Current Methods/Methods Development in the Literature 20

Instrumental Methods

Methods for the detection of steroid estrogens and alkylphenols (APs) in aqueous, soil, and biosolids matrices abound and have been reviewed in articles such as those published by (Kuster et al., 2004) and (Petrovic and Barcelo, 2004). Many of the methods use

GC/MS(/MS) or liquid chromatography with tandem mass spectrometry (LC/MS/MS), though there are other methods that incorporate ultraviolet (UV) or fluorescence detection with LC systems. Detection limits are generally in the very low (often less than 1 ng/L) range for LC/MS/MS (Benijts et al., 2003, Rodriguez-Mozaz et al., 2004a) and GC/MS/MS (Fine et al., 2003) analysis of aquatic matrices and less than 1 ng/g for soils (Ternes et al.,

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2002). Aqueous extraction methods rely on pre-concentration such as solid phase extraction (SPE) (Fine et al., 2003, Liu et al., 2004b), solid phase microfiber extraction (Basheer et al., 2005, Braun et al., 2003, Carpinteiro et al., 2004), or liquid-liquid extraction (Soliman et al., 2004). Extraction of EASs from solids (e.g., soils, sludge, biosolids, sediment) has typically revolved around Soxhlet extraction or other solvent extraction techniques such as sonication, shake-flask, microwave-assisted extraction, and accelerated solvent extraction (ASE), though there doesn’t appear to be much difference between the methods in terms of overall

extraction efficiency/recovery (Babic et al., 1998, Gan et al., 1999, Hollender et al., 2003, Liu et al., 2004a).

Though current published methods offer sensitive techniques for low level detection of EASs, none of the methods has demonstrated that the use of one or two surrogate standards is adequate for the accurate quantitation of a suite of compounds across a variety of matrices. Different chemical processes may cause analytes to behave differently during extraction and analysis, especially when matrix effects are considered. The use of “isotopic dilution” [using deuterated surrogates for the target analytes to account for recovery and instrumental

response in mass spectrometry as in EPA Method 1668 (Telliard, 1999)] appears to be a promising avenue for method development. Isotopic dilution has been successfully applied to the analysis of estrogens in groundwater, swine lagoon, and septic samples (Conn et al., 2006, Ferguson et al., 2001, Fine et al., 2003, Swartz et al., 2006) but has not been validated for use in multiple matrices nor for NPs, E1, E2, E3, and EE2 simultaneously. Due to the significant expense of deuterated standards, it would be prudent to determine if a single surrogate (or a reduced number of surrogates) would be able to compensate for extraction efficiency and instrument response. All other methods for the compounds mentioned above

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have used either a single deuterated surrogate or other non-labeled surrogate and have not demonstrated applicability across matrices nor have they adequately shown that the single surrogate behaves the same as the target analytes in extraction or analysis (Benijts et al., 2002, Ternes et al., 2002). Additionally, the methods either tended to be unable to clean the samples adequately for GC/MS analysis (based on laboratory trials using the published methods) or the methods were too long and complex to be useful for a large number of samples.

Bioassays for Estrogenicity

The yeast estrogen screen (YES) assay was first used for the measurement of estrogenic activity in environmental samples and was originally applied to the comparison of NP isomers (Routledge and Sumpter, 1996). Briefly, a common yeast strain, Saccharomyces cerevisiae, was transfected with the human estrogen receptor (hER) and with a reporter gene (lac-Z) that codes for the production of the enzyme, β-galactosidase. The β-galactosidase is secreted by the yeast proportionally to the presence of the estrogenic agent and interacts with a color forming agent, chlorophenol red-β-D-galactopyranoside (CPRG), changing it from yellow to red. Yeasts are cultured in micro-well plates in the presence of sample extracts. On the same plate, a dilution series of known concentration of E2 is used for “calibration” together with a negative control with no sample or standard presence. Color change is measured at 540 nm while yeast growth in solution is measured at 650 nm. Corrected measurements are calculated from A540 – A650. The EC50, or the concentration at which half

of the maximum absorbance is reached for a given dilution series, is used to compare the relative estrogenic activity of compounds/samples. There have been reports of interferences

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from humic acids and false positives due to the presence of androgenic compounds (Tanghe et al., 1999), yet the supplier of the yeast for this study has not found that to be the case (De Boever et al., 2001). For the purposes of this study, a positive response from a septic effluent or groundwater sample may still be indicative of endocrine activity and therefore warrants the use of the YES assay.

While the YES assay is a powerful tool that offers low detection limits, it is not without its limitations. Several studies have reported discrepancies between YES-calculated estradiol equivalent concentration and an instrumental assay of individual chemical compounds in heavily matrix-impacted mixtures (Cespedes et al., 2004, Shappell et al., 2007), though this discrepancy can be somewhat accounted for by adjusting the data to include relative

potencies of each analyte (Aerni et al., 2004). It is important that any data presented demonstrate the feasibility of the technique in terms of detection limits and the ability to match estradiol equivalents with instrumental measurements in septic effluent samples.