Soil Toxicity Testing
The soil used in this experiment was collected from a site contaminated by Zn and Pb milling and smelting operations near the city of Blackwell in north-central Oklahoma. The soil contained 2.63 mmol Cd/kg, 2.39 mmol Pb/kg, and 195 mmol Zn/kg. Clay content and pH of the study soil were 240 g/kg and 6.4, respectively. Three chemical immobilization amendments were examined: a lime-stabilized municipal biosolid (LSB), North Carolina rock phosphate (RP), and an anaerobically digested municipal biosolid (SS). Physical and chemical characteristics of the amendments are previously described in Gradwohl (1998). Each amendment was thoroughly incorporated into soil (100 g/kg soil) in plastic tubs. The amount of amendment applied was determined from a preliminary study where amendments were added at 10, 30, 100, and 300 g/kg to the Blackwell contaminated soil (Basta et al., 2001). The lowest application that resulted in lettuce growth for some treatments was 100 g/kg. All soil amendments were performed in triplicate. Soil moisture was adjusted to field capacity (0.33 bar, ca. 250 g water/kg) and the soils were incubated at 27°C for 90 d. Soil moisture was maintained and the soils were thoroughly mixed at weekly intervals. An uncontaminated Taloka soil (fine, mixed, thermic Mollic Albaqualt) was used as a reference soil. The Taloka reference soil received the same three chemical immobilization amendments as the metal-contaminated smelter soil.
A 14-d toxicity test using the earthworm E. fetida was conducted according to a standard protocol (American Society for Testing and Materials, 1997), with noted exceptions, to assess the ability of chemical immobilization amendments to reduce metal bioavailability. For each soil–amendment combination, three 100-g soil replicates were moistened to approximately 80% water holding capacity 24 h prior to the addition of five earthworms per replicate. Mature (clitellate) earthworms weighing approximately 0.2 to 0.4 g were obtained from in-house cultures and allowed to depurate culture bedding from their gastrointestinal tracts for 24 h before exposure to the soil–amendment combinations. Testing was conducted in environmental chambers maintained at 20 ± 1°C under constant light. Earthworm mortality was monitored on a geometric time scale (e.g., 2, 4, 8, 16, 24, 36, 48 h) for the first 48 h (when most mortality occurred), then daily for the remainder of the test. Earthworms were judged to be dead upon observing no movement after gentle stimulation with a blunt probe.
Metal Extraction Using Ion-Exchange Membranes
Ion-exchange membranes were simultaneously exposed to separate soil–amendment combination replicates under the same conditions as in the earthworm toxicity test. Plant Root Simulator (PRS; Western Ag Innovations, Saskatoon, SK, Canada) anion-exchange membranes (polystyrene cross-linked with divinylbenzene) were the ion-exchange membranes used. Plant Root Simulators were converted before use to chelating ion-exchange membranes by complexation of the anionic membrane surface with disodium-DTPA (Liang and Schoenau, 1996). Individual PRSs were then buried in 100 g (dry weight) soil replicates, and moistened as described above for soil–amendment combinations for earthworm exposure. During burial, soils were gently packed around each PRS membrane to ensure that 100% of the membrane's surface was in direct contact with moist soil. After a 1-h exposure period, PRSs were removed from soil, rinsed thoroughly with deionized water to remove soil, and eluted for 1 h with 20 mL of 0.5 M trace metal-grade HNO3 in separate, self-sealing plastic bags. The eluent was analyzed for Cd, Pb, and/or Zn using flame and/or graphite furnace atomic absorption spectroscopy, and the PRSs were regenerated for reuse (Liang, 1994). Two PRS replicates per soil were exposed alongside the earthworm toxicity tests in the same environmental chamber on the first day of the earthworm exposure periods. After regeneration, the PRSs were exposed again on the seventh day. Quality assurance–quality control (QA–QC) procedures included PRS blanks and spikes, conducted with water-rinsed PRSs during the acid elution step.
Soil Analyses
Upon termination of the toxicity tests and PRS exposures, individual soil–amendment combination replicates were stored at -40°C in self-sealing plastic bags until physical and chemical parameters could be measured. Soil pH for all earthworm and PRS soil replicates was measured in the supernatant of a settled 0.01 M CaCl2 soil slurry (10 g dry weight soil/20 mL solution) according to Hendershot et al. (1993). Organic matter content was measured by wet digestion with chromic acid (Yeomans and Bremner, 1988) followed by colorimetric determination (Heanes, 1984). Total metal concentrations of soils were obtained by wet digestion of 1.0 g (dry weight) soil using 5 mL concentrated trace metal-grade HNO3. The digests were then heated to dryness, resolubilized in 15 mL 0.5 M trace metal-grade HNO3, filtered, and brought to a 50-mL volume with 0.5 M trace metal-grade HNO3. To measure weak electrolyte extractable metals, 1.0 g (dry weight) of each soil replicate from the earthworm tests was combined with 20 mL 0.1 M Ca(NO3)2, mixed in a rotary mixer for 4 h at 23°C, and centrifuged at 2500 x g for 15 min. The resulting supernatant was then filtered with a 0.45-µm membrane filter and acidified with 0.5 mL concentrated trace metal-grade HNO3. Total metal digests and Ca(NO3)2 extractions were analyzed by atomic absorption spectroscopy. Quality assurance–quality control measures included duplicate analyses, metal spikes, blanks, and analyses of standard reference soil Sandy Soil B (CRM-SA-B, Environmental Express, Mt. Pleasant, SC). Total metal analyses of the standard reference soil were within performance acceptance limits determined by USEPA 3050 digestion procedures (USEPA, 1995).
Data Analysis
All data were checked for homogeneity of variance and normality and transformed as appropriate to meet requirements for ANOVA (Sokal and Rohlf, 1995). Soils were classified as nonlethal if the 14-d cumulative mortality rate was 
15%. Toxicity of the Blackwell smelter soil–amendment combinations was assessed by comparing arcsine square root-transformed percent cumulative mortality (Newman, 1995) at each mortality observation. Plant Root Simulator data were analyzed according to a split-plot (repeated measures) test design (Steel et al., 1997). Data not satisfying assumptions for analysis of variance (ANOVA) were analyzed nonparametrically using the Kruskal–Wallis test. Fisher's protected least significant difference (LSD) multiple comparison procedure was also employed to further elucidate differences between means (
= 0.05).
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