Laboratory studies were conducted at the University of Maryland Aquatic Pathobiology Center and at the North Carolina State University Center for Applied Aquatic Ecology, in coordination with the US Army, to test the biomonitoring system under controlled conditions. Several studies were conducted with bluegill sunfish (at the UM Aquatic Pathobiology Center) and with Tilapia hybrids (at NCSU) using the same biomonitoring hardware as used in the field studies on the Chicamacomico River.
These studies included a control baseline exposure, an exposure to minor temperature fluctuations, exposure to hypoxia, and exposure to a model harmful algal bloom toxin, brevetoxin. Six to eight fish were exposed in each study. The data provided are averages of 6-8 fish. The endpoints of the biomonitoring studies included ventilatory rate (number of respirations per minute; ventilatory depth (the force of each "breath" indicated as a voltage signal); cough rate (the number of forceful respiratory movements, or coughs, per minute; and Movement (biological signals indicating movement of the whole fish within the chamber). Brevetoxin-exposed fish were also examined for altered neurologic function in the central nervous system. Data from these studies may be seen by clicking on the corresponding links below:
Control Baseline Study. Fish were maintained within the exposure chambers for over 250 hours. Although there are some minor fluctuations in ventilatory rate and ventilatory depth, the fish did not exhibit any "out of control" behavior, as might set of a biomonitoring system alarm.
Temperature Fluctuation Study. The temperature
fluctuation study collected chamber data for over 120 hours. We used a
water delivery system that caused daily 4†C changes in water temperature
(from 19†C rising slowly to 24†C, then relatively more abruptly
back down to 19†C). With each daily rise in water temperature there
was an associated minor elevation in ventilatory rate and depression in
ventilatory depth. Cough rate and %Movement did not appear notably effected.
Note the major ventilatory rate and ventilatory depth spikes at 98 hours;
this was caused by one of the investigators simply walking into the exposure
Hypoxia Studies. Hypoxia was chosen as a stressor since it is a common cause of aquatic animal stress and fish kills along the U.S. Atlantic coast, and it is relatively easy to experimentally execute. The experimental design utilized 7 out of 8 chambers of the system for fish exposure and collection of biological data. The 8th chamber was used for monitoring dissolved oxygen, pH and ammonia. The figure below exemplifies a reduction of dissolved oxygen over several hours. Note the inverse relationship between dissolved oxygen in the water and the ventilatory response of the fish, and that this response is rapidly reversible.
We induced 5 discrete dissolved oxygen (DO) depressions (i.e., hypoxic events) at 98, 117, 162, 235 and 288 hours after test initiation. This was accomplished by stopping water flow to the exposure chambers. Reduction in DO, therefore, was caused by the biological oxygen demand of the fish respiring in the chambers. DO concentrations in the exposure chambers were reduced by approximately 50% while ammonia and pH remained within acceptable limits. Biological responses (see immediately below) indicated significant elevations in ventilatory rates with minor associated depressions in ventilatory depth and elevations in cough rate. In a field application these discrete responses would have triggered an alarm status from the system's computer. Note that the stress of up to 60% reduction in DO appears to cause reversible responses in the parameters measured in this study.
Brevetoxin Studies. A 60 minute brevetoxin
(PbTx2) exposure was conducted after 4 days of baseline acclimation at
Neurotoxicology Studies. This aspect of the project was done in collaboration with the Program for Human Health and the Environment at the University of Maryland School of Medicine, with support from the US EPA STAR Program. We developed a novel system to better understand the mechanisms of environmental neurotoxins and to evaluate brevetoxin as a model biotoxin. This was accomplished by detecting radiolabeled 2-deoxyglucose, a non-metabolizable form of glucose (glucose is an important energy source used by brain cells). Control and brevetoxin-exposed fish were exposed to 2-deoxyglucose and then were humanely put to sleep. The brains of these fish were examined for the radiolabel of the alternate form of glucose. The figure below shows three bluegill brain cross sections. The image on the left and in the middle are control fish (no brevetoxin). The image on the right is from a fish exposed to brevetoxin. These images indicate that there is some alteration of energy utilization in brevetoxin-exposed fish. By analyzing the areas of the brain that are most affected, we can better understand the mechanism by which various neurotoxins act.
Exposure to Pfiesteria cultures. The graph below illustrates the response of tilapia in the fish biomonitoring system to toxic Pfiesteria cultures. These laboratory efforts were conducted at the NCSU Center for Applied Aquatic Ecology directed by Dr. JoAnn Burkholder and Dr. Howard Glasgow. The green line represents the biological response during times when the group of 8 fish did not show significant stress. When the line turns yellow, "significant stress" is occurring with at least 6 out of the 8 fish in the group responding above a threshold level. The red line is a subset of the yellow line that indicates the number of fish that are "severely stressed" or dead. The data show a rapid alarm response to Pfiesteria (6 or more of 8 fish showing stress) approximately two hours after test initiation (blue triangle). A brief drop-off in response is followed by a second alarm that initiated approximately 8 hours after the test started and that continued for the duration of the exposure.