Evaluation Of A Method For Ballast Water Risk-Release Assessment Using A Protist Surrogate

University of Minnesota M.S. thesis. August 2015. Major: Water Resources Science. Advisor: Euan Reavie. 1 computer file (PDF); v, 41 pages.Aquatic invasive species cost the United States billions of dollars annually and are most often introduced via ships' ballast tanks. The International Maritime Organization (IMO) adopted a set of standards that sets limits on the number of viable organisms that can be discharged with ballast water. These standards require scientific research to determining the risk-release relationship (i.e. the number of propagules needed for an invasion). To examine the risk-release relationship a surrogate invader, the diatom Melosira varians, was used. Varying densities of the surrogate were added to 19-L mesocosms containing water from Duluth-Superior Harbor. Each mesocosm was sampled weekly for four weeks and the response of the phytoplankton community was measured via cell counts. Population responses varied by starting conditions but M. varians establishment was generally noted at higher inoculation densities. These findings suggest the mesocosm approach can inform the understanding of risk-release relationships

Morphological and Molecular Techniques to Support Detection and Management of Ballast-Borne AIS

Aquatic invasive species (AIS) released from ship ballast water threaten aquatic ecosystems. A unique opportunity to couple molecular and microscopic techniques is investigated to understand risk-release relationships in a controlled setting using mesocosms and surrogate protist species. This research aims to: Evaluate the relationship between release density and establishment using a wider variety of protist surrogates; Track surrogate and ambient community species using morphological and molecular techniques; Initiate a framework to support on-ship compliance testing methods using eDNA.Supported by USDOT award DTMA9117H00002 to the University of Wisconsin Superior's Lake Superior Research Institute

Rosemount Parks Invasive Species Management and Education Plan

Report, presentation, and poster completed by students enrolled in EnEd 5325: Sustainability Issues Investigation, taught by Ken Gilbertson in fall 2014.This project was completed as part of a year-long partnership between the City of Rosemount and the University of Minnesota’s Resilient Communities Project (http://www.rcp.umn.edu). Many communities throughout Minnesota are struggling to manage the growth of invasive species in their parks, open spaces, and on private property. Two invasive species on the rise in the City of Rosemount are emerald ash borer and common buckthorn. The goal of this project was to explore invasive species management options and examine how education can play a role in the management of invasive species in the community. In collaboration with city project lead Tom Schuster, Parks Supervisor for the City of Rosemount, a team of students in EnEd 5325: Sustainability Issues Investigation conducted a literature review, provided recommendations for the development of an invasive species and invasive pest management plan, and created a sustainability education plan for the management of emerald ash borer and common buckthorn in Carroll’s Woods and Schwarz Pond. A final report, presentation, and poster from the project are available.This project was supported by the Resilient Communities Project (RCP), a program at the University of Minnesota that convenes the wide-ranging expertise of U of M faculty and students to address strategic local projects that advance community resilience and sustainability. RCP is a program of the Center for Urban and Regional Affairs (CURA). More information at http://www.rcp.umn.edu.Aliff, Meagan; Bryan, Rebecca; Burton, Brent; Gill, Cian; Hagsten, Kate; Johnson, Megan. (2014). Rosemount Parks Invasive Species Management and Education Plan. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/180460

B-QUA

This technical report presents findings from bench-scale verification tests evaluating the performance of the B-QUA Quick Ballast Water Monitoring Kit, hereafter B-QUA, in freshwater. B-QUA was developed by LuminUltra Technologies Ltd. of New Brunswick, Canada. Researchers began conducting the bench-scale evaluation in October 2019 and ending in February 2020 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The monitoring kit utilizes adenosine triphosphate (ATP) and size fractionation to quantify living organisms in marine, brackish, and freshwater. The measurement of ATP is one of the indicative analyses to test for gross compliance with the D-2 ballast water management standard under the International Maritime Organization’s (IMO) Ballast Water Management (BWM) Convention, which applies to countries outside of the United States, including Canada (IMO, 2015). Two phases of testing were done. Phase I testing was completed in two water types using cultured organisms in the three regulated size classes, utilizing the pathogen indicator organisms Escherichia coli and Enterococcus faecium, the algae Haematococcus pluvialis and the zooplankton Ceriodaphnia dubia and Daphnia magna. Phase II testing was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of Lake Superior in two of the regulated size classes. Phase I testing showed high correlation of B-QUA’s luminometer output (i.e., cATP values) with microscopic counts for the algae Haematococcus pluvialis (≥10 μm to 50 μm size class) in both water types. However, the B-QUA system was unable to detect E. coli or E. faecium (<10 μm size class) at levels above the D2 regulatory value in either water type in Phase I. Phase II correlation between B-QUA cATP values and microscopic counts was good for natural assemblages of phytoplankton and zooplankton in Duluth-Superior harbor water.This work was supported by the United States Maritime Administration (United States Department of Transportation; Washington, D.C) and the United States Environmental Protection Agency’s Great Lakes Restoration Initiative

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

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)