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2003 Progress Report: Evaluation of Chemical and Biological Assays as Indicators of Toxic Metal Bioavailability in Soils
EPA Grant Number: R829418E02Title: Evaluation of Chemical and Biological Assays as Indicators of Toxic Metal Bioavailability in Soils
Investigators: Pierzynski, G. M.
Current Investigators: Pierzynski, G. M. , Lydy, Michael J. , Schneegurt, Mark
Institution: Kansas State University
Current Institution: Kansas State University , Southern Illinois University - Carbondale , Wichita State University
EPA Project Officer: Winner, Darrell
Project Period: September 24, 2001 through September 23, 2003 (Extended to February 23, 2005)
Project Period Covered by this Report: September 24, 2002 through September 23, 2003
Project Amount: $141,500
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (2000)
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Description:
Objective:The overall objectives of this research are to examine a series of biological and chemical assays to compare their effectiveness in adequately including the effects of bioavailability in dose-response assessments; and to explore the use of soil amendments for reducing metal bioavailability. The specific objectives of this research project are to:
1. Measure the effects of trace metals, individually and in mixtures, on particular biological endpoints and establish dose-response relationships for a range of indicator species. These assays will involve plant, microbial, and invertebrate species. The endpoints will include bioaccumulation, mortality, reproduction, growth, and biochemical measures.
2. Compare biological and chemical indicators of soil trace element availability that correlate with expected availability in amended and unamended contaminated soils and soil mixtures. Different levels of bioavailability are expected when uncontaminated control soils are supplemented with trace metals from different sources such as metal salts and highly contaminated mine waste materials. We will determine which of our biological and chemical assays are the most reliable and consistent measures of availability over a range of metal concentrations, metal sources, and bioavailabilities.
3. Develop soil-amendment combinations that reduce metal bioavailability and compare results from different dose-response assays in soils of known metal concentration and bioavailability levels to similar assays with contaminated soils for which we will not have bioavailability information and for which bioavailability will have changed because of the addition of soil amendments.
Progress Summary:Objective 1 of this research project addresses measuring the effects of trace metals on particular biological endpoints for invertebrate species, including bioaccumulation, mortality, reproduction, growth, and biochemical measures. The use of multiple endpoints was examined using two species of worms: Eisenia fetida and Lumbricus terrestris. The mortality and reproduction endpoints were examined in both active and time critical repository soils and a control soil from the region. Acute mortality (7 days and 28 days) did not occur in either of the repository soils. Reproductive endpoints were briefly examined, as was the effect of feeding on reproductive output for E. fetida. There were no differences in reproductive output of E. fetida among time-critical repository (TCR) and control soil mixtures (0, 12.5, 25, 50, 75, and 100 percent TCR). Because of the lack of response with these endpoints, we focused primarily on the bioavailability of Cd, Pb, and Zn to E. fetida because we were able to measure uptake to steady-state conditions of all three metals in both E. fetida and L. terrestris.
A refinement of the previously used methods for bioaccumulation experiments was added. The length of gut-clearance time (4 or 6 hours) for E. fetida for Pb uptake from TCR soil by E. fetida had no effect on Pb body burdens; therefore, 4-hour gut-clearance times were used for all experiments. Three phosphorus amendments, monocalcium phosphate (MCP), tricalcium phosphate (TCP), and rock phosphate (RP), were initially used at a rate of 5,000 mg P/kg soil. Zeolite, a molecular sieve, also was used as a potential amendment option. These amendments were added to dry soil and were allowed to mix via rolling for 1 hour. Deionized water was added to bring the moisture content up to 20 percent. The soils were mixed for 1 hour per day for a 13-day incubation period. No significant differences in Pb and Zn bioaccumulation were measured in MCP, TCP, and zeolite-amended ATR soils. RP amendments elevated Pb concentrations, possibly because of contaminated phosphorus. Zn concentrations decreased with RP amendment. Significant differences from nonamended soils were measured for Cd bioaccumulation with all amendments. Because the MCP, TCP, and RP amendments were not producing the desired decrease in bioaccumulation, we used the super triple phosphate (STP) amendments, which were previously found to decrease bioavailability of Pb and Zn from a different sample of active repository soil and U.S. Department of Agriculture soil from a specific contaminated residential area (Maenpaa, et al., 2002). Amendment with STP-decreased Pb, Zn, and Cd bioaccumulation from ATR and Pb and Zn bioaccumulation from TCR soil compared with nonamended soils. Future plans include L. terrestris bioassays, geochemical modeling of the soil for estimates of available metal, and collaborative studies using chemical extraction methods.
The focus of the microbial portion of the project has been on the development and application of dose-response assays using bacteria as the reporter organisms. Microbial assays pose a different set of challenges than those based on macroscopic organisms. Once mixed with a soil matrix, it is not possible to simply recapture the microbes by separating them from soil particles. What seem to be straightforward assays, such as measurements of growth inhibition, become nearly intractable problems. The laboratory has spent a considerable amount of effort finding ways around these difficulties.
Three types of dose-response assays using microbes have been developed. These include measurements of culture density, metals accumulation, and respiration. Assay development was initially confounded by difficulties in sterilizing soils. Standard chloroform fumigation was not effective enough, nor was typical autoclaving. The soils need to be sterile so that exogenous reporter bacteria can be added and not confounded by existing populations in the soil. This is particularly important when using mixtures of soils that differ in composition and in their indigenous microbial communities. This problem has been overcome through extensive autoclaving in flats where the soil can be spread out.
Initial studies began with determining the effects of metals; specifically Pb, Zn, and Cd, when added in the form of pure chemical compounds. In liquid culture, no significant toxic effects were observed for metals at relevant concentrations (1,000 ppm or more) on a wide variety of bacterial species. Microbes were isolated from contaminated test soils and other soils, but none showed significant toxicity as measured by growth of the culture. After pursuing several possible lines of research, it was discovered that rich media (such as Luria-Bertoni) and standard minimal mineral media were not suitable for these assays. A much more dilute solution is now being used for maintaining cells during exposure assays. The test bacteria, either Pseudomonas fluorescens or Bacillus subtilis, now show significant growth inhibition in the presence of relevant levels of Pb and Zn. The pure chemical work has been extended to include mixtures of chemical compounds with control uncontaminated soils. Again, significant growth inhibition was observed because of metal addition; however, the control soil had some inhibitory effects, and the experiments are being repeated using mixtures of sand.
Soils obtained from contaminated field sites have been used in dose-response experiments, where bacterial growth is measured by dilution plate counts. Soil with higher levels of metals are more inhibitory than soils with lower levels of metals. These studies are being extended by cutting the contaminated soils with sand to obtain varying levels of metals. This set of experiments will be followed by a set of similar assays where the contaminated soils have been treated with the amendments under study. Some work with pure chemicals has used mixtures of metals at the same concentrations and ratios as those in the contaminated field soils.
The growth experiments have been conducted in parallel with a similar series of experiments measuring a different response—respiration. Clark-type electrodes are used to measure oxygen concentrations in soil slurries or liquid cultures of bacteria exposed to various chemicals or contaminated soils. Oxygen decreases rapidly in untreated cells because of respiration. Cultures treated with metals have lower respiratory activity. Measurements of the accumulation of metals by bacteria have been hampered because the bacteria cannot be separated from the soils. Because both the earthworm and plant portions of the project will use accumulation measurements, it was worth pursuing this avenue with microbes for comparison. A significant amount of time was spent developing a system where bacteria are held in dialysis tubing and then exposed to bathing solutions with pure compounds or soil slurries. There were significant problems with contamination that were solved, but it was found that the dialysis tubing was not allowing sufficient exchange of small molecules upon extended incubation. A new system is being employed¾two chambers are separated by a glass microfiber filter that will not clog as easily. Bacteria can be grown in one chamber, and contaminated soil slurries can be held in the other. This system is being used to generate accumulation data.
Despite significant technical challenges, substantial volumes of data are now being generated with the systems developed. Now that the methods are available, assays can be run rapidly. There is still some development on other assays, particularly an assay that measures photosynthetic rates in cyanobacteria. In collaboration with the Kansas State University group, we will be testing our slurries and metal mixtures with ion-exchange test strips, as will all the laboratories involved. This will give us a better mechanism for comparison and integration of the different data sets. The synthesis of the data sets will allow us to address project goals and use these assays to examine the effectiveness of soil amendments on the bioavailability of toxic metals in soils.
Future Activities:We will conduct L. terrestris bioassays, geochemical modeling of the soil for estimates of available metal, and collaborative studies using chemical extraction methods.
Journal Articles on this Report: 2 Displayed | Download in RIS Format
| Other project views: | All 11 publications | 5 publications in selected types | All 5 journal articles |
| Type | Citation | ||
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Maenpaa KA, Kukkonen JVK, Lydy MJ. Remediation of heavy metal-contaminated soils using phosphorus: evaluation of bioavailability using an earthworm bioassay. Archives of Environmental Contamination and Toxicology 2002;43(4):389-398. |
R829418E02 (2003) R829418E02 (Final) |
not available |
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Zwonitzer JC, Pierzynski GM, Hettiarachchi GM. Effects of phosphorus additions on lead, cadmium, and zinc bioavailabilities in a metal-contaminated soil. Water Air and Soil Pollution 2003;143(1-4):193-209. |
R829418E02 (2003) R829418E02 (Final) R825549C047 (Final) |
not available |
ecological risk assessment, zinc, cadmium, lead, earthworms, heavy metals, soil contamination, assessment methods, biodegradation, biological assay, bioremediation, bioremediation of soils, biotechnology, catalysts, chemical contaminants, contaminated sediment, dose-response, engineering, environmental technology, human exposure, human health risk, metals. , POLLUTANTS/TOXICS, TREATMENT/CONTROL, Scientific Discipline, Waste, Health, Remediation, Risk Assessments, Technology, Chemicals, Chemistry, Environmental Engineering, Environmental Chemistry, Contaminated Sediments, bioavailability, heavy metals, biodegradation, catalysts, biotechnology, engineering, human health risk, contaminated sediment, remediation technologies, biological assay, dose-response, environmental technology, metals, bioremediation of soils, bioremediation, assessment methods, chemical contaminants
Progress and Final Reports:
Original Abstract
Final Report