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Physicochemical and microbial preservation of colloid characteristics of natural water samples. I: Experimental conditions
Pergamon PII: soo43-1354(%)ooo%4
Wm. Res. Vol. 30, No. 9, pp. 2178-2184, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/96 515.00 + 0.00
PHYSICOCHEMICAL AND MICROBIAL PRESERVATION OF COLLOID CHARACTERISTICS OF NATURAL WATER SAMPLES. I: EXPERIMENTAL CONDITIONS YU-WE1 CHEN* and JACQUES BUFFLEt@ Department of Inorganic, Analytical and Applied Chemistry, University of Geneva, Sciences II, 30 quai Ernest-Ansennet, CH-1211 Geneva 4, Switzerland (First received March 1995; accepted in revised form April 1996)
Abstract-Physical and chemical characterization of colloids in natural waters is a major issue to understand the mechanism of circulation of pollutants in natural waters. Although many researchers are interested in colloid interactions, they are not yet well understood, and little is known on the appropriate experimental conditions needed to perform reliable and environmentally significant analysis. The following study is dedicated to the development of well-controlled analytical conditions. It is shown that drastic degradation of the water sample can happen during sample handling, if experimental conditions are inappropriate. The study also points out that multidisciplinary cooperation is necessary in the study of environmental problems. This paper is split in two parts for editorial reasons. The first part of this series reports experimental conditions, used to follow colloid stability reported in the second part of the series. Copyright 0 1996 Elsevier Science Ltd Key words-olloids, natural waters, characterizations, contaminations, experimental conditions, storage, biodegradation, agitation, coagulation
INTRODUCTION
In aquatic systems, particles and colloids play a key role in the regulation of concentrations of vital and detrimental compounds (Stumm, 1992; Sigg et al., 1987). New available data suggest that submicrometer particles and macromolecules, denoted below as colloids, are of greatest importance because of their very large specific surface area (Newman et al., 1994). These colloids include inorganic solids, organic macromolecules, the debris of organisms, as well as viruses and bacteria, aggregates of these various components ~~ut?le and van Leeuwen, 1992, 1993). Little is known about their detailed composition and properties in natural aquatic systems. Thorough characterization of aquatic colloids and interpretation of data are difficult and imply taking into account a number of possible artifacts in the following key steps: (1) collection of a raw water sample, (2) sample handling and storage and (3) fractionation of particles and colloids (by size or other properties). These steps may drastically alter the nature and properties of colloidal materials under study.
tAuthor to whom all correspondence should be addressed [Fax: (41) 22 702 60691. *Present address: Department of Chemistry and Biochemistry, Laurentian University Ramsey Lake Road, Sudbury, Ontario, P3E 2C6, Canada.
The main below.
perturbations
to consider
are
listed
(4 Contaminations
by colloids and/or bacteria from the vessels, tubings, atmosphere etc., or loss of colloidal material by adsorption on the vessel walls. Total colloidal content of surface waters is generally less than 0.1-1.0 mg.dm-); contaminations and losses are therefore serious problems. (b) Slow coagulation of the colloidal components which modifies the whole size distribution of colloids, consequently their other properties (e.g. their settling rate). (cl Microbial changes (increase or decrease of population density, change of the nature of the dominant microbes). In addition to being part of the colloidal pool themselves, microbes may provoke degradation of non-living colloids (in particular the organic macromolecules), or aggregation of existing colloids, by releasing metabolic products such as enzymes or polysacchat-ides. The death of microbes will also increase the content of the debris of the organisms. Obviously, the colloidal pool of a water sample is a very delicate system which may be easily perturbed. In fact, perturbation starts at the very moment of sample collection. Indeed, due to coagulation processes and microbial activities, a colloidal system is not static but in continuous evolution. The following perturbations by sample handling and remediations have been reported.
2178
Preservation
of aquatic
-Too long storage periods is a problem because of coagulation and microbial growth. Some authors have used chemicals as anticoagulants (e.g. sodium hexametaphosphate, Gallegos and Menzell, 1987) or antibiotics (e.g. HgC12, Wells and Goldberg, 1991, 1992; or NaNn, Fox, 1988), but no systematic study of their overall effect on a natural colloidal system has been performed before. -Drying and freeze-drying can prevent bacterial growth, but they may induce changes in colloid and aggregate structures (de Mora and Harrison, 1983). In particular the drying and dehydration under high vacuum of unprotected aquatic aggregates deposited on the electron microscopy grid (Wells and Goldberg, 1992) may cause drastic structural changes (Leppard, 1992). -Artefacts introduced by filtration have been studied in more detail (Buffle et al., 1992). It has been clearly shown that the rather high flow rates normally used in colloid fractionation causes retention of colloids on the filter surface due to surface coagulation and not by size sieving. This effect becomes small at very low flow rates, but coagulation of colloid may then occur in the filtration cell due to the very long filtration time used. Contamination problems from filters in trace metals analysis (Truitt and Weber, 1979) and in DOC analysis (Buffle et al., 1982) have been studied carefully, but information on contaminations by particulate and colloidal matter is scarce. Similarly, there is no systematic study of the various sources of bacterial contaminations in the context of the characterization of aquatic colloids. The goal of this work is then to evaluate the most important factors which may affect the freshwater colloids after sample collection, and during sample handling. The following studies were systematically carried out and are reported below: contaminations by colloids and bacteria analytical systems; ??temperature effect during sedimentation; ??
by
colloid samples. I.
2179
changes of colloidal structures during storage and analytical operation; ??bacterial growth.
??
For editorial reasons, this report is split in two parts: the first part describes the choice of optimum experimental conditions used, and the second reports the results of the stability of colloids. Because of the complexity of the system studied, the interconnections of the various factors, and the analytical difficulties due to the low concentration of submicrometer colloids in fresh waters, only a semi-quantitative description can be given. However, this work, by reporting the relative importance of the major processes, discusses the major criteria for performing systematic studies of aquatic colloids in well-controlled conditions. The colloids of three rivers have been studied and compared: those of the Arve and RhBne rivers in Geneva and those of the Rhine river in Base1 (Switzerland). These rivers possess different characteristics. The Rhone river, at the outlet of the Lake Leman, has the lowest colloid concentration, due to previous sedimentation in the lake, but the organic to inorganic colloid concentration ratio is the highest. The Arve river directly flows from Alps (France) and has a relatively large particle load, dominated by sand, clay minerals, and calcium carbonates. The colloid content of the Rhine river is intermediate between the other two rivers. EXPERIMENTAL
Composition of the jresh waters studied The sampling sites on the RhBne and Arve rivers were in the middle of the bridges “Pont de Sous-Terre” and “Passerelle de 1’Ecole de Medecine”, respectively in Geneva (Switzerland). The Rhine river samples were collected at the middle of the river on a boat, immediately downstream of the “Birsfelden” dam in Base1 (Switzerland). In all cases, the samples were collected 0.5-1.0 m below the water surface. The particle loads of these rivers varied seasonally in the range 1.9-> 1000, 0.1-7, and 5-700 mg.dm-’ for the Arve, the RhBne and the Rhine rivers, respectively. The major characteristics of the three rivers are given in Table I (Perret et al., 1994; Chen and ButBe 1994; Chen, 1993; CIPRP, 1986; SIGSEL, 1993).
Table I. The maior characteristics of the three rivers studied RhBne Flow rate
(m’/s) TOC of river water bg Wm’) Suspended particulate material
(msidm’) Colloid concentration ( < 1pm) (mgidm’) Dissolved 0: (mgidm’) DH Conductivity (25°C) W/cm)
166-319 (248) 0.761.47 (1.15) 0.134.31 (1.78)
6.61-13.64 (9.9) 7.9-8.09 (8.02) 277-3 I5 (293)
Arve
I2-840 (79) 0.54-2.77
(1.20) 1.90-> 1000 (96) 2.7-7.06+ (4.3) 7.55-l I.46 (10.1) 7.50-8.46 (8.17) 240-528 (388)
The values in parentheses are means over the year 1993. *From Chen (1993). ‘The value in this parenthesis is the mean between March and May, 1992
Rhine 501-2459 (1059) 2.6-6.4 (3.8) 5.43-700.6’ (8.8) 0.6-46.9’ (0.9) 8.4-12.5 (10.6) 7.6-8. I (7.90) 288-374 (331)
2180 Apparatus
Y.-W. Chen and J. Buffle and reagents
Garden watering pump Marina Jolly 800 with high flow rate (1 dm’.s-I) was used to collect water samples. Home-made plexiglass tanks (30 dm3, height 12 cm) were then used at the sampling location for sedimentation of the largest particles (ra few micrometers) under natural gravity. They were thermally insulated and their cover included nine sampling tubes to allow quick, gentle and homogeneous sampling of the top surface layer, at a well-controlled depth (1.0-2.5 cm from the top surface, see Perret et al., 1994 for details). The following fractionation apparatus were subsequently used: centrifuged Hereaus 2.0 RS with swing-out bucket and thermostated conditions; ultracentrifuge Beckman L 7-55 with swing-out rotor SW 30 and thermostated conditions; home-made plexiglass filtration cells with and without stirring bar (47 mm and 90 mm); corresponding Nucleopore Polycarbonate membranes with pore sizes of 0.8 and 0.05 pm and Amicon Diaflo PM10 membranes with diameters of 47 mm. Light scattering (LS) was measured on line with the Fluorimeter Hitachi-Merck F-1050 (with white light and a flow-through cell) at the output of the filtration cell. Off-line LS were measured with a fluorimeter Perkin-Elmer 204. Photon correlation spectroscopy (PCS) was performed with a Malvem Zetasizer III with a 1 W Ar laser (Innova 70). DOC was measured with a Total Organic Analyzer Xertex Dohrmann DC 80. Transmission and scanning electron microscope (TEM and SEM) pictures were obtained with the Apparatus Zeiss EM 109 and JEOL JSM 6400, respectively. Laminary flow Clean Bench ADS (class 100) was used in the preparation of all the samples. All containers were high-density polyethylene or polypropylene except those used for bacteriological studies in which case glass containers were used. All plastic containers, the sampling bucket, the filtration cells, tubings and membranes were carefully cleaned by washing successively with detergent and 2 M HNOI, sterilized as much as possible by washing with 75% alcohol, and then rinsed thoroughly with an abundance of sterilized Mill&Q water. The glass containers were cleaned by careful washing as above, then sterilized by autoclave. The following reagents were used: NaN, (>99.5%; Fluka), sodium hexametaphosphate ((Na,P03)6,s (Na20)o.s; Fluka, pro anal.), trycase-soja (soy) broth (pH 7.3) (bioMerieux, HgClt (pro anal. Fluka), standards of latex beads (Interfacial Dynamics) for PCS calibration, helium and nitrogen gas (pro anal.). TEM grids were prepared by placing four grids at the bottom of centrifugation tube and filling with 11 cm3 of the centrifuged water sample. The tubes were ultracentrifuged at 30,000 rpm (124,000g) according to the method described by Nomizu et al. (1988) and Perret et al. (1994). Ultracentrifugation was performed at 5 k 2°C for 12 hours and started not more than 3 hours after sampling from the Arve river. Physical meaning of the measured parameters
LS at 90”, PCS and TEM were intensively used in this work. LS only gives a global signal depending on particle size, shape and concentration. However, the change of LS during an experiment is useful to get quick information on changes in size and/or concentration. When calibrated with standard solutions of latex beads, the absolute values of LS allow an estimation of the colloid concentration in the sample of interest. Because it is a simple and low-cost technique, usable on the field and allowing continuous real-time monitoring, it is very useful to monitor the change of a water sample during its processing. PCS, on the other hand, is a more delicate and sophisticated technique, which allows determination of the particle size distributions in the range 50 nm-5 pm. The limitations in the interpretation of PCS data applied to water samples are discussed in
detail in Filella et al. (1996) and Perret et al. (1994). In this work the reported sizes correspond to the means of the intensity-averaged sizes determined at several angles. Because of the large chemical and physical heterogeneity of aquatic colloids, the size values reported here from PCS measurements should only be considered as semiquantitative values. Their changes with experimental conditions, however, give important information on the colloidal system studied. The standard plate counting agar (PCA) method has been used for all studies of bacterial evolutions during storage and stirring experiments, and for bacterial contamination studies. This method (Clesceri et al., 1989) allows enumeration of a broad spectrum of aerobic bacteria. Since aerobic species are the dominant species in oxygenated river waters, this method has been used as an estimate of the total number of bacteria in the samples studied. In the present paper, this number is estimated by the number of colonies formed per cubic centimeter of water sample. Water sampling and fractionation
After collection, the water sample was fractionated by sedimentation as mentioned earlier, to eliminate the largest particles (> 2 pm) because only such fractionated samples are stable for a few days and because we were interested specifically in submicrometer colloids. Hereafter this fractionated sample is called “sedimented sample”. In the Arve and Rhine rivers, the submicrometer colloids represent less than 10% of total particle loads. Since the “signals” (LS, PCS and TEM) due to large particles are much more intense than those of small-size materials, they mask the signals of colloids. Thus, any study of the latter requires an initial separation from larger particles. The following overall fractionation procedure has been applied to samples unless otherwise stated. Two aliquots of the freshly collected water sample (30 dm’) were allowed to sediment under natural gravity in two 30 drn’ sedimentation tanks described previously for 2 hours, at 5 f 2°C. The water layer between 1.0 and 2.5 cm from the surface was then carefully and evenly collected with a peristaltic pump and mixed. 4 drna of this supernatant was then centrifuged at 4000 rpm (3700g) at 5 + 2°C for 2 hours. After centrifugation, the supematants in each centrifugation bottle was collected slowly with a peristaltic pump to avoid any mixing of the bottom part of the bottles. This bottom part (- 25% of the total volume) was rejected, and all the centrifuged water samples were mixed. It has been shown (Newman et al., 1994; Perret et al., 1994) that with this procedure, the fraction collected contains very little colloidal material with a size larger than about 1 pm. STABILIZING COMPOUNDS Sodium hexametaphosphate (SHMP) has been used by many authors as an anticoagulant because it forms complexes with some cations (Toy, 1973) in particular with the major cations of fresh waters such as Ca2+ and Mg*+. Electrolyte concentration is then lowered which reduces the coagulation rates of colloids (Ali et al., 1984). The systematic effect of SHMP on aquatic colloids, however, has not been studied. Our results have shown that SHMP indeed prevents coagulation, but it also induces disaggregation of existing aggregates &hen, 1993). In particular, PCS measurements of the Arve samples, fractionated by centrifugation for 30 min at 2000 tpm, indicated average particle sizes of 300-400 nm in the presence of 1.5 mM SHMP and of 800-900 nm in its absence. The rather large
2181
Preservation of aquatic colloid samples. I.
(~2-3 mM) of SHMP or any other anticoagulant required to complex the whole of Ca*+ and Mg?+ of the water sample may also be a problem for other reasons (analytical interferences, influence on bacterial activity, change of chemical speciation of minor cations when this is of interest). HgClz and NaNz have been tested systematically as antibiotic agents by using the Arve and Rhone river waters and Trycase soja broth as culture medium (Chen, 1993). Water samples with HgCh concentrations increasing from 2.5 x lo-’ to 2.5 x 10e3 M, and with NaN, concentrations of 0.451 mM have been tested individually. The antibiotic effect has also been studied as function of its contact time with the water sample. The results showed that at least _ 3 x 10m4M of HgClr had to be used to achieve an efficient bacteriostatic effect after a contact time of a few hours, which is comparable to the results reported by Robinson and Tuovinen (1984). For NaN,, even the highest concentration (51 mM) was not able to give any visible effect on bacterial activity. NaN, is clearly not efficient enough and other problems related to the use of this compound have been discussed by Rozycki and Bartha (198 1). HgClz also has drawbacks at concentrations > 3 x 10m4M: it poisons the catalyst used in TOC analysis, and it may also form a colloidal precipitate of Hg(OHh at natural pH, thus interfering with the study of natural colloids. Clearly, such results suggest that these antibiotics should not be used for the purpose of characterizing colloids. Other anticoagulants or antibiotics might be studied in detail. It is expected, however, that many of them will produce chemical, biological or physical modification of the colloidal system under study. These considerations justify performing systematic studies to find out in which conditions and how long
concentration
a natural colloidal system may be considered as stable, without the addition of any preservative agent. This is reported in Part II of this series.
CONTAMINATIONS
The DOC contaminations by membranes have already been studied in particular for the products of Schleicher & Schuell (0.45 pm and 0.2 pm), Millipore and Nuclepore polycarbonate (0.8, 0.4 and 0.05 pm) and Amicon Diaflo (PM10 and XM300) (Chen, 1993; Buffle et al., 1982) and they will not be discussed here. It was found that it is necessary to pass a minimum of 40cm3.cm-’ of sterilized bidistilled water through the membranes to avoid further DOC contamination in the filtrates and that Amicon membranes release much more DOC than other types of membranes. Contamination by particles and colloids from jilters and frits
Atmospheric colloidal contaminants are the best known. They may be avoided by working on a clean bench and minimizing the contact of samples with the atmosphere. However, apart from the atmosphere, the major source of colloid contaminations in aquatic colloid studies are the filters and frits when filtration is used. These contaminants are rarely discussed. Figure 1 shows LS intensity as function of the volume of Milli-Q water passed through polypropylene frits (diameter = 90 mm). The results show that new frits release many more particles than used frits and that high flow rates, therefore larger hydraulic washing forces, are preferable to efficiently clean out the adsorbed particles from a frit. Ultrasonic washing may also be helpful.
Milli-Q water
0
1
2
3
4
Washing Volume / liter Fig. 1.LS intensity due to contaminant particles released by polypropylene frits (4 = 90 mm) during their washing by filtering freshly produced Millipore water. The CV value of the measurement was _ 10% when LS intensity > 10; 1: New frit; 2 and 3: used frits; 1 and 3: filtration flow rate = 300 _ 400 cm’.mn-‘; 2: flow rate = 50 _ 80 cm’.mn-‘. Full line: LS intensity of Mini-Q water.
2182
Y.-W. Chen and J. Buthe
Amicon PM10 ___)___
? ,
PC
Ndele
, , , ‘: J ‘,’ : ’ :
1
3
10
30
100
300
1,000
3.000
Washing Volume /mL Fig. 2. LS intensity due to contaminant particles released by Nuclepore polycarbonate filter (0.05 mm, Q = 90 mm) and Amicon Diaflo” PM10 filter (4 = 47 mm) during filtration of Mini-Q water. CV value
of the measurement was m 10% when LS intensity > 10, and m 100% when LS intensity < 10.
Particle and colloid contaminations by filtration membranes have been investigated with Nuclepore polycarbonate (0.05 pm, diameter = 90 mm) and Amicon Diaflo PM10 membrane (diameter = 47 mm) by measuring the LS intensity of filtered Mini-Q water (Fig. 2), and observing the membrane surface by SEM. Figure 2 shows that the particle release of the Nuclepore membrane is negligibly small compared to Amicon membranes. SEM observations of both membranes before and after washing by Milli-Q water confirmed the result from LS: particles were rarely visible at the Nuclepore surface. On the contrary, non-washed Amicon PM10 membranes are covered by a protective layer of organic material in
which many particles and bacteria were imbedded. After washing by Milli-Q water, the protective layer is removed and many particles are released. Note that LS intensities of Figs 1 and 2 are comparable to those of Figs 1 and 2(a) in Part II. Therefore, particle contaminations may be quite significant compared to aquatic colloids when specific precautions are not taken: in some cases LS intensities due to contaminations were as high as the signals provided by natural colloids. Contamination by bacteria Contamination by bacteria is a crucial problem when studying aquatic colloids. The major sources of
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. ..’ . ..’
fresh MM-Q water -+-fresh bidistilled water fresh d&$ziater _..+.._
1
I ?
I 0
2
4
1
I 10
12
Storage fime / da; Fig. 3. Bacterial evolution in three analytically pure waters. They all were stored at 20°C in semi-transparent autoclaved glass bottles and exposed to laboratory light. The viable count* means that a visible colony has been developed from each individual organism and/or each initial colony of organisms (CFU = colony-forming units). None of the three waters was initially sterilized, but before collecting 200 cm3 water samples in the autoclaved glass bottles, the tubing systems were flushed with newly produced waters for 20 min, 10 min and several hours for deionized water, Milli-Q water and bistilled water, respectively.
Preservation of aquatic colloid samples. I. bacterial contaminations and remediations are as follows.
the
corresponding
(9 Atmospheric
contaminations: they can be minimized by using a clean bench for any sample handling, as mentioned for particle contaminations, and by shortening the operating time. (ii) Vessel and tubing walls: it is of key importance that any material in contact with the water sample be previously sterilized. This requirement has an important impact on the choice of procedure used to characterize the colloid sample; i.e. the minimum sample handling and the minimum of contacts with vessels and tubings are preferable. In that respect, centrifugation is much preferable to filtration as a fractionation procedure. Whenever possible, glass material is also preferable to plastic since more efficient sterilization can be achieved by autoclaving. (iii) “Pure waters”: in any sample handling, pure water (Milli-Q or deionized or distilled water) is required for washing or dilution steps. Unless they are carefully sterilized, these waters contain bacteria which proliferate quickly during storage. Figure 3 shows the growth rate of bacterial Mini-Q water, bidistilled water and deionized water. The three waters were not sterilized, but freshly collected after very careful washing of the tubing of the water-producing apparatus. 250 cm3 of each water sample was stored at 20°C in semi-transparent and initially autoclaved glass bottles, and bacterial counting in these waters was done using the PCA method. The choice of the above storage conditions was to mimic the usual conditions of pure water storage in chemical analytical laboratories. Figure 3 shows that in all cases bacterial growth became significant after a few days. Thus unsterilized waters must be used with great caution. Bidistilled water was found to be preferable to Mini-Q or deionized water.
REFERENCES
Ali W., O’Melia, C. R. and Edzwald J. K. (1984) Colloidal stability of particles in lakes: measurement and significance. War. Sri. Technol. 17, 701-712. Beckett R. and Hart B. T. (1993) Use of field-flow fractionation techniques to characterize aquatic particles, colloids and macromolecules. In Environmental Particles, Vol. II (Edited by Buffle J. and Van Leeuwen H. P.). Lewis, Chelsea, MI. Buffle J., Deladoey P., Zumstein J. and Haerdi W. (1982) Analysis and characterization of natural organic matters in freshwaters: I. Study of analytical techniques. Schweiz. 2. Hydrol. 44(Z), 325-362.
Buffle J., Perret D. and Newman M. (1992) The use of filtration and ultrafiltration for size fractionation of aquatic particles, colloids, and micromolecules. In Environmental Particles, Vol. I (Edited by Bufle J. and Van Leeuwen H. P.). Lewis, Chelsea, MI.
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Buffle J. and Van Leeuwen H. P. (Eds) (1992, 1993) Environmental Particles, Vols I and II. Lewis, Chelsea, MI. Chen Y.-W. (1993) Physicochemical Characterization of Colloids in River Waters and Studies of Experimental Conditions. Ph.D. Thesis. University of Geneva, p. 169. Chen Y.-W. and Buffle J. (1994) Chemical composition determination of suspended mineral particle fractions of the Arve river with ICP-AES. Inrernar. J. Environ. Anal. Chem. 57, 125-133. CIPRP (Commission Internationale pour la Protection des Eaux de Rhin contre la Pollution) (1986) Tableaux numC riques des analyses physico-chimique des eaux du Rhin. Publisher: Commission Internationale pour la Protection des Eaux du Rhin contre la Pollution, BP 309, D-5400, Coblence. Clesceri L. S., Greenberg A. B. and Trussell R. R. (Eds) (1989) Standard Methods for Examination of Water and Wastewater, 17th Edn. Published jointly by American Public Health Association, American Water Works Association and Water Pollution Control Federation, Washington, D.C. Degueldre C., Baeyens B., Geoerlich W., Riga J., Verbist J. and Stadelman P. (1989) Colloids in water from a subsurface fracture in granitic rock, Grimsel test site, Switzerland. Geochim. Cosmoschim. Acta 53, 603-610.
de Mora S. J. and Harrison R. M. (1983) Review paper: The use of physical separation techniques in trace metal speciation studies. war. Res. 17, 723-733. Filella M.. Zhana J.. Newman M. and Buffle J. (1996) Capabilities of-photon correlation spectroscopy is ai analytical tool for size distribution measurement of natural submicron colloidal suspensions. CoNoid and Surfaces A.
Fox L. E. (1988) The solubility of colloidal ferric hydroxide and its relevance to iron concentrations in river water. Geochim. Cosmochim. Acra 52, 771-777.
Gallegos C. L. and Menzel R. G. (1987) Submicron size distribution of inorganic suspended solids in turbid waters by photon correlation spectroscopy. War. Resour. Res. 23 (4), 596602.
Leppard G. G. (1992) Evaluation of electron microscopic techniques for the description of aquatic colloids. In Environmental Particles, Vol. I (Edited by Buffle J. and Van Leeuwen H. P.), pp. 231-289. Lewis, Chelsea, MI. Newman M., Filella M., Chen Y.-W., Negre J.-C., Perret D. and Buffle J. (1994) Submicron particles in the Rhine river. Comparison of field observations and model predictions. War. Res. 28, 107-118. Nomizu T., Goto K. and Mizuike A. (1988) Electron microscopy of nanometer particles in freshwater. Anal. Chem. 60 (23), 2653-2656..
Perret D.. Newman M.. Nirrre J.-C.. Chen Y.-W. and Buffle J: (1994) Submi&on>articles’in the Rhine river, Part I, Physico-chemical characterization. Wat. Res. 1, 91-106. Robinson J. B. and Tuovinen 0. H. (1984) Mechanism of microbial resistance and detoxification of mercury and organomercury compounds: physiological, biochemical and genetic analyses. Microbial. Rev. 48 (2), 95-124.
Rozycki M. and Bartha R. (1981) Problems associated with the use of azide as an inhibitor of microbial activity in soil. AL&. Environ. Microbial. 41. 833-836. Sigg L., &rm M. and Kistler D. (1487) Vertical transport of heavy metals by settling particles in Lake Zurich. Limnol. Oceanogr. 32,
112-I30.
SIGSEL (Services Industriels de Genbve, Services des Eaux Laboratories) (1993) Rapports des analyses physicochimiques des eaux de I’Arve et du RhBne.
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Stumm W. (1992) Chemistry of the Solid-Water
InterfuceProcesses at the Mineral- Water and Particle- Water Interface in Natural Systems. John Wiley 8~ Sons, Inc.,
Truitt R. E. and Webe.r J. H. (1979) Trace metal ion filtration losses at pH 5 and pH 7. Anal. Chem. 51,
New York, p. 428. Toy D. F. (1973) Phosphorous. In Comprehensive Inorganic Chemistry, Vol. II (Edited by Bailar J. C., Emeleus H. J., Nyholm R. and Trotman-Dickenson A. F.). Pergamon Press, Oxford.
Wells M. L. and Goldberg E. D. (1991) Occurrence of small colloids in sea water. Nature 353 (26) 342-344. Wells M. L. and Goldberg E. D. (1992) Marine submicron particles. Marine Chem. 40, 5-18.
2057-2059.
Wm. Res. Vol. 30, No. 9, pp. 2178-2184, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/96 515.00 + 0.00
PHYSICOCHEMICAL AND MICROBIAL PRESERVATION OF COLLOID CHARACTERISTICS OF NATURAL WATER SAMPLES. I: EXPERIMENTAL CONDITIONS YU-WE1 CHEN* and JACQUES BUFFLEt@ Department of Inorganic, Analytical and Applied Chemistry, University of Geneva, Sciences II, 30 quai Ernest-Ansennet, CH-1211 Geneva 4, Switzerland (First received March 1995; accepted in revised form April 1996)
Abstract-Physical and chemical characterization of colloids in natural waters is a major issue to understand the mechanism of circulation of pollutants in natural waters. Although many researchers are interested in colloid interactions, they are not yet well understood, and little is known on the appropriate experimental conditions needed to perform reliable and environmentally significant analysis. The following study is dedicated to the development of well-controlled analytical conditions. It is shown that drastic degradation of the water sample can happen during sample handling, if experimental conditions are inappropriate. The study also points out that multidisciplinary cooperation is necessary in the study of environmental problems. This paper is split in two parts for editorial reasons. The first part of this series reports experimental conditions, used to follow colloid stability reported in the second part of the series. Copyright 0 1996 Elsevier Science Ltd Key words-olloids, natural waters, characterizations, contaminations, experimental conditions, storage, biodegradation, agitation, coagulation
INTRODUCTION
In aquatic systems, particles and colloids play a key role in the regulation of concentrations of vital and detrimental compounds (Stumm, 1992; Sigg et al., 1987). New available data suggest that submicrometer particles and macromolecules, denoted below as colloids, are of greatest importance because of their very large specific surface area (Newman et al., 1994). These colloids include inorganic solids, organic macromolecules, the debris of organisms, as well as viruses and bacteria, aggregates of these various components ~~ut?le and van Leeuwen, 1992, 1993). Little is known about their detailed composition and properties in natural aquatic systems. Thorough characterization of aquatic colloids and interpretation of data are difficult and imply taking into account a number of possible artifacts in the following key steps: (1) collection of a raw water sample, (2) sample handling and storage and (3) fractionation of particles and colloids (by size or other properties). These steps may drastically alter the nature and properties of colloidal materials under study.
tAuthor to whom all correspondence should be addressed [Fax: (41) 22 702 60691. *Present address: Department of Chemistry and Biochemistry, Laurentian University Ramsey Lake Road, Sudbury, Ontario, P3E 2C6, Canada.
The main below.
perturbations
to consider
are
listed
(4 Contaminations
by colloids and/or bacteria from the vessels, tubings, atmosphere etc., or loss of colloidal material by adsorption on the vessel walls. Total colloidal content of surface waters is generally less than 0.1-1.0 mg.dm-); contaminations and losses are therefore serious problems. (b) Slow coagulation of the colloidal components which modifies the whole size distribution of colloids, consequently their other properties (e.g. their settling rate). (cl Microbial changes (increase or decrease of population density, change of the nature of the dominant microbes). In addition to being part of the colloidal pool themselves, microbes may provoke degradation of non-living colloids (in particular the organic macromolecules), or aggregation of existing colloids, by releasing metabolic products such as enzymes or polysacchat-ides. The death of microbes will also increase the content of the debris of the organisms. Obviously, the colloidal pool of a water sample is a very delicate system which may be easily perturbed. In fact, perturbation starts at the very moment of sample collection. Indeed, due to coagulation processes and microbial activities, a colloidal system is not static but in continuous evolution. The following perturbations by sample handling and remediations have been reported.
2178
Preservation
of aquatic
-Too long storage periods is a problem because of coagulation and microbial growth. Some authors have used chemicals as anticoagulants (e.g. sodium hexametaphosphate, Gallegos and Menzell, 1987) or antibiotics (e.g. HgC12, Wells and Goldberg, 1991, 1992; or NaNn, Fox, 1988), but no systematic study of their overall effect on a natural colloidal system has been performed before. -Drying and freeze-drying can prevent bacterial growth, but they may induce changes in colloid and aggregate structures (de Mora and Harrison, 1983). In particular the drying and dehydration under high vacuum of unprotected aquatic aggregates deposited on the electron microscopy grid (Wells and Goldberg, 1992) may cause drastic structural changes (Leppard, 1992). -Artefacts introduced by filtration have been studied in more detail (Buffle et al., 1992). It has been clearly shown that the rather high flow rates normally used in colloid fractionation causes retention of colloids on the filter surface due to surface coagulation and not by size sieving. This effect becomes small at very low flow rates, but coagulation of colloid may then occur in the filtration cell due to the very long filtration time used. Contamination problems from filters in trace metals analysis (Truitt and Weber, 1979) and in DOC analysis (Buffle et al., 1982) have been studied carefully, but information on contaminations by particulate and colloidal matter is scarce. Similarly, there is no systematic study of the various sources of bacterial contaminations in the context of the characterization of aquatic colloids. The goal of this work is then to evaluate the most important factors which may affect the freshwater colloids after sample collection, and during sample handling. The following studies were systematically carried out and are reported below: contaminations by colloids and bacteria analytical systems; ??temperature effect during sedimentation; ??
by
colloid samples. I.
2179
changes of colloidal structures during storage and analytical operation; ??bacterial growth.
??
For editorial reasons, this report is split in two parts: the first part describes the choice of optimum experimental conditions used, and the second reports the results of the stability of colloids. Because of the complexity of the system studied, the interconnections of the various factors, and the analytical difficulties due to the low concentration of submicrometer colloids in fresh waters, only a semi-quantitative description can be given. However, this work, by reporting the relative importance of the major processes, discusses the major criteria for performing systematic studies of aquatic colloids in well-controlled conditions. The colloids of three rivers have been studied and compared: those of the Arve and RhBne rivers in Geneva and those of the Rhine river in Base1 (Switzerland). These rivers possess different characteristics. The Rhone river, at the outlet of the Lake Leman, has the lowest colloid concentration, due to previous sedimentation in the lake, but the organic to inorganic colloid concentration ratio is the highest. The Arve river directly flows from Alps (France) and has a relatively large particle load, dominated by sand, clay minerals, and calcium carbonates. The colloid content of the Rhine river is intermediate between the other two rivers. EXPERIMENTAL
Composition of the jresh waters studied The sampling sites on the RhBne and Arve rivers were in the middle of the bridges “Pont de Sous-Terre” and “Passerelle de 1’Ecole de Medecine”, respectively in Geneva (Switzerland). The Rhine river samples were collected at the middle of the river on a boat, immediately downstream of the “Birsfelden” dam in Base1 (Switzerland). In all cases, the samples were collected 0.5-1.0 m below the water surface. The particle loads of these rivers varied seasonally in the range 1.9-> 1000, 0.1-7, and 5-700 mg.dm-’ for the Arve, the RhBne and the Rhine rivers, respectively. The major characteristics of the three rivers are given in Table I (Perret et al., 1994; Chen and ButBe 1994; Chen, 1993; CIPRP, 1986; SIGSEL, 1993).
Table I. The maior characteristics of the three rivers studied RhBne Flow rate
(m’/s) TOC of river water bg Wm’) Suspended particulate material
(msidm’) Colloid concentration ( < 1pm) (mgidm’) Dissolved 0: (mgidm’) DH Conductivity (25°C) W/cm)
166-319 (248) 0.761.47 (1.15) 0.134.31 (1.78)
6.61-13.64 (9.9) 7.9-8.09 (8.02) 277-3 I5 (293)
Arve
I2-840 (79) 0.54-2.77
(1.20) 1.90-> 1000 (96) 2.7-7.06+ (4.3) 7.55-l I.46 (10.1) 7.50-8.46 (8.17) 240-528 (388)
The values in parentheses are means over the year 1993. *From Chen (1993). ‘The value in this parenthesis is the mean between March and May, 1992
Rhine 501-2459 (1059) 2.6-6.4 (3.8) 5.43-700.6’ (8.8) 0.6-46.9’ (0.9) 8.4-12.5 (10.6) 7.6-8. I (7.90) 288-374 (331)
2180 Apparatus
Y.-W. Chen and J. Buffle and reagents
Garden watering pump Marina Jolly 800 with high flow rate (1 dm’.s-I) was used to collect water samples. Home-made plexiglass tanks (30 dm3, height 12 cm) were then used at the sampling location for sedimentation of the largest particles (ra few micrometers) under natural gravity. They were thermally insulated and their cover included nine sampling tubes to allow quick, gentle and homogeneous sampling of the top surface layer, at a well-controlled depth (1.0-2.5 cm from the top surface, see Perret et al., 1994 for details). The following fractionation apparatus were subsequently used: centrifuged Hereaus 2.0 RS with swing-out bucket and thermostated conditions; ultracentrifuge Beckman L 7-55 with swing-out rotor SW 30 and thermostated conditions; home-made plexiglass filtration cells with and without stirring bar (47 mm and 90 mm); corresponding Nucleopore Polycarbonate membranes with pore sizes of 0.8 and 0.05 pm and Amicon Diaflo PM10 membranes with diameters of 47 mm. Light scattering (LS) was measured on line with the Fluorimeter Hitachi-Merck F-1050 (with white light and a flow-through cell) at the output of the filtration cell. Off-line LS were measured with a fluorimeter Perkin-Elmer 204. Photon correlation spectroscopy (PCS) was performed with a Malvem Zetasizer III with a 1 W Ar laser (Innova 70). DOC was measured with a Total Organic Analyzer Xertex Dohrmann DC 80. Transmission and scanning electron microscope (TEM and SEM) pictures were obtained with the Apparatus Zeiss EM 109 and JEOL JSM 6400, respectively. Laminary flow Clean Bench ADS (class 100) was used in the preparation of all the samples. All containers were high-density polyethylene or polypropylene except those used for bacteriological studies in which case glass containers were used. All plastic containers, the sampling bucket, the filtration cells, tubings and membranes were carefully cleaned by washing successively with detergent and 2 M HNOI, sterilized as much as possible by washing with 75% alcohol, and then rinsed thoroughly with an abundance of sterilized Mill&Q water. The glass containers were cleaned by careful washing as above, then sterilized by autoclave. The following reagents were used: NaN, (>99.5%; Fluka), sodium hexametaphosphate ((Na,P03)6,s (Na20)o.s; Fluka, pro anal.), trycase-soja (soy) broth (pH 7.3) (bioMerieux, HgClt (pro anal. Fluka), standards of latex beads (Interfacial Dynamics) for PCS calibration, helium and nitrogen gas (pro anal.). TEM grids were prepared by placing four grids at the bottom of centrifugation tube and filling with 11 cm3 of the centrifuged water sample. The tubes were ultracentrifuged at 30,000 rpm (124,000g) according to the method described by Nomizu et al. (1988) and Perret et al. (1994). Ultracentrifugation was performed at 5 k 2°C for 12 hours and started not more than 3 hours after sampling from the Arve river. Physical meaning of the measured parameters
LS at 90”, PCS and TEM were intensively used in this work. LS only gives a global signal depending on particle size, shape and concentration. However, the change of LS during an experiment is useful to get quick information on changes in size and/or concentration. When calibrated with standard solutions of latex beads, the absolute values of LS allow an estimation of the colloid concentration in the sample of interest. Because it is a simple and low-cost technique, usable on the field and allowing continuous real-time monitoring, it is very useful to monitor the change of a water sample during its processing. PCS, on the other hand, is a more delicate and sophisticated technique, which allows determination of the particle size distributions in the range 50 nm-5 pm. The limitations in the interpretation of PCS data applied to water samples are discussed in
detail in Filella et al. (1996) and Perret et al. (1994). In this work the reported sizes correspond to the means of the intensity-averaged sizes determined at several angles. Because of the large chemical and physical heterogeneity of aquatic colloids, the size values reported here from PCS measurements should only be considered as semiquantitative values. Their changes with experimental conditions, however, give important information on the colloidal system studied. The standard plate counting agar (PCA) method has been used for all studies of bacterial evolutions during storage and stirring experiments, and for bacterial contamination studies. This method (Clesceri et al., 1989) allows enumeration of a broad spectrum of aerobic bacteria. Since aerobic species are the dominant species in oxygenated river waters, this method has been used as an estimate of the total number of bacteria in the samples studied. In the present paper, this number is estimated by the number of colonies formed per cubic centimeter of water sample. Water sampling and fractionation
After collection, the water sample was fractionated by sedimentation as mentioned earlier, to eliminate the largest particles (> 2 pm) because only such fractionated samples are stable for a few days and because we were interested specifically in submicrometer colloids. Hereafter this fractionated sample is called “sedimented sample”. In the Arve and Rhine rivers, the submicrometer colloids represent less than 10% of total particle loads. Since the “signals” (LS, PCS and TEM) due to large particles are much more intense than those of small-size materials, they mask the signals of colloids. Thus, any study of the latter requires an initial separation from larger particles. The following overall fractionation procedure has been applied to samples unless otherwise stated. Two aliquots of the freshly collected water sample (30 dm’) were allowed to sediment under natural gravity in two 30 drn’ sedimentation tanks described previously for 2 hours, at 5 f 2°C. The water layer between 1.0 and 2.5 cm from the surface was then carefully and evenly collected with a peristaltic pump and mixed. 4 drna of this supernatant was then centrifuged at 4000 rpm (3700g) at 5 + 2°C for 2 hours. After centrifugation, the supematants in each centrifugation bottle was collected slowly with a peristaltic pump to avoid any mixing of the bottom part of the bottles. This bottom part (- 25% of the total volume) was rejected, and all the centrifuged water samples were mixed. It has been shown (Newman et al., 1994; Perret et al., 1994) that with this procedure, the fraction collected contains very little colloidal material with a size larger than about 1 pm. STABILIZING COMPOUNDS Sodium hexametaphosphate (SHMP) has been used by many authors as an anticoagulant because it forms complexes with some cations (Toy, 1973) in particular with the major cations of fresh waters such as Ca2+ and Mg*+. Electrolyte concentration is then lowered which reduces the coagulation rates of colloids (Ali et al., 1984). The systematic effect of SHMP on aquatic colloids, however, has not been studied. Our results have shown that SHMP indeed prevents coagulation, but it also induces disaggregation of existing aggregates &hen, 1993). In particular, PCS measurements of the Arve samples, fractionated by centrifugation for 30 min at 2000 tpm, indicated average particle sizes of 300-400 nm in the presence of 1.5 mM SHMP and of 800-900 nm in its absence. The rather large
2181
Preservation of aquatic colloid samples. I.
(~2-3 mM) of SHMP or any other anticoagulant required to complex the whole of Ca*+ and Mg?+ of the water sample may also be a problem for other reasons (analytical interferences, influence on bacterial activity, change of chemical speciation of minor cations when this is of interest). HgClz and NaNz have been tested systematically as antibiotic agents by using the Arve and Rhone river waters and Trycase soja broth as culture medium (Chen, 1993). Water samples with HgCh concentrations increasing from 2.5 x lo-’ to 2.5 x 10e3 M, and with NaN, concentrations of 0.451 mM have been tested individually. The antibiotic effect has also been studied as function of its contact time with the water sample. The results showed that at least _ 3 x 10m4M of HgClr had to be used to achieve an efficient bacteriostatic effect after a contact time of a few hours, which is comparable to the results reported by Robinson and Tuovinen (1984). For NaN,, even the highest concentration (51 mM) was not able to give any visible effect on bacterial activity. NaN, is clearly not efficient enough and other problems related to the use of this compound have been discussed by Rozycki and Bartha (198 1). HgClz also has drawbacks at concentrations > 3 x 10m4M: it poisons the catalyst used in TOC analysis, and it may also form a colloidal precipitate of Hg(OHh at natural pH, thus interfering with the study of natural colloids. Clearly, such results suggest that these antibiotics should not be used for the purpose of characterizing colloids. Other anticoagulants or antibiotics might be studied in detail. It is expected, however, that many of them will produce chemical, biological or physical modification of the colloidal system under study. These considerations justify performing systematic studies to find out in which conditions and how long
concentration
a natural colloidal system may be considered as stable, without the addition of any preservative agent. This is reported in Part II of this series.
CONTAMINATIONS
The DOC contaminations by membranes have already been studied in particular for the products of Schleicher & Schuell (0.45 pm and 0.2 pm), Millipore and Nuclepore polycarbonate (0.8, 0.4 and 0.05 pm) and Amicon Diaflo (PM10 and XM300) (Chen, 1993; Buffle et al., 1982) and they will not be discussed here. It was found that it is necessary to pass a minimum of 40cm3.cm-’ of sterilized bidistilled water through the membranes to avoid further DOC contamination in the filtrates and that Amicon membranes release much more DOC than other types of membranes. Contamination by particles and colloids from jilters and frits
Atmospheric colloidal contaminants are the best known. They may be avoided by working on a clean bench and minimizing the contact of samples with the atmosphere. However, apart from the atmosphere, the major source of colloid contaminations in aquatic colloid studies are the filters and frits when filtration is used. These contaminants are rarely discussed. Figure 1 shows LS intensity as function of the volume of Milli-Q water passed through polypropylene frits (diameter = 90 mm). The results show that new frits release many more particles than used frits and that high flow rates, therefore larger hydraulic washing forces, are preferable to efficiently clean out the adsorbed particles from a frit. Ultrasonic washing may also be helpful.
Milli-Q water
0
1
2
3
4
Washing Volume / liter Fig. 1.LS intensity due to contaminant particles released by polypropylene frits (4 = 90 mm) during their washing by filtering freshly produced Millipore water. The CV value of the measurement was _ 10% when LS intensity > 10; 1: New frit; 2 and 3: used frits; 1 and 3: filtration flow rate = 300 _ 400 cm’.mn-‘; 2: flow rate = 50 _ 80 cm’.mn-‘. Full line: LS intensity of Mini-Q water.
2182
Y.-W. Chen and J. Buthe
Amicon PM10 ___)___
? ,
PC
Ndele
, , , ‘: J ‘,’ : ’ :
1
3
10
30
100
300
1,000
3.000
Washing Volume /mL Fig. 2. LS intensity due to contaminant particles released by Nuclepore polycarbonate filter (0.05 mm, Q = 90 mm) and Amicon Diaflo” PM10 filter (4 = 47 mm) during filtration of Mini-Q water. CV value
of the measurement was m 10% when LS intensity > 10, and m 100% when LS intensity < 10.
Particle and colloid contaminations by filtration membranes have been investigated with Nuclepore polycarbonate (0.05 pm, diameter = 90 mm) and Amicon Diaflo PM10 membrane (diameter = 47 mm) by measuring the LS intensity of filtered Mini-Q water (Fig. 2), and observing the membrane surface by SEM. Figure 2 shows that the particle release of the Nuclepore membrane is negligibly small compared to Amicon membranes. SEM observations of both membranes before and after washing by Milli-Q water confirmed the result from LS: particles were rarely visible at the Nuclepore surface. On the contrary, non-washed Amicon PM10 membranes are covered by a protective layer of organic material in
which many particles and bacteria were imbedded. After washing by Milli-Q water, the protective layer is removed and many particles are released. Note that LS intensities of Figs 1 and 2 are comparable to those of Figs 1 and 2(a) in Part II. Therefore, particle contaminations may be quite significant compared to aquatic colloids when specific precautions are not taken: in some cases LS intensities due to contaminations were as high as the signals provided by natural colloids. Contamination by bacteria Contamination by bacteria is a crucial problem when studying aquatic colloids. The major sources of
1LWOOO 100,ooo
../
I_* -;,.p+--“1.r .
,..’
/.t.”
..’
. ..’ . ..’
fresh MM-Q water -+-fresh bidistilled water fresh d&$ziater _..+.._
1
I ?
I 0
2
4
1
I 10
12
Storage fime / da; Fig. 3. Bacterial evolution in three analytically pure waters. They all were stored at 20°C in semi-transparent autoclaved glass bottles and exposed to laboratory light. The viable count* means that a visible colony has been developed from each individual organism and/or each initial colony of organisms (CFU = colony-forming units). None of the three waters was initially sterilized, but before collecting 200 cm3 water samples in the autoclaved glass bottles, the tubing systems were flushed with newly produced waters for 20 min, 10 min and several hours for deionized water, Milli-Q water and bistilled water, respectively.
Preservation of aquatic colloid samples. I. bacterial contaminations and remediations are as follows.
the
corresponding
(9 Atmospheric
contaminations: they can be minimized by using a clean bench for any sample handling, as mentioned for particle contaminations, and by shortening the operating time. (ii) Vessel and tubing walls: it is of key importance that any material in contact with the water sample be previously sterilized. This requirement has an important impact on the choice of procedure used to characterize the colloid sample; i.e. the minimum sample handling and the minimum of contacts with vessels and tubings are preferable. In that respect, centrifugation is much preferable to filtration as a fractionation procedure. Whenever possible, glass material is also preferable to plastic since more efficient sterilization can be achieved by autoclaving. (iii) “Pure waters”: in any sample handling, pure water (Milli-Q or deionized or distilled water) is required for washing or dilution steps. Unless they are carefully sterilized, these waters contain bacteria which proliferate quickly during storage. Figure 3 shows the growth rate of bacterial Mini-Q water, bidistilled water and deionized water. The three waters were not sterilized, but freshly collected after very careful washing of the tubing of the water-producing apparatus. 250 cm3 of each water sample was stored at 20°C in semi-transparent and initially autoclaved glass bottles, and bacterial counting in these waters was done using the PCA method. The choice of the above storage conditions was to mimic the usual conditions of pure water storage in chemical analytical laboratories. Figure 3 shows that in all cases bacterial growth became significant after a few days. Thus unsterilized waters must be used with great caution. Bidistilled water was found to be preferable to Mini-Q or deionized water.
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