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Graphene-based nanomaterials (GBNs) have been widely used in various fields nowadays. However, they are reported to be highly toxic to some aquatic organisms. However, the multi-organ toxicity caused by pristine graphene (pG) and graphene oxide (GO) to the developing zebrafish (Danio rerio) larvae or juvenile and the underlying mechanisms is not fully known. Therefore, in the present study, the effect of pG and GO with environmental concentrations (0, 5, 10, 15, 20, and 25 μg/L of pG; 0, 0.1, 0.2, 0.3, and 0.4 mg/mL of GO) on multi-organ system in developing zebrafish larvae was experimentally assessed. The pG and GO were found to accumulate in the brain tissue that also caused significant changes in the heart beat and survival rate. The sizes of hepatocytes were reduced. Altered axonal integrity, affecting axon length and pattern in "Tg(mbp:eGFP) transgenic lines" was also observed. In addition, the results indicated pathological effects in major organs and with disrupted mitochondrial structure was quite obvious. The pG and GO bioaccumulation leads to multi organ toxicity in zebrafish larvae. In future, the existence of the current study can be extrapolated to other aquatic system in general and in particularly to humans.

Vitellogenin (Vtg) is a specific phospho glycoprotein used as an environmental and steroidogenesis biomarker in fish. The present study shows the techniques to isolated IgY anti vitellogenin (Vtg) in Cichlasoma festae fish. Vtg was purified from adult males previously treated with estradiol and isolated from blood plasma using ion-exchange chromatography. Subsequently, Lohmann Brown hens were immunized at different doses, including three antigen booster doses. At day 45 post-immunization, posture eggs were collected, and successfully optimized the extraction of anti-Vtg -IgY antibody. The purified antibodies were validated with SDS-PAGE, detecting heavy and light chains of 70 kDa and 30 kDa, respectively. Further, we developed ELISA to estimate the Vtg specificity. Commercial anti-Vtg antibodies of Dorada (Sparus aurata) were used as a control, and immunoglobulins Y were validated for native species by Western Blot. The IgY anti-Vtg obtained by local conditions can be successfully used for programs in environmental monitoring and reproduction control in native aquatic species in Ecuador.

The multiple effect of pristine graphene (pG) toxicity on cardiovascular developmental defects was assessed using zebrafish as a model. Recently, the nanotoxicity is emerging as a critical issue, and it is more significant in embryogenesis. Especially, graphene and its derivatives have attracted a lot of interest in biomedical applications. However, very little is known about the toxic effects of pG which has been widely used carbon nanomaterial according to concentration and its effects on biological and cardiovascular development. In the present study, we examined the development of zebrafish embryos by exposing to pG (5, 10, 15, 20 and 25 μg/L) under different developmental toxicity end-points such as cardiotoxicity, cardiovascular defect, retardation of cardiac looping, apoptosis and globin expression analysis. For this, the developmental cardiotoxicity of pG at different concentrations and the specific cardiovascular defects thereof were elucidated for the first time. As a result, the exposure to pG was found to be a potential risk factor to cardiovascular system of zebrafish embryos. However, a further study on the variations of physical, molecular properties and mechanisms of nanotoxicity which vary depending on production method and surface functionalization is required. In addition, the potential risks of pG flakes to aquatic organisms and human health should be considered or checked before releasing them to the environment.

Contemporary research in epidemiology has found that being exposed to air pollution at an early stage of life has associations with both acute and chronic conditions of the multi-organs. Nevertheless, the reasons for this have yet to be fully explained. Because of this there is a need for a robust investigation into the damaging toxic influence of diesel particulate matter (PM

Graphene has been used in several fields covering from electronics to biomedicine, especially exhibiting a widespread variety of promising biological and biomedical applications. In the past decade, the biomedical applications of graphene have attracted much interest. However, the effect of pristine graphene (pG) toxicity in aquatic vertebrates has not been fully studied. Thus, in this study, the toxicity of pG was experimentally evaluated using developing zebrafish embryos as in vivo model system. To determine this, 4-hpf embryos were exposed to different concentrations of pG (1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg/L) and different early life-stage parameters were observed at 24, 48, 72, and 96 hpf. Through embryogenesis, pG was observed to induce significant embryonic mortality, delayed hatching, heartbeat, several morphological defects, pericardial toxicity, and bradycardia. Yolk sac edema and pericardial edema were induced by pG in developing embryos. These outcomes would provide new insights into the adverse effects of pG on the developing embryonic cardiac defects in vertebrates and highlight the probable natural environment and health hazards of pG flakes.

The study employed a green fluorescent vascular endothelium transgenic line, Tg (VEGFR2: GFP), to evaluate the effects of aristolochic acid (AA) at various concentrations on cardiovascular and fin imperfections in developing zebrafish embryos. The findings reveal that the intersegmental vessels (ISVs), dorsal longitudinal anastomotic vessels (DLAVs), and fin regeneration in the embryos were attenuated and underdeveloped in comparison to the control at 1, 5, 10, 20, 30, and 40 µM/L concentrations of AA. At 48 hours post-fertilization (hpf), pericardial sac edema, yolk sac edema, and slight trunk curvature were detected in the surviving larvae at 10 and 20 µM/L. These consequences were more pronounced at AA concentrations of 30 and 40 µM/L. A gradual reduction in survival, hatching, heart rate, length, and diameter of ISVs and DLAVs was observed at 10, 20, 30, and 40 µM/L when the embryos were 48 hpf. In addition, the fin redevelopment of the zebrafish embryos was also constrained in the treatment groups. The embryos exhibited heartbeats; however, blood circulation was terminated in response to the additive effect of AA at 10 and 20 µM/L concentrations. Furthermore, at higher AA levels, the embryos did not hatch. Instead, they died. The present study verified that AA has a significant toxic effect on zebrafish embryos. These outcomes may add to the existing knowledge of the possible toxicological impacts of AA and pave the path toward the sustainable development of treatments that promote human health.

Circulating levels of estradiol, vitellogenin and prostaglandins could be references of oocytes quality in the aquaculture industry. This study evaluated the performance of 17β-estradiol (E2) and vitellogenin (Vtg) levels in plasma and prostaglandins in oocytes (PGE 2) during the reproductive period of Oreochromis niloticus in a commercial farm in Ecuador. Adults fish females were treated with estradiol-synthetic (ES) and tilapia pituitary extracts (TP) during 25 days. For quantification of E2 and PGE 2, an enzyme-linked immunosorbent assay (ELISA) was used, while for Vtg relative quantification a Western blot analysis was performed. Circulating E2 concentrations were different during the first week of follow-up ( P < 0.05) resulting in different levels of Vtg per treatment ( P < 0.05) affecting on gonadal growth. The ovaries had an asynchronous development, where the mature oocytes proportions were higher with ES and TP than control. Relative fecundity was different between treatments ( P < 0.05), where TP generated 15.7 ± 5.8 oocytes per g of spawning female. Furthermore, concentrations of PGE 2 were different between treatments ( P < 0.05). The information reported in this study will be useful to improve the reproduction methods and have some biological signals before ovulation in tilapia commercial culture.

Bacteria were isolated by enrichment culture technique from groundnut (Arachis hypogaea L.) soils and tested for their ability to degrade monocrotophos in mineral salts medium under aerobic conditions in the laboratory. Based on some of the morphological and 16S rRNA gene sequence analysis, the isolates were identified as Rhodococcus phenolicus strain MCP1 and Rhodococcus ruber strain MCP-2. The initial (0-day) recovery of monocrotophos in the culture medium was 94%; and by the end of 4th day, about 21% of added monocrotophos was lost from the uninoculated medium. By the end of 1st, 2nd, 3rd and 4th day 13, 20, 24, 30% and 18, 33, 37, 45% of monocrotophos was degraded, by R. phenolicus strain MCP1 and R. ruber strain MCP-2 respectively, when the mineral salts medium was supplemented with monocrotophos as a C source. Simultaneously 12, 22, 26, 30% and 18, 26, 37, 40% of N-methylacetoacetamide a metabolite of monocrotophos was recovered in the media inoculated with the R. phenolicus strain MCP1 and R. ruber strain MCP-2 respectively, during the same period. Decrease in the amount of monocrotophos with a concomitant increase in the level of N-methylacetoacetamide clearly indicates the degradation of parent compound. Key words: Monocrotophos; N-methylacetoacetamide, biodegradation; isolation, identification.

Herbul black henna (hair dye) have been widely used as cosmetic agents to temporarily change the color of hair. Their use was tremendously increased in the past decade. Especially, hair dye containing paraphenylenediamine (PPD) is extensively used globally because of its abundance and low cost. PPD, one of the main chemicals in hair dye, is known as a toxin. Hair dye has various adverse effects, including negative impacts on human health, especially during pregnancy and on aquatic environment. Although a large amount of dyes has been released into the environment, studies on environmental toxicity of these substances are still poorly understood. Hence, the main objective of this study is to evaluate the potential cardiovascular toxicological effects of hair dye on zebrafish (Danio rerio) embryo as a model. After exposing zebrafish embryos to various concentrations (100, 200, 300, 400, 500, and 600 μM), their cardiotoxicity and other teratogenic effects were examined for various exposure periods ranged from 24 to 96 hpf. As a result, the tested embryos could not survive over intervals of 48 h after 72 and 96 hpf at higher concentrations (300, 400, 500, and 600 μM), except the lower concentration cases (100 and 200 μM) some embryos were survived, respectively. However, hair dye increases mortality and decreases yolk stalk length, heart rate and severely affects heart looping in zebrafish embryos. In addition, the dye was observed to induce cardiovascular defects in transgenic Tg (fli1a:EGFP) and Tg (flk1:EGFP) zebrafish embryos at environmentally realistic dye concentrations. However, further study using bioassays is fundamentally required to predict the toxicity of hair dyes, aside from providing the information on their safe levels for living organisms. Therefore, the present results would be vital for the ecological risk assessment of hair dye, particularly for freshwater aquatic ecosystem and human health.

Benzyl alcohol (BnOH) is an aromatic alcohol used worldwide as an excipient in foods, cosmetics, household products, and medications. Although BnOH is a bacteriostatic agent found in many parenteral preparations, this agent is responsible for precipitating the gasping syndrome in premature neonates. Increasing evidence of human exposure to BnOH and environmental contamination of BnOH requires a detailed toxicity assessment of this aromatic chemical. Few studies on the toxicity of BnOH have been reported on different animal models, but its developmental toxicity effects are not fully understood yet. Studies on the effects of BnOH on the specific endpoints of organ toxicity are rare. Thus, the present study aimed to examine the developmental toxicity effects of BnOH by using zebrafish (Danio rerio) embryo as a biological disease model. Four-hour post fertilization zebrafish embryos were exposed to BnOH for 72 h to assess BnOH toxicity on an ecological viewpoint. The median lethal concentrations of varying BnOH concentrations in zebrafish embryos were estimated. The embryonic toxicity induced by BnOH was revealed by the apoptosis in embryos and pathological alterations, such as increased mortality, inhibited hatching rate, and decreased somite number. Moreover, pericardial edema and string heartbeat were observed because of arrhythmia and cardiac malformation. The number of normal vessels in the head and trunk regions was remarkably reduced in transgenic zebrafish line Tg (Fli-1: EGFP). Morphological defects and yolk sac retention were related to the degenerated liver formation in Tg (Lfabp: dsRED). Furthermore, BnOH exposure led to the disruption of motor neuron axonal integrity and the alteration of the axon pattern in Tg (olig2: dsRED). In addition, the results exhibited the pathological effects of BnOH exposure on major organs. We believe that this study is the second to report the developmental organ toxicity of BnOH to zebrafish embryos. This study provides important information for further elucidating the mechanism of BnOH-induced developmental organ toxicity.

Due to the intensive use of pesticides within the greenhouse-rose production, remediation of polluted soils has become a hot topic for researchers in recent decades. Several bacterial strains having the ability to utilize various pesticides as a sole source of carbon and energy were isolated from pesticide-contaminated soils of Urcuqui, Ecuador. According to phenotypical, physiological, and biochemical characterizations, and 16S rRNA gene sequence analysis, the isolated pure bacterial cultures were identified as Pseudomonas putida sp. strain B1, Acinetobacter sp. strain B2 and Arthrobacter sp. strain B3. The bacterial isolates were used individually and mixed cultures were used in the laboratory and field experimentations for the degradation of various pesticides like Ridomil MZ 68 MG, Fitoraz WP 76, Cleaner, Decis 2.5 CE, Score 250 EC, Zero 5 EC, Bravo 720 SC, Meltatox, Mirage 45 CE and Teldor Combi at 50 mgL-1. The bacterial growth was monitored on both liquid culture medium (in laboratory) and field experiments by spectrophotometer method, Neubauer camera and colony-forming units. In addition, the degradation of individual pesticides (50 mgL-1) was determined by ultra-high performance liquid chromatography-tandem mass-spectrometry and UV-VIS spectrophotometer. The results showed that the highest growth rate of microbial consortium was observed during degradation of various pesticides than individual pure bacteria for both experiments. In addition, most pesticides were completely degraded by microbial consortium after 60 days. Key words: Bioremediation, spectrophotometer, bioreactors, pesticides, consortium.

This study was carried out to find out whether exposure to environmentally relevant concentration of chlorpyrifos (CP) modulates reproductive competence. To understand this, eight adult male and eight adult female zebrafish ( Danio rerio) were exposed to 200 µg/L of CP for 24, 48, 72, and 96 h. Vitellogenin levels did not show much change in female fish, whereas in male the levels increased with increasing exposure time thereby indicating estrogenecity of the toxicant. Attenuation of serum 11-ketotestosterone in male and serum 17β-estradiol in female was noticed in the exposed fish and thus signified interference of CP in the reproductive endocrine system. Structural damage common to both the gonads was vacuolization. Elongation of seminiferous tubules in testes and atretic follicles in ovary was also observed.

Ecuador generates lots of lingo cellulosic organic waste from cocoa, rice and corn agribusiness, and these products are rich in cellulose and can be used in fermentation processes by enzyme producing yeasts. In this study, the phytopathogenic fungus Moniliophthora roreri was isolated, characterized and the kinetics of production of cellulose was studied using rice husks, cocoa shell and cob as a growth inducer substance for a period of 20 days. The carboxymethylcellulose (CMC) was used as specific substrate to evaluate the kinetic parameters of the enzyme at different concentration of 0.5%, 1% and 1.5%; to evaluate the Vmax, Km, specific activity, kcatwe worked with the Michaelis-Menten and Lineweaver-Burk linearization and the crude enzyme was purified with ammonium sulfate. The enzyme production was increased on 10th, 15th and 20th day in rice hulls, cocoa husk and cob, respectively. There was also an increased enzyme activity and the kinetic parameters obtained from rice husk. The purification is a step to increase the specific activity of the enzyme in each of the extracts is under study.

The effect of the co-inoculation of arbuscular mycorrhizal fungi (AMF) and Azospirillum on micro-propagated banana seedlings development during their adaptation phase was determined. At the time of transplanting, banana seedlings were inoculated with an indigenous mycorrhizal inoculum containing 10 spores/g at four doses: 0, 50, 100 and 200 g. Seventy days after fungal inoculation, 20 ml of Azospirillum in four concentrations (0, 106, 107 and 108 CFU/ml) were applied. Finally, after 98 days from the start of the experiment a second dose (40 ml) of Azospirillum in the concentrations mentioned above was inoculated. Plants were harvested 5 months after transplanting and the growth and nutritional parameters were evaluated. The analysis of the data showed that banana plants co-inoculated with 200 g of AMF and 1.5E8 CFU/ml of Azospirillum presented greater development, an increase of 7 times in height, 4 times in perimeter, 16 times in leaf area, 12 times in aerial biomass, and 8 times in root biomass en relacion a las plantas control. The results achieved were due to synergism between fungus-bacteria when inoculated at higher doses, with lower doses stimulating growth is minimal. The co-inoculation in high doses demonstrates adequate support and cooperative effect between HMA and Azospirillum crops. In addition, co-inoculation promotes optimal nutritional status because microorganisms allowed plants achieve greater absorption of phosphorus and nitrogen relative to those treated with single inoculation and the control.sphorus and nitrogen relative to those treated with single inoculation and the control.

1 Department of Life Sciences, Universidad de las Fuerzas Armadas-ESPE, SangolquiPOBOX: 171-5-231B, Quito, Ecuador, South America. 2 Faculty of Prometeo Project, SENESCYT, Quito, Ecuador. 3 Shenzhen Key Laboratory of Advanced Materials, Department of Materials Science and Engineering, Shenzhen Graduate School, Harbin Institute of Technology, Shenzhen – 518055, China. 4 Department of Environmental Engineering, Universidad Estatal Amazonica, Puyo, Ecuador. 5 Loyola ICAM College of Engineering and Technology (LICET), Loyola campus, Chennai-600034, India.

1 Department of Life Sciences, Universidad de las Fuerzas Armadas-ESPE, SangolquiPOBOX: 171-5-231B, Quito, Ecuador, South America. 2 Department of Biotechnology, Universidad de las Americas UDLA, Quito, Ecuador. 3 Department of Zoology, University of Pune, Pune, India. 4 Department of Environmental Engineering, Universidad Estatal Amazonica, Puyo, Ecuador. 5 Loyola ICAM College of Engineering and Technology (LICET), Loyola campus, Chennai, India.

The ability of microalgae to remove nitrogen and phosphorus from wastewater has been used in recent years as an alternative treatment for discharges from livestock slurry, which generate a negative environmental impact on vulnerable ecosystems. With this background and the feasibility of using microalgae, we have evaluated the effect of Chlorella sp. and Synechocystis sp., in removing contaminants from the pig manure collected from El Prado ESPE. Slurry samples were collected, filtered and autoclaved, and the supernatant was further dilluted to three different concentrations of 40%, 60% and 80%. The microalgal growth and pollutants removal property was evaluated up to 15 days in batch culture. The cell density was determined by counting in a Neubauer hemocytometer, and the pollutants removal was analyzed by standard colorimetric methods. The microalgae Chlorella sp. showed a maximum cell growth of 1.70 ± 0.09 x107 cells/mL at 60% effluent concentration on day 6. While Synechocystis sp. showed a maximum growth of 1.04 ± 0.05 x107 cells/mL, at 60% concentration on day 9. On the other hand, there exists a competition when microalgae used as a consortium. The cell growth of Chlorella sp. was higher at all concentrations compared to Synechocystis sp.. Overall, efficiency of pollutant removal were between 40% and 90%, which demonstrate the feasibility of using microalgae in tertiary swine wastewater treatment.

The objective of this study was to determine the effect of application of pesticides on morphology, viability, and germination of pollen grains of Blackberry (Rubus glaucus Benth.) and Tree tomato (Solanum betaceum Cav.). The study was performed at Patate, Tungurahua province, Ecuador and was divided into two phases. Phase one dedicated to the study of morphology, viability, and identification of nutrient solution for better germination of pollen grains and phase two for the analysis of the effect of conventional, organic, and biological pesticides on pollen grain germination and pollen tube length. To study pollen morphology, pollens were extracted by hand pressure and was analyzed by optical and electron microscopy. The viable pollen grains were identified by staining with 1% acetocarmine. Even though Tree tomato and Blackberry pollen grains are morphologically similar, their exine shapes differ. We observed four times increase in pollen germination rate when suspended in nutrient solution (Sucrose with Boric acid) than control (water). Pollen grains under nutrient solution were subjected to different groups of pesticides for the period of 2, 4, and 6 h. With respect to pesticide affect, the Blackberry pollen grain germination followed the following order: Lecaniceb > Beauveb > Metazeb => Myceb > Control. However, the effect on Tree tomato pollen grains was as follows: Lecaniceb > Myceb > Cantus > Bacillus thuringiensis > Kripton > Control. As per as pollen grain germination is concerned, we observed that the chemical pesticides are more harmful than other pesticides. So, it is necessary to perform screening test for different pesticides and their effect on pollen grain germination before applying to the fields.

The objective of this study was to determine the effects of two insecticides, namely, acetamiprid and carbofuran on the enzymatic activities of arylamidase (as glucose formed from sinigrin) and myrosinase (as β-naphthylamine formed from L-leucine β-naphthylamide) in the black and red clay soils collected from a fallow groundnut (Arachis hypogaea L.) fields in the Anantapur District, Andhra Pradesh, India. The study was realized within the framework of the laboratory experiments in which the acetamiprid and carbofuran were applied to the soils at different doses (1.0, 2.5, 5.0, 7.5, 10.0 kg ha(-1)). Initially, the physicochechemical properties of the soil samples were analyzed. After 10 days of pesticide application, the soil samples were analyzed for the enzyme activities. Acetamiprid and carbofuran stimulated the arylamidase and myrosinase activities at lower concentrations after 10 days incubation. Striking stimulation in soil enzyme activities was noticed at 2.5 kg ha(-1), persists for 20 days in both the soils. Overall, higher concentrations (5.0-10.0 kg ha(-1)) of acetamiprid and carbofuran were toxic or innocuous to the arylamidase and myrosinase activities. Nevertheless, the outcomes of the present study clearly indicate that the use of these insecticides (at field application rates) in the groundnut fields (black and red clay soils) stimulated the enzyme (arylamidase and myrosinase) activities.

The powder of henna is extensively used as decorative skin paint for nail coloring and as a popular hair dye in Asian countries. Its human health risk is extensive, and it is frequently released as waste into the aquatic environment raising the concerns. Zebrafish (Danio rerio) embryos were employed to study the developmental effects of henna. Normal fertilized zebrafish embryos under standard water were selected for the control and test chambers. Three predetermined sublethal concentrations (100, 200, and 275 μM) of henna in 24-well cell culture plates were tested on 1-h postfertilized embryo (pfe) for 96 h. Observation for rates of survival and mortality was recorded; digital camera was used to image morphological anomalies of embryos with a stereomicroscope; and functional abnormalities at 24, 48, 72, and 96 h were performed. The hatching rates of embryos were reduced significantly when treated with 200 and 275 μM or higher concentrations of henna. Slow blood circulation in the whole body was observed with a median effect on hatching exposed to 200 and 275 μM of henna at 48-h pfe. At 72- and 96-h pfe, blood circulation was ceased in the whole body but still had a heartbeat. At 96-h pfe, pericardial sac edema, yolk sac edema, head deformation, spine crooked malformation, and tail malformation (bent tails or hook-like tails) were observed in the surviving larvae at 100 μM. In summary, exposure to henna at 100, 200, and 275 μM causes some altered morphological and physiological abnormalities including increased mortality, hatching delay, slow blood circulation, pericardial sac edema, yolk sac edema, abnormal body axes, twisted notochord, tail deformation, weak heartbeat, and growth retardation and was also detected in some treated embryos and groups having adverse effects on embryonic development of zebrafish provoking potential human developmental risk studies.