This work was funded by the U.S. Geological Survey's Ecosystems (Chesapeake Bay Environments and Fisheries) and Environmental Health (Contaminants Biology) programs and the West Virginia Department of Natural Resources. Use of trade names is for identification purposes only and does not imply endorsement by the U.S. government.

Methods described here have been approved by the Leetown Science Center&#39;s Institutional Animal Care and Use Committee.
1. Fish Collection
<ol>
	<li>Collect live fish with a minimum of stress. Use boat or backpack electrofishing, hook and line or nets.</li>
	<li>Hold fish in live wells or aerated containers until sampling. 
	NOTE: The American Fisheries Society has published a number of guides for fish collection, handling and anesthesia/euthanasia26,27,28. Wear gloves when handling fish.</li>
</ol>
2. Fish Necropsy
<ol>
	<li>Euthanize a fish.
	<ol>
		<li>Place fish in the anesthetic until opercular movement ceases and the fish loses equilibrium. After another 2&#8211;10 min the fish will be euthanized; however, this may also vary by species. 
		NOTE: Fish can be euthanized with a number of anesthetics (see Table of Materials for most commonly used). The method of euthanasia will depend on the laboratory measurements that will be conducted on tissues collected29.</li>
	</ol>
	</li>
	<li>Measure biometric characteristics.
	<ol>
		<li>Weigh the fish to the nearest gram.</li>
		<li>Measure the fish length to the nearest millimeter.
		<ol>
			<li>Measure the total length from the tip of the snout with the mouth closed to the end of the tail when pinched together.</li>
			<li>Measure the fork length from the fork in the tail to the tip of the snout, and the standard length from the tip of the snout to the end of the body (beginning of the tail).</li>
		</ol>
		</li>
		<li>Calculate the condition factor using the following formula: 
		Condition factor = (total body weight - gonad weight)/total length3. 
		Note: Gonad weight is subtracted from the total body weight since gonads can contribute significantly to the total body weight, particularly in prespawn female fish.</li>
	</ol>
	</li>
	<li>Obtain a blood sample. 
	NOTE: Blood is most commonly taken from the caudal vein but can also be withdrawn from the dorsal aorta or by cardiac puncture30.
	<ol>
		<li>Extract a peripheral blood sample from the caudal vein with a 22 or 23 G needle on a 1 to 5 mL syringe, depending on size of the fish. Insert the needle anterior to the caudal area below the lateral line (Figure&#160;1A and 1B). Angle it upwards until hitting the spine and then withdraw slightly. The vein is ventral to the overlying spine. 
		NOTE: If blood smears will be made or serum is required, no anticoagulant is used. In most cases, plasma will be collected and, hence, an anticoagulant such as sodium heparin, EDTA or lithium is used to coat the needle and syringe and is also in the blood collection tube (e.g., vacutainer).</li>
		<li>Remove the needle and place into a sharps disposal container prior to putting the blood into the collection tube. 
		NOTE: Blood can be held on ice but depending on subsequent analyses should be centrifuged as soon as possible30.</li>
		<li>If nuclear abnormalities or differential blood counts will be evaluated, immediately place a drop of blood on duplicate clean glass microscope slides. Back a second slide at a 45&#176; angle into the drop, which is then drawn across the surface by capillary action. Allow to air-dry31.</li>
		<li>Centrifuge blood at 1,500&#8211;2,500 x g for 15 min to sediment the cells. Remove plasma/serum with a sterile transfer pipet, aliquot into cryogenic vials, and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57946/57946fig1.jpg" /> 
Figure 1: Obtaining a blood sample from a fish.&#160;(A) A recently euthanized fish is laid on its side and the lateral line located. (B) A needle is inserted ventral to lateral line (arrow), angled upward until needle touches the backbone. It is then slightly withdrawn, and suction initiated to withdraw blood. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Conduct a necropsy-based health assessment on each fish. 
	NOTE: A number of publications illustrating and describing lesions and abnormalities are available32,33,34,35.
	<ol>
		<li>Document external abnormalities including lesions on body surface and fins (Figure 2), eyes and gills (Figure 3), external parasites such as leeches (Figure 2D), grubs or trematode metacercarial cysts (Figure&#160;2D, 3B) and gill parasites (Figure 3D). Document type, location and size of observed abnormalities on data sheets, as well as photographically, if possible.</li>
		<li>Open the abdominal cavity (Figure 4A) using a scissors by cutting from the anal area to the operculum and then removing the flap of muscle to expose the internal organs. 
		NOTE: If anterior kidney will be collected for immune function (see step 5 below) or samples collected for bacteriology or virology, the external body surface should be disinfected with 70% alcohol and those samples should be obtained before the necropsy performed. If tissues are only being used for visual observations, plasma analyses and histopathology sterile technique is not necessary.</li>
		<li>Document internal abnormalities (Figure 4) including general or focal discolorations of the various organs (Figure&#160;4B-4D), presence of raised areas (Figure 4E), cysts, parasites, and size abnormalities (enlarged, atrophied).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57946/57946fig2.jpg" /> 
Figure 2: Examples of visible lesions observed on body surface and fins of fish.&#160;(A) A small, slightly eroded lesion (arrow) on the lateral body surface. (B) A large reddened area (arrow) involving the caudal body surface. (C) Raised, black lesions (arrows) on the body surface and fins. (D) Leeches (white arrow) and small black spots (black arrows) on the fin. Scale bar = 3 mm. (E) A raised, multilobed, pale lesion (arrow) on the body surface. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57946/57946fig3.jpg" /> 
Figure 3: Examples of visible lesions of the gills and eyes of fish.&#160;(A) A pale area (arrow) within the lens of an eye. Scale bar = 5 mm. (B)&#160;White cysts (white arrows) and small black spots (black arrows) caused by trematode parasites on the operculum covering the gills (a). Scale bar = 1 cm.&#160;(C) A pale, eroded area (arrow) on the gill (a). Scale bar = 5 mm.&#160;(D)&#160;A gill that has been removed showing parasites (arrows) attached to the gill filaments. Scale bar = 2 mm. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57946/57946fig4.jpg" /> 
Figure 4:&#160;Examples of a necropsy and internal abnormalities of fish.&#160;(A) During a necropsy the fish is cut open (along the white arrow) and a flap of muscle (black arrow) removed to expose the gonad (a) and the spleen, being held by forceps&#160;and scissors. (B)&#160;Mottled liver (a), testes (b), intestine surrounded by adipose fat (c) and stomach (d). Scale bar = 5 mm. (C)&#160;Liver (a) with a dark red area (arrow), ovary (b) and intestines (c). Scale bar = 5 mm. (D)&#160;Liver with greenish discolored areas (arrows). Scale bar = 1 cm. (E) Example of a normal (a) and abnormal (b) testes with raised nodules. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Obtain hepatosomatic index (HSI).
	<ol>
		<li>Remove liver by severing the hepatic artery and the connective tissue of the anterior end. Gently lift out while trimming adhesions and other connections to intestine and adipose fat. Take care to not puncture the gallbladder. Weigh the liver. 
		NOTE: Some fish, such as cyprinids, do not have a discrete liver but rather hepatic tissue wrapped around intestines and other organs. For these species, it may not be possible to obtain liver weights.</li>
		<li>Calculate hepatosomatic index (HSI) using the formula: 
		HSI=liver weight/total body weight</li>
	</ol>
	</li>
	<li>Calculate the gonadosomatic index.
	<ol>
		<li>Remove the gonads and weigh it.</li>
		<li>Calculate gonadosomatic index (GSI) using the formula: 
		GSI = gonad weight/total body weight</li>
	</ol>
	</li>
</ol>
3. Preserve Tissues for Microscopic Pathology
NOTE: A number of fixatives including 10% neutral buffered formalin and Z-fix, a formalin-based fixative with zinc, can be used for preservation of tissue in the field. The latter is preferred if methods such as&#160;in situ&#160;hybridization or fluorescent antibody staining may be used.
<ol>
	<li>Carefully cut but do not pull out tissue samples. Keep individual tissue pieces &#60;2 cm in size and &#60;5 mm thick for proper fixation. As a rule of thumb, use approximately 10x more fixative by volume than the tissue for proper preservation. Place all tissue samples from one fish in the same leak-proof container of the appropriate size, depending on the size of fish being sampled.</li>
	<li>Place pieces of any external abnormalities in the fixative container. Also, include an adjacent piece of normal tissue. 
	NOTE: Improper handling such as compression or other mechanical damage, long exposure to air or sunlight, and freezing can cause artifacts.</li>
	<li>Cut at least five 3&#8211;4 mm thick pieces of liver from various regions and place into the fixative container. Include normal and abnormal areas, if observed.</li>
	<li>Depending on size, place a whole gonad or multiple pieces along one gonad into the fixative container.</li>
	<li>Place either whole organs, if small, or pieces of all other organs (spleen, anterior and posterior kidney, gills, heart, intestine and stomach) in the fixative container. If abnormal tissue is observed, preserve an adjacent piece of normal tissue as well.</li>
</ol>
4. Remove the Otoliths for Age Analyses
NOTE: Age can be an important variable in fish disease/fish health studies. While a number of structures, including scales and spines, have been used for age determination, most studies comparing structures have found the otoliths to give the best results36,37. Teleost fishes have three pairs of otoliths - lapillus, sagitta and asteriscus. Generally, the sagittal or lapillus otoliths are collected for aging although that may vary by species. Removal and aging techniques have been previously described38.
<ol>
	<li>Cut through the gill isthmus, and bend the head back. Strip away connective and muscular tissue around the inferior portions of the neurocranium to locate prootic bullae, a raised bony area.</li>
	<li>Score or cut with bone cutters and crack to expose the otoliths. They can be seen with the naked eye.</li>
	<li>Place otoliths in a labelled vial or a coin envelope and store at room temperature until analyzed for age by counting the rings or increments38. If placing in a vial, open cap once returned to the laboratory and allow to thoroughly dry prior to storage.</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57946/57946fig5.jpg" /> 
Figure 5: Removal of otoliths.&#160;(A)&#160;The isthmus is cut and the connective tissue and muscle pulled away to expose the base of spine and neurospinal area. (B)&#160;The bone is cracked to expose the otoliths. (C)&#160;Lapillar otoliths are removed. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Obtain Tissue for Immune Function Assays
NOTE: The anterior kidney is the major hematopoietic organ, the source of lymphocytes and macrophages for functional assays, and must be removed aseptically if cells will be cultured for functional assays, such as mitogenesis, phagocytic and killing ability of macrophages39,40.
<ol>
	<li>Spray the external surface of the fish with 70% ethanol. Use sterile scissors, a scalpel and forceps to open the abdominal cavity and remove the anterior kidney tissue, which is a dark red organ located anterior to the swim bladder.</li>
	<li>Place the anterior kidney sample in media (e.g., Leibovitz&#39;s L-15) to keep the cells alive. Homogenize kidney samples with a sterile hand-held tissue grinder (e.g., Tenbroeck tissue grinder) into single cell suspensions. Hold on wet ice until returned to the laboratory.</li>
</ol>
6. Preserve Tissue for Nucleic Acid Analyses
NOTE: If downstream molecular analysis will be conducted, such as gene expression using transcript abundance41&#160;or quantitative PCR42&#160;(polymerase chain reaction), place the pieces of tissue to be assessed in an appropriate preservative (e.g., RNAlater stabilization solution) as soon as possible.
<ol>
	<li>For RNA preservation, place two to three small (2&#8211;3 mm) pieces in the appropriate preservative at a 10:1 ratio of preservative volume to tissue. 
	NOTE: Samples should be shielded from sunlight or excessive heat and transported on wet ice.</li>
	<li>For DNA preservation, place two to three small pieces of tissue into 95% ethanol (10:1 ethanol to tissue by volume). Then hold the samples on wet ice and then store at -20 &#176;C.</li>
</ol>

The authors have nothing to disclose.

The necropsy-based assessment of fish health can be utilized on any fish species for which the investigator has an understanding of the normal appearance of both external and internal structures. Using a standardized approach allows for comparisons between sites and species as well as seasonal and temporal changes in a population. The findings can be used to identify effects associated with point and nonpoint sources of contaminants and to inform management actions. It can also be used to track improvements once management actions are initiated. The methodology can be modified to augment the documentation of visual external abnormalities in a variety of ways. Assessments, based only on visual observations, can be non-lethal, relatively inexpensive and data can be generated quickly for a large number of individuals. Consequently, they can be useful for exploratory or initial assessments, to monitor change over time or in combination with other indicators. If the length and weight of fish are measured during visual observations, the condition factor can also be calculated. Although assessments based only on visual observation do not provide information on cause or associated risk factors, long term trends of certain skin abnormalities45 and biometric parameters46 have indicated improvement in some areas associated with water quality improvements.
The necropsy-based assessment provides more information as internal organs are also examined and other condition factors such as hepatosomatic index and gonadosomatic index can be calculated. Goede and Barton22 developed a field necropsy method that included blood parameters, biometric factors, the percentage of abnormalities, and index values for specific abnormalities. A refinement of the method included a severity rating for some variables that allowed for calculation of a health assessment index that could be compared statistically23. This health assessment index has been used in regional site comparisons23,47,48 and in combination with other biological indicators including plasma and histopathological analyses in the U.S. Geological Survey&#39;s Biomonitoring of Environmental Status and Trends Program evaluating potential effects of contaminant exposure in large rivers nationwide49,50,51. A Fish Disease Index based on externally visible diseases and parasites, visible liver neoplasms and other histopathologically detected liver lesions has been developed and used extensively in the North Sea, Baltic Sea, and off Iceland. This index was found to be an important tool as an ecosystem health indicator52.
There are some critical factors in conducting the necropsy-based assessment on fish. First, assessments must be conducted on fish immediately after death. Changes in organ color and consistency can occur fairly rapidly after death. Additionally, some parasites may leave the host soon after death. Second, it is important to know what is normal for the species of interest. For instance, some fish normally have fatty and consequently, pale livers, while for most species a pale liver would be abnormal. It is also important to recognize seasonal changes that naturally occur. Some fish will have color changes or develop breeding tubercles during the spawning season.
The limitations of the necropsy-based assessment as a method for fish health assessment include the inability to 1) consistently identify the &#34;cause&#34; of specific lesions and 2) identify effects that may not be visible to the naked eye. These drawbacks can be overcome with the addition of histopathology, molecular or cultural identification of pathogens and parasites, and gene expression. For instance, a &#34;tumor&#34; or raised lesion (swelling) may be actual neoplasia or it may be a parasite, inflammation, edema or hyperplasia (increase in number of normal cells), caused by chemical exposure, infectious agents or other irritants. As shown in the representative results, definitive tumor or neoplasia diagnosis requires microscopic pathology to identify the lesion type and severity (i.e., benign or malignant). Assessment of white sucker external &#34;tumors&#34; by visual observation overestimated the prevalence, particularly at the reference site. Many of the raised lesions were not neoplasms but rather hyperplastic lesions. It is currently not known whether these hyperplastic lesions are pre-neoplastic. Conversely, the observation of raised nodules in the liver significantly underestimated the prevalence of liver neoplasms. Hence, collection of tissue for microscopic pathology was necessary to adequately address the potential for delisting.

Human activities have numerous adverse effects on aquatic environments. Fish inhabit various water bodies that the human population recreates in and often uses as a drinking water source and hence are important indicators of the health of the aquatic environment. Wild fish that live and reproduce in a particular habitat are exposed throughout their lives to various stressors including pathogens, parasites, poor water quality and chemical contaminants. Thousands of chemicals enter our waterways through industrial and human wastewater, suburban/urban stormwater and agricultural runoff. These complex mixtures of chemicals can have additive, synergistic or antagonistic effects on exposed organisms1,2,3. In addition, other environmental stressors such as elevated nutrients, elevated temperature, low dissolved oxygen or fluctuating pH can exacerbate the effects of chemical contaminants4,5. Environmental stressors can also influence infectious disease outcomes directly by increasing the number of infectious agents6, increasing the virulence of opportunistic pathogens7 or suppressing the immune response and disease resistance of the host8,9,10. For these reasons, there is increasing interest in biological or adverse effects monitoring11,12,13,14, utilizing fish and other aquatic organisms to identify populations and ecosystems at risk.
Adverse effects monitoring utilizes biomarkers at various levels of organization, from the organismal to the subcellular or molecular, to identify sublethal effects which may influence populations and be indicative of exposure to various stressors. Indicators at the organism level include visible abnormalities and conditions. Condition indices based on length and weight are calculated to evaluate the well-being or fitness of fish populations. The most common is Fulton&#39;s condition factors (K) = (weight/length3) 15. Another indicator is the presence of visible abnormalities. A variety of methods have been used in individual studies and monitoring programs to assess, document, and evaluate visible abnormalities. Assessment based only on external abnormalities, i.e., the proportion of individuals with disease, fin damage, tumors and skeletal anomalies, is one of the metrics for the index of biotic integrity (IBI) that evaluates community health16. A similar assessment termed DELTs (deformities, erosions, lesions, tumors) has also been used to evaluate the health of fish communities17. However, these methods only assess external visual abnormalities and not internal lesions or early sublethal indicators.
Necropsy-based assessments include external and internal observations and allow for the measurement of additional condition indices. Hepatosomatic index (liver weight/total body weight) has also been used as an indicator of fitness or energy reserves15 for which a higher index value indicates healthier fish. However, a number of studies have shown that hypertrophy or an increase in liver size occurs due to exposure to various contaminants metabolized by the liver18,19,20. In this case a higher index would be indicative of exposure to certain chemical classes. The gonadosomatic index (gonad weight/total body weight) is another condition index directed toward reproductive health21. Observations made during the necropsy-based assessment can be used to compare the prevalence of individual lesion types or percentage of normal individuals. However, they can also be used in a more quantitative health assessment22,23.
The standardized necropsy-based assessment described here can be used to augment the grossly visible assessment in multiple ways depending on the question(s) to be answered, expertise and other available resources. Our routine approach is to collect biometric data (length, weight, liver weight, gonad weight), blood for plasma/serum analyses, document external and internal visible abnormalities, preserve pieces of organs for microscopic analyses and collect otoliths for age analyses. The necropsy-based assessment plus age analysis and histopathology of various organs, allows for the calculation and comparison of various condition indices, prevalence of visible abnormalities, as well as microscopic tissue changes, by sex, age, site and sampling period. Additional tissue collections can be made for many other analyses including electron microscopy, bacteriology, virology, parasitology and chemical concentrations. These methods can also be part of more in-depth analyses used to diagnose the cause of fish kills24 or mortalities of captive fishes25. Methods for collection of tissue for two additional analyses, gene expression and functional immune analyses are illustrated.

<table border="0" cellpadding="0" cellspacing="0" style="width:855px;" width="856">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Folding tables</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Folding chairs</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dissecting boards</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Measuring board (in mm increments)</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Battery powered scale (in gm) for fish weight</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Battery powered scale (in mg) for organ weights</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dissecting forceps</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bone cutters</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Scalpel and blades</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Disposable gloves</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Buckets</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Leak-proof Nalgene bottles (250 ml)</td>
			<td style="width:215px;">ThermoFischer Scientific</td>
			<td style="width:119px;">02-924-5C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Vacutainer tubes with sodium heparin</td>
			<td style="width:215px;">ThermoFischer Scientific</td>
			<td style="width:119px;">02-689-6</td>
			<td>For blood collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Disposable&#160; 3 ml syringes with 23 gauge needle</td>
			<td style="width:215px;">ThermoFischer Scientific</td>
			<td style="width:119px;">14-826-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">1 &#8211; 2ml cryovials</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td>Used for plasma and RNAlater samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Invitrogen RNAlater Stabilization solution</td>
			<td style="width:215px;">ThermoFischer Scientific</td>
			<td style="width:119px;">AM7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Z-Fix Formaldehyde Zinc fixative</td>
			<td style="width:215px;">Anatech LTD</td>
			<td style="width:119px;">SKU-174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Tricaine-S (MS-222)</td>
			<td style="width:215px;">Syndel USA</td>
			<td style="width:119px;"></td>
			<td>fish anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Coin Envelopes</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td>for otoliths</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Pencils and pens</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">70% alcohol</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Data sheets</td>
			<td style="width:215px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

necropsy-based wild fish health assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

Great Lakes Areas of Concern (AOC) are geographic areas that were designated due to impairments of various beneficial uses. One of the beneficial use impairments (BUIs) at many AOC is the fish tumors or other deformities. Millions of dollars have been spent for remediation and restoration of each of these areas in order to delist the various BUIs and ultimately the AOC43. The criteria for delisting the fish tumor BUI differs from state to state (see epa.ohio.gov/portals/35/lakeerie/ohio_AOC_delisting_guidance.pdf and dnr.wi.gov/topic/GreatLakes/documents/SheboyganRiverFinalReport2008.pdf); however, as noted in the delisting documents, there is a requirement to determine the prevalence of liver tumors and in some cases skin tumors. In many cases, the prevalence is compared to a non-AOC reference site.
The fish tumor BUI was evaluated at three AOCs (St. Louis River, Milwaukee River and Sheboygan River) and a non-AOC reference site (Kewaunee River) on Lakes Superior and Michigan, utilizing a necropsy-based assessment of white sucker (Catostomus commersonii),&#160;followed by microscopic pathology of skin and liver tissue. Fish were collected from the Milwaukee, Sheboygan and Kewaunee rivers in 2012 and 201344 and from the St. Louis River in 2015 (unpublished data). Two hundred white suckers were assessed from Milwaukee, Kewaunee and St. Louis, and 193 from Sheboygan.
By definition, a tumor can be any swelling or raised area, although it is generally considered that a swelling caused by an abnormal growth of tissue with abnormal cells is either a benign or malignant neoplasm. White sucker collected from all sites exhibited a variety of external raised lesions including small, discrete white spots, larger white areas, slightly raised mucoid lesions and multilobed raised areas on the body surface and lips (Figure 6). Fish were weighed and measured to obtain a condition factor, external and internal abnormalities were documented, and skin and liver tissue was collected for histopathology.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57946/57946fig6.jpg" /> 
Figure 6: Raised skin lesions observed on white sucker from the Great Lakes.&#160;(A) A discrete white spot on the body surface. Scale bar = 5 mm. (B)&#160;A slightly raised mucoid (arrows) and multilobed lesions (a) on the posterior body surface. Scale bar = 1 cm. (C)&#160;A large, multilobed lesion on the body surface. Scale bar = 1 cm. (D)&#160;Numerous multi-lobed lesions on the lips. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The percent of fish with external tumors or raised discolored areas ranged from 15.5% at the St. Louis AOC to 58.0% at the Milwaukee AOC. In general, the discrete white spots were the least common visual lesion while the multilobed lip and body surface lesions were most common. The number of fish with observable liver nodules was low, ranging from 1.5% at Kewaunee and St. Louis to 2.5% at Milwaukee (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Rivers and Year Sampled</td>
		</tr>
		<tr>
			<td>Visible Lesions</td>
			<td>Kewaunee 2013</td>
			<td>St. Louis 2015</td>
			<td>Sheboygan 2012</td>
			<td>Milwaukee 2013</td>
		</tr>
		<tr>
			<td>Discrete white spots</td>
			<td>16</td>
			<td>3</td>
			<td>3.1</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Mucoid</td>
			<td>20</td>
			<td>9.5</td>
			<td>9.8</td>
			<td>30.5</td>
		</tr>
		<tr>
			<td>Multilobed</td>
			<td>22.5</td>
			<td>3</td>
			<td>29.5</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Total Raised Skin Abnormalitiesa</td>
			<td>46</td>
			<td>15.5</td>
			<td>38.3</td>
			<td>58</td>
		</tr>
		<tr>
			<td>Visible liver nodules</td>
			<td>1.5</td>
			<td>1.5</td>
			<td>1.6</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td colspan="5">aTotal number of fish with raised lesions. Some fish had multiple types of abnormalities.</td>
		</tr>
	</tbody>
</table>
Table 1: Necropsy-based Observations of White Sucker Collected at Great Lakes Areas of Concern and a Reference Site (Kewaunee River), Presented as a Percentage. 
Visual examination can be used to document the percent of fish with various abnormalities. However, to definitively diagnose the presence and type of neoplasia, tissues must be examined microscopically (histopathology). Upon microscopic examination, it was found that not all of the raised lesions were neoplastic. Many of the discrete white spots and the mucoid lesions, particularly at Kewaunee, were hyperplastic lesions rather than neoplasia (Table 2). Additionally, at Kewaunee and St. Louis, all of the skin tumors observed were benign papillomas. At Sheboygan and Milwaukee both papillomas and squamous cell carcinomas, malignant skin tumors, were observed (Table 2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Rivers Sampled</td>
		</tr>
		<tr>
			<td>Neoplasm Type</td>
			<td>Kewaunee 2013</td>
			<td>St. Louis 2015</td>
			<td>Sheboygan 2012</td>
			<td>Milwaukee 2013</td>
		</tr>
		<tr>
			<td>Papilloma</td>
			<td>21</td>
			<td>5.2</td>
			<td>30.5</td>
			<td>37.5</td>
		</tr>
		<tr>
			<td>Squamous cell carcinoma</td>
			<td>0</td>
			<td>0</td>
			<td>2.1</td>
			<td>10.5</td>
		</tr>
		<tr>
			<td>Total skin neoplasms</td>
			<td>21</td>
			<td>5.2</td>
			<td>32.6</td>
			<td>48</td>
		</tr>
		<tr>
			<td>Bile duct neoplasmsa</td>
			<td>2.5</td>
			<td>4</td>
			<td>6.2</td>
			<td>9.5</td>
		</tr>
		<tr>
			<td>Hepatic cell neoplasmsb</td>
			<td>1</td>
			<td>0</td>
			<td>2.1</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Total liver neoplasms</td>
			<td>3.5</td>
			<td>4</td>
			<td>8.3</td>
			<td>15.0c</td>
		</tr>
		<tr>
			<td colspan="3">aIncludes cholangioma and cholangiocarcinoma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">bIncludes hepatic cell adenoma and hepatic cell carcinoma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">cSome fish had both bile duct and hepatic neoplasms</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Microscopically Verified Neoplastic Lesions of White Sucker Collected at Great Lakes Areas of Concern and a Reference Site (Kewaunee River), Presented as a Percentage. 
The histopathological analysis also identified liver tumors that were not identified by visual observation. While only 1.5% of the fish collected from Kewaunee and St. Louis had visible liver nodules (Table 1), 3.5% and 4.0%, respectively, had microscopically identified neoplasms (Table 2). A larger difference was seen at Sheboygan (1.6% visible versus 8.3% microscopic) and Milwaukee (2.5% visible versus 15.0% microscopic). Microscopic examination also provides a differentiation of neoplasms of bile duct versus hepatic cell origin (Table 2) and benign versus malignant tumors.

Watch this Scientific Journal Video about Necropsy-based Wild Fish Health Assessment at JoVE.com

Necropsy-based Wild Fish Health Assessment

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

necropsy-based-wild-fish-health-assessment

Research

JoVE Journal

Environment

16.9K Views.  U.S. Geological Survey. The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Video: Necropsy-based Wild Fish Health Assessment

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread breeding distribution, that has been recently listed as endangered in some habitats of its range. Waterbird monitoring programs exist to track populations through time; however, some of the more intensive approaches require entering colonies and can be disruptive to nesting populations. This paper describes a protocol that utilizes a minimally invasive surveillance system to continuously monitor common tern nesting behavior in typical ground-nesting colonies. The video monitoring system utilizes wireless cameras focused on individual nests as well as over the colony as a whole, and allows for observation without entering the colony. The video system is powered with several 12 V car batteries that are continuously recharged using solar panels. Footage is recorded using a digital video recorder (DVR) connected to a hard drive, which can be replaced when full. The DVR may be placed outside of the colony to reduce disturbance. In this study, 3,624 h of footage recorded over 63 days in weather conditions ranging from 12.8 &#176;C to 35.0 &#176;C produced 3,006 h (83%) of usable behavioral data. The types of data retrieved from the recorded video can vary; we used it to detect external disturbances and measure nesting behavior during incubation. Although the protocol detailed here was designed for ground-nesting waterbirds, the principal system could easily be modified to accommodate alternative scenarios, such as colonial arboreal nesting species, making it widely applicable to a variety of research needs.

A Video Surveillance System to Monitor Breeding Colonies of Common Terns Sterna Hirundo

This paper describes a protocol that uses a remote video monitoring surveillance system to continuously monitor breeding colonies of ground-nesting waterbirds. The system includes five cameras monitoring individual nests and one camera monitoring the colony as a whole, and is powered by car batteries that are recharged via solar panels.

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...