Continuous-flow Bioassays Research Articles

Buffer-free integrative nanofluidic device for real-time continuous flow bioassays by ion concentration polarization.

To perform precision medicine in real-time, a sensor capable of continuously monitoring target biomolecules secreted from a patient under dynamic situations is essential. In this study, a novel portable device combining an aptamer probe and a nanofluidic component was developed, enabling the buffer-free continuous monitoring of small molecules in biological fluids. This integration is synergistic: the aptamer sensor is used to bind target biomolecules, triggering a fluorescence signal change, while the nanofluidic component is applied to achieve ion concentration polarization and convert serum into a clean buffer for aptamer signal regeneration. To demonstrate the system's versatility, we measured various adenosine triphosphate concentrations in human serum for hours with high sensitivity and specificity at minute temporal resolution. Our results demonstrate that this integrative device can be applied for the continuous measurement of target biomolecules and online signal regeneration in patient samples without the use of bulky clean buffer solutions for dynamic real-time healthcare.

Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3–mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.

Application of a Fluorescence-Based Continuous-Flow Bioassay to Screen for Diversity of Cytochrome P450 BM3 Mutant Libraries

A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants.

Effects of the insecticides carbofuran and fenvalerate on adenylate parameters in bluegill sunfish (Lepomis macrochirus)

Bluegills (Lepomis macrochirus) were exposed to two sublethal concentrations of carbofuran, a carbamate insecticide, and fenvalerate, a synthetic pyrethroid insecticide, in continuous flow bioassay systems for 30 days. Adenylate parameters (ATP concentration, total adenylates concentration, and adenylate energy charge) were monitored in gill, liver, muscle, and stomach tissues. There were significant decreases in adenylate energy charge of gill and stomach tissue relative to initial values after 10 days of exposure to 30 micrograms/L carbofuran. There was also a significant decline in adenylate energy charge in gills of fish exposed to 0.2 micrograms/L fenvalerate relative to initial values. These values returned to normal by the end of the experiment. The biological significance of these changes is uncertain. Adenylate parameters may not be sufficiently sensitive to reflect sublethal effects of pesticides which affect the nervous system of fishes.

ALGAL BIOASSAY AND GROSS PRODUCTIVITY EXPERIMENTS USING SEWAGE EFFLUENT IN A MICHIGAN WETLAND'

ABSTRACT: One component of the filamentous algal community of a northern fen ecosystem in central Michigan was studied under conditions of nutrient enrichment by secondarily treated sewage effluent during one growing season. The productivity of Cladophora spp. measured by continuous flow bioassay was 2.6 g dry weight m day at the site of effluent addition compared to 0.085 g m day at the control site. Under conditions of nutrient enrichment, uptake by bioassay Cladophora spp. averaged 12 mg m−2day−1for phosphorus and 55 mg m−2day−1for nitrogen, compared to 0.01 mg m−2 day−1and 0.16 mg m−2day−1for phosphorus and nitrogen, respectively, in the control area. At the end of the growing season approximately 4.3 g N m−2 and 0.96 g P m−2were immobilized in Cladophora algal biomass. Algal growth temporarily immobilized 3.0 percent of the nitrogen and 1.0 percent of the phosphorus added as sewage effluent. Gross productivity of surface water in the fen averaged 1.5 g O2m−2day−1at the nutrient enriched site, compared to 0.5 g O2 m−2day−1at the control area. Gross productivity, community respiration and reaeration constant values in the fen were similar to data collected by other researchers in shallow water aquatic systems, but only at the fertilized sites.

Lethal and Sublethal Toxicity of Drilling Fluids to Larvae of the American Lobster,Homarus americanus

The lethal and sublethal effects of five used, whole drilling fluids on the larval stages of the American lobster (Homarus americanus) were assessed in laboratory experiments using a continuous-flow bioassay. Although the five tested drilling fluids varied markedly in their toxicity, some were highly toxic, with LC50 values as low as 74 mg/L. Sublethal exposures to drilling fluids at concentrations as low as 10–50 mg/L resulted in reduced respiration rates, reduced O:N ratios, and increased protein:lipid ratios, demonstrating a change in energetics of the larval lobsters. Growth and development of the larvae were seriously impaired by exposure to three of the five drilling fluids at concentrations of 50 and 100 mg/L. The feeding rates were also significantly reduced after a 24-h exposure to 50 mg/L drilling fluid. Exposure of larvae to barite (a major component of drilling fluids) and to a field-collected, fine-grained sediment did not result in deleterious effects. We suggest that the chemical components and not the physical properties of the drilling fluids are primarily responsible for detrimental effects. From results of the chemical analyses of the tested drilling fluids, we consider that the adverse effects of these drilling fluids cannot be attributed to any one group of chemicals. For example, diesel oil, a known toxicant, was present in the more toxic drilling fluids; however, there was no direct correlation between the toxicity of a drilling fluid and diesel oil concentration. Phenolic compounds, various metals, and other components probably also contributed to the toxicity of these drilling fluids.

Lethal and sublethal exposure and recovery effects of ozone-produced oxidants on adult white perch ( Morone americana Gmelin)

Adult white perch ( Morone americana), acclimated to 15°C, were exposed to a series of ozone-produced oxidant (OPO) concentrations for 96 h using continuous flow bioassay techniques. Toxicity data were analyzed using both response surface modeling and standard probit regression. White perch were also exposed to a series of near and sublethal OPO concentrations, selected from the acute toxicity study, for 96 h and then placed in clean non-ozonated water for 14 days. Blood pH, hematocrit and gill histopathology were analyzed during exposure at 24, 48 and 96 h and after 4 and 14 days in the recovery period. Blood pH and hematocrit levels were analyzed statistically using standard ANOVA and multiple range tests. Histopathological effects were examined using both light microscopy and scanning electron microscopy. The 24-, 48- and 96-h LC 30's were 0.38, 0.26 and 0.20 mg OPO l −1, respectively. Blood pH was significantly reduced at concentrations ⩾0.15 mg OPO l −1 but not at 0.10 mg l −1 or lower concentrations. Hematocrit significantly increased at concentrations ⩾0.10 mg OPO l −1. Histopathological examination revealed minimal effects on gill tissue at 0.01 mg OPO l −1, moderate epithelial sloughing and heavy mucus production at 0.05 mg OPO l −1 and extreme tissue damage at concentrations ⩾0.10 mg l −1. Results from both the acute toxicity and the exposure and recovery study were compared with the effects of chlorine-produced oxidants (CPO) obtained from the literature. Both OPO and CPO appear to have similar effects on adult white perch.

The toxicity of jet fuels to fish—II: The toxicity of JP-8 to flagfish ( Jordanella floridae) and rainbow trout ( Salmo gairdneri) and golden shiners ( Notemigonus chysoleucas)

JP-8 is a widely-used commercial and military jet fuel which may reach the aquatic environment by several avenues. The toxicity of JP-8 to cold and warm water fish was investigated to help define the impact on fresh water species. Fish were exposed to the water soluble fraction (WSF) of JP-8 in static acute bioassays and continuous-flow bioassays of approx. 4 months duration. The goal was to determine the 96-h LC 50 and the long-term no effect level on the life cycle. The acute toxicity to a warm water fish was found to be unaffected by pH in the range of 7.3–9.1. The no effect level with respect to growth was lower in rainbow trout than in flagfish. Fuel accumulation and depuration from fish tissues are compared for the two species, as are the effects on egg hatching. The relative toxicity of JP-8 and JP-9 (previously reported) is discussed.

Mixing and separation device for continuous flow bioassays with coal liquids.

Efforts to increase United States' use of synthetic fuels, such as those derived from coal liquefaction, require examining potential toxicity of coal liquids that may eventually be released to the aquatic environment. Evaluation of potential long-term ecological effects must consider organism response to chronic exposure regimes. Since maintaining constant levels of the water soluble fraction (WSF) of coal liquids is complicated by presence of easily biodegradable phenolic compounds (DEGRAEVE et al. 1980), and since problems with low oxygen levels may occur with static tests, flow-through test systems must be employed. A serious problem with continuous flow studies of crude petroleum oils has been obtaining a consistent WSF. The test solution may contain dispersed oil and the character of the WSF may change during the testing period (CRADDOCK 1977). Although, batch-prepared WSFs of solvent refined coal (SRC) II materials were adequate for short-term continuous flow or static tests, a system was needed that would generate a stable WSF for long-term continuous flow bioassays. We developed a method to generate a reproducible stock solution for exposing aquatic organisms to sublethal concentrations of coal liquid WSFs under flowthrough conductions. Our apparatus extracts a primary WSF from a 2.9:1 blend of middle to heavy SRC II distillate, while separating and removing dispersed and floating insoluble materials. The WSF is efficiently extracted, thus reducing costs associated with waste treatment of the potentially hazardous materials. METHODS

Toxicity of underground coal gasification condenser water and selected constituents to aquatic biota.

The acute and embryo-larval toxicity of the Laramie Energy Technology Center's Hanna-3 underground coal gasification (UCG) condenser water and its constituents were studied in continuous-flow bioassays. The 96-hr LC50 dilution values for untreated Hanna-3 UCG condenser water were 0.1% for rainbow trout, 0.11% for fathead minnows and the 48-hr LC50 dilution forDaphnia pulicaria was 0.18%. Separate 96-hr acute tests with phenol, ammonia, and ammonia plus phenol showed that these two constituents, acting synergistically, were the major constituents affecting the acute toxicity of this coal conversion effluent to fishDaphnia pulicaria, on the other hand, was relatively insensitive to phenol exposure; the primary constituent of Hanna-3 UCG condenser water affecting this species was ammonia.

An apparatus for the continuous generation of stock solutions of hydrophobic chemicals.

A major technical difficulty associated with testing the toxicity to aquatic organisms of chemicals having low water solubility is the production of a stock solution of the chemical without the use of exogenous solubilizers or carriers. For highly toxic hydrophobic chemicals this problem has been approached by passing water through a column of chemical adsorbed to inert supports (CHADWICK & KIIGIMAGI 1968) o While this technique is somewhat effective, channelization of water in the column may lead to variation in the concentration of toxicant produced during long term exposure studies. VEITH & COMSTOCK (1975) reduced this variation by continuously recirculating the toxicant solution through the bed of substrate with a submersible pump. For many hydrophobic chemicals of moderate or low toxicity, the difficulties of producing a satisfactory stock solution are compounded by the fact that water concentrations of the chemical that are acutely toxic to fish may be near their limits of solubility~ Toxicity testing of these chemicals through continuous flow bioassay methods thus requires large volumes of stock solution of relatively high concentration. In addition, many toxicants are liquids at test temperatures and not amenable to application from a substrate bed.

Influence of three sediment types on copper toxicity to the polychaete Neanthes arenaceodentata

Adult male Neanthes arenaceodentata were exposed to 0.10±0.015 mg l-1 copper in the seawater of a continuous-flow bioassay system in the presence of a sand, a mud, a mixture of the sand and mud, and no sediment, to assess the influence of sediment type on Cu-induced mortality. The sediment type did influence mortality. The time to 50% mortality was 7.8 days without sediment, 36.5 days with sand, 54.5 days with the mixture, and 50.0 days with mud. There was no threshold Cu body burden that caused death. The mean Cu concentration per gram of N. arenaceodentata (≤24 h after death) without sediment was 270 μg, in sand 994 μg, in the mixture 1047 μg, and in mud 1464 μg. The differences in the toxic responses are discussed.

Acute toxicity of the water-soluble fraction of Cook Inlet crude oil to the Manila clam

Recognition of the inherent complexities of oil bioassays prompted the development of several apparatuses designed to deliver uniform solutions of the water-soluble components of crude oil and capable of system equilibration for both static and continuous-flow bioassays without the loss of the more volatile compounds and without the formation of emulsions. Clams from a relatively contaminated area exhibited greater resistance to the water-soluble fraction of Cook Inlet crude oil than specimens from a more pristine area. Behaviour was altered below lethal levels suggesting the possibility of using behavioural modifications as a concommitant criterion of toxicity with mortality. An increase in mortality with a longer exposure period and a significant rate of latent mortalities following the end of the 96-h exposure period suggest that future bioassays with bivalves should be determined over a longer exposure period.

Autoradiography on erythrokinesis and multihemoglobins in juvenile Salmo salar L. at various respiratory gas regimes.

The kinetics of erythropoiesis in 5--6 month old Salmo salar L. was correlated to 4 combinations of environmental PO2 and pH/PCO2. Given single doses of 55Fe at the start of continuous flow bioassays, the incorporation was examined in circulating red blood cells (RBC) by autoradiography on blood smears. The proportions of immature, labelled immature and labelled mature erythrocytes were calculated on samples taken at intervals up to 52 days. Compared to control fish kept at a PO2 corresponding to 90% air saturation and pH/PCO2 at 7.6/8 pH unit/mmHg, oxygen depletion to 50% air saturation stimulated the proliferation of RBC stem cells and enhanced RBC maturation. Ceteris paribus, sustained hypercapnia at a PCO2 in a respiratory water raised to 23 mm Hg (pH 7.1--7.2), stimulated proliferation but did not affect the output of mature RBC. Simultaneously lowered PO2 and raised PCO2 to the levels mentioned above obviously stressed the fish, as the effect of lowered PO2 per se was not manifested. Radioactivity was traced to all electrophoretically separable protein fractions from RBC hemolysates. The relevance of blood physiological criteria for probing the fitness or well being of fish in environmental respiratory stress conditions has been discussed.

Apparatus for the continuous dissolution of poorly water-soluble compounds for bioassays

An apparatus was designed and constructed to supply a continuous flow of water, saturated with soluble components of JP8 jet fuel. The product water, used to supply continuous flow bioassays, was found to be comparable in quality to that obtained with static dissolution techniques. The apparatus is constructed of inert materials and operates without pumps or other electro-mechanical regulating devices.

Influence of sediment in copper toxicity tests with the polychaete Neanthes arenaceodentata

Adult male Neanthes arenaceodentata were exposed to measured concentrations of 0.04, 0.06, 0.10, 0.16 and 0.26 mg l −1 Cu in a continuous-flow bioassay system, with and without a clean sand, to assess the influence of sand on Cu induced mortality. The presence of sand did affect the results. The 28-day LC50 was lower for polychaetes exposed without sand than those with sand, 0.044 and 0.10 mg l −5 Cu, respectively. The reasons for this difference are discussed.

The differential effects of free and combined chlorine on juvenile marine fish

The differential effects of free chlorine and chloramine on three species of juvenile marine fish have been investigated in continuous flow bioassay units. The toxicity of both chlorine forms to winter flounder, Pseudopleuronectes americanus, scup, Stenotomus versicolor, and killifish, Fundulus heteroclitus, appeared to be a threshold effect: an abrupt increase in mortality was observed over a narrow range of toxicant concentrations. The three species were similar in their responses to free chlorine, the more toxic of the two chlorine forms. However, there was a difference in chloramine toxicity among the three species tested; killifish were more susceptible than either of the other two species, probably reflecting differences in metabolic regulation or uptake rates. Behavioral aberrations—distended gills and erratic swimming behavior—of winter flounder and scup and significant reductions in standard respiration rates of killifish were observed with exposure to chlorine or chloramine concentrations approaching lethal levels.

Chlorinated cooling waters in the marine environment: Development of effluent guidelines

The effects of free chlorine and chloramine on stage I lobster larvae and juvenile killifish were investigated in continuous flow bioassay units. In comparing mortality and changes in standard respiration rates during and after exposure to either chlorine form, significant respiratory stress was observed with exposure to sublethal levels. Sublethal responses to free and combined chlorine should be considered when establishing regulations for chlorine residuals in cooling waters.

Assessment of the Acute Toxicity, Growth Impairment, and Flesh Tainting Potential of a Bleached Kraft Mill Effluent on Rainbow Trout (Salmo gairdneri)

Static and continuous-flow bioassays utilizing juvenile rainbow trout (Salmo gairdneri) were conducted on a northern Ontario bleached kraft mill effluent to assess the range of acute toxicity and related sublethal effects. The acute toxicity of the total mill effluent, as determined by static bioassay procedures, ranged from a 96-h LC50 value of 14–49.0% vol/vol. The results of these bioassays depended on testing methodology and effluent treatment systems employed by the mill when samples were collected. Continuous-flow bioassays conducted on the effluent Produced 96-h LC50 values of 21.8 and 24.8% vol/vol. Growth rate of juvenile rainbow trout (1–2 g) held under continuous-flow conditions for 18 days was significantly reduced at 6% vol/vol effluent concentration (P < 0.05). Flavor of yearling rainbow trout [Formula: see text] exposed to 3% effluent for 48 h was significantly impaired (P < 0.05). This effluent concentration, the lowest level impairing flavor represents approximately 0.12 of the 96-h LC50 derived from continuous-flow bioassays. No significant flavor impairment was detected in fish exposed for 144 h to a 2% effluent concentration Flavor impairment at a 3% vol/vol effluent concentration, the most sensitive sublethal response measured in this study, may also be the most critical response when assessing the economic impact of a kraft mill discharge to a freshwater ecosystem.

Fish toxicity of jet fuels—I. The toxicity of the synthetic fuel JP-9 and its components

The toxicity of the jet fuel JP-9 and its components RJ-4, RJ-5 and MCH was assessed in static bioassays on the warm water fish, golden shiner ( Notemigonus chrysoleucas). The 96-h LC 50 of emulsions was 2.0 mg 1 −1 for JP-9, 0.51 mg 1 −1 for RJ-4, 0.61 mg 1 −1 for RJ-5 and 72 mg 1 −1 for MCH. As determined by the 96-h LC 50 values unemulsified fuel (pools of fuel) JP-9 was 235 times less toxic, RJ-4 was 196 times less toxic, RJ-5 was 7700 times less toxic and MCH was 3.3 times less toxic than the corresponding emulsified materials. In continuous flow bioassays with the water soluble fraction of the fuel and its components the effect on egg hatchability and fry development of flagfish ( Jordanella floridae) and rainbow trout ( Salmo gairdneri) was studied. The no effect of level on flagfish egg hatchability was 0.23 mg 1 −1 for JP-9 and 0.05 mg 1 −1 for RJ-5. Concentrations of 0.83 mg 1 −1 MCH and 0.2 mg 1 −1 RJ-4 did not affect egg hatchability. In rainbow trout studies 97-day LC 50 values for RJ-4 and RJ-5 were 0.045 mg 1 −1 and 0.072 mg 1 −1, respectively, and 23-day LC 50 values for JP-9 and MCH were 0.26 mg 1 −1 and 1.3 mg 1 −1, respectively. The accumulation of fuels in fish bodies was studied and it was found that flagfish can tolerate a total body burden of 0.5 mg MCH g −1 wet weight without lethality. It was also found that body burdens of 0.43 mg RJ-4 g −1 and of 0.27 mg RJ-5 g −1 on a wet weight basis will produce 50% mortality in rainbow trout. Voiding of MCH from fish bodies occurs readily in fuel-free water, but RJ-4 and RJ-5 are retained in the tissues.