Final Report: Evaluation of Chemical and Biological Assays as Indicators of Toxic Metal Bioavailability in Soils

EPA Grant Number: R829418E02
Title: Evaluation of Chemical and Biological Assays as Indicators of Toxic Metal Bioavailability in Soils
Investigators: Pierzynski, G. M. , Lydy, Michael J. , Schneegurt, Mark
Institution: Kansas State University , Southern Illinois University - Carbondale , Wichita State University
EPA Project Officer: Hunt, Sherri
Project Period: September 24, 2001 through September 23, 2003 (Extended to February 23, 2005)
Project Amount: $141,500
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (2000) RFA Text |  Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)


The overall objective of this research project was 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 were to:

  1. Measure the effects of trace metals, individually and in mixtures, on particular biological endpoints and establishing dose-response relationships for a range of indicator species. These assays involved plant, microbial, and invertebrate species. Endpoints included 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 were expected when uncontaminated control soils were supplemented with trace metals from different sources like metal salts and highly contaminated mine waste materials. We attempted to 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 level to similar assays with contaminated soils for which we will not have bioavailability information and for which bioavailability will have changed because of soil amendments.

Summary/Accomplishments (Outputs/Outcomes):

Objective 1 addressed the measurement of 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 repository (ATR) and time-critical repository (TCR) soils and a control soil from the region. Acute mortality (7 day and 28 day) did not occur in either of the repository soils. Reproductive endpoints were examined briefly, as was the effect of feeding on reproductive output for E. fetida. There were no differences in reproductive output of E. fetida among TCR and control soil mixtures (0, 12.5, 25, 50, 75, and 100% TCR). Because of the lack of response with these endpoints, we focused primarily on the bioavailability of Cd, Pb, and Zn to E. fetida, as we were able to measure uptake to steady-state condition 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 over 192 hours had no effect of Pb body burdens, so 4 hour gut clearance times were used for all experiments. Three phosphorus amendments, monocalcium phosphate (MCP), tricalcium phosphate (TCP), and rock phosphate (RP), were used initially 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 allowed to mix via rolling for 1 hour, and then deionized water was added to bring the moisture content up to 20 percent. The soils were then 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. As the MCP, TCP, and RP amendments were not producing the desired decrease in bioaccumulation, we went back to using the super triple phosphate (STP) amendments that were found previously to decrease bioavailability of Pb and Zn from a different sample of active repository soil and a 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 recapture simply 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 hence differ 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 even 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 broth) and standard minimal mineral media were not suitable for these assays. A much more dilute solution now is being used for maintaining cells during exposure assays. The test bacteria, either Pseudomonasfluorescens 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. Here again, significant growth inhibition was observed because of metal addition. The control soil, however, 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. Soils 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 get 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 done 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 as a result of respiration. Cultures treated with metals have lower respiratory activity. Measurement of the accumulation of metals by bacteria has been hampered by the fact that the bacteria cannot be separated from the soils. Since both the earthworm and plant portions of the project will use accumulation measurements, it was worth pursuing this avenue with microbes for comparison. A good deal 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 then it became clear that the dialysis tubing was not allowing sufficient exchange of small molecules upon extended incubation. A new system is being employed where two 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.

A dose-response assay was developed based on respirometry of shake-flask slurries of contaminated soils mixed with bacterial culture. Contaminated soils repressed respiration rates, with chat being most inhibitory. Bioavailable standards were generated using Pb and Zn nitrate salts and sand and compared with field soils. Dose responses were observed with standards and with field soils. A variety of soil amendments were made to contaminated soil slurries. TSP and zeolite reduced some of the respiration inhibition observed with contaminated soils. Presumably, this indicates that the amendments made the metals in these soils less bioavailable. Bacterial growth with standard plates counts was used as a second dose-response assay. Similar results were obtained with this assay. A third assay based on a two-chamber apparatus where the bacterial culture and the soils were separated by a membrane was developed. This did not give consistent results, likely because of contamination issues.

Assessment of Zn phytoavailability can be predicted with routine soil extractants, but these methods generally do not perform well across a wide range of soils. The newly developed technique of diffuse gradients in thin films (DGT) has been employed to determine phytoavailable Cu concentrations, but its suitability for determining plant-available Zn concentrations has not been evaluated. A greenhouse study was conducted to assess the phytotoxicity thresholds and the phytoavailability of Zn to sorghum-sudan (Sorghum vulgare var. sudanese) grass by DGT, compared with CaCl2-extraction. A range of phytoavailable Zn concentrations were created by amending sand with ZnSO4 or with two different Zn mine wastes. Plant nutrients were added as Hoagland solution. In general, increasing Zn concentrations in the sand mixtures increased Zn adsorption by DGT and decreased the sorghum-sudan yield. A critical value for 90 percent of the control yield was chosen as an indicator of Zn toxicity. Critical values of DGT Zn, CaCl2 -extractable Zn, and plant tissue Zn were statistically similar across the three Zn sources. The performances of DGT and CaCl2 extraction for assessing Zn phytoavailability were similar. Shoot and root Zn concentrations of sorghum-sudan grass exceeded 500 mg kg-1 for many treatments. Ca-to-Zn ratios for shoots were less than 32, suggesting Zn phytotoxicity. The data suggested that Zn phytotoxicity can be induced with mine wastes, although further evaluation is needed to establish a link between mine waste and Zn phytotoxicity.

Pb is toxic to humans, especially to young children, as well as animals. Toxicity is strongly related to the bioavailability of Pb and soil-Pb bioavailability can be influenced by soil amendments. This study was conducted to evaluate the effects of Mn oxides and P on Pb bioaccessibility in five lead-contaminated soils or mine spoils from Kansas and Missouri. Fifteen treatments were used: Brazilian (BMN) and Amonone #4 (AM#4) (commercial Mn oxides), birnessite (BIR) and cryptomalene (CRYP) (synthetic Mn oxides), RP and TSP phosphorus sources, and combinations of each P source with the Mn oxides. A physiologically based extraction test (PBET) was used to evaluate time and treatment effects. All amendments significantly decreased Pb bioaccessibility compared with control, although treatment effects differed from soil to soil. In general, TSP, RP, and BIR treatments were most effective in decreasing Pb. Some amendments reduced the toxicity characteristic leaching procedure (TCLP) concentration for Pb to below the critical value of 5 mg L-1 but were not able to reduce TCLP-extractable Cd to below 1 mg L-1. Our results indicated that the use of P and Mn oxides for reducing soil Pb bioaccessibility in contaminated soils is promising.


The Tri-State mining region covers portions of Kansas, Missouri, and Oklahoma. Mining operations have left the soils contaminated with toxic metals such as Pb, Zn, and Cd, all priority pollutants posing particular hazards for children. The pathway analyses for select organisms typically used for dose-response assessments and risk assessment are plagued with difficulties. We have examined a series of biological and chemical assays to compare their effectiveness in including adequately the effects of availability in dose-response assessments. The test organisms include plants, oligochaete worms, and a variety of bacteria. Dose-response curves have been generated by observing endpoints having a reasonable expectation of indicating bioavailability including mortality, metabolic and growth measures, and metal bioaccumulation. The assays have been used to compare bioavailability in soils of known metal concentration and bioavailability levels with those of contaminated soils for which bioavailability is unknown. Significant differences were observed in bacterial growth rates and respiration that correlated positively with expected bioavailability and exposure levels. Chemical amendments of soil mixtures with phosphates or zeolite and added metal salts were used to generate a range of expected metal availabilities. The availability of Zn and Pb was decreased in soils amended with TSP, but not Ca phosphates or zeolites in bioaccumulation assays with E. fetida worms. The soil amendments induced a five-fold change in bioavailability in plants for Pb and a three-fold change in bioavailability for Zn and Cd using the DGT technique. Future work will include comparisons to chemical assays of availability including cation exchange strips. The goal is to determine which of the biological and chemical assays are the most reliable and consistent measures of bioavailability over a range of metal concentrations, metal sources, soil amendments, and availabilities.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 10 publications 4 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article 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
Journal Article Ownby DR, Galvan KA, Lydy MJ. Lead and zinc bioavailability to Eisenia fetida after phosphorus amendment to repository soils. Environmental Pollution. 2005;136(2):315-321. R829418E02 (Final)
not available
Journal Article Sonmez O, Pierzynski GM. Assessment of zinc phytoavailability by diffusive gradients in thin films. Environmental Toxicology and Chemistry 2005;24(4):934-941. R829418E02 (Final)
not available
Journal Article Sonmez O, Pierzynski GM. Phosphorus and manganese oxides effects on soil lead bioaccessibility: PBET and TCCP. Water, Air, & Soil Pollution 2005;166(1-4):3-16. R829418E02 (Final)
not available

Supplemental Keywords:

lead, zinc, cadmium, bioavailability, DGT,, Health, Scientific Discipline, Waste, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Contaminated Sediments, Remediation, Environmental Chemistry, Chemicals, Chemistry, Technology, Risk Assessments, Environmental Engineering, environmental technology, bioavailability, biodegradation, dose-response, contaminated sediment, remediation technologies, catalysts, chemical contaminants, biological assay, biotechnology, engineering, human exposure, bioremediation of soils, assessment methods, heavy metals, human health risk, metals

Relevant Websites: Exit

Progress and Final Reports:

Original Abstract
  • 2002
  • 2003 Progress Report
  • 2004