Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

Sub lethal mercuric chloride toxicity induced stress related alterations in the epithelial lining of foot of the freshwater mussel Lamellidens marginalis (Lamarck)

Sub lethal (0.2 ppm) mercuric chloride induced stress related histopathological alterations in the epithelial linings of foot (podium) of the edible freshwater mussel Lamellidens marginalis (Lamarck) were studied using histochemical techniques up to 60 days of exposure. The histomorphological changes were manifested only slowly and its intensity was somewhat proportional to the duration of exposure. The immediate response of the exposed mussels was the altered mucous secretion. There was a progressive incorporation of sulphated glycoproteins into the secretory contents of the mucous cells especially in the first half of the experiment. Marked histopathological changes including necrosis, appearance of pyknotic nuclei, sloughing of epithelial cells and appearance of non-tissue spaces, etc., started appearing during the later half of the experiment. The fag end of the experiment, which witnessed prominent histomorphological changes, was accompanied by highly decreased mucous secretion. KEYWORDS: heavy metal toxicity, mercuric chloride, Lamellidens marginalis, freshwater mussel, histopathology.

Comparative toxicity of antifouling compounds on the development of sea urchin

In the present study, embryotoxicity experiments using the sea urchin Lytechinus variegatus were carried out to better clarify the ecotoxicological effects of tributyltin (TBT) and triphenyltin (TPT) (the recently banned antifouling agents), and Irgarol and Diuron (two of the new commonly used booster biocides). Organisms were individually examined to evaluate the intensity and type of effects on embryo-larval development, this procedure has not been commonly used, however it showed to be a potentially suitable approach for toxicity assessment. NOEC and LOEC were similar for compounds of same chemical class, and IC10 values were very close and showed overlapping of confidence intervals between TBT and TPT, and between Diuron and Irgarol. In addition, IC10 were similar to NOEC values. Regardless of this, the observed effects were different. Embryo development was interrupted at the gastrula and blastula stages at 1.25 and 2.5 mu g l(-1) of TBT, respectively, whereas pluteus stage was reached with the corresponding concentrations of TPT. Furthermore, embryos reached the prism and morula stages at 5 mu g l(-1) of TPT and TBT, respectively. The effects induced by Irgarol were also more pronounced than those caused by Diuron. Pluteus stage was always reached at any tested Diuron concentration, while embryogenesis was interrupted at blastula/gastrula stages at the highest concentrations of Irgarol. Therefore, this study proposes a complementary approach for interpreting embryo-larval responses that may be employed together with the traditional way of analysis. Consequently, this application leads to a more powerful ecotoxicological assessment tool focused on embryotoxicity.

Sediment toxicity assessment of Guanabara Bay, Rio de Janeiro, Brazil.

Guanabara Bay (GB) comprises of estuarine and marine environments of high ecological and socio-economic relevance, together with port, industrial and urban areas. The anthropogenic activities produce environmental impacts, including the aquatic pollution. The sediment quality assessment is important to evaluate the effects of contamination, once sediments are a repository for most of the contaminants. In this Study, the quality of sediments from GB was evaluated, in rainy and dry periods, throughout the employment of acute toxicity tests with the amphipod Tiburonella viscana, and chronic bioassays with embryos of the sea-urchin Lytechinus variegatus. In the dry period, acute toxicity was found in the sediments from stations 1, 2 3 (NW) and 7 (near Guapimirim Environmental Protection Area). The bioassays with liquid phases showed effects, but were strongly influenced by the unionized ammonia levels, which were high in this period. In the rainy period, acute toxicity was found in sediments samples from stations 1, 2, 3, 6, 8, 10, 11, 12 and 15. Chronic toxicity could be clearly detected, as ammonia concentrations tended to be low in the most part of the samples. The results showed that the sediment toxicity is influenced by precipitation rates, which increase the input of contaminants to the Bay, and also allowed subdividing GB in three main zones: northwest (stations 1, 2, 3, 5), northeast (stations 6, 7, 8, 9) and centre-south (stations 10, 11, 12, 13, 14, 15). Results also showed that the quality of GB sediments is poor, and that toxicity tests could determine the combined effects of pollutants.

Acute and chronic toxicity of sediment samples from Guanabara Bay (RJ) during the rainy period

A Baía de Guanabara é um ambiente marinho-estuarino de grande relevância ecológica e sócio-econômica, e sujeita a uma ampla gama de impactos ambientais. O sedimento é o principal destino para a maioria das substâncias introduzidas nos corpos d'água, podendo fornecer uma medida integrada da qualidade ambiental, a qual pode ser avaliada por várias abordagens. Neste projeto, a qualidade de sedimentos da Baía de Guanabara foi por uma abordagem ecotoxicológica, por meio de testes de toxicidade aguda de sedimento integral, utilizando Tiburonella viscana, e testes de toxicidade crônica de água intersticial, elutriato e interface sedimento-água, utilizando embriões de Lytechinus variegatus. Os sedimentos foram coletados em 14 estações de amostragem. Nos testes crônicos houve efeitos significativos na maioria das amostras, enquanto os sedimentos coletados nas estações 1, 2, 3, 6, 8, 10, 11, 12 e 15 apresentaram também toxicidade aguda. Houve grande concordância entre os resultados dos diferentes testes, e sua integração mostrou que os sedimentos analisados encontram-se inadequados à vida aquática, indicando degradação ambiental na baía da Guanabara. Nesse contexto, o controle das fontes poluidoras e o gerenciamento dos múltiplos usos da baía devem ser implementados, no sentido da melhora da qualidade ambiental.

ACUTE TOXICITY of SODIUM SELENITE and SODIUM SELENATE TO TILAPIA, Oreochromis niloticus, FINGERLINGS

Selenium is an essential nutrient for many organisms, including fish. It can be released in the water by natural processes of dissolving rocks and minerals, and by the wastewater from industries and agricultural activities, which can increase its concentration in the environment, leading to toxic effects to the aquatic biota. Median Lethal Concentrations (LC(50-96h)) of two forms of selenium were estimated to fingerlings of Nile tilapia Oreochromis niloticus, focusing on estimating indicators for future environmental risk assessments in aquatic ecosystems contaminated with those elements, particularly for evaluate sources of water quality suitable for rearing tilapia. The results were: LC(50-96h) of sodium selenite (Na(2)SeO(3)) = 4.42 mg Se(4+) L(-1), and LC(50-96h) of sodium selenate (Na(2)SeO(4)) = 14,67 mg Se(6+) L(-1). According to those data, it was possible to classify sodium selenite as highly toxic and sodium selenate as moderately toxic to fingerlings of tilapia.

Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae)

Potassium permanganate is a chemical compound widely used in aquaculture for the control and removal of parasites, and in the prevention of diseases caused by bacteria and fungi. However, this compound can be toxic to fish, being a strong oxidant. Moreover, there is no consistent information in the literature about its toxicity to non-target organisms. The purpose of this study was to evaluate the acute toxicity (LC50;96h) of potassium permanganate for tilapia, Oreochromis niloticus, and to determine its toxic effects on nontarget organisms using ecotoxicological assays performed with the microcrustacean Ceriodaphnia dubia and with the green microalgae Pseudokirchneriella subcapitata. The results showed that the concentration of 1.81 mg L-1 of potassium permanganate caused acute toxic effect in tilapia fingerlings. The ecotoxicological assays demonstrated that concentrations above 0.12 mg L-1 can cause chronic toxic effects on non-target organisms, indicating possible deleterious effects on the food chain of the aquatic ecosystem that may receive the discharge of effluents released by fish cultures treated with this chemotherapy. All toxic concentrations determined in this study were below those recommended in the literature for the use of this chemotherapy in fish cultures, demonstrating that this type of therapy should be more carefully considered in order to avoid damage to the treated fish and to the environment. (C) 2011 Elsevier B.V. All rights reserved.

The Effect of Iron Oxide Nanoparticles on the Fate and Transformation of Arsenic in Aquatic Environments

Iron oxides and arsenic are prevalent in the environment. With the increase interest in the use of iron oxide nanoparticles (IONPs) for contaminant remediation and the high toxicity of arsenic, it is crucial that we evaluate the interactions between IONPs and arsenic. The goal was to understand the environmental behavior of IONPs in regards to their particle size, aggregation and stability, and to determine how this behavior influences IONPs-arsenic interactions. A variety of dispersion techniques were investigated to disperse bare commercial IONPs. Vortex was able to disperse commercial hematite nanoparticles into unstable dispersions with particles in the micrometer size range while probe ultrasonication dispersed the particles into stable dispersions of nanometer size ranges for a prolonged period of time. Using probe ultrasonication and vortex to prepare IONPs suspensions of different particle sizes, the adsorption of arsenite and arsenate to bare hematite nanoparticles and hematite aggregates were investigated. To understand the difference in the adsorptive behavior, adsorption kinetics and isotherm parameters were determined. Both arsenite and arsenate were capable of adsorbing to hematite nanoparticles and hematite aggregates but the rate and capacity of adsorption is dependent upon the hematite particle size, the stability of the dispersion and the type of sorbed arsenic species. Once arsenic was adsorbed onto the hematite surface, both iron and arsenic can undergo redox transformation both microbially and photochemically and these processes can be intertwined. Arsenic speciation studies in the presence of hematite particles were performed and the effect of light on the redox process was preliminary quantified. The redox behavior of arsenite and arsenate were different depending on the hematite particle size, the stability of the suspension and the presence of environmental factors such as microbes and light. The results from this study are important and have significant environmental implications as arsenic mobility and bioavailability can be affected by its adsorption to hematite particles and by its surface mediated redox transformation. Moreover, this study furthers our understanding on how the particle size influences the interactions between IONPs and arsenic thereby clarifying the role of IONPs in the biogeochemical cycling of arsenic.

Influence of Baculovirus anticarsia on the growth rate and survival of some nontarget aquatic organisms.

Recently, microbial pest control agents (MPCAs) have been worldwide used to reduce chemical pesticide use and to diminish the high risk of those compounds in the environment. Among various MPCAs, the nuclear polyhedrosis virus Baculovirus anticarsia is widely used in Brazil in the biological control of the velvet bean caterpillar. Although literature data do not show adverse effects of baculoviruses to nontarget organisms, it is necessary to evaluate their toxicity or patogenicity in order to study th environmental risk of those products and to register the formulations in the Brazilian Environmental Regularory Agency - IBAMA. In the presente work, the influence of a Baculovirus anticarsia formulation was evaluted to measure the consequences in the growth rateof the green algae Selenastrum capricornutum, the duckweed Lemna valdiviana and the microcrustacean Daphnia similis. The survival of the fish Hyphessobrycon scholzei exposed during 28 days was also evaluated. No significative adverse effects (P > 0.05) were observed in the test organisms which were exposed to 1-1000 times the maximum calculated pesticide concentration following a direct application to 15 cm layer of water.

Toxicity Of Fungal-generated Silver Nanoparticles To Soil-inhabiting Pseudomonas Putida Kt2440, A Rhizospheric Bacterium Responsible For Plant Protection And Bioremediation.

Silver nanoparticles have attracted considerable attention due to their beneficial properties. But toxicity issues associated with them are also rising. The reports in the past suggested health hazards of silver nanoparticles at the cellular, molecular, or whole organismal level in eukaryotes. Whereas, there is also need to examine the exposure effects of silver nanoparticle to the microbes, which are beneficial to humans as well as environment. The available literature suggests the harmful effects of physically and chemically synthesised silver nanoparticles. The toxicity of biogenically synthesized nanoparticles has been less studied than physically and chemically synthesised nanoparticles. Hence, there is a greater need to study the toxic effects of biologically synthesised silver nanoparticles in general and mycosynthesized nanoparticles in particular. In the present study, attempts have been made to assess the risk associated with the exposure of mycosynthesized silver nanoparticles on a beneficial soil microbe Pseudomonas putida. KT2440. The study demonstrates mycosynthesis of silver nanoparticles and their characterisation by UV-vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20 - a particle size distribution analyzer and TEM. Silver nanoparticles obtained herein were found to exert the hazardous effect at the concentration of 0.4μg/ml, which warrants further detailed investigations concerning toxicity.

In Vivo Evaluation Of The Genetic Toxicity Of Rubus Niveus Thunb. (rosaceae) Extract And Initial Screening Of Its Potential Chemoprevention Against Doxorubicin-induced Dna Damage.

Rubus niveus Thunb. plant belongs to Rosaceae family and have been used traditionally to treat wounds, burns, inflammation, dysentery, diarrhea and for curing excessive bleeding during menstrual cycle. The present study was undertaken to investigate the in vivo genotoxicity of Rubus niveus aerial parts extract and its possible chemoprotection on doxorubicin (DXR)-induced DNA damage. In parallel, the main phytochemicals constituents in the extract were determined. The animals were exposed to the extract for 24 and 48h, and the doses selected were 500, 1000 and 2000mg/kg b.w. administered by gavage alone or prior to DXR (30mg/kg b.w.) administered by intraperitoneal injection. The endpoints analyzed were DNA damage in bone marrow and peripheral blood cells assessed by the alkaline alkaline (pH>13) comet assay and bone marrow micronucleus test. The results of chemical analysis of the extract showed the presence of tormentic acid, stigmasterol, quercitinglucoronide (miquelianin) and niga-ichigoside F1 as main compounds. Both cytogenetic endpoints analyzed showed that there were no statistically significant differences (p>0.05) between the negative control and the treated groups with the two higher doses of Rubus niveus extract alone, demonstrating absence of genotoxic and mutagenic effects. Aneugenic/clastogenic effect was observed only at 2000mg/kg dose. On the other hand, in the both assays and all tested doses were observed a significant reduction of DNA damage and chromosomal aberrations in all groups co-treated with DXR and extract compared to those which received only DXR. These results indicate that Rubus niveus aerial parts extract did not revealed any genotoxic effect, but presented some aneugenic/clastogenic effect at higher dose; and suggest that it could be a potential adjuvant against development of second malignant neoplasms caused by the cancer chemotherapic DXR.

Engineered Nanoparticles And Organic Matter: A Review Of The State-of-the-art.

Growth in the development and production of engineered nanoparticles (ENPs) in recent years has increased the potential for interactions of these nanomaterials with aquatic and terrestrial environments. Carefully designed studies are therefore required in order to understand the fate, transport, stability, and toxicity of nanoparticles. Natural organic matter (NOM), such as the humic substances found in water, sediment, and soil, is one of the substances capable of interacting with ENPs. This review presents the findings of studies of the interaction of ENPs and NOM, and the possible effects on nanoparticle stability and the toxicity of these materials in the environment. In addition, ENPs and NOM are utilized for many different purposes, including the removal of metals and organic compounds from effluents, and the development of new electronic sensors and other devices for the detection of active substances. Discussion is therefore provided of some of the ways in which NOM can be used in the production of nanoparticles. Although there has been an increase in the number of studies in this area, further progress is needed to improve understanding of the dynamic interactions between ENPs and NOM.

Hemodynamic Changes With Two Lipid Emulsions For Treatment Of Bupivacaine Toxicity In Swines.

To compare the hemodynamic changes following two different lipid emulsion therapies after bupivacaine intoxication in swines. Large White pigs were anesthetized with thiopental, tracheal intubation performed and mechanical ventilation instituted. Hemodynamic variables were recorded with invasive pressure monitoring and pulmonary artery catheterization (Swan-Ganz catheter). After a 30-minute resting period, 5 mg.kg-1 of bupivacaine by intravenous injection was administered and new hemodynamic measures were performed 1 minute later; the animals were than randomly divided into three groups and received 4 ml.kg-1 of one of the two different lipid emulsion with standard long-chaim triglyceride, or mixture of long and medium-chain triglyceride, or saline solution. Hemodynamic changes were then re-evaluated at 5, 10, 15, 20 and 30 minutes. Bupivacaine intoxication caused fall in arterial blood pressure, cardiac index, ventricular systolic work index mainly and no important changes in vascular resistances. Both emulsion improved arterial blood pressure mainly increasing vascular resistance since the cardiac index had no significant improvement. On the systemic circulation the hemodynamic results were similar with both lipid emulsions. Both lipid emulsions were efficient and similar options to reverse hypotension in cases of bupivacaine toxicity.

Ecotoxicological Effects Of Carbofuran And Oxidised Multiwalled Carbon Nanotubes On The Freshwater Fish Nile Tilapia: Nanotubes Enhance Pesticide Ecotoxicity.

The interactions of carbon nanotubes with pesticides, such as carbofuran, classical contaminants (e.g., pesticides, polyaromatic hydrocarbons, heavy metals, and dyes) and emerging contaminants, including endocrine disruptors, are critical components of the environmental risks of this important class of carbon-based nanomaterials. In this work, we studied the modulation of acute carbofuran toxicity to the freshwater fish Nile tilapia (Oreochromis niloticus) by nitric acid treated multiwalled carbon nanotubes, termed HNO3-MWCNT. Nitric acid oxidation is a common chemical method employed for the purification, functionalisation and aqueous dispersion of carbon nanotubes. HNO3-MWCNT were not toxic to Nile tilapia at concentrations ranging from 0.1 to 3.0 mg/L for exposure times of up to 96 h. After 24, 48, 72 and 96 h, the LC50 values of carbofuran were 4.0, 3.2, 3.0 and 2.4 mg/mL, respectively. To evaluate the influence of carbofuran-nanotube interactions on ecotoxicity, we exposed the Nile tilapia to different concentrations of carbofuran mixed together with a non-toxic concentration of HNO3-MWCNT (1.0 mg/L). After 24, 48, 72, and 96 h of exposure, the LC50 values of carbofuran plus nanotubes were 3.7, 1.6, 0.7 and 0.5 mg/L, respectively. These results demonstrate that HNO3-MWCNT potentiate the acute toxicity of carbofuran, leading to a more than five-fold increase in the LC50 values. Furthermore, the exposure of Nile tilapia to carbofuran plus nanotubes led to decreases in both oxygen consumption and swimming capacity compared to the control. These findings indicate that carbon nanotubes could act as pesticide carriers affecting fish survival, metabolism and behaviour.