Introduction

In 2012, the Montana Department of Environmental Quality (MTDEQ) identified Lake Koocanusa (also referred to as “Koocanusa Reservoir”), a transboundary reservoir in northwestern Montana and southeastern British Columbia, Canada (fig. 1), as threatened by selenium contamination from sources outside the State of Montana’s jurisdiction or borders and subsequently listed the water body under section 303(d) of the U.S. Clean Water Act (33 U.S.C. 1251 et seq.) (MTDEQ 303(d) list [MTDEQ, 2018]). Research has determined that the dominant source of selenium to Lake Koocanusa is runoff from extensive metallurgical (nonthermogenic) coal mining operations (Storb and others, 2023), which are upstream from the reservoir in British Columbia. Selenium is biologically essential at low concentrations, but elevated environmental concentrations can cause toxicological and adverse biological effects (Lemly, 1999; Environment and Climate Change Canada, 2022; Montana Legislative Services Division, 2022). For example, trophic transfer and bioaccumulation of selenium in the aquatic food web can result in excess selenium being maternally transferred to eggs. In the egg, elevated selenium concentrations can impair egg viability and development of larval fish, resulting in physiological malformations (for example, spinal and fin deformities and teratogenic defects) and reduced egg survival and reduced growth, and survival of fish larvae and fry (Lemly, 1999; Johnson and others, 2020; Environment and Climate Change Canada, 2022). Fish populations within Lake Koocanusa, such as Oncorhynchus clarki lewisi (westslope cutthroat trout; Girard, 1856), Oncorhynchus mykiss (rainbow trout; Walbaum, 1792), Prosopium williamsoni (mountain whitefish; Girard, 1856), and Federally threatened Salvelinus confluentus (bull trout; Suckley, 1859; Hansen and others, 2002) declined considerably after the construction of the Libby Dam (Dunnigan and others, 2017; Presser and Naftz, 2020), while Lota lota (burbot; Linnaeus, 1758; Lee and others, 1980) populations have declined to near zero (Hardy and others, 2015; Dunnigan and others, 2017). The Kootenai River (spelled Kootenay in Canada) below Libby Dam supports the species described above and others including a population of the federally endangered Acipenser transmontanus (white sturgeon; Richardson, 1836; Duke and others, 2007). Moreover, burbot and westslope cutthroat trout are considered culturally important and have recreational value to Tribes in the region (Presser and Naftz, 2020). Considerable effort and resources have been dedicated to restoration of degraded habitats across the reservoir (Hardy and others, 2015), but water quality continues to contribute to declining populations (Dunnigan and others, 2023a).

Purpose and Scope

This report describes the processes, techniques, and methods used for fish tissue collection on Lake Koocanusa, including descriptions of postcollection processes, techniques, and methods for the preparation and analysis of fish tissue. In addition, the process includes data management and review procedures, including quality assurance (QA) and quality control (QC) procedures used by the U.S. Geological Survey (USGS) Wyoming-Montana Water Science Center and supporting analytical laboratories. Fish tissue collections began in 2021. Changes to collecting fish tissue could include the fish species or exact tissue type targeted for collection, timing of fish collection, or chemical constituents of interest.

Approach

The overall authority for the determination of fish collection and laboratory analysis of a tissue sample needs is collaborative decision between USGS Wyoming-Montana Water Science Center; Montana Fish, Wildlife and Parks (MTFWP); and the MTDEQ in coordination with the Koocanusa/Kootenai monitoring program. Fish species selected for sampling in 2021 included five or more individuals of the following fish species: rainbow trout, westslope cutthroat trout, Richardsonius balteatus (redside shiner; Richardson, 1836), Ptychocheilus oregonensis (northern pikeminnow; Richardson, 1836), Catostomus macrocheilus (largescale sucker; Girard 1856), and Mylocheilus caurinus (peamouth chub; Richardson, 1836; Page and others, 2023). The specific species targeted, timing and method of capture, and tissues harvested may change over time at the discretion of the researchers involved in this study. Fish collection is the responsibility of the MTFWP, leveraging existing fieldwork (Dunnigan and others, 2023b). MTFWP ensures all personnel who collect fish samples have appropriate training, are cognizant of onsite safety considerations, and maintain sample integrity throughout all collection procedures and field-processing steps. Postprocessing of fish, tissue sample preparation, sample submission for laboratory chemical analysis, tracking chains of custody, and data management is the responsibility of the USGS Wyoming-Montana Water Science Center.

All fish tissue samples are shipped to USGS or partner (non-USGS) laboratories for analysis; other laboratories may be added as needed. USGS laboratories described in this document include the Eastern Ecological Science Center Leetown Research Laboratory in Kearneysville, West Virginia; the Geology, Geophysics, and Geochemistry Stable Isotope Laboratory in Denver, Colorado; the National Research Program Ecology and Contaminants Project Laboratory in Moffett Field and Menlo Park, California; the Upper Midwest Water Science Center Mercury Research Laboratory in Madison, Wisconsin; and the Columbia Environmental Research Center in Columbia, Missouri. Non-USGS laboratories include the Center for Environmental Sciences and Engineering at the University of Connecticut in Storrs, Connecticut; Brooks Applied Laboratory in Bothell, Washington; the U.S. Environmental Protection Agency (EPA) Region 10 Manchester Environmental Laboratory in Port Orchard, Wash.; the Environmental Biogeochemistry Laboratory at the University of Montana in Missoula, Montana; and TrichAnalytics, Inc., in Saanichton, British Columbia, Canada. The addresses and contact information for USGS and non-USGS laboratories include the following:

Field Sampling and Design

The MTFWP leverages existing programs to collect desired fish species and sample numbers deemed necessary to address exposure and uptake of elemental contaminants by fish and into various fish tissues (for example, liver, ovary, and muscle). The sampling design, methods used, and data collected as part of fish collection in Lake Koocanusa and the Kootenai River downstream from Libby Dam have been previously described in part here and elsewhere (Dunnigan and others, 2023b) including descriptions of sampling season(s), sampling locations, collection methods, and required field data (that is, fish length, weight, sex, and maturity). The MTFWP is responsible for providing all field personnel with required training.

Fish collection efforts are categorized into two primary collection periods. The first collection period occurs during the MTFWP’s annual standardized fish population assessment, which monitors trends and status of the fish community in Lake Koocanusa. This effort uses gill nets placed at 14 standardized locations on the reservoir. This collection period is when MTFWP has historically collected the most fish for tissue analysis. Sexually mature fish are not targeted for collection but are identified once fish are captured. The targeted sample size during this collection period may be as many as 10 females of each species containing eggs to serve as a trend indicator of contaminant levels.

The second collection period employs a combination of gill netting, electrofishing, and angling to collect the targeted species at approximately weekly intervals leading to the peak of sexual maturity for each species. Sampling dates are identified annually based on water temperatures, which affect the timing of spawning activity and sexual maturity of the targeted fish species. Timing of sexual maturity can help provide a comprehensive evaluation of representative samples collected from the same water body over multiple years. Five female specimens of each species can be targeted for collection during each sampling event during this sampling period. Captured fish are humanely euthanized, minimally processed in the field, and placed immediately on ice until later frozen.

If field collections fail to achieve the target number of females or collect a male fish, these fish can be processed and analyzed according to methods and procedures outlined in this report. The target number of fish specimens per species is meant to minimize the number of fish harvested and provide enough data to develop an average concentration per sampling period. For example, burbot are purposely not targeted during some years but could be inadvertently collected. When possible, burbot are targeted by hoop nets for regular population estimates, and some burbot could be processed as described in this report.

Initial Sample Handling and Field Documentation

MTFWP maintains sample integrity for all steps of collection through field sample processing (Dunnigan and others, 2023b). Fish are handled with powder-free nitrile gloves at all phases of the sampling process. Sample collection information and field observations are documented using a field sampling form. Identification of fish to the species level are performed in the field by a MTFWP biologist. Samples that cannot be definitively identified in the field can be frozen for later identification in the laboratory. The MTFWP collection team measures total length and weight and notes the presence of any external abnormalities (for example, parasites, deformities, lesions, tumors, and eroded fins) on the field sampling form. A metric measuring board on a plastic or wooden base is used for length measurements. Total length is recorded to the nearest millimeter with the fish’s body positioned on its right side, mouth closed, and facing the observer’s left. Weight is recorded to the nearest gram by placing the fish into a precleaned plastic container set on a portable electronic balance. Standard field cleaning procedures are followed between each fish collected for all field equipment (EPA, 2015). When possible, gonads are removed in the field, weighed, and preserved to calculate the gonadosomatic index (GSI). The GSI is calculated by the formula (gonad weight/ [body weight–gonad weight]) (Jan and Jan, 2017). Individual gonads with sufficient weight are split equally into two parts—half are frozen to less than or equal to (≤) −20 °C for analytical chemistry analysis, and the other half are fixed in 10 percent neutral-buffered formalin (NBF) for histological analysis. Gonads identified for histological analysis are placed in a plastic, labeled collection jar, and covered with NBF to 10 times the tissue volume for initial tissue preservation. Prior to shipping, excess NBF volume is poured off leaving enough NBF to sufficiently cover the specimen. Frozen specimens cannot be used for histopathological analysis. Note that NBF is a hazardous material and should be handled with care by trained staff. When fish size could limit available gonad tissue sample weight, such as when working with redside shiners, gonads may not be removed in the field, and pairs of fish may be captured so that 5 females are available for chemical analysis and 5 females for histological evaluation. Any deviation from standard sampling practices is documented on the field sampling form.

Whole fish (ovary already dissected) are double bagged in sealable plastic zipper bags and placed on ice until delivered to MTFWP offices in Libby, Mont., where they are frozen to ≤−20 degrees Celsius (°C). Once frozen, fish should not be allowed to thaw until time of dissection by the USGS Wyoming-Montana Water Science Center. Fish may remain frozen for as much as 1 year before dissection. When not field extracted, gonads are removed in the WY–MT WSC labs, weighed, and preserved to calculate the GSI as described above.

Preparing for Dissection

Fish tissue resection and processing is performed by USGS WY–MT WSC staff. All fish processing activities are recorded in a laboratory tissue processing log data sheet (app. 2). Dissected fish tissue are weighed on a 4-place analytical balance if greater than 200 grams or a 5-place analytical balance if the weight is smaller than 200 grams. Analytical balances are certified annually and verified daily upon use. All surfaces, equipment, and cutting tools are decontaminated as appropriate between each fish tissue sample to avoid cross-contamination of samples. Clean plastic sheeting is placed on cutting surfaces, trays, and balances prior to placing the sample on the work area (EPA, 2015; Queensland Department of Environment and Science, 2018). Plastic covers and nitrile gloves are replaced after working with each fish or tissue sample to prevent cross-contamination between fish samples. Procedures for filleting and dissecting fish follow the EPA document “Technical Standard Operating Procedure for Fish Handling and Processing” (EPA, 2001), and additional details of the steps involved are provided in the next section. Any deviations from these procedures should be noted on the laboratory tissue processing log data sheet. All tissue samples are placed in a clean, labeled vial or zipper bag (according to procedures supplied by the USGS or supporting laboratory performing the analysis), and then placed into a second cleaned zipper bag and immediately placed in the laboratory freezer. Prior to dissection, a naming convention procedure should be created to identify each tissue sample and relate the sample back to each individual fish. Labeled sample containers are kept with the associated field and laboratory tissue processing sheets and chain of custody forms for the entirety of the project or shipped following laboratory-specified shipping procedures.

Fish Dissection

Sample Documentation and Chain of Custody

All collected fish tissue samples should be labeled with a unique alphanumerical sample identification (ID) number that relates back to the individual fish collected. Labels should contain, at a minimum, the sample ID number, species identification, sample date, and resection time. Tissue samples are maintained and stored at the USGS WY–MT WSC laboratory until shipped to an analytical laboratory for future analysis. Frozen samples are shipped to laboratories on dry ice and by the fastest shipping method possible. Refer to appendix 1 for the collection and processing flowchart.

A record of all fish tissue samples collected and shipped to other laboratories are maintained by the project chief or assigned project team member, with status updates to include received dates and laboratory assigned ID numbers in an electronic spreadsheet, to ensure the complete and timely receipt of analytical results.

Sample Preparation

Fish tissue samples are dried and homogenized by the WY–MT WSC in Helena, Mont., or by the individual laboratories receiving the samples depending on project and laboratory needs. Fish tissue is dried by freeze-drying or by dehydrator at a temperature no higher than 55 °C. The percentage of moisture (the quantity of water contained in the tissue) is calculated using the equation, moisture content (in percentage)=(wet weight of sample–dry weight of sample)/(wet weight of sample×100) (EPA, 2001). When freeze-drying the Wyoming-Montana Water Science Center will use a Labconco FreeZone® freeze-dry system with bulk tray dryer. The percentage of moisture calculations is notated in the WY–MT WSC laboratory notebook, and calculations and results should be provided as a machine-readable data file to other laboratories upon request.

Sample Analyses

The procedures for laboratory analyses of tissue samples are shown in table 1. Sample and QC results are compiled by the Wyoming-Montana Water Science Center and reported in a USGS ScienceBase data release (https://www.sciencebase.gov/catalog/). Redundancy of analytes among laboratories may be due to the need to use different methods to address the large differences in the weight of fish tissues analyzed and (or) changes in the availability of various laboratories to support this project.

Quality Assurance and Quality Control Procedures

The QA/QC procedures applicable to fish tissue processing, preservation, and preparation by the USGS WY–MT WSC include the following:

Data Quality Objectives

The data quality objectives for this study closely follow guidelines outlined by the U.S. Department of the Interior (2008) to ensure data of known and acceptable quality are obtained. Documentation of basic information such as compatible monitoring objectives and program design features, metadata (when, where, and how data will be collected and who collected and analyzed the data), ancillary information (explanatory variables and study-site characteristics), and QA/QC data released from the analytical laboratories is used to evaluate data quality and uncertainty.

Data Quality Assessment

Laboratory QC samples and results are reviewed and compared with those of the co-collected primary samples. Other factors considered in each data review include compliance with sample laboratory hold times and receipt conditions (samples arrive at the lab at the appropriate temperatures), results of laboratory blank sample analyses, laboratory detection limits, and adherence to laboratory control standards. Estimated values may be used for evaluation purposes, whereas rejected values are qualified, flagged, and not used in any interpretive analyses.

Data Management and Reporting

Any preparation and processing information, metadata, and (or) ancillary information recorded onto paper forms by the USGS WY–MT WSC laboratory personnel is scanned into electronic project files and considered original records. These data are combined with analytical data from the laboratories in electronic data files to maximize their preservation. The USGS WY–MT WSC site administrator maintains backups of all locally stored electronic information. Nonelectronic information, if any, is retained by the project chief or designated personnel. Analytical results and metadata that have been reviewed and approved is disseminated to the public through a USGS data release, interpretative report(s), and (or) other published documents.

Data Processing and Validation

Sample metadata, laboratory preparation and processing notes, and any ancillary information is combined with laboratory analytical data into electronic data files. Analytical data are any chemical, physical, or biological determination results released from the laboratory. All analytical results are reviewed for completeness, and questionable values are noted by the project chief or designated personnel. Analytical results from each laboratory are released in a Microsoft Excel spreadsheet format to the project chief and (or) designated personnel. These analytical results include any laboratory data qualifiers and (or) explanations in the laboratory note section of the spreadsheet. If data from more than one tissue sample are available, the results can be compared with previous results to identify obvious errors, such as decimal errors, and sample outliers. The analytical results and any comparisons noted are reviewed and recorded in the electronic project file by the project chief or designated personnel. “Reviewed and approved” results can be included in the USGS data release, and any laboratory data qualifiers can be indicated in the associated metadata.

Health and Laboratory Safety

A job hazard analysis has been prepared for this study and is provided to USGS WY–MT WSC laboratory personnel and reviewed prior to sample handling and processing activities. The job hazard analysis includes an assessment of job tasks; potential on-the-job exposures to chemical and biological hazards; unsafe acts, or conditions; required personal protective equipment and job responsibilities; and telephone contacts. A copy of the job hazard analysis is provided in appendix 2.