Rapid, semiautomated quantification of bacterial cells in freshwater by using a microfluidic device for on-chip staining and counting

A microfluidic device-based system for the rapid and semiautomated counting of bacteria in freshwater was fabricated and examined. Bacteria in groundwater and in potable water, as well as starved Escherichia coli O157:H7 spiked in pond water, were able to be on-chip stained and enumerated within 1 h using this system.     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.     

An RNA-DNA hybridization assay chip with electrokinetically controlled oil droplet valves for sequential microfluidic operations

A novel RNA-DNA hybridization microfluidic chip for detecting pathogens was developed. The on-chip sequential operations of reagent delivery and washing processes in the hybridization assay were performed by gravity-based pressure-driven flow controlled by a pair of electrokinetically controlled oil-droplet sequence valves (ECODSVs). Numerical method was used to simulate the fluidic processes of reagents in the complex microchannel network. Based on the parameters determined from the numerical simulations, a reasonable hybridization assay microfluidic chip was developed. The application of this on-chip assay to detect Salmonella was demonstrated. Significantly shortened assay time (25 min) and a 3-20-fold reduction in reagent/sample consumption were achieved. The detection limit was 10³ CFU/mL which is comparable to the conventional assay. With further development of automatic control and the improvement of the detection strategy, this microfluidic RNA-DNA hybridization assay technique has a potential for point-of-testing applications.     

Activated chemical defenses suppress herbivory on freshwater red algae

The rapid life cycles of freshwater algae are hypothesized to suppress selection for chemical defenses against herbivores, but this notion remains untested. Investigations of chemical defenses are rare for freshwater macrophytes and absent for freshwater red algae. We used crayfish to assess the palatability of five freshwater red algae relative to a palatable green alga and a chemically defended aquatic moss. We then assessed the roles of structural, nutritional, and chemical traits in reducing palatability. Both native and non-native crayfish preferred the green alga Cladophora glomerata to four of the five red algae. Batrachospermum helminthosum, Kumanoa holtonii, and Tuomeya americana employed activated chemical defenses that suppressed feeding by 30–60 % following damage to algal tissues. Paralemanea annulata was defended by its cartilaginous structure, while Boldia erythrosiphon was palatable. Activated defenses are thought to reduce ecological costs by expressing potent defenses only when actually needed; thus, activation might be favored in freshwater red algae whose short-lived gametophytes must grow and reproduce rapidly over a brief growing season. The frequency of activated chemical defenses found here (three of five species) is 3–20× higher than for surveys of marine algae or aquatic vascular plants. If typical for freshwater red algae, this suggests that (1) their chemical defenses may go undetected if chemical activation is not considered and (2) herbivory has been an important selective force in the evolution of freshwater Rhodophyta. Investigations of defenses in freshwater rhodophytes contribute to among-system comparisons and provide insights into the generality of plant–herbivore interactions and their evolution.     

Flocculation of microalgae in brackish and sea waters

Flocculation is an essential step in the concentration and harvesting of microalgae from aquatic media. Salinity of brackish water and sea water requires high flocculant dosages and renders flocculation less effective than in freshwater algal media. Experiments with the marine microalgae Isochrysis galbana and Chlorella stigmatophora showed that effective alum or ferric chloride flocculation was obtained only with dosages which are 5 to 10 times higher than the dosages required for the flocculation of freshwater microalgae. The flocculant dosages required for removing over 90% of the algae from suspensions were found to increase linearly with salinity as expressed in ionic strength. High salinity was found to inhibit flocculation with polyelectrolytes which are quite effective in freshwater algae flocculation. This inhibition was diminished at reduced salinity levels and effective flocculation was attained at salinity levels of 5 g/liter and below, which is typical of desert brackish water. Two methods were found to induce flocculation in sea water: (a) combining polyelectrolytes with inorganic flocculants such as ferric chloride or alum, and (b) ozone oxidation pretreatment followed by flocculation with inorganic flocculants.     

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.     

淡水藻类在监测水质和净化污水中的应用

淡水藻类作为水体中的初级生产者,分布广泛,适应性强,在水生生态系统食物链中占据着十分重要的地位,在水质监测中起着关键的作用。通过对藻类生长与水环境之间的相互关系进行简要的概述,探讨了pH值和氮磷对淡水藻类的生长的影响,以及淡水藻类的生长对外界环境的影响。藻类不但应用于水质监测,而且还能去除水体中的氮、磷等营养物质和其它有机物,对自然水域中的污水有良好的净化作用。重点论述淡水藻类在水质监测和污水净化中的作用以及利用淡水藻类来处理污水的方法。并提出了保护水资源的相关建议,为综合监测和治理水环境提供一定的理论依据和支持。     

Uncharted Diversity and Ecology of Saprolegniaceae (Oomycota) in Freshwater Environments

The fungal-like family Saprolegniaceae (Oomycota), also called “water mold,” includes mostly aquatic saprophytes as well as notorious aquatic animal pathogens. Most studies on Saprolegniaceae have been biased toward pathogenic species that are important to aquaculture rather than saprotrophic species, despite the latter’s crucial roles in carbon cycling of freshwater ecosystems. Few attempts have been made to study the diversity and ecology of Saprolegniaceae; thus, their ecological role is not well-known. During a survey of oomycetes between 2016 and 2021, we investigated the diversity and distribution of culturable Saprolegniaceae species in freshwater ecosystems of Korea. In the present study, members of Saprolegniaceae were isolated and identified at species level based on their cultural, morphological, and molecular phylogenetic analyses. Furthermore, substrate preference and seasonal dynamics for each were examined. Most of the species were previously reported as animal pathogens; however, in the present study, they were often isolated from other freshwater substrates, such as plant debris, algae, water, and soil sediment. The relative abundance of Saprolegniaceae was higher in the cold to cool season than that in the warm to hot season of Korea. This study enhances our understanding of the diversity and ecological attributes of Saprolegniaceae in freshwater ecosystems.     

Microfluidic bead-based enzymatic primer extension for single-nucleotide discrimination using quantum dots as labels

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5 pM, signal/noise > 3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.     

Development of a microfluidic chip-based plasmid miniprep

Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16 h, including the time needed for bacterial cell culture. Here we describe the development of a microfluidic chip (MFC)-based miniprep that uses on-chip lysis and trapping of large DNA in agarose to differentially separate plasmid DNA from the bacterial chromosome. Our approach greatly decreases both the time required for the miniprep itself and the time required for growth of the bacterial cultures because our on-chip miniprep uses 10⁵ times fewer E. coli cells. Because the quality of the isolated plasmid is comparable to that obtained using conventional miniprep protocols, this approach allows growth of E. coli and isolation of plasmid within hours, thereby making it ideal for rapid screening approaches. This MFC-based miniprep, coupled with recently demonstrated on-chip transfection capabilities, lays the groundwork for seamless manipulation of plasmids on MFC platforms.     

AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling

Growth of five aeroterrestrial green algal strains (Trebouxiophyceae) in response to changing water availabilities—caused by osmotic (ionic) and matric (desiccation) stresses—was investigated in comparison with a freshwater and a marine strain. All investigated algae displayed good growth under brackish conditions while four out of the five aeroterrestrial strains even grew well under full marine conditions (28–40 psu). The comparison between growth responses in liquid medium, on solid agarose, and on glass fiber filters at 100% air humidity indicated a broad growth tolerance of aeroterrestrial algae towards diminished water availability. While two aeroterrestrial strains even grew better on solid medium which mimics natural biofilm conditions, the aquatic strains showed significant growth inhibition under matric stress. Except Stichococcus sp., which contained the C6-polyol sorbitol, all other aeroterrestrial green algae investigated synthesized and accumulated the C5-polyol ribitol in response to osmotic stress. Using ¹³C NMR spectroscopy and HPLC, it could be verified that ribitol functions as an osmotically regulated organic solute. This is the first proof of ribitol in free-living aeroterrestrial green algae. The biochemical capability to synthesize polyols under environmental stress conditions seems to support algal life outside aquatic habitats.     

Design and Validation of DNA Libraries for Multiplexing Proximity Ligation Assays

Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.     

Nutritional quality of fish faeces is enhanced by highly unsaturated fatty acid-producing heterotrophic protozoa

Highly unsaturated fatty acids such as 20:5n3 (EPA) are both hormone precursors and cell membrane components, making them important nutrients for aquatic animals. Many animals must obtain EPA from their diets because they cannot synthesize enough EPA to meet their requirements, and algae are the main source of EPA in aquatic ecosystems. In a previous study, we detected EPA in the faeces of Danio rerio, a freshwater fish, even though the fish consumed a green algae diet that did not contain EPA. The objective of this study was to determine why EPA was detected in fish faeces. A significant positive relationship was detected between the number of heterotrophic protozoa and the concentration of EPA in the faeces, which suggests that this EPA was of protozoan origin. In addition, another experiment showed that protozoa adhered to faeces far more than the green algal diet remnants, which indicates that protozoa preferred to swarm on faeces. Furthermore, we cultured protozoa in an EPA-free medium and fed them a bacterial diet also lacking EPA, and found that Cyclidium sp. synthesized EPA de novo. The results demonstrate that protozoa produce essential fatty acids and enhance the nutritional quality of animal faeces in detritus-based food webs in freshwater ecosystems.     

Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring.     

Punch Card Programmable Microfluidics

Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word “PUNCHCARD MICROFLUIDICS” using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N2-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N₂-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with ³²P_i, and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Generation of Gradients on a Microfluidic Device: Toward a High-Throughput Investigation of Spermatozoa Chemotaxis

Various research tools have been used for in vitro detection of sperm chemotaxis. However, they are typically poor in maintenance of gradient stability, not to mention their low efficiency. Microfluidic device offers a new experimental platform for better control over chemical concentration gradient than traditional ones. In the present study, an easy-handle diffusion-based microfluidic chip was established. This device allowed for conduction of three parallel experiments on the same chip, and improved the performance of sperm chemotaxis research. In such a chip, there were six channels surrounding a hexagonal pool. The channels are connected to the hexagon by microchannels. Firstly, the fluid flow in the system was characterized; secondly, fluorescein solution was used to calibrate gradient profiles formed in the central hexagon; thirdly, sperm behavior was observed under two concentration gradients of progesterone (100 pM and 1 mM, respectively) as a validation of the device. Significant differences in chemotactic parameters were recognized between experimental and control groups (p < 0.05). Compared with control group, sperm motility was greatly enhanced in 1 mM group (p < 0.05), but no significant difference was found in 100 pM group. In conclusion, we proposed a microfluidic device for the study of sperm chemotaxis that was capable of generating multi-channel gradients on a chip and would help reduce experimental errors and save time in experiment.     

The xanthophyll cycle and NPQ in diverse desert and aquatic green algae

It has long been suspected that photoprotective mechanisms in green algae are similar to those in seed plants. However, exceptions have recently surfaced among aquatic and marine green algae in several taxonomic classes. Green algae are highly diverse genetically, falling into 13 named classes, and they are diverse ecologically, with many lineages including members from freshwater, marine, and terrestrial habitats. Genetically similar species living in dramatically different environments are potentially a rich source of information about variations in photoprotective function. Using aquatic and desert-derived species from three classes of green algae, we examined the induction of photoprotection under high light, exploring the relationship between nonphotochemical quenching and the xanthophyll cycle. In liquid culture, behavior of aquatic Entransia fimbriata (Klebsormidiophyceae) generally matched patterns observed in seed plants. Nonphotochemical quenching was lowest after overnight dark adaptation, increased with light intensity, and the extent of nonphotochemical quenching correlated with the extent of deepoxidation of xanthophyll cycle pigments. In contrast, overnight dark adaptation did not minimize nonphotochemical quenching in the other species studied: desert Klebsormidium sp. (Klebsormidiophyceae), desert and aquatic Cylindrocystis sp. (Zygnematophyceae), and desert Stichococcus sp. (Trebouxiophyceae). Instead, exposure to low light reduced nonphotochemical quenching below dark-adapted levels. De-epoxidation of xanthophyll cycle pigments paralleled light-induced changes in nonphotochemical quenching for species within Klebsormidiophyceae and Trebouxiophyceae, but not Zygnematophyceae. Inhibition of violaxanthin–zeaxanthin conversion by dithiothreitol reduced high-light-associated nonphotochemical quenching in all species (Zygnematophyceae the least), indicating that zeaxanthin can contribute to photoprotection as in seed plants but to different extents depending on taxon or lineage.     

Environmental DNA detection of aquatic invasive plants in lab mesocosm and natural field conditions

Aquatic invasive plant species cause negative impacts to economies and ecosystems worldwide. Traditional survey methods, while necessary, often do not result in timely detections of aquatic invaders, which can be cryptic, difficult to identify, and exhibit very rapid growth and reproduction rates. Environmental DNA (eDNA) is a relatively new method that has been used to detect multiple types of animals in freshwater and marine ecosystems through tissues naturally shed from the organism into the water column or sediment. While eDNA detection has proven highly effective in the detection of aquatic animals, we know less about the efficacy of eDNA as an effective surveillance tool for aquatic plants. To address this disparity, we designed mesocosm experiments with Elodea species to determine the ability to detect accumulation and degradation of the DNA signal for aquatic plants, followed by field surveillance of the highly invasive Hydrilla verticillata in freshwaters across several U.S. geographic regions. In both lab and field experiments, we designed a high sensitivity quantitative PCR assay to detect the aquatic plant species. In both experiments, plant eDNA detection was successful; we saw accumulation of DNA when plants were introduced to tanks and a decrease in DNA over time after plants were removed. We detected eDNA in the field in areas of known Hydrilla distribution. Employing eDNA detection for aquatic plants will strengthen efforts for early detection and rapid response of invaders in global freshwater ecosystems.     

Life cycle impacts of topsoil erosion on aquatic biota: case study on <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> forest

Purpose

This study illustrates the applicability of a framework to conduct a spatially distributed inventory of suspended solids (SS) delivery to freshwater streams combined with a method to derive site-specific characterisation factors for endpoint damage on aquatic ecosystem diversity. A case study on Eucalyptus globulus stands located in Portugal was selected as an example of a land-based system. The main goal was to assess the relevance of SS delivery to freshwater streams, providing a more comprehensive assessment of the SS impact from land use systems on aquatic environments.

Methods

The WaTEM/SEDEM model, which was used to perform the SS inventory, is a raster-based empirical erosion and deposition model. This model allowed to predict the amount of SS from E. globulus stands under study and route this amount through the landscape towards the drainage network. Combining the spatially explicit SS inventory with the derived site-specific endpoint characterisation factors of SS delivered to two different river sections, the potential damages of SS on macroinvertebrates, algae and macrophytes were assessed. In addition, this damage was compared with the damage obtained with the commonly used ecosystem impact categories of the ReCiPe method.

Results and discussion

The relevance of the impact from SS delivery to freshwater streams is shown, providing a more comprehensive assessment of the SS impact from land use systems on aquatic environments. The SS impacts ranged from 15.5 to 1234.9 PDF m³.yr.ha^?1.revolution^?1 for macroinvertebrates, and from 5.2 to 411.9 PDF.m³.yr.ha^?1.revolution^?1 for algae and macrophytes.For some stands, SS potential impacts on macroinvertebrates have the same order of magnitude than freshwater eutrophication, freshwater ecotoxicity, terrestrial ecotoxicity and terrestrial acidification impacts. For algae and macrophytes, most of the stands present SS impacts of the same order of magnitude as terrestrial ecotoxicity, one order of magnitude higher than freshwater eutrophication and two orders of magnitude lower than freshwater ecotoxicity and terrestrial acidification.

Conclusions

The SS impact results allow concluding that the increase of SS in the water column can cause biodiversity damage and that the calculated impacts can have a similar or even higher contribution to the total environmental impact than the commonly used ecosystem impact categories of the ReCiPe method. A wide application of the framework and method developed at a local scale will enable the establishment of a regionalised SS inventory database and a deep characterisation of the potential environmental impacts of SS on local aquatic environments.