Species- and element-specific patterns of metal flux from contaminated wetlands versus metals shed with exuviae in emerging dragonflies.

Dragonfly adults and their aquatic immature stages are important parts of food webs and provide a link between aquatic and terrestrial components. During emergence, contaminants can be exported into terrestrial food webs as immature adults fly away or be shed with their exuviae and remain in the wetland. Our previous work established metals accumulating in dragonfly nymphs throughout a contaminated constructed wetland designed to regulate pH and sequester trace metals from an industrial effluent line. Here, we evaluated the concentration and mass of metals leaving the wetland in flying emergents versus remaining in the wetland with the shed exuviae in 10 species of dragonflies belonging to 8 genera. Nine elements (Cu, Zn, Cd, Mn, V, Mg, Fe, Al, Pb) were evaluated that include essential and nonessential elements as well as trace and major metals. Metal concentrations in the emergent body and exuviae can differ by orders of magnitude. Aluminum, Fe, Mn, and Pb were largely shed in the exuviae. Vanadium and Cd were more variable among species but also tended to be shed with the exuviae. In contrast, Cu, Zn, and Mg showed a higher tendency to leave the wetland with an emerging dragonfly. Metals shed in dragonfly exuviae can moderate the transport of metals from contaminated wetlands. Taxonomic- and metal-specific variability in daily metal flux from the wetland depended upon concentration accumulated, individual body mass, and number of individuals emerging, with each factor's relative importance often differing among species. This illustrates the importance of evaluating the mass of metals in an individual and not only concentrations. Furthermore, differences in numbers of each species emerging will magnify differences in individual metal flux when calculating community metal flux. A better understanding of the variability of metal accumulation in nymphs/larvae and metal shedding during metamorphosis among both metals and species is needed.

Metal accumulation in dragonfly nymphs and crayfish as indicators of constructed wetland effectiveness.

Constructed wetland effectiveness is often assessed by measuring reductions of contaminant concentrations in influent versus departing effluent, but this can be complicated by fluctuations in contaminant content/chemistry and hydrology. We assessed effectiveness of a constructed wetland at protecting downstream biota from accumulating elevated metal concentrations-particularly copper and zinc in effluents from a nuclear materials processing facility. Contaminants distributed throughout a constructed wetland system and two reference wetlands were assessed using six dragonfly nymph genera (Anax, Erythemis, Libellula, Pachydiplax, Tramea, and Plathemis) as biomonitors. Additionally, the crayfish, Cambarus latimanus, were analyzed from the receiving and two reference streams. Concentrations of Cu, Zn, Pb, Mn, Cr, Cd, and Al were evaluated in 597 dragonfly nymph and 149 crayfish whole-body composite samples. Dragonfly genera varied substantially in metal accumulation and the ability to identify elevated metal levels throughout components of the constructed wetland. Genera more closely associated with bottom sediments tended to accumulate higher levels of metals with Libellula, Pachydiplax, and Erythemis often accumulating highest concentrations and differing most among sites. This, combined with their abundance and broad distributions make the latter two species suitable candidates as biomonitors for constructed wetlands. As expected, dragonfly nymphs accumulated higher metal concentrations in the constructed wetland than reference sites. However, dragonfly nymphs often accumulated as high of metal concentrations downstream as upstream of the water treatment cells. Moreover, crayfish from the receiving stream near the constructed wetland accumulated substantially higher Cu concentrations than from downstream locations or reference streams. Despite reducing metal concentrations at base flow and maintaining regulatory compliance, metal fluxes from the wetland were sufficient to increase accumulation in downstream biota. Future work should evaluate the causes of downstream accumulation as the next step necessary to develop plans to improve the metal sequestering efficiency of the wetland under variable flow regimes.

Basins, beaver ponds, and the storage and redistribution of trace elements in an industrially impacted coastal plain stream on the Savannah River Site, SC, USA.

Accumulation of eleven trace elements in sediment was evaluated throughout an industrially disturbed headwater stream on the Savannah River Site, SC, USA. Sampling began at upstream sedimentation basins at the margins of industrial areas, continued longitudinally downstream to a beaver pond representing a potential sink in the mid-reaches, and ended in downstream reaches. Additionally, sediment from beaver impacted areas in another industrially disturbed stream and a reference stream were analyzed to assess the natural tendency of these depositional features to settle out trace elements. We further compared trace element accumulation in sediment and biota from downstream reaches before and after an extreme rainy period to evaluate the potential redistribution of trace elements from sink areas. Trace elements accumulated in the headwater basins from which elements were redistributed to downstream reaches. The mid-reach beaver affected area sediments accumulated elevated concentrations of most analyzed elements compared to the free-flowing stream. The elevated accumulation of organic matter in these sink areas illustrated the effectiveness of reduced water velocity areas to settle out materials. The natural tendency of beaver ponds to accumulate trace elements and organic matter was further illustrated by sediments from the reference beaver pond accumulating higher concentrations of several elements than sediments from the free flowing section the stream impacted by industrial activity. However, concentrations in sediment from sedimentation basins and the beaver impacted area of the disturbed stream were highest. Trace elements and organic matter appeared to be redistributed from the sinks after the record rainy period resulting in increased trace element concentrations in both sediment and biota. These data suggest that assessments of contaminants in stream systems should include such slow-water, extreme depositional zones such as beaver impacted areas or basins to verify what contaminants may be pulsing through the stream.

Sediment and biota trace element distribution in streams disturbed by upland industrial activity.

Extensive industrial areas in headwater stream watersheds can severely impact the physical condition of streams and introduce contaminants. We compared 3 streams that received stormwater runoff and industrial effluents from industrial complexes to 2 reference streams. Reference streams provide a benchmark of comparison of geomorphic form and stability in coastal plain, sandy-bottomed streams as well as concentrations of trace elements in sediment and biota in the absence of industrial disturbance. We used crayfish (Cambarus latimanus, Procambarus raneyi, Procambarus acutus) and crane fly larvae (Tipula) as biomonitors of 15 trace elements entering aquatic food webs. Streams with industrial areas were more scoured, deeply incised, and less stable. Sediment organic matter content broadly correlated to trace element accumulation, but fine sediments and organic matter were scoured from the bottoms of disturbed streams. Trace element concentrations were higher in depositional zones than runs within all streams. Despite contaminant sources in the headwaters, trace element concentrations were generally not elevated in sediments of the eroded streams. However, element concentrations were frequently elevated in biota from these streams with taxonomic differences in accumulation amplified. In eroded, sand-bottomed coastal plain streams with unstable sediments, single snapshots of sediment trace element concentrations did not characterize well bioavailable trace elements. Biota that integrated exposures over time and space within their home ranges better detected bioavailable contaminants than sediment. Environ Toxicol Chem 2019;38:115-131. © 2018 SETAC.

Trace element accumulation in lotic dragonfly nymphs: Genus matters.

Constituents of coal combustion waste (CCW) expose aquatic organisms to complex mixtures of potentially toxic metals and metalloids. Multi-element trace element analyses were used to distinguish patterns of accumulation among 8 genera of dragonfly nymphs collected from two sites on a CCW contaminated coastal plain stream. Dragonfly nymphs are exceptional for comparing trace element accumulation in syntopic macroinvertebrates that are all predators within the same order (Odonata) and suborder (Anisoptera), but differ vastly in habitat use and body form. Sixteen trace element (Be, V, Cr, Ni, Cu, Zn, As, Se, Sr, Cd, Sb, Cs, Ba, Hg, Tl, and Pb) were analyzed and trophic position and basal carbon sources assessed with stable isotope analyses (C and N). Trophic positions varied within relatively narrow ranges. Size did not appear to influence trophic position. Trophic position rarely influenced trace element accumulation within genera and did not consistently correlate with accumulation among genera. Patterns between δ13C and trace element accumulation were generally driven by differences between sites. An increase in trace element accumulation was associated with a divergence of carbon sources between sites in two genera. Higher trace element concentrations tended to accumulate in nymphs from the upstream site, closer to contaminant sources. Influences of factors such as body form and habitat use appeared more influential on trace element accumulation than phylogeny for several elements (Ni, Ba, Sr, V, Be, Cd, and Cr) as higher concentrations accumulated in sprawler and the climber-sprawler genera, irrespective of family. In contrast, As and Se accumulated variably higher in burrowers, but accumulation in sprawlers differed between sites. Greater variation between genera than within genera suggests genus as an acceptable unit of comparison in dragonfly nymphs. Overall, taxonomic differences in trace element accumulation can be substantial, often exceeding variation between sites. Our results underscore the element and taxa specific nature of trace element accumulation, but we provide evidence of accumulation of some trace elements differing among dragonflies that differ in body form and utilize different sub-habitats within a stream reach.

Exposure to mercury and Aroclor 1268 congeners in least terns (Sternula antillarum) in coastal Georgia, USA.

Concentrations of total mercury and the rare PCB mixture Aroclor 1268 in least terns (Sternula antillarum), a colonially-nesting, piscivorous seabird, in the Turtle River estuary and other coastal sites in Georgia, USA, were investigated. The Turtle River estuary is the location of the Linden Chemical Plant (LCP) Superfund site, a site prioritized by USA law for immediate remediation, where industries released effluent containing these contaminants until 1994. Aroclor 1268 is a highly-chlorinated PCB mixture that was used and released exclusively at the LCP site and nowhere else in the south eastern USA. High concentrations of Aroclor 1268 congeners and mercury have been documented in biota local to LCP, but no studies report concentrations in high trophic level, piscivorous birds such as least terns. We collected feathers and feces from chicks, and eggs from adults, at nesting colonies along the Georgia coast to analyze contaminant loads (in dry weight ppb). Mean Aroclor 1268 mixture concentrations in eggs (≤16,329 ppb) were highest at colonies in and just outside LCP, and decreased with increasing distance (up to 110 km) from LCP, but the Aroclor 1268 signature congener mixture was present at all sites. Mercury concentrations in eggs (≤3370 ppb), feathers (≤5950 ppb), and feces (≤417 ppb), were present at all sites, but did not vary significantly among sites. This report confirms the extensive dispersal of Aroclor 1268 congeners (approximately 110 km north and 70 km south of its point source) via bioaccumulation and trophic transfer.

Spatial and taxonomic variation in trace element bioaccumulation in two herbivores from a coal combustion waste contaminated stream.

Dissimilarities in habitat use, feeding habits, life histories, and physiology can result in syntopic aquatic taxa of similar trophic position bioaccumulating trace elements in vastly different patterns. We compared bioaccumulation in a clam, Corbicula fluminea and mayfly nymph Maccaffertium modestum from a coal combustion waste contaminated stream. Collection sites differed in distance to contaminant sources, incision, floodplain activity, and sources of flood event water and organic matter. Contaminants variably accumulated in both sediment and biofilm. Bioaccumulation differed between species and sites with C. fluminea accumulating higher concentrations of Hg, Cs, Sr, Se, As, Be, and Cu, but M. modestum higher Pb and V. Stable isotope analyses suggested both spatial and taxonomic differences in resource use with greater variability and overlap between species in the more physically disturbed site. The complex but essential interactions between organismal biology, divergence in resource use, and bioaccumulation as related to stream habitat requires further studies essential to understand impacts of metal pollution on stream systems.

Variation in trace-element accumulation in predatory fishes from a stream contaminated by coal combustion waste.

Extensive and critical evaluation can be required to assess contaminant bioaccumulation in large predatory fishes. Species differences in habitat use, resource use, and trophic level, often influenced by body form, can result in diverging contaminant bioaccumulation patterns. Moreover, the broad size ranges inherent with large-bodied fish provide opportunity for trophic and habitat shifts within species that can further influence contaminant exposure. We compared contaminant bioaccumulation in four fish species, as well as two herbivorous invertebrates, from a coal combustion waste contaminated stream. Muscle, liver, and gonad tissue were analyzed from fish stratified across the broadest size ranges available. Effects of trophic position (δ (15)N), carbon sources (δ (13)C), and body size varied among and within species. Mercury and cesium concentrations were lowest in the invertebrates and increased with trophic level both among and within fish species. Other elements, such as vanadium, cadmium, barium, nickel, and lead, had greater levels in herbivorous invertebrates than in fish muscle. Sequestration by the fish livers averted accumulation in muscle. Consequently, fish liver tissue appeared to be a more sensitive indicator of bioavailability, but exceptions existed. Despite liver sequestration, within fishes, muscle concentrations of many elements still tended to increase by trophic level. Notable variation within some species was observed. These results illustrate the utility of stable isotope data in exploring differences of bioaccumulation within taxa. Our analyses suggest a need for further evaluation of the underlying sources of this variability to better understand contaminant bioaccumulation in large predatory fishes.

Multi-site analysis reveals widespread antibiotic resistance in the marine pathogen Vibrio vulnificus.

Vibrio vulnificus is a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. Therefore, few studies have verified the effectiveness of commonly prescribed antibiotics in V. vulnificus treatment. Here we screened 151 coastal isolates and 10 primary septicaemia isolates against 26 antimicrobial agents representing diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed for V. vulnificus infections, such as doxycycline, tetracycline, aminoglycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implications of our findings. Among environmental isolates, there were no consistent differences in the frequency of resistance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the importance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resistance factors.

Insights into the environmental resistance gene pool from the genome sequence of the multidrug-resistant environmental isolate Escherichia coli SMS-3-5.

The increasing occurrence of multidrug-resistant pathogens of clinical and agricultural importance is a global public health concern. While antimicrobial use in human and veterinary medicine is known to contribute to the dissemination of antimicrobial resistance, the impact of microbial communities and mobile resistance genes from the environment in this process is not well understood. Isolated from an industrially polluted aquatic environment, Escherichia coli SMS-3-5 is resistant to a record number of antimicrobial compounds from all major classes, including two front-line fluoroquinolones (ciprofloxacin and moxifloxacin), and in many cases at record-high concentrations. To gain insights into antimicrobial resistance in environmental bacterial populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 predicted to be involved in antimicrobial resistance were phenotypically characterized. Using recombinant vector clones from shotgun sequencing libraries, resistance to tetracycline, streptomycin, and sulfonamide/trimethoprim was assigned to a single mosaic region on a 130-kb plasmid (pSMS35_130). The remaining plasmid backbone showed similarity to virulence plasmids from avian-pathogenic E. coli (APEC) strains. Individual resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial isolates from multiple phylogenetic and geographic sources. Resistance to quinolones was assigned to several chromosomal loci, mostly encoding transport systems that are also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is therefore dependent both on determinants acquired from a mobile gene pool that is likely available to clinical and agricultural pathogens, as well, and on specifically adapted multidrug efflux systems. The association of antimicrobial resistance with APEC virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence through the extensive use of antibiotics.

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities.

The acquisition of new genetic material via horizontal gene transfer allows bacteria to rapidly evolve. One key to estimating the contribution of horizontal gene transfer to bacterial evolution is to quantify the abundance of mobile genetic elements (MGEs) in bacterial communities under varying degrees of selective pressure. We quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats, and in metal- or antibiotic-amended freshwater microcosms. The intI1 gene was more abundant in all contaminant-exposed communities indicating that relative gene transfer potential is higher in these communities. A second key to assessing the contributions of MGEs to bacterial evolution is to examine the structure and function of the MGE-associated gene pool. We determined that the gene cassette pool is a novel and diverse resource available for bacterial acquisition, but that contamination has no discernible effect on cassette richness. Gene cassette profiles were more similar within sites than among sites, yet bacterial community profiles were not, suggesting that selective pressures can shape the structure of the gene cassette pool. Of the 46 sequenced gene cassette products, 37 were novel sequences, while the 9 gene cassettes with similarity to database sequences were primarily to hypothetical proteins. That class 1 integrons are ubiquitous and abundant in environmental bacterial communities indicates that this group of MGEs can play a substantial role in the acquisition of a diverse array of gene cassettes beyond their demonstrated impact in mediating multidrug resistance in clinical bacteria.

Antibiotic resistance in the shellfish pathogen Vibrio parahaemolyticus isolated from the coastal water and sediment of Georgia and South Carolina, USA.

Vibrio parahaemolyticus is a gram-negative pathogen commonly encountered in estuarine and marine environments, and a common cause of seafood-related gastrointestinal infections. We isolated 350 V. parahaemolyticus strains from water and sediment at three locations along the Atlantic coast of Georgia and South Carolina during various seasons. These isolates were tested for susceptibility to 24 antibiotics. Isolate virulence was determined through PCR of tdh and trh genes. The breadth of resistance to antibiotics was unexpectedly high, with 24% isolates demonstrating resistance to 10 or more agents. A significant fraction of isolates were resistant to diverse beta-lactams, aminoglycosides, and other classes of antibiotics. Fifteen of the 350 strains possessed virulence genes, with no apparent correlation between virulence and site, sample type, or season of isolation. Antibiotic resistance was slightly reduced among the virulent strains. This study represents one of the largest surveys to date of the virulence and antibiotic resistance in environmental V. parahaemolyticus strains. The observed antibiotic susceptibility patterns suggest that current guidelines for the antibiotic treatment of non-cholerae Vibrio should be reevaluated and extended.

Novel tetracycline resistance determinant isolated from an environmental strain of Serratia marcescens.

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.

Coselection for microbial resistance to metals and antibiotics in freshwater microcosms.

Bacterial resistances to diverse metals and antibiotics are often genetically linked, suggesting that exposure to toxic metals may select for strains resistant to antibiotics and vice versa. To test the hypothesis that resistances to metals and antibiotics are coselected for in environmental microbial assemblages, we investigated the frequency of diverse resistances in freshwater microcosms amended with Cd, Ni, ampicillin or tetracycline. We found that all four toxicants significantly increased the frequency of bacterioplankton resistance to multiple, chemically unrelated metals and antibiotics. An ampicillin-resistant strain of the opportunistic human pathogen Ralstonia mannitolilytica was enriched in microcosms amended with Cd. Frequencies of antibiotic resistance were elevated in microcosms with metal concentrations representative of industry and mining-impacted environments (0.01-1 mM). Metal but not antibiotic amendments decreased microbial diversity, and a weeklong exposure to high concentrations of ampicillin (0.01-10 mg l-1) and tetracycline (0.03-30 mg l-1) decreased microbial abundance only slightly, implying a large reservoir of antibiotic resistance in the studied environment. Our results provide first experimental evidence that the exposure of freshwater environments to individual metals and antibiotics selects for multiresistant microorganisms, including opportunistic human pathogens.

Elevated microbial tolerance to metals and antibiotics in metal-contaminated industrial environments.

To test the hypothesis that industrial metal contaminants select for microorganisms tolerant to unrelated agents, such as antibiotics, we analyzed metal and antibiotic tolerance patterns in microbial communities in the intake and discharge of ash settling basins (ASBs) of three coal-fired power plants. High-throughput flow-cytometric analyses using cell viability probes were employed to determine tolerances of entire bacterioplankton communities, avoiding bias toward culturable versus nonculturable bacteria. We found that bacterioplankton collected in ASB discharges were significantly more tolerant to metal and antibiotic exposures than bacterioplankton collected in ASB intakes. Optical properties of microorganisms collected in ASB discharges indicated no defensive physiological adaptations such as formation of resting stages or excessive production of exopolymers. Thus, it is likely that the elevated frequency of metal and antibiotic tolerances in bacterioplankton in ASB discharges were caused by shifts in microbial community composition, resulting from the selective pressure imposed by elevated metal concentrations or organic toxicants present in ASBs.