Natural Resources Research Institute Technical Report

This report follows the same layout and data summaries as the report provided to the Lake Superior Steelhead Association (LSSA) in 2017 (Dumke and Wellard-Kelly, 2017). Thus, much of the text from introduction and methods sections are repeated because each report has been written as a stand-alone document. The Natural Resources Research Institute (NRRI) was contracted in 2017 by LSSA to conduct pre-treatment stream habitat, fish, invertebrate, and water chemistry surveys on three segments of the Knife River mainstem anticipated to have channel restoration work applied in the future. The reaches were named ‘Reach 4.5,’ ‘Reach 4_ED,’ and ‘Reach 4_CB’ to match section labels used in planning by LSSA. We also measured all the same parameters in an upstream reach not expected to undergo any treatment, which serves as the control for later comparisons. This before-after-control-impact (BACI) design is our standard for attributing changes over time to specific treatments applied to the stream and is very useful in evaluating changes caused by habitat improvement projects. In total, four river segments (Reach 4.5, Reach 4_ED, Reach 4_CB, and Reference) were surveyed by NRRI with the full suite of options. In addition, three upper-watershed reaches (Mcarthy, Red Dot, and White Landing) were surveyed via electrofishing only with the goal of detecting age0 Rainbow Trout presence, and two reaches on the Knife River mainstem were surveyed with rapid-response thermometers to detect ground-water inputs which would be important trout refuge during hot summer periods. We found that all reaches had water quality parameters acceptable for all salmonid species present in the Knife River watershed. In fact, we found a ground-water spring within one of temperature survey reaches. The pre-treatment reaches had lower MSHA habitat scores than the Reference, which was largely due to the presence of large eroding stream banks, but all reaches had fish habitat in the form of woody debris and pools. Brown Trout were present in the lower segments, but absent in upper watershed reaches. Brook Trout comprised more of the fish community as surveys progressed into the upper Knife River watershed, which is typical. Rainbow Trout were present in every electrofished river section, but only one age0 Rainbow Trout was collected within White Landing, and no age0 were detected in Red Dot. Red Dot and White Landing were not far apart, and the low capture of age0 Rainbow Trout indicates there was very little spawning activity, or poor spawning success, of Rainbow Trout in these upper sections during spring 2017. Macroinvertebrate communities were similar among the four reaches sampled for bugs, but the Reference had slightly fewer sensitive taxa, likely due to that reach being a steeper slope and dominated by larger boulders that were half-buried in the stream bed by smaller rocks (which offers fewer spaces between rocks for invertebrates to hide).Dumke, Josh; Wellard-Kelly, Holly. (2018). Results of Pre-Treatment Habitat and Biota Surveys from the Knife River, MN Watershed. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/204330

Resource Composition and Macroinvertebrate Resource Consumption in the Colorado River Below Glen Canyon Dam

Physical and biological changes to rivers induced by large dams can significantly alter downstream communities, decreasing the biotic integrity of these rivers. For example, completion of Glen Canyon Dam on the Colorado River in 1963 has altered the downstream ecosystem and contributed to the decline of native fish populations and dramatic changes in the macroinvertebrate communities. Physical changes associated with the dam may also influence the food resources supporting macroinvertebrate production, but this has not been previously measured. For example, autochthonous production is high in the clear tailwaters of the dam, but downstream tributary allochthonous carbon inputs are substantial and may provide an important food resource. I predict that macroinvertebrate diets will mirror these longitudinal changes in resource availability and may indicate how the dam has altered the macroinvertebrate food webs of this large river. I also predict that monsoon tributary flooding in the autumn and lower light availability in the winter, will amplify the longitudinal change in resource use by macroinvertebrates. I examined the diets of the most common macroinvertebrates (Simulium arcticum, Gammarus lacustris,Potamopyrgus antipodarum, and chironomids) at six sites below Glen Canyon Dam during all seasons. Macroinvertebrate diet composition was compared to the composition of the epilithon (rock faces), epicremnon (cliff faces) communities, and the suspended organic seston. Using these data, I calculated the relative contribution of autochthonous and allochthonous resources to macroinvertebrate production. To estimate the extent that tailwater primary production provides a subsidy to downstream consumers, during one season I identified algae to species in the various biofilms, seston and in macroinvertebrate diets to find species-specific tracers of tailwater production. In general, I found that macroinvertebrate diets tracked downstream changes in resource availability, and autochthonous resources were consumed in greater proportions in the tailwaters while more allochthonous resources were consumed downstream. The extent of diet shifts depended on consumer identity and season. I observed similar patterns in the resources that support macroinvertebrate production, despite the greater assimilation of autochthonous carbon. Allochthonous resources were most important during the monsoon season (July-September), when tributaries can contribute significant amounts of organic matter to the mainstem. Finally, using the species-specific indicators of tailwater production I estimated that less than seven percent of downstream macroinvertebrate production is attributable to tailwater algal production. This suggests that the tailwaters may not provide a significant subsidy to downstream food webs. These findings demonstrate that the influence of the Glen Canyon Dam is most evident in the tailwaters and as distance from the dam increases, tributary allochthonous carbon fuels food webs, which may be similar to pre-dam food webs

Evaluating a Most Probable Number Method for Assessing the Viability of Great Lakes Protists

To support type approval testing of ballast water management systems we evaluated freshwater viability assessments for protists from the Duluth-Superior harbor of western Lake Superior using the most probable number (MPN) method. Tests were performed using varying temperatures and growth media and were compared to standard microscopic methods for determining live organism densities. Tests were also performed focusing on growth series derived from harbor water, and during an actual land-based test of a treatment system being evaluated for efficacy. We determined that growth of protists during MPN experiments was especially favored under higher temperatures and a growth medium comprising a 50 % solution of Bold Modified Basal Media. This medium also supported the growth of the greatest number of protist taxa. Based on microscopic analysis of live protists use of a treatment system during land-based testing reduced protist densities from 554 – 3000 cells/mL in the untreated water to 12 – 52 cells/mL after treatment. Corresponding assessments using the MPN method estimated respective densities of 1651 – 6060 cells/mL and 0 – 2.8 cells/mL, indicating that MPN likely overestimated viable cells in ambient harbor samples while it underestimated cell densities in treated samples. As asserted in the MPN protocols we confirmed that MPN-estimated protist densities were similar to densities in the protist size class that includes only cells strictly 10 – 50 µm in minimum dimension; protist densities including cells <10 µm were much higher than MPN estimates. However, based on all evaluations of freshly acquired samples containing a wide range of starting densities there was no correlation between MPN- and microscopy-determined densities, regardless of size class. Based on all testing, certain protist taxa were poorly favored during MPN grow-out periods (e.g., the chrysophyte Mallomonas), while others (e.g., free-living centric diatoms) tended to thrive, though there was substantial variability in taxonomic selectivity among tests. These findings contribute important freshwater data to the field of efficacy testing of ballast water treatment systems

FASTBALLAST

This technical report presents findings from bench-scale verification tests evaluating the performance of the FastBallast compliance monitoring device in freshwater. FastBallast was developed by Chelsea Technologies Ltd. of Surrey, UK. The evaluation of the FastBallast compliance monitoring device began in August 2020 and ended in December 2020 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The FastBallast device employs Single Turnover Active Fluorometry (STAF) to rapidly quantify living organisms in ballast water samples in the ≥10 µm and <50 µm (nominally protists) regulated size class, providing an indication of compliance or exceedance of the International Maritime Organization (IMO) International Convention for the Control and Management of Ships’ Ballast Water and Sediments Regulation D-2 Ballast Water Performance Standard (2004). Verification testing composed of three phases in which results using the FastBallast device were compared to results using microscopic methods. Phase I testing was completed in two water types with laboratory-cultured organisms in the protist regulated size class, utilizing the single-celled protist Haematococcus pluvialis and colonial protist Scenedesmus quadricauda. Phase II testing was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of Lake Superior. Phase III testing was completed using Duluth-Superior harbor water an ambient organism before and after treatment with a ballast water treatment (BWT) technology during three land-based trials. Data from all phases were analyzed for precision, accuracy, and reliability. Quantification/detection limits were calculated using data from Phase I testing. Phase I testing showed that FastBallast was effective at quantifying single-celled protists but was less accurate at counting colonial protists. Increased turbidity and carbon content slightly impacted FastBallast results, however, both water types displayed strong correlations to microscopic counts. FastBallast results were lower than microscopic counts in all trials of Phase I. Phase II testing showed strong correlations between the FastBallast results and microscopic results of protists collected from the Duluth-Superior Harbor, however the counts reported by FastBallast were 4 to 10 times greater than the microscopic counts. Phase III testing showed FastBallast accurately measured uptake and treated discharge water from samples collected during a land-based BWT technology evaluation. FastBallast counts were more similar to the density of protist entities ≥10 µm in any dimension than they were to live density of individual protist cells comprising entities ≥10 µm in minimum dimension. The device was found to have minor operational issues but was found reliable for measuring organisms within the protist size class.LSRI-GWRC would like to thank Chelsea Technologies Ltd. (Surrey, UK) for their application to our laboratory-based testing program and for providing the FastBallast device and the expendable supplies for analysis. Mary Burkitt-Gray and Kevin Oxborough at Chelsea Technologies Ltd. provided operational training support prior to the start of testing and were also instrumental in helping to troubleshoot technical/operational issues that occurred during testing. This work was supported by a grant from the United States Department of Transportation Maritime Administration’s Maritime Environmental and Technical Assistance Program

Ballast Eye

This technical report presents findings from freshwater verification tests evaluating the performance of the Satake Ballast Eye Viable Organism Analyzer VOA1000K compliance monitoring device, hereafter Ballast Eye. Ballast Eye was developed by Satake Corporation of Hiroshima, Japan. The compliance monitoring device evaluation began in August 2020 and ended in December 2020 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. Ballast Eye estimates the number of viable organisms and associated risk based on IMO D-2 ballast water discharge standards in the ≥10 and <50 µm (nominally protists) and ≥50 µm (nominally zooplankton) regulated size classes by measuring the fluorescence pulse number from fluorescein diacetate (FDA) stained organisms within a water sample. The verification testing was composed of three phases. Phase I testing was completed in two water types with laboratory-cultured organisms in the two regulated size classes, utilizing the single-celled protist Haematococcus pluvialis and colonial protist Scenedesmus quadricauda, and the zooplankton Daphnia magna and Eucyclops spp. Phase II was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of western Lake Superior in the two regulated size classes. Phase III testing was completed using Duluth-Superior harbor water and ambient organisms before and after treatment with a ballast water treatment technology (BWT) during three land-based trials. Data from all phases were analyzed for precision, accuracy, and reliability. Quantification/detection limits were calculated for Phase I data. Phase I testing showed Ballast Eye was able to accurately estimate the number of zooplankton in high and low transparency water, while protist concentrations were not accurately determined. Phase II testing showed Ballast Eye was unable to accurately estimate the number or risk of ambient zooplankton or protists in Duluth-Superior harbor water. Phase III testing showed that Ballast Eye was able to accurately classify risk of ambient zooplankton or protists within uptake and treated discharge samples collected during land-based ballast water treatment technology testing at the Montreal Pier Facility located on the Duluth-Superior harbor.LSRI-GWRC would like to thank Satake Corporation (Hiroshima, Japan) and MOL Techno-Trade Ltd. (Tokyo, Japan) for their application to our laboratory-based testing program and for providing Ballast Eye and the expendable supplies for analysis. Hiroki Ishizuki, Yoshinori Tazoe, and Shinya Fushida provided operational training support prior to the start of testing and were instrumental in helping to troubleshoot technical/operational issues that occurred during testing. This work was supported by a grant from the United States Department of Transportation Maritime Administration’s Maritime Environmental and Technical Assistance Program

BallastWISE

This technical report presents findings from freshwater verification tests evaluating the performance of the MicroWISE BallastWISE compliance monitoring device, hereafter BallastWISE. BallastWISE was developed by MicroWISE, located in Ebeltoft, Denmark. The compliance monitoring device evaluation began in August 2020 and ended in December 2020, at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. BallastWISE utilizes separate chambers to enumerate organisms in each of two regulated size classes, ≥10 and <50 µm (nominally protists) and ≥50 µm (nominally zooplankton). Cameras and optical chambers capture video and track motility through software analysis for the zooplankton size class. Fluorescence microscopy evaluates chlorophyll containing organisms in addition to motility tracking in the protist size class. The verification testing was composed of three phases. Phase I testing was completed in two water types with laboratory-cultured organisms in the two regulated size classes, utilizing the single-celled protist Haematococcus pluvialis and colonial protist Scenedesmus quadricauda, and the zooplankton Eucyclops spp. and Daphnia magna. Phase II testing was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of Lake Superior in the two regulated size classes. Phase III testing was completed using Duluth-Superior Harbor water and ambient organisms before and after treatment with a ballast water treatment technology (BWT) during three land-based trials. Data from all phases were analyzed for precision, accuracy, and reliability. Quantification/detection limits were also calculated from Phase I data . Phase I testing showed BallastWISE was effective at quantifying single-celled protists to within about 20% of the microscopic counts, but undercounted colonial protists. Colonial protist entity counts were close to microscopic entity counts suggesting that individuals within the colonies were not resolved. High total suspended solids (TSS) and (DOC) may slightly reduce BallastWISE sensitivity to protists. BallastWISE overcounted zooplankton in both species tested in both high and low TSS/DOC by between 150% and 420%. Phase II testing from the Duluth-Superior Harbor showed BallastWISE counts of natural assemblages of protists strictly in the ≥10 and <50 μm size class to be slightly below microscopic counts by about 35% and with high precision. Zooplankton were overestimated by BallastWISE by roughly 40% and with considerably more variation compared to microscopic counts. Phase III testing showed low BallastWISE accuracy and precision in untreated protist and zooplankton samples. This may have been caused by organism densities higher than the device’s effective upper limit of detection in the zooplankton samples, but further investigation would be needed to determine the cause of low accuracy and precision in protist analysis. BallastWISE accurately measured treated protist samples as 0 cells/mL in agreement with strict microscopic counts, but overcounted treated zooplankton samples in 2 out of 3 tests, possibly due to the method of treatment. A number of operational issues made enumeration of zooplankton unreliable, but improvements (e.g., software updates, guidance on device operation) from the developer over the period of this assessment have already improved performance. BallastWISE shows promise as a useful device for detecting and measuring protists and zooplankton in the Great Lakes as additional improvements are made.LSRI-GWRC would like to thank MicroWISE (Ebeltoft, Denmark) for their application to our laboratory-based testing program and for providing the BallastWISE system and the expendable supplies for analysis. Pia Haecky, MicroWISE CEO, and Nicholas Blackburn, MicroWISE Software Development, provided operational training support prior to the start of testing and were also instrumental in helping to troubleshoot technical/operational issues that occurred during testing. This work was supported by the United States Maritime Administration (United States Department of Transportation; Washington, D.C)

Bawat Mk2

This technical report represents the shore-based evaluation of the Bawat Ballast Water Management System (BWMS) Mk2 – Mobile Treatment Unit, developed by Bawat A/S Agern Alle, 2970 Horsholm, Denmark (www.bawat.com). This work was conducted to evaluate the potential of the system to kill living organisms in the ballast water of ships to reduce the risk of aquatic nuisance species migration in the Laurentian Great Lakes. The Bawat Mobile Treatment system is designed to heat water killing the organisms carried in the water in a single pass through the treatment system. The single pass can be filling or discharging a ships ballast water and requires no retention period. Biological effectiveness was examined October 22–24, 2023 at the AMI Consulting Engineers facility in Superior, WI during three efficacy trials with a single pass of harbor water through the Bawat BWMS Mk2 – Mobile Treatment Unit. Effectiveness was assessed in terms of reducing live organisms in three size classes per unit volume: organisms ≥50 µm in minimum dimension (nominally zooplankton), organisms ≥10 and <50 µm in minimum dimension (nominally protists), and organisms <10 µm in minimum dimension (e.g., total coliform bacteria, Escherichia coli, and Enterococcus spp.). Samples were compared to the United States Coast Guard’s (USCG) Standards for Living Organisms in Ships’ Ballast Water Discharged in U.S. Waters (USCG, 2012) with a focus on the reduction in the number of propagules in the treated water. The Bawat BWMS Mk2 was found to be highly effective at reducing the densities of organisms in all three regulated size classes. Protist, zooplankton, E. coli and Enterococcus spp. densities on discharge were below the USCG ballast water discharge standard (BWDS) in all trials. Temperature of discharge water was approximately 5°C higher than uptake water, but other water quality parameters were minimally impacted by treatment.Lake Superior Research Institute; Great Waters Research Collaborative; Natural resources Research Institute, University of Minnesota Duluth; AMI Consulting Engineers; Balcer Taxonom

LAND-BASED EVALUATION OF THE EFFECTIVENESS OF THE BAWAT BALLAST WATER MANAGEMENT SYSTEM MK2-MOBILE TREATMENT UNIT

This technical report represents the land-based evaluation of the Bawat Ballast Water Management System (BWMS) Mk2 – Mobile Treatment Unit, developed by Bawat A/S Agern Alle, 2970 Horsholm, Denmark (www.bawat.com). This work was conducted to evaluate the potential of the system to be used as a flow-through water treatment method for the Laurentian Great Lakes, treating via heat treatment with one pass of water through the treatment system. The evaluation began in September 2021 and ended October 2021. All analyses were conducted at either the Montreal Pier Facility or the Lake Superior Research Institute (LSRI) at the University of Wisconsin-Superior (UWS), both located in Superior, WI, USA. Biological effectiveness was examined at the Montreal Pier Facility during a commissioning trial and four efficacy trials with a single pass of harbor water through the Bawat BWMS Mk2 – Mobile Treatment Unit. Harbor water was amended to achieve ETV Protocol challenge conditions. Effectiveness was assessed in terms of remaining live organisms in three size classes per unit volume: organisms ≥50 µm in minimum dimension (nominally zooplankton), organism entities ≥10 µm in any dimension and with cell sizes <50 µm in minimum dimension (nominally protists), and organisms <10 µm in minimum dimension (e.g., total culturable heterotrophic bacteria, total coliform bacteria, Escherichia coli, Enterococcus spp., and toxigenic Vibrio cholerae O1 and O139). Samples were compared to the United States Coast Guard’s (USCG) Standards for Living Organisms in Ships’ Ballast Water Discharged in U.S. Waters (USCG, 2012) with a focus on the reduction in the number of propagules in the treated water. The Bawat BWMS Mk2 was found to be highly effective at reducing the densities of organisms in all three regulated size classes. E. coli and Enterococcus spp., and Vibrio cholerae densities on discharge were below the USCG ballast water discharge standard (BWDS) in all trials. Protist densities were below the USCG BWDS in all but the final trial. Temperature of discharge water was approximately 5°C higher than uptake water, but other water quality parameters were minimally impacted by treatment

LAND-BASED EVALUATION OF THE EFFECTIVENESS OF THE OPTIMARIN BALLAST SYSTEM IN THE GREAT LAKES

This technical report presents the land-based evaluation of the Optimarin Ballast System, Model 334/340FX2. This work was conducted to evaluate the potential of the system to be used as a flow-through water treatment method for the Laurentian Great Lakes, treating via filtration and UV exposure on uptake and UV exposure again on discharge. The evaluation began September 2021 and ended October 2021. All analyses were conducted at either the Montreal Pier Facility or the Lake Superior Research Institute (LSRI) at the University of Wisconsin-Superior (UWS), both located in Superior, Wisconsin, USA. Biological effectiveness was examined during a commissioning trial and five efficacy trials with overnight retention of harbor water at the Montreal Pier Facility that was amended to achieve ETV Protocol challenge conditions. Effectiveness was assessed in terms of remaining live organisms in three size classes per unit volume: organisms ≥50 µm in minimum dimension (nominally zooplankton), organism entities ≥10 µm in any dimension and with cell sizes 95% compared to control discharge samples