The overall goal of this experiment is to test the salinity dependent toxicity of chemicals to aquatic organisms. This method can help answer key questions in the Ecotoxicology field, such as assessment of toxicity on the fresh water, brackish water, and sea water conditions. The main advantage of this technique, is that medaka are never asked to test toxicity of chemicals in different salinities.
Additionally, the implication of this technique extend to other therapy or diagnosis of chemicals toxicity. Preparation of commerical silver nanocolloids in solution, at various salinities, is covered in the text protocol. As is medaka culturing and egg collection.
Begin with rinsing six collections of Stage 21 medaka eggs, three times in test solution. Do this using ERMs, at a different concentration for each set of eggs. Now, from each rinsed collection, transfer 15 eggs to a different well of test solution, in a six-well plastic plate.
Set this experiment up in triplicate, washing every group separately. Wrap the plates in foil, and incubate them at 25 degrees Celcius, in the dark. Until the eggs hatch, or for 14 days, whichever comes sooner.
During the incubation, observe the eggs daily for any biological changes. And for any dead eggs. Change the test solution daily, along with making the observations.
On the sixth day of observation, count the heart rate of each embryo over 15 seconds. Also on the sixth day, measure the diameter of each embryo's eyes, in each experimental condition, using a micrometer. Whenever the embryos hatch into larvae, measure their full body lengths, using a micrometer.
Also, over the 14 days, keep count of the total eggs that hatch. To measure silver bioaccumulation, on the sixth day, collect the eggs'chorions, using the standard method. The dechorionated eggs can be analyzed, just as the SNC solution is analyzed, as described in the following section.
To isolate the soluble silver from each SNC solution. first, filter the mixture of colloids and soluble silver out, using a three kilodalton membrane. Then, spin down the loaded filter at 14, 000 Gs, for 10 minutes, in a refrigerated centrifuge.
From 50 microliters of the filtrate, measure the silver concentration by ICP-MS analysis. Load the aliquot into a 50 millileter Teflon beaker, and add two milliliters of ultrapure nitric acid to the beaker. Then, heat the mixture on a hot plate, at 110 degrees Celcius.
Over about three hours of heating, let the solution nearly dry out. Once the beaker has cooled, completely dissolve the organic solids. Add two millilters of ultrapure nitric acid, and 0.5 milliliters of hydrogen peroxide, to the beaker.
Then, heat the mixture again, until it has nearly dried out again. Now, dissolve the remaining residue in four milliliters of one percent ultrapure nitric acid, made in ultrapure water. Transfer the solution to a 15 milliliter centrifuge tube.
Repeat the process of dissolving the remaining solids in the Teflon beaker, two more times, cooling the three collections into a single tube. Now, measure the silver concentration in the sample, using ICP-MS. Use the manufacturer's recommended protocol to analyze the samples.
Be sure to use internal and external standards. The effect of salinity on SNC toxicity was easily apparent. The induction of deformity or death was clearly salinity-dependent.
Various physiological measurements were made. Heart rates were unaffected until the salinity reached 30 X ERM. Body length was significantly shorter at salinities of 15 and 30 X ERM.
Eye diameter dropped as well, consistent with body length. Hatching was less likely at 15 X ERM, and only occurred for two percent of eggs in 30 X ERM. Under the experimental solutions, water soluble metal complexes formed more readily as the salt concentration increased.
Examination of the metal complexes in the embryos after six days showed that they increased in concentration in the embryos as they increased in concentration in the solution. While attempting this procedure, it's important to remember to adjust the pH of the test conditions, because silver ion release is facilitated by acidic condition.
