Microplastic concentration, characterization, and size distribution in the Delaware Bay estuary.

Microplastic (MP) pollution has been widely reported across water matrices including in estuaries, which are important for the understanding of oceanic MPs. Estuaries can greatly alter the fate, transport, size distribution, and abundance of plastic pollution. The aim of this study was to quantify and characterize MP pollution in the Delaware Bay estuary USA, including the size distribution. Samples (N = 31) were collected from the mouth of the Delaware River to the coastal ocean including multiple frontal zones across two sampling campaigns (2019 and 2022). MP were extracted from the collected particles using wet peroxide oxidation and density separation with saturated sodium chloride. Particles collected on 500 µm mesh sieves were analyzed via Fourier transform infrared (FTIR) spectroscopy. Across all samples, 324 of the 1015 particles analyzed were MP, and 11 macroplastics were observed. MP concentrations ranged from below detection to 4.12 MP/m3 (mean 0.34 ± 0.80 MP/m3). No significant differences were observed between sampling sites; nonetheless, the two highest MP concentrations were observed when sampling along frontal zones with visible debris including macroplastics. Polyethylene (53%) and polypropylene (43%) were the most abundant polymers observed. The majority of the non-plastic particles were classified as particulate natural organic matter (82% of non-plastics). Particles from samples collected during 2022 (N = 864) also had color, morphology, and two size dimensions recorded. MP particle size was significantly associated with sampling site, with the coastal ocean sampling site generally having the smallest MPs. A correlation between total post-extraction particles and total plastic particles was observed. Aspect ratios for the plastics ranged from one to 40.7, with larger ratios for fibers, with a mean (±standard deviation) of 3.39 ± 4.72 (unitless). These aspect ratios can be used to select shape factors used to estimate the total volume of MP in the studied size range. Overall, these results can help inform fate, transport, and risk assessments related to estuarine plastic pollution.

Stormwater runoff microplastics: Polymer types, particle size, and factors controlling loading rates.

Stormwater runoff is a pathway of entry for microplastics (MPs, plastics <5 mm) into aquatic ecosystems. The objectives of this study were to determine MP size, morphology, chemical composition, and loading across urban storm events. Particles were extracted from stormwater samples collected at outfall locations using wet peroxide oxidation and cellulose digestion followed by analysis via attenuated total reflectance (ATR) FTIR. Concentrations observed were 0.99 ± 1.10 MP/L for 500-1000 µm and 0.41 ± 0.30 MP/L for the 1000-5000 µm size ranges. Seventeen different polymer types were observed. MP particle sizes measured using a FTIR-microscope camera indicated non-target size particles based on sieve-size classification, highlighting a potential source of error in studies reporting concentration by size class. A maximum MP load of 38.3 MP/m2 of upstream catchment was calculated. MP loadings had moderate correlations with both rainfall accumulation and intensity (Kendall τ = 0.54 and 0.42, respectively, both p ≤ 0.005). First flush (i.e. rapid wash-off of pollutants from watershed surfaces during rainfall early stages) was not always observed, and antecedent dry days were not correlated with MP abundance, likely due to the short dry periods between sampling events. Overall, the results presented provide data for risk assessment and mitigation strategies.

Quantitative and qualitative impacts of nitric acid digestion on microplastic identification via FTIR and Raman spectroscopy, implications for environmental samples.

Quantification and characterization of microplastics, synthetic polymers less than 5 mm in diameter, requires extraction methods that can reduce non-plastic debris without loss or alteration of the polymers. Nitric acid has been used to extract plastic particles from zooplankton and other biota because it completely digests tissue and exoskeletons, thus reducing interferences. While the impact of acid digestion protocols on several polymers has been demonstrated, advice for quantifying microplastic and interpreting their spectra following nitric acid digestion is lacking. Fourier transform infrared (FTIR) and/or Raman spectroscopy was performed on plastics from > 50 common consumer products (including a variety of textiles) pre- and post-nitric acid treatment. The percent match and assigned polymer were tabulated to compare the accuracy of spectral identification before and after nitric acid digestion via two open spectral analysis software. Nylon-66, polyoxymethylene, polyurethane, polyisoprene, nitrile rubber, and polymethyl methacrylate had ≥ 90% mass loss in nitric acid. Other less-impacted polymers changed color, morphology, and/or size following digestion. Thus, using nitric acid digestion for microplastic extraction can impact our understanding of the particle sizes and morphologies ingested in situ. Spectral analysis results were compiled to understand how often (1) the best-hit matches were correct (30-60% of spectra), (2) the best-hit matches exceeding the (arbitrary) threshold of 65% match were correct (53-78% of spectra), and (3) the best-hit matches for anthropogenic polymers were incorrectly identified as natural polymers (12-15% of spectra). Based on these results, advice is provided on how nitric acid digestion can impact microplastics as well as spectral interpretation.

Urban stormwater microplastic size distribution and impact of subsampling on polymer diversity.

Understanding not only microplastic (MP) concentration but also size distribution, morphology, and polymer profiles is desirable for stormwater, which is an important pathway of entry for MP into the aquatic environment. A challenge is that subsampling is often required for analysis of environmental samples and the impact of subsampling on the stormwater MP concentration determined and the polymer types identified is poorly characterized. To address this, MP were extracted from urban and suburban stormwater, including from green infrastructure. Fourier Transform Infrared microscopy was performed to characterize MP. In addition, particle dimensions and morphology were recorded. Varying the number of 63-250 µm particles subsampled per sample demonstrated the coefficient of variation for concentration (standard deviation/mean) for most samples was <0.3 when 20 particles (0.8-15% of total particles) or <0.2 when 30 particles (1.2-24% of total particles) per sample were analyzed. MP concentrations in the 63-250 µm size class ranged from 15 to 303 MP/L, one to two orders of magnitude greater than observed in previously reported paired samples from the 250-500 or 500-2000 µm size classes. A total of 25 plastic polymer types were observed across samples, more than observed in the large size classes. Spectral signatures of surface oxidation indicative of weathering were observed on most polyethylene, polypropylene, and polystyrene particles, which were the most abundant polymer types. Fragments were the dominant morphology with an average maximum length of 158 ± 92 µm. Overall, these results may help inform subsampling methods and be useful in future exposure assessments for aquatic organisms or design of MP removal technologies for urban and suburban stormwater.

Antibiotic-Resistant Bacteria and Resistance Genes in Isolates from Ghanaian Drinking Water Sources.

The control of infectious diseases is seriously threatened by the increase in the number of microorganisms resistant to antimicrobial agents. Antibiotic-resistant bacteria have also been identified in the water environment. A field study was performed sampling drinking water sources in seven districts of southern Ghana targeting boreholes, dams, hand-dug wells, and streams during baseflow conditions. Bacteria were isolated (N = 110) from a total of 67 water samples to investigate their antimicrobial susceptibility and to determine their carriage of select antibiotic resistance genes. Bacterial identification was performed using conventional selective media methods and the analytical profile index (API) method. Antibiotic susceptibility tests were carried out using the Kirby-Bauer method. Results indicated that all water sources tested were of poor quality based on the presence of fecal indicator organisms. The most commonly occurring bacterium isolated from water was Klebsiella spp. (N = 24, 21.8%), followed by E. coli (N = 23, 20.9%). Gram-negative bacteria isolates were most commonly resistant to cefuroxime (24.5%), while the Gram-positives were most commonly resistant to meropenem (21.3%). The highest rates of bacterial resistances to more than one antibiotic were observed in Klebsiella spp. (30.0%) followed by E. coli (27.8%). PCR was used to detect the presence of a select antibiotic resistance genes in the Gram-negative isolates. The presence of bla NDM-1, sull, tet(O), and tet(W) were observed in isolates from all water sources. In contrast, ermF was not detected in any of the Gram-negative isolates from any water source. Most (28.7%) of the resistance genes were observed in E. coli isolates. Reducing microbial contamination of the various water sources is needed to protect public health and to ensure the sustainability of this resource. This further calls for education of the citizenry.

Abundance, diversity, and host assignment of total, intracellular, and extracellular antibiotic resistance genes in riverbed sediments.

Human health risk assessment for environmental antibiotic resistant microbes requires not only quantifying the abundance of antibiotic resistance genes (ARGs) in environmental matrices, but also understanding their hosts and genetic context. Further, differentiating ARGs in intracellular and extracellular DNA (iDNA and eDNA) fractions may help refine our understanding of ARG transferability. The objectives of this study were to understand the (O1) abundance and diversity of extracellular, intracellular, and total ARGs along a land use gradient and (O2) impact of bioinformatics pipeline on the assignment of putative hosts for the ARGs observed in the different DNA fractions. Sediment samples were collected along a land use gradient in the Raritan River, New Jersey, USA. DNA was extracted to separate eDNA and iDNA and qPCR was performed for select ARGs and the 16S rRNA gene. Shotgun metagenomic sequencing was performed on DNA extracts for the different DNA fractions. ARG hosts were assigned via two different bioinformatic pipelines: network analysis of raw reads versus assembly. Results of the two pipelines were compared to evaluate their performance in terms of number and diversity of linkages and accuracy of in silico matrix spike host assignments. No differences were observed in the 16S rRNA gene normalized sul1 concentrations between the DNA fractions. The overall microbial community structure was more similar for iDNA and total DNA compared to eDNA and generally clustered by sampling site. ARGs associated with mobile genetic elements increased in iDNA for the downstream sites. Regarding host assignment, the raw reads pipeline via network analysis identified 247 ARG hosts as compared to 53 hosts identified by assembly pipeline. Other comparisons between the pipelines were made including ARG assignment to taxa containing waterborne pathogens and practical considerations regarding processing time.

Metabolically Active Prokaryotes and Actively Transcribed Antibiotic Resistance Genes in Sewer Systems: Implications for Public Health and Microbially Induced Corrosion.

Sewer systems are reservoirs of pathogens and bacteria carrying antibiotic resistance genes (ARGs). However, most recent high-throughput studies rely on DNA-based techniques that cannot provide information on the physiological state of the cells nor expression of ARGs. In this study, wastewater and sewer sediment samples were collected from combined and separate sanitary sewer systems. The metabolically active prokaryote community was evaluated using 16S rRNA amplicon sequencing and actively transcribed ARG abundance was measured using mRNA RT-qPCR. Three (sul1, blaTEM, tet(G)) of the eight tested ARGs were quantifiable in select samples. Sewer sediment samples had greater abundance of actively transcribed ARGs compared to wastewater. Microbiome analysis showed the presence of metabolically active family taxa that contain clinically relevant pathogens (Pseudomonadaceae, Enterobacteraceae, Streptococcaceae, Arcobacteraceae, and Clostridiaceae) and corrosion-causing prokaryotes (Desulfobulbaceae and Desulfovibrionaceae) in both matrices. Spirochaetaceae and methanogens were more common in the sediment matrix while Mycobacteraceae were more common in wastewater. The microbiome obtained from 16S rRNA sequencing had a significantly different structure from the 16S rRNA gene microbiome. Overall, this study demonstrates active transcription of ARGs in sewer systems and provides insight into the abundance and physiological state of taxa of interest in the different sewer matrices and sewer types relevant for wastewater-based epidemiology, corrosion, and understanding the hazard posed by different matrices during sewer overflows.

Comparison of residential dormitory COVID-19 monitoring via weekly saliva testing and sewage monitoring.

Wastewater surveillance has been used as a tool for COVID-19 outbreak detection particularly where there was not capability in place for routine and robust individual testing. Given clinical reports that earlier detection is possible following infection from throat/nasal samples compared to fecal samples for COVID-19 patients, the utility of wastewater testing where robust individual testing is possible is less clear. The objective of this study was to compare the results of weekly required COVID-19 saliva tests to weekly wastewater monitoring for residential buildings (i.e., dormitories) located across three college campuses capturing wastewater from 80 to 441 occupants per sampling location. Sampling occurred during the spring semester of the 2021 academic year which captured the third wave of SARS-CoV-2 cases in the study region. Comparison of the saliva and wastewater testing results indicated that the wastewater SARS-CoV-2 concentrations had a strong linear correlation with the previous week's percentage of positive saliva test results and a weak linear correlation with the saliva testing results surrounding the wastewater sampling (four days before and 3 days after). Given that no correlation was observed between the wastewater and the saliva testing from the following week, the weekly saliva testing captured spikes in COVID-19 cases earlier than the weekly wastewater sampling. Interestingly, the N1 gene was observed in buildings on all campuses, but N2 was observed in wastewater on only one of the campuses. N1 and N2 were also observed in sewer biofilm. The campus-specific challenges associated with implementation of wastewater surveillance are discussed. Overall, these results can help inform design of surveillance for early detection of SARS-CoV-2 in residential settings thereby informing mitigation strategies to slow or prevent the spread of the virus among residents in congregate living.

Inter-storm variation in microplastic concentration and polymer type at stormwater outfalls and a bioretention basin.

Microplastics (MP) are a commonly reported pollutant in the freshwater, marine, and soil environment. Few studies to date have reported MP concentrations and polymer types observed in stormwater, particularly not for catchments with separate storm sewers. The objectives of this study were to determine the microplastic concentration, polymer fingerprints, and the inter-storm variation of MP in two stormwater outfalls and a bioretention basin. Composite stormwater samples were collected at each site across three rain events each in catchments with urban and suburban land use. Particles 250 to 2000 µm were collected, separated into two sizes classes, treated with a wet peroxide oxidation, density separated with NaCl, and buoyant particles (fragments, films, and spheres) were collected for analysis with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Significant differences were observed in the total polymer concentrations and profiles between the sampling sites, potentially due to differences in land use within the catchments sampled, but not between size classes. The highest MP concentrations were observed in samples from the bioretention basin compared to the stormwater outfalls sampled, indicating the potential for green infrastructure to capture MP in the size range studied here. A weak but significant negative correlation was observed between cumulative rainfall (1.5 to 4.5 cm) and MP concentrations but no correlation was observed between antecedent dry days and MP concentrations. These data represent a conservative measure of MP concentrations given that fibers, particles <250 µm, and non-buoyant particles (i.e., density > 1.2 g/mL) were not targeted, but all targeted particles were analyzed with ATR-FTIR. Overall, these results presented provide insight into the loading and character (size, morphology, polymer type) of buoyant MP particles in stormwater that may be useful in designing mitigation strategies.

Quantification and composition of microplastics in the Raritan Hudson Estuary: Comparison to pathways of entry and implications for fate.

Comprehensive approaches are needed to understand accumulation patterns and the relative importance of pathways of entry for microplastics in the marine environment. Here, a highly urbanized estuarine environment was sampled along a salinity gradient from the mouth of the Raritan River, (New Jersey, USA) and into the Raritan Bay and the coastal ocean which are further influenced by discharge from the larger Hudson River. Polymers were characterized in two size classes by FTIR and/or Raman spectroscopy. The highest concentration of 500-2000 µm microplastic particles were observed in the mouth of the Raritan during summer low flow conditions, whereas the 250-500 µm microplastic particles were more prevalent in the bay and coastal ocean samples. These results were interpreted using fragmentation and mixing models to provide insight into the sources and fate of microplastics in this estuarine/coastal region. To investigate the potential pathways of entry into the system, samples were collected from various hydraulically connected storm water outfalls and the influent and effluent of wastewater treatment plants and polymer concentrations and types were compared to the estuarine samples. The concentrations of microplastics (500-2000 µm) ranged from 400 to 600 microplastics/m3 in storm water compared to <1-2.75 microplastics/m3 across the estuary. Of interest for analysis is the observed linear correlation between the total concentration of particles in a sample following oxidation and density separation and its microplastic concentration. Overall, the results presented reveal potentially important sources of microplastics in the estuarine environment and have implications for understanding the behavior, transport, and fate of microplastics under varying flow conditions and from estuaries with variable flushing times.

Sewer biofilm microbiome and antibiotic resistance genes as function of pipe material, source of microbes, and disinfection: field and laboratory studies.

Wastewater systems are recognized pathways for the spread of antibiotic resistant bacteria, but relatively little is known about the microbial ecology of the sewer environment. Sewer biofilm colonization by antibiotic resistance gene (ARG) carrying bacteria may impact interpretations of sewage epidemiology data, water quality during sewer overflows, and hazard to utility workers. The objectives of this research were to evaluate the (1) microbiome of real and simulated sewer biofilms and their potential to accumulate ARGs and (2) susceptibility of simulated sewer biofilms to bleach disinfection. First, biofilm samples were collected from sewer municipal systems. Next, an annular biofilm reactor was used to simulate the sewer environment while controlling the pipe material (concrete vs. PVC). The reactor was operated either as fed semi-batch with sewer sediment and synthetic wastewater (Sed-SB) or fed with a continuous flow of raw sewage (WW-CF). The abundance of ARGs, human fecal marker HF183, and 16S rRNA gene copies in these biofilm samples was measured with qPCR. Amplicon sequencing was performed to compare the prokaryotic diversity between samples. Finally, the susceptibility of reactor biofilm to a 4.6% bleach disnfection protocol was evaluated using viability qPCR and amplicon sequencing. Field and WW-CF biofilms contained the most ARG copies and the microbial community compositions varied between the different biofilm samples (field, Sed-SB, and WW-CF). Pipe material did not affect the abundance of ARGs in the reactor samples. However, log removal following bleach treatment suggested that the biofilm grown on PVC surface was primarily dislodged from the surface by the bleach treatment whereas more bacteria were lysed within the biofilm that remained on the concrete surface. Viable bacteria carrying ARGs were observed following 10 minutes of treatment. This study showed that sewer biofilms can accumulate bacteria carrying ARGs and that while bleach can reduce sewer biofilm density, the protocol tested here will not completely remove the biofilms.

Tagging the vanA gene in wastewater microbial communities for cell sorting and taxonomy of vanA carrying cells.

Failure to understand the microbial ecology driving the proliferation of antibiotic resistance in the environment prevents us from developing strategies to limit the spread of antibiotic resistant infectious disease. In this study, we developed for the first time a tyramide signal amplification-fluorescence in situ hybridization-fluorescence-activated cell sorting protocol (TSA-FISH-FACS) for the characterization of all vanA carrying bacteria in wastewater samples. Firstly, we validated the TSA-FISH protocol through microscopy in pure cultures and wastewater influent. Then, samples were sorted and quantified by FACS and qPCR. Significantly higher percentage tagging of cells was detected in vanA carrying pure cultures and wastewater samples spiked with vanA carrying cells as compared to vanA negative Gram positive strains and non-spiked wastewater samples respectively. qPCR analysis targeting vanZ, a regulating gene in the vanA cluster, showed its relative abundance was significantly greater in Enterococcus faecium ATCC 700221-spiked and positively sorted samples compared to the E. faecium spiked and negatively sorted samples. Phylogenetic analysis was then performed. Although further efforts are needed to overcome technical problems, we have, for the first time, demonstrated sorting bacterial-cells carrying antibiotic resistance genes from wastewater samples through a TSA-FISH-FACS protocol and provided insight into the microbial ecology of vancomycin resistant bacteria. Future potential applications using this approach will include the separation of members of an environmental microbial community (cultured and hard-to-culture) to allow for metagenomics on single cells or, in the case of clumping, targeting a smaller portion of the community with a priori knowledge that the target gene is present.

Factors associated with elevated levels of antibiotic resistance genes in sewer sediments and wastewater.

The sewer environment is a potential hotspot for the proliferation of antibiotic resistance genes (ARGs) and other hazardous microbial agents. Understanding the potential for ARG proliferation and retardation and/or accumulation in sewer sediments is of interest for protecting the health of sewage workers and the broader community in the event of sewer overflows as well as for interpreting sewage epidemiology data. To better understand this understudied environment for antibiotic resistance, a field survey was conducted to identify the factors that may control ARGs in sewer sediments and sewage. qPCR was performed for select ARGs and amplicon sequencing was performed for paired samples from combined and separate sanitary sewer systems. Metagenomic sequencing was performed on combined sewer sediments. The relative abundances of sul1, tet(O), tet(W), ermF, and vanA were higher in wastewater compared to sewer sediments, while NDM-1 was greater in sewer sediment and ermF was similar between the two matrices. NDM-1 was observed in sewer sediment but rarely above detection in wastewater in this study. This may indicate that larger/more frequent wastewater samples are needed for detection and/or that retardation and/or accumulation in sewage sediment may need to be considered when interpreting wastewater-based epidemiology data for ARGs. Random forest analyses indicated that season and conductivity were important variables and to a lesser extent so were pH, TSS, heavy metals, and sewer type for explaining the variance of the ARGs. These variables explained the 19-61% of the variance of sul1, tet(O), tet(G), and tet(W) quantified in wastewater. These variables performed less well for explaining the variance in sewer sediments (0.2-24%). Sewer sediment and wastewater had distinct microbial community structures and biomarkers for each are described. Metagenomics indicated that a high diversity of ARGs, including several of medical importance, were observed in the combined sewer sediment. This work provides insight into the complex sewer microbiome and the potential hazard posed by different sewer matrices.

Shifts in microbial community structure and function in surface waters impacted by unconventional oil and gas wastewater revealed by metagenomics.

Unconventional oil and gas (UOG) production produces large quantities of wastewater with complex geochemistry and largely uncharacterized impacts on surface waters. In this study, we assessed shifts in microbial community structure and function in sediments and waters upstream and downstream from a UOG wastewater disposal facility. To do this, quantitative PCR for 16S rRNA and antibiotic resistance genes along with metagenomic sequencing were performed. Elevated conductivity and markers of UOG wastewater characterized sites sampled downstream from the disposal facility compared to background sites. Shifts in overall high level functions and microbial community structure were observed between background sites and downstream sediments. Increases in Deltaproteobacteria and Methanomicrobia and decreases in Thaumarchaeota were observed at downstream sites. Genes related to dormancy and sporulation and methanogenic respiration were 18-86 times higher at downstream, impacted sites. The potential for these sediments to serve as reservoirs of antimicrobial resistance was investigated given frequent reports of the use of biocides to control the growth of nuisance bacteria in UOG operations. A shift in resistance profiles downstream of the UOG facility was observed including increases in acrB and mexB genes encoding for multidrug efflux pumps, but not overall abundance of resistance genes. The observed shifts in microbial community structure and potential function indicate changes in respiration, nutrient cycling, and markers of stress in a stream impacted by UOG waste disposal operations.

Influence of wastewater treatment plant discharges on microplastic concentrations in surface water.

The abundance of microplastic particles in the marine environment is well documented, but less is known about microplastics in the freshwater environment. Wastewater treatment plants (WWTPs) may not effectively remove microplastics allowing for their release to the freshwater environment. To investigate concentration of microplastic in fresh water and the impact of WWTP effluent, samples were collected upstream and downstream of four major municipal WWTPs on the Raritan River, NJ. Microplastics were categorized into three quantitative categories (500-2000 µm, 250-500 µm, 125-250 µm), and one semi-quantitative category (63-125 µm). Then, microplastics were classified as primary (manufactured in small size) or secondary (derived from larger plastics) based on morphology. The concentration of microplastics in the 125-250 and 250-500 µm size categories significantly increased downstream of WWTP. The smaller size classes, often not quantified in microplastic studies, were in high relative abundance across sampling sites. While primary microplastics significantly increased downstream of WWTP, secondary microplastic was the dominant type in the quantitative size categories (66-88%). A moderate correlation between microplastic and distance downstream was observed. These results have implications for understanding the fate and transport of microplastics in the freshwater environment.

Fecal Sterol and Runoff Analysis for Nonpoint Source Tracking.

Fecal pollution source identification is needed to quantify risk, target installation of source controls, and assess performance of best management practices in impaired surface waters. Sterol analysis is a chemical method for fecal source tracking that allows for differentiation between several fecal pollution sources. The objectives of this study were to use these chemical tracers for quantifying human fecal inputs in a mixed-land-use watershed without point sources of pollution and to determine the relationship between land use and sterol ratios. Fecal sterol analysis was performed on bed and suspended sediment from impaired streams. Human fecal signatures were found at sites with sewer overflow and septic inputs. Different sterol ratios used to indicate human fecal pollution varied in their sensitivity. Next, geospatial data was used to determine the runoff volumes associated with each land-use category in the watersheds. Fecal sterol ratios were compared between sampling locations and correlations were tested between ratio values and percentage of runoff for a given land-use category. Correlation was not observed between percentage of runoff from developed land and any of the five tested human-indicating sterol ratios in streambed sediments, confirming that human fecal inputs were not evenly distributed across the urban landscape. Several practical considerations for adopting this chemical method for microbial source tracking in small watersheds are discussed. Results indicate that sterol analysis is useful for identifying the location of human fecal nonpoint-source inputs.