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1.
Int J Biol Macromol ; 146: 560-568, 2020 Mar 01.
Article in English | MEDLINE | ID: mdl-31917985

ABSTRACT

In the present study, we report cytotoxic and antimicrobial potential of gold nanoparticles (Au NPs) synthesized using chitosan derived from squilla shell wastes. Here we adopted ecofriendly approach for the synthesis of Au NPs and characterized them using UV-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, atomic force microscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) methods. The UV-visible spectroscopic analysis revealed an absorption peak at 529 nm, which represents the Surface Plasmon Resonance of Au NPs. TEM analysis showed that the biosynthesized Au NPs were spherical in shape with an average size of 80 to 82 nm. Interestingly, the biosynthesized Au NPs showed antimicrobial activity against selected Gram-positive and negative bacterial isolates and also showed activity against fungal pathogens. Further, the cytotoxic effect of the synthesized Au NPs against MCF-7 cell lines was assessed by MTT assay with IC50 value of 250 µg mL-1. In addition, double staining of treated MCF-7 cells with acridine orange and ethidium bromide shows that the Au NPs exert apoptosis mediated cytotoxicity.


Subject(s)
Bacteria/drug effects , Chitosan/pharmacology , Fungi/drug effects , Gold/pharmacology , Metal Nanoparticles/chemistry , Animals , Anti-Infective Agents/pharmacology , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Cell Survival/drug effects , Humans , MCF-7 Cells , Metal Nanoparticles/ultrastructure , Microbial Sensitivity Tests , Microscopy, Atomic Force , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared
2.
J Parasit Dis ; 41(1): 188-192, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28316410

ABSTRACT

An ectoparasitic isopod, Gnathia sp. was found in the Gill chambers of Heniochus acuminatus collected from the Gulf of Mannar region, Southeast coast of India. The present study signifies the new record of Gnathia sp. an coral reef ectoparasitic isopod captured from the gill net during October 2014. Among the 36 specimens examined 5 specimens were infested with Pranzia larvae of Gnathia sp. The size of the isopods were ranged from 1.5 to 3.2 mm and the host fish length varied between 119 and 230 mm. They were specifically found attached to the gill chambers and no damage observed in the lamellar pattern.

3.
Biotechnol Appl Biochem ; 62(3): 424-32, 2015.
Article in English | MEDLINE | ID: mdl-25041078

ABSTRACT

Bioreduction of metal ions for the synthesis of stable nanoparticles (NPs) in physiological environment has been a great challenge in the field of nanotechnology and its application. In the present study, well-defined biofunctionalized gold nanoparticles (AuNPs) were developed following a biomimetic approach for an enhanced anticancer activity. The fucoxanthins-capped crystalline AuNPs showed a particle size of 14 nm. The temperature-mediated biosynthesized NPs were characterized by UV-vis, dynamic light scattering, high-resolution transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The cytotoxicity of the NPs was analyzed on liver (HepG2) and lung (A549) cancerous cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay infers that the biofunctionalized polyshaped AuNPs synthesized with an aqueous macroalgae extract showed a satisfactory anticancer effect on the cell lines, as evaluated by changes in cell morphology, cell viability, and metabolic activity. An altered cellular function and the morphology of cancer cell lines suggest a potential for in vivo application of AuNPs and the need to understand the interactions between nanomaterials, biomolecules, and cellular components. With continued improvements, these NPs may prove to be potential drug delivery vehicles for cancer therapy.


Subject(s)
Gold , Metal Nanoparticles , Seaweed/metabolism , Xanthophylls , Cell Survival/drug effects , Gold/chemistry , Gold/toxicity , Hep G2 Cells , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , Particle Size , Xanthophylls/chemistry , Xanthophylls/toxicity
4.
Environ Sci Pollut Res Int ; 21(16): 9604-16, 2014.
Article in English | MEDLINE | ID: mdl-24859697

ABSTRACT

Marine macroalgae have evolved a different mechanism to maintain physiological concentrations of essential metal ions and non-essential metals. The objective of the present work was to evaluate the antioxidant response and DNA damage of copper and cadmium ions in three halophytes, namely, Acanthophora spicifera, Chaetomorpha antennina, and Ulva reticulata. Accumulation of copper was significantly higher (P < 0.05) than that of cadmium. Biochemical responses showed that copper was considerably more toxic than cadmium (P < 0.05). Decreases in glutathione content and fluctuations of super oxide dismutase, catalase, and glutathione peroxidase activities were observed corresponding to time and concentration of exposure. Interestingly, it was also observed that antioxidant levels decreased as a result of metal accumulation, which may be due to free radicals generated by copper and cadmium in seaweeds. The present study also showed that copper and cadmium increased oxidative stress and induced antioxidant defense systems against reactive oxygen species. The order of toxicity for metals in the studied seaweeds was U. reticulata > A. spicifera > C. antennina. DNA damage index analysis supported that copper was significantly (P < 0.05) more toxic than cadmium. Bioaccumulation, biochemical responses, and DNA damage observed in the here analyzed marine macroalgae after exposure to selected metals indicate that these marine organisms represent useful bioindicators of marine pollution.


Subject(s)
Cadmium/chemistry , Cadmium/toxicity , Copper/chemistry , Copper/toxicity , Seaweed/drug effects , Antioxidants , Catalase/metabolism , DNA Damage , Free Radicals , Glutathione/metabolism , Metals/chemistry , Metals/toxicity , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species , Superoxide Dismutase/metabolism , Ulva/metabolism , Water Pollutants, Chemical/chemistry , Water Pollutants, Chemical/toxicity
5.
Bioprocess Biosyst Eng ; 37(9): 1859-69, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24671269

ABSTRACT

The main aim of this study was, using biomechanistic approach, to synthesize and characterize amperometric stable gold nanoparticles (AuNPs) under different pH conditions using UV Spec, dynamic light scattering and TEM with energy dispersive X-ray analysis. The biomolecules involved in conjugation and reduction were further characterized by Fourier transform infrared analysis. The pH stabilized nanoparticles were studied to determine the functional and molecular mechanism of cell death on liver cancer (HepG2) cell line and gastric cancer (YCC3) cell line. The zeta potential and TEM imaging demonstrated that AuNPs were spherical in nature and can pass through the cellular membrane because of their intrinsic properties of AuNPs to bind to carbon-bonded sulfhydryl (-C-SH or R-SH) group and, therefore, could interact with intracellular components of the cell which was confirmed through phase contrast microscopy. Altered molecular mechanism and cellular effects in different cancer cell suggest a potential for in vivo applications of gold nanomaterials.


Subject(s)
Gold/chemistry , Hydrogen-Ion Concentration , Marine Biology , Metal Nanoparticles , Neoplasms/pathology , Seaweed/metabolism , Cell Line, Tumor , Electrochemical Techniques , Humans , Microscopy, Electron, Transmission
6.
Ecotoxicology ; 22(7): 1111-22, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23836361

ABSTRACT

The present study investigated the acute toxicity, sub-lethal toxicity and biochemical response of naphthalene in fingerlings of milkfish Chanos chanos. The 96 h acute toxicity LC50 values for C. chanos exposed to naphthalene was 5.18 µg l(-1). The estimated no observed effect concentration and lowest observed effect concentration values for naphthalene in C. chanos were 0.42 and 0.69 µg l(-1) respectively for 30 days. The estimated maximum allowable toxicant concentration for naphthalene was 0.53 µg l(-1). Biochemical enzyme markers such as lipid peroxidation, catalase, glutathione S transferase and reduced glutathione were measured in gills and liver tissues of C. chanos exposed to sub-lethal concentrations of naphthalene. Fluctuation in lipid peroxidation and catalase level suggests that naphthalene concentrations play a vital role in induction of oxidative stress in fish. Induction of reduced glutathione level and inhibition of glutathione S-transferase level was observed in naphthalene exposed C. chanos suggesting that there may be enhanced oxidative damage due to free radicals. Increasing concentration increases in number of nuclear abnormalities. The formation of micronuclei and binucleated micronuclei induction by naphthalene confirm its genotoxic potential. The highest levels of DNA damage (% tail length) were observed at 1.24 µg l(-1) of naphthalene. The study suggests that biochemical enzymes, nuclear abnormalities and DNA damage index can serve as a biological marker for naphthalene contamination.


Subject(s)
DNA Damage/drug effects , Environmental Exposure/analysis , Naphthalenes/toxicity , Animals , Catalase/metabolism , Chemical Phenomena , Comet Assay , Fishes/metabolism , Gills/drug effects , Gills/metabolism , Glutathione/metabolism , Glutathione Transferase/metabolism , Inhibitory Concentration 50 , Lipid Peroxidation/drug effects , Liver/drug effects , Liver/metabolism , Micronucleus Tests , Oxidative Stress/drug effects , Toxicity Tests, Acute , Water Pollutants, Chemical/toxicity
7.
Bioprocess Biosyst Eng ; 36(4): 407-15, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22903592

ABSTRACT

Silver nanoparticles (Ag-NPs) are known to have inhibitory and fungicidal effects. Resistance against fungal infection has emerged as a major health problem in recent years, which needs great and immediate concern. Here, we report the extracellular biological synthesis of silver nanoparticles through a simple green route approach using a marine mangrove (Rhizophora mucronata) and silver nitrate. Aqueous extract of marine mangrove helped in reduction and was used as capping agent in biological synthesis. Nanoparticles were characterized using microscopy and spectroscopy techniques such as HRTEM, UV-Vis absorption spectroscopy and FTIR spectroscopy. X-ray diffraction analysis showed that the nanoparticles had face centered cubic structure with crystalline nature. FTIR spectroscopy showed the presence of different functional groups, such as hydroxyl and carbonyl, involved in the synthesis of nanoparticles. The antifungal activity of fluconazole and itraconazole was enhanced against the tested pathogenic fungi in the presence of Ag-NP and confirmed from increase in fold area of inhibition. This environmentally friendly method of biological synthesis can be easily integrated for various medical applications.


Subject(s)
Antifungal Agents/administration & dosage , Fungi/drug effects , Metal Nanoparticles/administration & dosage , Bioengineering , Biomimetic Materials , Biomimetics , Fluconazole/administration & dosage , Fungi/pathogenicity , Humans , Itraconazole/administration & dosage , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Microscopy, Electron, Transmission , Nanotechnology , Rhizophoraceae/metabolism , Silver/administration & dosage , Silver Nitrate/metabolism , Spectrometry, X-Ray Emission , Spectroscopy, Fourier Transform Infrared , X-Ray Diffraction
8.
Bull Environ Contam Toxicol ; 90(1): 60-8, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23196369

ABSTRACT

The feeding rate, growth rate and gross conversion efficiency were studied in milkfish Chanos chanos for 28 days of exposure to sub-lethal concentrations of anthracene (1.00, 2.00, 3.00, 6.00 and 12.0 µg l(-1)) and benzo [a] pyrene (0.30, 0.70, 1.40, 2.80 and 5.60 µg l(-1)) under continuous flow through bioassays. Based on survival and growth data, No Observed Effect Concentration; Lowest Observed Effect Concentration were estimated after 28 days, the values for anthracene were 2.03 and 3.09 µg l(-1), and the values for benzo [a] pyrene were 0.82 and 1.46 µg l(-1), respectively. Anthracene and benzo [a] pyrene exposure caused reduction in feeding and growth rate.


Subject(s)
Animal Nutritional Physiological Phenomena/drug effects , Anthracenes/toxicity , Benzo(a)pyrene/toxicity , Environmental Exposure , Feeding Behavior/drug effects , Fishes/physiology , Water Pollutants, Chemical/toxicity , Animals , Dose-Response Relationship, Drug , Environmental Monitoring , Fishes/growth & development , Gas Chromatography-Mass Spectrometry , India
9.
Chemosphere ; 90(3): 1158-66, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23062944

ABSTRACT

Chlorpyrifos (CPF) and carbendazim (CBZ) are widely used pesticide and fungicide in India. The 96 h LC(50) values of chlorpyrifos and carbendazim for the marine invertebrate Donax faba, were 247.72 µg L(-1) and 200.82 µg L(-1) respectively. CBZ was found to accumulate less than CPF. Gill, body and foot tissues were used as target organs in biomarker and genotoxic studies. The results showed that treatment with chlorpyrifos and carbendazim increased the MDA levels, decreased the GSH levels and changed the total protein, SOD, CAT, GPx and AChE activities in all the tissues compared to those of the control (p<0.05). These results suggest that lipid peroxidation can be a possible pathway for the toxicity of chlorpyrifos and carbendazim. The antioxidant enzyme levels decrease as a result of the consumption of enzymes to neutralize free radicals generated by CPF and CBZ. Comet tail was observed in the first three doses, in all the tissues, of which the mean tail length differed significantly (p<0.01) from those of the unexposed ones. D. faba can be used as an indicator organism to assess the genotoxic risks of chemical contamination in the marine environment using comet assay. Theses biomarkers can be used further to know these agrochemicals impact on coastal marine bivalves.


Subject(s)
Benzimidazoles/toxicity , Bivalvia/drug effects , Bivalvia/physiology , Carbamates/toxicity , Chlorpyrifos/toxicity , Fungicides, Industrial/toxicity , Pesticides/toxicity , Acetylcholinesterase/metabolism , Animals , Antioxidants/metabolism , Benzimidazoles/analysis , Bivalvia/cytology , Carbamates/analysis , Catalase/metabolism , Cell Survival/drug effects , Chlorpyrifos/analysis , Comet Assay , DNA Damage/drug effects , Fungicides, Industrial/analysis , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Lethal Dose 50 , Lipid Peroxidation/drug effects , Malondialdehyde/metabolism , Pesticides/analysis , Superoxide Dismutase/metabolism
10.
Ecotoxicol Environ Saf ; 75(1): 187-97, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21944957

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are common toxic pollutants found in the aquatic environment, and the assessment of their impact on biota is of considerable concern. The aim of the present research was to study the acute toxicity, bioaccumulation and biochemical response of milkfish Chanos chanos (Forsskal) to two selected PAHs: anthracene and benzo [a] pyrene. Acute toxicity test results were evaluated by the Probit analysis method and 96h LC(50) values for C. chanos exposed to anthracene was 0.030mgl(-1) and 0.014mgl(-1) for benzo [a] pyrene. Bioaccumulation concentration of anthracene was high when compared to benzo [a] pyrene. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (lipid peroxidation, LPO and catalase, CAT) and phase II biotransformation of xenobiotics (glutathione S transferase, GST and reduced glutathione, GSH) were measured to assess effects of selected PAHs. Anthracene and benzo [a] pyrene increase LPO and CAT level of C. chanos suggesting that these PAHs may induce oxidative stress. Both the PAHs inhibited AchE indicating that they have at least one mechanism of neurotoxicity in common: the disruption of cholinergic transmission by inhibition of AChE. An induction of C. chanos glutathione S-transferase (GST) activity was found in fish exposed to benzo [a] pyrene, while an inhibition was observed after exposure to anthracene. These results suggest that GST is involved in the detoxification of benzo [a] pyrene, but not of anthracene.


Subject(s)
Anthracenes/toxicity , Benzo(a)pyrene/toxicity , Water Pollutants, Chemical/toxicity , Acetylcholinesterase/metabolism , Animals , Biomarkers/analysis , Biomarkers/metabolism , Catalase/metabolism , Fishes , Glutathione/metabolism , Glutathione Transferase/metabolism , Lipid Peroxidation/drug effects , Oxidative Stress , Toxicity Tests, Acute
11.
Toxicol Mech Methods ; 22(2): 111-7, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21859359

ABSTRACT

Profenofos (EC(50)) is a persistent and toxic organophosphorus insecticide. Animals get exposed to profenofos via food and water. The present study was designed to explore the genotoxic effect of profenofos in the marine fish. The ubiquitously occurring marine fish, Therapon jarbua, was exposed to profenofos and its effect on DNA was measured using comet (single-cell gel electrophoresis) assay. DNA damage were scored using mean percentage of tail length and compared with the comet classes' viz., 0, 1, 2, 3, and 4. In the first three doses, the (21.5, 43.0 and 86.0 µg L(-1)) comets were observed, of which the mean tail length differed significantly (p < 0.01) from those of unexposed, but not from each other. The mean tail length values were significantly (p < 0.05) higher in gill than in matured erythrocytes. The result indicates that DNA strand breaks in T. jarbua were due to the genotoxic potential of profenofos. From the study, we suggest that T. jarbua may be used as an indicator organism to assess the genotoxic risks of profenofos contamination in marine environments using Comet assay as an identification tool. We infer that organophosphorus insecticides may be dangerous to the marine lives.


Subject(s)
DNA Damage , Fishes/genetics , Insecticides/toxicity , Organothiophosphates/toxicity , Animals , Comet Assay , Dose-Response Relationship, Drug , Lethal Dose 50
12.
J Environ Biol ; 33(4): 699-703, 2012 Jul.
Article in English | MEDLINE | ID: mdl-23359994

ABSTRACT

The marine fish Therapon jarbua was exposed to acute concentration of mercuric chloride (HgCl2). In static acute toxicity bioassays at 24, 48, 72 and 96 hr LC50 values were estimated for each concentrations such as control, 2, 1, 0.5, 0.25 and 0.125 ppm, respectively. DNAdamage (single-strand break) was also studied in gill, kidney and blood tissues at single-cell levels in the specimens exposed to different acute doses of HgCl2, by applying single-cell electrophoresis (comet assay). Dose-dependent responses were observed in DNA damage in all tissues. A comparison of DNA damage in all tissue at two concentration namely, 0.125 and 0.25 ppm indicated that the gill cells (maximum damage as 249.3 and 289.7 AU) were more sensitive to the heavy metal exposure than kidney (maximum 225.17 AU) and blood cells (maximum 200.3 AU). This study explored the utility of the comet assay for in vivo laboratory studies using fish for screening the genotoxic potential for various agents.


Subject(s)
Comet Assay/veterinary , DNA Damage/drug effects , Mercuric Chloride/toxicity , Perciformes/genetics , Animals , Dose-Response Relationship, Drug , Time Factors , Water Pollutants, Chemical/toxicity
13.
Asian Pac J Trop Med ; 4(10): 799-803, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22014736

ABSTRACT

OBJECTIVE: To identify the larvicidal activities of silver nano particles synthesised with Rhizophora mucronata (R. mucronata) leaf extract against the larvae of Aedes aegypti (Ae. aegypti) and Culex quinquefasciatus (Cx. quinquefasciatus). METHODS: In vitro larvicidal activities such as LC(50) and LC(90) were assessed for the Ae. aegypti and Cx. quinquefasciatus larval species. Further, characterisation such as UV, XRD, FTIR and AFM analysis were carried out for the synthesised silver nano particles. RESULTS: The LC(50) value of the synthesised silver nano particle was identified as 0.585 and 0.891 mg/L for Ae. aegypti and Cx. quinquefasciatus larvae respectively. Further, the LC(90) values are also identified as 2.615 and 6.291 mg/L for Ae. aegypti and Cx. quinquefasciatus species respectively. The synthesised silver nanoparticles have maximum absorption at 420 nm with the average size of 60-95 nm. The XRD data showed 2θ intense values with various degrees such as 37.10°, 47.66°, 63.97° and 70.01°. The FTIR data showed prominent peaks in (3 426.89, 2 925.49, 2 869.56, 2 346.95, 1 631.49, 1 031.73, 669.18 and 455.12) different ranges. CONCLUSIONS: The biosynthesis of silver nanoparticles with leaf aqueous extract of R. mucronata provides potential source for the larvicidal activity against mosquito borne diseases.


Subject(s)
Culicidae/drug effects , Metal Nanoparticles/toxicity , Plant Extracts/toxicity , Plant Leaves/chemistry , Rhizophoraceae/chemistry , Silver/toxicity , Aedes/drug effects , Animals , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Culex/drug effects , Larva/drug effects , Particle Size
15.
Ecotoxicology ; 14(7): 693-707, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16151610

ABSTRACT

The impact of distillery effluent on carbohydrate metabolism of Cyprinus carpio was studied at different days during exposure (7, 14, and 21 days) in the ambient temperature of 28 +/- 1 degrees C. Oxygen consumption in fish decreased with increasing effluent concentrations as well as duration of exposure. Effluent concentrations and exposure durations (days) had a significant effect on oxygen consumption of tested fish (p < 0.0005). Total carbohydrate, glycogen content and SDH enzyme activity in muscle, liver and brain tissues of C. carpio exposed to different sublethal concentrations decreased gradually and significantly. This was also the case with exposure duration. Reduction in glycogen content was greater in liver tissue i.e., 54.1% in 0.2% effluent concentration on the 21st day of exposure. However, serum glucose and lactic acid content showed an increasing trend with increase in effluent concentration and time of exposure. Unlike SDH, LDH enzyme activity of muscle, liver and brain tissues showed an increasing trend and the enhancement of enzyme activity was more in liver tissue (71.3%). From these results, it could be inferred that respiratory processes in C. carpio under distillery effluent stress was affected resulting in a shift towards anaerobiosis at organ level during sublethal intoxication.


Subject(s)
Alcoholic Beverages , Carbohydrate Metabolism/drug effects , Carps , Industrial Waste/adverse effects , Water Pollutants, Chemical/toxicity , Animals , Blood Glucose , Brain/metabolism , Glycogen/metabolism , L-Lactate Dehydrogenase/metabolism , Lactic Acid/blood , Lethal Dose 50 , Liver/metabolism , Muscles/metabolism , Oxygen/metabolism , Waste Disposal, Fluid
16.
J Environ Sci Eng ; 46(3): 186-93, 2004 Jul.
Article in English | MEDLINE | ID: mdl-16669308

ABSTRACT

Lichens are lower group of plants. They accumulate heavy metals and have been used as bio-indicators in pollution monitoring. Nine lichen species were collected from urban, rural and hilly areas in the southern part of Tamilnadu. Accumulation of metals differed with species and also with respect to their locations. Among the nine species, Pyxine petricola collected from urban areas showed greater accumulation of the heavy metals i.e., copper, zinc, lead, nickel, chromium and cadmium.


Subject(s)
Air Pollutants/pharmacokinetics , Lichens/chemistry , Metals, Heavy/pharmacokinetics , Environmental Monitoring , India
17.
Article in English | MEDLINE | ID: mdl-6138193

ABSTRACT

Metabolic rates associated with sustained, prolonged and critical swimming speeds were examined in 10 g trout exposed to 5% 96 hr LC50 (0.75 microgram X l-1) and 10% 96 hr LC50 (1.50 micrograms X l-1) at 12 degrees C. Permethrin did not influence the metabolic cost for swimming at sustained and prolonged speeds. Basal metabolic rate increased on initial exposure to permethrin reaching maximum values after 7 days and declined to the control level after 13 days in 5% and after 32 days in 10% 96 hr LC50. Critical swimming speeds were adversely affected in a manner reflective of the effects of permethrin on basal metabolic rate. Elevation in basal metabolic rate in fish exposed to permethrin was a result of increased energy requirements due to physiological stress, detoxication and tissue repair.


Subject(s)
Energy Metabolism/drug effects , Pyrethrins/toxicity , Salmonidae/metabolism , Trout/metabolism , Water Pollutants, Chemical/toxicity , Water Pollutants/toxicity , Adaptation, Physiological , Animals , Kinetics , Osmolar Concentration , Oxygen Consumption/drug effects , Permethrin , Swimming , Time Factors
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