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

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.