This work was financed by the research project: "NanoBioTech ambiente e salute. Progetto 2: Ambiente. Strumenti e metodi per il monitoraggio ecotossicologico delle Nanoparticelle" ("Nano-BioTechnology: environment and health. Project 2: Environment. Tools and methods for ecotoxicological monitoring of nanoparticles") granted to LM from Regione Lazio-Consorzio Hypatia. AR was funded by a postdoctoral grant from the University of Tor Vergata/Regione Lazio-Consorzio Hypatia under the framework of the previously cited project. An agreement with ISPRA-Tor Vergata University (N. 2015/52857) allowed the mutual use of facilities and the exchange of researchers.
The authors are indebted to Prof. Maria Cristina Thaller, our guardian angel for all microbial activities, for raising interest in the microbial world and for strongly helping to improve our research on the issue. The authors gratefully acknowledge Andrea Tornamb&#232; and Erika Magaletti for the precious collaboration with the ISPRA Lab of Phytoplankton ecology and ecotoxicology.

1. Preparation of Reagents/Materials
<ol>
	<li>Prepare (about 300) sterile 1.5 mL tubes for the serial dilution of bacterial suspensions, as well as 15 mL sterile tubes as test containers labeled with the test concentrations.</li>
	<li>Prepare 2% NaCl solution as the exposure medium and sterilize it. Alternatively, use sterilized synthetic or natural seawater, with salinity ranging from 5 to 40.</li>
	<li>Prepare tryptic soy agar (TSA) growth medium with 2% NaCl according to the label directions and considering the amount of NaCl already present in the medium.</li>
	<li>Pour the TSA (cool but still liquid) into the 90-mm Petri dishes previously labeled with the test concentration and exposure time, replicate number, and dilution factor; 19 mL is an appropriate volume.</li>
	<li>Prepare tryptic soy broth (TSB) growth medium according to the label directions. Add the appropriate amount of NaCl to obtain the same salinity as the exposure medium.</li>
	<li>Prepare a CuSO4 stock solution with double-distilled water and sterilize the (necessary) aliquot using a 0.22 &#181;m syringe filter. In case of environmental samples, prepare an appropriate interval of dilutions of the sample and sterilize them using a 0.22 &#181;m syringe filter.</li>
	<li>Prepare the test solutions in the 15 mL tubes labeled with the test concentrations. Fill the negative control with 5 mL of the exposure medium (2% NaCl). Fill the other tubes with the appropriate amount of exposure medium and CuSO4 stock solution to obtain the test concentrations in a 5 mL final volume.</li>
</ol>
2. Bacterial Inoculum Preparation
<ol>
	<li>12-18 h before the test, prepare a liquid fresh culture of Vibrio anguillarum. Using a sterile loop, select a single, well-isolated colony from an overnight culture on a solid medium (TSA). Inoculate a tube filled with 10 mL of TSB and incubate the bacterial culture at 25 &#176;C for 12-18 h.</li>
	<li>After 12-18 h, estimate the bacterial concentration of the inoculum spectrophotometrically. Vortex the inoculum and measure the optical density at&#160;600-nm wavelength, using TSB as blank.</li>
	<li>To obtain a known bacterial concentration, dilute 2 mL of the vortexed inoculum by adding the amount of TSB calculated by this formula: 
	TSB mL = [(O.D. / 0.14) * 2] - 2.</li>
	<li>Verify that the optical density of the diluted inoculum is 0.140 (&#177;0.005), which corresponds to the 0.5 point on the McFarland nephelometric standard.</li>
	<li>Centrifuge the diluted inoculum for 10 min at 3,000&#160;g. Eliminate the supernatant and re-suspend the microbial pellet in 1 mL of 2% NaCl solution (exposure medium).</li>
</ol>
3. Testing Exposure
<ol>
	<li>Add 150 &#181;L of the re-suspended bacterial inoculum to each tube, including the control. Incubate the V. anguillarum suspensions for 6 h at 25 &#176;C under darkness and in continuous shaking to avoid sedimentation.</li>
	<li>At the beginning (T0) and the end (T6) of the exposure time, carry out bacterial counting in all the exposed bacterial suspensions using colony forming unit (CFU) counting methods.</li>
	<li>Prepare serial dilutions of each exposed bacterial suspension in triplicate, applying a ten-fold dilution factor (up to 10-5). Add 100 &#181;L of each bacterial suspension to the corresponding tube&#160;already filled with 900 &#181;L of exposure medium (2% NaCl). Proceed with the serial dilution, vortexing at each step to re-suspend the bacteria.</li>
	<li>Plate 10 &#181;L of the 10-4 and 10-5 dilutions on TSA Petri dishes in the corresponding segment. Quickly let the drops glide in a small circle by rotating the plate. Incubate the plates at 25 &#176;C for 48 h.</li>
</ol>
4. CFU Counting
<ol>
	<li>After 48 h, count the colonies grown on the Petri dishes; plates harboring between 5 and 50 colonies are optimum for accurate counting.</li>
	<li>Calculate the number of viable bacteria per mL of each exposed bacterial suspension by applying the following formulas: 
	10-4 &#8594; CFU/mL = n&#176; CFU x 100 x 10,000 
	10-5 &#8594; CFU/mL = n&#176; CFU x 100 x 100,000</li>
	<li>Use the average of the counts obtained in parallel replicates to evaluate the mortality compared with the control. Calculate the mortality as a percentage with the following formula: 
	M% = 100 - [(N/C) * 100] 
	NOTE: Where N = number of CFU/mL grown after the exposure to the toxicant; C = number of CFU grown in the control medium.</li>
	<li>Calculate the LC50 (i.e., the concentration of toxicant that reduces the number of actively replicating bacteria by 50% per mL; CFU/mL) with non-linear regression analysis using an appropriate statistical software (see the table of materials). Run a one-way analysis of variance (ANOVA) followed by post-hoc pairwise t-tests to evaluate significant differences between treatments.</li>
</ol>

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Animal. 34 (4), 413-415 (2010).</li><li>Thompson, F., Iida, T., Swings, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiversity+of+Vibrios.">Biodiversity of Vibrios.</a> Microbiol. Mol. Biol. Rev. 68, 403-431 (2004).</li><li>Austin, B., Austin, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrionaceae+representatives:+characteristics+of+the+disease.">Vibrionaceae representatives: characteristics of the disease.</a> Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. 29-30, 108-115 (1999).</li><li>Larsen, J. 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The authors declare that they have no competing financial interests.

This work describes a new bioassay with the marine bacterium V. anguillarum that was successfully applied to assess the toxic effects of CuSO4, a reference toxicant, demonstrating a clear dose-response relationship. The marine bacterium V. anguillarum was chosen as a model organism because it is halotolerant, ubiquitous, and representative of marine ecosystems.
The test can be performed at a wide range of salinity values (5-40) and can use saline solutions and synthetic or natural seawaters as the exposure medium, as long as microorganisms can easily survive for the duration of the entire test. This allows for the analysis of different kind of samples, including brackish and marine environments.
No growth medium is required during the exposure phase, avoiding its interference with the contaminants8 and its possible influence on the biological response. The protocol is reliable, rapid, cost effective, and relatively easy. The procedure of liquid-to-plate micro-counts9 gives the advantage of using small (sample) volumes, although this implies a high degree of accuracy and robustness. The results of the three independent trials and replicates for each treatment show the high repeatability of this method. The use of bacterium as a biological model, as well as the adaptability of the technique, favor the ecological and environmental relevance of this procedure. Other critical technical issues are accuracy in the preparation of the bacterial inoculum and the sterility required in some steps of the procedure.
The proposed test is more rapid (6 h) than other marine ecotoxicological assays (24-96 h) and does not raise the ethical problems ensuing from the use of higher organisms. Furthermore, data on the reference toxicant show LC50 values comparable with those obtained with acute tests on other marine species10,11, demonstrating a good sensitivity. Among bacterial bioassays, the V. fischeri luminescence inhibition test is the most common and well-standardized12. This bioassay is very rapid (15-30 min) and valid for testing solid-phase samples, but it can be affected by colored and turbid samples, which interfere with luminescence measurements. Salinity is a limiting factor in the use of the abovementioned test, with 2% NaCl required13. On the contrary, the test proposed here with V. anguillarum gives affordable results at a wide range of salinity values, has no limitations in regard to turbid or colored samples, and requires less expensive equipment compared to the luminescence analyzers. A comparison between the results of our study and those available in the literature for V. fisheri14,15,16 shows comparable EC50 values, further supporting the effectiveness of this bioassay.
This bioassay assesses the reduction of the bacterial culturability, generally referred to as mortality, instead of population growth rate or enzymatic activity inhibition, which are used in the tests currently available for microorganisms. The LC50 calculation allows for comparison with other bioassays commonly applied to the ecotoxicological assessment of marine environments, which often have survival/mortality as the endpoint. An intercalibration exercise is urgently necessary to evaluate/confirm the reliability and reproducibility of this test and to support its standardization and use in regulatory protocols.
The increasing use of nanomaterials and their potential release in the environment imply the need for risk assessment17. However, classical (eco)toxicological approaches for these emerging contaminants seem not to give affordable results and may require some adaptations18. The characteristics of this new bioassay allow for its easy and useful application to the toxicity assessment of nanoparticles. In fact, the possibility of carrying out the assay at different salinities will give accounts of nanoparticle behavior under different ionic strengths, an environmental parameter variable that can significantly affect toxicity19. Furthermore, no use of growth medium and nutrients is particularly recommended in the ecotoxicity assessments of nanoparticles because organic substances can facilitate their absorption by increasing the toxic effects20 or can cause aggregation, reducing the bioavailable fraction and therefore their toxicity21.
In conclusion, the bioassay on Vibrio anguillarum is a promising tool for the risk assessment of classical and emerging contaminants, as well as for the assessment of the status of marine and brackish environments.

Ecotoxicological bioassays evaluate the toxicity of chemicals or environmental samples with standard biological models, integrating the effects of physical, chemical, and biological stressors on ecosystems. Due to the complexity of ecosystems, ecotoxicological risk assessments must consider a battery of bioassays that involve organisms from different trophic levels. Toxicity assays on laboratory animals may be expensive, time consuming, and ethically questionable. The drive to limit animal testing and develop alternative approaches (e.g., on bacteria and non-vertebrate animals) is now a pivotal issue, as reported in the framework of the current European legislation, including the EU Animal Protection Directive, the 7th Amendment to the EU Cosmetics Directive, and REACH.
Crustaceans, fish, and algae are largely used for toxicity measurements in the marine environment1. Bacteria are an important component of the ecosystem, and alterations to microbial communities can have significant effects on biogeochemical cycling and other critical ecosystem services. Toxicity testing based on microorganisms are gaining popularity because they are relatively quick, reproducible, and cheap and do not raise ethical issues2. The aim of this work is to describe an ecotoxicological protocol to evaluate the response of the marine bacterium Vibrio anguillarum (Listonella anguillarum, Vibrionaceae) when exposed to environmental contaminants.
V. anguillarum is a Gram-negative, short, curve-shaped rod bacterium (0.5 x 1.5 &#181;m) with a polar flagellum. Typically found in brackish or salt water, it is halotolerant, with an optimal salinity of about 20 and an optimal temperature between 25 and 30 &#176;C3. It has been chosen as an organism model due to its ubiquity and its important ecological roles in oceans worldwide4. Some serotypes of V. anguillarum are known to cause vibriosis in a variety of marine or brackish fish species5,6. For this, some steps of the experiment require standard microbiological practices, but no special safety equipment or precautions are needed. The proposed toxicity testing protocol uses the bacterial culturability (i.e., the capability to replicate and form colonies) as the endpoint and allows for the determination of the LC50, the concentration that causes a 50% reduction of bacteria actively replicating and forming colonies, after a 6-h exposure. In Vibrio, as in other microbes, this reduction, which we generally indicate as mortality, can partially be due to individuals in the viable but non-culturable (VBNC) phase7. In this study, we applied this method to measure the toxic effects of copper sulphate (CuSO4), a reference toxicant.
This method was developed to provide a suitable, microorganism-based test for the ecotoxic assessment of pollutants/chemical compounds, including emerging contaminants such as nanomaterials. The novelty of this protocol compared to existing methods used for microorganisms is mainly related to the exposure medium and the endpoint. In fact, the exposure is carried out in saline solution, avoiding any possible interference of the growth medium with the investigated contaminants, which may influence the biological response8. The endpoint is the reduction of bacterial culturability, which may be easily compared to other acute endpoints used for ecotoxicological screening in marine/brackish environments, based on survival/mortality. Moreover, the protocol uses the technique of liquid-to-plate micro-counts, already used on E. coli9, reducing volumes and thus the experimental effort (see steps 3.3 and 3.4 of the protocol for details).

<table><tbody><tr><td>&lt;i&gt;Vibrio anguillarum&lt;/i&gt; (strain AL 102, serotype O1)</td><td></td><td></td><td>Obtained from the laboratory collection of NOIFMA (Norway)</td></tr><tr><td>Tryptic Soy Agar&amp;nbsp;</td><td>Liofilchem</td><td>610052</td><td>Dehydrated Culture Media</td></tr><tr><td>Tryptic Soy Broth growth medium</td><td>Liofilchem</td><td>610053</td><td>Dehydrated Culture Media</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>209198</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S-3014</td><td></td></tr><tr><td>&lt;strong&gt;Name of Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Biosafety Laminar Flow Hood&amp;nbsp;</td><td>ESCO</td><td></td><td></td></tr><tr><td>Incubator&amp;nbsp;</td><td>Fratelli Galli</td><td>Mod. 2100</td><td></td></tr><tr><td>&lt;strong&gt;Name of Software&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Benchmark Dose Software&amp;nbsp;</td><td>US EPA</td><td>Benchmark Dose 2.4.0&amp;nbsp;</td><td>2012</td></tr></tbody></table>

ecotoxicological method with marine bacteria vibrio anguillarum to evaluate the acute toxicity of environmental contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

The results of three independent trials of exposing V. anguillarum to four concentrations of CuSO4 show a clear dose-response relationship and a significant decrease of bacteria actively replicating and forming colonies with an increasing concentration of the reference toxicant (Figure 1; ANOVA, F = 20.28, p &#60;0.001). The number of CFU/mL is significantly reduced at 1.25 mg/L (post-hoc Tukey&#39;s test, p &#60;0.05) compared with control (CNTR). Any culturable bacteria were detected at the highest concentration tested. No differences in CuSO4 toxicity were found along the wide salinity range of the exposure medium (i.e., 5, 20, or 35 g/L NaCl, data not shown). A representative output of the statistical analysis showing the non-linear regression is reported in Supplemental Figure 1. The LC50 values calculated for the three independent trials (Table 1) highlight the feasibility and reproducibility of this method.
<img alt="Figure 1" src="/files/ftp_upload/55211/55211fig1.jpg" /> 
Figure 1: Vibrio anguillarum exposed to CuSO4. The mean number of CFU/mL (CFU = colony forming unit) exposed to different concentration of CuSO4 for 6 h. The values represent the mean (&#177;SD) of three independent trials. The reductions of bacteria actively replicating and forming colonies with respect to the control (CNTR) are reported in the yellow boxes (as percentages). Significant differences with the control, based on a post-hoc Tukey&#39;s test, are indicated with asterisks (* = p &#60;0.05; ** = p &#60;0.01). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55211/55211fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/55211/55211table1.jpg" /> 
Table 1: Lethal concentration (LC50) values for Vibrio anguillarum exposed to CuSO4. Results of three independent trials and means (&#177; SD) are reported.
 
Supplemental Figure 1: Non-linear regression analysis. Representative output of a non-linear regression analysis (Logit-Hill model) performed on the results of the test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55211/suppl_fig_1.tif" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants at JoVE.com

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

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Nanyang Technological University

Research

JoVE Journal

Environment

11.0K Views.  Institute for Environmental Protection and Research (ISPRA), Rome (Italy).  Dit werk beschrijft een nieuw protocol voor de beoordeling van de ecotoxiciteit van verontreinigende stoffen, waaronder opkomende verontreinigingen zoals nanomaterialen, met behulp van de mariene bacterie Vibrio anguillarum . Deze methode laat de bepaling van de LC 50 of sterfte toe, de concentratie die een kweekbaarheid van 50% van de bacteriën veroorzaakt, na een 6 uur blootstelling. 

Video: Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

Chemistry

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Immunology and Infection

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Bioengineering

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Biology

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

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