Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism.

We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n = 35,584 total samples, 11,986 with ASD). Using an enhanced analytical framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate of 0.1 or less. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained to have severe neurodevelopmental delay, whereas 53 show higher frequencies in individuals ascertained to have ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In cells from the human cortex, expression of risk genes is enriched in excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory-inhibitory imbalance underlying ASD.

Predicting Splicing from Primary Sequence with Deep Learning.

The splicing of pre-mRNAs into mature transcripts is remarkable for its precision, but the mechanisms by which the cellular machinery achieves such specificity are incompletely understood. Here, we describe a deep neural network that accurately predicts splice junctions from an arbitrary pre-mRNA transcript sequence, enabling precise prediction of noncoding genetic variants that cause cryptic splicing. Synonymous and intronic mutations with predicted splice-altering consequence validate at a high rate on RNA-seq and are strongly deleterious in the human population. De novo mutations with predicted splice-altering consequence are significantly enriched in patients with autism and intellectual disability compared to healthy controls and validate against RNA-seq in 21 out of 28 of these patients. We estimate that 9%-11% of pathogenic mutations in patients with rare genetic disorders are caused by this previously underappreciated class of disease variation.

Environmental Impacts of Coal Combustion Residuals: Current Understanding and Future Perspectives.

On-site solid-waste impoundments, landfills, and receiving water bodies have served as long-term disposal sites for coal combustion residuals (CCRs) across the United States for decades and collectively contain billions of tons of CCR material. CCR components include fine particulate material, minerals, and trace elements such as mercury, arsenic, selenium, lead, etc., which can have deleterious effects on ecosystem functioning and public health. Effects on communities can occur through consumption of drinking water, fish, and other aquatic organisms. The structural failure of impoundments, water infiltration, leakage from impoundments due to poor construction and monitoring, and CCR effluent discharges to water bodies have in the past resulted in harmful environmental impacts. Moreover, the risks posed by CCRs are present to this day, as coal continues to account for 11% of the energy production in the United States. In this Critical Review, the legacy of CCR disposal and the concomitant risks posed to public health and ecosystems are assessed. The resiliency of CCR disposal sites in the context of increased frequency and intensity of storm events and other hazards, such as floods and earthquakes, is also evaluated. We discuss the current state of knowledge on the environmental fate of CCR-derived elements, as well as advances in and limitations of analytical tools, which can improve the current understanding of CCR environmental impacts in order to mitigate the associated risks. An assessment of the 2015 Coal Ash Final Rule is also presented, along with needs to improve monitoring of CCR disposal sites and regulatory enforcement.

Ranking Coal Ash Materials for Their Potential to Leach Arsenic and Selenium: Relative Importance of Ash Chemistry and Site Biogeochemistry.

The chemical composition of coal ash is highly heterogeneous and dependent on the origin of the source coal, combustion parameters, and type and configuration of air pollution control devices. This heterogeneity results in uncertainty in the evaluation of leaching potential of contaminants from coal ash. The goal of this work was to identify whether a single leaching protocol could roughly group high-leaching potential coal ash from low-leaching potential coal ash, with respect to arsenic (As) and selenium (Se). We used four different leaching tests, including the Toxicity Characteristic Leaching Protocol (TCLP), natural pH, aerobic sediment microcosms, and anaerobic sediment microcosms on 10 different coal ash materials, including fly ash, lime-treated ash, and flue gas desulfurization materials. Leaching tests showed promise in categorizing high and low-leaching potential ash materials, indicating that a single point test could act as a first screening measure to identify high-risk ash materials. However, the amount of contaminant leached varied widely across tests, reflecting the importance of ambient conditions (pH, redox state) on leaching. These results demonstrate that on-site geochemical conditions play a critical role in As and Se mobilization from coal ash, underscoring the need to develop a situation-based risk assessment framework for contamination by coal ash pollutants.

Induced pluripotent stem cell line generated from a patient with differences in sex development (DSD) and multiple genetic variants including a large deletion in NR5A1.

The nuclear receptor subfamily 5, Group A, Member 1 (NR5A1) gene encodes steroidogenic factor 1 (SF1), which is necessary for development of steroid hormone-producing tissues including the gonad and adrenal gland. An induced pluripotent stem cell line (iPSC) LCHi002-B was generated from a participant with differences (disorders) of sex development (DSD) and multiple genetic variants including a large deletion in NR5A1, and three single nucleotide changes in DYNC2H1, PDE4D, and ZFPM2. The line presented typical morphology, expressed stem cell markers, differentiated into three germ layers, had normal karyotype, was mycoplasma-free, and carried mutations in NR5A1, DYNC2H1, PDE4D, and ZFPM2.

The impact of coal combustion residue effluent on water resources: a North Carolina example.

The combustion of coal to generate electricity produces about 130 million tons of coal combustion residues (CCRs) each year in the United States; yet their environmental implications are not well constrained. This study systematically documents the quality of effluents discharged from CCR settling ponds or cooling water at ten sites and the impact on associated waterways in North Carolina, compared to a reference lake. We measured the concentrations of major and trace elements in over 300 samples from CCR effluents, surface water from lakes and rivers at different downstream and upstream points, and pore water extracted from lake sediments. The data show that CCR effluents contain high levels of contaminants that in several cases exceed the U.S. EPA guidelines for drinking water and ecological effects. This investigation demonstrates the quality of receiving waters in North Carolina depends on (1) the ratio between effluent flux and freshwater resource volumes and (2) recycling of trace elements through adsorption on suspended particles and release to deep surface water or pore water in bottom sediments during periods of thermal water stratification and anoxic conditions. The impact of CCRs is long-term, which influences contaminant accumulation and the health of aquatic life in water associated with coal-fired power plants.

Early Life Low-Calorie Sweetener Consumption Impacts Energy Balance during Adulthood.

Children frequently consume beverages that are either sweetened with sugars (sugar-sweetened beverages; SSB) or low-calorie sweeteners (LCS). Here, we evaluated the effects of habitual early life consumption of either SSB or LCS on energy balance later during adulthood. Male and female rats were provided with chow, water, and a solution containing either SSB (sucrose), LCS (acesulfame potassium (ACE-K) or stevia), or control (no solution) during the juvenile and adolescent periods (postnatal days 26-70). SSB or LCS consumption was voluntary and restricted within the recommended federal daily limits. When subsequently maintained on a cafeteria-style junk food diet (CAF; various high-fat, high-sugar foods) during adulthood, ACE-K-exposed rats demonstrated reduced caloric consumption vs. the controls, which contributed to lower body weights in female, but not male, ACE-K rats. These discrepant intakes and body weight effects in male ACE-K rats are likely to be based on reduced gene expression of thermogenic indicators (UCP1, BMP8B) in brown adipose tissue. Female stevia-exposed rats did not differ from the controls in terms of caloric intake or body weight, yet they consumed more SSB during CAF exposure in adulthood. None of the SSB-exposed rats, neither male nor female, differed from the controls in terms of total adult caloric consumption or body weight measures. The collective results reveal that early life LCS consumption alters sugar preference, body weight, and gene expression for markers of thermogenesis during adulthood, with both sex- and sweetener-dependent effects.

Exome sequencing of fetuses with congenital diaphragmatic hernia supports a causal role for NR2F2, PTPN11, and WT1 variants.

BACKGROUND: To identify genes associated with congenital diaphragmatic hernia (CDH) to help understand the etiology and inform prognosis. METHODS: We performed exome sequencing on fetuses with CDH and their parents to identify rare genetic variants likely to mediate risk. We reviewed prenatal characteristics and neonatal outcomes. RESULTS: Data were generated for 22 parent-offspring trios. Six Likely Damaging (LD) variants were identified in five families (23 %). Three LD variants were in genes that contain variants in other CDH cohorts (NR2F2, PTPN11, WT1), while three were in genes that do not (CTR9, HDAC6, TP53). Integrating these data bolsters the evidence of association of NR2F2, PTPN11, and WT1 with CDH in humans. Of the five fetuses with a genetic diagnosis, one was terminated, two underwent perinatal demise, while two survived until repair. CONCLUSIONS: Exome sequencing expands the diagnostic yield of genetic testing in CDH. Correlating CDH patients' exomes with clinical outcomes may enable personalized counseling and therapies.

Incorporating concentration-dependent sediment microbial activity into methylmercury production kinetics modeling.

In anoxic environments, anaerobic microorganisms carrying the hgcAB gene cluster can mediate the transformation of inorganic mercury (Hg(II)) to monomethylmercury (MMHg). The kinetics of Hg(II) transformation to MMHg in periphyton from East Fork Poplar Creek (EFPC) in Oak Ridge, TN have previously been modeled using a transient availability model (TAM). The TAM for Hg(II) methylation combines methylation/demethylation kinetics with kinetic expressions for processes that decrease Hg(II) and MMHg availability for methylation and demethylation (multisite sorption of Hg(II) and MMHg, Hg(II) reduction/Hg(0) oxidation). In this study, the TAM is used for the first time to describe MMHg production in sediment. We assessed MMHg production in sediment microcosms using two different sediment types from EFPC: a relatively anoxic, carbon-rich sediment with higher microbial activity (higher CO2 production from sediment) and a relatively oxic, sandy, carbon-poor sediment with lower microbial activity (lower CO2 production from sediment). Based on 16s rRNA sequencing, the overall microbial community structure in the two sediments was retained during the incubations. However, the hgcA containing methanogenic Euryarchaeota communities differed between sediment types and their growth followed different trajectories over the course of incubations, potentially contributing to the distinct patterns of MMHg production observed. The general TAM paradigm performed well in describing MMHg production in the sediments. However, the MMHg production and ancillary data suggested the need to revise the model structure to incorporate terms for concentration-dependent microbial activity over the course of the incubations. We modified the TAM to include Monod-type kinetics for methylation and demethylation and observed an improved fit for the carbon-rich, microbially active sediment. Overall our work shows that the TAM can be applied to describe Hg(II) methylation in sediments and that including expressions accounting for concentration-dependent microbial activity can improve the accuracy of the model description of the data in some cases.

Generation of two human induced pluripotent stem cell lines from a patient with complete androgen insensitivity syndrome with a hemizygous single nucleotide variant in the androgen receptor (AR) gene.

Complete Androgen Insensitivity Syndrome (CAIS) is a difference of sex development (DSD) caused by loss of function of the androgen receptor (AR) gene. Patients typically identify as female and have a 46,XY karyotype. Two induced pluripotent stem cell lines (iPSCs), LCHi001-A and LCHi001-B, were generated from a participant with CAIS with AR mutation: c.2698A>T (p.Ile900Phe). Both lines presented typical morphology, expressed stem cell markers, differentiated into three germ layers, had a normal 46,XY karyotype, were mycoplasma-free, and carried the expected mutation in AR. These iPSC lines are an important resource for studying CAIS pathogenesis and possible treatment options.

Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed.

The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennessee (United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.

Constructing and optimizing 3D atlases from 2D data with application to the developing mouse brain.

3D imaging data necessitate 3D reference atlases for accurate quantitative interpretation. Existing computational methods to generate 3D atlases from 2D-derived atlases result in extensive artifacts, while manual curation approaches are labor-intensive. We present a computational approach for 3D atlas construction that substantially reduces artifacts by identifying anatomical boundaries in the underlying imaging data and using these to guide 3D transformation. Anatomical boundaries also allow extension of atlases to complete edge regions. Applying these methods to the eight developmental stages in the Allen Developing Mouse Brain Atlas (ADMBA) led to more comprehensive and accurate atlases. We generated imaging data from 15 whole mouse brains to validate atlas performance and observed qualitative and quantitative improvement (37% greater alignment between atlas and anatomical boundaries). We provide the pipeline as the MagellanMapper software and the eight 3D reconstructed ADMBA atlases. These resources facilitate whole-organ quantitative analysis between samples and across development.

The research community needs precise, reliable 3D atlases of organs to pinpoint where biological structures and processes are located. For instance, these maps are essential to understand where specific genes are turned on or off, or the spatial organization of various groups of cells over time. For centuries, atlases have been built by thinly 'slicing up' an organ, and then precisely representing each 2D layer. Yet this approach is imperfect: each layer may be accurate on its own, but inevitable mismatches appear between the slices when viewed in 3D or from another angle. Advances in microscopy now allow entire organs to be imaged in 3D. Comparing these images with atlases could help to detect subtle differences that indicate or underlie disease. However, this is only possible if 3D maps are accurate and do not feature mismatches between layers. To create an atlas without such artifacts, one approach consists in starting from scratch and manually redrawing the maps in 3D, a labor-intensive method that discards a large body of well-established atlases. Instead, Young et al. set out to create an automated method which could help to refine existing 'layer-based' atlases, releasing software that anyone can use to improve current maps. The package was created by harnessing eight atlases in the Allen Developing Mouse Brain Atlas, and then using the underlying anatomical images to resolve discrepancies between layers or fill out any missing areas. Known as MagellanMapper, the software was extensively tested to demonstrate the accuracy of the maps it creates, including comparison to whole-brain imaging data from 15 mouse brains. Armed with this new software, researchers can improve the accuracy of their atlases, helping them to understand the structure of organs at the level of the cell and giving them insight into a broad range of human disorders.

Clinical impact of splicing in neurodevelopmental disorders.

Clinical exome sequencing is frequently used to identify gene-disrupting variants in individuals with neurodevelopmental disorders. While splice-disrupting variants are known to contribute to these disorders, clinical interpretation of cryptic splice variants outside of the canonical splice site has been challenging. Here, we discuss papers that improve such detection.

Development of a Novel Equilibrium Passive Sampling Device for Methylmercury in Sediment and Soil Porewaters.

We explored the concept of equilibrium passive sampling for methylmercury (MeHg) using the strategy developed for hydrophobic organic chemicals. Passive sampling should allow prediction of the concentration of the chemically labile fraction of MeHg in sediment porewaters based on equilibrium partitioning into the sampler, without modeling diffusion rates through the sampler material. Our goals were to identify sampler materials with the potential to mimic MeHg partitioning into animals and sediments and provide reversible sorption in a time frame appropriate for in situ samplers. Candidate materials tested included a range of polymers embedded with suitable sorbents for MeHg. The most promising were activated carbon (AC) embedded in agarose, thiol-self-assembled monolayers on mesoporous supports embedded in agarose, and cysteine-functionalized polyethylene terephthalate, which yielded log sampler-water partition coefficients of 2.8 to 5 for MeHgOH and MeHg complexed with dissolved organic matter (Suwannee River humic acid). Sampler equilibration time in sediments was approximately 1 to 2 wk. Investigation of the MeHg accumulation mechanism by AC embedded in agarose suggested that sampling was kinetically influenced by MeHg interactions with AC particles and not limited by diffusion through the gel for this material. Also, AC exhibited relatively rapid desorption of Hg and MeHg, indicating that this sorbent is capable of reversible, equilibrium measurements. In sediment:water microcosms, porewater concentrations made with isotherm-calibrated passive samplers agreed within a factor of 2 (unamended sediment) or 4 (AC-amended sediment) with directly measured concentrations. The present study demonstrates a potential new approach to passive sampling of MeHg. Environ Toxicol Chem 2020;39:323-334. © 2019 SETAC.

Ecosystem Controls on Methylmercury Production by Periphyton Biofilms in a Contaminated Stream: Implications for Predictive Modeling.

Periphyton biofilms produce a substantial fraction of the overall monomethylmercury (MMHg) flux in East Fork Poplar Creek, an industrially contaminated, freshwater creek in Oak Ridge, Tennessee. We examined periphyton MMHg production across seasons, locations, and light conditions using mercury stable isotopes. Methylation and demethylation rate potentials (km, trans av and kd, trans av , respectively) were calculated using a transient availability kinetic model. Light exposure and season were significant predictors of km, trans av , with greater values in full light exposure and in the summer. Season, light exposure, and location were significant predictors of kd, trans av , which was highest in dark conditions, in the spring, and at the upstream location. Light exposure was the controlling factor for net MMHg production, with positive production for periphyton grown under full light exposure and net demethylation for periphyton grown in the dark. Ambient MMHg and km, trans av were significantly correlated. Transient availability rate potentials were 15 times higher for km and 9 times higher for kd compared to full availability rate potentials (km, full av and kd, full av ) calculated at 1 d. No significant model for the prediction of km, full av or kd, full av could be constructed using light, season, and location. In addition, there were no significant differences among treatments for the full availability km, full av , kd, full av , or net MMHg calculated using the full availability rate potentials. km, full av was not correlated with ambient MMHg concentrations. The present results underscore the importance of applying transient availability kinetics to MMHg production data when estimating MMHg production potential and flux. Environ Toxicol Chem 2019;38:2426-2435. © 2019 SETAC.

Modifying emergency department electronic prescribing for outpatient opioid analgesia.

OBJECTIVES: The aim of this study was to examine how changing the electronic ordering sequences for opioid analgesics affected ED outpatient prescribing, and subsequent unused opioid tablets remaining in the community available for diversion. METHODS: A descriptive before and after study in adult patients prescribed an opioid analgesic by an ED prescriber for use in the outpatient setting. The hospital electronic prescribing system (FirstNet™) was modified to include smaller quantities of opioid analgesics for discharge. The change in quantity of opioid prescribed and change in quantity of opioid analgesic remaining in the community at follow up was measured pre- and post-intervention using a structured telephone interview. RESULTS: Pre- and post-intervention, 102 and 106 patients were interviewed, respectively. Percentage of prescriptions for oxycodone quantity five tablets increased from 3% to 32% and for quantity 20 tablets fell from 40% to 24% post-intervention. For paracetamol with codeine, prescriptions for quantity 10 tablets increased from 2% to 24% while for quantity 20 tablets fell from 98% to 76%. Mean number of tablets prescribed per patient fell from 13.8 (SD = 5.1) to 10.8 (SD = 5.6) for oxycodone and from 19.8 (SD = 1.5) to 17.6 (SD = 4.2) for paracetamol with codeine. Fifty-eight percent of patients in both pre- and post-intervention groups used half or less of the medication prescribed. CONCLUSION: Modification of an ED electronic prescribing system reduced overall quantities of opioid analgesics supplied and subsequently stored in the community but did not change the proportion of patients (>50%) who reported using half or less of their prescribed opioid medication.

Impact of dissolved organic matter on mercury and methylmercury sorption to activated carbon in soils: implications for remediation.

Activated carbon (AC) amendments have shown promise in reducing inorganic mercury (Hg(ii) complexes, "Hg") and methylmercury (MeHg) risk in contaminated soils. However, the effectiveness of AC in Hg and MeHg immobilization has varied among studies, suggesting that site biogeochemistry might dictate efficacy. In this study, we examined the effect of dissolved organic matter (DOM) on MeHg and Hg sorption to AC. We evaluated the impact of Suwannee River Humic Acid (SRHA) on sorption to AC directly using an isotherm approach and in a soil/AC mixture using slurry microcosms. Aqueous sorption coefficients to AC (log KAC) for Hg-SRHA and MeHg-SRHA complexes were one to two orders of magnitude lower (Hg-SRHA = 4.53, MeHgSRHA = 4.35) than those for chloride complexes (HgCl2 = 6.55, MeHgCl = 4.90) and more closely resembled the log KAC of SRHA (3.64). In anoxic, sulfidic soil slurries, the KAC for sulfide species appeared stronger than for chloride or SRHA species for both Hg and MeHg. AC significantly reduced porewater concentrations of both ambient MeHg and a fresh Me199Hg spike, and the addition of up to 60 mg L-1 SRHA did not reduce sorption to AC. The AC also reduced ambient Hg and 201Hg porewater concentrations, but as SRHA concentration increased, the magnitude of solid phase sorption decreased. Speciation modeling revealed that SRHA may have impacted Hg distribution to the solid phase by reducing HgS precipitation. This study highlights the need for site-specific evaluation of AC efficacy and the value in developing biogeochemical models of AC performance for Hg control.

Genome-wide de novo risk score implicates promoter variation in autism spectrum disorder.

Whole-genome sequencing (WGS) has facilitated the first genome-wide evaluations of the contribution of de novo noncoding mutations to complex disorders. Using WGS, we identified 255,106 de novo mutations among sample genomes from members of 1902 quartet families in which one child, but not a sibling or their parents, was affected by autism spectrum disorder (ASD). In contrast to coding mutations, no noncoding functional annotation category, analyzed in isolation, was significantly associated with ASD. Casting noncoding variation in the context of a de novo risk score across multiple annotation categories, however, did demonstrate association with mutations localized to promoter regions. We found that the strongest driver of this promoter signal emanates from evolutionarily conserved transcription factor binding sites distal to the transcription start site. These data suggest that de novo mutations in promoter regions, characterized by evolutionary and functional signatures, contribute to ASD.

An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder.

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.

Impacts of coal ash on methylmercury production and the methylating microbial community in anaerobic sediment slurries.

Mercury (Hg) associated with coal ash is an environmental concern, particularly if the release of coal ash to the environment is associated with the conversion of inorganic Hg to methylmercury (MeHg), a bioaccumulative form of Hg that is produced by anaerobic microorganisms. In this study, sediment slurry microcosm experiments were performed to understand how spilled coal ash might influence MeHg production in anaerobic sediments of an aquatic ecosystem. Two coal ash types were used: (1) a weathered coal ash; and (2) a freshly collected, unweathered fly ash that was relatively enriched in sulfate and Hg compared to the weathered ash. These ash samples were added to anaerobic sediment slurries constructed with a relatively pristine sediment (containing 0.03 mg kg-1 Hg) and a Hg-contaminated sediment (containing 0.29 mg kg-1 Hg). The results of these experiments showed negligible net production of MeHg in microcosms with no ash and in microcosms amended with the low sulfate/low Hg ash. In contrast, slurry microcosms amended with high sulfate/high Hg ash showed increases in total MeHg content that was 2 to 3 times greater than control microcosms without ash (p < 0.001). 16S amplicon sequencing of microbial communities in the slurries indicated that the coal ash addition generally increased the relative abundance of the methylating microbial community, including sulfate-reducing bacteria and iron-reducing bacteria species that are known to be efficient methylators of Hg. The stimulation of these microorganisms was likely caused by the release of substrates (sulfate and Fe) originating from the ash. Overall, the results highlight the need to incorporate both environmental parameters and coal ash characteristics into risk assessments that guide coal ash management and disposal.