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On 16 December 1998, the Environmental Protection Agency (EPA) finalized the Interim Enhanced Surface Water Treatment Rule. One of the purposes of this rule is to improve the control of microbial pathogens, including Cryptosporidium spp., in drinking water (10). As watershed characterization and management become more important to source water protection policies, it must be realized that the ability to detect and enumerate pathogenic protozoans in increasingly complex matrices is a paramount concern of both regulators and stakeholders. The effects of common types of interference, as well as complex matrix remediation techniques, must be thoroughly investigated. Critical issues, such as point and nonpoint source loading, environmental fate and transport, and evaluation of agricultural best management practices, all rely on accurate detection and enumeration of the target organisms.

The currently accepted method for detection and enumeration of Cryptosporidium and Giardia species is the filtration-immunomagnetic separation (IMS)-immunofluorescence assay (FA) method outlined in EPA Method 1623 (11). While this method represents a significant improvement over the ICR technique that was used previously (6), it should be recognized that it was developed primarily for untreated surface water and finished drinking water. Other researchers have shown that as the sample matrix becomes more complex, the level of recovery of Cryptosporidium spp. begins to decline due to various types of interference (4, 9). Therefore, if EPA Method 1623 is to be used to enumerate Cryptosporidium and Giardia species in complex matrices, such as agriculturally contaminated surface water, acid mine drainage, and raw sewage, it will be necessary to document the effects of common types of interference on the process and to develop and refine the method and/or the sample matrix in order to overcome the effects of the interference.

This study was undertaken to determine what, if any, effect dissolved iron has on the IMS-FA procedure. Dissolved iron was selected as a possible primary type of interference based on the following conclusions.

First, iron is extremely common in the environment. It is the second most abundant metal found in the earth's crust and is present primarily as iron ores (8). Iron is also commonly found in surface water and groundwater, where it is present primarily as either the ferrous [Fe(II)] or ferric [Fe(III)] aqueous ionic species (8).

Second, iron is commonly found in association with sewage and in wastewater treatment plants. At one location, raw and conventional activated sludge treated wastewater has been found to contain average iron concentrations between 0.5 and 20 mg/liter, and the concentrations in residual materials (sludge, ash) are between 5,000 and 30,000 mg/kg (Allegheny County Sanitary Authority, unpublished data). Iron compounds are commonly used in wastewater treatment plants for clarifying processes and for precipitation of phosphates. Elevated iron levels would be expected in the effluents of such treatment plants (1, 5).

Third, the solution chemistry of iron is such that it causes direct difficulties with the IMS-FA method. Soluble Fe(III) yields an acidic reaction by hydrolysis: 4Fe³⁺ + 12H₂O  4Fe(OH)₃ + 12H⁺ (http://www.dep.state.pa.us/dep/deputate /minres/bamr/amd/science_of_amd.htm). Since the immunomagnetic beads dissociate in an acid environment, we hypothesize that this reaction may interfere with the pH mechanisms of the IMS-FA procedure. In addition, iron in solution forms insoluble iron oxides and hydroxides that contribute to the overall turbidity of a matrix. It has been shown by other researchers that turbidity has an effect on the IMS-FA method (4).

Finally, other researchers have shown that there is a relationship between aqueous iron and biological surfaces (2, 12). Iron has been shown to interact with the amphoteric surface functional groups that are associated with the cell wall structural polymers of microorganisms (12). We hypothesize that this interaction may interfere with association of the immunomagnetic beads with the cyst surface or with the fluorescein isothiocyanate (FITC)-monoclonal antibody assay.

Matrix remediation through the use of EDTA (disodium salt) was also examined in this study. The chelating effects of EDTA for metals are well known, and many researchers have documented the chemistry of iron-EDTA complexes (3, 7, 13).

Experimental procedure. IMS recovery was performed in triplicate in lab-prepared deionized (DI) water containing various concentrations of dissolved iron. Two separate trials were performed with the initial concentration range. Each matrix was evaluated to determine its turbidity before spiking. Slides were enumerated by an FA. The entire experiment was repeated with EDTA (disodium salt) added in order to examine the remediation effects of a known trace metal chelator. Finally, additional concentrations of dissolved iron were studied in an attempt to gather more data for dissolved iron concentrations ranging from 4 to 400 mg/liter.

Dissolved iron matrix. A dissolved iron matrix was prepared in the laboratory by dissolving 1.45 g of anhydrous ferric chloride (catalog no. F-7134; Sigma) in 1 liter of lab-prepared pure DI water. The iron solution was passed through a sterile 0.45-µm-pore-size membrane filter (type HAWG047S1; Millipore). The filtrate was examined by using flame atomic absorption to determine the concentration of dissolved iron. The filtrate was also tested to determine its turbidity and pH. The stock dissolved iron matrix was serially diluted to provide the concentrations analyzed in this experiment.

IMS. Ten milliliters of each matrix was added to four Leighton tubes. One tube was used to monitor the separation pH after buffer was added. The remaining three tubes of each matrix were spiked with ~300 viable Cryptosporidium oocysts and ~300 viable Giardia cysts. The tubes were then subjected to the IMS procedure described in EPA Method 1623 (11) by using a GC Combo kit (catalog no. 730-02; Dynal AS, Oslo, Norway).

Slide staining and enumeration. Slides were prepared and stained by using the Merifluor Cryptosporidium-Giardia direct FA (catalog no. 250050; Meridan Diagnostics, Cincinnati, Ohio) and the general procedure outlined in EPA Method 1623 (11). Application of 4',6-diamidino-2-phenylindole (DAPI) was omitted. All slides were enumerated within 30 h of preparation.

Oocysts and cyst preparation. Viable Cryptosporidium parvum Iowa oocysts and viable Giardia duodenalis H3 cysts were obtained from Waterborne, Inc. (New Orleans, La.). The oocysts and cysts were shipped and stored in phosphate-buffered saline containing antibiotics at 2 to 8°C. The stock solutions were enumerated with a hemacytometer and were diluted to spike dose concentrations with sterile lab-prepared pure DI water. The oocysts and cysts that were utilized in the aged studies were stored at 2 to 8°C in phosphate-buffered saline containing antibiotics for approximately 90 days.

EDTA chelation. Approximately 0.1 g of EDTA (disodium salt; catalog no. BP 120-1 Fisher) was added to each Leighton tube after the Cryptosporidium oocysts and Giardia cysts were added. The tubes were agitated occasionally and left to react for 0.5 h. Then SL buffers were added, and the IMS-FA was performed exactly as described above. The pH of each matrix was determined after buffer was added.