Method development and laboratory intercomparison of an RP-HPLC-UV method for energetic chemicals in marine tissues.

Experiments were conducted to develop a method for the determination of a set of 17 military-relevant energetic compounds (including nitroaromatics, nitramines, and nitrate esters) in 5 types of marine tissues (Dungeness crab, Manila clam, starry flounder, sea cucumber, and geoduck) using reversed-phase high performance liquid chromatography with a UV detector (RP-HPLC-UV). Dry-ice grinding was evaluated and found to be an excellent method of sample homogenization prior to sample extraction and determination. An extract cleanup procedure based on solid-phase extraction was assessed. A cleanup procedure using solid phase extraction was adequate for the removal of interferences prior to HPLC analysis for the five marine tissue matrices tested. Mean method detection limits (MDLs) were estimated using two columns at two wavelengths (254 and 210 nm) and ranged from 17 to 293 µg/kg for the five tissue matrices tested. A six-laboratory intercomparison test was conducted to evaluate the performance of the method, each analyzing five marine tissue matrices fortified at three levels. The same marine tissues were used in the laboratory intercomparison study except Pacific halibut was substituted for starry flounder. Overall, USEPA Method 8330B modified for tissue analysis showed suitable detection capability, analytical accuracy, precision, sensitivity, linear range, and robustness for sixteen (16) of the seventeen (17) analytes, for all five (5) of the marine tissue matrices studied. The exception was tetryl that proved to be unstable for all matrices as has been found for soils and sediments.

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17ß-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish.

Modification to EPA Method 1623 to address a unique seasonal matrix effect encountered in some U.S. source waters.

U.S. Environmental Protection Agency (EPA) Method 1623 is designed to detect and determine concentrations of Cryptosporidium oocysts and Giardia cysts in water through concentration, immuno-magnetic separation (IMS), and immuno-fluorescence assay with microscopic examination. A seasonal interference with the method was observed in some municipal source waters collected from reservoirs and as reported to Shaw Environmental, Inc. in the summers of 2005, 2006, and 2007. This interference, which was not confined to a single region of the nation, caused clumping of the IMS beads during the acid dissociation of the IMS procedure in Method 1623. This effect lowered method recoveries for both Cryptosporidium and Giardia; however, the effect was more pronounced for Giardia. A heat dissociation technique (Ware et al., (2003) J. Microbiol. Methods 55, 575-583) was shown to be a viable option for samples which demonstrate the clumping matrix effect and improved Giardia recoveries in partially clumped samples. The heat dissociation application holds promise for fully clumped samples and warrants further investigation.

Preservation and analytical procedures for the analysis of chloro-s-triazines and their chlorodegradate products in drinking waters using direct injection liquid chromatography tandem mass spectrometry.

A direct injection, liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the analysis of the chloro-s-triazine herbicides and their degradates in finished drinking water. The target compounds in the method were selected based on their inclusion in a common mechanism group (CMG) because of their ability to induce a similar toxic effect through a common mechanism of toxicity. The target list includes the chloro-s-triazines (atrazine, simazine, cyanazine, and propazine) and their dealkylated degradates (desethylatrazine, desisopropylatrazine, and diaminochlorotriazine). Potential matrix effects are minimized by the use of individual isotopically enriched internal standards. Analyte stability in finished chlorinated drinking water samples is ensured through careful selection of proper dechlorinating and antimicrobial reagents and through buffering sample pH. In the absence of proper dechlorination, the target analytes were found to degrade over a short period of time, even under refrigerated storage conditions. The final method has adequate sensitivity to accurately detect all target analytes at or below 0.1 microg/L and displays sufficient precision and robustness to warrant publication as EPA Method 536.

An improved colorimetric method for chlorine dioxide and chlorite ion in drinking water using lissamine green B and horseradish peroxidase.

Lissamine Green B (LGB) was carefully selected as a potential candidate for the development of a new U.S. Environmental Protection Agency (EPA) method that is intended for use at water utilities to determine chlorine dioxide (ClO2) in drinking water. Chlorine dioxide reacts with LGB in aqueous solution to decrease the absorbance of LGB in direct proportion to the ClO2 concentration. LGB was confirmed to have adequate sensitivity, and to suffer less interference than other dyes reported in the literature. The stoichiometry for the reaction between LGB and ClO2 was found not to be 1:1 and is dependent on the LGB concentration. This required calibration of each LGB stock solution and prompted the investigation of alternate means of calibration, which utilized a horseradish peroxidase (HRP)-catalyzed conversion of chlorite ion (ClO2(-)) to ClO2. This approach allowed the simultaneous determination of ClO2(-) concentration, which is also required each day at water plants that use ClO2. Studies were conducted to characterize and carefully optimize the HRP-conversion of ClO2(-) to ClO2 in order to yield reaction conditions that could be accomplished in less than 30 min at modest cost, yet meet EPA's sensitivity and robustness requirements for routine monitoring. An assessment of method detection limit, linearity and slope (or sensitivity), precision, and accuracy in finished drinking water matrices indicated that this approach was suitable for publication as EPA Method 327.0.

Statistical procedures for determination and verification of minimum reporting levels for drinking water methods.

The United States Environmental Protection Agency's Office of Ground Water and Drinking Water has developed a single-laboratory quantitation procedure: the lowest concentration minimum reporting level (LCMRL). The LCMRL is the lowest true concentration for which future recovery is predicted to fall, with high confidence (99%), between 50% and 150%. The procedure takes into account precision and accuracy. Multiple concentration replicates are processed through the entire analytical method and the data are plotted as measured sample concentration (y-axis) versus true concentration (x-axis). If the data support an assumption of constant variance over the concentration range, an ordinary least-squares regression line is drawn; otherwise, a variance-weighted least-squares regression is used. Prediction interval lines of 99% confidence are drawn about the regression. At the points where the prediction interval lines intersect with data quality objective lines of 50% and 150% recovery, lines are dropped to the x-axis. The higher of the two values is the LCMRL. The LCMRL procedure is flexible because the data quality objectives (50-150%) and the prediction interval confidence (99%) can be varied to suit program needs. The LCMRL determination is performed during method development only. A simpler procedure for verification of data quality objectives at a given minimum reporting level (MRL) is also presented. The verification procedure requires a single set of seven samples taken through the entire method procedure. If the calculated prediction interval is contained within data quality recovery limits (50-150%), the laboratory performance at the MRL is verified.

Development of U.S. EPA method 527 for the analysis of selected pesticides and flame retardants in the UCMR survey.

Method 527 was developed to address the occurrence monitoring needs of the U.S. Environmental Protection Agency (EPA) under its second unregulated contaminant monitoring rule (UCMR 2). This method includes a wide range of semivolatile organic contaminants, including pesticides that were deferred during the first UCMR, flame retardants, and pyrethroid pesticides. This paper discusses the rationale for selection and inclusion of the various contaminants included in Method 527 and describes the challenges associated with developing analytical methods that will be used for the occurrence monitoring of such a diverse group of organic molecules. Method 527 employs solid-phase extraction with analysis by gas chromatography/ mass spectrometry (GC/MS). The final method preservation scheme requires the storage of samples in amber bottles buffered at pH 3.8 using citric acid to prevent degradation from acid-catalyzed hydrolysis and from UV light. Citric acid is also an effective antimicrobial reagent, preventing this mode of loss during storage. Ethylenediaminetetraacetic acid (EDTA) is added to remove transition metals such as copper, which was determined to degrade target analytes upon storage. Finally, free available chlorine (FAC), which is present in many finished waters and found to degrade a number of the targets, is removed using ascorbic acid. The final method meets all of the EPA UCMR survey requirements for sample storage, precision, accuracy, and sensitivity and will be proposed for use under the UCMR 2.

Challenges encountered in extending the sensitivity of US Environmental Protection Agency Method 314.0 for perchlorate in drinking water.

Concerns about the potential adverse health effects of perchlorate at concentrations below the minimum reporting level (MRL) of US Environmental Protection Agency (EPA) Method 314.0 (generally recognized as 4.0 microg/l) have led to an interest in increasing the sensitivity of the method. This work describes the use of 2 mm columns with a large-loop direct injection method, a column concentration technique and this concentration technique with a background reduction step, to increase the sensitivity for the analysis of trace levels of perchlorate in high ionic strength matrices. The concentrator columns studied were the Dionex TAC LP-1 and a new Dionex high capacity Cryptand concentrator column. The use of a surrogate to monitor trapping efficiency for the concentration technique and the use of confirmational columns to minimize the potential for false positives are also discussed. The large-loop direct injection method and the column concentration methods provided acceptable data when the samples were pre-treated with solid phase pretreatment cartridges. The background reduction technique did not provide acceptable data with either of the concentrator columns evaluated.

Optimizing the determination of haloacetic acids in drinking waters.

Three methods are currently approved by the US Environmental Protection Agency for the compliance monitoring of haloacetic acids in drinking waters. Each derivatizes the acids to their corresponding esters using either acidic methanol or diazomethane. This study was undertaken to characterize the extent of methylation of these analytes by these methods, and to fully optimize methylation chemistries to improve analytical sensitivity, precision and accuracy. The approved methods were shown to have little to no esterification efficiencies for the brominated trihaloacetic acids (HAA3). Methylation with acidic methanol was determined to be more efficient and rugged than methylation with diazomethane. A new higher boiling solvent, tertiary-amyl methyl ether, is reported which has significantly improved methylation efficiencies for HAA3. Additional modifications to the method have been made that improve method ruggedness. The revised method, EPA Method 552.3, outperforms the currently approved methods, especially for HAA3.

Validating sample preservation techniques and holding times for the approved compliance monitoring methods for haloacetic acids under the US EPA's stage 1 D/DBP rule.

Haloacetic acids (HAAs), which are formed during the disinfection of drinking waters with chlorine, are regulated by the US Environmental Protection Agency (EPA) under the Stage 1 Disinfectant/Disinfection Byproducts (D/DBP) Rule. Recently, three studies have been reported indicating that low concentrations of HAAs can also be formed during disinfection with chloramines. Methods currently approved for compliance monitoring under the Stage 1 Rule arrest the chlorine-mediated formation of HAAs by adding ammonium chloride, which forms chloramines. Studies were undertaken using an in-process water that favored the formation of HAAs with moderate total organic carbon concentration and high levels of chlorine to investigate the potential formation of HAAs under sample storage conditions. The ammonium chloride-quenched sample did form a small amount of HAAs, but total formation over a period equal to the 14-day sample storage time was less than 2 microg/l, whereas the unquenched samples increased 41 microg/l during the same period. Pour plate studies indicated that chlorinated drinking waters quenched with ammonium chloride are protected from microbial growth, which is an important additional advantage to this preservation scheme. The presence of a combined chlorine residual should prevent microbial degradation of HAAs in samples. These studies support the preservation protocols and the sample storage times promulgated for compliance monitoring under the Stage 1 D/DBP Rule.

Improving the performance of US Environmental Protection Agency Method 300.1 for monitoring drinking water compliance.

In 1998, the United States Environmental Protection Agency (EPA) promulgated the maximum contaminant level (MCL) for bromate in drinking water at 10 microg/l, and the method for compliance monitoring of bromate in drinking water was established under Stage 1 of the Disinfectants/Disinfection By-Products Rule (D/DBP) as EPA Method 300.1. In January 2002, the United States Food and Drug Administration (FDA) regulated the bromate concentration in bottled waters at 10 microg/l. EPA anticipates proposing additional methods, which have improved performance for bromate monitoring, in addition to EPA Method 300.1, in the Stage 2 DBP Rule. Until the Stage 2 Rule is promulgated, EPA Method 300.1 will continue to be the only method approved for compliance monitoring of bromate. This manuscript describes the work completed at EPA's Technical Support Center (TSC) to assess the performance of recently developed suppressor technologies toward improving the trace level performance of EPA Method 300.1, specifically for the analysis of trace levels of bromate in high ionic matrices. Three different types of Dionex suppressors were evaluated. The baseline noise, return to baseline after the water dip, detection limits, precision and accuracy, and advantages/disadvantages of each suppressor are discussed. Performance data for the three different suppressors indicates that chemical suppression of the eluent, using the AMMS III suppressor, is the most effective means to reduce baseline noise, resulting in the best resolution and the lowest bromate detection limits, even when a high ionic matrix is analyzed. Incorporation of the AMMS III suppressor improves the performance of EPA Method 300.1 at and below 5.0 microg/l and is a quick way for laboratories to improve their bromate compliance monitoring.

Improvements to EPA method 531.1 for the analysis of carbamates that resulted in the development of U.S. EPA Method 531.2.

This project is undertaken to fully optimize the U.S. Environmental Protection Agency Method 531.1 post-column chemistries and to incorporate recent advances in liquid chromatographic separation, post-column derivatization, and detection techniques. Sample preservation and storage stability studies establish citric acid as a suitable replacement for the caustic monochloroacetic acid in the current method and confirm its antimicrobial effectiveness. Performance of an alternate set of commercially available post-column reagents is also investigated. This research has resulted in the publication of Method 531.2, a high-performance liquid chromatographic direct injection method for the analysis of N-methylcarbamoyloximes and N-methylcarbamates using post-column derivatization and fluorescence detection.

US Environmental Protection Agency Method 326.0, a new method for monitoring inorganic oxyhalides and optimization of the postcolumn derivatization for the selective determination of trace levels of bromate.

The development of US Environmental Protection Agency (EPA) Method 317.0 provided a more sensitive, acceptable alternative to EPA Method 300.1 to be proposed as one of the recommended compliance monitoring methods for Stage II of the Disinfectants/Disinfection By-Products (DBP) Rule. This work was initiated to evaluate other postcolumn reagents (PCRs) that might be utilized to provide an additional, alternative method in order to augment compliance monitoring flexibility for inorganic oxyhalide DBP anions. Modifications of the method reported by Salhi and von Gunten, which included adjustment and optimization of flow-rates, reaction temperature, and delivery of the PCR, improved the method performance. Method 326.0 incorporates an acidic solution of potassium iodide containing catalytic amounts of molybdenum(VI) as the PCR and provides acceptable precision and accuracy for all analytes and a postcolumn bromate detection limit in reagent water of 0.17 microg/l.

The application of tris buffer and copper sulfate for the preservation of phenylurea pesticides analyzed using U.S. EPA method 532 in the UCMR Survey.

A high performance liquid chromatographic method was developed to meetthe U.S. Environmental Protection Agency's (EPA) Unregulated Contaminant Monitoring Rule (UCMR) Survey need for the analysis of phenylurea pesticides in drinking waters. Many of these phenylurea compounds were demonstrated to degrade rapidly in the presence of the residual chlorine disinfectant in drinking waters. This degradation was halted by the addition of a tris buffer, which was initially chosen to optimize the sample pH prior to extraction. Copper sulfate was found to prevent the regrowth of microorganisms in surface waters, which was observed upon dechlorination. Tris buffer provided the additional benefit of keeping the copper sulfate preservative in solution even in groundwater matrices that caused precipitation of copper in its absence. A C18 solid phase, in cartridge or disk form, was used to efficiently extract target compounds from the preserved drinking water matrices. A 21-day storage stability study, together with precision and accuracy studies, demonstrated thatthis method had suitable sensitivity, selectivity, accuracy, precision, and ruggedness for use in the EPA's UCMR drinking water occurrence survey.