WI DNR Field Procedures Manual
Intranet Edition

Scope

In situ bioaccumulation tests are used to assess if sediment borne contaminants are potentially bioavailable to aquatic biota under the conditions in the field. An in situ test bypasses the problem of possibly skewed results caused by sediment manipulation during collection and laboratory testing. Disrupting the physical and chemical integrity of the sediment can change the bioavailability of contaminants. In situ tests cannot, however, control for biouptake of contaminants that might come from water or food, or other factors such as temperature that might affect bioavailability and bioaccumulation.

Many different methods can be used to test for bioaccumulation in situ. This section offers one method utilizing caged fathead minnows, and should provide the first-time field person with a guide to plan ahead and avoid many pitfalls that might compromise the quality of the study results. With common sense and an understanding of the principles, this method can be modified to fit different sites and study objectives. Variations include the use of other fish or invertebrate species and suspending the cages in the water column rather than resting on the sediment.

Equipment

Method

1. Preparation and Considerations

   1. The most commonly used exposure times used for bioaccumulation in fathead minnows are 10 and 28 (or 30 days) from the time of placement to the time of collection. For most bioavailable contaminants, equilibrium will not be achieved by 30 days, so tissue concentrations will increase throughout the test period.
   2. Twenty grams (wet weight) of fish tissue are needed for sample to be analyzed for chemical contaminants. Most adult fathead minnows weigh 1-3 grams. At least twice as many fish as are needed should be ordered for each site. This leaves some room for fish mortality.
   3. A source of uncontaminated, healthy fish is essential to the ability to discern significant biouptake of contaminants, especially if the sediment contaminant levels are moderate. Finding a reliable source of uncontaminated fish can be difficult, but is necessary. The concentration of PCBs in fish from a single hatchery can change throughout the year. PCBs have also been detected in laboratory raised fish and fish food used at hatcheries. Contamination with metals (esp. mercury) has occurred during a two week holding period while being fed frozen brine shrimp that itself contained mercury. The level of mercury in these fish was significant and possibly occluded or shadowed any bioaccumulation of mercury that might have originated in the sediment.
   4. When planning the analyses and budget, it is a requirement that enough analysis dollars to analyze "blank" (control samples, time = 0) and reference site tissue samples be available. Without a reliable "blank" sample, there is no way to know if fish tissue concentrations increase during the testing period from the time of placement to (time=0) to the time of collection (usually time=10 or 28 days). Fish tissue should be collected for analysis immediately before the rest of the fish are placed at the sediment sites to determine the pre-exposure contaminant concentrations. Remember that any other containers or food introduced to the fish can be a source of contamination.
   5. Make sure enough minnow cages are available for the sampling period.
   6. Make sure the cages are sufficiently weighted or anchored to remain in position during rough water or currents.
   7. Arrange for the most convenient time and place for the delivery of the fish to reduce the necessary holding time and stress. A method to keep fish cool and the water aerated will reduce fish stress and loss.
   8. A placement and sampling plan including quality control measures should be written down to avoid pitfalls and unreliable data.
   9. Choose sites containing soft sediments. Unless the study objectives specifically dictate otherwise, sites for in situ bioaccumulation tests should contain fairly soft sediments to allow the fish to interact directly with the sediment through the mesh of the cage. Bioaccumulative contaminants are most often associated with fine sediments, and unless they partition readily into the water column, must make direct contact with the test organism to be bioaccumulated.
   10. Choose sites where there will be adequate water to completely submerge the cages even in low flow. Also, some sites in silty rivers or streams may lead to a mortal buildup of silt in and around the cage.
   11. Sites should not be located near a known toxics discharger if a measure of bioaccumulation from this point source is not desired.
2. Placing Cages

   1. If direct interaction between the fish and the sediment is desired, cages should be placed on a soft sediment deposit rather than hard sand or rubble.
   2. Anchor the boat so that the sediment where the cages will be is not disturbed, and the boat is directly over the cage sites.
   3. Upon arrival at the site, begin acclimating fish to the site water (both chemically and thermally) by adding site water every few minutes to the fish, until they are in at least 50% site water. The more difference there is between the fish water and site water temperatures, the more gradual should be the process.
   4. Fill a tub large enough to hold a cage with water, and place a cage in it, so that the fish will be in water while the cage is being filled.
   5. When the fish are acclimated to the site water, fill the cage with the appropriate number of fish, and secure the lid with cable ties or wire.
   6. Record the number of fish placed and any observations.
   7. Attach sufficient line to the cage and lower to the bottom gently so that the bottom of the cage or one full side is lying directly on the bottom. The line can be attached to a stationary object or to a buoy for retrieval of the cages. Leave enough extra line from the cages to the surface float so wind and wave action will not move the cages. A separate anchor for the buoy with a line slightly shorter than the cage lines should help prevent this.
   8. Repeat with any addition cages for that site. Replicate cages should be at least a few feet apart from each other and should not affect the flow of water around the other cages.
3. Collecting Fish

   1. Be aware of all possible routes of sample contamination especially during the collection procedures. All equipment and gloved or bare hands that touch the fish should be totally clean. Be aware of a possible petroleum slick from the boat motor; approach site and anchor boat so motor is downwind and/or downstream from the cages and work area. Ideally, the person operating the motor should not contact the sampling equipment or fish unless some cleansing procedure is used. All equipment should be washed or very well rinsed between sites, especially if some sites are obviously contaminated or an oil sheen develops when the sediment is disturbed.
   2. Take and record any field measurements before raising the cages and disturbing the site.
   3. Raise a cage and place into a tub of clean, fresh (same temperature) site water. If subsampling and some fish will remain at the site until a later time, fish should be held only as long as necessary to minimize stress.
   4. Make observations about the general health or appearance of the fish and the number dead and alive and record. The overall well-being of the fish at a site as well as mortality may be a clue to some toxicity.
   5. If subsampling, count and collect the necessary live fish in as random fashion as possible with a clean net, and place the fish in fresh aluminum foil and double wrap. Dead fish and dropped fish should not be included as part of the sample. Remember to leave enough fish for the final or subsequent samples with the possibility of some additional mortality.
   6. Tape the package closed and label appropriately: Site ID, contents (# and species), date, collector's name, analysis type (inorganic or organic).
   7. Place packaged fish on dry ice as soon as possible to freeze and preserve fish for later analysis. Fish tissue must be kept frozen until delivered to the lab for analysis.

Documentation

1. Laboratory Sheets - Laboratory form #'s 3200-82 and 3200-83 are specifically for fish tissue and must be delivered with the fish tissue samples to the State Lab of Hygiene. Record and keep a copy of sample information (number of samples, sites, field IDs, blanks, etc.) in addition to the lab sheets that will be sent with the samples to the lab. The lab sheets will not be sent back with the data, and the information will aid in tracking samples at the lab to see if analyses have been completed or not. This information will be keyed into the new Fish/Sediment Contaminant data base.
2. Field Measurements - It is important to measure and record parameters at each site that can affect bioaccumulation at the site. These might include: water temperature near the sediment, dissolved oxygen near the sediment, pH, water depth, conductivity, light attenuation, current velocity. Knowing these parameter can provide further information along with the data about the potential for bioaccumulation in situ. Also refer to section 701.3 Field Observations and Measurements.

References

Ankley, G.T. et al. 1992. Bioaccumulation of PCBs from sediments by oligochaetes and fishes: comparison of laboratory and field studies. Can. J. Fish. Aquat. Sci. 49:2080-2085.

Brunson, E.L., G.T. Ankley, G.A. Burton, F.J. Dwyer, C.G. Ingersoll, P.F. Landrum, H. Lee II, and G.L. Phipps. 1994 (In preparation). Bioaccumulation Kinetics and field validation of whole-sediment exposures with the oligochaete Lumbriculus variegaetus.

EPA. 1989. Guidance manual: bedded sediment bioaccumulation tests. ERL-N Pacific Ecosystems Branch. EPA/600/x-89/302.

Mac, M.J. and C.J. Schmitt. 1992. Sediment bioaccumulation testing with fish. In Allen B.G. (Ed.), Sediment Toxicity Assessment. Lewis Publishers. Boca Raton, Florida. pp 295-312.