We are grateful to the SPHERE group of the UAntwerpen and the Department of crop protection of the Ugent for analysis of water samples. Support during this project was provided by the Excellence Center &#39;Eco and socio-evolutionary dynamics (PF/10/007) of the KU Leuven Research Fund. AFG (11Q0516N) and ESJT (FWO-SB151323) were funded as doctoral and TP (12F0716N) as post-doctoral fellow by FWO Flanders (Fonds Wetenschappelijk Onderzoek).

All methods described here have been approved by the Ethical committee of KULeuven.
1. Hatching and General Maintenance of N. furzeri 
<ol>
	<li>Prepare fish medium (pH 7) at a temperature of 14 &#176;C and add purified Type II water, with added standardized salts, to a conductivity of 600 &#181;S/cm (24 &#176;C).</li>
	<li>Select eggs from the GRZ (Gona-Rhe-Zhou) laboratory line that have been stored under standardized conditions13. Select eggs in the DIII s...

<ol>
	<li>European-Chemicals-Bureau. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TAPIR Three point three-A Project for the Information Requirements of REACH. Final Report-2 August 2005. Scoping study on the development of a Technical Guidance Document on information requirements on intrinsic properties of substances (RIP 3.3-1). , (2005).</li><li>Philippe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+sensitivity+to+copper+of+a+promising+ecotoxicological+model+species,+the+annual+killifish+Nothobranchius+furzeri.">Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri.</a> Ecotoxicol Environ Saf. , 26-35 (2017).</li><li>Noyes, P. D., Lema, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forecasting+the+impacts+of+chemical+pollution+and+climate+change+interactions+on+the+health+of+wildlife.">Forecasting the impacts of chemical pollution and climate change interactions on the health of wildlife.</a> Current Zoology. 61 (4), 669-689 (2015).</li><li>Consommateurs, S. S. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Health &amp; Consumer Protection Directorate-General European Commission. 4, (2002).</li><li>Commission, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+document+on+aquatic+ecotoxicology.+Under+Council+directive+91/414/EEC.">Guidance document on aquatic ecotoxicology. Under Council directive 91/414/EEC.</a> SANCO/3268/2001 Rev 4. 2002b. , (2002).</li><li>. Ecological Effects Test Guidelines, OPPTS 850.1500 Fish life cycle toxicity Available from: <a target="_blank" href="https://www.epa.gov/sites/production/files/2015-07/documents/850-1500.pdf">https://www.epa.gov/sites/production/files/2015-07/documents/850-1500.pdf</a> (1996)</li><li>Philippe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+sensitivity+to+copper+of+a+promising+ecotoxicological+model+species,+the+annual+killifish+Nothobranchius+furzeri.">Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri.</a> Ecotoxicol Environ Saf. , (2017).</li><li>Ankley, G. T., Villeneuve, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fathead+minnow+in+aquatic+toxicology:+past,+present+and+future.">The fathead minnow in aquatic toxicology: past, present and future.</a> Aquatic Toxicology. 78 (1), 91-102 (2006).</li><li>Pola&#269;ik, M., Bla&#382;ek, R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+breeding+of+the+short-lived+annual+killifish+Nothobranchius+furzeri.">Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri.</a> Nature Protocols. 11 (8), 1396-1413 (2016).</li><li>Reichwald, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Sex+Chromosome+Evolution+and+Aging+from+the+Genome+of+a+Short-Lived+Fish.">Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.</a> Cell. 163 (6), 1527-1538 (2015).</li><li>Valenzano, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Turquoise+Killifish+Genome+Provides+Insights+into+Evolution+and+Genetic+Architecture+of+Lifespan.">The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.</a> Cell. 163 (6), 1539-1554 (2015).</li><li>Shedd, T. R., Widder, M. W., Toussaint, M. W., Sunkel, M. C., Hull, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+annual+killifish+Nothobranchius+guentheri+as+a+tool+for+rapid+acute+toxicity+screening.">Evaluation of the annual killifish Nothobranchius guentheri as a tool for rapid acute toxicity screening.</a> Environ. Toxicol. Chem. 18 (10), 2258-2261 (1999).</li><li>Platzer, M., Englert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nothobranchius+furzeri:+a+model+for+aging+research+and+more.">Nothobranchius furzeri: a model for aging research and more.</a> Trends Genet. 32 (9), 543-552 (2016).</li><li>Watters, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+and+distribution+of+Nothobranchius+fishes.">The ecology and distribution of Nothobranchius fishes.</a> J Am Killifish Assoc. 42, 58-61 (2009).</li><li>Op de Beeck, L., Verheyen, J., Stoks, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+magnifies+the+impact+of+a+pesticide+in+a+warming+world+by+reducing+heat+tolerance+and+increasing+autotomy.">Competition magnifies the impact of a pesticide in a warming world by reducing heat tolerance and increasing autotomy.</a> Environ Pollut. 233, 226-234 (2018).</li><li>Patra, R. W., Chapman, J. C., Lim, R. P., Gehrke, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+three+organic+chemicals+on+the+upper+thermal+tolerances+of+four+freshwater+fishes.">The effects of three organic chemicals on the upper thermal tolerances of four freshwater fishes.</a> Environ. Toxicol. Chem. 26 (7), 1454-1459 (2007).</li><li>Beitinger, T. L., Bennett, W. A., McCauley, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+tolerances+of+North+American+freshwater+fishes+exposed+to+dynamic+changes+in+temperature.">Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature.</a> Environ Biol Fishes. 58 (3), 237-275 (2000).</li><li>Cellerino, A., Valenzano, D. R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+bush+to+the+bench:+the+annual+Nothobranchius+fishes+as+a+new+model+system+in+biology.">From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology.</a> Biological Reviews. , (2015).</li></ol>

This work describes a new bioassay using Nothobranchius furzeri, an emerging model organism, to study the individual and combined long-term effects of toxicants and other stressors. The presented protocols were successfully applied to measure the sensitivity of the species to an array of toxicants (copper, cadmium, 3,4-dichloroaniline, and chlorpyrifos). Due to its fast life-cycle, this vertebrate model allows for assessment of sublethal and transgenerational effects within four months. Another major advantage o...

Sensitivity profiles of an array of species to strategically selected toxicants have been described1 for the European REACH legislation (Registration, Evaluation, Authorization, and Restriction of Chemicals). Acute or short-term toxicity tests were mostly used for this purpose as they give a quick indication of a species&#39; sensitivity. However, in their natural environment, organisms are exposed over much longer periods and full life-cycles or even several generations could be affected2. Moreover, organisms in polluted environments are typically exposed to more than one stressor at a time, which may interact with each other, possibly resulting in synergistic effects3. Hence, safe concentrations calculated based on acute, single stressor toxicity tests may underestimate the actual risks imposed by toxicants in natural environments. It is, therefore, advisable to also study the chronic and multigenerational effects of sublethal concentrations of toxicants in an environmentally relevant context as advocated by the European Commission4,5 and the USEPA (United States Environmental Protection Agency)6,7. Especially in vertebrate research, the costs in terms of labor, money, and time are high when performing chronic and multigenerational exposure studies because of the relatively long lifespan of vertebrates compared to invertebrate model organisms. Therefore, it is advisable to choose the most appropriate fish model organism, depending on the research question. Furthermore, a wide array of vertebrate species should be available in order to test the generality of responses across species to be able to adapt regulations based on the most sensitive species. For now, there is a need to develop new, efficient protocols with vertebrate model species characterized by short life-cycles to lower the costs of performing chronic and multigenerational exposures on vertebrates7,8.
The turquoise killifish Nothobranchius furzeri is an interesting fish model to use in such long-term exposure experiments because of its short maturation time and life-cycle (generation time less than 4 weeks9). This means that ecologically relevant endpoints such as maturation time and fecundity can be studied within a short time frame compared to other fish models7. Furthermore, these fish produce drought-resistant, dormant eggs that remain viable for several years when stored under standard conditions, thereby eliminating the need for a continuous culture9. In ecotoxicological studies, this also implies that replicate fish can all be hatched at the exact same moment, resulting in time synchrony for all animals, even among batches of eggs produced at different times. We advise using the laboratory GRZ strain to perform exposure experiments. This strain performs well under laboratory conditions, is homozygous (except for sex chromosomes) and the genome is well characterized10,11.
In ecotoxicological studies, it is important to select the appropriate range of test concentrations. Several complementary methods can be used to this end. The nominal concentration range can be based on the sensitivity of a related species, such as Nothobranchius guentheri12. Alternatively, the range can be based on the sensitivity of standard fish models, such as zebrafish (Danio rerio)2 that have a comparable sensitivity to most toxicants (Philippe et al. (in review)). In combination, with both of these options, a range finding experiment should be conducted to select the nominal concentration range. For acute testing, researchers should aim for concentration treatments with 100% mortality, intermediate mortality and 0% mortality after 24 h of exposure to the toxicant. For chronic testing, it is advisable to run the range finding experiment for two weeks to verify if larval mortality in the condition with the highest test concentrations does not exceed 10% during this reference period.
The protocol can serve as a baseline to perform acute and chronic exposure to waterborne pollutants on N. furzeri, examining potential effects of stressors both at the individual and cellular level. It can also be used to perform multi-stressor research to accommodate a higher ecological relevance, mixing different toxic compounds or studying interactive effects between pollution and other natural stressors (e.g. predation) or anthropogenic stressors (e.g. warming due to climate change).

<table>
	<tbody>
		<tr>
			<td>purified water Type 1 (milli Q)</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sea Salt</td>
			<td>Instant Ocean</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2L plastic tank</td>
			<td>SAVIC</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>1L plastic tank (spawning)</td>
			<td>Avamoplast</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>nets</td>
			<td>Aqua bilzen</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>2L glass jars</td>
			<td>Sepac-Flacover</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>0,5L glass jars</td>
			<td>Sepac-Flacover</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>Artemia eggs</td>
			<td>Ocean Nutrition</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>chironomus</td>
			<td>Ocean Nutrition</td>
			<td></td>
			<td>frozen</td>
		</tr>
		<tr>
			<td>tricaine</td>
			<td>Sigma aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petri dishes</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pipettes</td>
			<td>MLS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>FST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;m mesh sieve</td>
			<td>/</td>
			<td></td>
			<td>self-made</td>
		</tr>
		<tr>
			<td>microcentrifuge tube (2ml)</td>
			<td>BRAND</td>
			<td></td>
			<td>To store fish in freezer</td>
		</tr>
		<tr>
			<td>glass vials</td>
			<td>Sigma aldrich</td>
			<td></td>
			<td>For water analysis</td>
		</tr>
		<tr>
			<td>weighing boat</td>
			<td>MLS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jiffy 7c pellets</td>
			<td>Jiffy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>Gilac</td>
			<td></td>
			<td>for Ctmax</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Air liquide</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>digital thermometer</td>
			<td>Testo AG</td>
			<td>testo 926</td>
			<td></td>
		</tr>
		<tr>
			<td>HETO therm heater</td>
			<td>Anker Schmitt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>calibrated balance</td>
			<td>Mettler-Toledo AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>platform for camera</td>
			<td>/</td>
			<td></td>
			<td>self-made</td>
		</tr>
		<tr>
			<td>Multiparameter kit</td>
			<td>HACH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80&#176;C)</td>
			<td>Panasonic Ultra low temperature freezer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fysio</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>homogenisation buffer</td>
			<td>VWR</td>
			<td></td>
			<td>0.1&#160;M TRIS&#8211;HCl, pH 8.5, 15&#160;% polyvinyl pyrrolidone, 153&#160;&#181;M MgSO4&#160;and 0.2&#160;% Triton X-100</td>
		</tr>
		<tr>
			<td>chloroform:methanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glyceryl tripalmitate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>amyloglucosidase</td>
			<td>Sigma Aldrich</td>
			<td>A7420</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose assay reagent</td>
			<td>Sigma Aldrich</td>
			<td>G3293</td>
			<td></td>
		</tr>
		<tr>
			<td>Biorad protein dye</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtiter plate</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>384 microtiter plates</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml glass tubes</td>
			<td>Fiers</td>
			<td></td>
			<td>For fat analysis</td>
		</tr>
		<tr>
			<td>2,5ml eppendorf tubes</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>homogeniser</td>
			<td>Ultra-turrax TP 18/10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>photospectrometer</td>
			<td>Infinite M200 TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>heater for glass tubes</td>
			<td>Hach COD REACTOR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf Centrifuge 5415 R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Bumako</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The results of the acute exposure of N. furzeri to different concentrations of copper, calculated as in 2.5.2, show cleardose-response relationships (Figure 1). There is an increase in mortality with increasing toxicant concentration. LC50 values decrease over time, meaning that with decreasing concentrations, more time passes before 50% of the replicates die. For detailed results on the acute and chronic exposure of N. furzeri to...

Watch this Scientific Journal Video about Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri at JoVE.com

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.

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 ...

The overall goal of this experiment is to study the effects of toxicants on the turquoise killifish Nothobranchius furzeri. This method can help answer key questions in the field of ecotoxicology, such as the long term effects of stressors on important life-history traits, being maturation time, growth, or fecundity. The main advantage of this experiment is that we use a vertebrate with a very fast life-cycle, resulting in time and cost-efficient experiments.These killifish are ideal models to study the impact of long-term and multi generational effects of stressors. Ultimately, they will contribute towards better understanding of environmental impact of toxicants. Though this method can provide new insights into the combined effects of toxicants and temperature, it can also be applied to other stressors such as UV radiation, food deprivation and PH alterations.Killifish are challenging to work with, because they're a new model system in ecotoxicology, and because they're sensitivity to a lot of pollutants is still unknown. We developed the idea for this experimental method when we first learned about the exceptional short life span of killifish, and their capacity to produce dormant eggs that can be stored for years on the shelf. Visual demonstration of this method is critical as the type of the experiment, as well as handling of the individuals, can alter the results.To begin, select gona ray zoo, or GRZ eggs, in the D3 stage, which are recognizable by the presence of golden eyes. And use a soft pair of tweezers to gently transfer them to a plastic two-liter tank. Add one centimeter of the hatching medium at 12 degrees celsius, and let the water temperature gradually adjust to room temperature.After 24 hours, feed the hatchlings a concentrated dose of freshly hatched artemia nauplii, and increase the water depth to five centimeters by adding fish medium. 48 hours post-hatching, transfer healthy, buoyant larvae to individual containers with exposure medium, and toxicant concentrations as described in the text protocol. Each morning and evening, check fish for mortality, stress, and sickness, and calculate LC50 values according to the text protocol.Perform a chronic exposure trial by filling individual containers with the correct exposure medium, and add an air tube to aerate the jar. Transfer fish into the jars for the remainder of the experiment. From two to 23 days post-hatching or DPH, feed the fish artemia nauplii twice per day ad libitum.Then, after supplementing with chopped chironomus larvae until 37 DPH, use frozen chironomus larvae to feed the fish ad libitum twice per day, every day, moving forward. To determine growth, measure the body size on a weekly basis, by transferring fish to a Petri dish filled with medium from the reservoir. Take four to five size-calibrated dorsal view pictures of the fish at a fixed height.And use a spatial measuring program to digitally analyze them. From 15 DPH onwards, visually inspect the male fish for coloration, to determine maturation. Check the fins for the first signs of nuptial coloration, and use the day at which fist coloration was visible as a proxy for male maturation time.From 30 DPH onwards, check fecundity by setting up a one-liter spawning tank for mature male and female couple within each treatment, using exposure medium from the male's aquarium, and supplement it with spawning substrate. Transfer both the male and female into the spawning tank. And while minimizing human activity or disturbance around the spawning containers, allow them to spawn for two hours.Afterwards, without unnecessary mixing of the water, which would whirl up the eggs in the spawning substrate, gently transfer fish back to their original housing containers. Filter out the eggs by pouring the spawning substrate over a 500 micrometer mesh. Count the eggs and use a pipette, to transfer them to damp peat moss in Petri dishes.Remove dead eggs daily. After a week, use sealing film to seal the Petri dish. And store it in a temperature-controlled incubator at 28 degrees celsius, with a 14 to 10 light to dark cycle, for immediate development to the D3 phase.For long-term conservation, store the eggs in constant darkness at 17 degrees celsius, upon which eggs enter the dormant phase, and remain viable for multiple years. To recruit fish from the dormant eggs, transfer the eggs to 28 degrees celsius, and a 14 to 10 light to dark cycle, for approximately three weeks, to allow them to develop to the D3 phase. To measure the critical thermal maximum, or CT max of adult fish, begin with a continuously circulating water bath, heated at a constant rate of 0.33 degrees celsius per minute.Add several one-liter aquaria, for each individual fish. Start the trail by adding the fish to the aquarium, when the water has reached the experimental rearing temperature of the fish. Use a digital thermometer to monitor the temperature in the one-liter aquaria of the CT max bath, every five minutes.When the fish fails to maintain a dorsal ventral upright position, or starts twitching heavily, measure the temperature in the one-liter aquarium, which is the critical maximal temperature. Then, transfer the fish back to its experimental housing for recovery. As shown in this graph, acute exposure of enfurzeri to different concentrations of copper, caused an increase in mortality with increasing toxicant concentration.LC50 values decrease over time, meaning that with decreasing concentrations, more time passes before 50%of the replicates die. In chronic exposure assay, using water-born copper, fish are shorter in length, as measured after three weeks when raised at the highest concentration tested. In this experiment using water borne chlorpyrifos or CPH, when raised at 28 degrees celsius, fish produced less eggs when exposed to the highest CPF concentration, however, at 30 degrees celsius, control fish produced more eggs than fish exposed to either concentration of CPF, indicating that the combination of CPF and an increase in temperature affected fecundity more than each stressor separately.Reported here is the mean age at which the first signs of coloration appeared in enfurzeri males, exposed to CPF at 28 and 30 degrees celsius. After exposure to four micrograms per liter of CPF, the male fish took 18%longer to mature than control fish. Measuring the CT max of fish reared at either 24 or 28 degrees celsius, and exposure to the pollutant 3, 4-DCA revealed that erratic swimming, increased opercular movement, and loss of ability to remain in dorsoventrally upright position, occurred at a lower temperature when fish were raised at 24 degrees celsius.Once mastered, this protocol, including the effects on fecundity, can be measured in two months. To measure the effects on the whole reproductive period or even a following generation, the protocol can be extended, but can still be performed in six months.

protocol-for-acute-chronic-ecotoxicity-testing-turquoise-killifish

Research

JoVE Journal

Environment

7.4K vistas.  University of Leuven. The overall goal of this experiment is to study the effects of toxicants on the turquoise killifish Nothobranchius furzeri. This method can help answer key questions in the field of ecotoxicology, such as the long term effects of stressors on important life-history traits, being maturation time, growth, or fecundity. The main advantage of this experiment is that we use a vertebrate with a very fast life-cycle, resulting in time and cost-efficient experiments.These killifish are ideal m...