Mark R. Servos

Pharmaceuticals and Personal Care Products in the Environment: What Are the Big Questions?

Background: Over the past 10–15 years, a substantial amount of work has been done by the scientific, regulatory, and business communities to elucidate the effects and risks of pharmaceuticals and personal care products (PPCPs) in the environment. Objective: This review was undertaken to identify key outstanding issues regarding the effects of PPCPs on human and ecological health in order to ensure that future resources will be focused on the most important areas. Data sources: To better understand and manage the risks of PPCPs in the environment, we used the “key question” approach to identify the principle issues that need to be addressed. Initially, questions were solicited from academic, government, and business communities around the world. A list of 101 questions was then discussed at an international expert workshop, and a top-20 list was developed. Following the workshop, workshop attendees ranked the 20 questions by importance. Data synthesis: The top 20 priority questions fell into seven categories: a) prioritization of substances for assessment, b) pathways of exposure, c) bioavailability and uptake, d) effects characterization, e) risk and relative risk, f ) antibiotic resistance, and g) risk management. Conclusions: A large body of information is now available on PPCPs in the environment. This exercise prioritized the most critical questions to aid in development of future research programs on the topic.

Antidepressants and their metabolites in municipal wastewater, and downstream exposure in an urban watershed.

Antidepressants are a widely prescribed group of pharmaceuticals that can be biotransformed in humans to biologically active metabolites. In the present study, the distribution of six antidepressants (venlafaxine, bupropion, fluoxetine, sertraline, citalopram, and paroxetine) and five of their metabolites was determined in a municipal wastewater treatment plant (WWTP) and at sites downstream of two WWTPs in the Grand River watershed in southern Ontario, Canada. Fathead minnows (Pimephales promelas) caged in the Grand River downstream of a WWTP were also evaluated for accumulated antidepressants. Finally, drinking water was analyzed from a treatment plant that takes its water from the Grand River 17 km downstream of a WWTP. In municipal wastewater, the antidepressant compounds present in the highest concentrations (i.e., >0.5 microg/L) were venlafaxine and its two demethylation products, O- and N-desmethyl venlafaxine. Removal rates of the target analytes in a WWTP were approximately 40%. These compounds persisted in river water samples collected at sites up to several kilometers downstream of discharges from WWTPs. Venlafaxine, citalopram, and sertraline, and demethylated metabolites were detected in fathead minnows caged 10 m below the discharge from a WWTP, but concentrations were all < microg/kg wet weight. Venlafaxine and bupropion were detected at very low (<0.005 microg/L) concentrations in untreated drinking water, but these compounds were not detected in treated drinking water. The present study illustrates that data are needed on the distribution in the aquatic environment of both the parent compound and the biologically active metabolites of pharmaceuticals.

Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route

The attachment of thiolated DNA to gold nanoparticles (AuNPs) has enabled many landmark works in nanobiotechnology. This conjugate chemistry is typically performed using a salt-aging protocol where, in the presence of an excess amount of DNA, NaCl is gradually added to increase DNA loading over 1-2 days. To functionalize large AuNPs, surfactants need to be used, which may generate difficulties for downstream biological applications. We report herein a novel method using a pH 3.0 citrate buffer to complete the attachment process in a few minutes. More importantly, it allows for quantitative DNA adsorption, eliminating the need to quantify the number of adsorbed DNA and allowing the adsorption of multiple DNAs with different sequences at predetermined ratios. The method has been tested for various DNAs over a wide range of AuNP sizes. Our work suggests a synergistic effect between pH and salt in DNA attachment and reveals the fundamental kinetics of AuNP aggregation versus DNA adsorption, providing a novel means to modulate the interactions between DNA and AuNPs.

Surface Science of DNA Adsorption onto Citrate-Capped Gold Nanoparticles

Single-stranded DNA can be adsorbed by citrate capped gold nanoparticles (AuNPs), resulting in increased AuNP stability, which forms the basis of a number of biochemical and analytical applications, but the fundamental interaction of this adsorption reaction remains unclear. In this study, we measured DNA adsorption kinetics, capacity, and isotherms, demonstrating that the adsorption process is governed by electrostatic forces. The charge repulsion among DNA strands and between DNA and AuNPs can be reduced by adding salt, reducing pH or by using noncharged peptide nucleic acid (PNA). Langmuir adsorption isotherms are obtained, indicating the presence of both adsorption and desorption of DNA from AuNPs. While increasing salt concentration facilitates DNA adsorption, the desorption rate is also enhanced in higher salt due to DNA compaction. DNA adsorption capacity is determined by DNA oligomer length, DNA concentration, and salt. Previous studies indicated faster adsorption of short DNA oligomers by AuNPs, we find that once adsorbed, longer DNAs are much more effective in protecting AuNPs from aggregation. DNA adsorption is also facilitated by using low pH buffers and high alcohol concentrations. A model based on electrostatic repulsion on AuNPs is proposed to rationalize the DNA adsorption/desorption behavior.

Intersex and reproductive impairment of wild fish exposed to multiple municipal wastewater discharges.

The Grand River watershed in Ontario, Canada, receives and assimilates the outflow of 29 Municipal Wastewater Effluent (MWWE) discharges which is a mixture of domestic and industrial wastes. The purpose of this study was to investigate the cumulative impact of multiple sewage discharges on populations of wild fish. In field studies, responses of fish populations and individual fish responses in terms of growth (condition factor), reproduction (in vitro sex steroid production, gonadosomatic indices, histology [cellular development and intersex]) were assessed upstream and downstream of two municipal discharges. Fish [Greenside Darters Etheostoma blennioides and Rainbow Darters E. caeruleum] collected downstream of two municipal wastewater plants had the potential to have greater growth (longer and heavier) when compared to reference fish collections regardless of sex. Fish were not assimilating additional anthropogenic resources into energy storage (increased condition, liver somatic index). Impacts on ovarian development appeared to be minor with no differences in growth, steroid production or cellular development. Sewage exposed male fish were experiencing impairment in the capacity to produce testosterone and 11-ketotestosterone in vitro, and in cellular development (GSI, intersex). Male darters of both species collected in the upstream agricultural region demonstrated no evidence of intersex whereas our urban reference sites had incidence of intersex of up to 20%. Rates of intersex were elevated downstream of both sewage discharges studied (33% and>60%, respectively). Lower rates of intersex at the intermediate sites, and then increases downstream of second sewage discharge suggests that fish populations have to potential to recover prior to exposure to the second sewage effluent. Pre-spawning darters demonstrated dramatically higher incidence of intersex in the spring at both urban reference sites (33% and 50%, respectively), and increased more so downstream of the near-field and far-field exposure sites (60% and 100%, respectively). These findings suggest that the compounds released in STP effluents have a tendency to act on the male reproductive system. These effects may become more pronounced as projected human population growth will require the aquatic environment to assimilate an increasing amount of sewage waste.

Ultrahigh Nanoparticle Stability against Salt, pH, and Solvent with Retained Surface Accessibility via Depletion Stabilization

For many applications, it is desirable to stabilize colloids over a wide range of buffer conditions while still retaining surface accessibility for adsorption and reaction. Commonly used charge or steric stabilization cannot achieve this goal since the former is sensitive to salt and the latter blocks the particle surface. We use depletion stabilization in the presence of high molecular weight polyethylene glycol (PEG) to stabilize a diverse range of nanomaterials, including gold nanoparticles (from 10 to 100 nm), graphene oxide, quantum dots, silica nanoparticles, and liposomes in the presence of Mg(2+) (>1.6 M), heavy metal ions, extreme pH (pH 1-13), organic solvents, and adsorbed nucleosides and drugs. At the same time, the particle surface remains accessible for adsorption of both small molecules and macromolecules. Based on this study, high loading of thiolated DNA was achieved in one step with just 2% PEG 20,000 in 2 h.

Hydrothermal growth of free standing TiO2 nanowire membranes for photocatalytic degradation of pharmaceuticals

Highly entangled TiO(2) nanowires were directly synthesized by hydrothermal growth on Ti substrates at 180 °C utilizing various organic solvents to oxidize Ti. The growth mechanism, microstructure and phase transition of TiO(2) nanowire membranes were investigated in detail. TiO(2) nanowires, with diameters of 10-20 nm and lengths up to 100 μm, show a phase transition from Type-B to anatase by annealing at 700 °C. Robust, free standing TiO(2) nanowire membranes with millimeter level thickness can be cleaved from Ti substrates or directly prepared from thin Ti foils. These porous TiO(2) membranes, while effective for mechanical microfiltration, can also photocatalytically degrade pharmaceuticals such as trimethoprim under UV irradiation.

Fathead minnow (Pimephales promelas) reproduction is impaired in aged oil sands process-affected waters.

Large volumes of fluid tailings are generated during the extraction of bitumen from oil sands. As part of their reclamation plan, oil sands operators in Alberta propose to transfer these fluid tailings to end pit lakes and, over time, these are expected to develop lake habitats with productive capabilities comparable to natural lakes in the region. This study evaluates the potential impact of various oil sands process-affected waters (OSPW) on the reproduction of adult fathead minnow (Pimephales promelas) under laboratory conditions. Two separate assays with aged OPSW (>15 years) from the experimental ponds at Syncrude Canada Ltd. showed that water containing high concentrations of naphthenic acids (NAs; >25 mg/l) and elevated conductivity (>2000 μS/cm) completely inhibited spawning of fathead minnows and reduced male secondary sexual characteristics. Measurement of plasma sex steroid levels showed that male fathead minnows had lower concentrations of testosterone and 11-ketotestosterone whereas females had lower concentrations of 17β-estradiol. In a third assay, fathead minnows were first acclimated to the higher salinity conditions typical of OSPW for several weeks and then exposed to aged OSPW from Suncor Energy Inc. (NAs ∼40 mg/l and conductivity ∼2000 μS/cm). Spawning was significantly reduced in fathead minnows held in this effluent and male fathead minnows had lower concentrations of testosterone and 11-ketotestosterone. Collectively, these studies demonstrate that aged OSPW has the potential to negatively affect the reproductive physiology of fathead minnows and suggest that aquatic habitats with high NAs concentrations (>25 mg/l) and conductivities (>2000 μS/cm) would not be conducive for successful fish reproduction.

Tissue-Specific In Vivo Bioconcentration of Pharmaceuticals in Rainbow Trout (Oncorhynchus mykiss) Using Space-Resolved Solid-Phase Microextraction

The space-resolved solid-phase microextraction (SR-SPME) technique was employed to study the tissue-specific bioconcentration of pharmaceuticals in live fish. The segmented design of the SPME fibers allowed for the simultaneous determination of pharmaceutical residues in fish dorsal-epaxial muscle and adipose tissue with a single SPME fiber. The miniaturized fiber endowed the technique with high spatial resolution allowing for quantification of analytes within adjacent, relatively small tissues of immature rainbow trout. The pre-equilibrium sampling and kinetic calibration approach yielded efficient and accurate quantitation of pharmaceuticals in fish tissue. The ability of the SPME method to repeatedly sample the same fish circumvents problems arising from interanimal variation, thus improving the precision of generated bioconcentration kinetic profiles. In vivo monitoring with SR-SPME was validated with in vitro liquid extraction of tissue samples using methanol. Of the nine compounds evaluated, five (atrazine, gemfibrozil, carbamazepine, ibuprofen, and fluoxetine) bioconcentrated in adipose and muscle tissue over the eight exposure days. Although the accumulation of analytes in both tissues was positively correlated, each compound partitioned with differing affinities as modified by their hydrophobicity and unique molecular structure. Water samples analyzed using the SPME technique yielded results similar to those determined by solid-phase extraction (SPE); however, SPME was more rapid and operationally much simpler. This study illustrates the application conditions for in situ SR-SPME while demonstrating the potential of these miniaturized SPME fibers for simultaneous in vivo repeated sampling of multiple tissues.

Instantaneous attachment of an ultrahigh density of nonthiolated DNA to gold nanoparticles and its applications.

The last 16 years have witnessed the landmark development of polyvalent thiolated DNA-functionalized gold nanoparticles (AuNPs) possessing striking properties within the emerging field of nanobiotechnology. Many novel properties of this hybrid nanomaterial are attributed to the dense DNA shell. However, the question of whether nonthiolated polyvalent DNA-AuNP could be fabricated with a high DNA density and properties similar to those of its thiolated counterpart has not been explored in detail. Herein, we report that by simply tuning the pH of the DNA-AuNP mixture an ultrahigh capacity of nonthiolated DNA can be conjugated to AuNPs in a few minutes, resulting in polyvalent DNA-AuNP conjugates with cooperative melting behavior, a typical property of polyvalent thiolated DNA-functionalized AuNPs. With this method, large AuNPs (e.g., 50 nm) can be functionalized to achieve the colorimetric detection of sub-nanometer DNA. Furthermore, this fast, stable DNA loading was employed to separate AuNPs of different sizes. We propose that a large fraction of the attached DNAs are adsorbed via one or a few terminal bases to afford the high loading capacity and the ability to hybridize with the complementary DNA. This discovery not only offers a time- and cost-effective way to functionalize AuNPs with a high density of nonthiolated DNA but also provides new insights into the fundamental understanding of how DNA strands with different sequences interact with AuNPs.