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LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Number of Pages: 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
GREAT WATERS RESEARCH COLLABORATIVE:
GREAT LAKES SHIP BALLAST MONITORING PROJECT
TECHNICAL REPORT
May 31, 2018
Allegra Cangelosi*1, Olivia Anders1, Mary Balcer 2, Kimberly Beesley1, Lana Fanberg1, Steven Gebhard1,
Matthew Gruwell 3, Ivor Knight3, Nicole Mays2, Marylee Murphy1, Christine Polkinghorne1, Kelsey Prihoda1,
Euan Reavie 4, Deanna Regan1, Elaine Ruzycki4, Heidi Saillard1, Heidi Schaefer1, Tyler Schwerdt 5,
Matthew TenEyck1, Karada Tudor1, Tony Venditto5
Compiled By:
Allegra Cangelosi
GWRC Principal Investigator
Signature
Date
Cleared for Issue By:
Matthew TenEyck
Director of LSRI
Signature
Date
*First Author; To Whom Correspondence May be Addressed (e-mail: acangel1@uwsuper.edu)
1 Lake Superior Research Institute, University of Wisconsin-Superior, Belknap & Catlin Ave, Superior, WI, 54880
2 Independent Contractor
3 Department of Biology, Penn State Erie, The Behrend College, Erie, PA 16563
4 Natural Resources Research Institute, University of Minnesota-Duluth, 5013 Miller Trunk Highway, Duluth, MN, 55811
5 AMI Consulting Engineers, 91 Main Street, Superior, WI, 54880
Allegra
Cangelosi
Digitally signed by
Allegra Cangelosi
Date: 2018.05.31
14:08:24 -04'00'
Matthew
TenEyck
Digitally signed by
Matthew TenEyck
Date: 2018.05.31
13:02:17 -05'00'
5/31/18
5/31/18
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 2 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
ACKNOWLEDGMENT
The Great Waters Research Collaborative (GWRC) wishes to thank the Officers and Crews of the United
States and Canadian Flagged laker vessels which participated in this study. The Officers and Crews
consistently welcomed the research team members on board their ships, accommodated their research
needs, and assured their safety during our sampling events. We also thank the port-side personnel who
granted and processed our research team’s access to the ships. We thank Tom Rayburn of the Lake
Carriers’ Association, and Paul Topping of the Chamber of Marine Commerce for reviewing our project
plan, connecting our team with the participating ship owners, and otherwise advising throughout the
project. We thank the Minnesota Pollution Control Agency for providing input on their needs and
preferences with respect to study design. We thank the members of the GWRC Advisory Committee for
their input and informational support throughout this project. Finally, we are deeply grateful to the
United States Maritime Administration, and the United States Environmental Protection Agency for
funding this work through the Great Lakes Restoration Initiative.
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 3 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
EXECUTIVE SUMMARY
This Technical Report, developed by the Great Waters Research Collaborative (GWRC), presents
methods and findings from the Great Lakes Ship Ballast Monitoring Project (Project), a two-year effort
supported by the United States Environmental Protection Agency’s (USEPA’s) Great Lakes Restoration
Initiative via the Maritime Administration. The Lake Carriers’ Association requested that the GWRC team
conduct this project to help it meet a requirement to execute a study evaluating risk associated with
laker ballast water discharge in USEPA Vessel General Permit (VGP) 2013 Part 6.15.5.b., in response to
Minnesota's 401 certification of VGP2013. The overarching goal of the Project was to characterize
aquatic organism densities and community composition in Great Lakes ships’ ballast water (uptake and
discharge) and analyze target species presence/absence in selected source water and receiving ports.
Specifically, the Project generated information on Great Lakes vessels’ ballast water regarding:
• Densities of a target organism, Hemimysis anomala, i.e., the “bloody red shrimp”, and other
Great Lakes non-indigenous species in ballast uptake and discharge;
• Presence/absence of the H. anomala CO1 genetic marker in a subset of source and discharge
ports and ballast uptake and discharge;
• Densities and community composition of planktonic organisms (i.e., zooplankton and protists) in
ballast uptake and discharge;
• Water quality/chemistry of ballast uptake and discharge; and
• Densities of pathogen indicators Escherichia coli and Enterococcus spp. in ballast discharge.
Eight Canadian and United States bulk carriers participated in the study. Sampling events occurred
during the 2017 calendar year and focused on ballast operations resulting in discharges of water
sourced from locations in the lower four Great Lakes to western Lake Superior, including:
• Fifteen Discharge Sampling Events: GWRC sampled 15 ship discharges to western Lake
Superior loaded from various locations in the lower four lakes.
• Four Voyage-Wide Sampling Events: Four of the sampled discharges to western Lake Superior
were associated with “voyage-wide” sampling, including associated source harbor water, ballast
uptake, and receiving water.
• One Uptake-Only Sampling Event: One stand-alone uptake sampling event occurred in central
Lake Erie; GWRC was unable to couple with a WLS discharge sampling event.
In summary, this research found laker ballast water from the lower four Great Lakes that was destined
to, or directly in, discharge to western Lake Superior ports contained non-indigenous species of aquatic
organisms not previously recorded in Lake Superior, and in one case, in the Great Lakes. In voyage-wide
sampling events, evidence of Project-relevant non-indigenous species were found in the source harbors,
the ballast uptake and ballast discharge. The Project detected these specimens though it surveyed only a
fraction of the ship ballast water destined or discharged to western Lake Superior in 2017, only a small
portion of the target ballast uptake/discharge events, and only snapshots in time of the shipping season.
Next research steps should focus on practicability and efficacy evaluations of best ballast water
management alternatives for the laker ships, as well as further characterization of the risk-release
relationship for aquatic invasive species in the Great Lakes.
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 4 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
TABLE OF CONTENTS
ACKNOWLEDGMENT .............................................................................................................................................. 2
EXECUTIVE SUMMARY ........................................................................................................................................... 3
TABLE OF CONTENTS .............................................................................................................................................. 4
LIST OF FIGURES ..................................................................................................................................................... 5
LIST OF TABLES ...................................................................................................................................................... 6
ABBREVIATIONS AND ACRONYMS ......................................................................................................................... 7
1. INTRODUCTION .................................................................................................................................................. 8
2. TEST VESSELS AND VESSEL PREPARATION ........................................................................................................ 11
3. EXPERIMENTAL DESIGN AND METHODS ........................................................................................................... 12
3.1. SHIPS/VOYAGES/BALLAST EVENTS SAMPLED ............................................................................................................ 12
3.2. RESEARCH METHODS ............................................................................................................................................ 16
3.2.1. Ballast Uptake and Ballast Discharge Sample/Data Collection ............................................................. 16
3.2.2. Source and Receiving Water Sample/Data Collection ........................................................................... 21
3.3. SAMPLE PROCESSING AND ANALYSIS........................................................................................................................ 25
3.3.1. Water Chemistry and Water Quality Analysis ........................................................................................ 25
3.3.2. Biological Sample Analysis ...................................................................................................................... 25
3.3.3. Quality Assurance and Quality Control................................................................................................... 26
4. RESULTS ........................................................................................................................................................... 27
4.1. CHARACTERISTICS OF LAKER BALLAST WATER DISCHARGED TO WESTERN LAKE SUPERIOR ................................................. 27
4.1.1. Vessel and Shipboard Sampling System Operational Data .................................................................... 27
4.1.2. Non-Indigenous Species and Target Organism (Hemimysis Anomala) Results ..................................... 30
4.1.3. Background Biological, Physical/Chemical Characteristics .................................................................... 36
4.2. VOYAGE-WIDE SAMPLING (FOUR VOYAGES) ............................................................................................................. 40
4.2.1. Vessel and Shipboard Sampling System Operational Data .................................................................... 41
4.2.2. Non-Indigenous Species and Target Organism (Hemimysis Anomala) Results ..................................... 45
4.2.3. Background Biological, Physical/Chemical Characteristics .................................................................... 48
5. DISCUSSION AND CONCLUSION ........................................................................................................................ 55
6. REFERENCES ..................................................................................................................................................... 58
APPENDIX ............................................................................................................................................................ 60
LSRI/GWRC/TR/GLSBM/1
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
LIST OF FIGURES
Figure 1. Example Sample Port. ................................................................................................................................... 11
Figure 2. Geographic Overview Great Lakes Ship Ballast Monitoring Project Sampling Events. ................................ 13
Figure 3. Generalized Schematic of the Shipboard Sampling System ......................................................................... 18
Figure 4. Photo Showing Operation of the Shipboard Sampling System On Board a Test Vessel. .............................. 19
Figure 5. Photo Showing the Shipboard Sampling System’s Plankton Net Component On Board a Test Vessel. ....... 19
Figure 6. Generalized Schematic of Ballast Uptake and Source Water Sampling Site Locations for “Voyage-Wide”
Sampling Exercises, i.e., Trials 6, 11, 12 and 13. Note: Sites 1 and 2 were not sampled during Trial 6. ..................... 23
Figure 7. Generalized Schematic of Ballast Discharge and Receiving Water Sampling Site Locations for “Voyage-
Wide” Sampling Exercises, i.e., Trials 6, 11, 12 and 13. ............................................................................................... 24
Figure 8. Total Density and Percent Composition of Zooplankton in Ballast Discharge Samples. .............................. 36
Figure 9. Number of Taxa found in Ballast Discharge (D) Samples. ............................................................................ 37
Figure 10. Density and Percent Composition of Live Zooplankton in Ballast Discharge Samples. .............................. 38
Figure 11. Histograms of Protist Densities (Upper) and Proportions (Lower) in Discharge Samples. Grouping
Reflects Major Divisions of the Organisms. ................................................................................................................. 39
Figure 12. Density and Percent Composition of Zooplankton in Paired Ballast Uptake and Discharge Samples........ 48
Figure 13. Histograms of Protist Assemblage Composition by Major Divisions of Organisms Showing Densities
(Upper) and Proportions (Lower) in Voyage-Wide Uptake Samples. For Comparison, Voyages with Paired Uptake
and Discharge Samples also have Discharge Samples Shown. .................................................................................... 49
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
LIST OF TABLES
Table 1. Sampling Event Locations, Dates and Associated Ballast Hold Times. N/A = Not Applicable because Not
Sampled. Voyage-Wide = Sampling Occurred at both Uptake and Discharge over the course of the voyage; Ballast
Uptake-Only and Ballast Discharge–Only = Sampling occurred only for operation specified over the course of the
voyage.......................................................................................................................................................................... 14
Table 2. Operational, Water Quality/Chemistry and Biological Data/Samples and Measurements Collected/Taken
During Ballast Uptake and Ballast Discharge Sampling Events. N/A = Not Applicable. ............................................... 20
Table 3. Sample Site Characteristics, Water Quality/Chemistry and Biological Data/Samples and Measurements
Collected/Taken During Source Water and/or Receiving Water Sampling Events. N/A = Not Applicable. ................. 22
Table 4. Ballast Discharge Trials: Summary of Vessel and Shipboard Sampling System Operational Parameters.
Note: Trial 3 was an Uptake-Only Sampling Event and is not Presented in this Table. P= Port, S = Starboard, N/A=
Not Applicable (Not Collected). ................................................................................................................................... 28
Table 5. Minimum Number of Specimens per Non-Indigenous Species (NIS) Taxon (#/m3) that would Need to be
Present for Detection in Ballast Uptake and Discharge Samples Given Volumes Sampled ........................................ 31
Table 6. Summary of Measured Biological Parameters from Ballast Discharge to Western Lake Superior................ 32
Table 7. Summary of Information on Project-Relevant of Non-Indigenous Species in the Great Lakes. .................... 34
Table 8. Ballast Uptake Trials: Summary of Vessel and Shipboard Sampling System Operational Parameters. ........ 42
Table 9. Source and Receiving Water Samples: Summary of Location and Site Characteristics. ............................... 44
Table 10. Summary of Biological Parameters for Voyage-Wide Trials. ....................................................................... 46
Table 11. Occurrence of Hemimysis anomala DNA and Specimens in Samples Across Voyage-Wide Trial Sampling
Events. DL = Detection Level. ...................................................................................................................................... 47
Table 12. Voyage-Wide Trials: Summary of Chemistry and Water Quality Parameters (Average ± Standard
Deviation). N/A = Not Applicable (Not Collected). ...................................................................................................... 51
Table 13. Density of Zooplankton (#/m3) in Shipboard Ballast Uptake (U) and Discharge (D) Samples. .................... 61
Table 14. Density of Live Zooplankton (#/m3) in Shipboard Discharge Samples. D = Discharge. ............................... 68
Table 15. Density of Protists (cells/mL) in Shipboard Ballast uptake (U) and Discharge (D) Samples......................... 70
Table 16. Ballast Discharge Trials: Summary of Water Chemistry and Water Quality Parameters (Average ±
Standard Deviation). .................................................................................................................................................... 83
Table 17. Ballast Uptake Trials: Summary of Water Chemistry and Water Quality Parameters (Average ± Standard
Deviation). NM = Not Measured. ................................................................................................................................ 85
Table 18. Source Water Trials: Summary of Chemistry and Water Quality Parameters. NC = Not Collected. ........... 86
Table 19. Receiving Water Trials: Summary of Chemistry and Water Quality Parameters. ....................................... 87
Table 20. Top Twenty Sources and Volumes of Ballast Water Discharged to Western Lake Superior from Other
Great Lakes Ports in 2017. ........................................................................................................................................... 88
LSRI/GWRC/TR/GLSBM/1
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
ABBREVIATIONS AND ACRONYMS
%T: Percent Transmittance
ANSI: American National Standards Institute
BMP: Best Management Practices
BWMS: Ballast Water Management System
cfu: Colony Forming Unit
DNA: Deoxyribonucleic Acid
DOC: Dissolved Organic Carbon
eDNA: Environmental Deoxyribonucleic Acid
Ft: Feet
GLNPO: Great Lakes National Program Office
GLSLSS: Great Lakes and St. Lawrence Seaway System
GPS: Global Positioning System
GWRC: Great Waters Research Collaborative
IMO: International Maritime Organization
LSRI: Lake Superior Research Institute
MDL: Method Detection Limits
NBIC: National Ballast Information Clearinghouse
NOAA: National Oceanic and Atmospheric Administration
NIS: Nonindigenous Species
NPOC: Non-Purgeable Organic Carbon
PI: Principal Investigator
POM: Particulate Organic Matter
QAQC: Quality Assurance and Quality Control
QAPP: Quality Assurance Project Plan
RL: Reporting Limit
SOP: Standard Operating Procedure
SSS: Shipboard Sampling System
TQAP: Test/Quality Assurance Plan
TSS: Total Suspended Solids
USEPA: United States Environmental Protection Agency
VGP: Vessel General Permit
WLS: Western Lake Superior
LSRI/GWRC/TR/GLSBM/1
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
1. INTRODUCTION
This Great Waters Research Collaborative (GWRC) Technical Report presents methods and findings from
the Great Lakes Ship Ballast Monitoring Project (Project), a project funded by the United States
Environmental Protection Agency’s (USEPA’s) Great Lakes Restoration Initiative via the Maritime
Administration and carried out in cooperation with several Great Lakes ship owners and operators. The
Lake Carriers’ Association requested that the GWRC team conduct this project to help it meet a
requirement to execute a study evaluating risk associated with laker ballast water discharge in USEPA
Vessel General Permit (VGP) 2013 Part 6.15.5.b., in response to Minnesota's 401 certification of
VGP2013. The Minnesota Pollution Control Agency approved the GWRC study for that purpose. The
overarching goal of the Project was to characterize aquatic organism densities and community
composition—with particular attention to the presence of non-indigenous species (NIS) not previously
reported in Lake Superior—in Great Lakes ships’ ballast water (uptake and discharge) and, for a subset
of voyages, associated source and receiving port water. Test vessels were eight Canadian and United
States lakers. Ballast uptake and source water sampling locations comprised ports in the lower four
lakes. Ballast discharge and receiving water sampling locations were ports in western Lake Superior
(WLS). For purposes of this research, WLS comprises points west of Silver Bay on the north shore of the
western arm of Lake Superior and wraps around to points east of Sand Bay on the south shore of the
western arm of Lake Superior (Figure 2). The area includes the active ports of Superior, Wisconsin;
Duluth, Minnesota; Two Harbors, Minnesota; and Silver Bay, Minnesota. All other locations in Lake
Superior are described simply as Lake Superior sites.
The Project defined lakers as vessels that operate exclusively on the Laurentian Great Lakes and are
confined to operations upstream of the waters of the St. Lawrence River east of a thumb line drawn
from Cap de Rosiers to West Point, Anticosti Island, and west of a line along 63 W. longitude from
Anticosti Island to the north shore of the St. Lawrence River. Lakers are distinct from salties, or
oceangoing vessels, in that salties are not confined to operations within the Great Lakes and enter/exit
the Great Lakes from the Gulf of St. Lawrence. The Gulf of St. Lawrence is the outlet of the Laurentian
Great Lakes via the mouth of the St. Lawrence River into the Atlantic Ocean.
The purpose of the Project, and sampling exercises associated with it, was to understand characteristics
and trends with respect to organism movement into Lake Superior from other locations in the
Laurentian Great Lakes. Ship owners agreed to participate in the study, and asked that ship identities
and locations be coded. To that end, all results from the monitoring exercises are reported in summary
form to assure that individual ships are not identifiable as sources of specific sampled organisms. Dates
of sample collection also are reported by month rather than day.
Project objectives were to generate and analyze information regarding:
• Transit and seasonal-related alterations in the presence/absence of a target organism
Hemimysis anomala, i.e., the “bloody red shrimp”, and other Great Lakes NIS, in ballast uptake
and discharge water;
• The densities and community composition of planktonic organisms (i.e., zooplankton and
protists) and density of the pathogen indicator bacteria Escherichia coli and Enterococcus spp. in
the ballast uptake and/or ballast discharge of Great Lakes vessels; and
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
• Transit and seasonal-related alterations in water quality/chemistry, and zooplankton and protist
densities and communities in Great Lakes ballast uptake versus ballast discharge.
Sampling events took place primarily from July 2017 through December 2017. The concentration of
experimental activity in the second half of 2017, and that specific ballast tanks could not be individually
sampled during both uptake and discharge (laker ships often load and unload ballast water into two or
more ballast tanks simultaneously rather than ballasting/deballasting one tank at a time), made
seasonal and transit-related alterations described above impossible to assess. Therefore, rather than
assess patterns, this Technical Report provides descriptive characterizations of uptake and discharges vis
a vis the parameters listed. Specifically, the report presents findings relative to:
• Characteristics of laker ballast water discharged to WLS from non-Lake Superior source ports,
including:
o Vessel and Shipboard Sampling System (SSS) operational information;
o NIS and target organism (H. anomala) detections;
o Background biological, physical/chemical characteristics.
• Voyage-wide characteristics of laker ballast water, including non-Lake Superior source water,
uptake water, discharge water and WLS receiving water; including:
o Vessel and SSS operational information;
o NIS and target organism (H. anomala) detections; and
o Background biological, physical/chemical characteristics.
All research activities were consistent with the GWRC’s Shipboard Quality Assurance Project Plan (QAPP;
LSRI, 2017) and Lake Superior Research Institute (LSRI) standard operating procedures (SOPs). A Project-
specific GWRC Test/Quality Assurance Plan (TQAP), executed and agreed by all involved parties, guided
overall research activities and assured conformance to technical and quality system requirements.
Samples were characterized in terms of general water quality/chemistry and biota, including the
presence of organisms in taxa not previously detected and reported in Lake Superior. Genetic detection
tools were employed to detect the presence of the target NIS, H. anomala, a native of the Ponto-
Caspian region of eastern Europe that was first reported in the Great Lakes (Lakes Ontario and Michigan)
in 2006 by researchers from the National Oceanic and Atmospheric Administration (NOAA; Kipp et al.,
2017). At the commencement of the Project, this species had been found in samples collected from all
of the Great Lakes with the exception of Lake Superior (Kipp et al., 2017).6
Ballast uptake and/or discharge densities of zooplankton, protists, E. coli and Enterococcus spp.7 from
laker ships to WLS were calculated. Community composition of organisms entrained in ballast uptake
and discharge samples were characterized. Ballast uptake and discharge water physical/chemical
characteristics were characterized and compared to that of corresponding ballast source water and
receiving ports. Finally, ballast uptake and discharge samples were examined specifically for NIS not yet
6 After the completion of the Project’s sampling events, H. anomala was collected in samples from the St. Louis River, near Allouez Bay,
Wisconsin. https://nas.er.usgs.gov/queries/CollectionInfo.aspx?SpeciesID=2627&State=WI&HUCNumber=4010301; accessed 26 April 2018.
7 Though not in the original study plan, analysis of Enterococcus spp. was added because as with analysis E. coli, it is typical of assessments of
ballast discharge and provides useful general information of discharge water quality.
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
recorded in WLS, and the presence/absence of the CO1 gene of the target organism, H. anomala, was
evaluated in ballast uptake, source water, ballast discharge and receiving water.
Notably, it was not an objective of this Project to determine risk of establishment or invasion associated
with NIS detected in laker uptake, or discharge, or associated source and receiving water. Such an
assessment, if possible at all, would require a different experimental design. In this study, source and
receiving water assessments, which occurred shortly before or after the sampled ballast event, and at
varying distances upstream or downstream of the ballasting location, were not designed to deliver
direct cause and effect information relevant to that particular ballasting event. Instead they were
intended to determine whether there was evidence of an established, breeding population of the H.
anomala species already in the vicinity of the ship operations. Further, the genetic and microscopic
analyses were not designed to conclusively distinguish live/dead status of detected organisms/material
in this study. However, detection prevalence in discharge and across harbor sampling sites, and the
condition of individual specimens in microscopic analysis, can provide clues as to how recently
organisms were vital.
GWRC identified sampling opportunities based on trade route and voyage timing. The goal was to
concentrate most of the sampling on ships whose voyages plied from areas in the lower lakes in which
H. anomala is known to occur, to WLS. Other sampling events were distributed across other ship
voyages and vessels of opportunity. Overall the following sampling events took place associated with 16
different ship voyages:
∗ Fifteen Discharge Sampling Events: GWRC sampled 15 ship discharges to WLS loaded from
various locations in the lower four lakes.
∗ Four Voyage-Wide Sampling Events: Four of the sampled discharges were associated with
“voyage-wide” sampling events. That is, along with the ballast discharge, the associated source
harbor water, ballast uptake, and receiving water were sampled. All voyage-wide sampling
occurred on ship voyages from southern Lake Michigan to WLS.
∗ One Uptake-Only Sampling Event: One stand-alone uptake sampling event occurred in central
Lake Erie; GWRC was unable to couple with a WLS discharge sampling event.
This Technical Report summarizes Project methods, including test vessels, and vessel preparation;
experimental design and methods; ships, voyages and ballast events sampled; quality assurance and
quality control (QAQC) procedures; and Project results, discussion, recommendations and conclusions.
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
2. TEST VESSELS AND VESSEL PREPARATION
In 2016 the Project team8 developed a ship sampling approach and solicited volunteer laker vessels from
both United States and Canadian fleets for the study. Participating ship owners volunteered a total of 18
potential test vessels (i.e., ten United States lakers and eight Canadian lakers), all of which were self-
unloaders.
GWRC provided technical support to participating ship owners and operators to facilitate their
installation of ballast water sample ports. Based on GWRC design recommendations, ship owners
installed sample ports on the ballast mains in the best available locations for uptake and discharge
sampling. Owing to cargo routes, vessel availability and sampling team logistics, not all of the 18 vessels
equipped with sample ports were sampled. In keeping with the Project’s goal of focusing on ballast
water characteristics rather than individual ships, participating vessels were assigned codes for purposes
of data reporting.
All of the volunteer vessels installed a 4 inch steel ANSI flange in a segment of the ship’s ballast line
which served as many ballast tanks as possible. The sample port flanges were covered with blind flanges
when not in use for sampling. Immediately prior to sampling, a pitot-like sample port was installed by
ship personnel into the sample flange. Some ships installed an optional return flow port in the ballast
main to return filtered sample water back in-line. Engineering best judgement guided identification of
the sample point locations; GWRC personnel inspected vessel piping, analyzed fluid dynamics, and
recommended the best position for sample uptake and discharge ports. The vessel owners and
operators installed the sample points with the blind flanges consistent with the design; GWRC supplied
the sample ports. The length of bent elbow pipe varied depending on the diameter of the ballast main.
The length was chosen to reach the central third of the ballast main. The sample port was also equipped
with a ball valve pipe (Figure 1).
Figure 1. Example Sample Port.
8 Previously identified as the Great Ships Initiative of the Northeast-Midwest Institute.
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
3. EXPERIMENTAL DESIGN AND METHODS
3.1. SHIPS/VOYAGES/BALLAST EVENTS SAMPLED
Table 1 summarizes sampling event locations, dates, ballast water source regions, and ballast water hold
times. Figure 2 shows the voyage routes subject to sampling. All data are presented in categorical rather
than specific terms in keeping with the Project’s goal of focusing on the general characteristics of
locations and ballast water subject to movement as opposed to specific ships. For these trials, a random
subset of the water volume subject to ballasting during port operations was sampled during each
sampling event. Consequently, the same water mass was not subject to both uptake and discharge
sampling. Further, in some cases, a smaller secondary ballasting event took place from an interim port
between the port of uptake and the port of discharge where sampling occurred (Table 1). These
secondary uptake volumes ranged from 18.3 to 40.5 percent of the total. Though at times substantial,
these secondary ballasting operations did not interfere with the project objectives of assessing NIS
movements by laker ships from the lower four Great Lakes to Lake Superior. The Project objectives did
not include any estimation of a rate of organism transfer from the lower lakes to Lake Superior which
might be affected by dilution with interim Lake Superior uptakes. Nor did it include any geographic
constraints on the source of water from the lower lakes that was transferred to Lake Superior, which
might be affected by mixing of water from different lower lakes locations.
Of the ballast discharges sampled, estimated hold times (from end of initial uptake in the source system
until the beginning of discharge in WLS) ranged from 3 to 6 days (Table 1). For the voyage-wide sampling
events, the ballast hold times ranged from 3 to 4 days (Table 1).
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Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
Figure 2. Geographic Overview Great Lakes Ship Ballast Monitoring Project Sampling Events.
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Great Waters Research Collaborative.
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Table 1. Sampling Event Locations, Dates and Associated Ballast Hold Times. N/A = Not Applicable because Not Sampled. Voyage-Wide = Sampling Occurred
at both Uptake and Discharge over the course of the voyage; Ballast Uptake-Only and Ballast Discharge–Only = Sampling occurred only for operation
specified over the course of the voyage.
Trial
#
Date
(Mo/Yr) Sampling Target
Ballast Uptake/Source Water Ballast Discharge/Receiving Water
Initial Uptake /
Source Water
Port Location
Locations of
Secondary
Uptakes into
Sampled
Tanks1
Percentage
of Water
from
Secondary
Uptake in
Sampled
Tanks
Percentage of
Water
Discharged at
Intermediate
Locations from
Sampled Tanks
Discharge /
Receiving Water
Location
Estimated
Hold
Time from
Initial
Uptake1
Estimated
Hold Time
from Most
Recent
Uptake1
1 Jan-17
Ballast Discharge-
Only
Southern Lake
Michigan
Eastern Lake
Superior
20.2% N/A
Western Lake
Superior
6 Days 4 Days
2 Jul-17
Ballast Discharge-
Only
Eastern Lake
Erie
N/A N/A N/A
Western Lake
Superior
4 Days N/A
3 Aug-17
Ballast Uptake-
Only
Central Lake
Erie
N/A N/A N/A N/A N/A N/A
4 Aug-17
Ballast Discharge-
Only
Central Lake
Erie
Lake Superior 18.3% N/A
Western Lake
Superior
5 Days 2 Days
5 Aug-17
Ballast Discharge-
Only
Southern Lake
Michigan
N/A N/A N/A
Western Lake
Superior
4 Days N/A
6 Sep-17 Voyage-Wide Southern Lake
Michigan
St. Mary's
River, Lake
Superior
29.3% N/A Western Lake
Superior 3 Days 1 Day
7 Sep-17 Ballast Discharge-
Only
Southern Lake
Michigan
St. Mary's
River, Lake
Superior
23.0% N/A Western Lake
Superior 3 Days 1 Day
8 Sep-17 Ballast Discharge-
Only St. Clair River
Eastern Lake
Superior, Lake
Superior
40.5% 40.5% Western Lake
Superior 3 Days 1 Day
9 Oct-17
Ballast Discharge-
Only
Detroit River N/A N/A N/A
Western Lake
Superior
4 Days N/A
10 Oct-17
Ballast Discharge-
Only
Southern Lake
Michigan
2
Eastern Lake
Superior
21.3% 14.8%
Western Lake
Superior
3 Days 2 Days
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Trial
#
Date
(Mo/Yr) Sampling Target
Ballast Uptake/Source Water Ballast Discharge/Receiving Water
Initial Uptake /
Source Water
Port Location
Locations of
Secondary
Uptakes into
Sampled
Tanks1
Percentage
of Water
from
Secondary
Uptake in
Sampled
Tanks
Percentage of
Water
Discharged at
Intermediate
Locations from
Sampled Tanks
Discharge /
Receiving Water
Location
Estimated
Hold
Time from
Initial
Uptake1
Estimated
Hold Time
from Most
Recent
Uptake1
11 Oct-17 Voyage-Wide Southern Lake
Michigan2
St. Mary's
River, Eastern
Lake Superior
20.5% 16.1% Western Lake
Superior 3 Days 1 Day
12 Oct-17 Voyage-Wide Southern Lake
Michigan2
St. Mary's
River, Eastern
Lake Superior,
Lake Superior
19.8% 16.1% Western Lake
Superior 3 Days <1 Day
13 Nov-17 Voyage-Wide
Southern Lake
Michigan
N/A N/A N/A
Western Lake
Superior
4 Days N/A
14 Nov-17
Ballast Discharge-
Only
Northern Lake
Michigan
N/A N/A N/A
Western Lake
Superior
3 Days N/A
15 Dec-17
Ballast Discharge-
Only
Western Lake
Erie
St. Mary's River 24.9% N/A
Western Lake
Superior
3 Days 2 Days
16 Dec-17
Ballast Discharge-
Only
Lake Ontario N/A N/A N/A
Western Lake
Superior
5 Days* N/A
1 Data sourced from National Ballast Information Clearinghouse (NBIC, 2018).
2 Uptake occurred in two locations in the same harbor area within 1.5 miles. GWRC sampled the first of these uptake operations
* Data provided via personal communication between the Project Principal Investigator and the ship captain.
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3.2. RESEARCH METHODS
This section summarizes methods for source water, ballast uptake, ballast discharge and receiving water
sample/data collection and analysis.
3.2.1. BALLAST UPTAKE AND BALLAST DISCHARGE SAMPLE/DATA COLLECTION
The Project team collected representative samples of ballast uptake and discharge water masses during
routine ship operations. The vessel’s crew facilitated these sampling events by ensuring adequate space
and power sources and informing GWRC personnel on ballast operational events. Sample types and
volumes varied depending upon sampling objectives and comprised:
∗ Continuous in-line samples filtered through a plankton net with a minimum target volume of 2
m3 targeting larger organisms, mainly zooplankton (Table 2);
∗ Continuous in-line whole water samples (“seep samples”) of up to 8 L in volume (Table 2)
targeting protists and microbes (Table 2); and
∗ Grab samples of up to 1 L in volume collected at the beginning, middle and end of the ballast
sampling operation (Table 2), targeting physical/chemical properties of the water.
The samples were collected using either the active or passive version of the SSS (Figures 3-5; Table 2).
The active version pumped sample flow from the ballast main and returned it to the main if a return
port was provided. In the passive version, the ballast line pressure powered the sample flow and the
filtered ballast water is discharged to the bilge.
GWRC interviewed the vessel’s crew during discharge sampling events to determine the date and port
of the last uptake event. GWRC personnel in the control room recorded the water height (soundings) of
each ballast tank periodically into the Great Lakes Ship Ballast Monitoring Project: Ballast Tank Height
Measurements datasheet. The goal was to record a minimum of three soundings for each tank
ballasted/deballasted during each sampling event: one at the start, one in the middle, and one at the
end of GWRC sampling. Typically, soundings were taken far more often, i.e., every ten to fifteen minutes
throughout each event. The times of the first and last soundings did not always line up with the start
and end of sampling due to communication lags.
GWRC engineering staff used the soundings recorded during each sampling event to estimate the
volume of ballast water subject to sampling. The estimated volume of ballast water sampled was
calculated as the volume change of all tanks ballasting/deballasting during the sampling event. When
soundings were not recorded at the beginning and end of sampling linear interpolation between
available soundings was used to estimate beginning and end volumes. Changes in vessel list and trim
were not considered when estimating tank volumes. In some cases, current sounding tables were not
available to support conversion of tank heights to volumes, and in these instances volumes were
estimated based on ship drawings.
Project personnel collected samples according to LSRI/SOP/GWRC/12 – Sample Collection Procedures for
Ballast Water Monitoring. The SSS sample pitot delivered a continuous side flow from the ballast main
directed into a 35 µm plankton net for sampling of organisms ≥ 50 µm (i.e., zooplankton). GWRC
personnel controlled the flow rate to deliver a target minimum sample volume of 2.0 m3 of water.
Beginning with Trial 10, GWRC began collecting an additional larger-volume zooplankton sample using a
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plankton net with a larger pore size (400 µm). This larger volume sample was added to the TQAP with
the acquisition of a larger pore net, and allowed collection and enumeration of sparser organisms in the
size range of the target NIS, H. anomala. For these samples, an additional 3.0 m3 of ballast water was
filtered. Collection of this second zooplankton sample was only possible when a return port was
installed in the vessel’s ballast line, and when sufficient time remained during cargo loading/unloading
to allow for another ~60 minutes of sampling. Seep samples were collected into a 19 L carboy from a
side-stream of the sample water flow, branching off upstream of the plankton net. Seep sample water
was used to assess protist density and taxonomic composition, as well as E. coli and Enterococcus spp.
densities and the presence/absence of H. anomala eDNA. Whole water grab samples were collected for
characterization of water quality/chemistry via a dedicated side port located off the main sample line.
A multiparameter sonde (YSI EXO2 Multiparameter Instrument and EXO Handheld Display; YSI
Incorporated; Yellow Springs, Ohio) was used to measure temperature, conductivity, salinity (via
algorithm), turbidity, pH, dissolved oxygen, chlorophyll a (green algae) and phycocyanin accessory
pigment (blue-green algae). While a sonde measures in situ chlorophyll a and phycocyanin, it is not as
accurate as an extractive technique. Some sources of inaccuracy can be minimized by combining
extractive analysis of the samples with the sonde readings of the same samples and applying a
correction factor. Due to the constraints of the sampling events, a correction factor was not determined,
therefore the uncorrected values obtained can be used for relative comparison purposes only and not
actual concentrations. The sonde was calibrated weekly according to LSRI/SOP/FS/39 – Calibration,
Deployment, and Storage of YSI EXO Series Multiparameter Water Quality Sondes.
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------------------------------------------------------------------------------------------------------------------------------------------
Figure 3. Generalized Schematic of the Shipboard Sampling System
(Top Diagram Shows Active Version; Bottom Diagram Shows Passive Version).
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Figure 4. Photo Showing Operation of the Shipboard Sampling System On Board a Test Vessel.
Figure 5. Photo Showing the Shipboard Sampling System’s Plankton Net Component On Board a Test Vessel.
LSRI/GWRC/TR/GLSBM/1
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Table 2. Operational, Water Quality/Chemistry and Biological Data/Samples and Measurements Collected/Taken During Ballast Uptake and Ballast
Discharge Sampling Events. N/A = Not Applicable.
Sampling Event Category Parameter Number of Samples/Measurements
Per Sampling Event
Target Sample
Volume Sample Type
Ballast Uptake or
Ballast Discharge
Vessel Operations Ballast Tank Electronic Soundings Minimum of 3 N/A Vessel Log
Shipboard Sampling
System Operations
Plankton Net Flow Rate Continuous N/A Plankton Net
Plankton Net Volume Continuous 2.0 to 5.0 m3 Plankton Net
Seep Sampler Volume Continuous
5 L (uptake);
8 L (discharge)
Seep Sampler
Water Quality/
Chemistry
Temperature, Conductivity, Salinity (via
algorithm), Turbidity, pH, Dissolved Oxygen,
Chlorophyll a (green algae), Phycocyanin
Accessory Pigment
3 (Beginning, Middle, End) 500 mL Grab Sample Line
Percent Transmittance, Total Suspended
Solids, Particulate Organic Matter, Mineral
Matter
3 (Beginning, Middle, End) 1 L Grab Sample Line
Non-Purgeable Organic Carbon, Dissolved
Organic Carbon 3 (Beginning, Middle, End) 125 mL Grab Sample Line
Biology
All Zooplankton: Total Density and Taxonomic
Composition; Live Density for selected
discharge samples
1: Uptake; 1: Discharge 2.0 m3 Plankton Net
(35 µm mesh)
Larger Volume Sample for Hemimysis
anomala: Total Density (for Trials 10 – 16 and
vessels having a return port installed in ballast
main)
1: Uptake, 1: Discharge
(beginning with Trial 10) 3.0 m3 Plankton Net
(400 µm mesh)
Environmental DNA: Presence of CO1 Gene of
Hemimysis anomala
3
(beginning with Trial 6)
1 L Seep Sampler
Protists: Total Density and Taxonomic
Composition
2: Uptake; 2: Discharge 500 mL Seep Sampler
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3.2.2. SOURCE AND RECEIVING WATER SAMPLE/DATA COLLECTION
Source water and receiving water samples/data were collected in association with the four voyage-wide
trials (Table 3), within 20 hours of ballast uptake or discharge. Collection sites included a location in
close proximity to the test vessel, and up to three sites within 0.5 mile of the test vessel’s docking
location, typically within the port facility9 (Figures 6 - 7).
Longitude/latitude measurements were taken via Global Positioning System (GPS) devices or derived
from interpolation on georeferenced aerial photos. The sampling locations are reported relative to
location of the test vessel. Water depth was measured using a portable sonar sensor transducer
(Venterior VT-FF001 Portable Fish Finder) and weather conditions were qualitatively categorized
through observation.
Whole water grab samples for characterization of water quality/chemistry (i.e., percent transmittance,
%T; total suspended solids, TSS; particulate organic matter, POM; non-purgeable organic carbon, NPOC;
and dissolved organic carbon, DOC) were collected from a depth of approximately 1 meter below the
water surface. Temperature, conductivity, salinity (via algorithm), turbidity, pH, dissolved oxygen,
chlorophyll a (green algae) and phycocyanin accessory pigment (blue-green algae) were measured using
the multiparameter sonde. The sonde was calibrated weekly according to LSRI/SOP/FS/39 – Calibration,
Deployment, and Storage of YSI EXO Series Multiparameter Water Quality Sondes. Finally, three replicate
whole water samples were collected in sterile bottles attached to a sampling pole for presence/absence
determination of the CO1 gene of H. anomala according to LSRI/SOP/GWRC/13 – Processing and
Shipping Samples for Environmental DNA Analysis.
9 In one case, Trial 6, source water was collected outside the port facility at a location approximately five miles from the test vessel’s docking
location.
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Table 3. Sample Site Characteristics, Water Quality/Chemistry and Biological Data/Samples and Measurements Collected/Taken During Source Water
and/or Receiving Water Sampling Events. N/A = Not Applicable.
Sampling Event Category Parameter Number of Samples/Measurements Per
Sampling Event
Target Sample
Volume Sample Location
Source Water or
Receiving Water
Location/Sampling Site
Characteristics
Longitude/Latitude Up to 4 sites N/A Dock Wall
Estimated Distance to Test
Vessel
Up to 4 sites N/A Dock Wall
Water Depth Up to 4 sites N/A Dock Wall
Observational Weather
Conditions
Up to 4 sites N/A Dock Wall
Water Quality/ Chemistry
Temperature, Conductivity,
Salinity (via algorithm), Turbidity,
pH, Dissolved Oxygen,
Chlorophyll a (green algae),
Phycocyanin Accessory Pigment
1 per Site N/A – In Situ Dock Wall
Percent Transmittance, Total
Suspended Solids,
Particulate Organic Matter,
Mineral Matter
1 per Site 1 L Dock Wall
Non-Purgeable Organic Carbon,
Dissolved Organic Carbon 1 per Site 125 mL Dock Wall
Biology
Environmental DNA:
Presence of CO1 Gene of
Hemimysis anomala
3 per Site 1 L Dock Wall
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Great Waters Research Collaborative.
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Figure 6. Generalized Schematic of Ballast Uptake and Source Water Sampling Site Locations for “Voyage-Wide”
Sampling Exercises, i.e., Trials 6, 11, 12 and 13. Note: Sites 1 and 2 were not sampled during Trial 6.
6
6
2,000 Meters
La
ke
M
ic
hi
g
an
6
6
Trial
Site 3
Site 1
Site 2
Ship
11
11
11
250 Meters
Ship
Site 1
Site 2
Ship
12
12
12
250 Meters
Site 1
Site 2
Ship
13
13
13
Sampling Location
Trial
Sampling
Location
11
11
Trial
Sampling
Location
12
12
250 Meters
Trial
Sampling
Location
13
13
Lake Michigan Lake Michigan Lake Michigan
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Figure 7. Generalized Schematic of Ballast Discharge and Receiving Water Sampling Site Locations for “Voyage-
Wide” Sampling Exercises, i.e., Trials 6, 11, 12 and 13.
6
Site 2
Site 3
6
6
6
200 Meters 6
Lake Superior
Site 1
Ship
Trial 6
Sampling
Location
Site 1
Ship and
Site 2
13
1313
13
13
Lake Superior
Trial 13
Sampling
Location
200 Meters
11
Site 2
Site 3
11
11
11
Lake Superior
11
Site 1
Ship
200 Meters
Sampling
Location
Trial 11
12
Site 2
Site 3
12
12
200 Meters
Lake Superior
12
Sampling
Location
Trial 12
12
Site 1
Ship
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3.3. SAMPLE PROCESSING AND ANALYSIS
3.3.1. WATER CHEMISTRY AND WATER QUALITY ANALYSIS
Laboratory-based analysis of %T of ultraviolet light at 254 nm took place following LSRI/SOP/SA/69 –
Laboratory Determination of Percent Transmittance of Light in Water at 254 nm. %T was measured on
both filtered and unfiltered aliquots of each sample collected. Analysis of TSS and POM occurred
according to LSRI/SOP/SA/66 – Analyzing Total Suspended Solids, Particulate Organic Matter, and
Mineral Matter (MM). MM defined as the difference between TSS and POM, was calculated for each
sample following analysis of TSS and POM. The reporting limit (RL) for TSS and POM analyses was
between 1.25 (800 mL filtered) and 5.00 mg/L (200 mL filtered). Sample analyses for NPOC and DOC
were conducted according to LSRI/SOP/SA/47 – Procedures for Measuring Organic and Inorganic Carbon
in Aqueous Samples (DOC is a proxy for dissolved organic matter). Method detection limits (MDLs) were
determined for water quality analyses according to LSRI/SOP/SA/35 – Procedures for Determination of
Method Detection Limit and Limit of Quantification. Any deviations to these methods were recorded and
assessed according to LSRI-GWRC QAQC processes (Section 3.3.3).
3.3.2. BIOLOGICAL SAMPLE ANALYSIS
The taxonomic diversity and total density of zooplankton in ballast water uptake and discharge samples
was determined by examination of subsamples from preserved samples using either a compound or
dissecting microscope in accordance with the USEPA Great Lakes National Program Office (GLNPO)
procedure LG 403 (USEPA, 2016). A minimum of 400 microzooplankton (i.e., rotifers, copepod nauplii,
and dreissenid mussel veligers) and 400 to 1,600 macrozooplankton (i.e., cladocerans, and copepod
juveniles and adults) were targeted for examination from each sample. Larger organisms, including
mysids, amphipods, and the cladocerans Bythotrephes and Cercopagis, were enumerated from the
entire sample. The condition of the specimen was observed and only whole specimens indicating they
were alive or recently alive when collected were included in the count. For eight of the samples (i.e.,
Trial 1, 4, 8, 9, 10 and 15 discharges, and Trial 6 and 8 uptakes), adult harpacticoid copepods were
removed from the entire sample in order to increase the detection level of these macrozooplankton
taxa. Detection levels for each taxon were calculated as the density of organisms that would be in 1 m3
of the original water sample if a single specimen was found in the volume of water that was targeted for
examination for that particular taxon. The detection levels for microzooplankton, which were examined
from relatively small subsamples, are much higher than the detection levels determined for H. anomala
which were enumerated from the entire sample (Table 5). In some trials, the presence of harpacticoid
copepods were noted in the extra portion of the sample that was targeted for examination of larger
organisms. In these cases, the presence of the harpacticoids was noted in the sample, but they were not
quantitatively enumerated and densities were not calculated.
The density of live zooplankton in ballast discharge samples was determined according to
LSRI/SOP/GWRC/19 – Zooplankton Sample Analysis for Ship Monitoring Projects. Live analyses were only
conducted for samples that could be delivered to the analysts within four hours of sample collection,
and were executed on a relatively small volume of sample water to a coarse taxonomic level. When live
analyses were possible, live density of major taxonomic groups was determined by counting the number
of dead organisms in a subsample and then killing the rest of the organisms and performing a total
count of the same subsample. Live density was determined by subtracting the number of dead
organisms from the total number of organisms.
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Total protist densities and taxonomic diversity analysis of ballast uptake and discharge samples took
place following the “Preserved Protist Sample Analysis (Utermöhl, 1958)” method outlined in
LSRI/SOP/GWRC/4 – Site-Specific Validation of CMFDA/FDA Stain and Determination of Protist
Concentration in Ballast Water Samples. In addition, to provide a detailed assessment of diatom
assemblages, water samples were digested in strong acid to remove the organic matrix and isolate
diatom valves. Diatom remains were then plated on microslides and assessed using oil-immersion light
microscopy at 1250 X magnification. This method, which allows for fine taxonomic assessment of
diatoms, is detailed in a SOP developed by the USEPA (i.e., SOP LG401, section 6.6; 2010).
Analysis of E. coli in ballast discharge followed LSRI/SOP/SA/56 – Detection and Enumeration of Total
Coliforms and E. coli using IDEXX’s ColilertTM. Analysis of Enterococcus spp. in ballast discharge samples
was conducted according to LSRI/SOP/SA/62 – Detection and Enumeration of Enterococcus using
Enterolert™.
Samples for analysis of the CO1 gene of H. anomala were collected and processed within 24 hours
according to LSRI/SOP/GWRC/13 – Collection and Processing of Environmental DNA Samples. Following
processing, filters were submerged in Longmire’s Buffer and stored in microcentrifuge tubes at -20°C.
Preserved filters were held until the end of the 2017 Great Lakes shipping season, and then were
shipped overnight on ice to Pennsylvania State University – Behrend for analysis. Analysis was
conducted according to Knight et al. (2018).
Any deviations to these methods were recorded and assessed according to LSRI-GWRC QAQC processes
(Section 3.3.3).
3.3.3. QUALITY ASSURANCE AND QUALITY CONTROL
All sample collection, handling, analysis, and data management activities were conducted according to
the LSRI’s Quality Management System as outlined in the LSRI Quality Management Plan (2017) and the
GWRC Shipboard QAPP (2017). Consistent with the TQAP and the GWRC Shipboard QAPP (LSRI, 2017),
any methodological deviations from the planned methods, which occurred during the course of the
testing period were recorded and evaluated in deviation forms and are archived at LSRI. All TQAP and
SOP deviations were assessed by the Project’s Principal Investigator. None of the reported deviations are
significant enough to render any trial findings reported here invalid. Several deviations required
procedural improvements to LSRI-GWRC SOPs for future use. These preventive actions were deemed
appropriate by the Project’s Principal Investigator.
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4. RESULTS
Results are presented here in order of their relevance to the research objective, starting with biological
and physical characteristics of laker ship ballast discharges to WLS. Detections of H. anomala (by
microscope and DNA analyses), and other NIS (by microscope only), are presented along with associated
organism community composition and physical/chemical data from the ballast discharges. Next, results
of the four voyage-wide sampling events are presented, including measurements of the associated
source system, ballast uptake, ballast discharge and the receiving system sampling events (plus one
stand-alone ballast uptake sampling event). Detection levels in the tests varied by trial, taxonomic group
and sampling approach, and are also reported here.
4.1. CHARACTERISTICS OF LAKER BALLAST WATER DISCHARGED TO WESTERN LAKE SUPERIOR
Fifteen ship ballast discharges to WLS were sampled between January and December 2017. All but one
(which occurred in January 2017) took place July through December of 2017 (Table 4). Collectively, we
sampled over 78,000 m3 of the total 586,000 m3 of ballast that was discharged from the targeted vessels
during these sampling events (Table 4).
4. 1.1. VESSEL AND SHIPBOARD SAMPLING SYSTEM OPERATIONAL DATA
GWRC never sampled the entire duration of the discharge; each given regular zooplankton sampling
event was 24-61 minutes (Table 4). In total, during each sampling event, between 5 and 53% of the
ballast water on board the ship was sampled during discharge (Table 4). The number of individual ballast
tanks sampled during each event varied from two to sixteen (Table 4). Between 1,200 and 11,300 m3 of
ballast water was subject to sampling during each individual discharge event for regular zooplankton
samples (Table 4). GWRC collected sample water at 1.4 to 3.4 m3/hr (Table 4) to obtain sample sizes
ranging from 0.91 to 2.07 m3 for regular zooplankton samples. For five sampling events, an additional
2,900 to 11,500 m3 of water was subjected to sampling over a period of 32 to 49 minutes for detection
of H. anomala (Table 4). The additional sample sizes were 2.07 to 3.08 m3 (Table 4).
LSRI/GWRC/TR/GLSBM/1
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Table 4. Ballast Discharge Trials: Summary of Vessel and Shipboard Sampling System Operational Parameters. Note: Trial 3 was an Uptake-Only Sampling
Event and is not Presented in this Table. P= Port, S = Starboard, N/A= Not Applicable (Not Collected).
Parameter
Trial
1 2 4 5 6 7 8 9 10 11 12 13 14 15 16
Date (Month, Year) Jan-
2017
Jul-
2017
Aug-
2017
Aug-
2017
Sep-
2017
Sep-
2017
Sep-
2017
Oct-
2017
Oct-
2017
Oct-
2017
Oct-
2017
Nov-
2017
Nov-
2017
Dec-
2017
Dec-
2017
Ballast Tank(s)
Sampled
(Tanks were
Discharged
Simultaneously)
4P, 4S
1P
2P
3P
4P
5P
1P, 1S
2P, 2S
3P, 3S
4P, 4S
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
7P, 7S
8P, 8S
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
7P, 7S
2P, 2S
6P, 6S
7P, 7S
8P, 8S
1P, 1S
2P, 2S
3S
4P, 4S
6P, 6S
1P, 1S
2P, 2S
3P, 3S
4P, 4S
All
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
7P, 7S
8P, 8S
5P, 5S
2S, 2P
6S, 6P
7S, 7P
8S, 8P
Forward
Draft
1P, 1S
2P, 2S
Shipboard Sampling
System Used
Active Passive Passive Passive Passive Passive Passive Passive Active Active Active Active Active Passive Active
Shipboard Sampling
System Flow Rate
(m
3
/hour)
2.63 1.91 1.76 2.58 2.03 2.64 1.86 2.02 3.04 2.98 2.66 2.95 1.43 2.93 3.36
Regular Zooplankton
Sampling Duration (Hr:
min)
0:24 0:59 1:01 0:48 1:00 0:45 0:55 0:51 0:41 0:38 0:45 0:40 0:37 0:40 0:35
Estimated Volume
Discharged During
Regular Sampling (m
3
)
2,665 Not
Known1 7,420 8,818 7,585 11,308 9,564 1,171 4,318 4,742 7,466 9,565 Not
Known1 4,216 Not
Known1
Regular Zooplankton
Sample Volume (m3) 1.04 1.86 1.78 2.04 2.02 1.96 2.00 1.70 2.07 1.97 1.98 2.02 0.91 2.04 1.99
Seep Sample
Volume (L) 4 11 7 10.5 10.5 12 9.5 8 8.5 9.5 9.5 9.5 12.5 12 12
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Parameter
Trial
1 2 4 5 6 7 8 9 10 11 12 13 14 15 16
Larger Volume
Hemimysis Sample:
Ballast Tanks Sampled2
(Ballast Tanks were
Discharged
Simultaneously)
N/A N/A N/A N/A N/A N/A N/A N/A
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5S, 5P
6S, 6P
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
1P, 1S
2P, 2S
3P, 3S
4P, 4S
5P, 5S
6P, 6S
7P, 7S
8P, 8S
N/A N/A Not
Known
Larger Volume
Hemimysis Sample:
Shipboard Sampling
System Flow Rate
(m
3
/hour)
N/A N/A N/A N/A N/A N/A N/A N/A 4.11 3.88 2.66 3.7 N/A N/A 2.91
Larger Volume
Hemimysis Sample:
Duration (Hr: min)
N/A N/A N/A N/A N/A N/A N/A N/A 0:45 0:32 0:43 0:49 N/A N/A 0:46
Larger Volume
Hemimysis Sample:
Volume Discharged
During Sampling (m
3
)
N/A N/A N/A N/A N/A N/A N/A N/A 4,628 2,872 4,974 11,469 N/A N/A Not
Known1
Larger Volume
Hemimysis Sample
Volume (m
3
)²
N/A N/A N/A N/A N/A N/A N/A N/A 3.08 2.07 3.02 3.03 N/A N/A 2.24
Total Ballast Volume
on Ship (m3)3 53,606 17,655 48,646 39,596 53,606 46,444 40,436 13,656 53,606 53,606 53,606 39,596 15,876 40,3784 15,8765
Percent of Volume that
was Subsampled for
Detection of H.
anomala specimens
4.97% Not
Known1 15.2% 22.3% 14.2% 24.4% 23.6% 8.6% 16.7% 12.5% 23.2% 53.1% Not
Known1 10.4% Not
Known1
1 Ballast discharge volumes were not calculated because electronic sounding measurements were either insufficient or could not be recorded.
2 Large zooplankton sample was added to the test plan beginning with Trial 10. Trial 14 did not include this sample because the vessel had completed cargo loading operations during the regular
zooplankton sample collection period. Trial 15 did not include this sample because the vessel’s ballast main was not equipped with a return port. Trial 16 did include the large zooplankton
sample, but ballast volume is not known because soundings were not able to be recorded.
3 Data sourced from National Ballast Information Clearinghouse (NBIC, 2018).
4 Forward draft volume not included in NBIC data.
5 Estimate. The precise volume discharge is not available for this event. Volume listed is from a previous discharge operation in the same port from the same ballast tanks.
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4. 1.2. NON- I NDIGENOUS SPECIES AND TARGET ORGANISM (HEMIMYSIS ANOMALA) RESULTS
The density of H. anomala specimens in ballast water discharged to ports in WLS was determined for the
15 separate discharge events between January and December 2017 (Table 4). The Regular Zooplankton
Samples and any Larger Volume Hemimysis Samples were examined in their entirety for H. anomala
specimens. The volume of water examined for specimens from each discharge trial (regular sample
volume + larger sample volume) ranged widely from 0.91 (Trial 14) to 5.15 m3 (Trial 10, Table 4), with
the largest sample volumes in Trials 10, 11, 12, 13 and 16, when the additional sample was collected
with a larger mesh net to increase total sample size for H. anomala (Table 4).
This analysis could confirm presence of H. anomala in instances in which it was detected; it could not
confirm a complete absence of H. anomala in samples in which it was not detected. In samples in which
there was no detection, any concentrations of these specimens were lower than the reported detection
level. The detection limits vary with sample volume analyzed with greater volumes analyzed leading to a
lower detection limit (Table 5). Across trials, microscopic analysis detection limits ranged from 0.19 to
1.1 organisms m-3 (Table 5).
H. anomala specimens were found in the ballast discharge samples from Trials 10, 11 and 13 which were
large volume samples collected during October and November 2017 (Table 6). In all three of these cases,
water discharged to WLS had been loaded from ports in southern Lake Michigan where H. anomala has
been established since 2006 (Table 7). H. anomala densities in these samples ranged from 0.2 to 3.3
organisms m-3 (Table 6).
Discharge samples from Trials 6 through 16 were also analyzed for the CO1 gene of H. anomala (Table
6). H. anomala DNA was detected in all of the samples in which specimens were found (i.e., Trials 10, 11
and 13), as well as in samples from Trials 7, 14 and 15, such that six discharge events out of eleven
analyzed had detectable H. anomala DNA (Table 6). The discharge samples which tested positive for
H. anomala DNA were associated with primary and/or secondary uptake events from southern Lake
Michigan, northern Lake Michigan, Lake Erie and the St. Mary’s River (Table 6). H. anomala DNA was not
detected in Trials 8, 9 and 16, which were associated with uptake events from the St. Clair River, Detroit
River, and Lake Ontario, respectively (Table 6).DNA was also absent from samples from two of the six
discharges (Trials 6 and 12) associated with uptakes in southern Lake Michigan.
In addition to H. anomala, the Project found four additional NIS taxa of zooplankton not previously
reported in Lake Superior in samples of ballast water discharged to WLS (Table 6). The benthic
harpacticoid copepods Nitokra hibernica, Heteropsyllus nunni, and Schizopera borutzkyi and the
cyclopoid copepod Thermocyclops crassus were found in concentrations ranging from 0.5 to 3.0
organisms m-3 in samples from nine of the 15 discharge events (i.e., Trials 1, 4, 5, 8, 9, 10, 11, 13, and 15;
Tables 6 and 7). Specimens of Nitrokra hibernica also were observed in Larger Volume Hemimysis
Samples from Trials 12 and 16 (Tables 6 and 7). However, because they were not H. anomala, the target
of the large volume sample analysis, their densities were not calculated (Table 6).
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Table 5. Minimum Number of Specimens per Non-Indigenous Species (NIS) Taxon (#/m3) that would Need to be
Present for Detection in Ballast Uptake and Discharge Samples Given Volumes Sampled
(i.e., Project Detection Level).
Trial - Event
Microzooplankton NIS
Taxon (#/m3)
Macrozooplankton NIS Taxon
(#/m3) Hemimysis anomala
Regular Count Regular Count Entire Sample Entire Sample
1 - Discharge 12 1.92 0.96 0.96
2 - Discharge 33 17.16 0.54
3 - Uptake 498 9.50 0.59
4 - Discharge 497 4.50 0.56 0.56
5 - Discharge 308 1.96 0.49 0.49
6 - Uptake 85 13.06 0.41
6 - Discharge 120 7.94 0.50
7 - Discharge 118 8.15 0.51
8 - Discharge 37 1.00 0.50 0.50
9 - Discharge 60 1.18 0.59 0.59
10 - Discharge 74 3.87 0.48 0.19*
11 - Uptake 32 3.54 0.44 0.19*
11- Discharge 30 2.03 0.25*
12 - Uptake 12 2.15 0.20*
12 - Discharge 26 2.02 0.20*
13 - Uptake 7 0.59 0.59
13 - Discharge 4 0.50 0.20*
14 - Discharge 340 1.10 1.10
15 - Discharge 8 1.96 0.49 0.49
16 - Discharge 24 8.02 0.24*
* Indicates larger sample volume collected and analyzed for Hemimysis anomala.
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Table 6. Summary of Measured Biological Parameters from Ballast Discharge to Western Lake Superior.
DL = Detection Level, see Table 5 for values for each trial; N/A = Not Applicable (Not Collected).
Hn = Heteropsyllus nunni, Nh = Nitokra hibernica, Sb = Schizopera borutzkyi, Tc = Thermocyclops crassus
* Organism was present in the sample, but not in the portion that was enumerated.
**At least one replicate value was less than the DL of 1 MPN/100 mL. Half of DL was used to calculate the average of the replicates.
Trial
Parameter 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16
Primary Uptake
Location
Southern
Lake
Michigan
Lake
Erie Lake Erie
Southern
Lake
Michigan
Southern
Lake
Michigan
Southern
Lake
Michigan
St Clair
River
Detroit
River
Southern
Lake
Michigan
Southern
Lake
Michigan
Southern
Lake
Michigan
Southern
Lake
Michigan
Northern
Lake
Michigan
Lake
Erie
Lake
Ontario
Secondary
Uptake
Location
Eastern
Lake
Superior
N/A Lake
Superior N/A
St.
Mary’s
River,
Lake
Superior
St. Mary’s
River,
Lake
Superior
Eastern
Lake
Superior,
Lake
Superior
N/A
Eastern
Lake
Superior
St.
Mary’s
River,
Eastern
Lake
Superior
St. Mary’s
River,
Eastern
Lake
Superior,
Lake
Superior
N/A N/A
St.
Mary’s
River
N/A
Date (Month,
Year)
Jan-
2017
Jul-
2017
Aug-
2017
Aug-
2017
Sep-
2017
Sep-
2017 Sep-2017 Oct-
2017
Oct-
2017
Oct-
2017
Oct-
2017
Nov-
2017
Nov-
2017
Dec-
2017
Dec-
2017
Zooplankton:
Total Density
(#/m
3
)
5,000 49,600 208,000 186,000 83,400 63,900 26,600 37,500 42,800 18,700 14,100 2,600 6,800 8,000 25,700
Zooplankton:
Percent Live 58% 72% 74% NA NA 53% NA 72% NA 66% NA 63% NA 83% NA
Hemimysis
anomala (#/m3) < DL < DL < DL < DL < DL < DL < DL < DL 3.3 2.7 < DL 0.2 < DL < DL < DL
CO1 Gene of
Hemimysis
anomala
N/A N/A N/A N/A Not
Present Present Not
Present
Not
Present Present Present Not
Present Present Present Present Not
Present
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Trial
Parameter
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
Other
Nonindigenous
Species Not
Previously
Reported from
Lake Superior
(#/m
3
)
Nh 1.9 < DL Nh 1.7 Tc 2.4 < DL < DL Hn 0.5
Nh 3.0
Nh 1.8
Sb 0.6 Nh 1.9 Sb 2.0 Nh* Hn 0.5 < DL Hn 1.5
Nh 1.5 Nh*
Protists: Total
Density
(Cells/mL)
210 285 1,002 967 1,622 1,248 2,247 368 1,775 1,634 2,084 1,614 22,713 856 1,074
Escherichia
coli: Density
(MPN/100 mL)
N/A 1.0** < 1** < 1 < 1** 7 1.4** 5.6 189.8 3.4 2.4 1.7 51.4 21.2 114.4
Enterococcus
spp.: Density
(MPN/100 mL)
N/A 4.2 1.2** 2.7 < 1 5.5 1.3 1 133.4 1.0** 2.7 13.6 92.5 404.6 19.7
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Table 7. Summary of Information on Project-Relevant of Non-Indigenous Species in the Great Lakes.
Taxon Common
Name
Length
(mm) Photo Native
Range
Year and Location
of First Record in
the Great Lakes
Current Distribution
in the Great Lakes
Reference
Hemimysis
anomala
Bloody Red
Shrimp 6-13 mm
Freshwater
margins of
Black,
Azov,and
Ponto-
Caspian
Seas
2006. Southeastern
Lake Ontario and
channel from
Muskegon Lake to
Lake Michigan
Established in Lakes
Ontario, Michigan,
Erie and Huron.
Observed in Superior
Harbor of Lake
Superior in 2017
Kipp, R.M., A.
Ricciardi, J. Larson, A.
Fusaro, and T.
Makled, 2018
Heteropsyllus
nunni
Harpacticoid
Copepod 0.5 mm
Atlantic
coast of
North
America
1996. Lake Michigan
Established in Lakes
Michigan, Huron, and
St. Clair
U.S. Geological
Survey, 2018,
Nitokra hibernica Harpacticoid
Copepod
0.5 - 0.75
mm
Black and
Caspian
Seas,
European
coast of
Atlantic,
Arctic and
Baltic Seas
1972. Mouth of
Niagara River, Lake
Ontario
Established in Lakes
Erie, Huron,
Michigan, and
Ontario
Kipp, R.M., A.J.
Benson, J. Larson,
T.H. Makled, and A.
Fusaro, 2018
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Paraleptastacus
wilsoni
Harpacticoid
Copepod
0.45-0.48
mm
Atlantic
coast of
North
America
2017. Southern Lake
Michigan
Collected in ballast
uptake from
Southern Lake
Michigan
This Report
Schizopera
borutzkyi
Harpacticoid
Copepod 0.5-0.6 mm
Black Sea
Basin 1988. Lake Michigan Established in Lakes
Erie and Michigan
Kipp, R.M., J. Larson,
T.H. Makled, and A.
Fusaro, 2018
Thermocyclops
crassus
Cyclopoid
Copepod 0.7-1.1 mm
Eurasia 2014. Lake Erie Established in Lake
Erie
Sturtevant, R., and P.
Alsip, 2018
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4. 1 .3. BACKGROUND BIOLOGICAL, PHYSICAL/CHEMICAL CHARACTERISTICS
Zooplankton: The background densities of zooplankton in ballast water discharged to WLS ranged from
2,600 to 208,000 organisms m-3 (Table 6) and included a mixture of rotifers, copepods, cladocerans,
dreissenid veligers and a few primarily benthic taxa (Appendix Table 13) during the experimental period,
which mainly ranged from July – December, 2017 (Trial 1 took place in January, 2017). Highest densities
(> 150,000 m3) in sampled discharges were observed in August (Trials 4 and 5) when rotifers were at
their peak abundance and comprised up to 80% of the zooplankton community (Figure 8). Dreissenid
mussel veligers were common in samples collected from August through October irrespective of source
water location. The density of cladocerans was greatest in the July discharge sample from Trial 2 which
contained water from Lake Erie (Figure 8). Copepod nauplii, juvenile copepodids, and adults were
common in all samples analyzed, and dominated the late fall and winter zooplankton community (Figure
8).
Figure 8. Total Density and Percent Composition of Zooplankton in Ballast Discharge Samples.
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The diversity of zooplankton in ballast water discharged to WLS was high, with 139 distinct taxa found
during the sampling period (Appendix Table 13). Individual samples contained 23 to 56 taxa, with the
greatest diversity among rotifers and copepods (Figure 9). The discharge sample of ballast originating
from northern Lake Michigan (Trial 14) was unique in the limited diversity of rotifers in the sample. A
number of taxa that are generally found associated with the bottom sediment were collected, including
fourteen species of harpacticoid copepods, three of which have not previously been reported from Lake
Superior.
Figure 9. Number of Taxa found in Ballast Discharge (D) Samples.
The density and percentage of live zooplankton in ballast water discharge samples was determined for
the eight discharges for which samples were delivered to the analysts within four hours of sample
collection (Appendix Table 14). The density of live organisms ranged from 2,200 to 190,000 m-3 which
was 58 to 84% of the total density observed (Table 6). Mortality was highest for soft-bodied rotifers such
as Polyarthra which are easily damaged by ballast pumps and sample handling (Appendix Table 14) but
overall community composition of live zooplankton (Figure 10) was similar to that of the total
zooplankton (Figure 8).
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Figure 10. Density and Percent Composition of Live Zooplankton in Ballast Discharge Samples.
Protists: Protist densities in discharges ranged from 210 cells/mL (Trial 1) to 22,713 cells/mL (Trial 14)
(Table 6). Assemblages were a mixture of algal groups, though at higher densities the assemblages were
dominated by small-celled cyanophytes (Figure 11). Chrysophyte algae also dominated in several
samples, followed by diatoms (especially in Trials 1 and 2) and cryptophytes. Green algae were rare and
dinoflagellates occurred only occasionally. Two discharges stand out from the rest. Trial 9 had a fairly
low density (368 cells/mL) and contained a high proportion of ciliates. Trial 14’s discharge contained
high concentrations of protists, indicating that the ballast tanks were likely filled during a bloom period
in northern Lake Michigan, an observation that is backed by the highest chlorophyll a measurement in
this study (Water Chemistry/Water Quality, below; Appendix Table 16). High densities in that sample
were largely driven by the cyanophytes Microcystis and Aphanocapsa (Figure 11; Appendix Table 15).
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Figure 11. Histograms of Protist Densities (Upper) and Proportions (Lower) in Discharge Samples. Grouping
Reflects Major Divisions of the Organisms.
Microbes: The indicator microorganisms (E. coli and Enterococcus spp.) that were only analyzed in
ballast discharge did not vary significantly between source water locations. E. coli concentrations in
discharge samples ranged from < 1 E. coli per 100 mL to 189.8 E. coli per 100 mL (Table 6). Values for
each trial were below the Ballast Water Discharge Standard of < 250 colony forming units (cfu) per 100
mL set forth in the U.S. Code of Federal Regulations (Title 33; 121.1511.3).
Enterococcus spp. concentrations ranged from < 1 per 100 mL to 404.6 per 100 mL (Table 6). Two of the
trials (Trial 10 from southern Lake Michigan and Trial 15 from Lake Erie) had enterococci concentrations
over the acceptable Ballast Water Discharge Standard of < 100 cfu per 100 mL set forth in Code of
Federal Regulations (Title 33; 121.1511.3). The remaining trials had enterococci concentrations below
the required guideline.
Water Chemistry/Water Quality: Table 16 of the Appendix summarizes the discharge water chemistry
and water quality parameters captured by both sondes in situ and analysis equipment in the laboratory
across the 15 ballast discharges to WLS. Values were generally similar, except for Trial 14 discharge of
water that was ballasted from northern Lake Michigan which contained distinct water chemistry/quality
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values across all parameters except temperature and pH (Appendix Table 16). This trial was responsible
for the lowest numbers in the range for %T values and for the highest numbers measured of turbidity,
phycocyanin (blue-green) algae pigment, and TSS, among other parameters.
Water temperature varied between 3.4°C and 22.2°C in the discharge samples reflecting regional and
seasonal differences. pH was slightly basic and did not vary much with values between 7.64 and 8.18.
Dissolved oxygen concentrations were generally near saturation levels and ranged from a low value of
7.57 mg/L to 12.99 mg/L.
Chlorophyll a (uncorrected) values were generally quite low (0.06 to 1.95 µg/L) but reached a high of 4.2
µg/L during Trial 14 from northern Lake Michigan. Phycocyanin ranged from 0.06 and 0.59 µg/L
(uncorrected) for all samples except Trial 14 which had a value of 2.45 µg/L, likely due to the presence of
blue green algae.
Turbidity was generally quite low (1.00 to 3.98 FNU). Higher turbidity levels occurred in Trial 15’s
discharge from western Lake Erie (9.2 FNU) and Trial 14’s discharge from northern Lake Michigan (49.8
FNU). As expected, water transparency was inversely related to turbidity with filtered percent
transmittance values ranging from 56.1% to 95.9% and unfiltered portions of the sample displaying
values between 31.5% and 94.9%.
TSS values varied from below the detection level to 4.1 mg/L in all samples except those from Trial 14
which had a high of 92.6 mg/L. The concentration of MM correlated well with TSS, with concentrations
less than 3.3 mg/L for all samples except Trial 14 with a value of 76.4 mg/L.
POM was below the detection levels for all discharges except Trial 14 which had a POM content of 16.3
mg/L. NPOC and DOC values ranged between 1.9 and 8.7 mg/L with the highest values recorded from
Trial 14. NPOC measured was comprised nearly entirely of DOC.
4.2. VOYAGE-WIDE SAMPLING (FOUR VOYAGES)
Four voyage-wide trials took place during the project period. These trials included sampling of 1) one or
more southern Lake Michigan harbor water sites associated with a ballast uptake, 2) the uptake itself, 3)
ballast discharge into WLS, and 4) two to three sites within the receiving harbor. Sampling took place in
keeping with the TQAP, in general, with the following exceptions:
• One uptake sampling event in central Lake Erie in August (Trial 3) could not be paired with
planned discharge sampling event in WLS, for logistical reasons. The data are nonetheless
included to show background conditions of another port that contributes water to WLS.
• It was not practicable to sample the source water system near to the vessel or ballasting time
during the Trial 6 uptake. Samples were collected over a mile from the ship, and 13 hours prior
to ballasting (Figure 6, Table 9). Therefore these samples did not represent the water
characteristics of the uptake berth or the time of ballasting.
• In three of the four voyage-wide trials, there was a secondary uptake of ballast water en route
to WLS (Table 1). The interim uptake events which took place in Lake Superior or the St. Mary’s
River, were not sampled.
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4. 2.1. VESSEL AND SHIPBOARD SAMPLING SYSTEM OPERATIONAL DATA
Ballast water uptake samples were collected on five dates between August and November 2017 (Table
8). Subsamples of ballast uptake were collected for 0.5 to 1.5 hours during ballast operation at flow
rates of 2.5 to 3.1 m3/hr (Table 8). The ballast volumes subject to regular sampling ranged from >860 m3
to 4,700 m3 of uptake water (Table 8). An additional, larger sample volume was also collected targeting
H. anomala in Trials 11 and 12 (Table 8). Even with this second zooplankton sample, the total amount of
water sampled during each of the five uptake events was only a fraction (> 2.1 to > 13.5%) of the total
volume ballasted during cargo off-loading operations (i.e., approximately 40,000 m3 for each uptake, see
Table 8).
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Table 8. Ballast Uptake Trials: Summary of Vessel and Shipboard Sampling System Operational Parameters.
P= Port, S = Starboard, N/A = Not Applicable (Not Collected).
Parameter
Trial
3 6 11 12 13
Date (Month, Year) Aug-2017 Sept-2017 Oct-2017 Oct-2017 Nov-2017
Sampling Location Lake Erie
Southern
Lake Michigan
Southern
Lake Michigan
Southern
Lake Michigan
Southern
Lake Michigan
Ballast Tank(s) Sampled 2S
5S
2P, 2S, 3P, 3S
4P, 4S, 5P, 5S
6P, 6S
3P, 3S
5P, 5S
6P, 6S
3P, 3S
5P, 5S
6P, 6S
5P, 5S
Shipboard Sampling System
Used
Passive Passive Passive Active Active
Shipboard Sampling System
Flow Rate (m
3
/hour)
2.55 2.50 3.09 2.49 3.00
Sampling Duration (Hr: min) 00:40 00:59 00:40 0:45 0:31
Volume Ballasted During
Sampling (m
3
)
> 862* 1,459 > 3,223* 4,305 4,689
Regular Zooplankton
Sample Volume (m3) 1.69 2.45 2.26 1.86 1.69
Seep Sample Volume (L) 10.5 6.5 5 6 10
Larger Volume Hemimysis
Sample: Ballast Tanks
Sampled
N/A N/A
1P, 1S, 3S,
4P, 4S,
5P, 5S
Not Known; No Soundings N/A
Larger Volume Hemimysis
Sample: Shipboard Sampling
System Flow Rate (m
3
/hour)
N/A N/A 3.77 4.32 N/A
Larger Volume Hemimysis
Sample: Duration (Hr: min) N/A N/A 0:47 0:42 N/A
Larger Volume Hemimysis
Sample: Volume Ballasted
During Sampling (m
3
)
N/A N/A 2,547 Not Known, No Soundings N/A
Larger Volume Hemimysis
Sample Volume (m3) N/A N/A 3.02 3.02 N/A
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Parameter
Trial
3 6 11 12 13
Total Volume Ballasted (m3)1 40,630 42,632 42,632 42,632 39,596
Percent of Volume Sampled
(Large Zooplankton Sample) >2.1%** 3.4% >13.5%** >10.1%** 11.8%
1 Data sourced from National Ballast Information Clearinghouse (NBIC, 2018).
*Entire volume not recorded due to operational error.
**Based on recorded volume which is less than total volume.
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Table 9. Source and Receiving Water Samples: Summary of Location and Site Characteristics.
Sampling
Event Parameter Trial
6 11 12 13
Source
Water
Sample Collection
Time Relative to
Ballast Sampling
-13 hours +2.5 hours +2.5 hours -1 hour
Location Southern Southern Southern Southern
Lake Michigan Lake Michigan Lake Michigan Lake Michigan
Site Designation Site 3 Site 1 Site 2 Site 1 Site 2 Site 1 Site 2
Site Description West of the slip along the shore Behind vessel
towards lake
In front of vessel,
towards shore
Behind vessel,
towards lake
From shore at
interior of slip
Behind
vessel
towards lake
Behind
vessel
towards
lake
Distance to
Ballasting Ship (m) 8,486 40 200 215 950 115 450
Water Depth (m) 0.61 9.8 9.3 10.2 8.5
Not
Recorded
8.5
Receiving
Water
Sample Collection
Time Relative to
Ballast Sampling
+7.5 hours +20 hours +16.5 hours -2 hours
Ballast Hold Time 3 days 3 days 3 days 4 days
Site Designation Site 1 Site 2 Site 3 Site 1 Site 2 Site 3 Site 1 Site 2 Site 3 Site 1 Site 2
Site Description
Behind
vessel,
towards
lake
In front
of
vessel,
towards
shore
Offshore
Behind
vessel
towards
lake
Near shore at
interior of slip Offshore
Behind
vessel,
towards
lake
In front of
vessel, towards
shore
Offshore
In front of
vessel,
towards
shore
Behind
vessel at
end of slip
Distance to
Deballasting Ship
(m)
3 3 550 10 140 493 4 29 502 480 50
Water Depth (m) 7.5 10 5.5 10.7 3.3 4.5 11.9 8.1 5.5 1 12.8
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 45 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
4. 2.2. NON- I NDIGENOUS SPECIES AND TARGET ORGANISM (HEMIMYSIS ANOMALA) RESULTS
Source harbor, ballast uptake, ballast discharge and receiving harbor samples were analyzed for H.
anomala DNA (i.e., CO1 gene). Ballast uptake and discharge samples also were analyzed microscopically
for H. anomala and other NIS specimens (Table 10). When the voyage included secondary ballast
uptakes only the voyage’s initial source harbor and uptake event were sampled. A stand-alone uptake
sample event occurred in central Lake Erie (Trial 3) because we were unable to sample the discharge
from that trial (Table 10).
Source Harbor Water: Source harbor samples showed positive results for H. anomala DNA from at least
one sampling site close to the ship’s berth (Trials 11, 12 and 13; Table 11) during the voyage-wide trials.
Harbor sampling for one voyage-wide trial, Trial 6, could not occur closer than 5 miles away from the
ship’s berth due to private property and logistical constraints (Table 11), and that sampling event did not
yield positive results for H. anomala DNA. However, sampling of the same harbor much closer to the
ship berth in two subsequent voyage-wide trials did detect H. anomala DNA.
Ballast Uptake: Both H. anomala specimens and H. anomala DNA were detected in all four uptake event
samples, with specimen densities ranging from 0.2 to 2.4 organisms m-3 (Table 10). In addition,
specimens of other NIS, previously unreported in Lake Superior (Table 7), also were present in all four
voyage-wide sampling events’ uptake samples (Table 10). Specifically, the harpacticoid copepod
Schizopera borutzkyi was present in all four uptakes from Lake Michigan; and Heteropsyllus nunni was
found in three of the four Lake Michigan-based uptake samples (Table 10). Nitokra hibernica was found
in Lake Michigan uptake water in Trials 6, 11 and 13, as well as in uptake water from the central basin of
Lake Erie (Trial 3; Table 10). Paraleptastacus wilsoni was found in two of the Lake Michigan uptake
samples; there are no previous records for this estuarine/marine species in the Great Lakes (Table 10).
Ballast Discharge to WLS: There were detections of NIS in three out of the four ballast discharges to WLS
from the voyage-wide trials (Table 10). Discharge samples from two trials (i.e., Trials 11 and 13)
contained both H. anomala specimens and DNA (Table 10). Trial 11 discharge samples also contained
Schizopera borutzkyi specimens at a concentration of 2.0 organisms per m-3; Nitokra hibernica was
present in the discharge from Trial 12 (specimens were noted but not enumerated because the volume
in which the detections occurred targeted larger macroplankton); and Trial 13 also contained
Heteropsyllus nunni at a concentration of 0.5 organisms m-3 (Table 10).
Receiving Harbor: H. anomala DNA was detected in receiving water samples in three out of four voyage-
wide receiving harbor sampling events, specifically, Trials 6, 11, and 12, but not 13 (Table 11). The
detections were associated with sampling sites located within 30m of the discharge site; receiving
harbor samples taken at the sampling locations furthest from the ship did not show signs of H. anomala
DNA for any trials (Table 11). The receiving harbor samples from Trial 13, the sole receiving system
sampling event in which there were no detections of H. anomala DNA across sampling sites, were also
the only receiving system samples collected prior to the ship discharge sampling event as opposed to
within one day afterward.
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 46 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
Table 10. Summary of Biological Parameters for Voyage-Wide Trials.
P = Present but Not Enumerated, DL = Detection Level, N/A = Not Applicable (Not Collected).
Trial
Parameter Sampling
Event
3
6
11
12
13
Lake Erie
Southern
Lake Michigan
Southern
Lake Michigan
Southern
Lake Michigan
Southern
Lake Michigan
Zooplankton: Total
Density (#/m3)
Uptake 250,000 93,000 22,000 11,100 1,700
Discharge N/A 83,400 18,700 14,100 2,600
Hemimysis anomala
(#/m3)
Uptake < DL 0.4 0.4 0.2 2.4
Discharge N A < DL 2.7 < DL 0.2
Other Introduced
Taxa Not Previously
Reported from Lake
Superior (#/m3)
Uptake 19.0 Nitokra hibernica
1.2 Heteropsyllus nunni
22.9 Nitokra hibernica
0.8 Paraleptastacus wilsoni
29.0 Schizopera borutzkyi
0.4 Heteropsyllus nunni
0.9 Nitokra hibernica
3.1 Schizopera borutzkyi
P Schizopera borutzkyi
1.2 Heteropsyllus nunni
5.3 Nitokra hibernica
1.8 Paraleptastacus wilsoni
4.7 Schizopera borutzkyi
Discharge N A < DL 2.0 Schizopera borutzkyi P Nitokra hibernica 0.5 Heteropsyllus nunni
Protists: Total Density
(Cells/mL)
Uptake 210 285 1,002 967 1,623
Discharge N/A 1,622 1,634 2,084 1,614
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 47 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
Table 11. Occurrence of Hemimysis anomala DNA and Specimens in Samples Across Voyage-Wide Trial Sampling Events. DL = Detection Level.
Trial
6 11 12 13
Source Water Samples
Source Site Site 3 Site 1 Site 2 Site 1 Site 2 Site 1 Site 2
Source Site DNA Result Not Detected Present Present Not Detected Present Present Present
Uptake Samples
Uptake DNA Result Present Present Present Present
Uptake Density of
Specimens (#/m3) 0.4 0.4 0.2 2.4
Discharge Samples
Discharge DNA Result Not Detected Present Not Detected Present
Discharge Density of
Specimens (#/m3) < DL* 2.7 < DL* 0.2
Receiving Water
Samples
Receiving Site Site 1 Site 2 Site 3 Site 1 Site 2 Site 3 Site 1 Site 2 Site 3 Site 1 Site 2
Receiving Site DNA Result Present Not
Detected
Not
Detected Present Not
Detected
Not
Detected Present Present Not
Detected
Not
Detected
Not
Detected
*See Table 5 for detection levels for Hemimysis anomala specimens for each trial.
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 48 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
4. 2.3. BACKGROUND BIOLOGICAL, PHYSICAL/CHEMICAL CHARACTERISTICS
Zooplankton: Table 10; Appendix Table 13; and Figure 12 summarize zooplankton data from the paired
uptake and discharge events during Trials 6, 11, 12, and 13 from Lake Michigan, as well as the single
uptake from Lake Erie (Trial 3). Total zooplankton densities were generally similar for each of the paired
uptake and discharge events (Appendix Table 13) although the percentage of rotifers in the samples was
often higher in the discharge samples than in uptake samples (Figure 12). The disparate rotifer numbers
may have resulted from sampling differing portions of the ballast water mass on uptake versus
discharge, or rotifer reproduction during the three-day period between ballast uptake and discharge.
Figure 12. Density and Percent Composition of Zooplankton in Paired Ballast Uptake and Discharge Samples.
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 49 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
Protists: Protist densities varied widely across locations, generally increasing over the course of the year
(Table 15), a trend that is largely attributed to an increase in cyanophytes such as Microcystis and
Aphanocapsa. Although the earliest densities (Trials 3 and 6) were low, they were dominated by diatoms
which is typical of spring assemblages in the Great Lakes. [Trials 11 and 12 had comparative uptake and
discharge samples. Those densities declined by about half upon discharge due mostly to the loss of
cyanophytes. The other comparative set (Trial 13) indicated no notable difference between uptake and
discharge samples.] Although only a few uptake samples (Trials 3, 6, 11, 12, 13) were analyzed for
protists, samples contained a mixture of taxa similar to discharge samples. Uptakes for Trials 11 and 12
had densities higher than 1,000 cells/mL and were dominated by cyanophytes (mostly Aphanocapsa).
Trials 11 and 12 densities declined by about half upon discharge due mostly to the loss of cyanophytes
(Table 10). The other comparative set (Trial 13) indicated no notable difference between uptake and
discharge samples (Table 10).
Figure 13. Histograms of Protist Assemblage Composition by Major Divisions of Organisms Showing Densities
(Upper) and Proportions (Lower) in Voyage-Wide Uptake Samples. For Comparison, Voyages with Paired Uptake
and Discharge Samples also have Discharge Samples Shown.
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 50 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
Water Chemistry/Water Quality: Water chemistry and water quality measurements were determined
for samples from source harbors in southern Lake Michigan, ballast uptakes, ballast discharges, and
from receiving harbors in WLS in the four voyage-wide trials (Table 12; Appendix Tables 16-19). An
uptake sample also was collected from central Lake Erie (Trial 3), but no paired harbor or discharge
samples were collected for this trial. During Trials 6, 11, and 12, additional water was ballasted from
sites in Lake Superior between initial uptake and discharge, potentially influencing water quality in the
ballast tanks during transit.
Turbidity, TSS and MM measurements were generally higher in ballast uptake and the nearby source
water than in the harbor water some distance from the ship (Table 12), possibly as a result of bottom
sediment being suspended during docking and ballasting operations. Levels of all three parameters
dropped during transit, presumably associated with material settling in the ballast tanks, or the
influence of interim uptake operations. Resulting concentrations in ballast discharges were similar to, or
less than, those of the receiving waters.
The temperature of the source harbor and ballast uptake samples were quite similar to each other in a
given voyage, and showed seasonal variation from 24.3°C in August (Appendix Tables 17 and 18) to
11.5°C in November. Ballast water temperatures generally cooled approximately 3-5°C during transit
through Lake Superior, but were still 2- 9°C warmer than the receiving harbors where they were
discharged (Table 12).
The sondes recorded uncorrected chlorophyll a levels of 0.19 to 1.96 µg/L and uncorrected phycocyanin
levels of 0.07 to 0.53 µg/L in the paired ballast uptake and discharge samples (Table 12). Chlorophyll a
levels dropped by approximately 0.8 µg/L between uptake and discharge for Trials 6 and 11, which may
have been due to algae mortality or settling during the three-day transit, or dilution with interim ballast
uptake water from Lake Superior. The low chlorophyll a and phycocyanin levels generally in the samples
indicated that algal blooms were not occurring in the source harbors during these paired trials between
September and November. A late season diatom bloom in the receiving waters of WLS during Trial 13 in
November likely contributed to high chlorophyll a levels, reaching 8.47 µg/L, in the receiving harbor in
this trial.
The transparency of both filtered and unfiltered water samples from source harbors, uptake and
discharge samples and receiving waters was generally quite high with 84.5 to 97.0% transmittance at
254 nm (Table 12). The only exception was the receiving water of WLS during Trial 13 which had very
low transparency (7.9 - 10.8%T).
Organic carbon, measured as NPOC and DOC, was fairly low and did not fluctuate much between source
harbor, uptake, discharge and receiving water samples, ranging from 1.9 to 2.9 mg/L (Table 12). The
only exception was the receiving water from Trial 13 which had high organic carbon levels (17.0-17.7
mg/L as NPOC).
Most of the other water quality parameters were similar between source harbor, uptake, discharge, and
receiving water sampling events, including pH, and dissolved oxygen (Table 12). The pH was slightly
basic with uptake values ranging from 7.82 to 8.18. Dissolved oxygen levels remained near saturation
(Appendix Tables 16-19).
LSRI/GWRC/TR/GLSBM/1
Date of Issue: May 31, 2018
Page 51 of 88
Great Waters Research Collaborative.
Lake Superior Research Institute, University of Wisconsin-Superior
Table 12. Voyage-Wide Trials: Summary of Chemistry and Water Quality Parameters (Average ± Standard Deviation). N/A = Not Applicable (Not Collected).
Parameter Trial 6
Source: Site 3 Uptake Discharge Receiving: Site 1 Receiving: Site 2 Recei