Potential biomarkers of endocrine and habitat disruption identified via RNA-Seq in Salvelinus fontinalis with proximity to fracking operations in Pennsylvania headwater stream ecosystems.

Unconventional natural gas development (fracking) has been a rapidly expanding technique used for the extraction of natural gas from the Marcellus Shale formation in Pennsylvania. There remains a knowledge gap regarding the ecological impacts of fracking, especially regarding the long-term health of native Brook trout (Salvelinus fontinalis) populations. During the summer of 2015, Brook trout were sampled from twelve streams located in forested, northwestern Pennsylvania in order to evaluate the impacts of fracking on Brook trout. Four stream sites were undisturbed (no fracking activity), three had a developed well pad without fracking activity, and five had active fracking with natural gas production. Liver tissue was isolated from two to five fish per stream and underwent RNA-Seq analysis to identify differentially expressed genes between ecosystems with differing fracking status. Data were analyzed individually and with samples pooled within-stream to account for hierarchical data structure and variation in sample coverage within streams. Differentially expressed and differentially alternatively spliced genes had functions related to lipid and steroid metabolism, mRNA processing, RNA polymerase and protein regulation. Unique to our study, genes related to xenobiotic and stress responses were found as well as potential markers for endocrine disruption and saline adaptation that were identified in watersheds with active fracking activity. These results support the utility of RNA-Seq to assess trout health and suggest detrimental impacts of fracking on sensitive trout populations.

Impact of Patient and Family Involvement in Long-Term Outcomes.

Surviving a critical illness can have long-term effects on both patients and families. These effects can be physical, emotional, cognitive, and social, and they affect both the patient and the family. Family members play a key role in helping their loved one recover, and this recovery process can take considerable time. Transferring out of an intensive care unit, and discharging home from a hospital, are important milestones, but they represent only the beginning of recovery and healing after a critical illness. Recognizing that these challenges exist both for patients and families is important to improve critical illness outcomes.

Microbial Communities Associated With Passive Acidic Abandoned Coal Mine Remediation.

Acid mine drainage (AMD) is an environmental issue that can be characterized by either acidic or circumneutral pH and high dissolved metal content in contaminated waters. It is estimated to affect roughly 3000 miles of waterways within the state of Pennsylvania, with half being acidic and half being circumneutral. To negate the harmful effects of AMD, ∼300 passive remediation systems have been constructed within the state of Pennsylvania. In this study, we evaluated the microbial community structure and functional capability associated with Middle Branch passive remediation system in central PA. Sediment and water samples were collected from each area within the passive remediation system and its receiving stream. Environmental parameters associated with the remediation system were found to explain a significant amount of variation in microbial community structure. This study revealed shifts in microbial community structure from acidophilic bacteria in raw AMD discharge to a more metabolically diverse set of taxa (i.e., Acidimicrobiales, Rhizobiales, Chthoniobacteraceae) toward the end of the system. Vertical flow ponds and the aerobic wetland showed strong metabolic capability for sulfur redox environments. These findings are integral to the understanding of designing effective passive remediation systems because it provides insight as to how certain bacteria [sulfate reducing bacteria (SRBs) and sulfur oxidizing bacteria (SOBs)] are potentially contributing to a microbially mediated AMD remediation process. This study further supports previous investigations that demonstrated the effectiveness of SRBs in the process of removing sulfate and heavy metals from contaminated water.

Surface Water Microbial Community Response to the Biocide 2,2-Dibromo-3-Nitrilopropionamide, Used in Unconventional Oil and Gas Extraction.

Production of unconventional oil and gas continues to rise, but the effects of high-density hydraulic fracturing (HF) activity near aquatic ecosystems are not fully understood. A commonly used biocide in HF, 2,2-dibromo-3-nitrilopropionamide (DBNPA), was studied in microcosms of HF-impacted (HF+) versus HF-unimpacted (HF-) surface water streams to (i) compare the microbial community response, (ii) investigate DBNPA degradation products based on past HF exposure, and (iii) compare the microbial community response differences and similarities between the HF biocides DBNPA and glutaraldehyde. The microbial community responded to DBNPA differently in HF-impacted versus HF-unimpacted microcosms in terms of the number of 16S rRNA gene copies quantified, alpha and beta diversity, and differential abundance analyses of microbial community composition through time. The differences in microbial community changes affected degradation dynamics. HF-impacted microbial communities were more sensitive to DBNPA, causing the biocide and by-products of the degradation to persist for longer than in HF-unimpacted microcosms. A total of 17 DBNPA by-products were detected, many of them not widely known as DBNPA by-products. Many of the brominated by-products detected that are believed to be uncharacterized may pose environmental and health impacts. Similar taxa were able to tolerate glutaraldehyde and DBNPA; however, DBNPA was not as effective for microbial control, as indicated by a smaller overall decrease of 16S rRNA gene copies/ml after exposure to the biocide, and a more diverse set of taxa was able to tolerate it. These findings suggest that past HF activity in streams can affect the microbial community response to environmental perturbation such as that caused by the biocide DBNPA.IMPORTANCE Unconventional oil and gas activity can affect pH, total organic carbon, and microbial communities in surface water, altering their ability to respond to new environmental and/or anthropogenic perturbations. These findings demonstrate that 2,2-dibromo-3-nitrilopropionamide (DBNPA), a common hydraulic fracturing (HF) biocide, affects microbial communities differently as a consequence of past HF exposure, persisting longer in HF-impacted (HF+) waters. These findings also demonstrate that DBNPA has low efficacy in environmental microbial communities regardless of HF impact. These findings are of interest, as understanding microbial responses is key for formulating remediation strategies in unconventional oil and gas (UOG)-impacted environments. Moreover, some DBNPA degradation by-products are even more toxic and recalcitrant than DBNPA itself, and this work identifies novel brominated degradation by-products formed.

Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania.

Unconventional oil and gas (UOG) extraction, also known as hydraulic fracturing, is becoming more prevalent with the increasing use and demand for natural gas; however, the full extent of its environmental impacts is still unknown. Here we measured physicochemical properties and bacterial community composition of sediment samples taken from twenty-eight streams within the Marcellus shale formation in northeastern Pennsylvania differentially impacted by hydraulic fracturing activities. Fourteen of the streams were classified as UOG+, and thirteen were classified as UOG- based on the presence of UOG extraction in their respective watersheds. One stream was located in a watershed that previously had UOG extraction activities but was recently abandoned. We utilized high-throughput sequencing of the 16S rRNA gene to infer differences in sediment aquatic bacterial community structure between UOG+ and UOG- streams, as well as correlate bacterial community structure to physicochemical water parameters. Although overall alpha and beta diversity differences were not observed, there were a plethora of significantly enriched operational taxonomic units (OTUs) within UOG+ and UOG- samples. Our biomarker analysis revealed many of the bacterial taxa enriched in UOG+ streams can live in saline conditions, such as Rubrobacteraceae. In addition, several bacterial taxa capable of hydrocarbon degradation were also enriched in UOG+ samples, including Oceanospirillaceae. Methanotrophic taxa, such as Methylococcales, were significantly enriched as well. Several taxa that were identified as enriched in these samples were enriched in samples taken from different streams in 2014; moreover, partial least squares discriminant analysis (PLS-DA) revealed clustering between streams from the different studies based on the presence of hydraulic fracturing along the second axis. This study revealed significant differences between bacterial assemblages within stream sediments of UOG+ and UOG- streams and identified several potential biomarkers for evaluating and monitoring the response of autochthonous bacterial communities to potential hydraulic fracturing impacts.

Impacts of Glutaraldehyde on Microbial Community Structure and Degradation Potential in Streams Impacted by Hydraulic Fracturing.

The environmental impacts of hydraulic fracturing, particularly those of surface spills in aquatic ecosystems, are not fully understood. The goals of this study were to (1) understand the effect of previous exposure to hydraulic fracturing fluids on aquatic microbial community structure and (2) examine the impacts exposure has on biodegradation potential of the biocide glutaraldehyde. Microcosms were constructed from hydraulic fracturing-impacted and nonhydraulic fracturing-impacted streamwater within the Marcellus shale region in Pennsylvania. Microcosms were amended with glutaraldehyde and incubated aerobically for 56 days. Microbial community adaptation to glutaraldehyde was monitored using 16S rRNA gene amplicon sequencing and quantification by qPCR. Abiotic and biotic glutaraldehyde degradation was measured using ultra-performance liquid chromatography--high resolution mass spectrometry and total organic carbon. It was found that nonhydraulic fracturing-impacted microcosms biodegraded glutaraldehyde faster than the hydraulic fracturing-impacted microcosms, showing a decrease in degradation potential after exposure to hydraulic fracturing activity. Hydraulic fracturing-impacted microcosms showed higher richness after glutaraldehyde exposure compared to unimpacted streams, indicating an increased tolerance to glutaraldehyde in hydraulic fracturing impacted streams. Beta diversity and differential abundance analysis of sequence count data showed different bacterial enrichment for hydraulic fracturing-impacted and nonhydraulic fracturing-impacted microcosms after glutaraldehyde addition. These findings demonstrated a lasting effect on microbial community structure and glutaraldehyde degradation potential in streams impacted by hydraulic fracturing operations.

Response of Aquatic Bacterial Communities to Hydraulic Fracturing in Northwestern Pennsylvania: A Five-Year Study.

Horizontal drilling and hydraulic fracturing extraction procedures have become increasingly present in Pennsylvania where the Marcellus Shale play is largely located. The potential for long-term environmental impacts to nearby headwater stream ecosystems and aquatic bacterial assemblages is still incompletely understood. Here, we perform high-throughput sequencing of the 16 S rRNA gene to characterize the bacterial community structure of water, sediment, and other environmental samples (n = 189) from 31 headwater stream sites exhibiting different histories of fracking activity in northwestern Pennsylvania over five years (2012-2016). Stream pH was identified as a main driver of bacterial changes within the streams and fracking activity acted as an environmental selector for certain members at lower taxonomic levels within stream sediment. Methanotrophic and methanogenic bacteria (i.e. Methylocystaceae, Beijerinckiaceae, and Methanobacterium) were significantly enriched in sites exhibiting Marcellus shale activity (MSA+) compared to MSA- streams. This study highlighted potential sentinel taxa associated with nascent Marcellus shale activity and some of these taxa remained as stable biomarkers across this five-year study. Identifying the presence and functionality of specific microbial consortia within fracking-impacted streams will provide a clearer understanding of the natural microbial community's response to fracking and inform in situ remediation strategies.

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations.

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal behavior questions. The protocol described here provides a simple, reproducible, and controlled method to manipulate DO to study behavioral response in aquatic organisms resulting from hypoxic and anoxic conditions. While performing degasification of water with nitrogen is commonly used in laboratory settings, no explicit method for ecological (aquatic) application exists in the literature, and this protocol is the first to describe a protocol to degasify water to observe organismal response. This technique and protocol were developed for direct application for aquatic macroinvertebrates; however, small fish, amphibians, and other aquatic vertebrates could be easily substituted. It allows for easy manipulation of DO levels ranging from 2 mg/L to 11 mg/L with stability for up to a 5 min animal-observation period. Beyond a 5 min observation period water temperatures began to rise, and at 10 min DO levels became too unstable to maintain. The protocol is scalable to the study organism, reproducible, and reliable, allowing for rapid implementation into introductory teaching labs and high-level research applications. The expected results of this technique should relate dissolved oxygen changes to behavioral responses of organisms.

Marcellus and mercury: Assessing potential impacts of unconventional natural gas extraction on aquatic ecosystems in northwestern Pennsylvania.

Mercury (Hg) is a persistent element in the environment that has the ability to bioaccumulate and biomagnify up the food chain with potentially harmful effects on ecosystems and human health. Twenty-four streams remotely located in forested watersheds in northwestern PA containing naturally reproducing Salvelinus fontinalis (brook trout), were targeted to gain a better understanding of how Marcellus shale natural gas exploration may be impacting water quality, aquatic biodiversity, and Hg bioaccumulation in aquatic ecosystems. During the summer of 2012, stream water, stream bed sediments, aquatic mosses, macroinvertebrates, crayfish, brook trout, and microbial samples were collected. All streams either had experienced hydraulic fracturing (fracked, n = 14) or not yet experienced hydraulic fracturing (non-fracked, n = 10) within their watersheds at the time of sampling. Analysis of watershed characteristics (GIS) for fracked vs non-fracked sites showed no significant differences (P > 0.05), justifying comparisons between groups. Results showed significantly higher dissolved total mercury (FTHg) in stream water (P = 0.007), lower pH (P = 0.033), and higher dissolved organic matter (P = 0.001) at fracked sites. Total mercury (THg) concentrations in crayfish (P = 0.01), macroinvertebrates (P = 0.089), and predatory macroinvertebrates (P = 0.039) were observed to be higher for fracked sites. A number of positive correlations between amount of well pads within a watershed and THg in crayfish (r = 0.76, P < 0.001), THg in predatory macroinvertebrates (r = 0.71, P < 0.001), and THg in brook trout (r = 0.52, P < 0.01) were observed. Stream-water microbial communities within the Deltaproteobacteria also shared a positive correlation with FTHg and to the number of well pads, while stream pH (r = -0.71, P < 0.001), fish biodiversity (r = -0.60, P = 0.02), and macroinvertebrate taxa richness (r = -0.60, P = 0.01) were negatively correlated with the number of well pads within a watershed. Further investigation is needed to better elucidate relationships and pathways of observed differences in stream water chemistry, biodiversity, and Hg bioaccumulation, however, initial findings suggest Marcellus shale natural gas exploration is having an effect on aquatic ecosystems.

Assessing impacts of unconventional natural gas extraction on microbial communities in headwater stream ecosystems in Northwestern Pennsylvania.

Hydraulic fracturing and horizontal drilling have increased dramatically in Pennsylvania Marcellus shale formations, however the potential for major environmental impacts are still incompletely understood. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community structure of water, sediment, bryophyte, and biofilm samples from 26 headwater stream sites in northwestern Pennsylvania with different histories of fracking activity within Marcellus shale formations. Further, we describe the relationship between microbial community structure and environmental parameters measured. Approximately 3.2 million 16S rRNA gene sequences were retrieved from a total of 58 samples. Microbial community analyses showed significant reductions in species richness as well as evenness in sites with Marcellus shale activity. Beta diversity analyses revealed distinct microbial community structure between sites with and without Marcellus shale activity. For example, operational taxonomic units (OTUs) within the Acetobacteracea, Methylocystaceae, Acidobacteriaceae, and Phenylobacterium were greater than three log-fold more abundant in MSA+ sites as compared to MSA- sites. Further, several of these OTUs were strongly negatively correlated with pH and positively correlated with the number of wellpads in a watershed. It should be noted that many of the OTUs enriched in MSA+ sites are putative acidophilic and/or methanotrophic populations. This study revealed apparent shifts in the autochthonous microbial communities and highlighted potential members that could be responding to changing stream conditions as a result of nascent industrial activity in these aquatic ecosystems.