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Standardized methods for Grand Canyon fisheries research 2015

This document presents protocols and guidelines to persons sampling fishes in the Grand Canyon, to help ensure consistency in fish handling, fish tagging, and data collection among different projects and organizations. Most such research and monitoring projects are conducted under the general umbrella of the Glen Canyon Dam Adaptive Management Program and include studies by the U.S. Geological Survey (USGS), U.S. Fish and Wildlife Service (FWS), National Park Service (NPS), the Arizona Game and Fish Department (AGFD), various universities, and private contractors. This document is intended to provide guidance to fieldworkers regarding protocols that may vary from year to year depending on specific projects and objectives. We also provide herein documentation of standard methods used in the Grand Canyon that can be cited in scientific publications, as well as a summary of changes in protocols since the document was first created in 2002.

Grand Canyon↗

Load estimator (LOADEST): a FORTRAN program for estimating constituent loads in streams and rivers

LOAD ESTimator (LOADEST) is a FORTRAN program for estimating constituent loads in streams and rivers. Given a time series of streamflow, additional data variables, and constituent concentration, LOADEST assists the user in developing a regression model for the estimation of constituent load (calibration). Explanatory variables within the regression model include various functions of streamflow, decimal time, and additional user-specified data variables. The formulated regression model then is used to estimate loads over a user-specified time interval (estimation). Mean load estimates, standard errors, and 95 percent confidence intervals are developed on a monthly and(or) seasonal basis. The calibration and estimation procedures within LOADEST are based on three statistical estimation methods. The first two methods, Adjusted Maximum Likelihood Estimation (AMLE) and Maximum Likelihood Estimation (MLE), are appropriate when the calibration model errors (residuals) are normally distributed. Of the two, AMLE is the method of choice when the calibration data set (time series of streamflow, additional data variables, and concentration) contains censored data. The third method, Least Absolute Deviation (LAD), is an alternative to maximum likelihood estimation when the residuals are not normally distributed. LOADEST output includes diagnostic tests and warnings to assist the user in determining the appropriate estimation method and in interpreting the estimated loads. This report describes the development and application of LOADEST. Sections of the report describe estimation theory, input/output specifications, sample applications, and installation instructions.

Techniques and Methods↗

Characterization of the host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy, and immunological techniques

The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta ‐susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.

Journal of Fish Diseases↗

Temperature

The “National Field Manual for the Collection of Water-Quality Data” (NFM) provides guidelines and procedures for U.S. Geological Survey (USGS) personnel who collect data used to assess the quality of the Nation’s surface-water and groundwater resources. This chapter, NFM A6.1, provides guidance and protocols for the measurement of temperature of air, of a surface-water body or in groundwater, which include the scientific basis of the measurement, selection and maintenance of equipment, calibration verification, troubleshooting, and procedures for measurement and reporting. It updates and supersedes USGS Techniques of Water-Resources Investigations, book 9, chapter A6.1, version 2.0, by Franceska D. Wilde. Temperature of air and water is routinely measured when water samples are collected, is often measured continually at USGS streamgages, and is a parameter regularly measured during laboratory and field experiments. The field method for measuring temperature described in this chapter is applicable to air and most natural waters. Before 2017, the NFM chapters were released in the USGS Techniques of Water-Resources Investigations series. Effective in 2018, new and revised NFM chapters are being released in the USGS Techniques and Methods series; this series change does not affect the content and format of the NFM. More information is in the general introduction to the NFM (USGS Techniques and Methods, book 9, chapter A0) at https://doi.org/10.3133/tm9A0 . The authoritative current versions of NFM chapters are available in the USGS Publications Warehouse at https://pubs.usgs.gov/ . Comments, questions, and suggestions related to the NFM can be addressed to nfm@usgs.gov .

Techniques and Methods↗

U.S. Geological Survey groundwater toolbox, a graphical and mapping interface for analysis of hydrologic data (version 1.0): user guide for estimation of base flow, runoff, and groundwater recharge from streamflow data

This report is a user guide for the streamflow-hydrograph analysis methods provided with version 1.0 of the U.S. Geological Survey (USGS) Groundwater Toolbox computer program. These include six hydrograph-separation methods to determine the groundwater-discharge (base-flow) and surface-runoff components of streamflow—the Base-Flow Index (BFI; Standard and Modified), HYSEP (Fixed Interval, Sliding Interval, and Local Minimum), and PART methods—and the RORA recession-curve displacement method and associated RECESS program to estimate groundwater recharge from streamflow data. The Groundwater Toolbox is a customized interface built on the nonproprietary, open source MapWindow geographic information system software. The program provides graphing, mapping, and analysis capabilities in a Microsoft Windows computing environment. In addition to the four hydrograph-analysis methods, the Groundwater Toolbox allows for the retrieval of hydrologic time-series data (streamflow, groundwater levels, and precipitation) from the USGS National Water Information System, downloading of a suite of preprocessed geographic information system coverages and meteorological data from the National Oceanic and Atmospheric Administration National Climatic Data Center, and analysis of data with several preprocessing and postprocessing utilities. With its data retrieval and analysis tools, the Groundwater Toolbox provides methods to estimate many of the components of the water budget for a hydrologic basin, including precipitation; streamflow; base flow; runoff; groundwater recharge; and total, groundwater, and near-surface evapotranspiration.

Techniques and Methods↗

MODFLOW–USG version 1: An unstructured grid version of MODFLOW for simulating groundwater flow and tightly coupled processes using a control volume finite-difference formulation

A new version of MODFLOW, called MODFLOW–USG (for UnStructured Grid), was developed to support a wide variety of structured and unstructured grid types, including nested grids and grids based on prismatic triangles, rectangles, hexagons, and other cell shapes. Flexibility in grid design can be used to focus resolution along rivers and around wells, for example, or to subdiscretize individual layers to better represent hydrostratigraphic units. MODFLOW–USG is based on an underlying control volume finite difference (CVFD) formulation in which a cell can be connected to an arbitrary number of adjacent cells. To improve accuracy of the CVFD formulation for irregular grid-cell geometries or nested grids, a generalized Ghost Node Correction (GNC) Package was developed, which uses interpolated heads in the flow calculation between adjacent connected cells. MODFLOW–USG includes a Groundwater Flow (GWF) Process, based on the GWF Process in MODFLOW–2005, as well as a new Connected Linear Network (CLN) Process to simulate the effects of multi-node wells, karst conduits, and tile drains, for example. The CLN Process is tightly coupled with the GWF Process in that the equations from both processes are formulated into one matrix equation and solved simultaneously. This robustness results from using an unstructured grid with unstructured matrix storage and solution schemes. MODFLOW–USG also contains an optional Newton-Raphson formulation, based on the formulation in MODFLOW–NWT, for improving solution convergence and avoiding problems with the drying and rewetting of cells. Because the existing MODFLOW solvers were developed for structured and symmetric matrices, they were replaced with a new Sparse Matrix Solver (SMS) Package developed specifically for MODFLOW–USG. The SMS Package provides several methods for resolving nonlinearities and multiple symmetric and asymmetric linear solution schemes to solve the matrix arising from the flow equations and the Newton-Raphson formulation, respectively.

Techniques and Methods↗

A comparative study of surface waves inversion techniques at strong motion recording sites in Greece

Surface wave method was used for the estimation of Vs vs depth profile at 10 strong motion stations in Greece. The dispersion data were obtained by SASW method, utilizing a pair of electromechanical harmonic-wave source (shakers) or a random source (drop weight). In this study, three inversion techniques were used a) a recently proposed Simplified Inversion Method (SIM), b) an inversion technique based on a neighborhood algorithm (NA) which allows the incorporation of a priori information regarding the subsurface structure parameters, and c) Occam's inversion algorithm. For each site constant value of Poisson's ratio was assumed (ν=0.4) since the objective of the current study is the comparison of the three inversion schemes regardless the uncertainties resulting due to the lack of geotechnical data. A penalty function was introduced to quantify the deviations of the derived Vs profiles. The Vs models are compared as of Vs(z), Vs30 and EC8 soil category, in order to show the insignificance of the existing variations. The comparison results showed that the average variation of SIM profiles is 9% and 4.9% comparing with NA and Occam's profiles respectively whilst the average difference of Vs30 values obtained from SIM is 7.4% and 5.0% compared with NA and Occam's.

Conference Paper↗

Determination of human-health pharmaceuticals in filtered water by chemically modified styrene-divinylbenzene resin-based solid-phase extraction and high-performance liquid chromatography/Mass Spectrometry

In 1999, the Methods Research and Development Program of the U.S. Geological Survey National Water Quality Laboratory began the process of developing a method designed to identify and quantify human-health pharmaceuticals in four filtered water-sample types: reagent water, ground water, surface water minimally affected by human contributions, and surface water that contains a substantial fraction of treated wastewater. Compounds derived from human pharmaceutical and personal-care product use, which enter the environment through wastewater discharge, are a newly emerging area of concern; this method was intended to fulfill the need for a highly sensitive and highly selective means to identify and quantify 14 commonly used human pharmaceuticals in filtered-water samples. The concentrations of 12 pharmaceuticals are reported without qualification; the concentrations of two pharmaceuticals are reported as estimates because long-term reagent-spike sample recoveries fall below acceptance criteria for reporting concentrations without qualification. The method uses a chemically modified styrene-divinylbenzene resin-based solid-phase extraction (SPE) cartridge for analyte isolation and concentration. For analyte detection and quantitation, an instrumental method was developed that used a high-performance liquid chromatography/mass spectrometry (HPLC/MS) system to separate the pharmaceuticals of interest from each other and coextracted material. Immediately following separation, the pharmaceuticals are ionized by electrospray ionization operated in the positive mode, and the positive ions produced are detected, identified, and quantified using a quadrupole mass spectrometer. In this method, 1-liter water samples are first filtered, either in the field or in the laboratory, using a 0.7-micrometer (μm) nominal pore size glass-fiber filter to remove suspended solids. The filtered samples then are passed through cleaned and conditioned SPE cartridges at a rate of about 15 milliliters per minute. Excess water is eliminated from the cartridge sorbent bed by passing air through the cartridges, and the analytes retained on the SPE bed are eluted from the cartridge sequentially, first with methanol, followed by acidified methanol, and combined in collection tubes. This sample extract then is reduced from about 10 milliliters (mL) to about 0.1 mL (or 100 microliters) under a stream of purified nitrogen gas with the collection tubes in a heated (40°C) water bath. The reduced extracts then are fortified with an internal standard solution (when using internal standard quantitation), brought to a final volume of 1 mL with an aqueous ammonium formate buffer solution, and filtered through a 0.2-μm Teflon syringe filter as they are transferred into vials for instrumental analysis. Instrumental analysis by the HPLC/MS procedure permits determination of individual pharmaceutical concentrations from 0.005 to 1.0 microgram per liter, based on the lowest and the highest calibration standards routinely used. The reporting levels for this method are compound dependent, and have been experimentally determined based on the precision of quantitation of compounds from eight fortified organic-free water samples in single-operator experiments. The method detection limits and interim reporting levels for the compounds determined by this method were calculated from recoveries of the pharmaceuticals from reagent-water samples amended at 0.05 microgram per liter, and ranged between 0.0069 and 0.0142 microgram per liter, and 0.015 and 0.10 microgram per liter, respectively. Concentrations for 12 compounds are reported without qualification, and for two compounds are reported as qualified estimates. After initial development, the method was applied to more than 1,800 surface-, ground-, and wastewater samples from 2002 to 2005 and documented in a number of published studies. This research application of the method provided the opportunity to collect a large data set of ambient environmental concentrations and also permitted the collection of an extensive set of reagent blanks and spike quality-control (QC) samples. This multiple-year set of QC samples enabled further evaluation of method performance under multiple operator and multiple instrument conditions typical of routine laboratory operation. These results are an important part of the entire data set contained in this report because they document method performance over an extended time. Because sample matrix can substantially affect method performance, inclusion of environmental matrix-spike samples is required as a routine component of study plan quality control. Method performance has been measured by long-term tracking of observed recoveries from fortified organic-free water samples processed with environmental samples (laboratory reagent spikes), as well as by observed recoveries from multiple fortified environmental water samples. The fortified environmental samples included surface water, wastewater effluent-dominated surface water, and ground water, fortified at two environmentally relevant concentrations and corrected for ambient environmental concentrations. Because the responses of individual pharmaceuticals vary as a function of proton affinity, the ionization efficiency, and thus relative response, of each pharmaceutical, the quality-control surrogate compounds, and the quantitation internal standard can be suppressed or enhanced by the presence of the sample matrix. As a result, several quality-control sample types are required to properly interpret the ambient environmental concentrations of pharmaceuticals in aqueous samples. The quality-control sample types and results include laboratory reagent spikes and laboratory reagent blanks to provide insight into the performance of the method in the absence of a sample matrix, and matrix-spike recovery samples and replicate environmental samples, collected from representative sample matrix types within the aquatic system under study.

Techniques and Methods↗

A cryopreservation method for Pasteurella multocida from wetland samples

A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

California, Nebraska, Nevada↗

Methods for the preparation and analysis of solids and suspended solids for total mercury

The methods documented in this report are utilized by the Wisconsin District Mercury Lab for analysis of total mercury in solids (soils and sediments) and suspended solids (isolated on filters). Separate procedures are required for the different sample types. For solids, samples are prepared by room-temperature acid digestion and oxidation with aqua regia. The samples are brought up to volume with a 5 percent bromine monochloride solution to ensure complete oxidation and heated at 50C in an oven overnight. Samples are then analyzed with an automated flow injection system incorporating a cold vapor atomic fluorescence spectrometer. A method detection limit of 0.3 ng of mercury per digestion bomb was established using multiple analyses of an environmental sample. Based on the range of masses processed, the minimum sample reporting limit varies from 0.6 ng/g to 6 ng/g. Suspended solids samples are oxidized with a 5 percent bromine monochloride solution and held at 50?C in an oven for 5 days. The samples are then analyzed with an automated flow injection system incorporating a cold vapor atomic fluorescence spectrometer. Using a certified reference material as a surrogate for an environmental sample, a method detection limit of 0.059 ng of mercury per filter was established. The minimum sample reporting limit varies from 0.059 ng/L to 1.18 ng/L, depending on the volume of water filtered.

Techniques and Methods↗

Bayesian modeling of non-stationary, univariate, spatial data for the Earth sciences

Some Earth science data, such as geochemical measurements of element concentrations, are non-stationary—the mean and the standard deviation vary spatially. It is important to estimate the spatial variations in both statistics because such information is indicative of geological and other Earth processes. To this end, an estimation method is formulated as a Bayesian hierarchical model. The method represents the spatially varying mean and the spatially varying standard deviation with basis functions; this formulation implicitly accounts for a spatially varying covariance function. A unique advantage of this method is that it can map the mean, the standard deviation, quantiles, and exceedance probabilities. The method is demonstrated by mapping titanium concentrations, which are measured in the coastal plain of the southeastern United States. Various checks demonstrate that the model fits the data and that the estimated statistics are geologically plausible.

Techniques and Methods↗

Selection and application of microbial source tracking tools for water-quality investigations

Microbial source tracking (MST) is a complex process that includes many decision-making steps. Once a contamination problem has been defined, the potential user of MST tools must thoroughly consider study objectives before deciding upon a source identifier, a detection method, and an analytical approach to apply to the problem. Regardless of which MST protocol is chosen, underlying assumptions can affect the results and interpretation. It is crucial to incorporate tests of those assumptions in the study quality-control plan to help validate results and facilitate interpretation. Detailed descriptions of MST objectives, protocols, and assumptions are provided in this report to assist in selection and application of MST tools for water-quality investigations. Several case studies illustrate real-world applications of MST protocols over a range of settings, spatial scales, and types of contamination. Technical details of many available source identifiers and detection methods are included as appendixes. By use of this information, researchers should be able to formulate realistic expectations for the information that MST tools can provide and, where possible, successfully execute investigations to characterize sources of fecal contamination to resource waters.

Techniques and Methods↗

Chapter A6.2. Dissolved oxygen

The “National Field Manual for the Collection of Water-Quality Data” (NFM) provides guidelines and procedures for U.S. Geological Survey (USGS) personnel who collect data used to assess the quality of the Nation’s surface water and groundwater resources. This chapter, NFM A6.2, provides guidance and protocols for the measurement of dissolved oxygen, which include the scientific basis of the measurement, selection and maintenance of equipment, calibration, troubleshooting, and procedures for measurement and reporting. It updates and supersedes USGS Techniques of Water-Resources Investigations, book 9, chapter A6.2, version 3.0, by Stewart A, Rounds, Franceska D. Wilde, and George F. Ritz. Dissolved oxygen is routinely measured when water samples are collected, is often continually measured at USGS streamgages, and is a parameter regularly measured during laboratory and field experiments. The field method for measuring dissolved oxygen described in this chapter is applicable to most natural waters. Before 2017, the USGS NFM chapters were released in the USGS Techniques of Water-Resources Investigations series. Effective in 2018, new and revised NFM chapters are being released in the USGS Techniques and Methods series; this series change does not affect the content and format of the NFM. More information is in the general introduction to the NFM (USGS Techniques and Methods, book 9, chapter A0—U.S. Geological Survey, 2018) at https://doi.org/10.3133/tm9A0 . The authoritative current versions of NFM chapters are available in the USGS Publications Warehouse at https://pubs.er.usgs.gov . Comments, questions, and suggestions related to the NFM can be addressed to nfm@usgs.gov .

Techniques and Methods↗

dMODELS, a MATLAB software package for modeling crustal deformation near volcanic centers and active faults using Global Navigation Satellite System data—User guide

dMODELS is a MATLAB software package that implements the most common analytical models used to interpret deformation measurements near faults and active volcanic centers. This manual focuses on inversion of deformation data from the Global Navigation Satellite System (GNSS). The included case studies emphasize the GNSS inversion component of the software. Source models include pressurized spherical, spheroidal, and horizontal sill (penny-crack) magma reservoirs in a homogeneous, elastic, isotropic, flat half-space. A topography correction is available for the spherical source. Dikes and faults are described following the mathematical notation for the rectangular dislocations in a homogeneous, elastic, flat half-space. Equations have been reviewed for typographical errors present in the original literature and verified against finite-element method numerical models. GNSS data from the 2006 eruption at Augustine Volcano, Alaska; the 1998–2000 unrest at Taal Volcano, Philippines; and the 2009 earthquake in L’Aquila, Italy, are used to demonstrate the application of the software package.

Techniques and Methods↗

SWB Version 2.0—A soil-water-balance code for estimating net infiltration and other water-budget components

The U.S. Geological Survey’s Soil-Water-Balance (SWB) code was developed as a tool to estimate distribution and timing of net infiltration out of the root zone by means of an approach that uses readily available data and minimizes user effort required to begin a SWB application. SWB calculates other components of the water balance, including soil moisture, reference and actual evapotranspiration, snowfall, snowmelt, canopy interception, and crop-water demand. SWB is based on a modified Thornthwaite-Mather soil-water-balance approach, with components of the soil-water balance calculated at a daily time step. Net-infiltration calculations are computed by means of a rectangular grid of computational elements, which allows the calculated infiltration rates to be imported into grid-based regional groundwater-flow models. SWB makes use of gridded datasets, including datasets describing hydrologic soil groups, moisture-retaining capacity, flow direction, and land use. Climate data may be supplied in gridded or tabular form. The SWB 2.0 code described in this report extends capabilities of the original SWB version 1.0 model by adding new options for representing physical processes and additional data input and output capabilities. New methods included in SWB 2.0 allow for direct gridded input of externally calculated water-budget components (fog, septic, and storm-sewer leakage), simulation of canopy interception by several alternative processes, and a crop-water demand method for estimating irrigation amounts. New input and output capabilities allow for grids with differing spatial extents and projections to be combined without requiring the user to resample and resize the grids before use.

Techniques and Methods↗

Track tube construction and field protocol for small mammal surveys with emphasis on the endangered Pacific pocket mouse (Perognathus longimembris pacificus)

Track tubes are used to identify small animals by their tracks. Animals that are small enough to fit into the tubes walk over ink pads and onto cardstock paper to obtain bait within the tube, leaving their footprints. The tracking tubes described in this document are designed to be set on the ground with free access and exit at either end with additional design components for stability, durability, and efficiency. They are also designed to prevent dirt from getting onto the ink pads and to decrease the ability of birds and other mammals to pull out track cards or bait. We describe detailed methods for constructing, setting and checking track tubes, as well as measuring and identifying small mammal prints for a small mammal study. The protocols described are for monitoring the Pacific pocket mouse (PPM); however, this method can be applied to many small mammal species that have uniquely identifiable tracks in relation to co-occurring species. We have deployed track tubes for over 5 years on Marine Corps Base Camp Pendleton for PPM discovery efforts and to monitor the three extant PPM populations on Base. We have shown that nightly detection probability is similar to that of live-trapping, but the track tubes can be checked weekly or bi-monthly. We use this passive and economical method to assess timing of annual emergence and torpor, seasonal activity, and localized colonization and extinction events. Using this method, we can model occupancy dynamics in relation to habitat and disturbance covariates that directly inform management and support a monitoring and management feedback loop for this species.

California↗

Determination of wastewater compounds in whole water by continuous liquid-liquid extraction and capillary-column gas chromatography/mass spectrometry

A method for the determination of 69 compounds typically found in domestic and industrial wastewater is described. The method was developed in response to increasing concern over the impact of endocrine-disrupting chemicals on aquatic organisms in wastewater. This method also is useful for evaluating the effects of combined sanitary and storm-sewer overflow on the water quality of urban streams. The method focuses on the determination of compounds that are indicators of wastewater or have endocrine-disrupting potential. These compounds include the alkylphenol ethoxylate nonionic surfactants, food additives, fragrances, antioxidants, flame retardants, plasticizers, industrial solvents, disinfectants, fecal sterols, polycyclic aromatic hydrocarbons, and high-use domestic pesticides. Wastewater compounds in whole-water samples were extracted using continuous liquid-liquid extractors and methylene chloride solvent, and then determined by capillary-column gas chromatography/mass spectrometry. Recoveries in reagent-water samples fortified at 0.5 microgram per liter averaged 72 percent ? 8 percent relative standard deviation. The concentration of 21 compounds is always reported as estimated because method recovery was less than 60 percent, variability was greater than 25 percent relative standard deviation, or standard reference compounds were prepared from technical mixtures. Initial method detection limits averaged 0.18 microgram per liter. Samples were preserved by adding 60 grams of sodium chloride and stored at 4 degrees Celsius. The laboratory established a sample holding-time limit prior to sample extraction of 14 days from the date of collection.

Techniques and Methods↗

Colorimetric determination of nitrate plus nitrite in water by enzymatic reduction, automated discrete analyzer methods

This report documents work at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) to validate enzymatic reduction, colorimetric determinative methods for nitrate + nitrite in filtered water by automated discrete analysis. In these standard- and low-level methods (USGS I-2547-11 and I-2548-11), nitrate is reduced to nitrite with nontoxic, soluble nitrate reductase rather than toxic, granular, copperized cadmium used in the longstanding USGS automated continuous-flow analyzer methods I-2545-90 (NWQL laboratory code 1975) and I-2546-91 (NWQL laboratory code 1979). Colorimetric reagents used to determine resulting nitrite in aforementioned enzymatic- and cadmium-reduction methods are identical. The enzyme used in these discrete analyzer methods, designated AtNaR2 by its manufacturer, is produced by recombinant expression of the nitrate reductase gene from wall cress (Arabidopsis thaliana) in the yeast Pichia pastoris. Unlike other commercially available nitrate reductases we evaluated, AtNaR2 maintains high activity at 37&deg;C and is not inhibited by high-phenolic-content humic acids at reaction temperatures in the range of 20&deg;C to 37&deg;C. These previously unrecognized AtNaR2 characteristics are essential for successful performance of discrete analyzer nitrate + nitrite assays (henceforth, DA-AtNaR2) described here. Method detection levels (or limits; MDL) estimated for standard- and low-level DA-AtNaR2 nitrate + nitrite methods were 0.02 milligrams nitrogen per liter (mg-N/L) and 0.002 mg-N/L, respectively, which are comparable to 2010 NWQL long-term MDLs of the continuous-flow analyzer, cadmium-reduction methods (henceforth, CFA-CdR) they replace. Typically, reagent-water blanks for standard- and low-level DAAtNaR2 nitrate + nitrite methods are one half MDL or less. Nitrate + nitrite concentration differences for between-day replicates were 3 percent or less at or above 5 times the MDL and were as great as 35 percent near the MDL. Typically, nitrate spike recoveries from reagent water, surface water, groundwater, and high-phenolic-content, humic-acid-amended reagent water were 100&plusmn;20 percent. In addition to operational details and performance benchmarks for these new DA-AtNaR2 nitrate + nitrite assays, this report also provides results of interference studies for common inorganic and organic matrix constituents at 1, 10, and 100 times their median concentrations in surface-water and groundwater samples submitted annually to the NWQL for nitrate + nitrite analyses. Paired t-test and Wilcoxon signed-rank statistical analyses of results determined by CFA-CdR methods and DA-AtNaR2 methods indicate that nitrate concentration differences between population means or sign ranks were either statistically equivalent to zero at the 95 percent confidence level (p &ge; 0.05) or analytically equivalent to zero-that is, when p < 0.05, concentration differences between population means or medians were less than MDLs.

Techniques and Methods↗