Environ. Sci. Technol. 2000, 34, 2388-2392
The Use of Hydrophobic Resins as Analogues for Sediment Testing K. SIMKISS,* P. A. EDWARDS, M. A. M. LAWRENCE, N. A. DAVIES, AND M. G. TAYLOR School of Animal & Microbial Sciences, The University of Reading, Reading RG6 6AJ, U.K.
Many of the pollutants that enter the aquatic environment become adsorbed onto the surface of particles and are carried out of the water column into the sediments. This phenomenon plays a major part in decontaminating the water supply but results in significant concentrations of toxicants entering the habitat of benthic organisms. It had been thought until fairly recently that the sediments acted as a sink that removed these chemicals from interaction with the biota, but more recent work has shown that a number of these materials remain bioavailable. Estimating the significance of this problem has been problematic as sediments show considerable spatial and temporal diversity in the variety and distribution of their properties. The purpose of this work was to explore the use of artificial particles with known surface properties to study these pollutant-particle-animal interactions. By identifying the factors involved in the binding and release of contaminants from particles in a controlled way it was hoped to learn something of the mechanisms involved in the natural environment. The results of this study indicate that there are some simple correlations between the relevant distribution coefficients of the pollutants, their environmental fate, and the complex behavior and assay of natural sediments.
Introduction It is now generally accepted that many pollutants in the aquatic environment are removed from the water column by particles and become trapped in sediments. Traditionally sediments were considered to be sinks for such materials and up until 20 years ago such deposits were thought to be biologically inert. This concept has had to be abandoned, however, as evidence has accumulated that such contaminated sediments are significant factors in the degradation of surface waters and a threat to benthic communities (1). Hydrophobic pollutants such as many of the biocides that become accumulated in sediments may not only induce toxicological effects within benthic organisms but they may also bioaccumulate in the body and pass up food chains. There is, therefore, an urgent need to be able to assess the factors that are involved in the binding and release of these materials in sediments and to quantify their likely impact on the environment. The scavenging of trace materials from the water column is associated with the presence of organic films on the surfaces of the sedimenting particles. These films are typically of biological origin, being derived from bacterial exopolymers, cell walls, and plant products. Their composition in terms * Corresponding author phone: 0044 118 931 8460; fax: 0044 118 931 0180; e-mail:
[email protected]. 2388
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of reactive ligands is relatively simple and is characterized by carboxylate and phenolic ligands attached to a complex mixture of hydrophobic compounds (2, 3). It should, therefore, be relatively easy to model such surfaces and thus predict their reactivity in the natural environment. To date, little effort has been put into this approach, and most of the work in sediment toxicity has tended to emphasize the complexity of the substrates in terms of the variations in particle size, clay type and content, cation exchange capacity, redox activity, pH, and anaerobic profile (4). The elaborate composition of natural sediments is not the only complicating factor in assessing their potential for toxic interactions with the biota. One of the standard toxicity testing systems has been based on “water-only” exposure. This is a relatively easy test to perform, and it has been argued that it is a valid assay as the effect of adding sediment to the system may be considered as largely due to its effect on pore water which is in equilibrium with materials on particle surfaces. Only recently has it been accepted that for some benthic invertebrates the alimentary tract may also provide a direct and important route of uptake (5). It is against this background that we have recently advocated a much simpler and more direct approach to assessing the bioaccumulation of hydrophobic pollutants by benthic organisms (6, 7). The technique uses resin beads with well-defined surface characteristics of the type used in high performance liquid chromatography (HPLC) columns as a substitute for the biofilms on sediment particles. Such HPLC beads are available in a variety of forms in known and controlled size ranges. In the test system that we have been using these resin particles are mixed with acid washed sand that is sieved to a size range that is too large to be ingested by the test organism. The beads are equilibrated with the contaminant to be tested, and the organism is exposed for a fixed period of time prior to depuration and analysis of the organism to determine the relevant bioaccumulation. This approach enables complete control over the size and surface properties of the particles, the composition of the pore water, the hydraulics of the sediment, and the feeding habits of the organism. To obtain a “proof of concept” for this approach we have compared the results obtained from these model particles with those derived from a natural sediment. This should indicate how effective the artificial system is in predicting the properties of the organic carbon material that is considered to be the main fraction responsible for the accumulation of lipophilic materials in natural sediments. The organic material in the test sediment was assumed to be typical of the complex mixtures of molecules that occur in natural systems. Thus the proof of concept was based on the proposition that it is the relatively simple hydrophobicity of this organic matrix that dictates its partitioning properties but that these might be modified by simple ionic interactions with particular hydrophobic pollutants.
Materials and Methods Organisms and Particles. The test organism was the oligochaete worm L. variegatus. It was cultured and used as described previously (6). Specimens in the size range of 1-2 cm were selected so as to minimize animals undergoing asexual reproduction by architomy during the test period (5). Two types of resin particle were used. These were Toyopearl SP 650M which has an anionic sulfonyl group on a three carbon alkyl chain and Toyopearl Phenyl 650M which contains a surface-bound phenyl group. Both particle surfaces were presented on the same strongly hydrophilic polymeric 10.1021/es9912630 CCC: $19.00
2000 American Chemical Society Published on Web 05/04/2000
TABLE 1. Chemical Properties of Contaminants (12, 13)a chemical 2,4-dichlorophenol pentachlorophenol pyrene trifluralin fenpropidin cyproconazole a
solubility molecular wt log Ko/w (mg/L) 163 266 202 335 273 292
3.23 5.24 5.13 4.83 2.59 2.91
vss 80 ins 0.22 530 140
p Ka 7.85 4.71 10.1 neutral pH 3.5-10
vss ) very slightly soluble; ins ) insoluble.
backbone derived from ethylene glycol and methyl methacrylate so as to produce beads that were 40-60 µm diameter. These particles were mixed with acid washed sand that had been sieved to be 100-300 µm in size. All the materials were washed before use. A natural sediment was used for comparison. This came from a standard sample collected and analyzed by WRc (Water Research Centre, Medmenham, U.K.). It was passed through a 1 mm sieve and had an organic carbon content of 1.7% and a particle size distribution of 73% below 63 µm. This material was used in an identical way to the artificial particles. Chemicals. The contaminants that were used were all radiolabeled with 14C and consisted of 2,4-dichlorophenol (2,4-DCP), pentachlorophenol (PCP), pyrene (all supplied by Sigma), and three biocides, Fenpropidin (a morpholine analogue), Cyproconazole (a triazole), and Trifluralin (a derivative of 2,6-dinitroaniline) all kindly donated by Novartis (Basle). A summary of the chemical properties of these materials is given in Table 1. Partitioning Data. Preliminary experiments were undertaken to determine the percentage of contaminant that was bound to each type of particle, and the particles were then dosed so that they gave equilibrated water concentrations of 10-7 mol/L for the chlorophenols or 10-9 mol/L for the other less soluble materials. The partition coefficient (Kd) defined as mol g-1 solid/mol g-1 solution was determined from the adsorption isotherms by plotting the initial slopes for each type of particle at water concentrations in the range of the test system. For this analysis the resin particles were shaken over a 4 h period prior to analysis. This was well in excess of the equilibrium times for all these artificial particles but not applicable to the natural sediment. In a strict sense the partition coefficient is a measure of the equilibrium of a chemical between an aqueous and a defined solid phase. Natural sediments are, of course, mixtures of a range of different materials and therefore do not have a simple partition coefficient so that the measured values are the sum of a number of different interactions. Thus, some of the components that lie deep beneath the surface of a natural particle may not equilibrate with the water phase for weeks or months so that the system cannot be accurately defined (8, 9). In the specific case of the sediment used in these experiments most of the adsorption occurred within 2 days, and an apparent equilibrium was established within 5 days. For these reasons all samples in the bioaccumulation experiments were equilibrated by shaking for 24 h and allowing a further 24 h for equilibration. Results obtained with the natural sediment are expressed as “apparent Kd” and relate to a 48 h time scale relevant to the types of desorption effects that will occur during the passage of sediment through the alimentary tract of benthic organisms. At least eight different concentrations of each contaminant were used in determining the Kd (10) and covered the concentration ranges used in the bioaccumulation tests.
Test Procedures. The concentrations used for the bioaccumulation experiments were chosen to be well below the saturation level and outside the ranges associated with toxicity. There was no mortality associated with the use of the contaminants. The test system consisted of 0.5 g of reactive particles with 1.5 g of sand and 20 mL of dechlorinated tap-water in an incubator set at 20 ( 0.5 °C (7). The particles were equilibrated prior to use with sufficient contaminant to give a water-phase concentration of between 10-7 and 10-9 mol/L. The contaminants were analyzed using a Packard 2250CA Tricarb liquid scintillation spectrometer, and concentrations were derived from a knowledge of their specific activities. Animals were exposed to the pollutants for 48 h in a static system, depurated by feeding in clean sediment for 24 h, killed, weighed, solubilized in Soluene 350, and counted in Hionic Fluor scintillation fluid (Packard). The concentration of chemical in solution was measured at the start and end of each bioaccumulation experiment. If there was a significant change of more than 10%, the final concentration was used to standardize the biological accumulation factor (7). This was defined as mol contaminant‚g-1 animal‚48 h-1/mol contaminant‚mL-1 water. Animals that had been exposed to contaminants for 48 h were killed and extracted with organic solvents. The extracts were dissolved in methanol and analyzed using a Varian 9010 HPLC linked to a Packard 500TR flow scintillation spectrometer to search for potential breakdown products that might have been derived from the contaminant molecules during the test procedures. Mass balance calculations were not undertaken with the natural sediments because of the problems of quantitatively recovering trace quantities of radioisotope from these complex mixtures. Recoveries for experiments using artificial resin beads were in the range of 92-103%.
Results and Discussion Contaminant Bioaccumulation. The oligochaete worm, L. variegatus, has been identified as a convenient test organism for assessing the toxicity and bioaccumulation of sedimentassociated contaminants (11). Its general biology and feeding activity has been well studied (5, 12). There is general agreement that the throughput time for ingested food is 12 h or less and that depuration periods of this length remove sediment from the alimentary tract. The 24 h time used in the current depuration period may underestimate the assimilated dose of pollutant as some of the body burden may be progressively removed during this process (13), but in these experiments it is assumed that this effect is similar for all treatments. No significant concentrations of metabolites of the test materials were detected by the HPLC analysis of the worms after their exposure to the pollutants for 48 h. It was, therefore, concluded that the 14C analyses of the worms represented the bioaccumulation of the parent compounds. The extent to which the contaminants were bioaccumulated in the depurated worm L. variegatus are shown in Table 2 for animals exposed via water, water with sand, or water with reactive particles. The data in Tables 1 and 2 provide a coherent series of analyses on the physicochemical properties of the contaminants and their bioaccumulation from a variety of substrates into the oligochaete L. variegatus. These data can be used to test a number of hypotheses on the factors involved in the uptake of pollutants from sediments. Contaminant Properties: Octanol/Water Coefficients. There is extensive literature on the penetration of lipid-soluble molecules into cells that date back over 100 years to Ernst Overton’s original observations on the cell membrane (16). The compound octanol is a good analogue for the hydrophobic properties of the cell membrane, and the octanol/ VOL. 34, NO. 12, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Chemical Contaminants with Their Partition Coefficients (mol g-1 Solid/mol g-1 Solution) and Bioaccumulation Factors (mol contaminant‚g-1 Animal‚48 h-1/mol Contaminant‚mL-1 Water) for L. variegatus Exposed to Water Only or to Water Containing Various Particlesa chemical
water only
Kd bioaccumulation
14.4 ( 10.7
Kd bioaccumulation
39.5 ( 2.6
Kd bioaccumulation
240.2 ( 14.8
Kd bioaccumulation
179.2 ( 25.1
Kd bioaccumulation
289.7 ( 24.1
Kd bioaccumulation
9.6 ( 1.7
a
sand
WRc sediment
2,4-Dichlorophenol 10 24 32.2 ( 1.8 24.1 ( 4.4 Pentachlorophenol 1 21 124.7 ( 9.6 453.3 ( 44.2 Pyrene 57 1081 362 ( 47.1 702.3 ( 140. Trifluralin 72 535 89.9 ( 12.6 107.6 ( 11.6 Fenpropidin 33 532 205.5 ( 6.5 153.1 ( 39.9 Cyproconazole 16 16 7.8 ( 1.6 6.5 ( 0.9
phenyl 650M
SP 650M
138 101.2 ( 15.5
45 17.1 ( 3.2
161 334.1 ( 36.1
7 305 ( 39.6
5412 638.4 ( 90.7
1742 429.7 ( 25.9
436 44.8 ( 4.2
308 60.3 ( 8.3
171 165.5 ( 19.1
807 42.4 ( 4.9
192 15.9 ( 2.2
47 14.0 ( 1.5
Values are means ( S.E.M for n ) 10.
FIGURE 1. The bioaccumulation (mol‚g-1 L. variegatus 48 h-1/mol‚ g-1 water) of various contaminants in relation to the log octanol/ water partition coefficient (Ko/w). There is no significant correlation in this 48 h test. Cyp ) Cyproconazole; 2,4-DCP ) 2,4-dichlorophenol; Fenprop ) Fenpropidin; PCP ) pentachlorophenol; Triflur ) Trifluralin. water partitioning of an organic molecule is frequently taken to be a good indicator of the likely penetration of a pollutant into an organism (17). In Figure 1 the log Ko/w of six contaminants is plotted against their bioaccumulation from water into L. variegatus. The results show that there is no significant relationship (r 2 ) 0.004) and no detectable slope. There may be a number of reasons for this. The first is that in a water-only test the worms show erratic locomotory behavior. This is normally abated when the worms bury themselves in the sediment, but the effect of this activity in a water-only test is to pass fresh contaminant over the animal’s body thereby modifying its uptake. The second factor worth noting is that lipophilic molecules that contain polar groups will assume specific orientations at hydrophobic/ hydrophilic interfaces such as those produced by octanol/ water, and these may cause interactions at the external 2390
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FIGURE 2. A comparison of the bioaccumulation (mol‚g-1 L. variegatus 48 h-1/mol‚g-1 water) of a variety of contaminants as shown by a water only or a “water and sediment” test. surfaces of the worm (18). Whatever the explanation it is clear that the octanol/water partition coefficient is not a good predictor of the bioaccumulation of a compound into an aquatic oligochaete over the time period of 48 h. This result throws considerable doubt on the use of water-only tests for predicting bioaccumulation into sedimentary organisms. This problem is emphasized in Figure 2 where the uptake of six contaminants is compared between a water-only system and water containing a natural sediment. The linear regression is not significant (r 2 ) 0.141) showing that such tests are poor predictors of the potential impact of pollutants on benthic organisms. It is clear from Figure 2 that the presence of sediment is responsible for a large increase in the bioaccumulation of pyrene and PCP above that observed in a water-only system. All the evidence suggests that this increase is due to the additional effect of the ingestion of the contaminated sediment and the subsequent release of the pollutant from these particles into the alimentary tract of the organism. The same phenomenon has previously been reported for pyrene ingested by L. variegatus (5) and for the
FIGURE 3. A comparison of the bioaccumulation (mol‚g-1 L. variegatus 48 h-1/mol‚g-1 water) of different contaminants from a natural sediment with the apparent partition coefficient (Kd ) mol kg-1 sediment/mol kg-1 water). The correlation coefficient, r 2 ) 0.39. uptake of PCP in terms of the Kd in a pollutant/particlesurface interaction (7). Particle Effects: Sediment Partition Coefficients. The partitioning of nonionic organic compounds onto sediment particles is intended to give an indication of how a chemical could be adsorbed into the organic carbon content of the sediment (19). The use of this latter factor emphasizes that it is the hydrophobic organic material (cell membranes, humic acids, bacterial biopolymers etc.) that is largely responsible for the accumulation of lipophilic contaminants in the sediment. These interactions should be represented by the sediment partition coefficient (Kd; defined as mol contaminant‚kg-1 solid/mol contaminant‚kg-1 solution i.e., a concentration ratio). In Figure 3 the bioaccumulation of these contaminants is plotted against the data for the “apparent” Kd values of the natural sediment. The relationship is not significant (p ) 0.18) with a correlation coefficient r 2 ) 0.39. As in Figure 2 this result emphasizes that two routes of uptake are probably involved with aqueous and particlebound components. The interpretation of the natural sediment effect is, however, complex since these substrates are mixtures of a wide range of particles, and selective feeding organisms including L. variegatus may utilize fractions with very different properties from the bulk phase upon which measurements of Kd are usually based. It will be apparent from these analyses that the bioaccumulation of contaminants from an aqueous, natural sediment system cannot be satisfactorily predicted from either the octanol/water partition coefficient of the contaminant or the apparent partition coefficient of the natural sediment. This suggests that there may be more specific and complex interactions at the water/particle-surface interface which need to be either investigated in detail or modeled using chemical analogues as suggested in the present work. Hydrophobic Contaminant/Hydrophobic Surface Interactions. Contaminants in aqueous solution can be expected to orientate so as to maximize any polar interactions with the solvent and minimize the exposure of hydrophobic regions. On approaching a hydrophobic region any structured water layers between the two surfaces will need to be displaced in a dehydration reaction that facilitates the interactions of van der Waal forces (18). Thus any simulation
FIGURE 4. A comparison of the bioaccumulation (mol‚g -1 L. variegatus 48 h-1/mol‚g-1 water) of different contaminants from natural sediment and a hydrophobic particle (Phenyl). The correlation coefficient, r 2 ) 0.95. Slope of line 1.15 ( 0.13. of the adsorption of ionic contaminants upon hydrophobic surfaces and their subsequent absorption by benthic organisms should model these polar and nonpolar interactions as closely as possible. These influences can be simulated by comparing resin particles with simple hydrophobic surfaces (Toyopearl Phenyl) or hydrophobic surfaces with a negatively charged end group (Toyopearl SP) to mimic the effects of natural sediments. In Figure 4 the bioaccumulation of contaminants from a system containing a natural sediment is compared with the uptake from a similar system containing a particle with a phenyl coated surface. There is a highly significant relationship (p ) 0.001) between these two sets of data with a correlation coefficient r 2 ) 0.95. The additional effect of a negative charge on the hydrophobic particle Toyopearl SP can be seen in Figure 5. The relative positions of the charged contaminants (cationic Fenpropidin; anionic pentachlorophenol) move relative to the other contaminants, but the relationship remains highly significant (p < 0.001) with a correlation coefficient of r 2 ) 0.98. Potential Value of Artificial Particles. The results of this study show that artificial particles can be used to give a laboratory test system that is as predictable of bioaccumulation as a natural sediment. The system as it is used in the current study normalizes the procedure around a constant concentration of contaminant in the water phase so that there should be no variation in the concentration of contaminant between overlying and pore water in this study. This facilitates the investigation of the relative importance of ingested sediment and pore water as routes for the bioaccumulation of pollutants. As an example of this we refer to a study on the uptake of pyrene by L. variegatus that has previously been investigated in a natural sediment (5). It was found that over an 8 day exposure period approximately 61% of the body burden was due to absorption from the alimentary tract which was, therefore, the dominant route for uptake. In our test system natural sediment gave a bioaccumulation value of 702 of which 240 was attributed to water and 462 to the sediment i.e., over a 2 day test period 65% of the accumulated dose came from ingestion of the natural sediment. The corresponding figure for phenyl-coated beads was 62% (Table 2). This confirms the observation that for the pollutant pyrene the quantity of material accumulated through the alimentary tract is roughly twice that from water VOL. 34, NO. 12, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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should also be possible to subject these systems to all the additional variables (redox, pH, etc.) that concern environmentalists in predicting what will happen under particular natural conditions to the bioavailability of particular biocides. These results imply that there is a close association between the chemistry of contaminants, the surface properties of particles, and their availability to the biota. It should, therefore, also be possible to use this approach to devise a simple chromatography column with retention times that will give good predictions of the potential bioaccumulation of biocides from natural sediments.
Acknowledgments This work was supported by the Environmental Diagnostics program of the U.K. Natural Environment Research Council. We also thank WRc (plc) for their collaboration and Novartis for their kind gift of radiolabeled products.
Literature Cited
FIGURE 5. A comparison of the bioaccumulation (mol‚g-1 L. variegatus 48 h-1/mol‚g-1 water) of different contaminants from a natural sediment and a hydrophobic particle with a negative charge (SP 650M). The correlation coefficient r 2 ) 0.98. Slope of line 1.54 ( 0.11. alone and confirms the similarities between a natural sediment and the artificial particles used in the current study. It is usual in sediment toxicity studies to normalize the bioaccumulation data to the organic carbon content of the substrate since this is regarded as the most important sorption site for nonionic hydrophobic contaminants. Unfortunately organic carbon and any adsorbed contaminants are not uniformly distributed throughout the different particle fractions in a natural sediment. Furthermore many benthic organisms are selective feeders, and ingest fractions of the substrate that have entirely different properties from the bulk phase (20). This can lead to anomalous results whereby the feces of the test organism may contain up to eight times the organic carbon content of the bulk sediment. In the case of the experiments reported in this work the natural sediment contained 1.7% organic carbon and when compared with Toyopearl Phenyl particles under identical conditions (Figure 4) gave a slope of 1.15 ( 0.13 again suggesting that these particles behaved similarly to the naturally ingested material and enabling some comparisons to be made with the normalized organic carbon data used by environmentalists. The system described in this work gives rates of bioaccumulation similar in magnitude and routes of uptake (aqueous and particulate) to those of natural sediments. It also facilitates the study of other variables such as anionic or cationic charges. Given the robustness of this system it
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Received for review November 9, 1999. Revised manuscript received March 10, 2000. Accepted March 20, 2000. ES9912630
