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Influence of particle characteristics and organic matter content on the bioavailability and bioaccumulation of pyrene by clams
Environmental Pollution 121 (2003) 115–122 www.elsevier.com/locate/envpol
Influence of particle characteristics and organic matter content on the bioavailability and bioaccumulation of pyrene by clams N.R. Verrengia Guerreroa,b,*, M.G. Taylorb, E.A. Widera, K. Simkissb a
Biomarkers Lab., Dept. of Biological Chemistry, Faculty of Exact and Natural Sciences, University of Buenos Aires, 4 piso, Pab. II, 1428, Buenos Aires, Argentina b School of Animal and Microbial Sciences, The University of Reading, PO Box 228, Reading RG6 6AJ, UK Received 25 July 2001; accepted 15 March 2002
‘‘Capsule’’: An experimental model with artificial particles and humic acids describes bioavailability of sediment-bound pyrene to clams. Abstract Hydrophobic chemicals are known to associate with sediment particles including those from both suspended particulate matter and bottom deposits. The complex and variable composition of natural particles makes it very difficult therefore, to predict the bioavailability of sediment-bound contaminants. To overcome these problems we have previously devised a test system using artificial particles, with or without humic acids, for use as an experimental model of natural sediments. In the present work we have applied this experimental technique to investigate the bioavailability and bioaccumulation of pyrene by the freshwater fingernail clam Sphaerium corneum. The uptake and accumulation of pyrene in clams exposed to the chemical in the presence of a sample of natural sediment was also investigated. According to the results obtained, particle surface properties and organic matter content are the key factors for assessing the bioavailability and bioaccumulation of pyrene by clams. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Artificial particles; Humic acids; Particle-bound contaminants; Pyrene bioavailability; Sphaerium corneum
1. Introduction Hydrophobic and persistent environmental chemicals tend to concentrate on sediment particles, from both suspended matter and bottom deposits, at levels that greatly exceed those found in the water column. For this reason, sediment-bound contaminants pose a particular risk to the whole aquatic ecosystem, especially when the particles are ingested by benthic organisms. Most classical monitoring programs include analyses of total levels of contaminants in the soluble aqueous fraction, particulate suspended matter, and bottom sediments. It is not always possible, however, from this data to assess the bioavailability of sediment-bound contaminants due to the complex and variable composition of natural particles. In addition, their interactions with the various chemical pollutants are very difficult to predict.
* Corresponding author. Tel./fax: +54-114-576-3342. E-mail address: [email protected] (N.R. Verrengia Guerrero).
During the past decade sediment quality criteria have received increasing attention from regulatory agencies (US EPA, 1994; ASTM, 1995). The use of control sediments has become an important tool to assess and predict the bioavailability of particle-bound contaminants under laboratory conditions (Ingersoll, 1995). Several types of formulated sediments have been proposed, consisting of different kinds of sand, silt and clay (Suedel and Rogers, 1994; Naylor and Rodrigues, 1995; Kemble et al., 1999). In addition to these natural materials, the use of artificial particles as analogues for aquatic sediments has also been suggested (Simkiss, 1995; Fleming et al., 1998). In particular, commercial materials commonly used as chromatographical resins have been selected because their size, backbone structure, and active groups on their surfaces are well characterised and standardised by the manufacturers (Davies et al., 1999a,b; Simkiss et al., 2000). However, this model may be considered too simple, since it may not reflect fully the complexity of natural sediments. Thus, artificial particles will clearly not show the full range of ligands that are present in natural organic
0269-7491/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(02)00197-5
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matter (Fleming et al., 1998; Kemble et al., 1999). For these reasons a variety of formulated sediments have been proposed containing organic matter such as composted cow manure, peat moss, maple and cereal leaves, a-cellulose, and humic acids (US EPA, 1994; Kemble et al., 1999). Among these materials, humic acids constitute the most important and natural source of organic matter in sediments from aquatic systems (Rand et al., 1995) and they have the added advantage that they can be obtained in a consistent form as a commercial product. For these reasons, we have devised an experimental model consisting of artificial particles and humic acids for modelling the uptake and bioaccumulation of pentachlorophenol by the freshwater fingernail clam Sphaerium corneum (Verrengia Guerrero et al., 2001). In this work we applied these same experimental protocols to investigate the uptake and accumulation of pyrene. This compound was selected as an example of polycyclic aromatic hydrocarbons, substances of great concern as environmental pollutants. Pyrene has a similar hydrophobicity as pentachlorophenol, with a pKow=5.13 (Xia and Ball, 1999) and 5.1 (Tomlin, 1997) respectively, but unlike the chlorophenol it is a neutral compound. The uptake and bioaccumulation of pyrene by clams exposed to a sample of natural sediment was also determined to compare these values with those obtained from animals exposed to artificial particles with or without humic acids. In addition, the resulting accumulation data were compared with the amount of pyrene sorbed onto the different test systems, using the corresponding particle–water partition coefficients (Kd).
2. Materials and methods 2.1. Materials The stock solution contained radiolabelled [14C] pyrene (specific activity 21.72105 Bq/mol, purity > 99%). The compound was obtained from Sigma-Aldrich Company (Poole, Dorset, United Kingdom) and used without further purification. The following resins were selected as artificial particles: Dowex 18400, an anionic exchanger (SigmaAldrich Company, Poole, Dorset, United Kingdom); Toyopearl SP-650 M, a cationic exchanger, and Toyopearl Phenyl 650 M, a resin designed for studying hydrophobic interactions (Fisher, Loughborough, United Kingdom). The backbone of Dowex particles was formed from a cross-linked copolymer of styrene and divinylbenzene. Both Toyopearl particles had the same backbone structure consisting of polymers of ethylene glycol and methyl methacrylate. Additionally, a non-commercial particle based on a tallow substituted clay was used in the study. These particles
were synthesized from dimethylditallow-ammonium chloride and montmorillonite clay (DMDTA clay) according to Lawrence (1996). Functional groups and particle size for each substance are presented in Table 1. Although some of the functional groups are not likely to occur in the natural particles, the artificial particles selected may reflect some of the possible interactions that occur in the natural environment, such as charge effects and hydrophobic interactions. The particle sizes were all in the range 40–63 mm, ensuring that they could be ingested by the animals. Commercially available humic acids (HA) were obtained from Fluka Chemie AG, Switzerland, (product number 53680). The composition reported by the manufacturer was 46.63% of carbon content, 4.30% of hydrogen content and 0.72% of nitrogen content. The selected concentration of 20 mg HA/l was prepared by dissolving the humic material in dechlorinated tap water, which was previously filtered through activated charcoal to remove any possible endogenous organic matter. Solutions were filtered through a 0.45-mm membrane filter to remove any remaining particles prior to use (Verrengia Guerrero et al., 2000). It is considered that a level of about 6 mg/l is the average concentration of humic acids dissolved in natural freshwaters (Benson and Long, 1991). Therefore, the selected value was rather higher than that, but more closely related to the organic matter content usually present in natural sediments (Ingersoll, 1995). The sample of natural sediment was collected from a non-contaminated site at the ARC Study Centre, Milton Keynes, United Kingdom, by representatives of the Water Research Centre, Medmenham, United Kingdom. After collection the sample was sieved through a 1-mm filter to remove large objects and any endogenous organisms. The sediment was homogenised in a cement mixer before being stored at 4 C. The organic matter content was 1.7% with 70% of the particles in the size range < 63 mm. Dry weight was 50% of the wet weight. Table 1 Some characteristics of the particles used Particle
Groups in the active sites
Particle size
Dowex 18400
60 mm
Toyopearl SP
40 mm
Toyopearl Phenyl
40 mm
DMDTA claya a
<53 mm
DMDTA clay: dimethylditallow-ammonium chloride substituted montmorillonite clay.
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Pyrene concentration in this natural sediment was below the detection limit of 0.10 nmol/g dry weight (A. Conrad, personal communication).
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in control clams were 420 pmol/g wet tissue. There was no mortality in any of the exposures. 2.4. Pyrene analyses
2.2. Organism selected Freshly collected clams, S. corneum, were obtained from Sciento, Manchester, United Kingdom. Once in the laboratory, they were maintained as previously described (Verrengia Guerrero et al., 2001). Bivalves were allowed to acclimatise to the laboratory conditions for at least 7 days before conducting the bioassays (FAO, 1987). For all the experiments animals of similar size (0.5 0.1 cm shell length) were selected. The ability of clams to tightly close their valves in response to an external stimulus was selected as health criteria (FAO, 1987, Verrengia Guerrero, 1995). 2.3. Bioassays All the bioassays were performed in triplicate in static conditions without renewal of the water medium at 8.5 0.5 C. Six animals were placed in 250-ml borosilicate glass beakers, containing 100 ml of 14.2 nmol pyrene/l in dechlorinated tap water, and exposed for 6; 24; 30; 48, and 54 h. During the treatments animals were starved as recommended by the Food and Agriculture Organisation (1987). After these periods, the clams were transferred to clean 250-ml glass beakers containing 100 ml of dechlorinated tap water with a drop of Liquifry No. 1 as food and allowed to depurate for 24 h to purge gut contents. In this procedure the water medium was renewed approximately 2.5 h before the end of that period to avoid reabsorption of any depurated chemical. For the rest of the experiments six animals were exposed for 48 h to 14.2 nmol/l of pyrene, under similar conditions, in the presence or absence of particles and/ or humic acids. The experiments were performed in parallel allowing direct comparisons of the results. After the treatments animals were again allowed to depurate as described above. The particulate test systems consisted of 100 mg (dry weight) of each kind of particle suspended in 100 ml of dechlorinated tap water plus 14.2 nmol pyrene/l. For the systems containing humic material, the particles were suspended in 100 ml of the humic acid solution plus 14.2 nmol pyrene/l. Each system was equilibrated by stirring for 4 h prior to the experiments, except for the sample of natural sediment for which a period of 48 h was required. For the bioassays, clams were placed in the beakers on a stainless steel mesh with continuous gentle stirring to ensure a homogeneous suspension of the particles during the 48-h exposure period. Control organisms that were not exposed to the radiolabelled pyrene were used as blanks. Levels of pyrene
Animals were anaesthetised by cooling them to 0 C for 4–6 min and then sacrificed by opening the valves. The whole body soft tissue was carefully removed, weighed, and placed in a glass vial (20 ml). The tissues were solubilised by adding 1 ml of Soluene 350 (Packard, Pangbourne, Berkshire, United Kingdom) and heated in an oven at 50 C overnight. After the digestion procedure, 10 ml of Hionic Fluor (Packard, Pangbourne, Berkshire, United Kingdom) were added to each vial. Pyrene concentration was determined from the specific activity of the stock solution after counting the samples in a Packard 2250 CA Tricarb Liquid Scintillation Analyser (LSA; Packard Instruments, Meridan, CT, USA) and correcting for chemical and colour quenching. 2.5. Particle–water partition coefficients (Kd) Batch adsorption experiments were used to determine the particle partition coefficients. Eight different concentrations of radiolabelled [14C] pyrene were used, using dechlorinated tap water from the stock solution. Experiments were performed in duplicate at a solid concentration of 1 g/l in conical flasks that were stirred continually. The artificial particles were shaken over a 4-h period prior to analyses. Previous experiments had shown that this period was well in excess of the equilibrium times for all the artificial particles but it was not applicable to the natural sediment where a 48-h period had to be used (Simkiss et al., 2000). The amount of pyrene adsorbed onto each kind of particles was determined as the difference between the initial and the equilibrium concentration. Three millilitres of samples were taken and centrifuged at 3000 rpm for 15 min. Triplicate aliquots of 0.1 ml of the supernatant were transferred to plastic vials and 5.0 ml of Insta gel (Packard, Pangbourne, Berkshire, United Kingdom) were added. Pyrene analyses were performed by liquid scintillation counting as described above for tissue analyses. 2.6. Statistical analyses The data were statistically evaluated applying analysis of variance (ANOVA) tests to study the differences among treatments (Sokal and Rohlf, 1997). The analyses of the sample populations were performed by comparing pyrene levels in clams maintained in systems with water only and the different types of particles, with or without humic acids. To investigate differences within the treatments the Scheffe´ method was used
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(Sokal and Rohlf, 1997). All the results were considered significant at =0.05.
3. Results 3.1. Bioaccumulation of pyrene versus time of exposure The levels of pyrene accumulated by S. corneum after different periods of exposure are presented in Fig. 1. The data show a linear increase in the pyrene accumulated with the time of exposure, with a highly significant regression (r2=0.9427) during the studied periods. 3.2. Pyrene bioaccumulation in test systems without humic acids Fig. 2 shows the levels of accumulation and uptake of pyrene by clams exposed in the test systems containing either water or water in the presence of the different artificial particles, or natural sediment. All the artificial particles tested induced a significant decrease in the levels of pyrene bioaccumulated when compared with the test system containing water only (P < 0.05). In addition, there were significant differences (P< 0.05) between the amount of pyrene accumulated by clams exposed to the different particle test systems. The highest accumulation was observed in clams exposed using Toyopearl Phenyl resin followed by uptake from Toyopearl SP resin. The lowest levels of accumulation were found in the test systems containing DMDTA clay and Dowex particles which gave very similar results (P > 0.05). In contrast to this the presence of natural sediment resulted in the largest accumulation of pyrene in the clams. The value was almost five times higher than in the system containing water only (P < 0.05).
Fig. 1. Values of pyrene bioaccumulation by Sphaerium corneum after different periods of exposure to 14.2 nmol/l of the chemical. Data for pyrene concentration are expressed as the mean valuestandard deviation (error bars).
3.3. Pyrene bioaccumulation in test systems with humic acids Results for the uptake and accumulation of pyrene by clams exposed to the solution of humic acids with or without artificial particles are presented in Fig. 3. The highest levels of accumulation were observed for the systems containing humic acid solution only and Toyopearl SP particles. Between them no significant differences were found (P > 0.05). In decreasing order lower levels were observed in presence of Toyopearl Phenyl followed by DMDTA clay and, finally, Dowex particles. These last values were significantly different (P < 0.05). 3.4. Relationship between bioaccumulation and Kd Table 2 shows the particle–water partition coefficients (Kd) obtained for pyrene and each kind of particle, both in the presence or absence of the humic acid solution. The amount of pyrene sorbed onto the artificial particles can be calculated from the Kd values. The results show that the adsorption follows the series Dowex> DMDTA clay> Toyopearl Phenyl> Toyopearl SP, for the test systems containing the particles without humic material, and the series DMDTA clay> Dowex> Toyopearl Phenyl> Toyopearl SP, for the test systems containing the particles with humic material. The pH values for each test system were also included, showing that no significant differences were found among them (P> 0.05).
4. Discussion The present experiments differ from those performed by Davies et al. (1999a,b) and Simkiss et al. (2000) who
Fig. 2. Values of pyrene bioaccumulation by Sphaerium corneum exposed to water only, artificial particles, or a sample of natural sediment (mean valuesSD) without humic acids. T-SP: Toyopearl SP; T-Phenyl: Toyopearl Phenyl; DMDTA clay: dimethylditallowsubstituted montmorillonite clay; WR sed: Water Research natural sediment.
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N.R. Verrengia Guerrero et al. / Environmental Pollution 121 (2003) 115–122 Table 2 Values of Kd and pH for the different test systems Without humic acids solution
Just water Toyopearl SP Toyopearl Phenyl DMDTA claya Dowex Natural sediment a
With humic acids solution
Kd
pH
Kd
pH
– 1742 5412 14876 22184 1081
8.30 0.08 8.29 0.05 8.31 0.06 8.29 0.06 8.29 0.07 8.28 0.04
– 3133 5616 25701 23437 –
8.280.07 8.270.07 8.300.06 8.280.04 8.320.07 –
DMDTA clay: dimethylditallow-ammonium chloride substituted montmorillonite clay.
Fig. 3. Values of pyrene bioaccumulation by Sphaerium corneum exposed to humic acids solution, or artificial particles plus humic acids solution (mean valuesSD). T-SP: Toyopearl SP; T-Phenyl: Toyopearl Phenyl; DMDTA clay: dimethylditallow-substituted montmorillonite clay.
used a similar approach but standardised their treatments by using conditions that held the pollutants at a constant concentration in the aqueous phase. In contrast to that approach, the test systems described here contained the same total amount of pyrene in all treatments, allowing direct comparisons of accumulation by clams. We have previously shown that more than 90% of the pyrene remained as the parent compound in S. corneum treated for 48 h (Verrengia Guerrero et al., 2002) so that the differences in bioaccumulation must be due to the influence of variations in partitioning within the test systems. Two aspects of this partitioning deserve particular attention. First, it is clear from the Kd values given in Table 2 that virtually all the pyrene becomes partitioned onto the particles used in these experiments. Thus the very significant uptake of pyrene from water only (Fig. 2) will be reduced to less than 0.1% of that value by the presence of the solid phases. This demonstrates the importance of sediments in removing pollutants from the water column. At the same time, however, benthic
organisms will be exposed to these contaminated particles and Fig. 2 clearly demonstrates that the pyrene can remain in an available form from ingested particles. A second aspect of the significance of the partitioning effect is also shown in Fig. 2. The Toyopearl SP particles have a weak partition coefficient (Kd =1742) so that a relatively small concentration of pyrene will enter the alimentary tract on the surface of these particles, i.e. the ingested dose will be small. The Toyopearl Phenyl particles bind a higher proportion of pyrene (Kd=5412) so that a larger dose is provided when these particles are ingested and the data show that the clams accumulate more of the pyrene under these circumstances. When the partitioning of the pyrene onto particles is very high, as with the DMDTA clay (Kd=14876) and Dowex resin (Kd=22184), the clams will swallow a large dose of the pollutant, but it will accumulate very little if the contaminant remains tightly bound to the particle surface being biologically unavailable. In these experiments, therefore, variations in the Kd of the particles define the window of bioavailability for pyrene when it is adsorbed onto the surface of ingested sediments. As pyrene is a neutral and hydrophobic compound with a pKow=5.13 (Xia and Ball, 1999), ionic interactions with the active groups of both Toyopearl SP and Dowex, cationic and anionic exchanger resins respectively, are not likely to be significant. Instead, Toyopearl Phenyl resin has surface bound phenyl groups that may show weak hydrophobic interactions with pyrene so that the contaminant would be partly able to desorb from the particles in the digestive tract of the animals, promoting the highest bioaccumulation. Toyopearl SP has propyl sulphonate as active groups and the same backbone structure as Toyopearl Phenyl. It could be hypothesised that a certain proportion of pyrene could also interact with the backbone of the resin. DMDTA clay is a more complex particle with three possible sites for interactions with substrates. In the outer-lying surface there are oxygen and hydroxide groups. In the interlamellar area one half is occupied by the highly hydrophobic dimethylditallow-ammonium
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groups, which are introduced during the preparation of the particles. The other half contains sodium and calcium ions. It has been demonstrated that expansive clays like montmorillonite allow the interlamellar penetration and entrapment of organic substances (Witkowski et al., 1987). Therefore hydrophobic interactions between pyrene and the interlayer groups of dimethylditallow-ammonium could have been established within the constraints of steric interactions. The sorption of the compound onto these particles was shown to be strong and negligible amounts could be desorbed since clams were not able to accumulate significant pyrene from these particles. The high value of Kd for pyrene and Dowex particles showed that most of the compound was sorbed onto this resin, perhaps by interactions with the backbone, but the basic mechanism still remains unclear. It is obvious from the data shown in Fig. 2 that the natural sediment does not fit easily into the partitioning interpretation that has just been invoked. There are two possible explanations for this. The first is that the partition coefficient Kd is really only meaningful for a single defined surface. Natural sediments are typically heterogeneous in inorganic and organic composition. Inorganic components are constituted mainly of rock fragments and clay minerals usually coated with oxides and hydroxides of aluminum, iron and manganese. These metal compounds act as weak basics conferring some positive charges to the sediment surfaces (Witkowski et al., 1987; Bradford and Horowitz, 1988; Ingersoll, 1995). Most of the organic material can be described as humic acids. Humic substances are natural compounds that can derive from vegetable and animal tissues (Rand et al., 1995). Their chemical structures are still not well defined but on their surfaces they have a great number of carboxylic acids, phenol and alcoholic hydroxyls, and carbonyls as principal functional groups (Schulten et al., 1991). These substances have negative charges that usually dominate the surface charge of the sediment particles (Witkowski et al., 1987; Bradford and Horowitz, 1988). It is also considered that these negative charges produce the deactivation of the metal oxide and metal hydroxide sites (Bradford and Horowitz, 1988). Probably the most important mechanism of organic contaminants association with sediments is hydrophobic bonding or partitioning of non-polar hydrocarbon moieties with hydrocarbon portions of the natural organic matter (Bradford and Horowitz, 1988). This fact may be especially valid for hydrophobic contaminants like pyrene. Therefore, in a natural sediment there will be a large range of components each with a different partition coefficient so that the averaged value that is measured experimentally is relatively meaningless. This is particularly true if the test organism is a selective feeder that
may ingest components of the sediment with quite specific Kd values that differ considerably from those of the ‘bulk phase’. An alternative explanation for the large bioavailability of pyrene from the natural sediment relates to this same phenomenon since the ingested particles may be selected for their digestability (Davies et al., 1997; Ward et al., 1998). Clearly if the partitioning phase had a degradable surface this could also facilitate the release and assimilation of the pyrene. As the highest value of accumulation was obtained from the natural sediment this may indicate that the organic materials in the sediment became degraded within the alimentary tract of the clam leading to the release and absorption of the pyrene within the animal. The resin beads, being resistant to breakdown, would not show this effect. The second set of experiments shown in Fig. 3 were intended to study the impact of adding a complex naturally occurring organic component (humic acid) to the test systems. Several features are apparent from these results. It is immediately clear that direct uptake from the water increases dramatically in the presence of humic acid. When animals were exposed simultaneously to each kind of particle in the presence of the humic acid solution, the bioaccumulation of pyrene was increased. It reached the highest levels in clams exposed to Toyopearl SP, followed in decreasing order by Toyopearl Phenyl, DMDTA clay, and finally Dowex resin (Fig. 3). According to the Kd values (Table 2), the amount of pyrene adsorbed onto Toyopearl SP and DMDTA clay particles in the presence of humic acids increased to almost twice the values found in the test systems without humic material. This was in contrast to the results with Toyopearl Phenyl and Dowex resins where the values were not significantly affected by the presence of humic acids. The bioaccumulation of pyrene by clams was, however, much more markedly increased, reaching values approximately 21 times higher in the case of Toyopearl SP; 15 times higher for DMDTA clay, and 5.6 times higher for Toyopearl Phenyl. Only in the case of the Dowex particles was bioaccumulation not modified. These results show that for the test systems containing artificial particles plus humic acids, bioaccumulation by clams did not depend exclusively on the amount of chemical adsorbed onto the particles. In these systems the organic content of the system showed a stronger influence on the bioavailability of the contaminant. Clearly the influence of the organic matter was somehow modulated by the particle surface properties, but it also seems likely that as with the natural sediment there was an enhanced uptake of pyrene from the humic acid coated particles once they entered the alimentary tract. Humic acid solutions are known to influence the uptake and accumulation of both metal and organic substances (Bradford and Horowitz, 1988; Verrengia Guerrero et al., 2000). Most previous studies have
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shown that different types of dissolved organic matter (including humic acids from both natural and commercial sources) induce a decrease in the bioaccumulation of organic chemicals by aquatic organisms (Benson and Long, 1991; Haitzer et al., 1999). Nevertheless, general rules are very difficult to establish. The data compiled by Haitzer et al. (1998) showed that in about one fourth of the studies it produced enhanced accumulation. The interactions between organic substances and dissolved organic matter can depend on several factors such as concentration and type of dissolved organic matter, type of chemical, test organism, exposure time, pre-test contact time, pH, temperature, etc. (Benson and Long, 1991; Haitzer et al., 1998). Although several hypotheses have been proposed to explain the interactions between natural organic matter and polycyclic aromatic hydrocarbons, the true mechanism still remains unclear (Kenworthy and Hayes, 1997). It has been suggested that these interactions would occur by hydrophobic bonding due to van der Waals forces (Kenworthy and Hayes, 1997). On the other hand, it is considered that humic substances are found naturally as micelle-like structures formed either by intra or intermolecular aggregates of humic substances (Engebretson et al., 1996; Piccolo et al., 1996). Our results showed that bioaccumulation of pyrene from the humic acid solution and also from the Toyopearl SP resin plus humic acids, was quite similar to the levels found in clams exposed to the sample of natural sediment. As the humic acid concentration used was 20 mg/l, the total organic carbon content in the test systems (containing 100 mg of artificial particles in 100 ml of test solution) was 2%, quite similar to the total organic content found in the sample of natural sediment (1.7%). Therefore, Toyopearl SP resin plus humic acids appeared to be a suitable experimental analogue for determining the bioavailability and bioaccumulation of pyrene by S. corneum clams.
5. Conclusions In agreement with previous studies using filter feeding organisms such as S. corneum, the uptake of contaminants was related to particle surface properties (Davies et al., 1997; Ward et al., 1998). Our results demonstrated that the uptake and bioaccumulation of uncharged organic contaminants could be rationalised taking into account the functional groups, the backbone structure, and also the organic matter content of the artificial particles. In general, natural sediments are normally characterised by the presence of negative charges and a given proportion of organic matter, constituted principally by humic acids. Bioaccumulation of pyrene by S. corneum from the test system containing Toyopearl SP
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(with negative charged functional groups) plus humic acids was quite similar to the value obtained from the sample of natural sediment. Therefore this artificial system appears to be a suitable experimental analogue for natural particles even for uncharged and hydrophobic substances like pyrene.
Acknowledgements This work was supported by the Environmental Diagnostics Program of the Natural Environmental Research Council (UK). We are very grateful to Monique A.M. Lawrence for her assistance with the Kd determinations. Dr. N.R.V.G. was a postdoctoral fellow from the FOMEC Programme of the University of Buenos Aires. Dr E.A.W. is member of the Scientific Research Career of Argentine National Council of Scientific and Technical Research (CONICET).
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Suedel, B.C., Rogers Jr., J.H., 1994. Development of formulated reference sediments for use in freshwater and estuarine sediment tests. Environmental Toxicology and Chemistry 13, 1163–1175. Tomlin, C.D.S., (Ed.) 1997. Pesticide Manual: A World Compendium. British Crop Protection Council,Farnham, Surrey, UK. US EPA, United States Environmental Protection Agency, 1994. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates, 600/ R-94/024. Verrengia Guerrero, N.R., 1995. Contaminantes Meta´licos en el Rio de La Plata: Monitoreo del Sistema Acua´tico y Estudio de Algunos Efectos To´xicos en Moluscos Bivalvos por Medio de Bioensayos. PhD thesis. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina. Verrengia Guerrero, N.R., Nahabedian, D.E., Wider, E.A., 2000. Analyisis of some factors that may modify the bioavailability of cadmium and lead by Biomphalaria glabrata. Environmental Toxicology and Chemistry 19, 2779–2787. Verrengia Guerrero, N.R., Taylor, M.G., Wider, E.A., Simkiss, K., 2001. Modelling pentachlorophenol (PCP) bioavailability and bioaccumulation by the freshwater fingernail clam Sphaerium corneum using artificial particles and humic acids. Environmental Toxicology and Chemistry 20, 2910–2915. Verrengia Guerrero, N.R., Taylor, M.G., Davies, N.A., Edwards, P.A., Lawrence, M.A.M., Simkiss, K., Wider, E.A., 2002. Evidence of differences in the biotransformation of organic contaminants in three species of freshwater invertebrates. Environmental Pollution 117, 523–530. Ward, J.E., Levinton, J.S., Shumway, S.E., Cucci, T., 1998. Site of particle selection in a bivalve mollusc. Nature 390, 131–132. Witkowski, P.J., Smith, J.A., Fusillo, T.V., Chiou, C.T. (Eds.), 1987. A Review of Surface–Water Sediment Fraction and their Interactions with Persistent Manmade Organic Compounds. Circular 993, United States Geological Survey, Denver, USA. Xia, G.S., Ball, W.P., 1999. Adsorption-partitioning uptake of nine low-polarity organic chemicals on a natural sorbent. Environmental Science and Technology 33, 262–269.
Influence of particle characteristics and organic matter content on the bioavailability and bioaccumulation of pyrene by clams N.R. Verrengia Guerreroa,b,*, M.G. Taylorb, E.A. Widera, K. Simkissb a
Biomarkers Lab., Dept. of Biological Chemistry, Faculty of Exact and Natural Sciences, University of Buenos Aires, 4 piso, Pab. II, 1428, Buenos Aires, Argentina b School of Animal and Microbial Sciences, The University of Reading, PO Box 228, Reading RG6 6AJ, UK Received 25 July 2001; accepted 15 March 2002
‘‘Capsule’’: An experimental model with artificial particles and humic acids describes bioavailability of sediment-bound pyrene to clams. Abstract Hydrophobic chemicals are known to associate with sediment particles including those from both suspended particulate matter and bottom deposits. The complex and variable composition of natural particles makes it very difficult therefore, to predict the bioavailability of sediment-bound contaminants. To overcome these problems we have previously devised a test system using artificial particles, with or without humic acids, for use as an experimental model of natural sediments. In the present work we have applied this experimental technique to investigate the bioavailability and bioaccumulation of pyrene by the freshwater fingernail clam Sphaerium corneum. The uptake and accumulation of pyrene in clams exposed to the chemical in the presence of a sample of natural sediment was also investigated. According to the results obtained, particle surface properties and organic matter content are the key factors for assessing the bioavailability and bioaccumulation of pyrene by clams. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Artificial particles; Humic acids; Particle-bound contaminants; Pyrene bioavailability; Sphaerium corneum
1. Introduction Hydrophobic and persistent environmental chemicals tend to concentrate on sediment particles, from both suspended matter and bottom deposits, at levels that greatly exceed those found in the water column. For this reason, sediment-bound contaminants pose a particular risk to the whole aquatic ecosystem, especially when the particles are ingested by benthic organisms. Most classical monitoring programs include analyses of total levels of contaminants in the soluble aqueous fraction, particulate suspended matter, and bottom sediments. It is not always possible, however, from this data to assess the bioavailability of sediment-bound contaminants due to the complex and variable composition of natural particles. In addition, their interactions with the various chemical pollutants are very difficult to predict.
* Corresponding author. Tel./fax: +54-114-576-3342. E-mail address: [email protected] (N.R. Verrengia Guerrero).
During the past decade sediment quality criteria have received increasing attention from regulatory agencies (US EPA, 1994; ASTM, 1995). The use of control sediments has become an important tool to assess and predict the bioavailability of particle-bound contaminants under laboratory conditions (Ingersoll, 1995). Several types of formulated sediments have been proposed, consisting of different kinds of sand, silt and clay (Suedel and Rogers, 1994; Naylor and Rodrigues, 1995; Kemble et al., 1999). In addition to these natural materials, the use of artificial particles as analogues for aquatic sediments has also been suggested (Simkiss, 1995; Fleming et al., 1998). In particular, commercial materials commonly used as chromatographical resins have been selected because their size, backbone structure, and active groups on their surfaces are well characterised and standardised by the manufacturers (Davies et al., 1999a,b; Simkiss et al., 2000). However, this model may be considered too simple, since it may not reflect fully the complexity of natural sediments. Thus, artificial particles will clearly not show the full range of ligands that are present in natural organic
0269-7491/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(02)00197-5
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matter (Fleming et al., 1998; Kemble et al., 1999). For these reasons a variety of formulated sediments have been proposed containing organic matter such as composted cow manure, peat moss, maple and cereal leaves, a-cellulose, and humic acids (US EPA, 1994; Kemble et al., 1999). Among these materials, humic acids constitute the most important and natural source of organic matter in sediments from aquatic systems (Rand et al., 1995) and they have the added advantage that they can be obtained in a consistent form as a commercial product. For these reasons, we have devised an experimental model consisting of artificial particles and humic acids for modelling the uptake and bioaccumulation of pentachlorophenol by the freshwater fingernail clam Sphaerium corneum (Verrengia Guerrero et al., 2001). In this work we applied these same experimental protocols to investigate the uptake and accumulation of pyrene. This compound was selected as an example of polycyclic aromatic hydrocarbons, substances of great concern as environmental pollutants. Pyrene has a similar hydrophobicity as pentachlorophenol, with a pKow=5.13 (Xia and Ball, 1999) and 5.1 (Tomlin, 1997) respectively, but unlike the chlorophenol it is a neutral compound. The uptake and bioaccumulation of pyrene by clams exposed to a sample of natural sediment was also determined to compare these values with those obtained from animals exposed to artificial particles with or without humic acids. In addition, the resulting accumulation data were compared with the amount of pyrene sorbed onto the different test systems, using the corresponding particle–water partition coefficients (Kd).
2. Materials and methods 2.1. Materials The stock solution contained radiolabelled [14C] pyrene (specific activity 21.72105 Bq/mol, purity > 99%). The compound was obtained from Sigma-Aldrich Company (Poole, Dorset, United Kingdom) and used without further purification. The following resins were selected as artificial particles: Dowex 18400, an anionic exchanger (SigmaAldrich Company, Poole, Dorset, United Kingdom); Toyopearl SP-650 M, a cationic exchanger, and Toyopearl Phenyl 650 M, a resin designed for studying hydrophobic interactions (Fisher, Loughborough, United Kingdom). The backbone of Dowex particles was formed from a cross-linked copolymer of styrene and divinylbenzene. Both Toyopearl particles had the same backbone structure consisting of polymers of ethylene glycol and methyl methacrylate. Additionally, a non-commercial particle based on a tallow substituted clay was used in the study. These particles
were synthesized from dimethylditallow-ammonium chloride and montmorillonite clay (DMDTA clay) according to Lawrence (1996). Functional groups and particle size for each substance are presented in Table 1. Although some of the functional groups are not likely to occur in the natural particles, the artificial particles selected may reflect some of the possible interactions that occur in the natural environment, such as charge effects and hydrophobic interactions. The particle sizes were all in the range 40–63 mm, ensuring that they could be ingested by the animals. Commercially available humic acids (HA) were obtained from Fluka Chemie AG, Switzerland, (product number 53680). The composition reported by the manufacturer was 46.63% of carbon content, 4.30% of hydrogen content and 0.72% of nitrogen content. The selected concentration of 20 mg HA/l was prepared by dissolving the humic material in dechlorinated tap water, which was previously filtered through activated charcoal to remove any possible endogenous organic matter. Solutions were filtered through a 0.45-mm membrane filter to remove any remaining particles prior to use (Verrengia Guerrero et al., 2000). It is considered that a level of about 6 mg/l is the average concentration of humic acids dissolved in natural freshwaters (Benson and Long, 1991). Therefore, the selected value was rather higher than that, but more closely related to the organic matter content usually present in natural sediments (Ingersoll, 1995). The sample of natural sediment was collected from a non-contaminated site at the ARC Study Centre, Milton Keynes, United Kingdom, by representatives of the Water Research Centre, Medmenham, United Kingdom. After collection the sample was sieved through a 1-mm filter to remove large objects and any endogenous organisms. The sediment was homogenised in a cement mixer before being stored at 4 C. The organic matter content was 1.7% with 70% of the particles in the size range < 63 mm. Dry weight was 50% of the wet weight. Table 1 Some characteristics of the particles used Particle
Groups in the active sites
Particle size
Dowex 18400
60 mm
Toyopearl SP
40 mm
Toyopearl Phenyl
40 mm
DMDTA claya a
<53 mm
DMDTA clay: dimethylditallow-ammonium chloride substituted montmorillonite clay.
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Pyrene concentration in this natural sediment was below the detection limit of 0.10 nmol/g dry weight (A. Conrad, personal communication).
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in control clams were 420 pmol/g wet tissue. There was no mortality in any of the exposures. 2.4. Pyrene analyses
2.2. Organism selected Freshly collected clams, S. corneum, were obtained from Sciento, Manchester, United Kingdom. Once in the laboratory, they were maintained as previously described (Verrengia Guerrero et al., 2001). Bivalves were allowed to acclimatise to the laboratory conditions for at least 7 days before conducting the bioassays (FAO, 1987). For all the experiments animals of similar size (0.5 0.1 cm shell length) were selected. The ability of clams to tightly close their valves in response to an external stimulus was selected as health criteria (FAO, 1987, Verrengia Guerrero, 1995). 2.3. Bioassays All the bioassays were performed in triplicate in static conditions without renewal of the water medium at 8.5 0.5 C. Six animals were placed in 250-ml borosilicate glass beakers, containing 100 ml of 14.2 nmol pyrene/l in dechlorinated tap water, and exposed for 6; 24; 30; 48, and 54 h. During the treatments animals were starved as recommended by the Food and Agriculture Organisation (1987). After these periods, the clams were transferred to clean 250-ml glass beakers containing 100 ml of dechlorinated tap water with a drop of Liquifry No. 1 as food and allowed to depurate for 24 h to purge gut contents. In this procedure the water medium was renewed approximately 2.5 h before the end of that period to avoid reabsorption of any depurated chemical. For the rest of the experiments six animals were exposed for 48 h to 14.2 nmol/l of pyrene, under similar conditions, in the presence or absence of particles and/ or humic acids. The experiments were performed in parallel allowing direct comparisons of the results. After the treatments animals were again allowed to depurate as described above. The particulate test systems consisted of 100 mg (dry weight) of each kind of particle suspended in 100 ml of dechlorinated tap water plus 14.2 nmol pyrene/l. For the systems containing humic material, the particles were suspended in 100 ml of the humic acid solution plus 14.2 nmol pyrene/l. Each system was equilibrated by stirring for 4 h prior to the experiments, except for the sample of natural sediment for which a period of 48 h was required. For the bioassays, clams were placed in the beakers on a stainless steel mesh with continuous gentle stirring to ensure a homogeneous suspension of the particles during the 48-h exposure period. Control organisms that were not exposed to the radiolabelled pyrene were used as blanks. Levels of pyrene
Animals were anaesthetised by cooling them to 0 C for 4–6 min and then sacrificed by opening the valves. The whole body soft tissue was carefully removed, weighed, and placed in a glass vial (20 ml). The tissues were solubilised by adding 1 ml of Soluene 350 (Packard, Pangbourne, Berkshire, United Kingdom) and heated in an oven at 50 C overnight. After the digestion procedure, 10 ml of Hionic Fluor (Packard, Pangbourne, Berkshire, United Kingdom) were added to each vial. Pyrene concentration was determined from the specific activity of the stock solution after counting the samples in a Packard 2250 CA Tricarb Liquid Scintillation Analyser (LSA; Packard Instruments, Meridan, CT, USA) and correcting for chemical and colour quenching. 2.5. Particle–water partition coefficients (Kd) Batch adsorption experiments were used to determine the particle partition coefficients. Eight different concentrations of radiolabelled [14C] pyrene were used, using dechlorinated tap water from the stock solution. Experiments were performed in duplicate at a solid concentration of 1 g/l in conical flasks that were stirred continually. The artificial particles were shaken over a 4-h period prior to analyses. Previous experiments had shown that this period was well in excess of the equilibrium times for all the artificial particles but it was not applicable to the natural sediment where a 48-h period had to be used (Simkiss et al., 2000). The amount of pyrene adsorbed onto each kind of particles was determined as the difference between the initial and the equilibrium concentration. Three millilitres of samples were taken and centrifuged at 3000 rpm for 15 min. Triplicate aliquots of 0.1 ml of the supernatant were transferred to plastic vials and 5.0 ml of Insta gel (Packard, Pangbourne, Berkshire, United Kingdom) were added. Pyrene analyses were performed by liquid scintillation counting as described above for tissue analyses. 2.6. Statistical analyses The data were statistically evaluated applying analysis of variance (ANOVA) tests to study the differences among treatments (Sokal and Rohlf, 1997). The analyses of the sample populations were performed by comparing pyrene levels in clams maintained in systems with water only and the different types of particles, with or without humic acids. To investigate differences within the treatments the Scheffe´ method was used
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(Sokal and Rohlf, 1997). All the results were considered significant at =0.05.
3. Results 3.1. Bioaccumulation of pyrene versus time of exposure The levels of pyrene accumulated by S. corneum after different periods of exposure are presented in Fig. 1. The data show a linear increase in the pyrene accumulated with the time of exposure, with a highly significant regression (r2=0.9427) during the studied periods. 3.2. Pyrene bioaccumulation in test systems without humic acids Fig. 2 shows the levels of accumulation and uptake of pyrene by clams exposed in the test systems containing either water or water in the presence of the different artificial particles, or natural sediment. All the artificial particles tested induced a significant decrease in the levels of pyrene bioaccumulated when compared with the test system containing water only (P < 0.05). In addition, there were significant differences (P< 0.05) between the amount of pyrene accumulated by clams exposed to the different particle test systems. The highest accumulation was observed in clams exposed using Toyopearl Phenyl resin followed by uptake from Toyopearl SP resin. The lowest levels of accumulation were found in the test systems containing DMDTA clay and Dowex particles which gave very similar results (P > 0.05). In contrast to this the presence of natural sediment resulted in the largest accumulation of pyrene in the clams. The value was almost five times higher than in the system containing water only (P < 0.05).
Fig. 1. Values of pyrene bioaccumulation by Sphaerium corneum after different periods of exposure to 14.2 nmol/l of the chemical. Data for pyrene concentration are expressed as the mean valuestandard deviation (error bars).
3.3. Pyrene bioaccumulation in test systems with humic acids Results for the uptake and accumulation of pyrene by clams exposed to the solution of humic acids with or without artificial particles are presented in Fig. 3. The highest levels of accumulation were observed for the systems containing humic acid solution only and Toyopearl SP particles. Between them no significant differences were found (P > 0.05). In decreasing order lower levels were observed in presence of Toyopearl Phenyl followed by DMDTA clay and, finally, Dowex particles. These last values were significantly different (P < 0.05). 3.4. Relationship between bioaccumulation and Kd Table 2 shows the particle–water partition coefficients (Kd) obtained for pyrene and each kind of particle, both in the presence or absence of the humic acid solution. The amount of pyrene sorbed onto the artificial particles can be calculated from the Kd values. The results show that the adsorption follows the series Dowex> DMDTA clay> Toyopearl Phenyl> Toyopearl SP, for the test systems containing the particles without humic material, and the series DMDTA clay> Dowex> Toyopearl Phenyl> Toyopearl SP, for the test systems containing the particles with humic material. The pH values for each test system were also included, showing that no significant differences were found among them (P> 0.05).
4. Discussion The present experiments differ from those performed by Davies et al. (1999a,b) and Simkiss et al. (2000) who
Fig. 2. Values of pyrene bioaccumulation by Sphaerium corneum exposed to water only, artificial particles, or a sample of natural sediment (mean valuesSD) without humic acids. T-SP: Toyopearl SP; T-Phenyl: Toyopearl Phenyl; DMDTA clay: dimethylditallowsubstituted montmorillonite clay; WR sed: Water Research natural sediment.
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Just water Toyopearl SP Toyopearl Phenyl DMDTA claya Dowex Natural sediment a
With humic acids solution
Kd
pH
Kd
pH
– 1742 5412 14876 22184 1081
8.30 0.08 8.29 0.05 8.31 0.06 8.29 0.06 8.29 0.07 8.28 0.04
– 3133 5616 25701 23437 –
8.280.07 8.270.07 8.300.06 8.280.04 8.320.07 –
DMDTA clay: dimethylditallow-ammonium chloride substituted montmorillonite clay.
Fig. 3. Values of pyrene bioaccumulation by Sphaerium corneum exposed to humic acids solution, or artificial particles plus humic acids solution (mean valuesSD). T-SP: Toyopearl SP; T-Phenyl: Toyopearl Phenyl; DMDTA clay: dimethylditallow-substituted montmorillonite clay.
used a similar approach but standardised their treatments by using conditions that held the pollutants at a constant concentration in the aqueous phase. In contrast to that approach, the test systems described here contained the same total amount of pyrene in all treatments, allowing direct comparisons of accumulation by clams. We have previously shown that more than 90% of the pyrene remained as the parent compound in S. corneum treated for 48 h (Verrengia Guerrero et al., 2002) so that the differences in bioaccumulation must be due to the influence of variations in partitioning within the test systems. Two aspects of this partitioning deserve particular attention. First, it is clear from the Kd values given in Table 2 that virtually all the pyrene becomes partitioned onto the particles used in these experiments. Thus the very significant uptake of pyrene from water only (Fig. 2) will be reduced to less than 0.1% of that value by the presence of the solid phases. This demonstrates the importance of sediments in removing pollutants from the water column. At the same time, however, benthic
organisms will be exposed to these contaminated particles and Fig. 2 clearly demonstrates that the pyrene can remain in an available form from ingested particles. A second aspect of the significance of the partitioning effect is also shown in Fig. 2. The Toyopearl SP particles have a weak partition coefficient (Kd =1742) so that a relatively small concentration of pyrene will enter the alimentary tract on the surface of these particles, i.e. the ingested dose will be small. The Toyopearl Phenyl particles bind a higher proportion of pyrene (Kd=5412) so that a larger dose is provided when these particles are ingested and the data show that the clams accumulate more of the pyrene under these circumstances. When the partitioning of the pyrene onto particles is very high, as with the DMDTA clay (Kd=14876) and Dowex resin (Kd=22184), the clams will swallow a large dose of the pollutant, but it will accumulate very little if the contaminant remains tightly bound to the particle surface being biologically unavailable. In these experiments, therefore, variations in the Kd of the particles define the window of bioavailability for pyrene when it is adsorbed onto the surface of ingested sediments. As pyrene is a neutral and hydrophobic compound with a pKow=5.13 (Xia and Ball, 1999), ionic interactions with the active groups of both Toyopearl SP and Dowex, cationic and anionic exchanger resins respectively, are not likely to be significant. Instead, Toyopearl Phenyl resin has surface bound phenyl groups that may show weak hydrophobic interactions with pyrene so that the contaminant would be partly able to desorb from the particles in the digestive tract of the animals, promoting the highest bioaccumulation. Toyopearl SP has propyl sulphonate as active groups and the same backbone structure as Toyopearl Phenyl. It could be hypothesised that a certain proportion of pyrene could also interact with the backbone of the resin. DMDTA clay is a more complex particle with three possible sites for interactions with substrates. In the outer-lying surface there are oxygen and hydroxide groups. In the interlamellar area one half is occupied by the highly hydrophobic dimethylditallow-ammonium
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groups, which are introduced during the preparation of the particles. The other half contains sodium and calcium ions. It has been demonstrated that expansive clays like montmorillonite allow the interlamellar penetration and entrapment of organic substances (Witkowski et al., 1987). Therefore hydrophobic interactions between pyrene and the interlayer groups of dimethylditallow-ammonium could have been established within the constraints of steric interactions. The sorption of the compound onto these particles was shown to be strong and negligible amounts could be desorbed since clams were not able to accumulate significant pyrene from these particles. The high value of Kd for pyrene and Dowex particles showed that most of the compound was sorbed onto this resin, perhaps by interactions with the backbone, but the basic mechanism still remains unclear. It is obvious from the data shown in Fig. 2 that the natural sediment does not fit easily into the partitioning interpretation that has just been invoked. There are two possible explanations for this. The first is that the partition coefficient Kd is really only meaningful for a single defined surface. Natural sediments are typically heterogeneous in inorganic and organic composition. Inorganic components are constituted mainly of rock fragments and clay minerals usually coated with oxides and hydroxides of aluminum, iron and manganese. These metal compounds act as weak basics conferring some positive charges to the sediment surfaces (Witkowski et al., 1987; Bradford and Horowitz, 1988; Ingersoll, 1995). Most of the organic material can be described as humic acids. Humic substances are natural compounds that can derive from vegetable and animal tissues (Rand et al., 1995). Their chemical structures are still not well defined but on their surfaces they have a great number of carboxylic acids, phenol and alcoholic hydroxyls, and carbonyls as principal functional groups (Schulten et al., 1991). These substances have negative charges that usually dominate the surface charge of the sediment particles (Witkowski et al., 1987; Bradford and Horowitz, 1988). It is also considered that these negative charges produce the deactivation of the metal oxide and metal hydroxide sites (Bradford and Horowitz, 1988). Probably the most important mechanism of organic contaminants association with sediments is hydrophobic bonding or partitioning of non-polar hydrocarbon moieties with hydrocarbon portions of the natural organic matter (Bradford and Horowitz, 1988). This fact may be especially valid for hydrophobic contaminants like pyrene. Therefore, in a natural sediment there will be a large range of components each with a different partition coefficient so that the averaged value that is measured experimentally is relatively meaningless. This is particularly true if the test organism is a selective feeder that
may ingest components of the sediment with quite specific Kd values that differ considerably from those of the ‘bulk phase’. An alternative explanation for the large bioavailability of pyrene from the natural sediment relates to this same phenomenon since the ingested particles may be selected for their digestability (Davies et al., 1997; Ward et al., 1998). Clearly if the partitioning phase had a degradable surface this could also facilitate the release and assimilation of the pyrene. As the highest value of accumulation was obtained from the natural sediment this may indicate that the organic materials in the sediment became degraded within the alimentary tract of the clam leading to the release and absorption of the pyrene within the animal. The resin beads, being resistant to breakdown, would not show this effect. The second set of experiments shown in Fig. 3 were intended to study the impact of adding a complex naturally occurring organic component (humic acid) to the test systems. Several features are apparent from these results. It is immediately clear that direct uptake from the water increases dramatically in the presence of humic acid. When animals were exposed simultaneously to each kind of particle in the presence of the humic acid solution, the bioaccumulation of pyrene was increased. It reached the highest levels in clams exposed to Toyopearl SP, followed in decreasing order by Toyopearl Phenyl, DMDTA clay, and finally Dowex resin (Fig. 3). According to the Kd values (Table 2), the amount of pyrene adsorbed onto Toyopearl SP and DMDTA clay particles in the presence of humic acids increased to almost twice the values found in the test systems without humic material. This was in contrast to the results with Toyopearl Phenyl and Dowex resins where the values were not significantly affected by the presence of humic acids. The bioaccumulation of pyrene by clams was, however, much more markedly increased, reaching values approximately 21 times higher in the case of Toyopearl SP; 15 times higher for DMDTA clay, and 5.6 times higher for Toyopearl Phenyl. Only in the case of the Dowex particles was bioaccumulation not modified. These results show that for the test systems containing artificial particles plus humic acids, bioaccumulation by clams did not depend exclusively on the amount of chemical adsorbed onto the particles. In these systems the organic content of the system showed a stronger influence on the bioavailability of the contaminant. Clearly the influence of the organic matter was somehow modulated by the particle surface properties, but it also seems likely that as with the natural sediment there was an enhanced uptake of pyrene from the humic acid coated particles once they entered the alimentary tract. Humic acid solutions are known to influence the uptake and accumulation of both metal and organic substances (Bradford and Horowitz, 1988; Verrengia Guerrero et al., 2000). Most previous studies have
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shown that different types of dissolved organic matter (including humic acids from both natural and commercial sources) induce a decrease in the bioaccumulation of organic chemicals by aquatic organisms (Benson and Long, 1991; Haitzer et al., 1999). Nevertheless, general rules are very difficult to establish. The data compiled by Haitzer et al. (1998) showed that in about one fourth of the studies it produced enhanced accumulation. The interactions between organic substances and dissolved organic matter can depend on several factors such as concentration and type of dissolved organic matter, type of chemical, test organism, exposure time, pre-test contact time, pH, temperature, etc. (Benson and Long, 1991; Haitzer et al., 1998). Although several hypotheses have been proposed to explain the interactions between natural organic matter and polycyclic aromatic hydrocarbons, the true mechanism still remains unclear (Kenworthy and Hayes, 1997). It has been suggested that these interactions would occur by hydrophobic bonding due to van der Waals forces (Kenworthy and Hayes, 1997). On the other hand, it is considered that humic substances are found naturally as micelle-like structures formed either by intra or intermolecular aggregates of humic substances (Engebretson et al., 1996; Piccolo et al., 1996). Our results showed that bioaccumulation of pyrene from the humic acid solution and also from the Toyopearl SP resin plus humic acids, was quite similar to the levels found in clams exposed to the sample of natural sediment. As the humic acid concentration used was 20 mg/l, the total organic carbon content in the test systems (containing 100 mg of artificial particles in 100 ml of test solution) was 2%, quite similar to the total organic content found in the sample of natural sediment (1.7%). Therefore, Toyopearl SP resin plus humic acids appeared to be a suitable experimental analogue for determining the bioavailability and bioaccumulation of pyrene by S. corneum clams.
5. Conclusions In agreement with previous studies using filter feeding organisms such as S. corneum, the uptake of contaminants was related to particle surface properties (Davies et al., 1997; Ward et al., 1998). Our results demonstrated that the uptake and bioaccumulation of uncharged organic contaminants could be rationalised taking into account the functional groups, the backbone structure, and also the organic matter content of the artificial particles. In general, natural sediments are normally characterised by the presence of negative charges and a given proportion of organic matter, constituted principally by humic acids. Bioaccumulation of pyrene by S. corneum from the test system containing Toyopearl SP
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(with negative charged functional groups) plus humic acids was quite similar to the value obtained from the sample of natural sediment. Therefore this artificial system appears to be a suitable experimental analogue for natural particles even for uncharged and hydrophobic substances like pyrene.
Acknowledgements This work was supported by the Environmental Diagnostics Program of the Natural Environmental Research Council (UK). We are very grateful to Monique A.M. Lawrence for her assistance with the Kd determinations. Dr. N.R.V.G. was a postdoctoral fellow from the FOMEC Programme of the University of Buenos Aires. Dr E.A.W. is member of the Scientific Research Career of Argentine National Council of Scientific and Technical Research (CONICET).
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