W. Matthew Henderson

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Photochemical transformation of graphene oxide in sunlight.

Graphene oxide (GO) is promising in scalable production and has useful properties that include semiconducting behavior, catalytic reactivity, and aqueous dispersibility. In this study, we investigated the photochemical fate of GO under environmentally relevant sunlight conditions. The results indicate that GO readily photoreacts under simulated sunlight with the potential involvement of electron-hole pair creation. GO was shown to photodisproportionate to CO2, reduced materials similar to reduced GO (rGO) that are fragmented compared to the starting material, and low molecular-weight (LMW) species. Kinetic studies show that the rate of the initially rapid photoreaction of GO is insensitive to the dissolved oxygen content. In contrast, at longer time points (>10 h), the presence of dissolved oxygen led to a greater production of CO2 than the same GO material under N2-saturated conditions. Regardless, the rGO species themselves persist after extended irradiation equivalent to 2 months in natural sunlight, even in the presence of dissolved oxygen. Overall, our findings indicate that GO phototransforms rapidly under sunlight exposure, resulting in chemically reduced and persistent photoproducts that are likely to exhibit transport and toxic properties unique from parent GO.

Analysis of perfluorinated carboxylic acids in soils II: optimization of chromatography and extraction.

With the objective of detecting and quantitating low concentrations of perfluorinated carboxylic acids (PFCAs), including perfluorooctanoic acid (PFOA), in soils, we compared the analytical suitability of liquid chromatography columns containing three different stationary phases, two different liquid chromatography-tandem mass spectrometry (LC/MS/MS) systems, and eight combinations of sample-extract pretreatments, extractions and cleanups on three test soils. For the columns and systems we tested, we achieved the greatest analytical sensitivity for PFCAs using a column with a C(18) stationary phase in a Waters LC/MS/MS. In this system we achieved an instrument detection limit for PFOA of 270 ag/microL, equating to about 14 fg of PFOA on-column. While an elementary acetonitrile/water extraction of soils recovers PFCAs effectively, natural soil organic matter also dissolved in the extracts commonly imparts significant noise that appears as broad, multi-nodal, asymmetric peaks that coelute with several PFCAs. The intensity and elution profile of this noise is highly variable among soils and it challenges detection of low concentrations of PFCAs by decreasing the signal-to-noise contrast. In an effort to decrease this background noise, we investigated several methods of pretreatment, extraction and cleanup, in a variety of combinations, that used alkaline and unbuffered water, acetonitrile, tetrabutylammonium hydrogen sulfate, methyl-tert-butyl ether, dispersed activated carbon and solid-phase extraction. For the combined objectives of complete recovery and minimization of background noise, we have chosen: (1) alkaline pretreatment; (2) extraction with acetonitrile/water; (3) evaporation to dryness; (4) reconstitution with tetrabutylammonium-hydrogen-sulfate ion-pairing solution; (5) ion-pair extraction to methyl-tert-butyl ether; (6) evaporation to dryness; (7) reconstitution with 60/40 acetonitrile/water (v/v); and (8) analysis by LC/MS/MS. Using this method, we detected in all three of our test soils, endogenous concentrations of all of our PFCA analytes, C(6) through C(10)-the lowest concentrations being roughly 30 pg/g of dry soil for perfluorinated hexanoic and decanoic acids in an agricultural soil.

Development and application of a physiologically based pharmacokinetic model for triadimefon and its metabolite triadimenol in rats and humans

A physiologically based pharmacokinetic (PBPK) model was developed for the conazole fungicide triadimefon and its primary metabolite, triadimenol. Rat tissue:blood partition coefficients and metabolic constants were measured in vitro for both compounds. Pharmacokinetic data for parent and metabolite were collected from several tissues after intravenous administration of triadimefon to male Sprague-Dawley rats. The model adequately simulated peak blood and tissue concentrations but predicted more rapid clearance of both triadimefon and triadimenol from blood and tissues. Reverse metabolism of triadimenol to triadimefon in the liver was explored as a possible explanation of this slow clearance, with significant improvement in model prediction. The amended model was extrapolated to humans using in vitro metabolic constants measured in human hepatic microsomes. Human equivalent doses (HEDs) were calculated for a rat no observable adverse effect level (NOAEL) dose of 3.4mg/kg/day using area under the concentration curve (AUC) in brain and blood for triadimefon and triadimenol as dosimetrics. All dosimetric-based HEDs were 25-30 fold above the human oral reference dose of 0.03mg triadimefon/kg/day, but did not account for intra-human variability or pharmacodynamic differences. Ultimately, derivations of this model will be able to better predict the exposure profile of these and other conazole fungicides in humans.

Multiwalled Carbon Nanotube Dispersion Methods Affect Their Aggregation, Deposition, and Biomarker Response

To systematically evaluate how dispersion methods affect the environmental behaviors of multiwalled carbon nanotubes (MWNTs), MWNTs were dispersed in various solutions (e.g., surfactants, natural organic matter (NOM), and etc.) via ultrasonication (SON) and long-term stirring (LT). The two tested surfactants [anionic sodium dodecyl sulfate (SDS) and nonionic poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEO-PPO-PEO) triblock copolymers (Pluronic)] could only disperse MWNTs via ultrasonication; while stable aqueous SON/MWNT and LT/MWNT suspensions were formed in the presence of the two model NOMs (Suwannee river humic acid and fulvic acid). Due to the inherent stochastic nature for both methods, the formed MWNT suspensions were highly heterogeneous. Their physicochemical properties, including surface charge, size, and morphology, greatly depended upon the dispersant type and concentration but were not very sensitive to the preparation methods. Aggregation and deposition behaviors of the dispersed MWNTs were controlled by van der Waal and electrostatic forces, as well as other non-DLVO forces (e.g., steric, hydrophobic forces, etc.). Unlike the preparation method-independent physicochemical properties, LT/NOM-MWNTs and SON/NOM-MWNTs differed in their fathead minnow epithelial cell metabolomics profiles.

Metabolomic Response of Human Embryonic Stem Cell–Derived Germ-like Cells After Exposure to Steroid Hormones

To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC exposure. Human embryonic stem cells can differentiate into numerous functional cell types including germ-like cells (GLCs). These cells possess characteristics indicative of a germ cell state, suggesting they offer a novel system to investigate the consequences of chemical exposure on normal germ cell processes. To characterize these processes, a metabolomic-based approach was employed to determine the response of GLCs following exposure to 0.001, 0.01, 0.1, 1, 10, or 100µM estradiol, testosterone, or progesterone for 48h. Following exposure, cellular extracts underwent gas chromatography coupled with mass spectrometry analysis. Models were then constructed using principal component analysis on acquired spectra to discriminate among steroid hormones as well as doses for each hormone. t-test comparisons generated a preliminary list of metabolites that were statistically significant in GLCs biochemical response to these steroid hormones. Steroid hormone exposures caused fluxes in intracellular pathways such as amino acid synthesis and metabolism, fatty acid synthesis, as well as cholesterol and lipoprotein metabolism. Further pathway analysis, based on these identified metabolites, will aid in modeling the response of GLCs to endogenous steroid hormones and allow for identification of biomarkers delineating germ cell-based developmental and reproductive pathways.

Measurement of steroids in rats after exposure to an endocrine disruptor: mass spectrometry and radioimmunoassay demonstrate similar results.

INTRODUCTION Commercially available radioimmunoassays (RIAs) are frequently used to evaluate the effects of endocrine disrupting chemicals (EDCs) on steroidogenesis in rats. Currently there are limited data comparing steroid concentrations in rats as measured by RIAs to those obtained using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This study evaluates the concordance of serum and urine steroid concentrations as quantified by select RIA kits and LC-MS/MS following exposure to an EDC, atrazine (ATR). METHODS Adult male rats were orally dosed with ATR (200 mg/kg/day) or methylcellulose (1%, vehicle control) for 5 days. Serum was collected and separated into aliquots for analysis. Serum was assayed by RIA for androstenedione (ANDRO), corticosterone (CORT), estradiol (E2), estrone (E1), progesterone (P4), and testosterone (T). Serum was extracted prior to LC-MS/MS analysis with positive electrospray ionization in multiple-reaction monitoring mode for ANDRO, CORT, P4, and T. E1 and E2 concentrations were quantified similarly by LC-MS/MS, following derivatization with dansyl chloride. To compare CORT values from urine, pregnant adult rats were orally dosed with either ATR (100 mg/kg/day) or methylcellulose for 5 days (i.e., gestational days 14-18). Urine samples were collected daily and assayed for CORT by RIA and LC-MS/MS as described above. RESULTS Data analyses demonstrated significant agreement between the two detection methods as assessed by Pearson product-moment correlation coefficient, Bland-Altman analysis, and the interclass correlation coefficient. No statistically significant differences were observed between RIA and LC-MS/MS means for any of the steroids assayed. DISCUSSION These findings indicate a significant correlation between the measurement of steroids within rat serum and urine using RIA kits and LC-MS/MS. Differences in the absolute measurements existed, but these were not statistically significant. These findings indicate that steroids may be reliably measured in rat biological media using RIAs or LC-MS/MS.

Gender and species differences in triadimefon metabolism by rodent hepatic microsomes

Understanding the potential differences in metabolic capacity and kinetics between various common laboratory species as well as between genders is an important facet of chemical risk assessment that is often overlooked, particularly for chemicals which undergo non-cytochrome P450 mediated metabolism. The use of physiologically based pharmacokinetic (PBPK) models to better describe chemical exposure is made more powerful by incorporation of high quality in vitro kinetic data. To this end, metabolism of the conazole fungicide triadimefon was studied in hepatic microsomes of both genders of SD rats and CD-1 mice. Triadimefon depletion and triadimenol formation were measured in each type of microsomes. Michaelis-Menten regressions were applied to metabolic data and V(MAX) and the Michaelis constant (K(M)) values calculated. Male SD rats metabolized triadimefon more rapidly than female SD rats or either gender of CD-1 mouse. K(M) values were in the micromolar range, indicating the possibility of competitive inhibition with endogenous substrates. Intrinsic clearances derived from kinetic parameters indicate that triadimefon metabolism is blood-flow limited in all organisms studied with the possible exception of female rat. The in vitro half-life method was investigated as a less resource intensive method for the derivation of intrinsic clearance, and was found to be useful as a complement to the traditional Michaelis-Menten approach.

Soil organic matter content effects on dermal pesticide bioconcentration in American toads (Bufo americanus)

Pesticides have been implicated as a major factor in global amphibian declines and may pose great risk to terrestrial phase amphibians moving to and from breeding ponds on agricultural landscapes. Dermal uptake from soil is known to occur in amphibians, but predicting pesticide availability and bioconcentration across soil types is not well understood. The present study was designed to compare uptake of 5 current-use pesticides (imidacloprid, atrazine, triadimefon, fipronil, and pendimethalin) in American toads (Bufo americanus) from exposure on soils with significant organic matter content differences (14.1% = high organic matter and 3.1% = low organic matter). We placed toads on high- or low-organic matter soil after applying individual current-use pesticides on the soil surface for an 8-h exposure duration. Whole body tissue homogenates and soils were extracted and analyzed using liquid chromatography-mass spectrometry to determine pesticide tissue and soil concentration, as well as bioconcentration factor in toads. Tissue concentrations were greater on the low-organic matter soil than the high-organic matter soil across all pesticides (average ± standard error; 1.23 ± 0.35 ppm and 0.78 ± 0.23 ppm, respectively), and bioconcentration was significantly higher for toads on the low-organic matter soil (analysis of covariance p = 0.002). Soil organic matter is known to play a significant role in the mobility of pesticides and bioavailability to living organisms. Agricultural soils typically have relatively lower organic matter content and serve as a functional habitat for amphibians. The potential for pesticide accumulation in amphibians moving throughout agricultural landscapes may be greater and should be considered in conservation and policy efforts. Environ Toxicol Chem 2016;35:2734-2741.

Biomarker analysis of liver cells exposed to surfactant-wrapped and oxidized multi-walled carbon nanotubes (MWCNTs)

Carbon nanotubes (CNTs) have great potential in industrial, consumer, and mechanical applications, based partly on their unique structural, optical and electronic properties. CNTs are commonly oxidized or treated with surfactants to facilitate aqueous solution processing, and these CNT surface modifications also increase possible human and ecological exposures to nanoparticle-contaminated waters. To determine the exposure outcomes of oxidized and surfactant-wrapped multiwalled carbon nanotubes (MWCNTs) on biochemical processes, metabolomics-based profiling of human liver cells (C3A) was utilized. Cells were exposed to 0, 10, or 100ng/mL of MWCNTs for 24 and 48h; MWCNT particle size distribution, charge, and aggregation were monitored concurrently during exposures. Following MWCNT exposure, cellular metabolites were extracted, lyophilized, and buffered for (1)H NMR analysis. Acquired spectra were subjected to both multivariate and univariate analysis to determine the consequences of nanotube exposure on the metabolite profile of C3A cells. Resulting scores plots illustrated temporal and dose-dependent metabolite responses to all MWCNTs tested. Loadings plots coupled with t-test filtered spectra identified metabolites of interest. XPS analysis revealed the presence of hydroxyl and carboxyl functionalities on both MWCNTs surfaces. Metal content analysis by ICP-AES indicated that the total mass concentration of the potentially toxic impurities in the exposure experiments were extremely low (i.e. [Ni]≤2×10(-10)g/mL). Preliminary data suggested that MWCNT exposure causes perturbations in biochemical processes involved in cellular oxidation as well as fluxes in amino acid metabolism and fatty acid synthesis. Dose-response trajectories were apparent and spectral peaks related to both dose and MWCNT dispersion methodologies were determined. Correlations of the significant changes in metabolites will help to identify potential biomarkers associated with carbonaceous nanoparticle exposure.