Phytoremediation of cadmium-trichlorfon co-contaminated water by Indian mustard (Brassica juncea): growth and physiological responses.

In this study, the morphological and physiological responses of Brassica juncea to the stresses of Cadmium (Cd) and trichlorfon (TCF), and the phytoremediation potential of B. juncea to Cd and TCF were investigated under hydroponics. Results showed that Cd exhibited strong inhibition on biomass and root morphology of B. juncea as Cd concentration increased. The chlorophyll a fluorescence intensity and chlorophyll content of B. juncea decreased with the increased Cd concentration, whereas the malondialdehyde and soluble protein contents and superoxide dismutase activity increased. TCF with different concentrations showed no significant influence on these morphological and physiological features of B. juncea. The biomass and physiological status of B. juncea were predominantly regulated by Cd level under the co-exposure of Cd and TCF. B. juncea could accumulate Cd in different plant parts, as well as showed efficient TCF degradation performance. A mutual inhibitory removal of Cd and TCF was observed under their co-system. The present study clearly signified the physiological responses and phytoremediation potential of B. juncea toward Cd and TCF, and these results suggest that B. juncea can be used as an effective phytoremediation agent for the Cd-TCF co-contamination in water.

Combined pollution of heavy metals and pesticides in agricultural water systems is a common phenomenon. In previous phytoremediation studies, limited information is available on the co-contamination of heavy metals and pesticides. In this study, we aimed to investigate the concentration-dependent morphological and physiological characteristics of B. juncea under single and co-stress of Cd and trichlorfon (TCF), and the phytoremediation ability of B. juncea to remove Cd and TCF through hydroponic experiment. B. juncea exhibited efficient removal performance of Cd and TCF alone and simultaneous exposure of both pollutants, indicating that B. juncea is an effective phytoremediation agent for the Cd-TCF co-contaminated water.

Assessing the effect of ß-glucan diets on innate immune response of tilapia macrophages against trichlorfon exposure: an in vitro study.

The widespread use of pesticides in some areas where fish species such as tilapia are farmed may cause damage to the environment and affect commercial fish and therefore, human health. Water leaching with the pesticide trichlorfon, during the fumigation season in the field, can affect water quality in fish farms and consequently affect fish health. At the same time, the use of immunomodulatory compounds such as ß-glucan supplied in the diet has become widespread in fish farms as it has been shown that improves the overall immune response. The present research examines the immunomodulatory impacts observed in macrophages of Nile tilapia (Oreochromis niloticus) after being fed a diet supplemented with ß-glucan for 15 days, followed by their in vitro exposure to trichlorfon, an organophosphate pesticide, at concentrations of 100 and 500 µg mL-1 for 24 h. The results showed that ß-glucan diet improved the viability of cells exposed to trichlorfon and their antioxidant capacity. However, ß-glucan did not counteract the effects of the pesticide as for the ability to protect against bacterial infection. From the present results, it can be concluded that ß-glucan feeding exerted a protective role against oxidative damage in cells, but it was not enough to reduce the deleterious effects of trichlorfon on the microbicidal capacity of macrophages exposed to this pesticide.

Detection of insecticides by Tetronarce californica acetylcholinesterase via expression and in silico analysis.

The acetylcholinesterase (AChE) is involved in termination of synaptic transmission at cholinergic synapses and plays a vital role in the insecticide detection and inhibitor screening. Here, we report the heterologous expression of an AChE from Tetronarce californica (TcA) in Escherichia coli (E. coli) as a soluble active protein. TcA was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized TcA were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of TcA. The results showed that TcA exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized TcA demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free TcA was observed with trichlorfon, whereas immobilized TcA showed reduced IC50 values towards tested insecticides by 3 to 180-fold. Molecular docking analysis revealed the interaction of trichlorfon, acephate, isoprocarb, λ-cyhalothrin, and fenpropathrin in the active site gorge of TcA, particularly mediated through the formation of hydrogen bonds and π-π stacking. Therefore, TcA expressed heterologously in E. coli is a promising candidate for applications in food safety and environmental analysis. KEY POINTS: • T. californica AChE was expressed solubly in prokaryotic system. • The biochemical properties of free/immobilized enzyme were characterized. • The sensitivity of enzyme to insecticides was evaluated in vitro and in silico.

Efficacy of chlorine, sodium chloride and trichlorfon baths against monogenean Dawestrema cycloancistrium parasite of pirarucu Arapaima gigas.

This study investigated the efficacy of sodium chloride (SC) and trichlorfon (T) against Dawestrema cycloancistrium and its physiological effects on Arapaima gigas. The efficacy of chlorine (C) as a prophylactic disinfectant was also evaluated. In vitro test with 15 treatments were: SC 4, 8, 10, 12, 14 g/L, T0.1, 0.4, 0.8, 1.6, 3.2 mg/L, and C500, 1000, 3000, 6000 mg/L. Scanning electron microscopy was performed to evaluate parasite damage. The in vivo test was as follows: control, 4 h short baths, once a day, for four consecutive days (SC12 g/L, T5 mg/L); 24 h long baths, for 2 days in 24 h intervals (SC10 g/L, T5 mg/L). In vitro exposure to SC12 and 14 g/L caused 100% mortality of monogeneans at 45 and 60 min, while at T3.2 and 1.6 mg/L 100% of monogeneans died at 30 and 60 min, respectively. In vitro exposure to C resulted in complete mortality after 2-5 min exposure. The SC and T LD50-96 h were 9.9 g/L and 9.73 mg/L, respectively. All in vivo treatments presented efficacy above or close to 90%, with low survival in the long baths. C, starting at 500 mg/L for 5 min, can be used as a disinfectant. Short baths with SC12 g/L and T5 mg/L are recommended for D. cycloancistrium infestations in Arapaima.

The Combined Use of Copper Sulfate and Trichlorfon Exerts Stronger Toxicity on the Liver of Zebrafish.

In aquaculture, copper sulphate and trichlorfon are commonly used as disinfectants and insecticide, sometimes in combination. However, improper use can result in biotoxicity and increased ecological risks. The liver plays a crucial role in detoxification, lipid metabolism, nutrient storage, and immune function in fish. Selecting the liver as the main target organ for research helps to gain an in-depth understanding of various aspects of fish physiology, health, and adaptability. In the present study, zebrafish were exposed to Cu (0.5 mg/L) and Tri (0.5 mg/L) alone and in combination for 21 days. The results demonstrate that both Cu and Tri caused hepatocyte structure damage in zebrafish after 21 days of exposure, with the combination showing an even greater toxicity. Additionally, the antioxidant and immune enzyme activities in zebrafish liver were significantly induced on both day 7 and day 21. A transcriptome analysis revealed that Cu and Tri, alone and in combination, impacted various physiological activities differently, including metabolism, growth, and immunity. Overall, Cu and Tri, either individually or in combination, can induce tissue damage by generating oxidative stress in the body, and the longer the exposure duration, the stronger the toxic effects. Moreover, the combined exposure to Cu and Tri exhibits enhanced toxicity. This study provides a theoretical foundation for the combined use of heavy metal disinfectants and other drugs.

Developmental Neurotoxicity of Trichlorfon in Zebrafish Larvae.

Trichlorfon is an organophosphorus pesticide widely used in aquaculture and has potential neurotoxicity, but the underlying mechanism remains unclear. In the present study, zebrafish embryos were exposed to trichlorfon at concentrations (0, 0.1, 2 and 5 mg/L) used in aquaculture from 2 to 144 h post fertilization. Trichlorfon exposure reduced the survival rate, hatching rate, heartbeat and body length and increased the malformation rate of zebrafish larvae. The locomotor activity of larvae was significantly reduced. The results of molecular docking revealed that trichlorfon could bind to acetylcholinesterase (AChE). Furthermore, trichlorfon significantly inhibited AChE activity, accompanied by decreased acetylcholine, dopamine and serotonin content in larvae. The transcription patterns of genes related to acetylcholine (e.g., ache, chrna7, chata, hact and vacht), dopamine (e.g., drd4a and drd4b) and serotonin systems (e.g., tph1, tph2, tphr, serta, sertb, htrlaa and htrlab) were consistent with the changes in acetylcholine, dopamine, serotonin content and AChE activity. The genes related to the central nervous system (CNS) (e.g., a1-tubulin, mbp, syn2a, shha and gap-43) were downregulated. Our results indicate that the developmental neurotoxicity of trichlorfon might be attributed to disorders of cholinergic, dopaminergic and serotonergic signaling and the development of the CNS.

Trichlorfon is effective against Dawestrema cycloancistrium and does not alter the physiological parameters of arapaima (Arapaima gigas): A large Neotropical fish from the Amazon.

This study investigated the in vitro and in vivo efficacy of trichlorfon against Dawestrema cycloancistrium, as well as its physiological effects on arapaima. Naturally parasitized arapaima gill arches were exposed in vitro to 100, 250, 500 and 750 mg/L of trichlorfon and a control group (only distilled water), in triplicate. Parasites were monitored, and mortality was used to determine the median effective concentration (EC50 ). The 750 mg/L concentration demonstrated 100% in vitro efficacy against D. cycloancistrium after 60 min, while the intermediate (500 mg/L) and the lowest (100 and 250 mg/L) tested concentrations were completely efficient after 90 and 130 min, respectively. The EC50-1h of trichlorfon for D. cycloancistrium was determined at 171.73 mg/L. Parasitized arapaima juveniles were exposed to a control group and 150 mg/L of trichlorfon in triplicate. Fish were exposed to two therapeutic baths for 60 min with 24-h intervals between treatments. Therapeutic baths with 150 mg/L of trichlorfon were 92.99% effective against D. cycloancistrium and did not bring about haematological alterations (erythrogram, white blood cell count, thrombogram, plasma glucose and total proteins). Therefore, 150 mg/L of trichlorfon can be used in therapeutic baths to control and treat D. cycloancistrium infestations with no physiological impairments for arapaima.

Colorimetric assay based on peroxidase-like activity of dodecyl trimethylammonium bromide-tetramethyl zinc (4-pyridinyl) porphyrin for detection of organophosphorus pesticides.

A simple and sensitive colorimetric assay for detecting organophosphorus pesticides (OPs) was developed based on 3,3',5,5'-tetramethylbenzidine (TMB)/hydrogen peroxide (H2O2)/dodecyl trimethylammonium bromide (DTAB)-tetramethyl zinc (4-pyridinyl) porphyrin (ZnTPyP). In this system, based on the peroxidase-like activity of DTAB-ZnTPyP, H2O2 decomposes to produce hydroxyl radicals, which oxidize TMB, resulting in blue oxidation products. The OPs (trichlorfon, dichlorvos, and thimet) were first combined with DTAB-ZnTPyP through electrostatic interactions. The OPs caused a decrease in the peroxidase-like activity of DTAB-ZnTPyP due to spatial site blocking. At the same time, π-interactions occurred between them, and these interactions also inhibited the oxidation of TMB (652 nm), thus making the detection of OPs possible. The limits of detection for trichlorfon, dichlorvos, and thimet were 0.25, 1.02, and 0.66 µg/L, respectively, and the corresponding linear ranges were 1-35, 5-45, and 1-40 µg/L, respectively. Moreover, the assay was successfully used to determine OPs in cabbage, apple, soil, and traditional Chinese medicine samples (the recovery ratios were 91.8-109.8%), showing a great promising potential for detecting OPs also in other complex samples.

Determination of trichlorfon using a molecularly imprinted electrochemiluminescence sensor on multi-walled carbon nanotubes decorated with silver nanoparticles.

Considering the limitations associated with existing methods for the detection of trace amounts of trichlorfon, this paper proposes a novel molecularly imprinted electrochemiluminescence (ECL) sensor for the detection of trichlorfon by utilizing the double enhancement effect of trichlorfon and Ag nanoparticles supported by multi-walled carbon nanotubes (MWCNTs/Ag NPs) in a luminol-H2O2 ECL system. Here, trichlorfon was electropolymerized on the surface of the MWCNT/Ag NP-modified gold nanoelectrode with o-phenylenediamine to prepare the molecularly imprinted polymer-based sensor. After eluting the trichlorfon, imprinted holes for the identification of trichlorfon were retained on the sensor, which were used as signal switches to obtain different ECL intensities through the adsorption of different concentrations of trichlorfon. The ECL signal of the sensitized luminol-H2O2 was doubly enhanced by the MWCNTs/Ag and trichlorfon, improving the sensitivity of the sensor. The trichlorfon concentration was positively correlated with the enhanced ECL intensity of the sensor in the range 5.0 × 10-8-5.0 × 10-11 mol L-1, and the detection limit of trichlorfon was 3.9 × 10-12 mol L-1. Moreover, the proposed sensor was successfully applied to the detection of trichlorfon residues in real samples, and the recovery ranged between 91.8 and 109%. A molecularly imprinted electrochemiluminescence sensor for trichlorfon detection by utilizing the double enhancement effect of trichlorfon and Ag nanoparticles supported by multi-walled carbon nanotubes in a luminol-H2O2 ECL system. The dual enhancement of the ECL signal improved the sensitivity of the sensor.

A novel signal amplified electrochemiluminescence biosensor based on MIL-53(Al)@CdS QDs and SiO2@AuNPs for trichlorfon detection.

An ultrasensitive electrochemiluminescence (ECL) biosensor was developed based on MIL-53(Al)@CdS QDs and SiO2@AuNPs for trichlorfon detection. Metal-organic frameworks (MOFs) were used as a loading platform that provided a large surface area to load targets and modified materials onto the electrode. At the same time, SiO2@AuNPs loaded plenty of AuNPs which effectively increased the ECL resonance energy transfer between the CdS QDs, so that the ECL signal was strongly quenched and resulted in an amplified response. In the range of 10-11-10-4 M, the ECL response showed a linear relationship with the concentration (logarithm) of trichlorfon, and the detection limit was 5.1 × 10-12 M (S/N = 3). When the biosensor was applied to detect trichlorfon in lettuce, broccoli, cucumber, and chives, the recoveries obtained from the spiked samples were 97%-105%, 102%-104%, 100%-104%, and 98%-104%, respectively. Thus, this novel ECL biosensor has potential applications for the analysis of trichlorfon in food samples.

Temperature-dependent pharmacokinetics of trichlorfon in common carp (Cyprinus carpio L.) after bath immersion therapy.

The common carp (Cyprinus carpio L.) is one of the most important freshwater fish species. As C. carpio culture has escalated, bacterial and parasitic infections have become a real threat to the industry. Antibacterial and antiparasitic treatments are provided for infection control in C. carpio. However, adequate vaccines have not yet been developed. Trichlorfon (TCF), an organophosphate, is an antiparasitic agent used in aquaculture to treat external parasites. However, there are few pharmacokinetic (PK) studies on its use in fish. This study investigated the residue elimination and temperature-dependent PK characteristics of TCF in C. carpio at 15°C and 25°C after 30 mg/L TCF bath immersion for 30 min. TCF residue concentrations in plasma and muscle tissues were determined using liquid chromatography-tandem mass spectrometry and further analyzed using a noncompartmental model. Temperature significantly affected specific PK parameters. Increasing the temperature from 15°C to 25°C shortened the elimination half-life from 36.07 to 22.72 h. The time to reach the maximum plasma TCF residue concentration (Cmax ) (Tmax ) remained the same (0.5 h), but Cmax increased from 67.72 to 70.76 µg/L. The area under the plasma concentration-time curve decreased from 1,057.31 to 962.14 h∙µg/L. The muscle TCF Cmax was 446.99 µg/L with a corresponding Tmax of 0.5 h at 25°C, and 267.53 µg/L, with a corresponding Tmax of 1.0 h at 15°C. The temperature-sensitive PK parameters, such as increased in Cmax and decreased elimination and distribution rates, significantly affected the plasma TCF residue concentration and its overall exposure to increasing temperature. Temperature affected the therapeutic outcomes of TCF treatment in C. carpio and likely other freshwater fish. Therefore, proper dosing regimens should take temperature into consideration.

Effects of trichlorfon organophosphate on the morphology of the gills and liver of Pseudoplatystoma corruscans.

Possible changes in the morphology of the gills and liver of P. corruscans after exposure to TCF were evaluated. The fish were distributed into five groups in triplicate (n = 10 in each group: 0.0; 0.125; 0.25; 0.5; 1.0 mg/L of the commercial product NEGUVON®, for 96 h. TCF induced a high histopathological index associated with circulatory disorders (congestion and aneurysm), as well as progressive changes (lamellar hyperplasia, capillary dilation, epithelial detachment and edema) at the highest concentrations (0.25; 0.5 and 1.0 mg/L). In addition to the histopathological disturbances, there was an increase in the levels of neutral glycoproteins and alterations in the histomorphometry of the secondary lamellae (total height, apical region, median, basal and interlamellar) at the highest concentrations. In the liver, the alterations were more intensified, in addition to circulatory (congestion) and regressive disturbances (loss of hepatocyte architecture and accumulation of intracellular substances), progressive alterations (focal necrosis) were observed, indicating a high degree of hepatic tissue involvement. The results indicate that the use of TCF concentrations above 0.25 mg/L promotes histopathological, histomorphometric and histochemical changes in the gills and liver of P. corruscans, imposing a biological risk that affects the aquatic environment and the health of fish.

Chronic trichlorfon stress induces differential transcriptome expression and interferes with multifunctional pathways in the brain of Rana chensinensis.

Trichlorfon is widely used to control pest insects and various parasitic infestations in agriculture, aquaculture and human medicine. However, the long-term widespread use and overuse of trichlorfon poses risks to public and environmental health. Thus, the aim of this study was to evaluate the interference of trichlorfon on gene transcription patterns in the brain of Rana chensinensis with 4 weeks treatment under control conditions and 0.1 mg/L exposure. In total, 102,013 unigenes were obtained from the brain tissue of R. chensinensis, and 874 differentially expressed genes (DEGs) were identified. Functional annotation indicated that out of 118,643 unigenes, 45,600 (44.7%) were annotated in the Nr, Nt, the Swiss-Prot, KEGG, COG, and GO databases. The differential expression patterns of 4 genes associated with neural activity were selected and validated by quantitative polymerase chain reaction (qPCR). The results revealed that except for the canonical cholinesterase-based mechanism, trichlorfon could act on other receptors and alter certain types of neuronal ion channels as the major target sites. All of these effects ultimately cause disorders of multifunctional pathways and other neurotransmitter pathways in the host. The results further our understanding of the mechanisms underlying nontarget effects of organophosphate insecticides (OPs) through multitargets studies.

Bioremediation of cadmium-trichlorfon co-contaminated soil by Indian mustard (Brassica juncea) associated with the trichlorfon-degrading microbe Aspergillus sydowii: Related physiological responses and soil enzyme activities.

Soil co-contaminated with heavy metals and organics is often difficult to remediate. In this study, pot experiments were conducted to investigate the concurrent removal of cadmium (Cd, two levels: CdL [10 mg kg-1] and CdH [50 mg kg-1]) and trichlorfon (TCF, 100 mg kg-1) from co-contaminated soil by comparing the following remediation methods: natural remediation (NR), soil inoculated with Aspergillus sydowii (AS), soil planted with Brassica juncea (BJ), and soil planted with B. juncea and inoculated with A. sydowii (BJ-AS). The physiological responses of B. juncea and soil enzyme activities after remediation were also studied. B. juncea grew well in co-contaminated soil at both Cd levels. The biomass and chlorophyll content of B. juncea in CdH soil were lower than those in CdL soil, whereas the malondialdehyde content and activities of catalase, peroxidase and superoxide dismutase of B. juncea in CdH soil were higher than those in CdL soil. Cd accumulation in B. juncea was high in CdH soil, whereas high Cd removal efficiency was observed in CdL soil. TCF could be thoroughly degraded within 35 days in NR at both Cd-level soils. AS, BJ and BJ-AS promoted TCF degradation and enhanced the activities of catalase, urease, sucrase and alkaline phosphatase in soil compared with the NR. BJ-AS showed the highest phytoextraction ratio (3.32% in CdL and 1.34% in CdH soil) and TCF degradation rate (half-life of 2.18 and 2.37 days in CdL and CdH soil, respectively). These results demonstrate that BJ-AS could effectively remove Cd and TCF from soil and is thus a feasible technology for the bioremediation of these co-contaminated soil.

Protective effects of diet containing rutin against trichlorfon-induced muscle bioenergetics disruption and impairment on fatty acid profile of silver catfish Rhamdia quelen.

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage.

Quinolone and Organophosphorus Insecticide Residues in Bivalves and Their Associated Risks in Taiwan.

Bivalves, such as freshwater clams (Corbicula fluminea) and hard clams (Meretrix lusoria), are the most extensive and widely grown shellfish in land-based ponds in Taiwan. However, few studies have examined the contamination of bivalves by quinolone and organophosphorus insecticides. Thus, we adapted an established procedure to analyze 8 quinolones and 12 organophosphorus insecticides using liquid and gas chromatography-tandem mass spectrometry. Surveys in Taiwan have not noted high residual levels of these chemicals in bivalve tissues. A total of 58 samples of freshwater or hard clams were obtained from Taiwanese aquafarms. We identified 0.03 mg/kg of enrofloxacin in one freshwater clam, 0.024 mg/kg of flumequine in one freshwater clam, 0.02 mg/kg of flumequine in one hard clam, 0.05 mg/kg of chlorpyrifos in one freshwater clam, 0.03 mg/kg of chlorpyrifos in one hard clam, and 0.02 mg/kg of trichlorfon in one hard clam. The results indicated that 5.17% of the samples had quinolone insecticide residues and 5.17% had organophosphorus residues. However, the estimated daily intake (EDI)/acceptable daily intake quotient (ADI) indicated no significant risk and no immediate health risk from the consumption of bivalves. These results provide a reference for the food-safety screening of veterinary drugs and pesticides in aquatic animals. Aquatic products should be frequently screened for residues of prohibited chemicals to safeguard human health.

Integrated biomarker parameters response to the toxic effects of high stocking density, CuSO4, and trichlorfon on fish and protective role mediated by Angelica sinensis extract.

The present study explored the protective role of dietary the extract of Angelica sinensis (EAs) on high density, CuSO4, or trichlorfon-treated Crucian carp (Carassius auratus auratus). Firstly, the study showed that the optimum density for growth and growth inhibition was 0.49 and 0.98 fish L-1 water, respectively. Dietary EAs relieved the high density-induced growth inhibition in Crucian carp. The appropriate concentration of EAs for recovery of growth was estimated to be 4.30 g kg-1 diet in high-density fish. Moreover, high density decreased both digestive and absorptive enzyme activities and increased lipid oxidation in digestive organs, suggesting the ability of high density to induce oxidative damage. However, dietary EAs inhibited the oxidative damage through elevating ROS scavenging ability and enzymatic antioxidant activity in digestive organs. Secondly, our data demonstrated that the appropriate concentration of CuSO4 to induce the decrease in feed intake (FI) was 0.8 mg Cu L-1 water. Dietary EAs returned to FI of Crucian carp treated with CuSO4. The appropriate concentration of EAs for recovery of FI was estimated to be 4.25 g kg-1 diet. Moreover, dietary EAs suppressed the CuSO4-induced decrease in digestion and absorption capacity and increase in protein metabolism in digestive organs of Crucian carp. Finally, the present results suggested that dietary EAs inhibited the trichlorfon-induced rollover (loss of equilibrium) in Crucian carp. The appropriate concentration of EAs for inhibition of rollover was estimated to be 4.18 g kg-1 diet. Moreover, trichlorfon stimulated not only the decrease in energy metabolism but also lipid and protein oxidation, suggesting that trichlorfon caused loss of function and oxidative damage in muscles of fish. However, dietary EAs improved muscular function and inhibited oxidative damage via quenching ROS and elevating non-enzymatic and enzymatic antioxidant activity in muscles of trichlorfon-induced fish. So, EAs could be used as an inhibitor of high density, CuSO4, and trichlorfon stress in fish.

Neurotoxic effects of sublethal concentrations of cyanobacterial extract containing anatoxin-a(s) on Nauphoeta cinerea cockroaches.

The detection of cyanotoxins, such as the anatoxin-a(s), is essential to ensure the biological safety of water environments. Here, we propose the use of Nauphoeta cinerea cockroaches as an alternative biological model for the biomonitoring of the activity of anatoxin-a(s) in aquatic systems. In order to validate our proposed model, we compared the effects of a cyanobacterial extract containing anatoxin-a(s) (CECA) with those of the organophosphate trichlorfon (Tn) on biochemical and physiological parameters of the nervous system of Nauphoeta cinerea cockroaches. In brain homogenates from cockroaches, CECA (5 and 50 µg/g) inhibited acetylcholinesterase (AChE) activity by 53 ±â€¯2% and 51 ±â€¯7%, respectively, while Tn (5 and 50 µg/g) inhibited AChE activity by 35 ±â€¯4% and 80 ±â€¯9%, respectively (p < 0.05; n = 6). Moreover, CECA at concentrations of 5, 25, and 50 µg/g decreased the locomotor activity of the cockroaches, diminishing the distance travelled and increasing the frequency and duration of immobile episodes similarly to Tn (0.3 µg/g) (p < 0.05, n = 40, respectively). CECA (5, 25 and 50 µg/g) induced an increase in the leg grooming behavior, but not in the movement of antennae, similarly to the effect of Tn (0.3 µg/g). In addition, both CECA (50 µg/200 µl) and Tn (0.3 µg/200 µl) induced a negative chronotropism in the insect heart (37 ±â€¯1 and 47 ±â€¯8 beats/min in 30 min, respectively) (n = 9, p > 0.05). Finally, CECA (50 µg/g), Tn (0.3 µg/g) and neostigmine (50 µg/g) caused significant neuromuscular failure, as indicated by the monitoring of the in vivo neuromuscular function of the cockroaches, during 100 min (n = 6, p < 0.05, respectively). In conclusion, sublethal doses of CECA provoked entomotoxicity. The Tn-like effects of CECA on Nauphoeta cinerea cockroaches encompass both the central and peripheral nervous systems in our insect model. The inhibitory activity of CECA on AChE boosts a cascade of signaling events involving octopaminergic/dopaminergic neurotransmission. Therefore, this study indicates that this insect model could potentially be used as a powerful, practical, and inexpensive tool to understand the impacts of eutrophication and for orientating decontamination processes.

Effects of low doses Trichlorfon exposure on Rana chensinensis tadpoles.

Trichlorfon is an organophosphate insecticide widely used in aquaculture and agriculture. Little is known about the effects of long-term of low doses trichlorfon exposure on amphibians. In this study, we investigated the effects of low doses trichlorfon on Rana chensinensis tadpoles after exposure to 0.01, 0.1, and 1.0 mg/L trichlorfon for 2 and 4 weeks. Survival, growth, development and mortality were monitored regularly over the course of exposure. The results showed that trichlorfon led to a decrease in tadpole survival. Reductions in growth and disruptions to the development of tadpoles were observed in trichlorfon treatments. Morphological abnormalities of affected tadpoles included axial flexures, skeletal malformations and lateral kinks. Trichlorfon increased the frequency of micronucleus (MN) formation in circulating erythrocytes of tadpoles exposed for 2 weeks to 0.1 and 1.0 mg/L trichlorfon. At all concentrations, an enhanced frequency of MN formation was observed in tadpoles exposed for 4 weeks. Exposure to trichlorfon induced other nuclear abnormalities such as lobed and notched nuclei only in tadpoles exposed to 1.0 mg/L trichlorfon for 4 weeks. In addition, exposure to trichlorfon within the 0.01-1.0 mg/L range increased the genetic damage index in hepatic tissues in all treatments. Apoptosis-associated DNA fragmentation in hepatic tissues occurred in a weak ladder-like pattern. This study presents evidence of low doses trichlorfon effects on amphibians, highlighting the properties of this organophosphate insecticide that jeopardize nontarget species exposed to trichlorfon.

Transcriptome analysis of Rana chensinensis liver under trichlorfon stress.

Trichlorfon is a selective organophosphate insecticide that is widely applied in aquaculture and agriculture for control of various parasites. However, repeated and excess applications of trichlorfon often lead to water pollution and threaten non-targeted species. Our previous studies showed that trichlorfon could cause oxidative stress, lipid peroxidation and hepatic lesions in the liver of Rana chensinensis, but the related molecular mechanisms remain unclear. To explore the interference of trichlorfon in gene transcription, the differentially expressed genes in the liver of R. chensinensis exposed to trichlorfon were characterized using the RNA-seq platform. A search of all unigenes against non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, Kyoto Encyclopaedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases resulted in 22,888, 21,719, 20,934, 16,923, 7375 and 15,631 annotations, respectively, and provided a total of 27,781 annotated unigenes. Among the annotated unigenes, 16,923 were mapped to 257 signalling pathways. A set of 3329 differentially expressed unigenes was identified by comparison of the two groups in liver. Notably, relative expression of metabolism-related genes, including both up- and down-regulated genes, were also validated by qPCR. The present study depicts the high degree of transcriptional complexity in R. chensinensis under trichlorfon stress and provides new insights into the molecular mechanisms of organophosphate insecticide toxicology. Some of these metabolism-responsive genes could be useful for understanding the toxicological mechanism of trichlorfon on non-target aquatic organisms and will contribute to the conservation of aquatic life.