Name: quantifying fish swimming behavior in response to acute exposure of aqueous copper using computer assisted video and digital image analysis
Uploaded: 2016-02-26T13:00:00
Duration: 16 min 21 s
Description: Watch this Scientific Journal Video about Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis at JoVE.com

We thank the staff in the Ecology Branch, Toxicology Branch and Environmental Chemistry Branch of the U.S. Geological Survey, Columbia Environmental Research Center for technical and analytical assistance. Funding provided in part by Teck American Incorporated through an agreement with the US Environmental Protection Agency (USEPA) Region 10 with funds provided by USEPA to US Geological Survey (USGS) through the Department of Interior Central Hazmat Fund.

1. Setup Diluter for Larval Sturgeon Exposure to Aqueous Copper Concentrations
<ol>
	<li>Set up exposures using a flow-through system such as a modified Mount and Brungs12 diluter system following guidelines by the ASTM International13,14,15.</li>
	<li>Select 25 &#181;g/L as the high copper concentration based on previous test results where effects were observed around 4-6 &#181;g/L. Use reagent grade copper II sulfate pentahydrate (&#62;98% purity) and mix up a test stock solution. 
	NOTE: Example concentration series for the exposures are 25, 12.5, 6.25, 3.125, 1.0625, and 0 &#181;g/L. Using 50% serial dilutions starting with 25 &#181;g/L as the high concentration will target a range of concentrations observed to cause behavioral impairment.</li>
	<li>Prepare the test stock solution in a volumetric flask 48 hours prior to the initiation of the exposure and set to deliver to spike the diluter mixing chamber using an automated syringe dispenser.</li>
	<li>Use a spreadsheet template to determine the weight of chemical to be added to 1 L of deionized water that will result in 25 &#181;g/L copper concentration, when diluter mixing chamber is spiked with 1 ml of the test stock solution. 
	NOTE: Figure 1 depicts an example of a spreadsheet template for chemical stock preparation for diluter toxicity tests.</li>
	<li>Weigh out 195 mg of reagent grade copper II sulfate pentahydrate using an analytical balance and pour into a 1 L volumetric flask and mix with 1 L of deionized water for a stock concentration of 48.65 mg/L.</li>
	<li>Place the intake tube from the automated syringe dispenser into the test stock solution and set the spike volume to 1 ml and turn the diluter on by flipping the power switch and let cycle for 48 hr allow it to equilibrate to the corresponding copper concentration prior to stocking sturgeon.</li>
	<li>Attach an in-line 4-way flow splitter16 to each delivery line to partition water flow to each of the four replicate exposure chambers in the diluter water bath. Gravity feed the water. Cycle the water through the diluter when the power to the diluter turns on and a solenoid valve is opened allowing water to flow to the dilution tanks. Set chiller unit to 15 &#176;C and turn on water pump to circulate water in the diluter water bath. 
	NOTE: This process is regulated by the use of an automated timer.</li>
	<li>Set the diluter to cycle every 30 min using the automated timer and to deliver 250 ml of test water with each cycle, resulting in 12 volume additions per day to each replicate test chamber.</li>
	<li>Select exposure test chambers based on the size of the sturgeon to maintain acceptable loading rate which is &#60;10 g fish wet weight/L in any chamber at any given time. For example, to conduct exposures with 30 day-post-hatch (dph) white sturgeon (average weight in grams 0.17 g) use 12 x 21.5 cm2 glass jars with a 4 cm hole in the side. Cover this side with a 30 micron mesh stainless steel screen to allow for flow through of the test water. The volume of test water in the exposure jars is 1 L.</li>
	<li>Using a 50 ml plastic syringe take two replicate 50 ml water samples at each concentration for a total of 12 samples and dispense test water into 100 ml glass beakers and measure dissolved oxygen (DO), temperature, conductivity, pH, alkalinity, hardness, total ammonia, major cations, major anions and dissolved organic carbon using standard commercial equipment and follow the manufacturer&#39;s instructions. 
	NOTE: samples should be taken at the initiation and end of exposure.</li>
	<li>To collect sub-samples for chemical analysis, use a 25 ml plastic syringe, draw up approximately 24 ml of test water from the exposure chambers using a sipper straw attached to the syringe instead of a needle.</li>
	<li>Remove the sipper straw from the syringe and place a polypropylene filter cartridge housing a 0.45 &#181;m pore size, polyethersulfone membrane on the plastic syringe.</li>
	<li>Push 4 ml of test water through the filter and dispose.</li>
	<li>Dispense the remaining 20 ml of test water through the filter into an acid-cleaned polyethylene bottle and acidify to 1% volume/volume with high-purity, 16 M nitric acid for storage of up to 3 months. 
	NOTE: Samples for chemical analysis should be taken at the initiation, middle and end of exposure to confirm copper concentrations.</li>
	<li>Perform chemical analysis using inductively coupled plasma mass spectrometry following U.S. Environmental Protection Agency Method 6020a17</li>
	<li>After taking all water samples and the diluter is cycling, stock 10 (random, haphazardly) sturgeon in each replicate test chamber. Collect the sturgeon from the culture tank where they are housed using a small mesh non-abrasive net. Place sturgeon in a small bucket with culture water. A total of 240 fish are needed to start the exposure.</li>
	<li>Do not feed the fish during the exposure. 
	NOTE: Please refer to Figure 1 for a visual setup of the diluter layout.</li>
	<li>Read the test every day for the duration of the exposure and record fish mortality and monitor swimming behavior. 
	NOTE: Other endpoints to look for include lethargy, loss of equilibrium, changes in respiration, changes in pigmentation, position fish are in the water column, hiding activity and any other abnormalities that can be visually identified. 
	NOTE: Read the test at the same time each day for consistency.</li>
	<li>Measure and quantify swimming activity (time fish spent moving, velocity and the distance moved) using a commercially available digital tracking software program.</li>
</ol>
2. Observations and Mortality Counts During Exposure
<ol>
	<li>Visually inspect each test chamber and note mortality and observations of abnormal behavior using a behavioral checklist (Table 1) on the datasheets approximately at the same time daily during the 96 hr exposure, preferably in the morning. 
	NOTE: Behaviors that are strikingly, uncharacteristically, subjectively, qualitatively different from the controls are considered abnormal. Optimally the observer is unaware of the treatments. 
	NOTE: The loss of equilibrium is defined as the inability of fish to maintain an upright position within the water column and immobility is defined as the inability of fish to move or swim unless prodded. Other abnormalities such as lethargy, hyperactivity, increases or decreases in respiration, color changes, tremors, spasms, bloated abdomens, position in the water column and any other unusual swimming patterns should also be recorded on the data sheet. 
	NOTE: Refer to Video 1 for examples of abnormal behavior.</li>
	<li>Record and remove dead sturgeon daily.</li>
	<li>Using a hand held Dissolved Oxygen (DO) meter with probe measure the dissolved oxygen in situ and record water temperature in two replicates of each exposure concentration and record on the data sheet.</li>
</ol>
3. Videotaping Swimming Activity
<ol>
	<li>Capture video data samples using a hand-held video camera mounted to a tripod positioned directly overhead of the testing chamber to document behavioral abnormalities.</li>
	<li>To quantify swimming activity, cut a piece of PVC pipe 13 cm in diameter and 13 cm tall to use as the test arena (Figure 1). Place the PVC pipe in the diluter within each corresponding copper concentration exposure tanks. Use the area within the PVC pipe as the test arena as this is large enough for sturgeon to swim about freely.</li>
	<li>At the end of the 96 hr exposure, randomly subsample 5 surviving sturgeon from each copper concentration to measure for swimming activity and place them in to the test arena using a small mesh net. 
	NOTE: In the higher test concentrations where sturgeon mortality was prevalent, any remaining surviving sturgeon should be used to measure swimming activity and in some cases might be less than 5.</li>
	<li>After placing the fish in to the test arena, allow the fish to acclimate for a period of 30 min. 
	NOTE: Successful, error free analysis of the video requires a high contrast image of the fish against a background with a minimum of structure that might obscure or hide the image of the fish. The image of the fish must be in good focus and must be free of surface glare or free of distortions due to moving water so the diluter system must be turned off.</li>
	<li>After 30 min, turn the video camera ON and set to REC to record the swimming activity for a period of 2 min.</li>
	<li>Euthanize the fish after taking the video recordings to determine behavior.</li>
	<li>Place sturgeon in a concentrated solution of tricaine methanesulfonate (MS222) water for at least 10 min to allow for cessation of opercular movement. 
	NOTE: A concentration of at least 250 mg/L is recommended and might be much higher for certain species.</li>
	<li>Place the euthanized sturgeon in a plastic zip lock bag and place in the freezer for disposal at a later time. Turn the video camera OFF and transfer all video files to a computer for post processing using digital tracking software.</li>
</ol>
4. Measures of Swimming Activity from Video Playback
<ol>
	<li>Locate the data sheet and video data files for the experiment to be analyzed. Convert video files to a compatible format the digital analysis software can handle.</li>
	<li>Upload all files to be processed into software. Open tracking software by clicking on the icon. Click on the &#34;New default experiment&#34; under the &#34;Create a new experiment&#34; option on main screen.</li>
	<li>Enter name for experiment in &#34;New Experiment&#34; dialog box that appears on screen. Choose location experiment file is to be saved. Click &#34;OK&#34;. Choose &#34;Experiment Settings&#34; option under &#34;Setup&#34;.Choose &#34;From video file&#34; under &#34;Video Source&#34;.</li>
	<li>Select &#34;1&#34; for &#34;Number of arenas&#34;. Select &#34;3&#34; for &#34;Number of subjects per arena&#34;. Select &#34;Center-point detection&#34; under &#34;Tracked Features&#34;. Select desired units.</li>
	<li>Select &#34;Trial list&#34; option under &#34;Setup&#34;. Click &#34;Add Videos&#34; at top of screen. Choose &#34;Alphabetical order&#34; under &#34;Sort order&#34; option on &#34;Add Videos&#34; dialog box that appears on screen. Click &#34;Browse&#34;. Navigate to folder where video files are located.</li>
	<li>Highlight all video files. Click &#34;Open&#34;. Click &#34;Add Variable&#34; at top of screen. Enter &#34;Concentration&#34; into &#34;Label&#34; box. Enter &#34;Copper concentration in micrograms/L&#34; into &#34;Description&#34; box.</li>
	<li>Choose &#34;Numerical&#34; from drop down list for &#34;Type&#34;. Click on &#34;Predefined Values&#34; box. Choose &#34;Define Individual values&#34; option in the &#34;Predefine Numerical Values&#34; dialog box that appears.</li>
	<li>Enter &#34;0&#34;, &#34;3&#34;, &#34;6&#34;, &#34;13&#34;, &#34;25&#34;, and &#34;50&#34; in &#34;Predefined Value&#34; space. Click &#34;Add &#62;&#62;&#34; between each number addition. Uncheck &#34;Allow other values&#34; option. Click &#34;OK&#34;.</li>
	<li>Choose &#34;Trial&#34; from drop down menu in &#34;Scope&#34; box. Enter appropriate concentration for each trial in boxes provided. Click &#34;Add Variable&#34; at top of screen. Enter &#34;Replicate&#34; into &#34;Label&#34; box. Enter &#34;Replicate number&#34; into &#34;Description&#34; box. Choose &#34;Numerical&#34; from drop down list for &#34;Type&#34;.</li>
	<li>Click on &#34;Predefined Values&#34; box. Choose &#34;Define Individual values&#34; option in the &#34;Predefine Numerical Values&#34; dialog box. Enter &#34;1&#34;, &#34;2&#34;, &#34;3&#34; and &#34;4&#34; in &#34;Predefined Value&#34; space. Click &#34;Add &#62;&#62;&#34; between each number addition. Uncheck &#34;Allow other values&#34; option.Click &#34;OK&#34;.</li>
	<li>Choose &#34;Trial&#34; from drop down menu in &#34;Scope&#34; box. Enter appropriate replicate number for each trial in boxes provided. Choose &#34;Arena Settings&#34; under &#34;Setup&#34; tab at top of screen. Name first setting &#34;Trial 1&#34;. Click &#34;Grab Background Image&#34; option from &#34;Arena Settings (Trial 1)&#34; dialog box.</li>
	<li>Click &#34;Browse&#34; on the &#34;Grab Background Image&#34; dialog box. Locate video file for Trial 1 and click &#34;Open&#34;. Click &#34;Grab&#34; option in the &#34;Grab Background Image&#34; dialog box after video appears. Click the white circle icon near top of screen under &#34;Arena Settings&#34;.</li>
	<li>Manipulate circle that appears so that entire swimming area is enclosed in the circle. Hatch marks appear where the arena area is defined. Click &#34;Calibration Scale&#34; icon near top of screen under &#34;Arena Settings&#34;. Left click on one edge of arena. Hold and drag mouse over to opposite end of arena. Release left click.</li>
	<li>Enter &#34;10.5&#34; in &#34;Real world distance&#34; box in the &#34;Calibration Distance&#34; dialogue box that appears. Click &#34;OK&#34;. If necessary, adjust calibration line so that it spans the entire diameter of circular arena.</li>
	<li>Click &#34;Validate Arena Settings&#34; option on &#34;Arena Settings (Trial 1)&#34; dialogue box. Address any issues if settings are not validated. Right click &#34;Arena Settings&#34; under &#34;Setup&#34; option from the &#34;Experiment Explorer&#34; toolbar on left of screen and choose &#34;New&#34; from menu.</li>
	<li>Repeat steps 4.11-4.15 until arena settings have been created for each trial. Be sure to choose the appropriate video file for each trial. Choose &#34;Detection Settings&#34; under &#34;Setup&#34; option from the &#34;Experiment Explorer&#34; toolbar on left of screen.</li>
	<li>Choose &#34;Dynamic Subtraction&#34; from dropdown menu under &#34;Method&#34; in the &#34;Detection Settings: Detection Settings 1&#34; dialogue box that appears.Choose different fill colors for each subject under &#34;Subject Identification&#34; in the &#34;Detection Settings: Detection Settings 1&#34; dialogue box.</li>
	<li>Choose &#34;Select Video&#34; and locate video for Trial 1.Click &#34;Open&#34;.Select &#34;5.9941&#34; from &#34;Sample rate&#34; box under &#34;Video&#34; in the &#34;Detection Settings: Detection Settings 1&#34; dialogue box. Click &#34;Settings&#34; for the &#34;Reference image&#34; option under &#34;Detection&#34; in the &#34;Detection Settings: Detection Settings 1&#34; dialogue box.</li>
	<li>Click &#34;Start Learning (C)&#34; option in the &#34;Reference Image&#34; dialogue box.Wait for program to learn reference image. Once the image in &#34;Reference Image&#34; dialogue box appears without animals, click &#34;Use dynamic reference image&#34; under &#34;Acquisition settings&#34; within the dialogue box.</li>
	<li>Click &#34;Close&#34;. Choose &#34;Darker&#34; from the drop down menu for &#34;Subject is&#34; under &#34;Detection&#34; in the &#34;Detection Settings: Detection Settings 1&#34; dialogue box. Set smaller number to &#34;33&#34; and larger number to &#34;153&#34; for &#34;Dark contrast&#34; under &#34;Detection&#34; &#34;Detection Settings: Detection Settings 1&#34; dialogue box.</li>
	<li>Click &#34;Save Changes&#34; at bottom right of &#34;Detection Settings: Detection Settings 1&#34; dialogue box. Click the play button on the &#34;Playback Control&#34; dialogue box and confirm that software is successfully tracking animals as opposed to shadows or debris. Adjust numbers for &#34;Dark contrast&#34; as necessary.</li>
	<li>Once tracking is appropriate, click &#34;Save Changes&#34; at bottom right of &#34;Detection Settings: Detection Settings 1&#34; dialogue box. Choose &#34;Acquisition&#34; under &#34;Setup&#34; option from the &#34;Experiment Explorer&#34; toolbar on left of screen.</li>
	<li>Click &#34;Track next planned trial&#34; in the &#34;Acquisition Settings&#34; dialogue box. Confirm the correct trial, video and arena setting is displayed under &#34;Settings&#34; in the &#34;Acquisition Settings&#34; dialogue box.</li>
	<li>Check the &#34;Detection determines speed&#34; option in the &#34;Acquisition Control&#34; dialogue box. Click the button with the green circle enclosed in a white square to begin the acquisition process. Repeat steps 4.22-4.23 until all trials have been tracked.</li>
	<li>Click &#34;Data Profiles&#34; under &#34;Analysis&#34; option from the &#34;Experiment Explorer&#34; toolbar on left of screen.Choose &#34;Time&#34; under &#34;Nesting&#34; option in the &#34;Components&#34; toolbar. Adjust &#34;To&#34; to &#34;0:02:00&#34; under the &#34;Select track time interval&#34; heading in the &#34;Time&#34; dialogue box. Click &#34;OK&#34;.</li>
	<li>Drag the &#34;Nest&#34; box between the &#34;Start&#34; box and the &#34;Result 1&#34; box in the &#34;Data Profiles&#34; area on the right side of the screen. Click &#34;Analysis Profile&#34; under the &#34;Analysis&#34; option from the &#34;Experiment Explorer&#34; toolbar on left of screen. Click &#34;Velocity&#34; under the &#34;Distance and Time&#34; heading in the &#34;Dependent Variables&#34; toolbar that appears.</li>
	<li>Click &#34;Add&#34; on the &#34;Velocity&#34; dialogue box. Click &#34;Distance moved&#34; under the &#34;Distance and Time&#34; heading in the &#34;Dependent Variables&#34; toolbar. Click &#34;Add&#34; on the &#34;Distance moved&#34; dialogue box. Click &#34;Movement&#34; under the &#34;Individual Behavior&#34; heading in the &#34;Dependent Variables&#34; toolbar.</li>
	<li>Adjust &#34;Averaging interval&#34; to &#34;1&#34; under the &#34;Outlier filter&#34; heading in the &#34;Movement&#34; dialogue box. Adjust &#34;Start velocity&#34; to &#34;2.00&#34; and &#34;Stop velocity&#34; to &#34;1.75&#34; under the &#34;Threshold&#34; heading in the &#34;Movement&#34; dialogue box.</li>
	<li>Check both boxes for &#34;Moving&#34; and &#34;Not moving&#34; under the &#34;Calculate statistics for&#34; heading in the &#34;Movement&#34; dialogue box. Click &#34;Add&#34; at the bottom of the &#34;Movement&#34; dialogue box. Click &#34;Analysis Output&#34; under &#34;Results&#34; option in the &#34;Experiment Explorer&#34; toolbar on left of screen. Click &#34;Calculate&#34; at top of screen.</li>
	<li>Once dependent variables are calculated, click &#34;Export&#34; at top of screen. Choose destination folder in &#34;Export Analysis Output&#34; dialogue box. Select &#34;Excel&#34; from &#34;File type&#34; dropdown menu in the &#34;Export Analysis Output&#34; dialogue box. Click &#34;OK&#34;.</li>
	<li>Click &#34;Save Experiment&#34; under the &#34;File&#34; tab at the top of the screen. Close the digital tracking software. Import the data to a spreadsheet file and analyze using a commercial statistical analysis software package.</li>
</ol>

<ol>
	<li>Gerhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+behavioral+ecotoxicology-prospects+and+limitations.">Aquatic behavioral ecotoxicology-prospects and limitations.</a> Hum Ecol Risk Assess: An International Journal. 13 (3), 481-491 (2007).</li><li>Beitinger, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+reactions+for+the+assessment+of+stress+in+fishes.">Behavioral reactions for the assessment of stress in fishes.</a> J Great Lakes Res. 16, 495-528 (1990).</li><li>Beitinger, T. L., McCauley, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-animal+and+physiological+processes+for+the+assessment+of+stress+in+fishes.">Whole-animal and physiological processes for the assessment of stress in fishes.</a> J Great Lakes Res. 16, 542-575 (1990).</li><li>Dell'Omo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Behavioural Ecotoxicology. , (2002).</li><li>Little, E. E., Finger, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+behavior+as+an+indicator+of+sublethal+toxicity+in+fish.">Swimming behavior as an indicator of sublethal toxicity in fish.</a> Environ Toxicol Chem. 9, 13-19 (1990).</li><li>Rand, G. M., Rand, G. M., Petrocelli, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> Fundamentals of Aquatic Toxicology: Methods and Applications. , 221-256 (1985).</li><li>Calfee, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+sensitivity+of+white+sturgeon+(Acipenser+transmontanus)+and+rainbow+trout+(Onchorhynchus+mykiss)+to+copper,+cadmium,+or+zinc+in+water-only+laboratory+exposures.">Acute sensitivity of white sturgeon (Acipenser transmontanus) and rainbow trout (Onchorhynchus mykiss) to copper, cadmium, or zinc in water-only laboratory exposures.</a> Environ Toxicol Chem. 33 (10), 2259-2272 (2014).</li><li>Little, E. E., Calfee, R. D., Linder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+smelter+slag-contaminated+sediments+from+Upper+Lake+Roosevelt+and+associated+metals+to+early+life+stage+White+Sturgeon+(Acipenser+transmontanus+Richardson,+1836).">Toxicity of smelter slag-contaminated sediments from Upper Lake Roosevelt and associated metals to early life stage White Sturgeon (Acipenser transmontanus Richardson, 1836).</a> J Appl Ichthyol. , 1-11 (2014).</li><li>Wang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+sensitivity+of+white+sturgeon+(Acipenser+transmontanus)+and+rainbow+trout+(Onchorhynchus+mykiss)+to+cadmium,+copper,+lead+or+zinc+in+water-only+laboratory+exposures.">Chronic sensitivity of white sturgeon (Acipenser transmontanus) and rainbow trout (Onchorhynchus mykiss) to cadmium, copper, lead or zinc in water-only laboratory exposures.</a> Environ Toxicol Chem. 33 (10), 2246-2258 (2014).</li><li>Little, E. E., Dell'Omo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+measures+of+injuries+to+fish+and+aquatic+organisms:+regulatory+considerations.">Behavioral measures of injuries to fish and aquatic organisms: regulatory considerations.</a> Behavioural Ecotoxicology. , 411-431 (2002).</li><li>Little, E. E., Brewer, S. K., Schlenk, D., Benson, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+toxicity+in+fish.">Neurobehavioral toxicity in fish.</a> Target Organ Toxicity in Marine and Freshwater Teleosts New Perspectives: Toxicology and the Environment. Volume 2. , 139-174 (2001).</li><li>Mount, D. I., Brungs, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+dosing+apparatus+for+fish+toxicological+studies.">A simplified dosing apparatus for fish toxicological studies.</a> Water Res. 1, 21-29 (1967).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+performing+early+life-stage+toxicity+tests+with+fishes.">Standard guide for performing early life-stage toxicity tests with fishes.</a> Annual.Book of ASTM International Standards. Volume 11.06. , 1241-1305 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+measurement+of+behavior+during+fish+toxicity+tests.">Standard guide for measurement of behavior during fish toxicity tests.</a> Annual.Book of ASTM Standards. Volume 11.06. , 1711 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+conducting+acute+toxicity+tests+on+test+materials+with+fishes,+macroinvertebrates,+and+amphibians.">Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians.</a> Annual.Book of ASTM Standards. Volume 11.06. , 729-796 (2014).</li><li>Brunson, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+bioaccumulation+of+contaminants+from+sediments+from+the+upper+Mississippi+River+using+field-collected+oligochaetes+and+laboratory-exposed+Lumbriculus+variegatus.">Assessing bioaccumulation of contaminants from sediments from the upper Mississippi River using field-collected oligochaetes and laboratory-exposed Lumbriculus variegatus.</a> Arch Environ ConTox. 5, 191-201 (1998).</li><li>Brumbaugh, W. G., May, T. W., Besser, J. M., Allert, A. L., Schmitt, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+elemental+concentrations+in+streams+of+the+New+Lead+Belt+in+southeastern+Missouri,+2002-05.">Assessment of elemental concentrations in streams of the New Lead Belt in southeastern Missouri, 2002-05.</a> U.S. Geological Survey Scientific Investigations Report 2007-5057. , (2007).</li><li>Kane, A. S., Salierno, J. D., Gipson, G. T., Molteno, T. C. A., Hunter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+video-based+movement+analysis+system+to+quantify+behavioral+stress+responses+of+fish.">A video-based movement analysis system to quantify behavioral stress responses of fish.</a> Water Res. 38, 3993-4001 (2004).</li><li>Sprague, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+of+Copper-Zinc+Solutions+by+Young+Salmon+in+the+Laboratory.">Avoidance of Copper-Zinc Solutions by Young Salmon in the Laboratory.</a> JWater Pollut Control Fed. 36 (8), 990-1004 (1964).</li><li>Saunders, R. L., Sprague, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+copper-zinc+mining+pollution+on+a+spawning+migration+of+Atlantic+salmon.">Effects of copper-zinc mining pollution on a spawning migration of Atlantic salmon.</a> Water Res. 1 (6), 419-432 (1967).</li><li>Barron, M. G., Dell'Omo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+contaminants+altering+behavior.">Environmental contaminants altering behavior.</a> Behavioural Ecotoxicology. , 167-186 (2002).</li><li>Ellgaard, E. G., Guillot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+the+swimming+behavior+of+bluegill+sunfish,+Lepomis+macrochirus+rafinesque,+exposed+to+copper:+hypoactivity+induced+by+sublethal+concentrations.">Kinetic analysis of the swimming behavior of bluegill sunfish, Lepomis macrochirus rafinesque, exposed to copper: hypoactivity induced by sublethal concentrations.</a> J Fish Biol. 33, 601-608 (1998).</li><li>Drummond, R. A., Spoor, W. A., Olson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+short-term+indicators+of+sublethal+effects+of+copper+on+brook+trout,+Salvelinus+fontinalis.">Some short-term indicators of sublethal effects of copper on brook trout, Salvelinus fontinalis.</a> J Fish Res Board Can. 30, 698-701 (1973).</li><li>Hansen, J. A., Rose, J. D., Jenkins, R. A., Gerow, K. G., Bergman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinook+salmon+(Oncorhynchus+tshawytscha)+and+rainbow+trout+(Oncorhynchus+mykiss)+exposed+to+copper:+neurophysiological+and+histological+effects+on+the+olfactory+system.">Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss) exposed to copper: neurophysiological and histological effects on the olfactory system.</a> Environl Toxicol Chem. 18, 1979-1991 (1999).</li><li>Sandahl, J. F., Baldwin, D. H., Jenkins, J. J., Scholz, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensory+system+at+the+interface+between+urban+stormwater+runoff+and+salmon+survival.">A sensory system at the interface between urban stormwater runoff and salmon survival.</a> Environ Sci Technol. 41 (8), 2998-3004 (2007).</li><li>McIntyre, J. K., Baldwin, D. H., Beauchamp, D. A., Scholz, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+copper+exposures+increase+visibility+and+vulnerability+of+juvenile+coho+salmon+to+cutthroat+trout+predators.">Low-level copper exposures increase visibility and vulnerability of juvenile coho salmon to cutthroat trout predators.</a> Ecol Appl. 22 (5), 1460-1471 (2012).</li><li>Green, W. W., Mirza, R. S., Wood, C. M., Pyle, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+binding+dynamics+and+olfactory+impairment+in+fathead+minnows+(Pimephales+promelas).">Copper binding dynamics and olfactory impairment in fathead minnows (Pimephales promelas).</a> Environ Sci Technol. 44 (4), 1431-1437 (2010).</li></ol>

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Changes in behavior due to exposure to a contaminant are often used as an endpoint for sub-lethal toxicity, but can be difficult to measure. Generally, behavior responses are measured by visual observations and manual data analysis which requires a lot of time to process. However with advancing technology, methods for quantifying swimming activity have focused on using videography18 and motion analysis or digital tracking software which reduces the processing and analysis time. During analysis of the video captured data, quantifying swimming variables manually would have been very time consuming so the use of video data recordings and fish tracking software provided a more effective and efficient way for analyzing sturgeon swimming behavior. Although the procedure highlighted swimming behavior of a fish, customizing for other organisms such as amphibians and aquatic invertebrates would require simple modifications. Depending on what behavioral endpoints are being addressed, experimental design and camera systems can be developed for use with just about any commercially available tracking software package.
The method is demonstrated using dissolved copper, but is applicable to other aqueous contaminants or characteristics such as temperature or oxygen content. The protocols developed and presented in this paper utilized a simple digital video camera as the recording device. The digital files are easily transferred to a computer and uploaded in to the motion analysis software. The methods are constantly being modified and refined to streamline the quantification process. It is imperative that the video quality be in high definition in order for the analysis software to identify each individual fish for tracking. Any background that does not contrast with the fish will cause problems when trying to process the data files. Another common problem with two dimensional video tracking is identifying individuals when swimming paths cross. This can be corrected manually by identifying each fish during the path crossing and linking up the path segments within the software. Alternatively, total activity can be determined from each replicate chamber as a group average. Several individual chambers with each one containing one fish can be filmed in the same field of view to calculate movements of individual fish.
Currently we have upgraded to using a series of overhead surveillance cameras above the exposure chambers that are linked to a high definition digital video recording device (HD-DVR). However, the use of any camera system that can record high definition MPEG-4 videos will work. The HD-DVR can be set to record at a specified time and programmed for up to 7 days. This hands-off automated approach allows the capture of several videos at the same time to maintain consistency while minimizing external disturbances which could compromise fish behavior. The HD-DVR systems are connected to an internal network so transferring of files is relatively simple. While the automated camera system is a much improved technique for quantifying swimming behavior, it is still beneficial to conduct visual observations to serve as additional supporting information for documenting behavioral impairment during toxicity tests.
There is a long history of literature documenting altered fish behavior resulting from exposure to metals dating back to the early 1960s19,20,21. Copper has been shown to cause changes in activity levels such as hypoactivity in bluegill22 (Lepomis macrochirus rafinesque) and changes in locomotor and feeding activity of brook trout23 (Salvelinusfontinalis). At least some juvenile fish rely on their sense of smell to detect and avoid predators, and copper-induced chemosensory deprivation may impact behaviors related to detecting alarm chemicals24,25,26. The olfactory epithelium is damaged due to copper exposure thereby affecting sensory mechanisms which could result in disorientation, behavioral avoidance, reduced feeding and any other behaviors that are guided by olfaction27. These altered behaviors were consistent with what was observed during the exposures.
The swimming behavior of white sturgeon was greatly impacted during sublethal exposure to aqueous copper concentrations.. These results illustrate how behavior is affected at sublethal concentrations of copper and can be used as an indicator of toxic stress. The video based analysis proved effective in quantifying swimming behavior and also served as qualitative visual documentation of the severe impacts on sturgeon exposed to copper. The analysis software is also capable of quantifying other various behavioral endpoints. Please refer to Table 1 for a list. The exposure system can be modified to address each endpoint in real-time mode and can be used to quantify differences in behavior associated with exposure to various contaminants of concern.
The use of behavioral endpoints in aquatic toxicological research is increasingly employed and should be considered when evaluating the effects of contaminants because adaptive behavioral function is crucial in the determination of environmental injury9. The impacts of environmental contaminants to fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints in the absence of standard methods..
Swimming activity as quantified by using these methods can be rigorously monitored, is non-destructive with minimal stress to the organism and can be repeated. Swimming behavior is a valid and consistent index of sublethal toxicity that should be incorporated in test protocols to expand the sensitivity of standard toxicity tests5.

Understanding how contaminant exposure can affect behavior is at times very challenging and subjective. Behavior is typically defined as a series of overt, observable, whole-body activities that operate through the central nervous system and enable an organism to survive, grow, and reproduce. Changes in behavior due to exposure to a toxicant are among the most sensitive indicators of environmental stress often between 10-100 times more sensitive when compared to survival1. The majority of these studies examined swimming activity, ventilation, and foraging behavior of fish2,3,4. Swimming activity is the most frequently used sublethal endpoint in determining a behavioral change in response to a contaminant in toxicity tests5. Swimming variables include the frequency and duration of movement, speed and distance travelled, the frequency and angle of turns, position in the water column and the pattern of swimming. Swimming activity is an effective measure of swimming behavior when assessing sensitivity to a toxicant based on the criteria proposed in Chapter 9 by Rand6 in the book Fundamentals of Aquatic Toxicology.
This paper presents a toxicological study as an example of how the toxicity of copper to early life-stage white sturgeon (Acipenser transmontanus) at various stages of early development in water-only exposures relative to sturgeon swimming behavior was evaluated and illustrates methods for quantifying swimming behavior.
In previous studies, adverse behavioral responses were evident with early onset during the first few days of acute and chronic exposures to copper and became progressively more severe over exposure duration and concentration7,8,9. The magnitude and timing of the onset of these behavioral responses are likely sufficient to limit long-term survival and thus are of concern given the implications for recruitment failure10. In order to accurately interpret the significance of this sensitivity to metal exposure methods and procedures were developed for quantifying the time course and extent of behavioral impairments relative to copper concentrations.
Test protocols for behavioral function and development were established by analyzing overhead video samples of sturgeon in exposure chambers. The video samples provided qualitative assessment of development and function among copper treatments throughout the exposure period. Behavior and shelter-seeking activity were evaluated during exposures to aqueous copper concentrations to determine the time to effect and to characterize the temporal sequence of impairment that affects shelter seeking, lethargy, swimming coordination, equilibrium, and respiration. In addition, video recordings were made of sturgeon subsampled from each replicate for the purpose of quantitatively documenting spontaneous swimming activity. These endpoints included measures of duration or the time spent moving, swimming speed and distance travelled5,11 using a commercially available digital image analysis software package. This software defines the outline of each image within a field of view and from this, defines the centroid for each image. The software then can track the position of each centroid in a frame by frame succession to determine the paths of movement.
This study complied with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR) and with all institutional guidelines for the humane treatment of the test organisms during culture and experimentation. Upon termination of the study, all fish were euthanized according to appropriate guidelines as approved by the Institutional Animal Care and Use Committee for the U.S. Geological Survey, Columbia Environmental Research Center.

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">copper II sulfate pentahydrate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">contaminant of concern</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">syringe dispenser</td>
			<td style="width:285px;">Hamilton MicroLab 600 Series</td>
			<td style="width:119px;"></td>
			<td>apparatus to spike chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">2-L volumetric flask</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td>container for holding stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">24-1.5 L glass jars</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td>test chamber for 2 dph sturgeon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">video camera</td>
			<td style="width:285px;">Sony Handycam HDR-CX550V</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">digital tracking software</td>
			<td style="width:285px;">Noldus Ethovision</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3-17&#34; flat screen monitors</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">24 surveillance cameras</td>
			<td style="width:285px;">Model CL101</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3-16 channel digital recording devices</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">DO meter</td>
			<td style="width:285px;">YSI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">pH meter</td>
			<td style="width:285px;">Orion 940</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ph probe</td>
			<td style="width:285px;">Orion&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ammonia meter</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ammonia probe</td>
			<td style="width:285px;">Orion</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">chiller unit</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">recirculating water pump</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying fish swimming behavior in response to acute exposure of aqueous copper using computer assisted video and digital image analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

Manually processing the visual observational data showed percent abnormalities increased with increasing copper concentration after just 72 hr of exposure initiated with 2 day post hatch (dph) sturgeon (Figure 2). The video samples documented the extreme impact of copper exposure on sturgeon swimming behavior (Video 2) and aided in defining the behavioral impairment as a result. In another example, sturgeon at 30 dph proved sensitive to copper exposure with a 96 hr median lethal effect concentration (LC50) of 40.3 &#181;g/L based on mortality only. However, when sublethal behavioral endpoints of loss of equilibrium and immobilization are included along with mortality the sensitivity increases with an estimated 96 hr median effect concentration (EC50) ranging from 2.4 to 5.0 &#181;g/L. Video documentation captured these sublethal effects and further validated human observations of behavioral abnormalities recorded during the exposure. The use of digital tracking software reduced the post processing time significantly when analyzing swimming activity. Fish swimming speed, the time spent moving, and the distance travelled all significantly decreased (Figure 3) with increasing copper concentration. Swimming paths were also reduced with increasing copper concentration (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/53477/53477fig1.jpg" /> 
Figure 1: Computer screenshot of a spreadsheet template used to determine test stock solution concentration. Chemical stock preparation for the diluter toxicity tests was determined using a spreadsheet template based on a target concentration. <a href="https://www.jove.com/files/ftp_upload/53477/53477fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53477/53477fig2.jpg" /> 
Figure 2: Diluter setup and experimental design. Various life-stages of white sturgeon were exposed to copper. The size of diluter used for the exposures was selected based on the size of the fish. (a) Early life-stage sturgeon were exposed using a small diluter setup and (b) older life-stage sturgeon were exposed using a large diluter setup.
<img alt="Figure 3" src="/files/ftp_upload/53477/53477fig3.jpg" /> 
Figure 3: Swimming behavior results from an early life-stage white sturgeon (Acipenser transmontanus) 72 hr copper exposure (starting with 30 day post hatch [dph] fish). Swimming activity endpoints (a) duration of movement among 30 dph white sturgeon; (b) speed of swimming; and (c) distance moved declined with increasing copper concentration among white sturgeon exposed for 96 hr. Asterisk indicates significant difference from control, error bars represent standard deviation.
<img alt="Figure 4" src="/files/ftp_upload/53477/53477fig4.jpg" /> 
Figure 4: Results from an early life-stage white sturgeon (Acipenser transmontanus) 96 hr copper exposure (starting with 2 day post hatch [dph] fish). Surviving white sturgeon at 2 dph exhibiting loss of equilibrium and immobilization after 72 hr of a 96 hr exposure with increasing copper concentration. Asterisk indicates significant difference from control, error bars represent standard deviation.
<img alt="Figure 5" src="/files/ftp_upload/53477/53477fig5.jpg" /> 
Figure 5: Example swimming path results from an early life-stage white sturgeon (Acipenser transmontanus) 96 hr copper exposure (staring with 30 day post hatch [dph] fish). Swimming paths of sturgeon digitized using digital tracking software from (a) control replicate (n = 5 fish) and (b) from a high treatment (50 &#181;g/L) replicate (n = 3 fish) after a 96 hr exposure. Note the number of swimming paths does not represent the number of fish present in the chamber because of some fish were inactive. <a href="https://www.jove.com/files/ftp_upload/53477/53477fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Quantifiable Parameter</td>
			<td>Observational Parameters</td>
		</tr>
		<tr>
			<td>Velocity</td>
			<td>Lethargy/Hyperactivity</td>
		</tr>
		<tr>
			<td>Distance Moved</td>
			<td>Loss of equilibrium</td>
		</tr>
		<tr>
			<td>Duration of time spent in zone</td>
			<td>Spasms/tremors/upside down</td>
		</tr>
		<tr>
			<td>Zone transition-number of times organism moves between selected zones</td>
			<td>Position in the water column</td>
		</tr>
		<tr>
			<td>Heading to point-calculates the deviation of the animal&#8217;s path towards a point of interest</td>
			<td>Respiration (fast/slow)</td>
		</tr>
		<tr>
			<td>Heading calculates the heading of the selected body point</td>
			<td>Coloration</td>
		</tr>
		<tr>
			<td>Turn angle-difference in heading between two samples</td>
			<td>Hiding</td>
		</tr>
		<tr>
			<td>Angular velocity-calculated by dividing the turn angle by the sample interval</td>
			<td></td>
		</tr>
		<tr>
			<td>Meander-calculated by dividing the turn angle by the distance moved. Used to compare turning in animals moving at different speeds</td>
			<td></td>
		</tr>
		<tr>
			<td>Time spent moving</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobility state-calculates the duration for which the complete area detected as animal is changing, even if the center point remains the same</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotation-one rotation is completed when the selected body point has a cumulative turn angle of 360&#176;. Turns in the opposite direction of less than the threshold are ignored.</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobility continuous-calculates the percentage of mobility for the complete area of the detected animal, even if the center point remains the same.</td>
			<td></td>
		</tr>
		<tr>
			<td>Distance between subjects-calculates distance between all actors and the selected receivers</td>
			<td></td>
		</tr>
		<tr>
			<td>Proximity-calculates the duration for which the actor is or is not in proximity to the receiver</td>
			<td></td>
		</tr>
		<tr>
			<td>Relative Movement</td>
			<td></td>
		</tr>
		<tr>
			<td>Net weighted movement-The movement of the actor to (positive) and from (negative) the receiver, weighted by the distance between them</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighted movement from-movement of the actor from the receiver, weighted by the distance between them.</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighted movement to- movement of the actor to the receiver, weighted by the distance between them</td>
			<td></td>
		</tr>
		<tr>
			<td>Trial control state- period between two events of the Trial Control elements, or within one element</td>
			<td></td>
		</tr>
		<tr>
			<td>Trial control event-moment in which an event within an element you have defined in Trial Control occurs.</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Behavioral endpoints quantified through digital tracking software. These endpoints can be used on individuals or groups and also serve as a list for visual observational checks of behavioral impairment during the exposure.
<img alt="Video 1" src="/files/ftp_upload/53477/53477movie1.jpg" /> 
<a href="https://www.jove.com/files/ftp_upload/53477/53477video1.avi">Video 1: Visual definition of abnormal behavior exhibited by affected white sturgeon. (Right click to download)</a> A daily observational check list was used to document abnormalities. The loss of equilibrium and immobilization were the most prevalent abnormalities observed during the exposures. Modified from Calfee et al.7
<img alt="Video 2" src="/files/ftp_upload/53477/53477movie2.jpg" /> 
<a href="https://www.jove.com/files/ftp_upload/53477/53477video2.avi">Video 2: Visual documentation highlighting an example of white sturgeon swimming behavior. (Right click to download)</a> White sturgeon swimming activity was greatly reduced with exposure to increasing copper concentration. The sturgeon depicted in this video are from a control, medium low, and a high treatment at the end of a 96 hr aqueous copper exposure. Although the white sturgeon were still alive it is evident the fish were greatly impaired in the treatments compared to the controls. Modified from Calfee et al.7

Watch this Scientific Journal Video about Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis at JoVE.com

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

The overall goal of this procedure is to demonstrate how to quantify behavioral responses in white sturgeon to sub-lethal exposure to aqueous copper. This method can help to answer key questions in the ecotoxicology field, such as how sub-lethal exposure to contaminants affects swimming behavior of fish. This method can provide important information for white sturgeon conservation.It can also be readily applied to other fish species, amphibians, and invertebrates. The main advantage of this technique is that the response is highly sensitive, provides an early indication of toxicity, and is relevant to organism survival. Initially this method may be challenging if the individual is unfamiliar with making behavioral measurements.Demonstrating the protocol will be my co-author Holly Puglis. First, prepare two liters of aqueous reagent grade copper two sulfate pentahydrate. Next, equilibrate the diluter system and exposure tanks.Place the intake tube from the automated syringe dispenser into the stock solution flask. Then, set the automated pump spike volume to one milliliter, and turn the diluter on. The system makes a 50%dilution series of five concentrations and a control.Through a four-way splitter, these solutions are delivered to the four replicate exposure chambers. Next, turn on the pump for the chiller set to maintain the water bath at 15 degrees Celsius. Now, using the automated timer on the diluter, set it to cycle every 30 minutes.The water level in the tanks is maintained by a stand pipe drain. Prepare the exposure chambers by putting a four centimeter hole in the side, and covering the hole with 30 micron stainless steel mesh. Be sure to select exposure chambers that hold at least one liter of water per 10 grams of fish.For example, an 89 day old white sturgeon weighs about 373 milligrams, and can thus be tested in a 28 centimeter by 13 point five centimeter by 25 centimeter square chamber. At the initiation, mid-point, and end of an experiment, collect 24 milliliter test solution sub-samples for chemical analysis. Use sipper straws attached to plastic syringes.Replace the sipper straw with a zero point four five micron polyethersulfone filter, and dispense four milliliters of test water through the filter. Dispose of this water. Then, filter the remaining 20 milliliters of water into an acid-cleaned polyethylene bottle.Acidify the sample with 16 molar nitric acid to 1%volume by volume. These samples may then be stored for up to three months. Once the test exposures are confirmed, stock three sturgeon in each replicate test chamber for about 172 milligrams of fish per chamber liter.Collect the sturgeon using a nonabrasive net, and transfer them to a small bucket. In total, 144 fish are needed to collect data from six test solutions. During the 96 hour exposure, do not feed the fish.Consistent daily observations must be made to score for mortality and abnormal behavior. The morning is the best time of day to do this, and ideally the observer is blind to the test conditions. Loss of equilibrium is defined as the inability of fish to maintain an upright position within the water column.Immobility is defined as the inability of fish to move or swim unless prodded. Also record any other abnormalities, such as lethargy, hyperactivity, increases or decreases in respiration, color changes, tremors, spasms, bloated abdomens, position in the water column, and any other unusual swimming patterns. Dead fish should always be removed immediately.Each day, also measure dissolved oxygen levels with a handheld meter from two water samples. At the end of the 96 hour exposure, make videos for swimming behavior analysis. Position the test arena within an exposure tank.Be sure to select a test arena that is large enough for the fish to swim freely. Directly above each test arena, set up a handheld video camera on a tripod. Now, from the treated fish, randomly select up to three survivors.Transfer three into the test arena using a small mesh net. Allow the fish to acclimate to the arena for about 30 minutes. While the fish acclimate, focus the image, minimize the glare distortions from moving water, and maximize the contrast of the fish against the background, adjusting the view so the fish is not obscured.Once the fish is acclimated, start the recording and collect two minutes of video data. After taking the video, euthanize and dispose of the fish. After converting the video files to the format needed for the digital analysis software, open the tracking software and select new default experiment under the create a new experiment option on the main screen.Fill in the prompts, and press okay. Then, go to the experiment settings option under set up, and choose from video file under video source. Set the number of arenas to one.Set the number of subjects per arena to three. Next, select center point detection under tracked features and select the desired units. Now, under set up, select the trial list option and click add videos at the top of the screen.Choose alphabetical order from the dialogue box. Click browse. Navigate to the folder where the videos are located, highlight the pertinent files, and click open.Now that the video files are selected, proceed with defining the scope of the experiment in the software. First, click add variable at the top of the screen and enter concentration into the label box. Enter copper concentration in micrograms per liter into the description box.Next, choose numerical from the drop down list for type. Click on the pre-defined values box, and choose the define individual values option from the dialogue box. Enter the concentrations used for the experiment, clicking add between each number addition.Then, uncheck the allow other values option and click okay. Choose trial from the drop down menu in the scope box. Enter the appropriate concentration for each trial in the boxes provided.Click add variable at the top of the screen. Enter replicate into the label box. Enter replicate number into the description box.Choose numerical from the drop down list for type. Next, click on the pre-defined values box and choose the define individual values option from the dialogue box. Enter one, two, three, and four, and un-check the allow other values option.Then, click okay. Now, choose the trial option from the drop down menu in the scope box. Enter the appropriate replicate number for each trial.The next task is to set up the arena spaces. Begin with choosing the arena settings under the set up tab, and name the first setting as trial one. From the dialogue box, select the grab background image option and click browse.Locate the video file for trial one and click open. After the video appears, click grab on the grab background image dialogue box. Next, click the white circle icon near the top of the screen under arena settings, and manipulate the circle that appears to surround the swimming area.Then, click the calibration scale icon under arena settings. Use a click-hold-drag motion to select the arena space. Now, under the real world distance box in the calibration distance, enter 10 point five.Then, click okay. If necessary, adjust the calibration line so that it spans the entire diameter of the circular arena. Click the validate arena settings option on the arena settings box, and address any issues in the settings that were not validated.Now, repeat this process until all the arenas are defined for each trial. Next, set the fish detection parameters. First, choose detection settings under the set up option from the experiment explore tool bar on the left of the screen.Then, choose dynamic subtraction from the drop down menu under method in the detection settings one dialogue box. Also, choose a fill color for each subject under subject identification in the dialogue box. Next, choose select video and locate the video for trial one.In the detection settings one dialogue, fill in the sample rate box under video with five point nine nine four one. In the same box, click settings for the reference image option under detection. Then, click the start learning C option in the reference image dialogue box.Once the image in the reference image dialogue box appears without animals, click use dynamic reference image under acquisition settings within the dialogue box and close this dialogue box. Now, choose darker from the drop down menu for subject is under detection in the detection settings one dialogue box. For the dark contrast under detection, set the smaller number to 33 and the larger number to 153.Then, click save changes at the bottom right. Now, confirm that that software is successfully tracking animals, as opposed to shadows or debris. Click the play button on the playback control dialogue box and adjust the numbers for dark contrast as needed.Be sure to save any adjustments made. The software is now set to collect data from the video, so the next task is to track the fish in each trial. First, choose acquisition under the setup option from the experiment explorer tool bar on the left of the screen.Next, click track next planned trial in the acquisition settings dialogue box, and confirm that the correct trial, video, and arena settings are all displayed under the settings in the acquisition settings dialogue box. Then, check the detection determines speed option in the acquisition control dialogue box. Click the button with the green circle enclosed in a white square to begin the acquisition process.Repeat this process until all the trials have been tracked. Now, the tracked objects being the fish can be analyzed. First, click data profiles under the analysis option from the experiment explorer tool bar on the left of the screen.Then, choose time under the nesting option in the components toolbar. Under the select track time interval setting in the time dialogue box, adjust the to value to two minutes and click okay. Then, drag the nest box between the start box and the result one box in the data profiles area on the right side of the screen.From the experiment explorer toolbar on the left of the screen, look under the analysis option and click analysis profile. Then, in the dependent variables toolbar, under the distance and time heading click velocity. Click add, and under the distance and time heading in the dependent variables toolbar, click distance moved.Then, click add on the distance moved dialogue box, and under the individual behavior heading in the dependent variables toolbar, click movement. In the movement dialogue box, look under the outlier filter heading and adjust the averaging interval to one. Then, under the threshold heading, adjust the start velocity to two point zero zero and adjust the stop velocity to one point seven five.Under the calculate statistics for heading, check both boxes for moving and not moving. Then, click add, and under the results option in the experiment explorer toolbar on the left of the screen click analysis output. Now, click calculate.Once the dependent variables are calculated, click export at the top of the screen. Choose the destination folder from the export analysis output dialogue box, and select Excel from the file type drop down menu. Then, click okay.Finally, click save experiment under the file tab at the top of the screen. Close the digital tracking software. Import the data to a spreadsheet file, and analysis it using a commercial statistical analysis software package.After just 72 hours of exposure to various copper concentrations, two days post-hatch sturgeon showed many observable abnormalities. There was an obvious impact on swimming behavior. Video data aided in defining this behavioral impairment.In further tests, sturgeon at 30 days post-hatch proved sensitive to 96 hour copper exposures. Swimming speed, the time spent moving, and the distance traveled all significantly decreased with increase in copper concentrations. Swimming paths were also reduced with increase in copper concentrations.After watching this video, you should have a good understanding on how to quantify behavior in a toxicity test. This technique can provide environmental managers a means of using behaviorally-defined criteria to document environmental injury and guide restoration. When conducting this procedure, it is important to remember to be consistent when recording behavioral observations.Other behaviors, such as feeding and aggression, can be quantified by following this procedure in order to answer additional questions about the long term impacts of contaminant exposure on survival.

Research

JoVE Journal

Neuroscience

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