Per- and polyfluoroalkyl substances (PFAS) in river discharge: Modeling loads upstream and downstream of a PFAS manufacturing plant in the Cape Fear watershed, North Carolina.

The Cape Fear River is an important source of drinking water in North Carolina, and many drinking water intakes in the watershed are affected by per- and polyfluoroalkyl substances (PFAS). We quantified PFAS concentrations and loads in river water upstream and downstream of a PFAS manufacturing plant that has been producing PFAS since 1980. River samples collected from September 2018 to February 2021 were analyzed for 13 PFAS at the upstream station and 43-57 PFAS downstream near Wilmington. Frequent PFAS sampling (daily to weekly) was conducted close to gauging stations (critical to load estimation), and near major drinking water intakes (relevant to human exposure). Perfluoroalkyl acids dominated upstream while fluoroethers associated with the plant made up about 47% on average of the detected PFAS downstream. Near Wilmington, Σ43PFAS concentration averaged 143 ng/L (range 40-377) and Σ43PFAS load averaged 3440 g/day (range 459-17,300), with 17-88% originating from the PFAS plant. LOADEST was a useful tool in quantifying individual and total quantified PFAS loads downstream, however, its use was limited at the upstream station where PFAS levels in the river were affected by variable inputs from a wastewater treatment plant. Long-term monitoring of PFAS concentrations is warranted, especially at the downstream station. Results suggest a slight downward trend in PFAS levels downstream, as indicated by a decrease in flow-weighted mean concentrations and the best-fitting LOADEST model. However, despite the cessation of PFAS process wastewater discharge from the plant in November 2017, and the phase-out of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) in North America, both fluoroethers and legacy PFAS continue to reach the river in significant quantities, reflecting groundwater discharge to the river and other continuing inputs. Persistence of PFAS in surface water and drinking water supplies suggests that up to 1.5 million people in the Cape Fear watershed might be exposed.

A New Perspective on Sustainable Soil Remediation-Case Study Suggests Novel Fungal Genera Could Facilitate in situ Biodegradation of Hazardous Contaminants.

Deciding upon a cost effective and sustainable method to address soil pollution is a challenge for many remedial project managers. High pressure to quickly achieve cleanup goals pushes for energy-intensive remedies that rapidly address the contaminants of concern with established technologies, often leaving little room for research and development especially for slower treatment technologies, such as bioremediation, for the more heavily polluted sites. In the present case study, new genomic approaches have been leveraged to assess fungal biostimulation potential in soils polluted with particularly persistent hydrophobic contaminants. This new approach provides insights into the genetic functions available at a given site in a way never before possible. In particular, this article presents a case study where next generation sequencing (NGS) has been used to categorize fungi in soils from the Atlantic Wood Industries Superfund site in Portsmouth, Virginia. Data suggest that original attempts to harness fungi for bioremediation may have focused on fungal genera poorly suited to survive under heavily polluted site conditions, and that more targeted approaches relying on native indigenous fungi which are better equipped to survive under site specific conditions may be more appropriate.

Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species.

Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer non-gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 µg of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one- or two-dimensional (1-D or 2-D) ultrapressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish ( Actinopterygii ) database, resulting in identification of 782 unique proteins by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9× in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.

Low birth weight as a risk factor for hypertension.

While the geographic and demographic disparities in the prevalence of hypertension have been recognized for decades, the reasons for these differences in disease risks remain unknown. The demographic and geographic patterns of hypertension are similar to those of low birth weight, giving support to the "Barker Hypothesis" which proposes a fetal origin of adult-onset disease. In fact, ecologic and observational studies throughout the world have detected significant associations of low birth weight and increased risks of hypertension. Nonetheless, the mechanisms for the association have not been fully described and documented. With some supportive evidence, proposed mechanisms include reduced nephrogenesis with a higher threshold for pressure natriuresis and greater susceptibility to progressive renal disease, impaired development of the endothelium, and increased sensitivity to glucocorticoids. Still, considerable work needs to be done to explain the birth weight/blood pressure relationship. The findings to date and the clinical significance warrant continued research in this intriguing area of study.

Effects of sewage-impacted sediment on reproduction in the benthic crustacean Leptocheirus plumulosus.

Several organic contaminants in sewage effluent have been shown to elicit an estrogenic response in juvenile fish. Comparatively little emphasis has been placed on assessing these effects in marine invertebrates, particularly benthic organisms inhabiting sediment where lipophilic contaminants tend to persist. The present study examined reproductive effects in the benthic crustacean Leptocheirus plumulosus exposed to sewage-impacted sediment from Jamaica Bay, New York. Data from chronic 28-day tests showed a 50% reduction in the average number of young (juveniles + embryos) produced per surviving female in exposures to sediment from Jamaica Bay (JB). Nonylphenol ethoxylate ('NPEO) concentrations at this site were measured at 44.2 microg/g dw, concentrations two orders of magnitude higher than reference site concentrations in central Long Island Sound (CLIS). Dose-response studies with nonylphenol (NP) amended reference sediment, however, did not significantly affect reproduction suggesting that other contaminants may have contributed to the effects observed.

Determination of steroid estrogens in wastewater by immunoaffinity extraction coupled with HPLC-electrospray-MS.

A new method, based on immunoaffinity extraction coupled with liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) is described for the determination of the steroid estrogens beta-estradiol (E2), estrone (E1), and alpha-ethynylestradiol (E2) in wastewater. The use of highly selective immunosorbents in sample preparation prior to analysis allows the removal of interfering sample matrix compounds present in the wastewater extracts that would otherwise cause severe ionization suppression of the estrogens during the electrospray process. In addition, immunoextraction removes much of the isobaric noise from the selected ion monitoring chromatograms, increasing the signal-to-noise ratios for analytes, and contributing to the low detection limits (0.18 and 0.07 ng/L for E2 and E1, respectively) achieved by the current method. The method was applied to analysis of estrogens in two wastewater effluents. Recoveries of E2 and E1 were excellent (>90%), while the nonimmunogen (but structurally related) analyte EE2 was not retained (recovery <2%) from effluent extracts by the immunosorbent. This illustrates the extreme selectivity of the immunoextraction purification step. Precision of the method was high, with relative standard deviations below 5%. Concentrations of E2 in wastewater varied from 0.77 to 6.4 ng/L, while concentrations of E1 were greater (1.6-18 ng/L).

Distribution and fate of neutral alkylphenol ethoxylate metabolites in a sewage-impacted urban estuary.

The distribution and fate of neutral metabolites of the alkylphenol ethoxylate (APEO) surfactants in an urbanized estuarine environment were examined utilizing a recently developed, highly sensitive LC-MS method. Results indicated that short ethoxyl-chain APEOs and alkylphenols (APs) were present in surficial sediments throughout the estuary at concentrations roughly correlated to the organic carbon content of the sediment and that the APEO mixture was dominated by nonylphenol ethoxylate (NPEOs) metabolites (0.05-30 microg/g), with lesser amounts of octylphenol ethoxylate metabolites (OPEOs)(<0.005-0.09 microg/ g) and halogenated nonylphenols (<0.001-0.03 microg/g). NPEO metabolites in surface water (0.22-1.05 microg/L) were also present at higher concentrations than OPEO metabolites (0.007-0.040 microg/L). APEO metabolite concentrations in both sediment and water showed a strong correlation with conventional sewage tracers, affirming a wastewater source of these contaminants. APEO distributions in surface waters within the estuary could be explained by a combination of post-discharge degradation and mixing with a seawater end-member enriched in OPEO metabolites. Measured in situ Koc values of APEO metabolites were comparable to previously reported values derived from field experiments but higher than Kow and Koc values derived from laboratory experiments. Results from the present work indicate that the fate of APEO metabolites entering the estuarine environment through discharge of wastewater is directed primarily by scavenging onto particles and subsequent burial in sediments, degradation during residence in the water column, and transport out of the estuary through advective and dispersive processes.

Analysis of nonylphenol and nonylphenol ethoxylates in environmental samples by mixed-mode high-performance liquid chromatography-electrospray mass spectrometry.

A new method is described based on mixed-mode high-performance liquid chromatography with electrospray mass spectrometry detection for comprehensive quantitative analysis of nonylphenol (NP) and nonylphenol ethoxylates (NPEOs) in wastewater and sediment. Efficient separation, reduced band broadening, and high sensitivity were achieved by employing a methanol-water gradient on a mixed-solvent gel filtration column designed for MS interfacing. Quantitative accuracy and precision of the method were improved by the use of custom-synthesized [13C6]NPEO analogs as isotope-dilution surrogate standards. Method detection limits for NP and individual NPEOs ranged from I to 55 pg injected on column.

Analysis of alkylphenol ethoxylate metabolites in the aquatic environment using liquid chromatography-electrospray mass spectrometry.

A quantitative method is described for the analysis of the metabolites of alkylphenol ethoxylate (APEO) surfactants in estuarine water and sediment samples using reversed-phase high-performance liquid chromatography with electrospray mass spectrometry detection. Nonyl- and octylphenols, nonyl- and octylphenol mono-, di-, and triethoxylates, halogenated nonylphenols, and nonylphenol ethoxycarboxylates were concentrated from water samples using a C18 solid-phase extraction procedure. A novel, continuous-flow, high-temperature, sonicated extraction system was developed to isolate APEO metabolites from sediment samples. Quantitative LC-MS was performed in the negative ion mode for nonylphenols, octylphenols, and halogenated nonylphenols and in the positive ion mode for nonyl- and octylphenol ethoxylates using selected ion monitoring with isotopically labeled surrogate standards. Recoveries for sediment and water analyses ranged between 78 and 94%, and detection limits for APEO metabolites were between 1 and 20 pg injected on column. This is a significant improvement over previously reported methods. Suppression of analyte response was encountered in the presence of matrix components in sediment samples, but this effect was eliminated by careful selection of surrogate and internal standards. Individual APEO metabolite concentrations of 1-320 ng/L and 5-2000 ng/g are reported for water and sediment samples, respectively, from Jamaica Bay, NY.

Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail.

The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly. Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation. Beginning with the onset of late protein synthesis, the cultures were labeled continuously with [(3)H]leucine to normalize against subsequent sample losses. After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with [(35)S]methionine, and then chased. Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation. Total (35)S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails. The inflection point in the sigmoidal (35)S-incorporation curve of each protein marks the maximal uptake of (35)S within that pool just before the effect of the chase becomes apparent and the curve begins to level off. The length of the delay in the apparent chase time reflects the position of that protein in the pathway. The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly. As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase. Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway. The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times.

Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion.

The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.

Development of separation systems for polynuclear aromatic hydrocarbon environmental contaminants using micellar electrokinetic chromatography with molecular micelles and free zone electrophoresis.

Of four systems available from the literature, based on cyclodextrins, dioctylsulfosuccinate, bile salts, and molecular micelles consisting of oligomers of undecylenic acid, the most successful separation system in our hands is based on the molecular micelles, oligomers of sodium undecylenic acid (OSUA). We have employed organic additives of acetonitrile, acetone, and tetrahydrofuran in achieving separations of polyaromatic hydrocarbons (PNAs) using molecular micelles. Generally, successful separations are achieved with 20-40% composition as the organic additive in an 8 mM borate buffer. We separated 16 PNAs with 20% tetrahydrofuran in a system of 8 mM borate and 0.125 g/10 mL (ca. 6.25 mM) of OSUA. Typical extracts of environmental samples contain additional analytes besides the typical 16 target compounds. Among these are the nitrogen-containing aromatics that can act as cations under conditions of low pH and additional compounds that can act as anions under basic conditions in free-zone electrophoresis. These additional classes of analytes are separated by capillary zone electrophoresis/laser-induced fluorescence detection using a frequency-doubled laser operated at 257 nm.

Capillary electrophoresis/laser-induced fluorescence detection of fluorescein as a groundwater migration tracer.

Capillary electrophoresis (CE) has been applied to the determination of the groundwater migration tracer dye fluorescein based on laser-induced fluorescence (LIF) detection and compared to determinations obtained with traditional spectrofluorimetry. Detection limits of injected dye in the low parts per trillion (ppt) ranges have been accomplished with both CE/LIF based on the Ar ion laser and with a spectrofluorimeter. This approach was used for a real-world problem in determining groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites by the Environmental Sciences Division in response to regional needs and as application of new analytical tools under development. Fluorescent dye was injected into source wells and then was determined in monitoring wells by extracting pads that adsorbed the dye or by directly determining the dye in the water using solid-phase extraction (SPE), a preconcentration technique. The approaches based on CE/LIF exhibits increased specificity over existing approaches due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water using solid-phase extraction and field-amplified injection techniques.

Identification of bacteriophage T4 virion proteins by transverse pore-gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dual amino acid labeling.

We have developed a horizontal N,N'-methylenebisacrylamide (Bis) acrylamide gradient sodium dodecyl sulfate (SDS) gel system that permits the evaluation of the purity of individual protein bands in complex mixtures. A Bis gradient gel is poured vertically and, after polymerization, reoriented horizontally. A single large sample spanning the top of the gel is then run down at right angles to the gradient. The relative mobility of a few proteins varies considerably from the rest, causing them to merge with and cross other bands as the Bis concentration changes. Band splitting revealed that several bands previously thought to represent a single species are actually comprised of comigrating proteins. Once the Bis/monomer concentration offering the best separation was identified, we sought a simple method for identifying the genetic origin of bands, since many proteins now migrated in new positions on the gel. We reasoned that if infected cells were simultaneously labeled with [35S]methionine and [3H]leucine and the purified virion proteins analyzed to determine their 35S/3H ratio, we could identify most proteins by comparing this ratio with one calculated from the T4 DNA sequence. Our expectations were realized, and we here report the separation and identification of all T4 virion proteins. Finally, we comment on the incorporation of various changes to the original Laemmli SDS-polyacrylamide gel formulations that have been reported in the literature.

Applications of capillary electrophoresis/laser-induced fluorescence detection to groundwater migration studies.

Capillary electrophoresis has been applied to the determination of groundwater migration based on laser-induced fluorescence (LIF) detection and traditional spectrofluorimetry. Detection limits of injected dye-fluorescent whitening agent (tinopal) in the low ppt ranges have been accomplished with both a spectrofluorometer and with CE/LIF based on the HeCd laser. The real-world problem was the determination of groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites. Fluorescent dyes were injected into wells and were discovered in monitoring wells by extracting pads that adsorbed the dye. The methodology based on CE/LIF exhibits increased specificity over existing methodology due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water directly using solid-phase extraction (SPE) and field-amplified injection techniques.