This work was funded, in part, by the Great Lakes Fishery Commission and the Annis Water Resources Institute. Use of trade, product, or firm names does not imply endorsement by the U. S. Government. This article is Contribution 1867 of the U. S. Geological Survey Great Lakes Science Center.

1. Laboratory Experiment
<ol>
	<li>Obtain the prey fish to be fed to the predator fish during the experiment. Preferably these prey fish should be captured in the field, frozen, and stored at about -30 &#176;C. Consider commercial fishing operations as a potential source for the prey fish.</li>
	<li>Introduce the predator fish into the laboratory tanks to be used for the experiment. Up to 15 predator fish have been introduced into each of 870-L tanks, and up to 30 predator fish have been introduced into each of 2,380-L tanks in previous studies16,18.</li>
	<li>Acclimate the predator fish to a diet of the selected prey fish. Once acclimated, the predator fish should remain on this diet for 1-3 months before beginning the experiment.</li>
	<li>Set aside samples of prey fish by randomly selecting 10 to 20 composite samples from the batch of prey fish. Number of prey fish in a composite sample could range from 3 to 100, depending on the size of the prey fish. Each composite sample should be double bagged, frozen, and stored at about -30 &#176;C.</li>
	<li>Initiate the experiment by sacrificing 30 to 50% of the fish in each of the tanks.
	<ol>
		<li>To euthanize the fish, mix 8 g of Finquel with 45 L of water in a large plastic container and then place the fish in the container with the Finquel solution.</li>
		<li>Once euthanized, place all of the sacrificed fish from one tank into a bag, then double bag, and store at about -30 &#176;C until time of the processing.</li>
		<li>Weigh each of the fish remaining in each of the tanks, and record the weights; an anesthetic will likely be needed to conduct the weighing.</li>
		<li>To anesthetize the fish, mix 4.6 g of Finquel with 45 L of water in a large plastic container, and then place the fish in the container with the Finquel solution.</li>
		<li>Wait a few minutes for the anesthetic to take effect before weighing the fish.</li>
	</ol>
	</li>
	<li>On each day of the experiment, thaw an appropriate amount of prey fish, and cut the prey fish into pieces weighing roughly 1 to 5 g. Weigh the quantity of prey fish to be placed in each of the tanks, then drop the prey fish pieces into each tank and allow the predator fish about 1 hr to feed. Then remove all uneaten food, allow the food to air dry for about 20 min, and then weigh the uneaten food for each of the tanks. Record the amount of food placed in the tank and the amount of uneaten food for each of the tanks each day. 
	NOTE: For the representative experiment, lake trout were fed as much food as they would consume during one feeding period each day18. However, the predator fish could also be placed on fixed rations16,19.</li>
	<li>Terminate the experiment by sacrificing all of the remaining predator fish in each of the tanks. To euthanize the fish, mix 8 g of Finquel with 45 L of water in a large plastic container and then place the fish in the container with the Finquel solution. Record the weight of each of the sacrificed fish. For reliable results, the experiment must run for at least 100 days, preferably for at least 130 days. Place all of the fish from a tank into one bag, then double bag, and store at about -30 &#176;C until time of processing.</li>
</ol>
2. Fish Homogenization
<ol>
	<li>Select a set of predator fish and/or prey fish composites for thawing. Allow the composites to partially thaw. Each composite may require 0.5 to 1 hr to homogenize.</li>
	<li>Using the appropriate-sized blenders, homogenize each of the composites. For each composite, place a sample (from 50 to 100 g) of the homogenate into a cleaned, acetone-rinsed, and labeled jar. Then cap the jar and store the jar at about -30 &#176;C until the time of processing.</li>
	<li>Wash all equipment used to homogenize the fish, and then properly rinse with distilled water and methanol, between samples.</li>
</ol>
3. Extraction
<ol>
	<li>Weigh 20.0 g of thawed homogenized fish tissue in a 200-ml beaker.</li>
	<li>Add approximately 40 g of sodium sulfate and mix well with spatula.</li>
	<li>Add surrogate spike solution containing congeners 30, 61, 161, and 166. Spike at a concentration that yields a final concentration of 20 ng/ml in the extract.</li>
	<li>Allow the sample to dry at RT while mixing every 20 min.</li>
	<li>Allow the sample to reach a consistency of dry sand, at which point the sample is ready for extraction.</li>
	<li>Set up the Soxhlet extraction apparatus with a 500-ml flask containing Teflon boil chips, Soxhlet, and condenser.</li>
	<li>Add the dried fish mixture to a glass thimble with a coarse-fritted disc bottom or paper thimble.</li>
	<li>Add 150 ml of 50% hexane and 50% dichloromethane to the beaker used for the sample and stir while scraping the walls of the beaker with a spatula.</li>
	<li>Transfer the solvent to the top of the Soxhlet with the flask attached and allow it to cycle through the Soxhlet and into the flask.</li>
	<li>Repeat a second time with 150 ml again.</li>
	<li>Place the Soxhlet with the attached flask onto the heating element and attach the condenser.</li>
	<li>Turn on the heating element and bring the solvent to a gentle boil, then extract for a minimum of 16 hr making sure that cold water is supplied to the condensers.</li>
	<li>After allowing the solvent to cool, check to see if any of the sample flasks contain water. For those flasks containing water, add sodium sulfate and swirl until the water is absorbed by the sodium sulfate.</li>
	<li>Concentrate the sample using a nitrogen sample concentrator or a Kaderna Danish (KD) glassware setup with a hot water bath.</li>
	<li>Allow the sample to evaporate to a volume of less than 2 ml, and then bring to a final volume of 5 ml by using small washings of hexane to transfer the sample from the glassware used to a 5-ml volumetric flask.</li>
	<li>Transfer to a 10-ml vial and label with sample information.</li>
</ol>
4. Extract Clean-up
<ol>
	<li>Prepare acidified silica gel by adding 44 g of concentrated sulfuric acid to 100 g of activated silica gel.</li>
	<li>Add 10 g of acidified silica gel into a small chromatography column containing a small plug of glass wool at the bottom.</li>
	<li>Add 1 ml of sample extract to the column after pre-cleaning the column with 10 ml of hexane.</li>
	<li>Elute the column with 20 ml of hexane and collect in tapered 20-ml glass tube.</li>
	<li>Place the glass tube on the nitrogen evaporator (N-Vap) apparatus under a stream of nitrogen and immersed in hot water.</li>
	<li>Evaporate to less than 1 ml but not to dryness.</li>
	<li>Remove from the N-Vap apparatus and transfer to 1-ml volumetric flask with small washings of hexane.</li>
	<li>Transfer to a 1.8 ml autosampler vial labeled with sample information.</li>
	<li>Spike 4 &#956;l of internal standard into the vial. The sample is now ready for analysis.</li>
</ol>
5. Analysis by Gas Chromatography &#8211; Mass Spectrometry Using Negative Chemical Ionization
<ol>
	<li>Use standards to calibrate the instrument: The standards are available in mixtures consisting of groups of well separated congeners. Mixes 1-5 consist of nearly all of the congeners found in Arochlors 1016, 1221, 1232, 1242, 1248, 1254, and 1260. Mix 1 is used as a multi-level calibration mix, and system linearity is confirmed by preparing at least five calibration levels at concentrations between 2 and 100 ng/ml. Mixes 2-5 are used as single point calibrations for each congener.</li>
	<li>Set up the chromatography &#8211; mass spectrometry system in the negative chemical ionization mode with hydrogen as the carrier gas (1 ml/min) and methane as the reagent gas.</li>
	<li>Use a fused-silica, capillary column (60 m &#215; 0.25 mm inner diameter) coated with DB-XLB at 0.25-&#956;m film thickness for separation. Program oven temperature from 60 to 212 &#176;C at 25 &#176;C/min, then to 260 &#176;C at 1 &#176;C/min, and then to 280 &#176;C at 4 &#176;C/min, with a final hold time of 4 min. Injector and transfer line temperatures should be set at 280 &#176;C. Inject 1 to 2 &#956;l of the sample using the splitless injection mode.</li>
	<li>Analyze all standards and samples by the internal standard method using 13C-labeled decachlorobiphenyl.</li>
	<li>Perform a check on the initial calibration by running a second source standard and Aroclors 1242 and 1260, and then compare predicted values for the Aroclor congeners with the observed amounts from this check procedure.</li>
	<li>Once the initial calibration procedure has been successfully accomplished, complete analysis of all samples. Run a calibration check every ten samples, using any of the calibration mixtures from the initial calibration.</li>
</ol>
6. Calculation of Net Trophic Transfer Efficiency
<ol>
	<li>Calculate net trophic transfer efficiency, &#947;, for each combination of tank and PCB congener using the following equation: 
	<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/51496/51496eq1.jpg" />, where [PCBf] is the average PCB congener concentration of the predator fish in the tank at the end of the experiment, Wf is the average weight of the predator fish in the tank at the end of the experiment, [PCBi] is the average PCB congener concentration of the predator fish in the tank at the start of the experiment, Wi is the average weight of the predator fish at the start of the experiment, and the amount of PCB congener ingested refers to the weight of the PCB congener ingested, on average, by each lake trout in the tank during the course of the experiment.</li>
	<li>Calculate the denominator in the above equation by multiplying the average concentration of the PCB congener in the prey fish composites by the average amount (weight) of the prey fish eaten per predator fish in the tank during the entire course of the experiment.</li>
</ol>

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For the most accurate estimates of &#947;, the experimenter must be able to accurately track both the amount of food placed in each of the tanks and the amount of uneaten food in each of the tanks during the course of the experiment. To accomplish this, the experimenter must be able to remove all of the uneaten food from the tanks and accurately determine its weight. In addition to accurate tracking of the food actually eaten by the predator fish, accurate estimation of &#947; may also depend on sufficient duration of th...

Of all of the factors affecting the rate at which fish accumulate contaminants, the efficiency with which fish retain contaminants from the food that they eat is one of the most important1-3. Risk assessment models have been developed to predict future risks to both people and wildlife eating contaminated fish under various scenarios of environmental contamination, and the reliability of these predictions critically depends on the accuracy of the estimates of the efficiency at which fish retain contaminants from their food4.
The efficiency with which the contaminant in the food ingested by the predator is transported through the gut wall is known as gross trophic transfer efficiency5. A portion of the quantity of the contaminant transported through the gut wall of the predator may eventually be lost through depuration and/or metabolic transformation. The efficiency with which the contaminant in the food ingested by the predator is retained by the predator, including any losses due to elimination and metabolic transformation, is known as net trophic transfer efficiency6.
Gross trophic transfer efficiency of organic contaminants to fish from their prey appears to vary with the contaminant&#8217;s chemical properties, including lipid affiliation as measured by the octanol-water partition coefficient, Kow3,7. According to an empirical relationship developed by Thomann3, gross trophic transfer efficiency is relatively high when log Kow is equal to a value between 5 and 6. Gross trophic transfer efficiency declines exponentially at a rate of 50% per unit of log Kow as log Kow increases from 6 to 10, according to the Thomann3 relationship.
Nevertheless, the gross and net trophic transfer efficiencies of polychlorinated biphenyl (PCB) congeners to fish from their prey do not appear to follow the Thomann3 relationship in most cases. Although the trophic transfer efficiencies of PCB congeners to lake whitefish (Coregonus clupeaformis) from its food followed the relationship proposed by Thomann8, trophic transfer efficiencies of PCB congeners were either just weakly related or not related at all to log Kow for Atlantic salmon (Salmo salar)9, rainbow trout (Oncorhynchus mykiss)10, coho salmon (Oncorhynchus kisutch)11, and northern pike (Esox lucius)11.
The overall goal of this study was to present a laboratory technique for estimating the net trophic transfer efficiencies of PCB congeners to a piscivorous fish from its prey. Lake trout (Salvelinus namaycush) was chosen as the piscivorous fish for our experiment because lake trout are relatively easy to maintain in laboratory tanks. Bloater (Coregonus hoyi) was selected as the prey fish to be fed to the lake trout because bloater is eaten by lake trout in its natural setting12. In addition, we determined whether the net trophic transfer efficiencies for lake trout estimated from our laboratory experiment followed the Thomann3 relationship. We also determined whether the degree of activity by the lake trout had a significant effect on net trophic transfer efficiency (&#947;) of the PCB congeners. Activity by lake trout in the Laurentian Great Lakes is believed to have recently increased because changes in the food webs have caused lake trout to allocate more energy toward searching for food13. Lake trout were forced to exercise in one set of tanks by subjecting these lake trout to relatively high flow rates, whereas the other lake trout were permitted to remain relatively inactive by subjecting them to relatively low flow rates. Finally, the specific details of our laboratory procedure that need to be carefully followed to ensure the highest degree of accuracy in the &#947; estimates and to make the laboratory results applicable to the field are discussed, as well as future directions for research building on our laboratory technique. Net trophic transfer efficiency can be estimated both in the laboratory and in the field, and advantages and disadvantages are associated with both approaches. Accuracy in the estimate of &#947; depends on the accuracy of the estimate of food consumption. The amount of food eaten by fish in the laboratory can be accurately determined when proper protocols are followed, whereas the amount of food eaten by fish in the field is typically estimated via bioenergetics modeling. Use of bioenergetics modeling to derive the amount of food eaten has the potential to introduce a substantial amount of uncertainty into the estimates of food consumption. Fish bioenergetics models have been shown to estimate food consumption with no detectable bias for the case of lake trout14,15, but considerable bias in bioenergetics model estimates of food consumption has been detected for the case of lake whitefish15,16. On the other hand, estimates of net trophic transfer efficiency estimated in the laboratory may not be applicable to the field due to a difference in feeding rates between the laboratory and the field17. Evidence from both the laboratory and the field suggest that feeding rate can influence &#947;14,17.
The methodology used in the present study for estimating &#947; in the laboratory is applicable to situations where the predator fish is fed prey fish, and the amount of prey fish eaten by the predator can be accurately tracked. To accomplish this, the experimenter must weigh all of the food before placement in the tank; and the experimenter must be able to remove all of the uneaten food from the tank, and then weigh the uneaten food. In addition, an adequate suite of mixers and blenders should be available to obtain a sufficient degree of homogenization of the samples of both predator and prey fish. Finally, the gas chromatography &#8211; mass spectrometry instrumentation used to determine the PCB congener concentrations must be capable of detecting and quantifying individual PCB congeners at relatively low concentrations.

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			<td height="21" style="height:21px;width:259px;">870-L fiberglass tanks</td>
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			<td style="width:167px;">Eugenol could also be used as an anesthetic.</td>
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			<td style="width:87px;">Wilmad-Lab Glass</td>
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			<td style="width:87px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:119px;">Cat. 211-4</td>
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			<td height="35" style="height:35px;width:259px;">Dichloromethane</td>
			<td style="width:87px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:119px;">Cat. 300-4</td>
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			<td height="21" style="height:21px;width:259px;">Silica gel</td>
			<td style="width:87px;">BDH</td>
			<td style="width:119px;">Cat. BDH9004-1KG</td>
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			<td height="35" style="height:35px;width:259px;">Labl Line 5000 mult-unit extraction heater</td>
			<td style="width:87px;">Lab Line Instruments</td>
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			<td height="35" style="height:35px;width:259px;">Agilent 5973 GC/MS with chemical ionization</td>
			<td style="width:87px;">Agilent</td>
			<td style="width:119px;">5973N</td>
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			<td height="52" style="height:52px;width:259px;">Internal standard solution&#160;</td>
			<td style="width:87px;">Cambridge Isotope Laboratories</td>
			<td style="width:119px;">EC-1410-1.2</td>
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			<td height="21" style="height:21px;width:259px;">PCB congener calibration standards</td>
			<td style="width:87px;">Accustandard</td>
			<td style="width:119px;">C-CSQ-SET</td>
			<td></td>
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			<td height="21" style="height:21px;width:259px;">DB-XLB column (60m x 0.25mm, 0.25 micron)</td>
			<td style="width:87px;">Agilent/ J&#38;W</td>
			<td style="width:119px;">122-1262</td>
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laboratory estimation of net trophic transfer efficiencies of pcb congeners to lake trout (salvelinus namaycush) from its prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Lake trout showed a substantial amount of growth during the experiment, as the initial lake trout mean weights ranged from 694 to 907 g while the final lake trout mean weights ranged from 853 to 1,566 g (Table 1). The average amount of food consumed by a lake trout during the course of the 135-day experiment ranged from 641 to 2,649 g. Mean PCB congener concentrations in the lake trout increased during the experiment, as mean PCB congener concentrations ranged from 0.01 to 7.14 ng/g (wet-weight basis) at...

Watch this Scientific Journal Video about Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey at JoVE.com

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

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JoVE Journal

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10.9K Views.  U. S. Geological Survey. A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

Video: Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

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 ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...