Biological-based techniques for real-time water-quality studies: Assessment of non-invasive (swimming consistency and respiration) and toxicity (antioxidants) biomarkers of zebrafish.

Swimming consistency and respiration of fish are recognized as the non-invasive stress biomarkers. Their alterations could directly indicate the presence of pollutants in the water ecosystem. Since these biomarkers are a routine process for fish, it is difficult to monitor their activity manually. For this reason, experts employ engineering technologies to create sensors that can monitor the regular activities of fish. Knowing the importance of these non-invasive stress biomarkers, we developed online biological behavior monitoring system-OBBMS and online biological respiratory response monitoring system-OBRRMS to monitor real-time swimming consistency and respiratory response of fish, respectively. We continuously monitored the swimming consistency and respiration (OCR, CER and RQ) of zebrafish (control and atrazine-treatments) for 7 days using our homemade real-time biological response monitoring systems. Furthermore, we analyzed oxidative stress indicators (SOD, CAT and POD) within the vital tissues (gills, brain and muscle) of zebrafish during stipulated sampling periods. The differences in the swimming consistency and respiratory rate of zebrafish between the control and atrazine treatments could be precisely differentiated on the real-time datasets of OBBMS and OBRRMS. The zebrafish exposed to atrazine toxin showed a concentration-dependent effect (hypoactivity). The OCR and CER were increased in the atrazine treated zebrafish. Both Treatment I and II received a negative response for RQ. Atrazine toxicity let to a rise in the levels of SOD, CAT and POD in the vital tissues of zebrafish. The continuous acquisition of fish signals is achieved which is one of the main merits of our OBBMS and OBRRMS. Additionally, no special data processing was done, the real-time data sets were directly used on statistical tools and the differences between the factors (groups, photoperiods, exposure periods and their interactions) were identified precisely. Hence, our OBBMS and OBRRMS could be a promising tool for biological response-based real-time water quality monitoring studies.

IR-based device to acquire real-time online heart ECG signals of fish (Cyprinus carpio) to evaluate the water quality.

Water quality monitoring is a challenging task due to continuous pollution. The rapid development of engineering technologies has paved the way for the development of efficient and convenient computer-based online continuous water-quality assessment techniques. Techniques based on biological-responses are gaining attention, worldwide. Different biosensors have been developed in recent years to monitor real-time biological responses to evaluate water-quality. The survival and function of various organs of the organism depends on the cardiac system. Alterations in the cardiac system could signify the occurrence/initiation of stress in the organism. We developed a real-time online cardiac function assessment system-OCFAS to acquire fish ECG-signals. We obtained P-wave, R-wave, T-wave, PR-intervals, QT-intervals and QRS-complex continuously, which did not affect the normal activities of carp. We exposed Cyprinus carpio to different concentrations (National Environmental Quality Standards) of ammonia for 48 h. Our OCFAS has precisely acquired the required ECG-signals. A real-time dataset reveals sensitivity to ammonia in carp ECG-indexes. Compared with the control group the P-wave, R-wave and T-wave were weaker in ammonia-treated groups. In contrast, the PR-intervals, QT-intervals and QRS-complex were prolonged in the ammonia-treatment groups. The self-organizing map signifies that the PR-intervals, the QRS-complex and the QT-intervals are consistent with environmental stress. Linear regression analysis also quantitatively signifies that the PR interval has the highest R2 value and the lowest SSE-value, followed by the QRS complex and the QT interval. A concentration-related effect was observed in the ammonia treated groups. The integrated biomarker response (IBRv2) index was used to determine the overall stress of ammonia on carp heart ECG-indexes. IBRv2 also supports the real-time response of carp to ammonia stress. Ammonia levels in the aquaculture and water environment require special attention to avoid its adverse effects on the health of aquatic biota. Our study emphasizes the importance of online real-time fish ECG for water-quality assessment.

Developmental toxicity of the emerging contaminant cyclophosphamide and the integrated biomarker response (IBRv2) in zebrafish.

The safety of cyclophosphamide (CP) in the early developmental stages is not studied yet; it is important to study the responses at these stages because they might have relevance to CP-administered humans. We studied the developmental toxicity of CP by analysing physiological, morphological, and oxidative stress, neurotransmission enzymes, gene expression and histological endpoints in zebrafish embryos/larvae. The study lasted for 120 hpf at environmentally relevant concentrations of CP. No visible alterations were noticed in the control group. Delayed hatching, slow heart rate, yolk sac oedema, pericardial oedema, morphological deformities, the incompetence of oxidative stress biomarkers, excessive generation of ROS, apoptosis, inhibition of neurotransmitters and histopathological anomalies were observed in CP-treated groups. These alterations were found to be concentration- and duration-dependent effects for physiological and morphological endpoints, whereas concentration-dependent effects were antioxidants, ROS, apoptosis and histological endpoints. Biomarkers and gene expression were standardised using the integrated biomarker response-IBRv2 index. The IBRv2 index showed a concentration-dependent behaviour. A non-lethal developmental and teratogenic effect was observed in CP-treated zebrafish embryos/larvae at the studied concentrations. The studied biomarkers are sensitive, and the responses are interrelated; thus, their responses are useful to assess veiled and unseen hazards of pharmaceuticals.

Efficiency of hematological, enzymological and oxidative stress biomarkers of Cyprinus carpio to an emerging organic compound (alphamethrin) toxicity.

Alphamethrin is one of the extensively used pyrethroids. Its non-specific mode-of-action might affect the non-target-organisms. Its toxicity data on aquatic organisms are lacking. We determined the toxicity (35 days) of alphamethrin (0.6 µg/L and 1.2 µg/L) on non-target-organisms by evaluating the efficiency of hematological, enzymological and antioxidants biomarkers of Cyprinus carpio. Compared with the control group, the efficiency of the biomarkers studied was significantly (p < 0.05) impaired in the alphamethrin treated groups. Alphamethrin-toxicity altered hematology, transaminases and the potency of LDH of fish. ACP and ALP activity and biomarkers of oxidative stress in the gills, liver and muscle tissues were affected. IBRv2 index reveals that the biomarkers were inhibited. The observed impairments were the toxicity effects of alphamethrin with respect to concentration and time. The effectiveness of biomarkers for alphamethrin toxicity was like the toxicity data available on other banned insecticides. Alphamethrin could cause multiorgan toxicity on aquatic organisms at µg/L level.

Immunotoxicity and Transcriptome Analyses of Zebrafish (Danio rerio) Embryos Exposed to 6:2 FTSA.

As a new alternative to perfluorooctane sulfonic acid (PFOS), 6:2 fluorotelomer sulfonic acid (6:2 FTSA) has been widely produced and used in recent years, and its concentration and frequency of detection in the aquatic environment and aquatic organisms are increasing. However, studies of its toxicity in aquatic biological systems are alarmingly scarce, and the relevant toxicological information needs to be improved. In this study, we investigated AB wild-type zebrafish (Danio rerio) embryos subjected to acute 6:2 FTSA exposure for immunotoxicity using immunoassays and transcriptomics. Immune indexes showed significant decreases in SOD and LZM activities, but no significant change in NO content. Other indexes (TNOS, iNOS, ACP, AKP activities, and MDA, IL-1ß, TNF-α, NF-κB, TLR4 content) all showed significant increases. These results indicated that 6:2 FTSA induced oxidative stress and inflammatory responses in zebrafish embryos and exhibited immunotoxicity. Consistently, transcriptomics showed that genes involved in the MAPK, TLR and NOD-like receptor signaling pathways (hsp70, hsp701, stat1b, irf3, cxcl8b, map3k8, il1b, tnfa and nfkb) were significantly upregulated after 6:2 FTSA exposure, suggesting that 6:2 FTSA might induce immunotoxicity in zebrafish embryos through the TLR/NOD-MAPK pathway. The results of this study indicate that the safety of 6:2 FTSA should be examined further.

A study to assess the health effects of an anticancer drug (cyclophosphamide) in zebrafish (Danio rerio): eco-toxicity of emerging contaminants.

Cyclophosphamide (CP) is widely used for treating various kinds of cancer. Because of its high intake, metabolism and excretion, these anticancer medications have been detected in the aquatic environment. There is very limited data on the toxicity and effects of CP on aquatic organisms. The present study aims to assess the toxic effect of CP on certain oxidative stress biomarkers (superoxide dismutase-SOD, catalase-CAT, glutathione peroxidase-GPx, glutathione-GSH, glutathione S-transferases-GST and lipid peroxidation-LPO), protein, glucose, metabolising enzymes (aspartate aminotransferase-AST, alanine aminotransferase-ALT), and ion-regulatory markers (sodium ions-Na+, potassium ions-K+, and chloride ions-Cl-), and histology in the gills and liver of Danio rerio at environmentally relevant concentrations (10, 100 and 1000 ng L-1). Exposure to CP for 42 days led to a significant decrease in SOD, CAT, GST, GPx and GSH levels in the gills and liver tissues of zebrafish. The level of lipid peroxidation in the gills and liver tissues of zebrafish was significantly increased compared to the control group. Chronic exposure significantly changes protein, glucose, AST, ALT, Na+, K+ and Cl- biomarkers. Fish exposed to different levels of CP showed necrosis, inflammation, degeneration and hemorrhage in the gills and hepatic tissues. The observed changes in the studied tissue biomarkers were proportional to both dose and time. In conclusion, CP at environmentally relevant concentrations causes oxidative stress, energy demand, homeostasis disturbances, and enzyme and histological alterations in the vital tissues of zebrafish. These alterations were similar to the toxic effects reported in mammalian models.

Triphenylmethane dye (C52H54N4O12) is potentially a hazardous substance in edible freshwater fish at trace level: toxicity, hematology, biochemistry, antioxidants, and molecular docking evaluation study.

Malachite green (C52H54N4O12) is a synthetic dye that is used in textile industries as a colorant and in aquaculture sectors to contain microbial damage. Aquatic contamination of malachite green (MG) has been reported globally. Fish is the highest trophic organism among aquatic inhabitants, highly sensitive to waterborne contaminants (metals, coloring agents, etc.). Toxicity of waterborne chemicals on nontarget organisms can be determined by assessing biomarkers. Assessing blood parameters and tissue antioxidants (enzymatic and nonenzymatic) is useful to evaluate MG toxicity. To initiate the MG toxicity data for freshwater fish (Cyprinus carpio), the median lethal toxicity was primarily evaluated. Then, hematological, blood biochemical (glucose, protein, and cholesterol) and tissue biochemical (amino acids, lipids), and vital tissue (gills, liver, and kidney) antioxidant capacity (CAT, LPO, GST, GR, POxy, vitamin C, and GSH) of C. carpio were analyzed under acute (LC50-96 h) and sublethal (Treatment I-1/10th and Treatment II-1/5th LC50-96 h) exposure periods (28 days). Molecular docking for MG with hemoglobin was also obtained. Biomarkers examined were affected in the MG-treated groups with respect to the control group. Significant changes (p < 0.05) were observed in hematology (Hb, RBCs, and WBCs), glucose, proteins, lipids and tissue CAT, LPO, and GST activities under acute MG exposure. In sublethal treatment groups, biomarkers studied were significant (p < 0.05) throughout the study period. The potential for MG binding to hemoglobin was tested in this study. MG is potentially a multiorgan toxicant. Literally a chemical that is harmful to the aquatic environment if safety is concerned.

Real-time determination of water status upon simultaneous zebrafish exposure to sublethal concentrations of CuSO4.

Water pollution from commonly occurring contaminants (metals, xenobiotics, etc.) is a serious global problem. Copper is a commonly occurring water contaminant. A variety of physiological and biological methods have been developed to monitor water quality. The assessment of biological responses is an effective method for identifying the harmful effects of contaminants on ecosystems. Fish is a highly recommended animal model in water quality monitoring. Swimming consistency (firmness) and respiratory metabolism (oxygen consumption rate, carbon dioxide excretion rate and respiratory quotient) are essential for fish to maintain body homeostasis toward coping with environmental stress. We exposed zebrafish to different concentrations (Treatment I-0.1 mg/L and Treatment II-1.58 mg/L) of CuSO4. We have continuously quantified the strength of behavior (swimming consistency) and physiological (respiratory rates) biomarkers for ten days using an online monitoring system of swimming behavior and external respiration. Swimming consistency and respiratory rates of zebrafish (p<0.05) decreased in the CuSO4-treated groups compared to the control group. Avoidance behavior has led to an endpoint behavior at copperiedus. The time-delayed toxic effect has resulted in CuSO4 treatment groups. We checked for swimming consistency aberration on the artificial neural array, Self-organizing map (SOM). Circadian rhythms were influenced by prolonged exposure to CuSO4 toxicity. A concentration- and duration-dependent behavior anomaly was noted in this study. Swimming behavior and respiratory metabolism patterns are sensitive non-invasive stress biomarkers for water quality monitoring studies.

IR-Based Novel Device for Real-Time Online Acquisition of Fish Heart ECG Signals.

We developed an infrared (IR)-based real-time online monitoring device (US Patent No: US 10,571,448 B2) to quantify heart electrocardiogram (ECG) signals to assess the water quality based on physiological changes in fish. The device is compact, allowing us to monitor cardiac function for an extended period (from 7 to 30 days depending on the rechargeable battery capacity) without function injury and disturbance of swimming activity. The electrode samples and the biopotential amplifier and microcontroller process the cardiac-electrical signals. An infrared transceiver transmits denoised electrocardiac signals to complete the signal transmission. The infrared receiver array and biomedical acquisition signal processing system send signals to the computer. The software in the computer processes the data in real time. We quantified ECG indexes (P-wave, Q-wave, R-wave, S-wave, T-wave, PR-interval, QRS-complex, and QT-interval) of carp precisely and incessantly under the different experimental setup (CuSO4 and deltamethrin). The ECG cue responses were chemical-specific based on CuSO4 and deltamethrin exposures. This study provides an additional technology for noninvasive water quality surveillance.

Assessment of eco-toxic effects of commonly used water disinfectant on zebrafish (Danio rerio) swimming behaviour and recovery responses: an early-warning biomarker approach.

Eco-toxicity profiles for commonly used disinfectants were lacking. Available traditional toxicity techniques have some limitations (assessments and ethical issues). Behaviour toxicology is a promising research area towards early warning and non-invasive approaches. We studied the potential eco-toxic effects of sodium hypochlorite (NaOCl) on the swimming behaviour of zebrafish. Zebrafish were exposed to different concentrations (Treatment I, Treatment II, Treatment III, and Treatment IV) of NaOCl for 360 h. Recovery study (144 h) was conducted for NaOCl treatment groups. The swimming behaviour of zebrafish was quantified efficiently using an online monitoring system (OMS). OMS dataset was processed for determination of behavioural differences by MATLAB and SPSS. Compared to the control group, the swimming strength of zebrafish under NaOCl treatments declined significantly (p < 0.001). Avoidance behaviour has occurred on zebrafish under NaOCl exposure periods. Furthermore, NaOCl toxicity also adjusted circadian rhythms on zebrafish. Zebrafish swimming strength was significantly (p < 0.001) improved under-recovery periods. Moreover, normal diurnal patterns have occurred. NaOCl could cause behavioural abnormalities in non-target organisms. Continuous exposure to common disinfectants could cause external and internal stress on non-target organisms, resulting in behavioural changes and circadian rhythm adjustments. Continuous changes in behavioural and circadian rhythms might reduce organisms' fitness and adaptation capacity. This study highlights (1) the importance of computer-based toxicity assessments, and (2) swimming behaviour is an early warning biomarker for eco-toxicity studies.

Synthetic organic chemicals (flame retardants and pesticides) with neurotoxic potential induced behavioral impairment on zebrafish (Danio rerio): a non-invasive approach for neurotoxicology.

Behavior responses of organisms can be used as a non-invasive method for neurotoxicology studies since it directly links the nervous system's functioning and biochemical activities. Among different behavioral activities, aquatic organisms' swimming behavior (fitness) is the essential factor for health assessment; thus, it is practiced routinely in neurotoxicological studies. Zebrafish (Danio rerio) are excellent models for neurotoxicology studies. Based on the above information, we hypothesized that zebrafish's swimming behavior is a potential biomarker for neurotoxic effect assessment. We exposed zebrafish (length, 3-4 cm; weight, 0.2-0.3 g) to different synthetic organic chemicals (organophosphorus flame retardants (tri-cresyl phosphate and cresyl diphenyl phosphate) and neurotoxic pesticides (cypermethrin and methomyl) for 15 days. For each test chemical, we chose two different concentrations (Treatment-I 5 µL/L and Treatment-II 25 µL/L) to study their eco-toxicity. The swimming strength of zebrafish was quantified using an online monitoring system. The swimming strength of zebrafish decreased under different treatments (Treatment-I (5 µL/L) and -II (25 µL/L)) of target chemicals. The circadian rhythm of zebrafish was predominantly not affected in this study. Higher neurotoxic effect (behavioral impairment) was observed in Treatment-II when compare to Treatment-I of organophosphorus flame retardants and pesticides groups. Responses of zebrafish under organophosphorus flame retardant (tri-cresyl phosphate and cresyl diphenyl phosphate) treatments were identical with pesticide (cypermethrin and methomyl) treatments. Based on the results, we conclude that swimming behavior could be an ideal non-invasive biomarker to assess waterborne contaminants' neurotoxic effect.

Responses of Cirrhinus mrigala to second-generation fluoroquinolone (ciprofloxacin) toxicity: Assessment of antioxidants, tissue morphology, and inorganic ions.

Ciprofloxacin drugs are a second-generation fluoroquinolone highly prescribed medication against various bacterial infections in human and aquaculture practices. These drugs are chemically designed to persist in the body long enough to achieve target objectives. Extensive usage has resulted in ciprofloxacin becoming a ubiquitous contaminant in the environment. Unfortunately, the ecotoxicological profiles for ciprofloxacin are scanty. This study was aimed to assess the ecotoxicity of ciprofloxacin at environmentally relevant concentrations (1 µg/L, and 1.5 µg/L) to a cultivable fish Cirrhinus mrigala. Responses of antioxidant enzymes, histological anomalies, and inorganic ion levels were studied. SOD activity in gill, liver, and kidney tissues was elevated in ciprofloxacin-exposed groups when compared with the control group. CAT activity was predominantly decreased in ciprofloxacin treated groups relative to the control group. GST activity in the ciprofloxacin treated groups was increased (except kidney tissues [Treatment I (1 µg/L)], and gill tissues fifteenth day) significantly (p < .05). The LPO level was elevated in the ciprofloxacin treatment groups throughout the study period (except Treatment II (1.5 µg/L) tenth day in kidney tissues). A series of histological anomalies were noticed in the gill, liver, and kidney tissues of the ciprofloxacin treated groups. Ciprofloxacin exposure caused a significant decrease of sodium, potassium, and chloride levels in the plasma of C. mrigala. A parallel among an imbalanced oxidative defense system, tissue structural changes, and alterations of plasma inorganic ions could be considered as a reliable biomarker for antibiotic toxicity study. This study could be a primary platform for further toxicity studies to understand the potential molecular impacts and adverse effects of ciprofloxacin on aquatic organisms.

The specification of zebrafish (Danio rerio) heart electrocardiogram index characteristic responses to different types of pollutants.

Water quality was highly affected by common pollutants. Metals, pesticides and small molecules are ubiquitous pollutants. Advancement in engineering technology (computer-based monitoring systems) increased the efficiency of quantifying toxicity of different chemicals in an organism. The cardiovascular system reflects internal and external stress of an organism, and electrocardiogram (ECG) data reliably measure external stress. As ECG data can accurately reflect the physiological conditions of organisms, and zebrafish (Danio rerio) are considered to be good models for cardiovascular research, it is hypothesized that ECG parameters of zebrafish could indicate the toxicity of water-borne chemicals. To achieve this, we treated zebrafish with different concentrations of target chemicals (CuSO4, C10H19O6PS2 and NH4Cl) for 48 h and ECG data were measured. P-wave, R-wave, T-wave, PR-interval, QRS-complex and QT-interval data were the focus of this study. The results of self-organizing maps and Pearson correlation analysis indicate that the QRS-complex can be used as an indicator for CuSO4 stress. The QT-interval could be used to assess the C10H19O6PS2 stress. The QT-interval and P-wave can be used to evaluate the NH4Cl stress. Responses of zebrafish ECG parameters were identical with other vertebrate model, and were specific to toxicant types. It is proved that zebrafish heart ECG index could be used as a potential indicator in early detection of environmental stress.

Organophosphorus-based chemical additives induced behavioral changes in zebrafish (Danio rerio): Swimming activity is a sensitive stress indicator.

Organophosphorus flame retardants (OPFRs) have been extensively used as chemical additives in polymer based consumer products. Among them, Isopropylphenyl phosphate (IPPP) and tripropyl phosphate (TPP) are predominant, which have potential to cause neuro-toxic effects on non-target organisms. As behavior (swimming activity) response is the first adjustment due to neurotoxic stress on the fitness of fish. In this study, the quantified swimming activity of zebrafish (Danio rerio) under IPPP and TPP exposure in an online monitoring system was investigated to assess the neurotoxin effects under long-term exposure periods, no swimming anomalies were observed in the control group. Whereas, in the OPFR exposures ((treatment I: 5 µg/L and treatment II: 25 µg/L), a series of anomalies were identified. Hyperactivity was shown in IPPP treatment I group (5 µg/L), whereas zebrafish swimming activity was declined throughout the study period in IPPP treatment II (25 µg/L), and TPP groups (5 µg/L and 25 µg/L) when compared to the control group. Circadian rhythm was not affected in the present study. The results of the present study indicated that the fitness of test individuals was a valid biomarker for eco-toxicity assessment under unescapable conditions. Hypoactivity of zebrafish signified the neurotoxic effects of IPPP and TPP. A concentration based improvement in swimming activity was observed under recovery conditions, which suggested that recovery capacity along with toxicity responses could be a comprehensive non-invasive technique to assess the eco-toxicity of waterborne chemicals.

Organophosphorus flame retardant induced hepatotoxicity and brain AChE inhibition on zebrafish (Danio rerio).

Organophosphorus flame retardants (OPFRs) are high production volume (HPV) chemicals. Recent reports reveal that OPFRs are ubiquitous in the environment. Unfortunately, the toxicity profiles for OPFRs on organisms remain limited. Hence, to illustrate the potential toxic effects of OPFRs at environmental relevant concentrations on aquatic biota, in the present study, we investigated biochemical, enzymological, antioxidants, and histological (at long-term study) changes of zebrafish tissues under short- (96 h) and long- (21 days) -term triphenyl phosphate (TPhP) exposure. The hepatic glucose production (except short-term TPhP treatment up to 48 h), aspartate transaminase, alanine transaminase, lactate dehydrogenase, reactive oxygen species generation, lipid peroxide, and catalase activities were found to be increased in TPhP exposed groups when compared to control groups (normal and solvent control groups). The hepatic protein content and sodium dismutase activity were declined in TPhP exposed groups. Likewise, brain tissue acetylcholinesterase activity was declined in TPhP exposed groups. The hepatic glutathione S-transferase activity increased after 24 h under short-term TPhP exposure (96 h), while under long-term exposure period (21 days) the enzyme activity was accelerated when compared to control groups. Long-term TPhP exposure resulted in a series of morphological anomalies in the hepatic tissues of zebrafish. Our study reveals that TPhP can potentially cause antioxidants imbalance, alterations in enzymological and biochemical profiles, and morphological anomalies in hepatic tissues of zebrafish. Moreover, TPhP could cause neurotoxic effects on zebrafish at studied concentrations. Our findings expand the available toxicity profiles for TPhP on aquatic biota and propose that zebrafish are a good indicator, and studied parameters are valid biomarkers in assessing the eco-toxicological effects of OPFRs.

Machine-learning-based detection of adaptive divergence of the stream mayfly Ephemera strigata populations.

Adaptive divergence is a key mechanism shaping the genetic variation of natural populations. A central question linking ecology with evolutionary biology is how spatial environmental heterogeneity can lead to adaptive divergence among local populations within a species. In this study, using a genome scan approach to detect candidate loci under selection, we examined adaptive divergence of the stream mayfly Ephemera strigata in the Natori River Basin in northeastern Japan. We applied a new machine-learning method (i.e., random forest) besides traditional distance-based redundancy analysis (dbRDA) to examine relationships between environmental factors and adaptive divergence at non-neutral loci. Spatial autocorrelation analysis based on neutral loci was employed to examine the dispersal ability of this species. We conclude the following: (a) E. strigata show altitudinal adaptive divergence among the populations in the Natori River Basin; (b) random forest showed higher resolution for detecting adaptive divergence than traditional statistical analysis; and (c) separating all markers into neutral and non-neutral loci could provide full insight into parameters such as genetic diversity, local adaptation, and dispersal ability.

Biochemical responses of a freshwater fish Cirrhinus mrigala exposed to tris(2-chloroethyl) phosphate (TCEP).

Freshwater fish Cirrhinus mrigala were exposed to tris(2-chloroethyl) phosphate (TCEP) with three different concentrations (0.04, 0.2, and 1 mg/L) for a period of 21 days. During the study period, thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) levels were significantly (p < 0.05) inhibited. The superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and lipid peroxidation (LPO) levels were increased significantly (p < 0.05) in gills, liver, and kidney tissues, whereas glutathione (GSH) and glutathione peroxidase (GPx) (except liver tissue) activities were inhibited when compared to the control group. Likewise, exposure to TCEP significantly (p < 0.05) altered the biochemical (glucose and protein) and electrolyte (sodium, potassium, and chloride) levels of fish. Light microscopic studies exhibited series of histopathological anomalies in the gills, liver, and kidney tissues. The present study reveals that TCEP at tested concentrations causes adverse effects on fish and the studied biomarkers could be used for monitoring the ecotoxicity of organophosphate esters (OPEs).

Simultaneous eco-toxicity assessment technique using an online monitoring system: effects of different environmental factors on swimming behavior of zebrafish (Danio rerio).

Environmental factors, such as photoperiod and temperature were the main limiting factors for the survival of organisms in the nature environment. Changes in environmental factors are well predicted but determining their effects on organisms are challenging hot topic in the field of eco-toxicology. Thus, technology based eco-toxicity assessment was focused worldwide. In this research, the effects of different temperatures (15 °C, 22 °C, 30 °C, 32 °C, and 35 °C) and photoperiods (dark and light periods) on the continuous behavior responses of Zebrafish (Danio rerio) were investigated using an online monitoring system (OMS). We designed a new fish chamber with sensors to measure the behavior responses of zebrafish under different conditions. Data obtained from the OMS could be assessed for factors such as difference in swimming behavior, circadian rhythm, and avoidance behavior using latest software (MATLAB). The observed behavior anomalies on zebrafish under different temperatures and continuous photoperiods were statically significant (p < 0.05). We conclude that the new designed fish chamber (behavior sensors) is good in sensing behavioral responses of zebrafish under different conditions. The fish behavior strength could be a potential biomarker to assess the effects of environmental factors. The present study would be a basic platform for assessing the effects of different stressors simultaneously on swimming behavior of zebrafish.

Biochemical and behavior effects induced by diheptyl phthalate (DHpP) and Diisodecyl phthalate (DIDP) exposed to zebrafish.

Both Diheptyl-phthalate (DHpP) and Diisodecyl-phthalate (DIDP) were used extensively as plasticizers. Recently, their occurrence in the environmental matrices and human body fluids have been reported. Unfortunately, these phthalate congeners are without basic toxicity profiles. Hence, we studied the toxic effects of both DHpP and DIDP in the median lethal concentration (LC50 96-h) on zebrafish (Danio rerio). We assessed swimming behavior strength and tissues biomarker responses including total antioxidants capacity (TAOC), transaminases, and acetylcholinesterase (AChE) enzyme. Fish exposed to phthalate congeners (Treatment-I and-II) for 15-days showed alterations on fish swimming behavior and circadian rhythm. At the end of the exposure period, both liver and heart tissue transaminases activities were found to be accelerated in DHpP and DIDP treated fish, when compared to control group. TAOC and AChE activities were found to be decreased in brain, gills, intestine, and muscle tissues of phthalate congeners treated fish than the control group. Alterations observed in the studied biomarkers were concentration-based response. Among treatment groups DHpP showed higher effects. Comparative studies on swimming behavior and biochemical activities were reasonable to know the swimming responses are mediated due to external stress or internal stress. More studies on molecular and biomarkers assessments are warranted on toxicity of emerging contaminants.

The potential assessment of green alga Chlamydomonas reinhardtii CC-503 in the biodegradation of benz(a)anthracene and the related mechanism analysis.

Benz(a)anthracene (BaA) is a polycyclic aromatic hydrocarbons (PAHs), that belongs to a group of carcinogenic and mutagenic persistent organic pollutants found in a variety of ecological habitats. In this study, the efficient biodegradation of BaA by a green alga Chlamydomonas reinhardtii (C. reinhardtii) CC-503 was investigated. The results showed that the growth of C. reinhardtii was hardly affected with an initial concentration of 10 mg/L, but was inhibited significantly under higher concentrations of BaA (>30 mg/L) (p < 0.05). We demonstrated that the relatively high concentration of 10 mg/L BaA was degraded completely in 11 days, which indicated that C. reinhardtii had an efficient degradation system. During the degradation, the intermediate metabolites were determined to be isomeric phenanthrene or anthracene, 2,6-diisopropylnaphthalene, 1,3-diisopropylnaphthalene, 1,7-diisopropylnaphthalene, and cyclohexanol. The enzymes involved in the degradation included the homogentisate 1,2-dioxygenase (HGD), the carboxymethylenebutenolidase, the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ubiquinol oxidase. The respective genes encoding these proteins were significantly up-regulated ranging from 3.17 fold to 13.03 fold and the activity of enzymes, such as HGD and Rubisco, was significantly induced up to 4.53 and 1.46 fold (p < 0.05), during the BaA metabolism. This efficient degradation ability suggests that the green alga C. reinhardtii CC-503 may be a sustainable candidate for PAHs remediation.