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>

Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

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.  ...

quantifying-fish-swimming-behavior-response-to-acute-exposure-aqueous

Research

JoVE Journal

Neuroscience

11.1K Views.  US Geological Survey. 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.

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

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

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

Summary

Explore More Videos

Chapters in this video

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

A Computer-assisted Multi-electrode Patch-clamp System

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats