Non-targeted identification and semi-quantitation of emerging per- and polyfluoroalkyl substances (PFAS) in US rainwater.

High-resolution mass spectrometry was used to screen for emerging per- and polyfluorinated alkyl substances (PFAS) in precipitation samples collected in summer 2019 at seven sites in the United States. We previously quantified the concentration of ten PFAS in the rainwater samples using the method of isotopic dilution (Pike et al., 2021). Nine of these targeted analytes belonged to the U.S. Environmental Protection Agency Regional Screening Level list, herein referred to as EPA-monitored analytes. In this new work, we identify emerging PFAS compounds by liquid chromatography quadrupole time-of-flight mass spectrometry. Several emerging PFAS were detected across all samples, with the most prevalent compounds being C3-C8 hydrogen-substituted perfluorocarboxylic acids (H-PFCAs) and fluorotelomer carboxylic acids (FTCAs). Concentrations of emerging PFAS were in the 10-1000 ng L-1 range (approximately 1-2 orders of magnitude greater than EPA-monitored PFAS) at all sites except Wooster, OH, where concentrations were even higher, with a maximum estimated ΣPFAS of 16 400 ng L-1. The elevated levels of emerging PFAS in the Wooster samples were predominantly even and odd chain-length H-PFCAs and FTCAs comprised of complex mixtures of branched isomers. This unique composition did not match any known manufactured PFAS formulation reported to date, but it could represent thermally transformed by-products emitted by a local point source. Overall, the results indicate that PFAS outside of the standard analyte lists make up a significant and previously unappreciated fraction of contaminants in rainwater collected within the central U.S.-and potentially world-wide-especially in proximity to localized point sources.

Contaminants of Emerging Concern in the Lower Volta River, Ghana, West Africa: The Agriculture, Aquaculture, and Urban Development Nexus.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments across all continents and are relatively well known in the developed world. However, few studies have investigated their presence and biological effects in low- and middle-income countries. We provide a survey of CEC presence in the Volta River, Ghana, and examine the microbial consequences of anthropogenic activities along this economically and ecologically important African river. Water and sediment samples were taken by boat or from shore at 14 sites spanning 118 km of river course from the Volta estuary to the Akosombo dam. Sample extracts were prepared for targeted analysis of antimicrobial CECs, N,N-diethyl-meta-toluamide, and per- and polyfluoroalkyl substances (PFAS; water only). Concurrent samples were extracted to characterize the microbial community and antibiotic-resistant genes (ARGs). Antibiotics and PFAS (PFAS, 2-20 ng/L) were found in all water samples; however, their concentrations were usually in the low nanograms per liter range and lower than reported for other African, European, and North American studies. N,N-Diethyl-meta-toluamide was present in all samples. The number of different genes detected (between one and 10) and total ARG concentrations varied in both water (9.1 × 10-6 to 8.2 × 10-3 ) and sediment (2.2 × 10-4 to 5.3 × 10-2 ), with increases in gene variety at sites linked to urban development, sand mining, agriculture, and shellfish processing. Total ARG concentration spikes in sediment samples were associated with agriculture. No correlations between water quality parameters, CEC presence, and/or ARGs were noted. The presence of CECs in the lower Volta River highlights their global reach. The overall low concentrations of CECs detected is encouraging and, coupled with mitigation measures, can stymie future CEC pollution in the Volta River. Environ Toxicol Chem 2022;41:369-381. © 2021 SETAC.

Correlation Analysis of Perfluoroalkyl Substances in Regional U.S. Precipitation Events.

Per- and polyfluoroalkyl substances (PFAS) are transported in the atmosphere, leading to both wet and dry deposition to the surface. The concentrations of 15 PFAS were measured at six locations in the Ohio-Indiana region of the U.S. during the summer of 2019 and compared to samples collected at a distant site in NW Wyoming. ΣPFAS concentrations ranged from 50-850 ng L-1, with trifluoroacetic acid (TFA) being the dominant compound (~90%). Concentrations of perfluorooctanoic acid (PFOA) and perfluorosulfonic acid (PFOS) were similar to amounts observed over the past 20 years, indicating persistence in the atmosphere despite regulatory action, and the newer species HFPO-DA (GenX) was also widely detected in rainwater. ANOVA modeling and correlation matrices were used to determine association of PFAS concentrations, location, and functional group and chain length. Statistically significant differences (p < 0.05) in PFAS profiles across sites separated by 10-100 km indicate that local point sources strongly contribute to wet deposition. This work introduces correlation plots for PFAS that allow rapid visual comparison of multi-analyte and multi-site data sets.

Correlating effluent concentrations and bench-scale experiments to assess the transformation of endocrine active compounds in wastewater by UV or chlorination disinfection.

Transformation of endocrine active compounds (EACs) by either chlorination (Cl-D) or UV disinfection (UV-D) was studied by field sampling and bench-scale validation studies. Field testing assessed concentration of 13 EACs in effluent at two Chicago area 250 MGD wastewater reclamation plants (WRP) over two years. One WRP uses chlorination/dechlorination while the other employs UV disinfection. Target compounds included bupropion, carbamazepine, citalopram, duloxetine, estradiol, estrone, fluoxetine, nonylphenol, norfluoxetine, norsertraline, paroxetine, sertraline, and venlafaxine. Concentrations of 9/13 target compounds were partially reduced after disinfection (5-65% reduction). None of the target compounds were fully transformed by either chlorination or UV treatment at the WRP scale. In bench-scale experiments each compound was spiked into deionized water or effluent and treated in a process mimicking plant-scale disinfection to validate transformations. Correlation was observed between compounds that were transformed in bench-testing and those that decreased in concentration in post-disinfection WRP effluent (10/13 compounds). A survey of potential reaction products was made. Chlorination of some amine containing compounds produced chloramine by-products that reverted to the initial form after dechlorination. Transformation products produced upon simulated UV disinfection were more diverse. Laboratory UV-induced transformation was generally more effective under stirred conditions, suggesting that indirect photo-induced reactions may predominate over direct photolysis.

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site.

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.

Selective detection of gas-phase TNT by integrated optical waveguide spectrometry using molecularly imprinted sol-gel sensing films.

A chemical sensor was developed to detect the explosive 2,4,6-trinitrotoluene (TNT) utilizing planar integrated optical waveguide (IOW) attenuated total reflection spectrometry. Submicron thick films of organically modified sol-gel polymers were deposited on the waveguide surface as the sensing layer. Sol-gels were molecularly imprinted for TNT using covalently bound template molecules linked to the matrix through 1 or 2 carbamate linkages. Upon chemical cleavage of the template and displacement of the TNT-like pendant groups from the matrix, shape-selective binding sites were created that possess a primary amine group. The amine was used to deprotonate bound TNT yielding an anionic form that absorbs visible light. Binding of TNT and subsequent conversion to the anion results in the attenuation of light propagating through the waveguide, thus creating a spectrophotometric device. Sensitivity can be achieved by taking advantage of the substantial pathlength provided by the use of single mode IOWs. The limit-of-detection to gas-phase TNT was found to be five parts-per-billion (ppbV) in ambient air at a flow rate of 40 mL min(-1) given a 60 s sampling time. The sensor is highly selective for TNT due to the selectivity of binding site recognition of TNT and the subsequent generation of the TNT anion. Response to TNT is not reversible which results in an integrating sensor device which, in theory, can improve the ability to detect small amounts of the explosive if the exposure time is sufficient in length.

Sensor for fluorene based on the incorporation of an environmentally sensitive fluorophore proximal to a molecularly imprinted binding site.

A fluorescence-based chemical sensor for fluorene was created by molecularly imprinting a sol-gel comprising the bridged silsesquioxane, bis(trimethoxysilylethyl)benzene. The template was covalently bound to the sol-gel matrix using a fluorene analogue functionalized silane. After chemical removal of template via cleavage of a carbamate linkage, an amine group was left that provided an attachment site for the environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD). Fluorene binding was detected by a change in NBD fluorescence intensity induced by a difference in the local polarity around the probe when the recognition site is filled. Such an approach eliminated response to nonspecific binding to the matrix. Sensing films deposited on glass slides were shown to have response times of <60 s and detection limits below 10 parts-per-trillion. Binding experiments demonstrated that the materials had good selectivity for fluorene over close structural analogues including naphthalene, fluoranthene, and anthracene. However, the sensing design is limited by a lack of reversibility following fluorene binding.

Transition state stabilization by six arginines clustered in the active site of creatine kinase.

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase.

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.

Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability.

Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for this quaternary structure are unclear, given that there is no evidence of active site communication and more primitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimer interface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directed mutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, and R147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatography and analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared to be an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants, R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although k(cat) was 60% of the wild-type enzyme. Unlike the R147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibrium mechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatine binding first. Substrate binding affinities were also significantly lower for the R147A mutant, but k(cat) was 11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showed that increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomeric mutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that protein stability is significantly decreased in the monomeric mutants compared to wild-type. Denaturation experiments measuring lambda(max) of the intrinsic fluorescence as a function of guanidine hydrochloride concentration helped confirm the quaternary structures and indicated that the general unfolding pathway of all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is not a prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.

Preferential extraction of hydrocarbons from fire debris samples by solid phase microextraction.

Headspace analysis by extraction/GC-MS is a common method of detecting volatile hydrocarbon accelerants in fire debris samples. Solid-phase microextraction was tested to determine if there is selective extraction of chemically distinct compounds. It was found that both the polydimethylsiloxane (PDMS) and Carboxen/PDMS solid phase microextraction fibers show preferential extraction of aliphatic or aromatic compounds from the headspace depending on fiber type and temperature. The Carboxen/PDMS fiber type showed particular (although not exclusive) selectivity for extraction of aromatic hydrocarbons. Other experimental considerations of SPME are noted.

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase.

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.

Development and characterization of molecularly imprinted sol-gel materials for the selective detection of DDT.

Molecularly imprinting sol-gel materials for DDT using both a noncovalent and a covalent approach was examined. A nonpolar porous sol-gel network was created through the use of the bridged polysilsesquioxane, bis-(trimethoxysilylethyl)benzene (BTEB), as the principal sol-gel component. Noncovalent molecular imprinting was deemed unsuccessful, presumably because of the lack of strong intermolecular interactions that can be established between the DDT and the sol-gel precursor. A covalent imprinting strategy was employed by generating a sacrificial spacer through the reaction of two 3-isocyanatopropyltriethoxysilanes with one of two different template molecules: 4,4'-ethylenedianiline (EDA) or 4,4'-ethylidenebisphenol (EBP). After formation of the sol-gel, the bonds linking the spacer template to the matrix were cleaved in a manner that generated a pocket of the appropriate size bordered by amine groups that could aid in the binding of DDT through weak hydrogen bonding interactions. Experiments indicated that DDT could be bound selectively by such an approch. To generate a sensor, an environmentally sensitive fluorescent probe, 7-nitrobenz-2-oxa-1,3-diazole, (NBD) located adjacent to the DDT binding site was used to transduce the binding of analyte. EDA-imprinted sol-gels, deposited as films on glass microscope slides, were shown to quantitatively detect DDT in water to a limit-of-detection of 50 ppt with a response time of <60 s. Repeat measurements could be made with the same sensing films after rinsing with acetone between each measurement. The EDA sensing material was selective for DDT and other structurally similar molecules. However, the sensing film design was limited by the relatively minor changes in fluorescence intensity upon binding DDT. This situation may be remedied by an alternative methodology that can facilitate attachment of the NBD fluorophore in an optimal position proximal to the binding pocket.