The overall goal of the following experiment is to estimate net trophic transfer efficiencies of PCB congeners to lake trout from its prey, and then determine if the degree of chlorination or the degree of lipid solubility of the PCB Congener has an effect on its net trophic transfer efficiency. This is achieved by first conducting a laboratory experiment in which lake trout are fed a natural food such as blo over a period of at least four months. As a second step extraction and cleanup is used to extract the PCBs from the lake trout and blo tissues and prepare the extracts for the quantification procedure.
Next gas chromatography mass spectrometry using negative chemical ionization is used in order to determine the PCB congener concentrations in the fish tissues. The results show that net trophic transfer efficiencies of PCB congeners to lake trout from its prey are not significantly affected by the degree of chlorination of the PCB congeners. The results also show that the lake trout activity does not appear to have a significant effect on net trophic transfer efficiency.
The main advantage of this technique over existing methods like injection of the contaminant directly into the SIVs fish or into the food of the SIVs fish, is that the SS fish accumulates the contaminant in a manner that best mimics the process of contaminant accumulation in the ferous fish in its natural setting. Visual demonstration of this method is critical as the concentration and extraction steps require great care to be performed correctly. These steps are best learned through visual observation.
Demonstrating this procedure will be Jim O'Keefe, a chemist from my laboratory. First thaw an appropriate amount of prey fish previously stored in a negative 30 degree Celsius freezer. Cut the thawed prey fish into pieces weighing roughly one to five grams using a chef knife.
Following this, weigh the quantity of prey fish to be placed in each of the tanks and drop the pieces into each tank. After allowing the predator fish to feed for about one hour, remove the ate food and allow it to air dry for about 20 minutes. Once the une food has been weighed, record the amount of food placed in each tank and the amount of ate food for each of the tanks.
After sacrificing and freezing the predator fish, select a set of predator, fish and or prey fish composites for thawing, and allow the composites to partially thaw using the appropriate sized homogenize. Each of the composites. For each composite place a 50 to 100 gram sample of the homogenate into a cleaned, acetone, rinsed, and labeled jar.
After capping the jar, store it at negative 30 degrees Celsius until the time of processing for the extraction. Weigh 20 grams of thawed homogenized fish tissue in a 200 milliliter beaker, then at approximately 40 grams of sodium sulfate and mix well with a spatula. Add a surrogate spike solution containing CONGENERS 30 61, 161, and 166 at a concentration that yields a final concentration of 20 nanograms per milliliter.
In the extract. Allow the sample to dry at room temperature while mixing Every 20 minutes after the sample has reached a consistency of dry sand. Set up a soli extraction apparatus with a 500 milliliter flask containing Teflon boil chips, a sock, sl, and a condenser.
Then add the dried fish mixture to a glass thimble with a coarse fritted disc bottom. Add 150 milliliters of a premixed solution of 50%hexane and 50%chloro methane to the beaker used for the sample and stir while scraping the walls of the beaker. With a spatula, transfer the solvent to the top of the solit, allowing it to cycle through the solit and into the flask.
After repeating the previous step, place the sock lit with the attached flask onto a heating element and attach a condenser. Next turn on the heating element, bringing the solvent to a gentle boil. Then extract the solvent for a minimum of 16 hours, making sure that cold water is supplied to the condensers.
Once the solvent has cooled, check to see if any of the sample flasks contain water. For the flasks containing water, add sodium sulfate and swirl until the water is absorbed. Following this, concentrate the sample using a nitrogen sample concentrator.
After the sample has a volume of less than two milliliters, transfer the sample to a five milliliter volumetric flask. Then bring the final volume to five milliliters by using five to seven small washings of hexane to transfer the residual sample from the previous glassware to the volumetric flask. At this point, transfer the sample to a 10 milliliter vial and label it with the sample information.
Prepare acidified silica gel by adding 44 grams of concentrated sulfuric acid to 100 grams of activated silica gel. Then add 10 grams of the acidified silica gel into a small chromatography column containing a small plug of glass wool at the bottom. After pre-cleaning the column with 10 milliliters of hexane, add one milliliter of the sample extract to it, elute the column with 20 milliliters of hexane and collect the sample in a tapered 20 milliliter glass tube.
Next, place the glass tube on a nitrogen evaporator or NVAP apparatus under a stream of nitrogen and immerse it in hot water. Once the sample has been concentrated to less than one milliliter, transfer it to a one milliliter volumetric flask. Then bring the final volume to one milliliter by using two to three small washings of hexane to transfer the residual sample from the tube to the volumetric flask.
Following this, transfer the sample to a 1.8 milliliter auto sampler vial labeled with the sample information. Add four microliters of the appropriate internal standard, which in this case is deca chloro fennel to the vial. You use the appropriate standards to calibrate the instrument.
Then set up the chromatography mass spectrometry system in the negative chemical ionization mode with hydrogen as the carrier gas at one milliliter per minute and methane as the reagent gas. Use a fused silica capillary column coated with DB XLB at 0.25. Micrometer film thickness for separation.
Inject one to two microliters of the sample using the split injection mode. At this point, analyze all the standards and samples by the internal standard method using carbon 13 labeled DECA chloro b fennel. Perform a check on the initial calibration by running a second source standard and arrow Chlor 1242 and 1260, and then compare predicted values for the arrow chlor congeners with the observed amounts from this check procedure.
Once the initial calibration procedure has been successfully accomplished, complete the analysis of all the samples. Run a calibration check every 10 samples using any of the calibration mixtures from the initial calibration. Lake Tre trout showed substantial growth as the initial lake trout.
Mean weights ranged from 694 to 907 grams while the final lake trout mean weights ranged from 853 to 1, 566 grams. Mean PCB Congener concentrations in the lake trout increased during the experiment for all of the PCB congeners. The mean net trophic transfer efficiency for the active lake trout did not significantly differ from the inactive lake trout.
Active lake trout retained the PCB congeners from the food that they had consumed with nearly the same efficiency as inactive lake trout for 66 of the 75 PCB congeners. The standard error about the mean estimate of net trophic transfer efficiency was small for six of the nine other PCB congeners. The standard errors about the mean estimate of net trophic transfer efficiency were fairly low as the degree of chlorination increased estimates of the net trophic transfer efficiency showed a slight decrease.
However, the net trophic transfer efficiency did not vary significantly with degree of chlorination of the PCB congeners AS log KOW increased, the net trophic transfer efficiency declined exponentially. This rate of decline was significantly different from zero, but was equal to 7%per unit of log KOW. After watching this video, you should have a good understanding of how to estimate net trophic transfer efficiencies of CB congeners to ous fish from their prey using a laboratory experiment in which the ous fish is fed a natural food.
Don't forget that working with organic solvents such as hexane and di chloro methane can be hazardous and precautions such as proper ventilation should always be taken when performing this procedure.
