Osmotic Behavior Research Articles

Analysis of the Effects of Temperature and Treatment Duration on the Resistance of Expansive Soil Improved with Lime in Baghdad, Iraq

Multiple studies have revealed the challenge of constructing infrastructure on expansive soils, including pipelines, roads, or buildings. This predicament stems from the uneven moisture distribution inherent to the specific soil type. Numerous methods, involving the addition of chemicals, have been employed to enhance the properties of clayey soils. This study introduces lime as a cation exchange material and demonstrates its capacity to improve the load-bearing characteristics, rendering it a more favorable option for engineering construction purposes. Lime's reaction with clay minerals and water produces calcium hydroxide, which subsequently reacts with the silica and alumina in the clay to form new compounds that promote stability. Additionally, lime helps reduce the soil's water-holding ability, thereby decreasing its swelling potential. This research will focus on evaluating the influence of temperature and treatment duration on the osmotic pressure behavior of chemically treated expansive clayey soils using lime. The swell meter test was utilized to develop lime-clay samples containing 7% lime by dry weight. These samples were then subjected to compression at temperatures ranging from 20 °C to 40 °C over a period of up to 28 days. The findings indicate that the pozzolanic reaction results in higher compressive strengths when tested at the upper limits of the temperature range in laboratory experiments. Therefore, the combined effects of temperature and curing duration play a positive role in improving the compressive strength of expansive soils.

Biophysical characterization and modeling of osmotic behavior of suspended HEPG2 cells

Biophysical characterization and modeling of osmotic behavior of suspended HEPG2 cells

Applied Electric Field Effects on Diffusivity and Electrical Double-Layer Thickness.

This study utilizes molecular dynamics (MD) simulations and continuum frameworks to explore electroosmotic flow (EOF) in nanoconfined aqueous electrolytes, offering a promising alternative to conventional micro-/nanofluidic systems. Although osmotic behavior in these environments is deeply linked to local fluid properties and interfacial dynamics between the fluid and electrolyte solutions, achieving a complete molecular-level understanding has remained challenging. The findings establish a linear relationship between electric field strength and fluid velocity, uncovering two distinct transport regimes separated by a critical threshold, with a markedly intensified flow in the second regime. It is demonstrated that rising electric field strengths significantly enhance water diffusion coefficients, supported by a detailed analysis of fluid hydration structures, the potential of mean force (PMF), and local stress tensors. Due to the applied electric field strength, the motion of ions and water accelerates, leading to the redistribution of ions and intensification of electrostatic forces. This expands the thickness of the electric double layer (EDL) and amplifies fluid diffusivity, thereby enhancing nanoscale fluid activity. These insights enhance the molecular-level understanding of EOF and define the stability of flow regimes, providing valuable guidelines for advancing nanofluidic technologies, such as drug delivery systems and lab-on-a-chip devices.

Osmotic absorption behaviors of imidazolium ILs in digestive system and their effective removal way

Considering the controversial safety and risk solutions of ionic liquids (ILs), more essential basic researches are required as their applied fields become broader and applied scale grows larger. Under this background, the permeation behaviors of six common imidazole ILs on porcine gastric and enteric membranes together with the feasibility of their removal by adsorption with montmorillonite were investigated in vitro. Through the single-factor investigation of different cations, anions and ionic liquid concentrations, the current results show that the IL of 1-ethyl-3-methylimidazolium bromide ([Emim]Br) has the strongest permeation capacity on the gastric membrane during the tests. The cumulative permeation amount of Qn can reach 0.72 mg/cm2, and the cumulative permeation percentage alsoreaches 95.77 %; the whole permeation process conforms to the Hixcon-Crowell model. As comparison, the maximum cumulative penetration percentage of [Emim]Br on enteric member was determined as 89.11 %, and related models was also compared. In order to remove the potential IL in digestive tract, montmorillonite was screened out from those popularily used antidotes in clinic and the adsorption of [Emim]Br in gastric fluid by montmorillonite has been proved more consistent with the second-order kinetics and Langmuir model. The results of molecular simulation, infrared spectrum (IR), X-ray diffraction (XRD) and thermogravimetric (TG) characterization exhibit that [Emim]Br has been successfully adsorbed into the interlayer of montmorillonite. As a whole, the study provided a scientific and effective way to diminish the hidden danger of ionic liquid entering the blood through the digestive system.

Theory-based cryopreservation mode of mesenchymal stromal cell spheroids

Cryopreservation of spheroids requires development of new improved methods. The plasma membranes permeability coefficients for water and cryoprotectants determine time characteristics of mass transfer through the cell membranes, and therefore the optimal modes of cells cryopreservation. Here we proposed an approach to cryopreservation of multicellular spheroids which considers their generalized characteristics as analogues of the membranes’ permeability coefficients of the individual cells. We have determined such integral characteristics of spheroids from mesenchymal stromal cells (MSCs) as osmotically inactive volume; permeability coefficients for water and Me2SO molecules and the activation energy of their penetration. Based on these characteristics, we calculated the osmotic behavior of multicellular spheroids under cooling conditions to select the optimal cooling rate. We also determined the optimal cooling rate of spheroids using the probabilistic model developed based on the two-factor theory of cryodamage. From the calculation it follows that the optimal cooling rate of the MSC-based spheroids is 0.75°С/min. To verify the obtained theoretical estimates, we conducted experiments on freezing MSC-based spheroids under different modes. The obtained results of primary viability screening indicate that freezing at a constant linear cooling rate of 0.75–1.0°С/min gives a good result. Theoretical prediction of the spheroid osmotic behavior during cooling provided the basis for experimental verification of varying the temperature to which slow cooling should be carried out before immersion in liquid nitrogen. Slow freezing of spheroids to −40 °C followed by immersion in liquid nitrogen was shown to preserve cells better than slow freezing to −80 °C. Obtained data allow more effective use of MSC-based spheroids in drug screening and regenerative medicine.

Gradient Hydrogels Spatially Trapped Optical Cell Profiling for Quantitative Blood Cellular Osmotic Analysis.

The quantitative exploration of cellular osmotic responses and a thorough analysis of osmotic pressure-responsive cellular behaviors are poised to offer novel clinical insights into current research. This underscores a paradigm shift in the long-standing approach of colorimetric measurements triggered by red cell lysis. In this study, we engineered a purpose-driven optofluidic platform to facilitate the goal. Specifically, creating photocurable hydrogel traps surmounts a persistent challenge─optical signal interference from fluid disturbances. This achievement ensures a stable spatial phase of cells and the acquisition of optical signals for accurate osmotic response analysis at the single-cell level. Leveraging a multigradient microfluidic system, we constructed gradient osmotic hydrogel traps and developed an imaging recognition algorithm, empowering comprehensive analysis of cellular behaviors. Notably, this system has successfully and precisely analyzed individual and clustered cellular responses within the osmotic dimension. Prospective clinical testing has further substantiated its feasibility and performance in that it demonstrates an accuracy of 92% in discriminating complete hemolysis values (n = 25) and 100% in identifying initial hemolysis values (n = 25). Foreseeably, this strategy should promise to advance osmotic pressure-related cellular response analysis, benefiting further investigation and diagnosis of related blood diseases, blood quality, drug development, etc.

Injectable peptide-glycosaminoglycan hydrogels for soft tissue repair: in vitro assessment for nucleus augmentation.

We report the development of peptide-glycosaminoglycan hydrogels as injectable biomaterials for load-bearing soft tissue repair. The hydrogels are injectable as a liquid for clinical delivery, rapidly form a gel in situ, and mimic the osmotic swelling behaviour of natural tissue. We used a new in vitro model to demonstrate their application as a nucleus augmentation material for the treatment of intervertebral disc degeneration. Our study compared a complex lab gel preparation method to a simple clinical benchtop process. We showed pH differences did not significantly affect gel formation, and temperature variations had no impact on gel performance. Rheological results demonstrated consistency after benchtop mixing or needle injection. In our in vitro disc degeneration model, we established that peptide augmentation could restore the native biomechanical properties. This suggests the feasibility of minimally invasive peptide-GAG gel delivery, maintaining consistent properties across temperature and needle sizes while restoring disc height and stiffness in vitro.

Osmotic behavior of t cells determined by flow imaging microscopy

Osmotic behavior of t cells determined by flow imaging microscopy

Cryogenic enrichment of Plasmodium falciparum gametocytes from spiked whole blood

Each individual cell type typically requires a unique set of conditions for optimal cryopreservation outcome, which relates to its specific response to cryoprotective agent (CPA) toxicity, osmotic behavior and sensitivity to ice crystallization. Cryopreservation of heterogenous cell populations is therefore exceedingly difficult as it requires separate and often conflicting conditions for each cell type. Conversely, these contrasting conditions could be utilized to favor cryogenic preference of a single cell population within a heterogenous sample, leading to its enrichment by elimination of remaining cells. To establish proof-of-concept for this overall approach, a protocol was developed for the cryogenic enrichment of Plasmodium falciparum gametocytes from whole blood. To accomplish this goal, we evaluated the effects of CPAs and cooling conditions during cryopreservation of whole blood samples spiked with P. falciparum gametocytes. We identified that cooling to −80 °C at a rate of −1 °C/min in the presence of 11 % glycerol selectively favors recovery of gametocytes. This protocol eliminates 95.3 ± 1.7 % of total blood cells and recovers 43.2 ± 6.5 % of parasites, leading to a 19-fold enrichment as assessed by microscopic examination of blood smears. This protocol is tunable, where gametocyte enrichment 900-fold may be feasible, however there is an apparent tradeoff in overall parasite recovery. Although translation of this protocol for point-of-care testing for malaria presents many challenges, the overall approach of cryogenic purification may prove useful for alternative diagnostic applications.

Understanding the Enhanced Osmotic Energy Conversion in Heterogeneous Membranes Using Engineered Branched Alumina Nanochannel Membranes

Heterogeneous membranes with two composite layers of distinct pore sizes have emerged as promising candidates for efficient osmotic energy extraction from salinity gradients. Previous studies about heterogeneous membranes report a strong link between the induced diode‐like rectification property and improved osmotic energy conversion performance. Nevertheless, the inherently large interfacial resistance of heterogeneous membranes may offset such enhancement. To further understand the osmotic energy conversion behavior in heterogeneous membranes, a series of the branched alumina nanochannel (BAN) membranes consisting of large stem channels interconnected with small branched channels are designed. The interconnected and orderly aligned structure of the stem and branched channels ensures high effective driving force and ion selectivity while reducing interfacial resistance at the same time. Experimental and simulation results confirm the rectification effect induced by the asymmetric pores of BAN, which can achieve an osmotic power ≈130% higher than that of the conventional cylindrical nanochannel membranes. Additionally, a power density as high as 5.42 W m−2 is obtained by mixing seawater and river water, surpassing most of the existing heterogeneous membranes and the commercial benchmark. The BAN membrane proposed in this work provides a promising platform for the development of highly efficient osmotic energy harvesting devices.

Biomimetic Design of 3D Fibrous Mesh Reinforced Hydrogel Replicating the Form and Function of the Intervertebral Disc

A Biomimetic Design of the Artificial Intervertebral Disc In article number 2200254 Wei Lei, Mingjie Liu, Yang Zhang, and co-workers fabricate biomimetic prototype of artificial intervertebral disc with nature-mimicking mechanics and structure. The design possesses an angle-ply fiber scaffold with multi-scale structural hierarchy, which exhibits osmotic behaviors, non-linear viscoelasticity and durability, enduring it with a great potential for total cervical disc replacement in future.

Quantitative assessment of intracellular/extracellular dimethyl sulfoxide concentrations during freezing with low-temperature confocal Raman micro-spectroscopy.

Although cryopreservation plays an indispensable role in the clinical application of cell therapy, the research on the osmotic behavior of cells during freezing is still at the level of theoretical models, and quantitative experimental data are still lacking. Therefore, the Raman spectra of dimethyl sulfoxide (DMSO) solutions with different standard concentrations (5%-80% v/v) were recorded experimentally to establish a quantitative evaluation method with the intensity ratio of different labeled peaks to the hydrogen bonding peak (as the internal standard) of water molecules in relation to different DMSO concentrations. By using this method, the characteristics of quantitative changes in intra- and extracellular concentrations under three different freezing methods were explored, including direct freezing, ice seeding freezing and vitrification. It was found that the intracellular concentration (@ -50 °C) after the ice seeding (@ -7 °C) freezing (1 °C min-1) method could reach 41.6%-49.2%, significantly higher than that of the direct freezing method (1 °C min-1 to -50 °C) of 32.4%-39.1%. Moreover, it is worth noting that the quantitative values of concentrations (@ -50 °C) of the ice seeding freezing are more consistent with the primary saturation curve of the DMSO solution. Thus, for the first time, it was revealed from the experimental data that the fundamental reason for the improvement of cell survival after ice seeding operation was pre-dehydration, higher concentration and smaller osmotic pressure difference between the inside and outside of the cell. These results also confirmed the validity of the famous two-factor hypothesis and more work will be carried out in depth.

Real-time computer assisted measurement of oocyte and embryo volume for assessment of transport parameters

Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols.

Delamination by Repulsive Osmotic Swelling of Synthetic Na-Hectorite with Variable Charge in Binary Dimethyl Sulfoxide-Water Mixtures.

Swelling of clays is hampered by increasing layer charge. With vermiculite-type layer charge densities, crystalline swelling is limited to the two-layer hydrate, while osmotic swelling requires ion exchange with bulky and hydrophilic organic molecules or with Li+ cations to trigger repulsive osmotic swelling. Here, we report on surprising and counterintuitive osmotic swelling behavior of a vermiculite-type synthetic clay [Na0.7]inter[Mg2.3Li0.7]oct[Si4]tetO10F2 in mixtures of water and dimethyl sulfoxide (DMSO). Although swelling in pure water is restricted to crystalline swelling, with the addition of DMSO, osmotic swelling sets in at some threshold composition. Finally, when the DMSO concentration is increased further to 75 vol %, swelling is restricted again to crystalline swelling as expected. Repulsive osmotic swelling thus is observed in a narrow composition range of the binary water-DMSO mixture, where a freezing point suppression is observed. This suppression is related to DMSO and water molecules exhibiting strong interactions leading to stable molecular clusters. Based on this phenomenological observation, we hypothesize that the unexpected swelling behavior might be related to the formation of different complexes of interlayer cations being formed at different compositions. Powder X-ray diffraction and 23Na magic angle spinning-NMR evidence is presented that supports this hypothesis. We propose that the synergistic solvation of the interlayer sodium at favorable compositions exerts a steric pressure by the complexes formed in the interlayer. Concomitantly, the basal spacing is increased to a level, where entropic contributions of interlayer species lead to a spontaneous thermodynamically allowed one-dimensional dissolution of the clay stack.

Safety Assessment of Channel Seepage by Using Monitoring Data and Detection Information

Seepage analysis has always been the focus of channel safety and stability research. Establishing a diagnosis method based on osmotic pressure monitoring data and combining the detection information to achieve osmotic safety is also an effective way to ensure the safety and stability of osmotic engineering. In this paper, a high-fill channel section of a water diversion project is taken as an example, and the study of osmotic safety is carried out by analyzing the engineering characteristics of linear engineering. High-fill channel sections were selected to study the temporal and spatial characteristics of various monitoring data reflecting the osmotic behavior of linear engineering; that is, these data reflect the time-varying regularity characteristics of the osmotic pressure value and the changing regularity of environmental variables. A single-point multifactor model of the monitoring data was established by establishing an evaluation index system, combining the monitoring index value method and the cloud model theory method according to the distribution law of the measured data and considering the uncertainty of the osmotic pressure data. Additionally, this model was integrated with the set pair analysis method to determine the monitoring data evaluation level; channel detection data information was collected, the abnormal detection of detection information was realized, and the monitoring data results were used to verify the detection results. In this way, an adaptive evaluation method reflecting the working behavior of high-filled channel sections is established, and a diagnostic technology for the safe operation of high-filled channel sections of linear engineering is proposed. The application results show that this method is suitable for engineering an osmotic safety assessment.

An understanding of oil–water replacement mechanism based on interfacial tension gradient during the well shut-in

Spontaneous imbibition behavior of fracturing fluid retained in reservoirs is considered to be one of the important mechanisms of spontaneous oil–water displacement in tight reservoirs. However, the mechanism of imbibition displacement in oil-wet rocks remains unclear. Inspired by the principle of the Marangoni effect, a mathematical model of oil displacement driven by an interfacial tension gradient in an oil-wet capillary tube was established, and the effects of osmotic pressure and hydraulic mechanical dispersion were coupled in the model. The results show that the key component of this mechanism is the breakdown of the initial capillary equilibrium state due to the diffusion of solute, which changes the pressure distribution in the capillary and drives the water to drive oil out of the pores. This can explain the spontaneous imbibition of low-salinity fracturing fluid into the oil-wet tight sample and displace oil without any external force, despite its low permeability. In addition, the related factors were analyzed, including initial concentration, contact angle, solute type, osmotic behavior, and size of the capillary. The results show that for an oil wet reservoir with high salinity, the connate water interfacial tension gradient plays an important role in oil displacement, and the coupling effect of osmotic pressure and hydromechanical dispersion considerably improves recovery. This can provide new insight into the mechanism of spontaneous imbibition of fracturing fluid.

INFLUENCE OF SOLUTIONS OF DIFFERENT OSMOLARITY ON MORPHOMETRIC PARAMETERS OF 15-32-CELLS BOVINE EMBRYOS

The illustrative material was obtained that allows to observe clearly the effect of different tonicity solutions on the morphology of bovine 5-days pre-compacted 15-17-cells embryos and 6-days early morulae obtained in vivo, depending on their technological state. The average values and rates of variation of the following morphometric parameters were determined in embryos at the stages of the developmental study: outer and inner zona pellucida (ZP) diameters, ZP thickness, embryonic cell mass (CM) diameter and individual embryonic cell diameters. The mentioned parameters were determined in each of the embryos consistently in intact, denuded and deblastomerised states in isotonic (0.29 Osm), hypertonic (1.5 Osm) and hypotonic (0.2 Osm) conditions. Manipulations with embryos were carried out in such a way that during consistent transfer through solutions of different osmolarity, it was possible to identify the dynamics of changes in the morphology and parameters of each of the embryos (in the intact state and after denudation) and of each of the individual embryonic cells (after deblastomerisation of the embryo). A decrease or increase in the size of the CM of embryos of the studied developmental stages during their successive transfer through solutions with different osmolarity occurred with the preservation of the approximate proportionality of their shape. After partial dehydration in hypertonia, the denuded embryos recovered their sizes mostly not fully relative to their initial sizes in the intact state in solutions of the appropriate osmolarity. Similar osmotic behavior was also observed in individual cells of deblastomerised embryos. The experimental data presented in the article make it possible to predict the behavior of embryos during micromanipulations, to determine the sequence, technique and conditions of operations with them, as well as the types, shape and sizes of microtools necessary for this.

Hydrogel composite mimics biological tissues.

A novel composite hydrogel was developed that shows remarkable similarities to load bearing biological tissues. The composite gel consisting of a poly(vinyl alcohol (PVA) matrix filled with poly(acrylic acid) (PAA) microgel particles exhibits osmotic and mechanical properties that are qualitatively different from regular gels. In the PVA/PAA system the swollen PAA particles "inflate" the PVA network. The swelling of the PAA is limited by the tensile stress Pel developing in the PVA matrix. Pel increases with increasing swelling degree, which is opposite to the decrease of the elastic pressure observed in regular gels. The maximum tensile stress Pmaxel can be identified as a quantity that defines the load bearing ability of the composite gel. Systematic osmotic swelling pressure measurements have been made on PVA/PAA gels to determine the effects of PVA stiffness, PAA crosslink density, and Ca2+ ion concentration on Pmaxel. It is found that Pmaxel increases with the stiffness of the PVA matrix, and decreases with (i) increasing crosslink density of the PAA and (ii) increasing Ca2+ ion concentration. Small angle neutron scattering (SANS) measurements indicate only a weak interaction between the PVA and PAA gels. It is demonstrated that the osmotic swelling pressure of PVA/PAA composite gels reproduces the osmotic behavior of healthy and osteoarthritic cartilage.

Theoretical approaches to determination of optimal cryopreservation regimens for cell spheroids of different cultivation terms

Background: Three-dimensional culture systems are unique platforms for studying complex biological processes in vitro. Cell-cell and cell-extracellular matrix interactions form a communication network of biochemical and mechanical signals, bring spheroids (SP) closer to native tissues and significantly distinguish them from monolayer cultures. It is important for cell technologies to develop methods for cryopreservation of 3D cultures, that allows creating the stocks of valuable cell samples, save time and materials, and prevent the loss of cultures due to technical failures, contamination, phenotype drift and aging. Objectives: Development of approaches to cellular spheroids cryopreservation. Determination of the permeability parameters of L929 cells spheroids at different cultivation periods for the theoretical assessment of optimal freezing regimens. Materials and methods: We have used L929 cells, which form SPs of different diameters and can be maintained for a long time in 3D conditions. To determine the integral filtration Lp and permeability for DMSO kp coefficients for SP at different periods of cultivation, the volumetric method was used. The study of the changes in the spheroids volume in time was carried out with a confocal microscope LSM 510 META. The numerical values of the integral SF permeability coefficients were determined by approximating the experimental data on the change in the relative volume of the SP versus the exposure time in the test solution with theoretical curves calculated on the basis of a physical and mathematical model for passive mass transfer between the spheroid and the environment, provided that they coincide as much as possible. Prediction of the osmotic behavior of spheroids under cooling conditions was carried out based on the differential equation describing the kinetics of changes in the relative cell volume during extracellular crystallization of a cryoprotective solution, substituting determined values of integral permeability coefficients Lp and kp and activation energies EAL and EAk into the model equations. The kinetics of changes in the extracellular solution concentration during freezing was set analytically by approximating the phase melting diagram of the DMSO solution. Results: The filtration and permeability for DMSO molecules coefficients in SP were determined and their significant decrease with a cultivation duration was shown. The activation energy values for the penetration of water and DMSO molecules into the SP were calculated and their dependence on the cultivation time was determined. Proceeding from the determined parameters of permeability, the dynamic of changes in the volume of SPs for different periods of cultivation at different rates of cooling was calculated. Conclusions: The optimal cooling modes of SP from L929 cells were in theory determined: for 7 days of cultivation — 1,5-2 °C/min with cooling to -80°C and subsequent immersion in nitrogen; for 14 and 21 days of cultivation — 0.5 °C/min to -40°C and subsequent immersion in nitrogen.

The effect of salinity on osmotic regulation and respiratory rate of the prawn Macrobrachium tenellum Smith, 1871 (Decapoda: Caridea: Palaemonidae)

Abstract The effect of salinity on oxygen consumption rate and hemolymph osmolarity of the palaemonid prawn Macrobrachium tenellum (Smith, 1871) maintained at 0, 5, 10, 15, 20 and 25 psu was analyzed. Oxygen consumption rate was measured in respiratory chambers and osmolality from samples of hemolymph. Oxygen consumption rose significantly beyond 15 psu, with individuals showing hyper regulatory behavior from 0 to 10 psu, being able to maintain its internal solutes concentration (426–504 mmol kg–1) higher than that of the water (153–348 mmol kg–1). They acted as hypo-regulators from 15 to 25 psu as their internal solute concentration (454–562 mmol kg–1) was lower than that of the water (459–744 mmol kg–1). The isosmotic point was 505 mmol kg–1 at 16 psu, and survival was high in all salinities. The osmotic behavior of M. tenellum allows it to successfully invade fresh water by keeping constant the ionic and osmotic concentrations of both extra- and intra-cellular solute concentrations, always above fresh water, but varying its O2 consumption as salinity changes. The implications of such adaptations for the dispersal of the species into freshwater habitats is discussed.