Grantee Research Project Results
Final Report: Digestive Solubilization of Sediment-Sorbed Contaminants: A Comparison of In Vitro and In Vivo Processes
EPA Grant Number: R825353Title: Digestive Solubilization of Sediment-Sorbed Contaminants: A Comparison of In Vitro and In Vivo Processes
Investigators: Weston, Donald P. , Penry, Deborah L. , Mayer, Larry M.
Institution: University of California - Berkeley , University of Maine
EPA Project Officer: Hahn, Intaek
Project Period: December 1, 1996 through November 30, 1999
Project Amount: $374,945
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
While it is clear that sediment-sorbed contaminants may be bioavailable, it is equally clear that not all of the particle-associated contaminant is bioavailable. In most cases, the majority of the ingested contaminant is not absorbed during gut passage. This may be one reason why bulk chemical analyses often fail to accurately predict the extent of bioaccumulation. Traditional chemical extraction techniques for sediment are intended to extract as much of the contaminant as possible. They rely on a strong organic solvent (for analysis of organics) or a strong acid (for analysis of metals). Partial extractions, such as by use of a weak acid, have been explored but none have been broadly accepted. Traditional extractions are not intended to mimic the processes occurring in animal guts. The acids or organic solvents used in chemical extractions bear little resemblance to the chemistry occurring in gut fluids. The time allowed for extraction does not consider digestive kinetics or the residence times commonly observed in the guts of deposit feeders.We have been developing an extraction procedure that is intended to more closely mimic in vivo digestive processes. It is not possible, at the present time, to synthesize a biologically realistic extractant, but we are able to recover the digestive fluid from large, deposit-feeding invertebrates. This fluid is then used for in vitro incubation of contaminated sediment, and the amount of contaminant solubilized during the incubation is determined.
The general goal of this project was to contrast in vitro digestive fluid solubilization with in vivo digestive processes and bioaccumulation. The in vitro technique allows us to answer mechanistic questions by manipulating sediment or digestive fluid characteristics in ways that would not be possible in vivo, but first we need to establish that in vitro solubilization provides meaningful information in the context of the whole animal's accumulation of sediment-sorbed contaminants. Included within this objective are contrasts between the novel digestive fluid solubilization approach and more traditional means of measuring bioavailability. This research also was intended to expand the approach to multiple species and varied substrate types.
Our specific objectives, as stated in the initial proposal, were to: (1) quantitatively compare in vitro digestive fluid solubilization to desorption of sediment-sorbed contaminants in vivo; (2) quantitatively compare in vitro digestive fluid solubilization to bioaccumulation of the contaminant within the organism's tissues; (3) determine if digestive fluid solubilization is a function of hydrophobicity and, if so, over what range of hydrophibicity does the technique provide meaningful data; and (4) determine the generality of the digestive fluid solubilization approach to a wide range of benthic invertebrate taxa.
Summary/Accomplishments (Outputs/Outcomes):
Work conducted under this grant and the results produced can best be summarized under four research components, which are described individually below:Research Element #1: Initial Investigations of Digestive Fluid Extraction. Our initial investigations, focusing on PAH, explored the variables that affect in vitro solubilization and compared this new procedure to more established methods for measuring bioavailability. Six sediments were spiked with phenanthrene and benzo[a]pyrene, and used for both in vitro extractions with digestive fluid of the polychaete Arenicola brasiliensis, and bioaccumulation experiments with the same species. Our results include:
- In vitro extraction decreased as organic carbon content of the sediment increased, a result that is consistent with earlier work on bioavailability.
- There was a 3-fold variation among individuals in the extent of contaminant solubilization by their digestive fluid. This variability was unrelated to enzyme activity, suggesting fluid sufactancy may be the more important variable determining solubilization.
- The proportion of PAH solubilized during in vitro extraction appeared comparable to that achieved in vivo based on preliminary results.
- There was good agreement on relative bioavailability of benzo[a]pyrene among the sediments as measured by in vitro extraction and more traditional measures such as steady state bioaccumulation and uptake clearance. Phenanthrene bioavailability showed poorer correlation, perhaps due to both a smaller data set and greater importance of uptake from the dissolved phase.
- The bioavailability of spiked polycyclic aromatic hydrocarbons (PAH) differed from that of equivalent in situ-contaminated PAH, but not in a predictable or consistent manner.
- A. brasiliensis appeared to retain fluid within the gut so that any given volume of fluid was used for repeated sequential digestion of many gut volumes of sediment.
- As a result of selective feeding, the PAH content of ingested sediment was increased about 20 percent relative to the bulk sediment.
- Absorption efficiency was comparable to in vitro extraction efficiency, suggesting solubilization rather than absorption is the critical constraint on uptake.
Results from these studies have been published in Weston and Mayer (1998a; 1998b).
Research Element #2: Digestion as a Route of Contaminant Uptake. This research element had a two-fold purpose. First, it was intended to compare solubilization in digestive fluid with absorption efficiency, a more traditional means to measure bioavailability. Secondly, it was intended to quantify the relative importance of sediment ingestion as a route of contaminant uptake (versus uptake of dissolved phase from surrounding water).
Five polycyclic aromatic hydrocarbons were used in this study, spanning a broad range of hydrophobicity (naphthalene, fluoranthene, phenanthrene, pyrene, and benzo[a]pyrene). Sediments spiked with these compounds were extracted in vitro using the digestive fluid of Arenicola brasiliensis. Concurrently, the polychaete Abarenicola pacifica was exposed to the five sediments and the in vivo absorption efficiency determined by the Cr51/C14 dual label technique. Because the dual label approach requires collection of the fecal material and A. brasiliensis feeding behavior does not allow this, it was necessary to do the in vivo experiments with A. pacifica and approximate its digestive intensity with gut fluid from the other arenicolid polycheate, A. brasiliensis.
In vitro solubilization and in vivo absorption efficiency both tended to decrease as hydrophobicity increased, though neither behaved linearly. Digestive fluid solubilization decreased from 46 percent (naphthalene) to 28 percent (benzo[a]pyrene), while absorption efficiency for the same compounds decreased from 24 percent to 5 percent. There was substantial difference between the proportion solubilized and that absorbed, with the absorbed fraction consistently being considerably smaller. It is possible that not all the solubilized contaminant is subsequently absorbed across the gut wall, but it also is possible that the dual label approach may not be accurately quantifying absorption efficiency. While often used, the approach does have some inherent untested assumptions, and further evaluation (see Research Element #4) is necessary to establish its validity.
The second purpose of these experiments was to establish the relative importance of particle-adsorbed and dissolved contaminants as sources for uptake. Using a well-established kinetic model, we estimated the body burden of each PAH that would have been attained by A. pacifica if all uptake were via ingestion and digestion. We then compared this predicted body burden with actual body burden, and attributed any shortfall in the predicted concentration to dissolved phase uptake. A paper has already been published containing the results for the benzo[a]pyrene (Weston et al., 2000), and a second paper with data for the other PAH is in preparation. Approximately three-quarters of the benzo[a]pyrene accumulation was via ingested sediment, and that proportion decreased as hydrophobicity decreased. This relationship is consistent with current conceptual understanding in the field, though these predictions had heretofore been largely untested.
Research Element #3: Interphyletic Comparison. The vast majority of our work with digestive fluid extraction has been done using fluid from arenicolid polychaetes (Arenicola brasiliensis or Arenicola marina). This choice is due to the availability of these species in large numbers and their large size, which makes collection of digestive fluid feasible; however, we felt it important to establish how arenicolid digestive fluid may differ from that of other invertebrates in contaminant extraction potential. From a more conceptual perspective, it was important to establish how bioavailability differs among species, as quantification of bioavailability in any given study has almost invariably been done with a single species.
A total of 18 invertebrate species representing 7 phyla were collected from Alaska, Washington, California, and Maine. Digestive fluid was extracted from each species, and used to extract sediments that had been spiked with either benzo[a]pyrene or zinc. Biochemical parameters of the fluid also were quantified including enzyme activities, surfactancy, pH, dissolved organic carbon, amino acids, and lipids.
Our results provided a striking illustration of the species dependency of bioavailability, for there were order-of-magnitude differences among the various digestive fluids in their ability to extract benzo[a]pyrene or zinc from a given sediment. For example, some digestive fluids extracted only 5 percent of the BaP from the sediment, whereas others extracted up to 45 percent. Thus, the often-used concept of "the bioavailable contaminant fraction" is meaningful only when placed in the context of the species used to define it. In general, echinoderm digestive fluid was the least able to solubilize particle-associated toxicants. Digestive fluids of polychaetes and echiurans were among the most effective extractants. Fluid from molluscs and a sipunculan tended to be intermediate.
Biochemical characterization of the various digestive fluids illustrated similar phylogenetic trends. Echinoderms could be characterized as "weak digesters," as indicated by low enzymatic activity and low surfactancy in their gut fluid. Polychaetes and echiurans tended to have highly colored gut fluids, with high enzymatic activity and surfactancy. A notable exception was the echinoderm, Parastichopus californicus, which shared low enzymatic activity with the other echinoderms, but was unique in having surfactant micelles. Its gut fluid was effective at solubilization of benzo[a]pyrene, but not zinc, as the former is a process likely to be highly surfactant-dependent.
In an attempt to discern the mechanisms behind pollutant solubilization in gut fluid, we attempted to correlate benzo[a]pyrene and zinc solubilization efficiency with the various biochemical parameters. Nearly all parameters showed a highly significant relationship due to their covariance. Thus, this approach was not helpful in establishing causality.
The primary paper describing the results of this study has been prepared for publication in Environmental Toxicology and Chemistry (Mayer et al., in press). A second study using these same digestive fluids has already been published (Chen et al., 2000), and a third is in preparation.
Research Element #4: Comparison of in vitro Solubilization to in vivo Absorption Efficiency. Our interphyletic comparison (Research Element #3) showed the gut fluids of some species to be far better than others in desorbing pollutants from sediment in vitro. It is important to demonstrate that this finding has implications for pollutant uptake at the whole animal level. Thus, we wanted to test two species, one of which had strong digestive capabilities and one of which was weaker, and determine if in vivo absorption efficiencies were predictable based on the in vitro extractions. A polychaete, Arenicola brasiliensis, and a holothuroid, Parastichopus californicus, were exposed to sediments spiked with benzo[a]pyrene and zinc. Based on the in vitro extractions, we would expect that absorption efficiency of benzo[a]pyrene would be high (and possibly quantitatively comparable to solubilization efficiency), but that A. brasiliensis would have a much higher zinc absorption efficiency than P. californicus.
This same experiment was valuable in addressing two other critical issues. First, we could get a direct measure of absorption efficiency by comparing pollutant concentration of sediment in the anterior gut (just ingested) with that in the posterior gut (fecal material). Both of the species used in these experiments are large, making it possible to dissect open the gut and remove sediment and/or fluid at various points along its length. This direct measurement of absorption efficiency could then be compared to both in vitro solubilization and the more typical indirect measure of absorption efficiency based on Cr51/C14 dual labeling. The results of Research Element #1, discussed above, raised the issue of whether the widely used Cr51/C14 dual label approach to quantifying absorption efficiency provides accurate values, and this experiment provided a means to validate the procedure.
A second benefit of this experimental approach was the ability to contrast in vitro with in vivo solubilization. In Research Element #1, we had preliminary data indicating that the concentration of pollutant achieved in digestive fluid during an in vitro extraction was comparable to that reached by fluid in the gut of an actively feeding organism. When dissecting sediment from along the length of the gut to determine absorption efficiency, we also could recover digestive fluid for this purpose. While our earlier work suggested a close agreement between in vitro and in vivo concentrations, that data set was limited to about six individuals of A. brasiliensis. These new experiments extended our observations to dozens of animals from two species. The two test species, A. brasiliensis and P. californicus, were exposed to five sediments widely varying in type and quantity of organic matter. These sediments were labelled with benzo[a]pyrene/Cr51 and zinc (separate treatments). After a brief period of exposure (usually 24 hours), the animals were removed from the sediment and processed as described above to obtain data on in vivo solubilization, absorption efficiency by foregut/hindgut comparison, and absorption efficiency by Cr51/C14. Data are currently being analyzed, and at least one paper will be prepared based on the results.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 5 publications | 5 publications in selected types | All 5 journal articles |
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Type | Citation | ||
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Chen Z, Mayer LM, Quetel C, Donard OFX, Self RFL, Jumars PA, Weston DP. High concentrations of complexed metals in the guts of deposit feeders. Limnology and Oceanography 2000;45(6):1358-1367 |
R825353 (Final) |
not available |
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Mayer LM, Weston DP, Bock MJ. Benzo[a]pyrene and zinc solubilization by digestive fluids of benthic invertebrates-A cross-phyletic study. Environmental Toxicology and Chemistry 2001;20(9):1890-1900. |
R825353 (Final) |
not available |
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Weston DP, Mayer LM. Comparison of in vitro digestive fluid extraction and traditional in vivo approaches as measures of polycyclic aromatic hydrocarbon bioavailability from sediments. Environmental Toxicology and Chemistry 1998;17(5):830-840. |
R825353 (Final) |
not available |
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Weston DP, Mayer LM. In vitro digestive fluid extraction as a measure of the bioavailability of sediment-associated polycyclic aromatic hydrocarbons: Sources of variation and implications for partitioning models. Environmental Toxicology and Chemistry 1998;17(5):820-829. |
R825353 (Final) |
not available |
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Weston DP, Penry DL, Gulmann LK. The role of ingestion as a route of contaminant bioaccumulation in a deposit-feeding polychaete. Archives of Environmental Contamination and Toxicology 2000;38(4):446-454. |
R825353 (Final) |
not available |
Supplemental Keywords:
sediments, bioavailability, risk assessment, polycyclic aromatic hydrocarbons, PAHs, metals., Scientific Discipline, Toxics, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Ecology, Contaminated Sediments, Environmental Chemistry, Chemistry, HAPS, Biology, contaminant transport, digestive solubilization, polychlorinated biphenyls (PCBs), in vivo, hydrocarbon, PCBs, benthic biota, contaminated sediment, in vitro, polychlorinated biphenyls, benthos-associated organismsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.