TESTS EXPOLORING THE EFFECTIVENESS OF ELECTRON BEAM TREATMENT FOR BALLAST WATER MANAGEMENT

This technical report presents findings from bench-scale tests evaluating the ability of electron beam treatment to inactivate aquatic organisms. This evaluation was the first attempt to assess the electron beam treatment as a potential treatment method for ballast water in the Laurentian Great Lakes. Researchers from the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA traveled to the Fermi National Accelerator Laboratory, hereafter Fermilab, to conduct the bench-scale evaluation in January 2022. The test apparatus is a stationary copper particle accelerator that supplies a radiation dose expected to cause mortality in standard test organisms. It has been previously demonstrated that electron beam treatment can cause direct damage by creating either single- or double-strand breaks in the DNA molecule beyond cell repair (Shehata, M.M.K. et al., 2011) or recovery (Lei, J. et al., 2020). All experimental exposures were conducted at Fermilab. The irradiation process was conducted by Fermilab staff in the Illinois Accelerator Research Center’s (IARC’s) Accelerator Applications Development and Demonstration (A2D2) machine. Doses ranged from 1 to 50 kGy. Dose effectiveness testing was completed in two water types using bacteria, algae, and zooplankton—Escherichia coli, Selenastrum capricornutum, Eucyclops spp., and Daphnia magna, respectively. The treatment caused complete mortality for E. coli in both water types at even the lowest dose employed. Eucyclops spp. and Daphnia magna also experienced complete mortality in both water types between 5 and 10 kGy. Selenastrum capricornutum was the most resistant to treatment effect in both water types, but did experience mortality at the highest doses. Algae examined a week after exposure for delayed mortality experienced high mortality rates at all doses

TEST EXPLORING THE EFFECTIVENESS OF THE ULTRASONICATOR INVASIVE SPECIES WATER TREATMNET FOR BALLAST WATER MANAGEMNET

This technical report presents findings from bench-scale evaluation of the ultrasonicator invasive species water treatment developed by UPIR LLC to inactivate aquatic organisms. This evaluation was the first attempt to assess this technology as a potential flow through or recirculating ballast water treatment method for use on ships within the Laurentian Great Lakes. The evaluation began in January 2022 and ended in February 2022. All analyses occurred at the Lake Superior Research Institute at the University of Wisconsin-Superior in Superior, Wisconsin, USA. According to the developer, the UPIR ultrasonicator invasive species water treatment system produces a unique cavitation and agitation environment within the treated water to destroy unwanted organisms. Biological effectiveness testing was completed with the bacteria, Escherichia coli and Enterococcus faecium, and algae, Selenastrum capricornutum, in dechlorinated laboratory water. The system was found to be ineffective in the treatment of bacteria and algae when the organisms were exposed to a single pass through the system at a flow rate of 6 gpm. However, when exposed to multiple passes through the system, the delayed mortality of bacteria increased, and effectiveness with algae remained low. Water quality parameters were minimally impacted by the ultrasonicator invasive species water treatment

EXPLORING OPTIONS FOR REAL-TIME WATER QUALITY MONITORING WITHIN GREAT LAKES PORTS

This technical report presents findings from the exploration of available environmental monitoring sensors, the piloting experience of long-term field deployment of the Proteus multiparameter sonde developed by Proteus Instruments, and the development of an accessible platform for viewing real-time data in support of uptake avoidance best management practices as defined by the U.S. Environmental Protection Agency’s 2013 Vessel General Permit. Project staff conducted a literature review and investigation of commercially available sensors and selected an instrument that consists of a multiparameter sonde—able to measure turbidity, chlorophyll-a, phycocyanin, and total coliform bacteria—, telemetry system, and solar panel. Long-term deployment of the Proteus began in April 2022 and ended in October 2022 at the Montreal Pier Testing Facility located on the Duluth-Superior Harbor in Superior, Wisconsin, USA. Data collected from the multiparameter sonde was transmitted and stored on Outpost.com and real-time data was made publicly available using Web AppBuilder for ArcGIS. This piloting experience has shown promise to provide real-time data in support of uptake avoidance practices

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

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

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

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

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 DN100 AND DN150 BALLAST SYSTEMS IN THE GREAT LAKES

This technical report presents the land-based evaluation of two Optimarin Ballast System models. The focus was primarily on the Model 68/340FX2 using a DN100 chamber but also includes data for one trial using the Model 334/340FX2 with a DN150 ultraviolet (UV) chamber. This work evaluated the potential of the systems to be used as a flow-through water treatment methods for the Laurentian Great Lakes, treating via filtration and UV exposure on uptake and UV exposure again on discharge. The evaluation began August 2022 and ended October 2022. 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 five efficacy trials, which included overnight retention of treated harbor water at the Montreal Pier Facility that had been amended to achieve ETV Protocol challenge conditions (NSF International, 2010). Trial 1 assessed performance of both systems consecutively during a short timeframe to ensure water quality was as similar as possible. In Trials 2-5, the Model 68/340FX2 with a DN100 UV chamber was tested. Biological effectiveness was assessed in terms of remaining live organisms in three size classes per unit volume: • Organisms ≥50 µm in minimum dimension (nominally zooplankton), • organisms ≥10 µm and <50 µm in minimum dimension (nominally protists), • and organisms <10 µm in minimum dimension (nominally bacteria; e.g., total culturable heterotrophic 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 (U.S. Coast Guard, 2012) with a focus on the reduction in the number of propagules in treated water versus control water. The Model 68/340FX2 Optimarin system using a DN100 UV chamber was found to be effective at reducing the densities of organisms in all three regulated size classes. The densities of zooplankton in treatment discharge samples did not meet the USCG Ballast Water Discharge Standard (BWDS) but were >98% lower than control discharge in all trials. The densities of protists in the treatment discharge samples were above the USCG BWDS in all trials but had decreased by >84% when compared to control discharge samples. All indicator bacteria (i.e., E. coli, Enterococcus spp., and Vibrio Cholerae) were below the USCG BWDS on uptake and discharge and decreased to less than the limit of detection after treatment. The Optimarin system Model 334/340FX2 using a DN150 ultraviolet (UV) chamber showed very similar trends to in all size classes when compared to testing completed in 2021 (Polkinghorne et al., 2022) and when compared to testing completed in 2022 with Model 68/340FX2 Optimarin system using a DN100 UV chamber