and Holloway, 1996; Norton et al., 1998), can be conducted, and composition analysis can be accomplished with intact aggregates. Large sampling devices, such as cameras and sediment traps, may disturb the natural shape and size of particles as a result of local changes in current speed (Kranck and Milligan, 1988; Kranck, 1993; Maldiney and Mouchel, 1996; Milligan, 1996) or shear stress (Bale, 1996; Eisma and Kalf, 1996; Katz et al., 1999; Knowles and Wells, 1996, 1998; Milligan, 1996). The SVPM is a small device, and is unlikely to disrupt flow significantly as it is lowered into the water, but may affect flow as a result of water passing through the small space between the solenoid plates. However, no flow artifacts are obvious in that the size and numbers of large flocs were similar between the SVPM and video methods (Figs. 5–7), despite sampling during flow from the upweller. Our ongoing flume experiments with clay-algal flocs will help to elucidate this influence further. Flow alteration may be less important if sampling for individual particle composition is the primary interest.
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Unlike large camera systems, the SVPM is small and manageable, and several devices can be installed on a hydrowire for sampling at different depths simultaneously. The devices can be attached to a rosette and triggered at different depths for water column profiles of particle distribution. Resulting images of the filters can be stored on film or digital media to provide longlived records of experimental data, unlike Multisizer samples, which must be analyzed fresh (Billones et al., 1999). The filters can be preserved, but ideally the samples should be observed without delay to minimize the risk of particle alteration due to drying or preservation effects. The coupling of SVPM particle collection and image analysis has the potential to provide valuable information on the nature of particles in suspension. The comparison between Multisizer and SVPM filter particle concentrations indicated that the SVPM accurately sampled particle load composed of sand grains. However, sand grains were not aggregated and were thus evenly distributed on filters. Trials with flocs suggested that despite indicating a wider size spectrum compared to video imaging, particle numbers per volume (ml) were again comparable between the two methods. Replicate sampling with multiple SVPMs or multiple deployments will be important in accurate determination of particle concentration, especially in the open ocean where particle concentration may be 51 mg l1. Caution should be extended in attempting to extrapolate particle concentrations from small sampling volumes. However, the SVPM’s ability to sample at low concentrations cannot be dismissed as it has yet to be tested. The goal of this research was to establish the SVPM more as a tool for sampling aggregate size distribution and as a means to obtain intact aggregates for composition analyses. An interest in flocculated SPM has prompted many studies of its roles in biological, chemical and geological processes in the ocean (Fowler and Knauer, 1986; Kranck and Milligan, 1988; Knowles and Wells, 1996). These aggregates are known to be important carriers for carbon and other elements and may contain rich microbial communities (Alldredge and Silver, 1988; Fowler and Knauer, 1986; Wotton, 1996). As a result, it is important to identify the components of these flocs with respect to distribution of organic matter and microbes (Alldredge and Silver, 1988; Schumann and Rentsch, 1998). Where particles are rare, the SVPM can still be useful by capturing them for the investigation of composition. Our particular area of interest is the use of the SVPM to understand trophic resources of benthic suspension feeders, wherein the definition of food quality is obscured by bulk water sampling, e.g. total chlorophyll a content. By capturing individual particles, the SVPM may provide valuable information on composition and assist in resolving questions about the relationship between food source and bivalve growth. A variety of other applications are apparent including zooplankton feeding, microbial colonization and decomposition of aggregates, and basic questions of floc structure such as organic/inorganic composition in relation to sinking rate and shear strength. In situ photography provides an opportunity to determine size distribution and other particle characteristics such as shape, volume, porosity and settling velocity (Cowen and Holloway, 1996; Syvitski et al., 1995), but fails to identify the composition of the aggregates. The SVPM allows sampling of particles with minimized disruption of the aggregates, and since the filters can be examined in the laboratory, composition analysis on a particle basis may be conducted. Photographs and image analysis may identify components such as microalgae and bacteria, and selective staining has the potential for quantifying the amount of carbohydrates, protein and lipid in the flocs. Schumann and Rentsch (1998) have used DTAF to stain polysaccharides in aggregates from plankton and sediment samples. SVPM investigations of floc compo-
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sition by selective staining and epifluorescence microscopy are currently underway in our laboratory. The SVPM has proved to be a device with enormous potential in the sampling and analysis of SPM and aggregated particles. The sand trials indicated that distributions quantified with the SVPM are comparable to those produced with the Multisizer. Trials with microalgae and cultivated flocs indicated that the SVPM is ideal for sampling aggregated particles, and is more effective in covering a wide size range than the Multisizer and in situ video photography. Camera systems that are available for in situ observation of SPM are limited by their resolution, but the SVPM surpasses that limitation via adjustments to other equipment, such as the microscope and image analysis software. The advantage of sampling particles with the SVPM is that filters with intact SPM specimens can be taken to the laboratory for other analyses such as floc composition using imaging techniques or staining procedures. The SVPM has several limitations specific to sampling small volumes and will not replace the current systems used for the determination of size distribution or concentrations. However the potential of the SVPM for better definition of size distribution of both large flocs and smaller particles, as well as collection of particles for analysis, provides an excellent complement to existing techniques. Moreover, it provides a means to view aggregates from an individual standpoint rather than simply as a bulk quantity. Acknowledgements We wish to thank Bryan Schofield, Paul Macpherson and Geoff MacIntyre for their assistance and advice with this work. This project was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic Grant to Paul Hill and J.G., and from an NSERC Undergraduate Research Award to M.-C.A.
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