Freshwater Mesocosms Research Articles

Silver nanoparticles alter planktonic community structure and promote ecosystem respiration in freshwater mesocosms

The widespread use of silver nanoparticles (AgNPs) has resulted in their release into the aquatic environment, which threatens the health of aquatic ecosystems. Although the ecotoxicological effects of AgNPs have been widely reported at individual and population levels, the impact of long-term exposure to AgNPs on community structure and ecosystem function in aquatic ecosystems remains poorly understood. Herein, the present study investigated the effects of long-term exposure (28 d) to environmentally relevant concentrations (1 μg/L and 10 μg/L) of AgNPs on the community structure and function of freshwater ecosystems by artificially constructed 28 mesocosms freshwater ecosystem in experimental greenhouses, using plastic water tanks and food web manipulation. The results showed that long-term exposure to AgNPs significantly altered the community structure of zooplankton, phytoplankton, and bacterioplankton in the aquatic ecosystem. Exposure to 10 μg/L AgNPs significantly reduced the zooplankton density (70.3%, p < 0.05) and increased the phytoplankton biomass and bacterial richness and diversity via a “top-down effect.” With regards to ecosystem function, AgNPs exposure significantly increased the respiration in freshwater ecosystems but did not have a significant effect on decomposition. The partial least squares path modeling (PLS-PM) further revealed that AgNPs may have a negative impact on ecosystem functions by reducing zooplankton community density and thus increasing phytoplankton biomass. This study is the first to show that long-term exposure to environmentally relevant concentrations of AgNPs leads to alterations in plankton community structure and promotes respiration in freshwater ecosystems. It emphasizes the need for assessing the environmental risk of long-term exposure to AgNPs at the ecosystem level.

Single-/Co-Driving of Tetracycline, Triclocarban and Zinc on Microbial Community, Resistome and Function in the Cyanobacteria-Blooming Freshwater Ecosystem.

Environmental concentrations of antimicrobials can inhibit Cyanobacteria, but little is known about their effects on Cyanobacteria-blooming freshwater ecosystem. Here, a 21 days' outdoor freshwater mesocosm experiment was established to study effects of single and combined tetracycline, triclocarban and zinc at environmental concentrations on microbial community, microbial function and antimicrobial resistance using amplicon- and metagenomic-based methods. Results showed that three chemicals reshaped the microbial community with magnified effects by chemical combinations. Relative abundance of Cyanobacteria was decreased in all chemical groups, especially from 74.5 to 0.9% in combination of three chemicals. Microbial community networks were more simplified after exposure. Proteobacteria and Bacteroidetes predominated in Cyanobacteria-degraded ecosystems, and their relative abundances were significantly correlated with antibiotic resistome, suggesting that they might host antibiotic resistance genes. Notably, relative abundance (copy per 16S rRNA gene) of total antibiotic resistome reached five to nine folds higher than the initial abundance in chemical-combined groups. The affected antibiotic resistance genes referred to a wide range of antibiotic classes. However, weak effects were detected on biocide/metal resistance and microbial virulence. Three chemicals posed complicated effects on microbial function, some of which had consistent variations across the groups, while some varied greatly in chemical groups. The findings highlight sensitivity of Cyanobacteria-blooming ecosystem to antimicrobials.

Combined stress of an insecticide and heatwaves or elevated temperature induce community and food web effects in a Mediterranean freshwater ecosystem

Ongoing global climate change will shift nature towards Anthropocene's unprecedented conditions by increasing average temperatures and the frequency and severity of extreme events, such as heatwaves. While such climatic changes pose an increased threat for freshwater ecosystems, other stressors like pesticides may interact with warming and lead to unpredictable effects. Studies that examine the underpinned mechanisms of multiple stressor effects are scarce and often lack environmental realism. Here, we conducted a multiple stressors experiment using outdoor freshwater mesocosms with natural assemblages of macroinvertebrates, zooplankton, phytoplankton, macrophytes, and microbes. The effects of the neonicotinoid insecticide imidacloprid (1 µg/L) were investigated in combination with three temperature scenarios representing ambient, elevated temperatures (+4 °C), and heatwaves (+0 to 8 °C), the latter two having similar energy input. We found similar imidacloprid dissipation patterns for all temperature treatments with lowest average dissipation half-lives under both warming scenarios (DT50: 3 days) and highest under ambient temperatures (DT50: 4 days) throughout the experiment. Amongst all communities, only the zooplankton community was significantly affected by the combined treatments. This community demonstrated low chemical sensitivity with lagged and significant negative imidacloprid effects only for cyclopoids. Heatwaves caused early and long-lasting significant effects on the zooplankton community as compared to elevated temperatures, with Polyarthra, Daphnia longispina, Lecanidae, and cyclopoids being the most negatively affected taxa, whereas Ceriodaphnia and nauplii showed positive responses to temperature. Community recovery from imidacloprid stress was slower under heatwaves, suggesting temperature-enhanced toxicity. Finally, microbial and macrofauna litter degradation were significantly enhanced by temperature, whereas the latter was also negatively affected by imidacloprid. A structural equation model depicted cascading food web effects of both stressors with stronger relationships and significant negative stressor effects at higher than at lower trophic levels. Our study highlights the threat of a series of heatwaves compared to elevated temperatures for imidacloprid-stressed freshwaters.

Fate and effects of a new generation fluorosurfactant (cC6O4) in freshwater mesocosms

Per- and polyfluorinated alkyl substances (PFAS) have raised international concerns due to their widespread use, environmental persistence and potential bioaccumulative and ecotoxicological effects. Therefore, the chemical industry has been dedicated to develop new generation fluorosurfactants which are aimed to replace the most concerning PFAS. Here we investigated the fate and effects of cyclic C6O4 (cC6O4), a compound used as alternative to long-chain perfluorocarboxylic acids, in freshwater mesocosms located in the Mediterranean region (Spain) over a period of 90 days. cC6O4 was applied as ammonium salt once at the following nominal concentrations: 0 µg/L (control), 1 µg/L, 20 µg/L, 400 µg/L, and 8,000 µg/L. The study shows that cC6O4 is relatively persistent in water (dissipation: 34–37 % after 90 days), has very low sorption capacity to sediments (sediment-water partition coefficient: 0.18–0.32 L/kg) and very limited bioconcentration (BCF: 0.09–0.94), bioaccumulation (BAF: 0.09–4.06) and biomagnification (BMF: 0.05–0.28) potential. cC6O4 did not result in significant adverse effects on aquatic populations and communities of phytoplankton and zooplankton at the tested concentrations. As for the macroinvertebrate community, the ephemeropteran Cloeon sp. showed a population decline at the highest test concentration on day 60 onwards, and a significant effect on the macroinvertebrate community was identified on the last sampling day at the same exposure level. Therefore, the calculated NOEC for cC6O4 in freshwater mesocosms exposed over a period of 90 days was 400 µg/L, which corresponded to a time weighted average concentration of 611 µg/L, given the water evaporation in the test systems. This concentration is about an order of magnitude higher than the highest exposure concentration monitored in freshwater ecosystems. Therefore, it can be concluded that cC6O4 poses insignificant ecological risks for freshwater plankton and macroinvertebrate communities given the current environmental exposure levels.

Effects of the fungicide azoxystrobin in two habitats representative of mediterranean coastal wetlands: A mesocosm experiment

This paper investigates the effects of the fungicide azoxystrobin, a compound widely used in rice farming, on aquatic communities representative of two habitats characteristic of Mediterranean wetland ecosystems: water springs and eutrophic lake waters. The long-term effects of azoxystrobin were evaluated on several structural (phytoplankton, zooplankton, macroinvertebrate populations and communities) and functional (microbial decomposition, macrophyte and periphyton growth) parameters making use of freshwater mesocosms. Azoxystrobin was applied in two pulses of 2, 20, 200 µg/L separated by 14 d using the commercial product ORTIVA (23 % azoxystrobin w/w). The results show that these two habitats responded differently to the fungicide application due to their distinct physico-chemical, functional, and structural characteristics. Although overall sensitivity was found to be similar between the two (lowest NOEC < 2 µg/L), the taxa and processes that were affected differed substantially. In general, the most sensitive species to the fungicide were found in the water spring mesocosms, with some species of phytoplankton (Nitzschia sp.) or macrocrustaceans (Echinogammarus sp. and Dugastella valentina) being significantly affected at 2 µg/L. In the eutrophic lake mesocosms, effects were found on phytoplankton taxa (Desmodesmus sp. and Coelastrum sp.), on numerous zooplankton taxa, on chironomids and on the beetle Colymbetes fuscus, although at higher concentrations. The hemipteran Micronecta scholtzi was affected in both treatments. In addition, functional parameters such as organic matter decomposition or macrophyte growth were also affected at relatively low concentrations (NOEC 2 µg/L). Structural Equation Modelling was used to shed light on the indirect effects caused by azoxystrobin on the ecosystem. These results show that azoxystrobin is likely to pose structural and functional effects on Mediterranean wetland ecosystems at environmentally relevant concentrations. Moreover, it highlights the need to consider habitat-specific features when conducting ecotoxicological research at the population and community levels.

Assessing ecological responses to exposure to the antibiotic sulfamethoxazole in freshwater mesocosms

Antibiotics are a contaminant class of worldwide concern as they are frequently detected in aquatic ecosystems. To better understand the impacts of antibiotics on aquatic ecosystems, we conducted an outdoor mesocosm experiment in which aquatic communities were exposed to different concentrations of the antibiotic sulfamethoxazole (0, 0.15, 1.5, 15 and 150 μg/L). These concentrations include mean (0.15 μg/L) and maximum detected concentrations (15 and 150 μg/L) in aquatic ecosystems worldwide. Sulfamethoxazole was applied once a week for eight consecutive weeks to 1530 L outdoor mesocosms in the Netherlands, followed by an eight-week recovery period. We evaluated phytoplankton-, bacterial- and invertebrate responses during and after sulfamethoxazole exposure and assessed impacts on organic matter decomposition. Contrary to our expectations, consistent treatment-related effects on algal and bacterial communities could not be demonstrated. In addition, sulfamethoxazole did not significantly affect zooplankton and macroinvertebrate communities. However, some effects on specific taxa were observed, with an increase in Mesostoma flatworm abundance (NOEC of <0.15 μg/L). In addition, eDNA analyses indicated negative impacts on the insects Odonata at a sulfamethoxazole concentration of 15 μg/L. Overall, environmentally relevant sulfamethoxazole concentration did not result in direct or indirect impairment of entire aquatic communities and ecological processes in our mesocosms. However, several specific macroinvertebrate taxa demonstrated significant (in)direct effects from sulfamethoxazole. Comparison of the results with the literature showed inconsistent results between studies using comparable, environmentally relevant, concentrations. Therefore, our study highlights the importance of testing the ecological impacts of pharmaceuticals (such as sulfamethoxazole) across multiple trophic levels spanning multiple aquatic communities, to fully understand its potential ecological threats.

Biotic and Abiotic Interactions in Freshwater Mesocosms Determine Fate and Toxicity of CuO Nanoparticles.

Transformation, dissolution, and sorption of copper oxide nanoparticles (CuO-NP) play an important role in freshwater ecosystems. We present the first mesocosm experiment on the fate of CuO-NP and the dynamics of the zooplankton community over a period of 12 months. Increasingly low (0.08-0.28 mg Cu L-1) and high (0.99-2.99 mg Cu L-1) concentrations of CuO-NP and CuSO4 (0.10-0.34 mg Cu L-1) were tested in a multiple dosing scenario. At the high applied concentration (CuO-NP_H) CuO-NP aggregated and sank onto the sediment layer, where we recovered 63% of Cu applied. For the low concentration (CuO-NP_L) only 41% of applied copper could be recovered in the sediment. In the water column, the percentage of initially applied Cu recovered was on average 3-fold higher for CuO-NP_L than for CuO-NP_H. Zooplankton abundance was substantially compromised in the treatments CuSO4 (p < 0.001) and CuO-NP_L (p < 0.001). Community analysis indicated that Cladocera were most affected (bk = -0.49), followed by Nematocera (bk = -0.32). The abundance of Cladocera over time and of Dixidae in summer was significantly reduced in the treatment CuO-NP_L (p < 0.001; p < 0.05) compared to the Control. Our results indicate a higher potential for negative impacts on the freshwater community when lower concentrations of CuO-NP (<0.1 mg Cu L-1) enter the ecosystem.

Persistence of human- and cattle-associated Bacteroidales and mitochondrial DNA markers in freshwater mesocosms

Accurate identification of the origins of non-point source pollution is essential for the effective control of fecal pollution. Host-associated Bacteroidales and mitochondrial DNA (mtDNA) markers have been developed to identify the sources of human and cattle fecal pollution. However, the differences in persistence between these two types of markers under different environmental conditions are still poorly understood. Here, we conducted mesocosm experiments to investigate the influence of indigenous microbiota and nutrients on the decay of Bacteroidales and mtDNA markers associated with humans and cattle. Raw sewage or cattle feces were inoculated into mesocosms containing natural eutrophic water, sterile eutrophic water or artificial freshwater. The Bacteroidales markers HF183 (human) and CowM3 (cattle) and mtDNA markers HcytB (human) and QMIBo (cattle) were quantified using the quantitative polymerase chain reaction (qPCR) assays. All markers but HF183 decreased the fastest in the presence of indigenous microbiota. Nutrients caused a decrease in the persistence of HF183; however, no significant nutrient effects were observed for HcytB, CowM3, and QMIBo. The time to reach one log reduction (T90) for HF183 and HcytB was similar; CowM3 reached T90 earlier than QMIBo in all the treatments but eutrophic water. E. coli persisted longer than both Bacteroidales and mtDNA markers in the mesocosms regardless of inoculum type. Additionally, 16S rRNA gene amplicon sequencing was used to determine the changes in bacterial communities accompanying the marker decay. Analysis using the SourceTracker software showed that bacterial communities in the mesocosms became more dissimilar to those in the corresponding inoculants over time. Our results indicate that environmental factors are important determinants of genetic markers' persistence, but their impact can vary depending on the genetic markers. The cattle Bacteroidales markers may be more suitable for determining recent fecal contamination than cattle mtDNA.

The Correlation between Genotype Richness of Submerged Macrophytes and Periphyton Biomass: A Mesocosm Study Based on Five Dominant Submerged Macrophytes from Yangtze River

Submerged macrophyte and periphyton are main primary producers which strongly interact with each other in clear water shallow lakes. In this study, the effects of genetic variation of the macrophyte species on periphyton biomass were studied in five submerged species. A two-year mesocosm study was conducted with four levels of genetic diversity (1, 4, 8 and 16 genotypes) for each submerged macrophyte, including 1600 individuals and 320 boxes in 20 mesocosms. Of the five submerged species, only Vallisneria spinulosa showed a positive correlation between its levels of genotype richness and the periphyton biomass. The correlation between genetic distance of genotypes and periphyton biomass was tested, which varied with the difference of seasons and species. In summary, we found that in freshwater mesocosms, the genetic diversity of submerged macrophytes may play a role in regulating the periphyton biomass, but the interaction between genetic diversity of macrophytes and periphyton biomass was not straightforward. This study will provide new insights into the interaction dynamics between the two primary producers in shallow lakes.

Matrix Matters: novel insights for the extraction, preparation, and quantitation of microplastics in a freshwater mesocosm study

The extraction and characterization of secondary microplastics, those formed through subjection to the environment, must continuously improve in accuracy and applicability in order to generate robust microplastics exposure and risk assessments. Currently, there is a dearth of reliable extraction and quantitation methods for solid microplastic particles in sediment with chemical specificity. Herein we present advances in methodologies to extract and quantify microplastic particles from sediment, utilizing an outdoor mesocosm to model a freshwater coastal environment. Model secondary microplastics of crosslinked polyurethane (PU) were studied in comparison to model secondary polypropylene (PP) microplastics. Techniques to characterize particles in sediment included pyrolysis gas chromatography mass spectrometry (py GC/MS), stereoscope microscopy, and scanning electron microscopy. To complement particle analysis, plastic-associated leachable molecules were extracted from sediment and analyzed semi-quantitatively by high performance liquid chromatography with high-resolution mass spectrometry (HPLC/HR-MS). After developing and optimizing extraction and analytical methods we quantitated PU microparticles by count and weight and discovered that model PU particles fell from the water column into the sediment over the course of a year, while no PP particles were located in sediment samples. In addition, target small molecules associated with the crosslinked PU were identified in sediment by HPLC/HR-MS but leachable molecules associated with polypropylene could not be identified in sediment samples. We share the new py GC/MS method to quantify highly crosslinked PUs in complex environmental matrices containing both inorganic and organic components. In the process of generating robust extraction methods for microplastics in sediment, we discovered important considerations for the quantitation of microplastics by py GC/MS and the impacts of sample matrix on the quantitation of PU and PP specifically. We provide guidance for the preparation of microplastics from complex environmental matrices (e.g., sediment and soil) for analysis by py GC/MS.Graphical

Zooplankton community turnover in response to a novel TiO2-coated nano-formulation of carbendazim and its constituents

Novel nanomaterial-based pesticide formulations are increasingly perceived as promising aids in the transition to more efficient agricultural production systems. The current understanding of potential unintended (eco)toxicological impacts of nano-formulated pesticides is scarce, in particular with regard to (non-target) aquatic organisms and ecosystems. The present study reports the results of a long-term freshwater mesocosm experiment which assessed responses of individual zooplankton taxa and communities to a novel TiO2-coated nano-formulation of the fungicide carbendazim. Population- and community trends were assessed and compared in response to the nano-formulation and its constituents applied individually (i.e. nano-sized TiO2, carbendazim) and in combination (i.e. nano-sized TiO2 & carbendazim). Minimal differences were observed between effects induced by the nano-formulation and its active ingredient (i.e. carbendazim) when applied at equivalent nominal test concentrations (4 μg L−1). Nano-sized TiO2 was found to affect zooplankton community trends when applied separately at environmentally realistic concentrations (20 μg L−1 nominal test concentration). However, when nano-sized TiO2 was applied in combination with carbendazim, nano-sized TiO2 was found not to alter effects on community trends induced by carbendazim. The findings of the current study provide an extensive and timely addition to the current body of work available on non-target impacts of nano-formulated pesticides.

Environment and time drive the links between the species richness and ecosystem multifunctionality from multitrophic freshwater mesocosms

Understanding the link between biodiversity and ecosystem functioning is imperative for ecosystem-based management. The role of multitrophic diversity in sustaining multifunctionality remains unclear, especially in highly diverse aquatic ecosystems. We performed a species-addition experiment consisting of one, two, three, and five species in simulated multitrophic ecosystems to evaluate biodiversity and ecosystem functioning relationships within and across trophic levels. Our results showed that there are positive species richness–productivity relationships within and across trophic levels. We found significant negative correlations between species richness and the cumulative variation of total phosphorus, and between species richness and ecosystem multifunctionality across trophic levels. Also, we found that the relationships between ecosystem multifunctionality and species richness within and across the trophic levels are mediated by a combination of environmental factors, including water temperature, dissolved oxygen, pH, irradiance, and time, rather than by species richness. Our results imply that species richness–ecosystem functioning relationships vary for different ecological functions; the individual ecosystem functions selected and the way multifunctionality calculated are critical when examining links between biodiversity and ecosystem multifunctionality. Our study highlights that multitrophic richness, such as for consumers, is crucial for driving ecosystem multifunctionality. Furthermore, our study implies that management practices for restoring the diversity of aquatic macrophytes in wetlands should consider not only macrophyte richness but also different functional groups and life-forms.

Contrasting the effects of microplastic types, concentrations and nutrient enrichment on freshwater communities and ecosystem functioning

Microplastics are now ubiquitous in freshwater environments. As most previous research has focused on species-specific effects of microplastics under controlled laboratory conditions, little is known about the impact of microplastics at higher levels of ecological organisation, such as freshwater communities and their associated ecosystem functions. To fill this knowledge gap, an outdoor experiment using 40 freshwater mesocosms, each 1.57 m3, was used to determine the effects of (i) microplastic type: traditional oil-based high-density polyethylene versus bio-based biodegradable polylactic acid, (ii) concentration of microplastic particles and (iii) nutrient enrichment. The two concentrations of microplastics used were equivalent to measured environmentally occurring concentrations and concentrations known to cause toxicological effects under laboratory conditions. Freshwater communities are also at increasing risk from nutrient enrichment, which can alter community composition in favour of competitively dominant taxa. The independent and interactive effects of these treatments on pelagic community structure (phytoplankton standing stock, taxonomic richness, and composition) and ecosystem functioning (periphyton productivity and leaf litter decomposition) were assessed. Taxonomic richness and community composition were not affected by exposure to the experimental treatments and there were no significant treatment effects on phytoplankton standing stock, periphyton productivity, total or microbial leaf litter decomposition. Overall, multiple microplastic exposures, crossed with nutrient addition had little impact on the structure and functioning of semi-natural freshwater ecosystems. These findings indicate that the negative impacts of microplastics predicted from species-specific studies may not be readily realised at the ecosystem scale.

The combined effects of filter-feeding bivalves (Cristaria plicata) and submerged macrophytes (Hydrilla verticillate) on phytoplankton assemblages in nutrient-enriched freshwater mesocosms.

Freshwater ecosystems are threatened by eutrophication, which causes persistent and harmful algal blooms. Filter-feeding bivalve mollusks and submerged macrophytes (SMs) alleviate the eutrophication effects by inhibiting phytoplankton biomass blooms. However, very little is known about whether and how the combined manipulation of filter-feeding bivalves and SMs control eutrophication and influence phytoplankton assemblages. Here, we performed a nutrient-enriched freshwater mesocosm experiment to assess the combined effects of the filter-feeding bivalve Cristaria plicata, a cockscomb pearl mussel, and the macrophyte Hydrilla verticillate on the biomass and composition of phytoplankton assemblages. We found that addition of C. plicata and H. verticillate decreased the water nutrient concentrations and suppressed overall phytoplankton biomass. Further, distinct differences in taxa between restoration and control treatments were observed and noticeably competitive exclusion of cyanobacteria in the restoration treatments occurred. An antagonistic interaction between filter-feeding bivalves and SMs was only detected for total cyanobacteria biomass demonstrating that a larger magnitude of SM restoration may override the effect of filter-feeding bivalves. Our results suggest that manipulation, through the addition of bivalves as grazers, associated with the restoration of SMs, is an efficient approach for reducing cyanobacterial blooms and alleviating eutrophication.

Matrix-driven environmental fate and effects of silver nanowires during printed paper electronics end of life

The fate and impact of silver nanowires incorporated in a paper-based nano-enabled product (NEP) was investigated in freshwater mesocosms.

Opposite effects of nutrient enrichment and an invasive snail on the growth of invasive and native macrophytes.

Many ecosystems are now co-invaded by alien plant and herbivore species. The evolutionary naivety of native plants to alien herbivores can make the plants more susceptible to the detrimental effects of herbivory than co-occurring invasive plants, in accordance with the apparent competition hypothesis. Moreover, the invasional meltdown hypothesis predicts that in multiply invaded ecosystems, invasive species can facilitate each other's impacts on native communities. Although there is growing empirical support for these hypotheses, facilitative interactions between invasive plants and herbivores remain underexplored in aquatic ecosystems. Many freshwater ecosystems are co-invaded by aquatic macrophytes and mollusks and simultaneously experience nutrient enrichment. However, the interactive effects of these ecological processes on native macrophyte communities remain an underexplored area. To test these effects, we performed a freshwater mesocosm experiment in which we grew a synthetic native community of three macrophyte species under two levels of invasion by an alien macrophyte Myriophyllum aquaticum (invasion vs. no invasion) and fully crossed with two levels of nutrient enrichment (enrichment vs. no enrichment) and herbivory by an invasive snail Pomacea canaliculata (herbivory vs. no herbivory). In line with the invasional meltdown and apparent competition hypotheses, we found that the proportional aboveground biomass yield of the invasive macrophyte, relative to that of the native macrophyte community, was significantly greater in the presence of the invasive herbivore. Evidence of a reciprocal facilitative effect of the invasive macrophyte on the invasive herbivore is provided by results showing that the herbivore produced greater egg biomass in the presence versus in the absence of M. aquaticum. However, nutrient enrichment reduced the mean proportional aboveground biomass yield of the invasive macrophyte. Our results suggested that herbivory by invader P. canaliculata may enhance the invasiveness of M. aquaticum. However, nutrient enrichment of habitats that already harbor M. aquaticum may slow down the invasive spread of the macrophyte. Broadly, our study underscores the significance of considering several factors and their interactions when assessing the impact of invasive species, especially considering that many habitats experience co-invasion by plants and herbivores and simultaneously undergo various other disturbances, including nutrient enrichment.

Aqueous naphthenic acids and polycyclic aromatic hydrocarbons in a meso-scale spill tank affected by diluted bitumen analyzed directly by membrane introduction mass spectrometry

With the increasing use of unconventional, heavy crude oils there is growing interest in potential impacts of a diluted bitumen (DB) spill in marine and freshwater environments. DB has the potential to release several toxic, trace organic contaminants to the water column. Here, the aqueous concentrations and compositions of two classes of organic contaminants, naphthenic acids (NAs) and polycyclic aromatic hydrocarbons (PAHs), are followed over 8 weeks after a simulated spill of DB (10 L) into a freshwater mesocosm (1200 L) with river sediment (2.4 kg). These complex samples contain biogenic dissolved organic matter, inorganic ions, petroleum contaminants, suspended sediments, and oil droplets. We report the first use of condensed phase membrane introduction mass spectrometry (CP-MIMS) as a direct sampling platform in a complex multi-phase mesocosm spill tank study to measure trace aqueous phase contaminants with little to no sample preparation (dilution and/or pH adjustment). CP-MIMS provides complementary strengths to conventional analytical approaches (e.g., gas- or liquid chromatography mass spectrometry) by allowing the entire sample series to be screened quickly. Trace NAs are measured as carboxylates ([M-H]-) using electrospray ionization and PAHs are detected as radical cations (M+•) using liquid electron ionization coupled to a triple quadrupole mass spectrometer. The DB-affected mesocosm exhibits NA concentrations from 0.3 to 1.2 mg/L, which rise quickly over the first 2 – 5 days , then decrease slowly over the remainder of the study period. The NA profile (measured as the full scan in negative-electrospray ionization at nominal mass resolution) shifts to lower m/z with weathering, a process followed by principal component analysis of the normalized mass spectra. We couple CP-MIMS with high-resolution mass spectrometry to follow changes in molecular speciation over time, which reveals a concomitant shift from classical ‘O2’ naphthenic acids to more oxidized analogues. Concentrations of PAHs and alkylated analogues (C1 – C4) in the DB-affected water range from 0 to 5 μg/L. Changes in PAH concentrations depend on ring number and degree of alkylation, with small and/or lightly alkylated (C0 – C2) PAH concentrations rising to a maximum in the first 4 – 8 days (100 – 200 h) before slowly decaying over the remainder of the study period. Larger and heavily alkylated (C3 – C4) PAH concentrations generally rise slower, with some species remaining below the detection limit throughout the study period (e.g., C20H12 class including benzo[a]pyrene). In contrast, a control mesocosm (without oil) exhibited NA concentrations below 0.05 mg/L and PAHs were below detection limit. Capitalizing on the rapid analytical workflow of CP-MIMS, we also investigate the impacts of sample filtration at the time of sampling (on NA and PAH data) and sample storage time (on NA data only).

Uptake and Transfer of Polyamide Microplastics in a Freshwater Mesocosm Study

Steadily increasing inputs of microplastics pose a growing threat to aquatic fauna, but laboratory studies potentially lack realism to properly investigate its effects on populations and ecosystems. Our study investigates the trophic and ontogenetic transfer of microplastics in a near-natural exposure scenario. The controlled outdoor freshwater mesocosms were exposed to polyamide (PA) 5–50 µm in size in concentrations of 15 and 150 mg L−1 and a control without microplastic addition. To verify the uptake of particles via the food chain, larvae and imagines of the midges Chaoborus crystallinus and C. obscuripes were examined, which feed on zooplankton during their larval stage. Larvae were captured after 117 days and imagines were caught in emergence traps that were emptied weekly. To detect the microparticles within the organisms, 200 larvae and 100 imagines per application were macerated and treated with fluorescent dye before investigation under a fluorescent microscope. We could detect up to 12 PA particles per individual larvae, while nearly no plastic was found in the imagines. This shows that, while Chaoborus sp. takes up microplastics via predation, most of the pollutant is egested through regurgitation and remains in the water, where it can further accumulate and potentially harm other organisms.

A Glyphosate-Based Herbicide Cross-Selects for Antibiotic Resistance Genes in Bacterioplankton Communities.

ABSTRACTAgrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, the herbicide glyphosate has the potential to indirectly select for antibiotic-resistant bacteria. Such cross-selection could occur if the same genes (encoding efflux pumps, for example) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added a glyphosate-based herbicide (GBH) to 1,000-liter mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS. Together, these results indicate that GBHs can cross-select for antibiotic resistance in natural freshwater bacteria.IMPORTANCE Glyphosate-based herbicides (GBHs) such as Roundup formulations may have the unintended consequence of selecting for antibiotic resistance genes (ARGs), as demonstrated in previous experiments. However, the effects of GBHs on ARGs remain unknown in natural aquatic communities, which are often contaminated with pesticides from agricultural runoff. Moreover, the resistance provided by ARGs compared to canonical mutations in the glyphosate target enzyme, EPSPS, remains unclear. Here, we performed a freshwater mesocosm experiment showing that a GBH strongly selects for ARGs, particularly multidrug efflux pumps. These selective effects were evident after just a few days, and the ability of bacteria to survive and thrive after GBH stress was predictable by the number of ARGs in their genomes and, to a lesser extent, by mutations in EPSPS. Intensive GBH application may therefore have the unintended consequence of selecting for ARGs in natural freshwater communities.

Effects of aquatic nitrogen pollution on particle-attached ammonia-oxidizing bacteria in urban freshwater mesocosms.

Ammonia-oxidizing bacteria (AOB) attached to aquatic particles are important participants in ammonia oxidation within hypereutrophic urban river systems. To explore the effects of aquatic nitrogen pollution on particle-attached AOB in urban river, we utilized laboratory mesocosms to investigate the responses of abundances and community structure of particle-attached AOB to ammonium (NH4+) and glycine (C2H5NO2) amendments. The abundance and community structure of particle-attached AOB were determined with quantitative real-time polymerase chain reaction (qRT-PCR) analysis and high-throughput sequencing based on the AOB amoA gene, respectively. Most of the bacterial amoA sequences from different treatments were affiliated with uncultured Nitrosomonadaceae bacterium, uncultured Nitrosomonadales bacterium, and uncultured Nitrosomonas sp., which are closely associated with organic pollution. The species richness and diversity of particle-attached AOB communities increased with increasing NH4+ and glycine concentrations. Treatment effects contributed significantly to the variance in particle-attached AOB communities. Although, glycine was completely transformed to ammonium within a few days and ammonium amendments would change the community structure of particle-attached AOB, the effect of glycine on the particle-attached AOB community was regulated by both the resulting ammonium concentration, as well as organic matter availability to the heterotrophic bacteria. Results suggested that high anthropogenic nitrogen loadings appeared to promote higher particle-attached AOB richness and diversity in the hypereutrophic urban river, but the effect of organic nitrogen on the particle-attached AOB community was different from the effect of inorganic nitrogen. This study informs ammonia oxidization mechanisms in the hypereutrophic urban rivers, which contributes to remediation/restoration strategies.