United States Environmental Protection Agency
Office of Water / Office of Wastewater Management/
Water Permits Division
Sampling Report for the
Vessel General Permitting Program
Pump Mortality Study
EPA 830-^15-003
April 2015
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Contents
CONTENTS
Page
SECTION 1 INTRODUCTION 1-1
1.1 Background 1-1
1.2 Objectives and Scope 1-2
1.3 Location Selection 1-2
SECTION 2 MORTALITY TESTING 2-1
2.1 Bench-Scale Testing Apparatus 2-1
2.2 Fish Mortality Testing 2-5
2.3 Fish Egg Mortality Testing 2-7
2.4 Quality Assurance/Quality Control 2-9
2.5 Deviations from the Sampling and Analysis Plan 2-9
SECTION 3 RESULTS AND DISCUSSION 3-1
3.1 Laboratory and Field Analytical Results 3-1
3.2 Data Analysis and Discussion 3-2
SECTION 4 DATA QUALITY 4-1
4.1 Analytical Quality Control 4-1
4.1.1 Completeness 4-1
4.1.2 Precision 4-1
4.1.3 Accuracy 4-3
4.2 Field Quality Control 4-5
4.2.1 Integrity of Stock Organisms 4-5
4.2.2 Variability 4-6
SECTION 5 REFERENCES 5-1
Appendices
Appendix A: Sampling and Analysis Plan
Appendix B: Fish and Fish Egg Stocking Permits
Appendix C: Photographs
Appendix D: Laboratory Mortality Data
Appendix E: Laboratory Quality Assurance Data
The EPA technical contact for this document is Ryan Albert (202) 564-0763.
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study List of Tables
LIST OF TABLES
Page
2-1 Sample Numbers and Start and Stop Times for the Fathead Minnow Replicates 2-5
2-2 Ambient and Aquarium Temperature Data for Fathead Minnow Testing 2-6
2-3 Ambient and Aquarium Hardness Data for Fathead Minnow Testing 2-6
2-4 Ambient and Aquarium Dissolved Oxygen Data for Fathead Minnow Testing 2-7
2-5 Fathead Minnow Egg Mortality Testing Start and Stop Times and Corresponding
Sample Numbers 2-8
2-6 Fathead Minnow Egg Temperature Data Throughout the Testing Period 2-8
2-7 Ambient and Fathead Minnow Egg Container Hardness Data 2-8
2-8 Ambient and Fathead Minnow Egg Container Dissolved Oxygen Data 2-9
2-9 Deviations from the Sampling and Analysis Plan 2-9
3-1 Fathead Minnow Mortality Data for the Control, Gravity Drain and Pump Tanks 3-1
3-2 Fathead Minnow Egg Mortality Data for the Control, Gravity Drain and
Pump Tanks 3-2
3-3 Weighted Average Percent Mortality for the Control, Gravity Drain and
Pump Test Tanks 3-2
4-1 Duplicate Sample Results and Calculated RPDs for Fathead Minnows 4-2
4-2 Duplicate Sample Results and Calculated RPDs for Fathead Minnow Eggs 4-3
4-3 Accuracy Data for Fathead Minnow Quality Assurance Subsamples 4-4
4-4 Accuracy Data for Fathead Minnow Eggs Quality Assurance Subsamples 4-5
4-5 Mortality Analysis Results for Fathead Minnow Eggs Prior to Testing 4-5
4-6 Averages of the Mean Percent Mortality Rates and 95% Confidence Intervals 4-7
11
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study List of Figures
LIST OF FIGURES
Page
1-1 Diagram of the LSNERR with Red Circle Indicating the Dock Location where the
Study Occurred 1-3
2-1 Pump System Testing Apparatus 2-2
2-2 Gravity Drain System Testing Apparatus 2-2
2-3 Control System Testing Apparatus 2-3
2-4 Layout of Testing System 2-3
3-1 Weighted Average Percent Mortality and 95% Confidence Interval of
Fathead Minnows 3-3
3-2 Weighted Average Percent Mortality and 95% Confidence Interval of
Fathead Minnow Eggs 3-3
in
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 1 - Introduction
SECTION 1
INTRODUCTION
EPA's vessel general permit requires several best management practices (BMPs) which
are thought to reduce the numbers of living organisms discharged in ballast water. The objective
of this bench-scale study is to explore the efficacy of one of those BMPs for Great Lakes bulk
carriers (Lakers1) by determining if emptying ballast tanks by pumping creates greater mortality
for larger organisms taken up and discharged in ballast water than emptying ballast tanks by
gravity. This study took place September 8 to 10, 2014 at the Lake Superior National Estuarine
Research Reserve (LSNERR) located in Superior, Wisconsin under the direction of the Office of
Wastewater Management of the U.S. Environmental Protection Agency (EPA).
Samples were collected in accordance with procedures specified in the Sampling and
Analysis Plan for the Vessel General Permitting Program Pump Mortality Study (SAP) and the
Quality Assurance Project Plan for Technical Support for the Vessel General Permitting Program -
Pump Mortality Study (QAPP). The SAP is provided in Appendix A of this report. Samples offish
and fish eggs for mortality analysis were collected by EPA's contractor Eastern Research Group, Inc.
(ERG) and analyzed on-site by Great Lakes Environmental Center (GLEC) (subcontractor to ERG).
Section 2.0 of this report describes the mortality testing methodology and deviations from
the SAP. Section 3.0 presents the analytical data collected during the sampling episode and
ERG's evaluation of the data. Section 4.0 describes the quality assurance and quality control
(QA/QC) procedures and results, and Section 5.0 presents references used in this document.
1.1 BACKGROUND
As the result of a 2006 court order, on February 6, 2009, EPA began permitting
discharges incidental to the normal operation of vessels operating in a capacity as a means of
transportation through the Vessel General Permit (VGP). The 2013 VGP (USEPA, 2013)
includes general effluent limits applicable to all discharges; general effluent limits applicable to
27 specific discharge streams; narrative water-quality based effluent limits; inspection,
monitoring, recordkeeping, and reporting requirements; and additional requirements applicable
to certain vessel types. Ballast water is one of the applicable vessel discharges controlled under
that permit.
During ballast water intake, a diverse community of live organisms present in both the
water column and seafloor sediments is entrained into a vessel's ballast tanks (Ruiz and Reid,
2007). When Lakers ballast in a Great Lakes' port which has been colonized by an aquatic
nuisance species (ANS), and then discharge their ballast in another Great Lakes' port, they have
the potential to spread ANS within the Great Lakes (Rup et al., 2010; Briski et al., 2012). In Part
2.2.3.3 of the 2013 VGP, EPA included several permit conditions for ballast water management
1 "Laker" is the common name for the large and uniquely designed and constructed dry bulk vessels (or carriers)
used to transport bulk material commodities throughout the Great Lakes system. U. S. flag Lakers usually only
transport goods on the four upper Great Lakes and connecting channels, as most are limited by their size from
transiting the Welland Canal. The primary commodities transported by the Lakers include iron ore pellets, coal,
grain, limestone, cement, sand, and salt.
1-1
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 1 - Introduction
for Lakers to reduce the likelihood of those vessels dispersing and spreading aquatic invasive
species. One of those requirements is for vessels to use their ballast pumps to empty their ballast
tanks, rather than gravity draining, to produce both shear and cavitational stresses on these
organisms, theoretically resulting in higher mortality. Although pumping ballast water rather
than gravity draining should result in additional organism mortality, EPA is aware of only one
experimental study (USCG, 2013a) to support the BMP. This study examined only larval fish
and did not investigate other life stages such as fish eggs that can also be drawn into ballast
tanks. As such, EPA is actively gathering data on the mortality caused by pumps to other life-
stages offish.
1.2 OBJECTIVES AND SCOPE
The objective of this bench-scale study was to determine if emptying ballast tanks by
pumping creates greater mortality for fish eggs and fish (minnows) than emptying ballast tanks
by gravity draining. Based on the results of the bench-scale testing, additional pilot or full scale
testing may be conducted under future work assignments.
The general approach of this bench-scale study included the following steps:
• Collect fish eggs and small minnows from a laboratory-raised culture;
• Place the organisms into two feed tanks and a control tank;
• Gravity drain one feed tank into a collection net and count the number of live and
dead organisms following gravity draining;
• Pump the second feed tank into a collection net and count the number of live and
dead organisms following pumping. The pumping rate was adjusted to simulate
the ballast pumping rate on a Laker;
• Determine the test handling mortality by analyzing live and dead organisms in the
control tank;
• Using statistical analysis, determine the differences in mortality between the
control, gravity draining and pumping for each organism type.
1.3 LOCATION SELECTION
EPA conducted the bench-scale study at the LSNERR located in Superior, Wisconsin
(see Figure 1-1). This facility was selected for the study due to its location on the shore of Lake
Superior's St. Louis River estuary (a major shipping port) and the availability of Lake Superior
estuary water for maintaining the organisms before and after testing. In addition, the facility
provided laboratory and dock space needed for the test tanks and analytical equipment.
1-2
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 1 - Introduction
Figure 1-1. Diagram of the LSNERR with Red Circle Indicating the Dock Location where the Study Occurred
1-3
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 2 - Mortality Testing
SECTION 2
MORTALITY TESTING
This section provides the detailed procedure that was used to conduct the bench-scale
organism mortality tests at LSNERR. The bench-scale testing procedure was divided into three
phases that included: (1) constructing the test apparatus including the tanks, piping, pumps, and
post discharge organism collection nets; (2) obtaining small fish (native fathead minnows) and
conducting the gravity drain, pump and control testing and live/dead sample analysis; and (3)
obtaining fish eggs (native fathead minnow eggs) and conducting the gravity drain, pump and
control testing and live/dead sample analysis. Testing for each organism group (fish eggs and
minnows) was conducted separately. All tests had multiple replicates. The following subsections
describe each of these three phases in greater detail.
Special certification requirements were required and obtained for this study to bring live
fathead minnow eggs spawned at EPA's Mid-Continent Ecological Division (MED) laboratory
in Duluth Minnesota to the LSNERR in Superior, Wisconsin test facility. Those included a Fish
Health Certification (FHC) from a veterinarian for EPA's laboratory supplying the fathead
minnow eggs and a Fish Import Permit from the Wisconsin Department of Agriculture to bring
the eggs to Wisconsin. In addition, Fish Stocking Permits were required from the Wisconsin
Department of Natural Resources for both the fathead minnow eggs and the fathead minnows
obtained from a local minnow supplier. Copies of the MED FHC, the Fish Import Permit, and
the Fish Stocking Permits are provided in Appendix B.
2.1 BENCH-SCALE TESTING APPARATUS
Figure 2-1, Figure 2-2, and Figure 2-3 are diagrams depicting the pump, gravity drain,
and control system testing apparatus, respectively. Figure 2-4 is a site layout showing the
arrangement of the test tanks and the sample receiving areas.
2-1
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
Live organisms
55-gallontesttank.
2" transfer hose
Pump
4" transfer hose
Net Support
Structure
/
30" diameter plankton net
Lake Bed
Figure 2-1. Pump System Testing Apparatus
Live organisms
55-gallon test tank
2" transfer hose
4" transfer hose
Dock
Net Support
Structure
30" diameter plankton net
Lake Bed
Figure 2-2. Gravity Drain System Testing Apparatus
2-2
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
Live organisms
Dock
55-ga lion test tankin estuarv
30" diameter plankton net insidetest lank
X
Lake Bed
Figure 2-3. Control System Testing Apparatus
Control Test Tank in Estuary
Plankton Net Inside Control Test Tank
Plankton Net
Plankton Net
End of Dock
Figure 2-4. Layout of Testing System
2-3
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 2 - Mortality Testing
The bench-scale testing system consisted of two 55-gallon open top plastic test tanks
placed on concrete blocks (see Photographs 47, 52, and 53 in Appendix C). Each test tank had a
diameter of 30 inches and a height of 35 inches. The pump and gravity drain tanks were fitted
with a 2-inch diameter valve beneath the tanks for draining or pumping. The pump and gravity
drain tanks were filled with water directly from the St. Louis River estuary using a pump. The
pump and gravity drain tanks were directed into the plankton nets placed in approximately 4 feet
of water in the estuary (see Photographs 2, 28, and 41 in Appendix C). The plankton nets had a
35 micron screen size (USEPA, 2010), were 30-inch diameter and were fitted with a 1-liter
plastic bottle on the bottom to capture the organisms rinsed from the nets.
The pump tank discharged below the water line into the plankton net to simulate a ballast
discharge through the sea chest. The gravity drain tank also discharged into the plankton net
below the water line to simulate gravity draining by Lakers.2 EPA placed the plankton nets in the
estuary rather than in another receiving tank to buffer the force of the water and minimize the
potential mortality that could be caused by the organisms contacting plankton nets during
discharge. In addition, to further reduce mortality caused by organisms being forced into the
plankton nets by water pressure, the pipe diameter on the pump tank discharge line was increased
from 2 inches to 4 inches. This reduced the pressure and force with which test water was
discharged into the plankton net to help counteract the force imposed by the pump. The gravity
drain tank also included a pipe size increase from 2 inches to 4 inches near the middle of the pipe
run in to provide consistency between the pump and gravity drain systems. To ensure against
"contamination" of the test net with organisms in the water (and escape of live organisms inside
the test nets), net supports were constructed to hold the nets approximately 8 inches above the
water level. See Photographs 27, 28, and 51 in Appendix C.
To evaluate organism mortality caused by handling, the control tank (third tank) was
submerged in the estuary adjacent to the pump and gravity drain collection nets. The control tank
was lined with a 35-micron plankton net and suspended by the lifting winch. The control tank
was submerged rather than drained to reduce possible organism mortality that could be caused by
the change in elevation from the dock to the sample receiving area in the estuary. This net was
also supported approximately 8 inches above the water level to guard against organism escape or
entrainment of additional organisms from estuary water (see Photographs 7 and 28 in Appendix
C).
To simulate ballast pump conditions from a Laker, EPA used a gasoline powered
centrifugal trash pump with 2-inch diameter hose connected to the valve on the bottom of the
pump tank (see Photograph 52 in Appendix C). The pump flow rate was calibrated by measuring
the time needed to reduce the volume of a full 55 gallon tank to its overflow weir located at the
tank bottom. Based on an average time of 35 seconds to empty the tank, EPA estimated the
pump flow rate to be approximately 83 gallons per minute (gpm) at the hydrostatic head
conditions observed during the study. For a 2-inch diameter flexible suction hose and a measured
flow rate of 83 gpm, the flow rate per area of hose was calculated to be 26.4 gpm/in2.
2 According to Mr. Noel Bassett with The American Steamship Company and Mr. Jim Weakley at the Lake Carriers
Association, American Lakers typically discharge ballast water through either their low or high sea chests below the
water line.
2-4
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
According to ballast system design data for Lakers (USCG, 2013b), ballast water
pumping rates range between 3,600 and 26,000 gpm resulting in a flow rate per area ranging
between 31 and 46 gpm/in2 depending on the diameter of the vessels ballast water piping. Other
published information suggests that actual ballast water flow through the piping of Lakers is 10
ft/second and therefore ballast water treatment system testing should be conducted at flows
ranging between 23 gpm and 40.1 gpm (Cangelosi et al., 2011). Although the flow per area for
this study was less than the flow per area for Lakers (26.4 gpm/in2), the flow rate (83 gpm) was
above the flow rates suggested for testing (Cangelosi et al., 2011); therefore, the flows for this
study appear appropriate for a bench-scale test to evaluate pump mortality.
2.2 FISH MORTALITY TESTING
Approximately 1,500 fathead minnows were obtained from Hayward Bait in Hayward,
Wisconsin, and were brought to the LSNERR in the early morning hours of September 9, 2014,
and placed in 3 aerated 10-gallon aquaria established in the on-site laboratory (see Photographs
15, 19, and 20 in Appendix C). The fathead minnows ranged in size from approximately 1 inch
in length to approximately 2.5 inches in length. Fathead minnows were selected for testing
because they are native to Lake Superior and the St. Louis River. These minnows also tend to be
relatively hardy, helping to ensure that there will be little handling and control mortality.
Mortality testing was conducted by first filling the pump and gravity drain tanks with
approximately 50 gallons of St. Louis River estuary water and then adding approximately 100
fathead minnows collected from the stock aquaria. Once the minnows were added, the drain
valves were opened and the minnows were allowed to flow by either gravity to a plankton net, or
through the operating trash pump and into a plankton net. For the control tank submerged in the
estuary, approximately 100 minnows were added directly into the net. Following a one hour
recovery period, the nets were raised and the minnows were collected into 1-liter plastic sample
bottles fastened to the bottom of the nets. The sample bottles were immediately removed from
the nets following sample collection and the bottles transferred to the on-site laboratory where
they were gently emptied into pre-labeled (control, pump and gravity) aerated aquaria containing
St. Louis River estuary water (see Photographs 35 through 38 in Appendix C). Mortality
assessment was conducted on-site by GLEC by removing subsamples of minnows from the
appropriate aquaria and examining the minnows either under the microscope or with the naked
eye to assess body or gill movement depending on fish size.
Table 2-1 shows the sample numbers and the times when the minnows were introduced
into the nets and removed from the nets for each of the five replicate samples.
Table 2-1. Sample Numbers and Start and Stop Times for the Fathead Minnow Replicates
Replicate
1
2
o
J
4
5
Control
In
9:24
10:38
11:51
12:59
14:07
Out
10:24
11:38
12:49
14:00
15:07
Gravity
In
9:26
10:42
11:53
13:01
14:10
Out
10:28
11:42
12:53
14:02
15:08
Pump
In
9:32
10:45
11:56
13:05
14:13
Out
10:33
11:46
12:56
14:05
15:11
Sample Numbers
FC1,FG1,FP1
FC2, FG2, FP2
FC3, FG3, FP3
FC4, FG4, FP4
FC5, FG5, FP5
2-5
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
Throughout the testing period, the temperature, hardness and dissolved oxygen
concentrations were monitored in the stock aquaria (aquaria 1 through 3) and the post discharge
aquaria (aquaria 4 through 6) to ensure these conditions would not impart mortality on the
minnows. Temperature data for each of the aquaria are provided in Table 2-2.
Table 2-2. Ambient and Aquarium Temperature Data for Fathead Minnow Testing
Time
8:45
9:50
10:50
12:00
13:07
14:16
Ambient
(°C)a
19.5
19
19.5
19.5
20
21
Stock
Aquaria 1
(°C)b
20
20
20
20.5
20.5
NA
Stock
Aquaria 2
(°C)b
20
20
20
20
20
NA
Stock
Aquaria 3
(°C)b
19.5
20
20
20
20
NA
Control
Aquaria 4
(°C)b
20
20
19.5
20
20
20.5
Pump
Aquaria 5
(°C)b
20
20
19.5
21
20
20
Gravity
Aquaria 6
(°C)b
19.5
19.5
20
20.5
20
20
NA - All fish removed after last replicate so no data were collected.
a Ambient temperature of the St. Louis River estuary adjacent to the plankton nets receiving the minnows.
b Aquaria 1 through 3 hold stock minnows prior to testing, and aquaria 4 through 6 hold minnows after testing.
The hardness of the ambient water placed in Aquaria 1 through 5 was also measured and
compared to the hardness of the water received from Hayward Bait (placed in Aquaria 6) to
determine if hardness adjustments were necessary to prevent osmotic shock when the minnows
were introduced to the St. Louis River estuary during testing. Hardness of the water from
Hayward Bait in which the minnows were raised was determined to be 50 ppm (as CaCOs) while
the hardness of the ambient water in Aquaria 1 through 5 ranged from 90 ppm to 115 ppm as
CaCOs. Because of the difference in hardness between the water received from Hayward Bait
and the ambient water in Aquaria 1 through 5, the hardness of the Hayward Bait water (Aquaria
6) was adjusted to 100 ppm (as CaCOs) by slowly adding commercially purchased mineral water
before transferring minnows to Aquaria 1 to 3 to prevent osmotic shock. Hardness data collected
during the entire testing period are provided in Table 2-3.
Table 2-3. Ambient and Aquarium Hardness Data for Fathead Minnow Testing
Time
9:00
11:00
13:34
Ambient
(ppm)a
130
100
120
Stock
Aquaria 1
(ppm)b
110
140
120
Stock
Aquaria 2
(ppm)b
90
100
110
Stock
Aquaria 3
(ppm)b
110
100
110
Control
Aquaria 4
(ppm)b
115
115
120
Pump
Aquaria 5
(ppm)b
115
110
120
Gravity
Aquaria 6
(ppm)b
50C
100
100
a Ambient hardness of the St. Louis River estuary adjacent to the plankton nets receiving the minnows.
b Aquaria 1 through 3 hold stock minnows prior to testing, and aquaria 3 through 6 hold minnows after testing.
0 Aquaria 6 was used as the original stock tank that all minnows were placed in after receipt from Hayward Bait.
Water from Hayward Bait was used to fill Aquaria 6 resulting in a stock tank hardness of 50 ppm. Hardness was
slowly adjusted in Aquaria 6 to approximately 100 ppm before transferring minnows to Aquaria 1 through 3 to
prevent osmotic shock.
2-6
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
The dissolved oxygen of the water in each aquaria was also measured along with the
dissolved oxygen of the St. Louis River estuary (ambient) to verify sufficient dissolved oxygen
was available to sustain the minnows. Dissolved oxygen data, measured by a direct reading
probe, for the estuary and the aquariums are provided in Table 2-4. The data show that dissolved
oxygen was present in the stock aquaria and the ambient water.
Table 2-4. Ambient and Aquarium Dissolved Oxygen Data for Fathead Minnow Testing
Time
8:45
9:50
10:50
12:00
13:08
14:18
Ambient
(mg/L)a
9.3
8.6
9.0
9.1
8.7
9.8
Stock
Aquaria 1
(mg/L)b
5.0
6.0
4.2
5.2
5.0
NAC
Stock
Aquaria 2
(mg/L)b
3.1
3.8
3.9
4.9
4.8
NAC
Stock
Aquaria 3
(mg/L)b
5.5
6.4
5.5
6.6
7.1
NAC
Control
Aquaria 4
(mg/L)b
NAC
NAC
8.7
9.0
9.1
9.8
Pump
Aquaria 5
(mg/L)b
NAC
NAC
8.2
8.0
9.0
9.6
Gravity
Aquaria 6
(mg/L)b
NAC
NAC
8.6
9.0
9.1
9.4
a Ambient dissolved oxygen of the St. Louis River estuary adjacent to the plankton nets receiving the minnows.
b Aquaria 1 through 3 hold stock minnows prior to testing, and aquaria 3 through 6 hold minnows after testing.
0 Aquaria did not contain minnows and therefore dissolved oxygen was not measured.
2.3 FISH EGG MORTALITY TESTING
Live fathead minnow eggs were obtained from the MED laboratory in Duluth on
September 10, 2014 for testing at the LSNERR. The eggs, hatched and raised by MED, were 3
days old, had a diameter of 1 millimeter, and included eyes and heartbeats that could be viewed
under the microscope to assess living/dead. According to researchers at MED, the 3-day old eggs
were within hours of hatching into larvae and were therefore most susceptible to environmental
stress. Fathead minnow eggs were selected for testing because they are native to Lake Superior
and the St. Louis River. See Photographs 50 and 55 in Appendix C.
For each test, the pump, gravity drain and control tanks were filled with approximately 50
gallons of St. Louis River estuary water and dosed with approximately 250 fathead minnow
eggs. Due to the number of available eggs, EPA conducted only three replicate tests with the
eggs (compared to five for fish). For each test, the eggs were introduced first into the control
tank, then to the gravity drain tank and finally to the pump tank. Dosing and discharge of the
three tanks occurred within a 15 minutes period for each replicate. For the gravity drain and
pump tanks, the tank drain valves were opened and discharged to the receiving nets within 1
minute of dosing the eggs into the tanks. Once the eggs entered the plankton nets suspended in
the estuary, the one hour recovery period began. Following the one-hour recovery period, the
plankton nets were raised and rinsed using a hand sprayer to wash the eggs into the 1-liter
receiving bottled fastened to the bottom of the nets. Once the nets were completely raised and
rinsed, the 1-liter receiving bottle was immediately taken into the laboratory for mortality
assessment under the dissecting microscopes (see Photographs 43 through 46 in Appendix C).
Table 2-5 shows the sample numbers and the times when the eggs were introduced into
the nets and removed from the nets for each of the three replicate samples.
2-7
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
Table 2-5. Fathead Minnow Egg Mortality Testing Start and Stop Times and
Corresponding Sample Numbers
Replicate
1
2
3
Control
In
9:44
11:05
12:15
Out
10:44
12:02
13:13
Gravity
In
9:58
11:13
12:20
Out
10:59
12:12
13:18
Pump
In
9:51
11:07
12:17
Out
10:52
12:07
13:16
Sample Numbers
EC1, EG1, EP1
EC2, EG2, EP2
EC3, EG3, EP3
Prior to testing, the temperature of the water in which the eggs were received (Stock
Bottle 1) was measured and compared to the temperature of the St. Louis River estuary to
determine if acclimation was required. As shown in Table 2-6, the temperature of the estuary
was 17.5°C, and the temperature of Stock Bottle 1 was 20°C prior to the start of testing. Since the
temperature difference between the water in the estuary and the water holding the eggs was less
than 5°C, no further temperature acclimation was required. Temperature data measured for the
St. Louis Rivers estuary and in the stock and sample bottles throughout testing are provided in
Table 2-6.
Table 2-6. Fathead Minnow Egg Temperature Data Throughout the Testing Period
Time
9:25
10:15
11:18
13:15
Ambient
oCa
17.5
17.5
17
16.5
Stock Bottle 1
oCa
20
19
20
NAb
Control Bottle 2
oCa
NAb
17
16
16.5
Gravity Bottle 3
oCa
NAb
17
16.5
16.5
Pump Bottle 4
oCa
NAb
17
16.5
16
a Bottle 1 contains stock fathead minnow eggs from MED prior to testing, and bottles 2 through 4 hold fathead
minnow eggs following testing.
b NA - no eggs in bottle so measurement was not obtained.
Water hardness was also measured using an aquarium hardness test kit and compared to
the hardness of the estuary to determine whether the eggs would need to be acclimated to prevent
osmotic shock upon entering the receiving nets. Hardness of the estuary was measured at 90 ppm
(as CaCOs), and the hardness of the water provided with the fathead minnow eggs was 100 ppm
(as CaCOs). Since the hardness of the estuary and the hardness of the water in which the eggs
were raised were within 10 percent, no hardness adjustments were made. Hardness data
measured during testing are provided in Table 2-7.
Table 2-7. Ambient and Fathead Minnow Egg Container Hardness Data
Time
9:25
11:20
Ambient
(ppm)
90
100
Stock Bottle 1
(ppm)a
100
110
Control Bottle 2
(ppm)a
NAb
90
Gravity Bottle 3
(ppm)a
NAb
90
Pump Bottle 4
(ppm)a
NAb
90
a Bottle 1 contains stock fathead minnow eggs from MED prior to testing, and bottles 2 through 4 hold fathead
minnow eggs following testing.
b NA - no eggs in bottle so measurement was not obtained.
2-8
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
The dissolved oxygen of the water in each bottle was also measured along with the
dissolved oxygen of the St. Louis River estuary (ambient) to verify sufficient dissolved oxygen
was available to sustain the eggs. Dissolved oxygen data, measured by a direct reading probe, for
the estuary and the bottles containing the eggs are provided in Table 2-8. The data show that
sufficient dissolved oxygen was present in the ambient water, the stock bottle containing the
stock of eggs prior to testing, and in the sample bottles of eggs after testing.
Table 2-8. Ambient and Fathead Minnow Egg Container Dissolved Oxygen Data
Time
9:25
10:15
11:20
13:15
Ambient
(mg/L)
9.8
10.3
9.3
9.9
Stock Bottle 1
(mg/L)a
7.8
8.2
7.3
NAb
Control Bottle 2
(mg/L)a
NAb
9.1
10.4
10.4
Gravity Bottle 3
(mg/L)a
NAb
10.1
10.6
10.1
Pump Bottle 4
(mg/L)a
NAb
9.7
9.4
9.8
a Bottle 1 contains stock fathead minnow eggs from MED prior to testing, and bottles 2 through 4 hold fathead
minnow eggs following testing.
b NA - no eggs in bottle so measurement not obtained.
2.4 QUALITY ASSURANCE/QUALITY CONTROL
Analytical quality control was measured by assessing overall data completeness, by
comparing mortality counts of consecutive subsamples (precision), and by comparing duplicate
mortality counts of the same subsample performed by two different analysts (accuracy). These
data are discussed in Section 4.1. Field quality control was evaluated by assessing the integrity of
the stock organisms and by measuring the variability among replicates for the control, gravity
drain, and pump tests. These results are discussed in Section 4.2.
2.5 DEVIATIONS FROM THE SAMPLING AND ANALYSIS PLAN
The study proceeded as specified in the SAP with the deviations described in Table 2-9.
Table 2-9. Deviations from the Sampling and Analysis Plan
Deviation
Description
Pump Flow
Rate
ERG intended to operate the pump flow rate at 32 gpm to simulate the testing protocols used by the
Great Ships Initiative (Cangelosi et al., 2011); however, the minimum flow that could be obtained
from the gasoline driven trash pump used for the study was 83 gpm (26.4 gpm/in2) at the head
pressure provided from the test tank. Although the flow rate from the trash pump used for this
study is significantly greater than that of pump flow rate used for testing in the Great Ships
Initiative protocols, the flow rate is slightly below the calculated unit area design maximum flow
rate (gpm/in2 of pipe) found on Lakers pumping ballast water, which ranges from 31 gpm/in2 to 46
gpm/in2 (USCG, 2013b). Since the pump flow rate used for this study (83 gpm, 26.4 gpm/in2) is
above the flow rate used by the Great Ships Initiative (32 gpm) but below the expected unit area
flow rate for Lakers (31 to 46 gpm/in2), the results should provide a reasonable estimate of the
mortality caused by pumping and gravity draining ballast water for the two organism types.
2-9
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 2 - Mortality Testing
Table 2-9. Deviations from the Sampling and Analysis Plan
Deviation
Description
Fathead
Minnow Egg
Test Replicates
ERG had intended to conduct 4 replicates using the 2,000 fathead minnow eggs provided by the
MED laboratory; however, only 3 replicate tests could be completed since more eggs were required
per test than originally planned. The SAP estimated that 3 eggs per gallon would be sufficient for
estimating mortality; however, after further evaluation, the sampling team decided to use
approximately 5 eggs per gallon to guarantee sufficient egg recovery in the plankton nets for
mortality analysis.
2-10
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 3 - Results and Discussion
SECTION 3
RESULTS AND DISCUSSION
This section presents the data collected during the pump mortality study. Mortality results
for fathead minnows and fathead minnow eggs are presented in Section 3.1. Section 3.2 provides
a summary of the data, including graphic representations along with a discussion regarding how
the data may be used to evaluate the impact of pumping ballast water rather than gravity draining
on the mortality of small fish and fish eggs. All raw mortality data provided by GLEC are
provided in Appendix D of this report.
3.1 LABORATORY AND FIELD ANALYTICAL RESULTS
Mortality results for the fathead minnows and the fathead minnow eggs are provided in
Table 3-1 and Table 3-2, respectively. The two tables present the live/dead counts for each tank
(control, gravity drain, and pump) and for each replicate. The estimated percent mortality for
each replicate and test tank is also included.
Table 3-1. Fathead Minnow Mortality Data for the Control, Gravity Drain and
Pump Tanks
Replicate
1
2
3
4
5
Condition
Dead Minnows
Live Minnows
Total Minnows
Percent Mortality
Dead Minnows
Live Minnows
Total Recovered Minnows
Percent Mortality
Dead Minnows
Live Minnows
Total Recovered Minnows
Percent Mortality
Dead Minnows
Live Minnows
Total Recovered Minnows
Percent Mortality
Dead Minnows
Live Minnows
Total Recovered Minnows
Percent Mortality
Control Tank
2
115
117
1.7%
0
152
152
0.0%
0
93
93
0.0%
0
154
154
0.0%
0
139
139
0.0%
Gravity Drain Tank
0
113
113
0.0%
0
121
121
0.0%
1
94
95
1.1%
0
127
127
0.0%
0
195
195
0.0%
Pump Tank
65
56
121
53.7%
51
67
118
43.2%
31
58
89
34.8%
45
51
96
46.9%
72
86
158
45.6%
3-1
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 3 - Results and Discussion
Table 3-2. Fathead Minnow Egg Mortality Data for the Control, Gravity Drain and
Pump Tanks
Replicate
1
2
3
Condition
Dead Eggs
Live Eggs
Total Recovered Eggs
Percent Mortality
Dead Eggs
Live Eggs
Total Recovered Eggs
Percent Mortality
Dead Eggs
Live Eggs
Total Recovered Eggs
Percent Mortality
Control Tank
2
127
129
1.6%
0
40
40
0.0%
0
1
1
0.0%
Gravity Drain Tank
1
26
27
3.7%
0
25
25
0.0%
1
56
57
1.8%
Pump Tank
1
124
125
0.8%
2
55
57
3.5%
0
38
38
0.0%
3.2 DATA ANALYSIS AND DISCUSSION
To analyze the mortality data for the minnows and eggs, EPA took the weighted average
mortality rate across the replicates (weighted by sample size of recovered organisms) and
calculated the 95% confidence intervals around these point estimates (see Table 3-3). EPA also
plotted the weighted average percent mortalities and confidence intervals for fathead minnows
and fathead minnow eggs for each of the test tanks (see Figure 3-1 and Figure 3-2, respectively).
(Note that negative mortality rate is not plausible, so the minimum lower bound is 0%.)
Table 3-3. Weighted Average Percent Mortality for the Control, Gravity Drain and
Pump Test Tanks
Organism
Fathead Minnows
Fathead Minnow Eggs
Weighted Average Percent Mortality and 95% Confidence Interval
Control Tank"
0.3% + 0.4%
1.2% +1.6%
Gravity Drain Tank"
0.2% + 0.3%
1.9% + 2.5%
Pump Tank"
45. 4% + 3. 4%
1.4% + 2.1%
a Average calculated from five replicates for fathead minnows and three replicates for fathead minnow eggs;
weighted by the number of fish in each tank.
3-2
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 3 - Results and Discussion
in
|
c
I
•a
(0
01
1
LL.
M—
o
£
(0
e
o
2
4-1
Ol
a
Ol
Q.
60%
50%
40%
30%
20%
10%
0%
-10%
I
Control Tank Gravity Drain Tank Pumped Tank
Figure 3-1. Weighted Average Percent Mortality and 95% Confidence Interval of
Fathead Minnows
1 5.00%
+•»
£ 4.00%
o
2 S 3'00%
1 5
2 o 2.00%
S 1
Ol ^
g 1-00%
Q.
g1 0.00%
k.
Ol
"* -1.00%
Control Tank Gravity Drain Tank Pumped Tank
Note that negative mortality rate is not plausible, so the minimum lower bound is 0%.
Figure 3-2. Weighted Average Percent Mortality and 95% Confidence Interval of
Fathead Minnow Eggs
3-3
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 3 - Results and Discussion
To evaluate the impact of pumping ballast water rather than gravity draining on the
mortality of small fish and fish eggs, EPA compared the weighted average percent mortality and
variability in the control against the weighted average percent mortality and variability of the
pump and gravity drain tests. Variability for each tank type and organism was measured using
the 95 percent confidence interval around the average; this generates the upper and lower
bounds. The average and upper bound control mortality were then compared to the average and
lower bound pump and gravity drain mortalities to determine if the pump and gravity drain tests
caused more mortality than the control. EPA also used the Wilcoxin signed-rank test, a non-
parametric statistical hypothesis test, to assess whether mortality was significantly greater in the
pump tank than in the gravity drain and control tanks (Wilcoxin, 1945).
Fathead Minnows
As indicated in Table 3-3 and depicted graphically in Figure 3-1, the upper bound of the
95 percent confidence interval on percent mortality for the fathead minnow control tank is 0.7%
(mean of 0.3% plus 0.4%) and the lower bound mortality rate for the gravity drain tank and
pump tank are 0% (0.2 minus 0.3%) and 41.9% (45.4% minus 3.4%), respectively. (Note that
negative mortality rate is not plausible, so the minimum lower bound is 0%). For the gravity
drain tank, the lower bound percent mortality is below the upper bound for the control tank
mortality, verifying there is no difference in mortality between the control and gravity drain tank
(i.e., gravity draining does not cause mortality). However, for the pump tank, the lower bound
percent mortality is much larger than the upper bound for the control tank (41.9% versus 0.7%)
verifying that pumping does impact mortality of fathead minnows. The Wilcoxin signed-rank
test also found that fathead minnow mortality was significantly greater in the pump treatment
than in both the gravity (p<0.0001) and control treatments (p<0.0001). Rates offish mortality
between control and gravity drain treatments were not significantly different.
One explanation as to why some minnows survived pumping while others were killed
may be linked to their introduction in to the impeller cavity of the pump head. Observations
made of the minnows discharged from the pump show that the minnows that died before
reaching the laboratory were completely dismembered or were swimming erratically after
discharge. Minnows that survived 1 hour after passing through the pump appeared healthy
immediately after passing through the pump. These observations suggest that if minnows enter
the pump's impeller cavity at the point where the impeller is passing the intake port, then they
will likely be injured or killed. If the minnows enter the impeller cavity between the impeller
blades, then they could be swept through the impeller cavity unharmed without contacting either
the impeller or the cavity walls, or experiencing excessive shear or cavitational stress.
The mortality data obtained from this study using adult fathead minnows are only
partially comparable to the study conducted by the U.S. Coast Guard (USCG) using Asian carp
larvae on the Illinois River in 2011 (USCG, 2013a). First, published literature indicates that
mortality offish larvae is higher than fry or adult fish in many marine or freshwater species
(Dahberg, 1979). Secondly, fathead minnows are a different species than Asian carp and possibly
less susceptible to the shear stress caused by centrifugal pumps.
Fathead Minnow Eggs
As indicated in Table 3-3 and depicted graphically in Figure 3-2, the upper bound of the
95 percent confidence interval on percent mortality for the fathead minnow egg control tank is
3-4
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 3 - Results and Discussion
2.8% (mean of 1.2% plus 1.6%) and the lower bound mortality rate for the gravity drain tank and
pump tank are 0% (1.9% minus 2.5%) and 0% (1.4% minus 2.1%), respectively. (Note that
negative mortality rate is not plausible, so the minimum lower bound is 0%.) Since the lower
bound for the gravity drain and pump tanks are both below the upper bound for the control, there
is no statistically significant difference in mortality between the control tank and the gravity
drain or pump tanks, verifying that neither gravity draining nor pumping increases the mortality
of fathead minnow eggs. The Wilcoxin signed-rank test also found that fathead minnow egg
mortality was not significantly different between the pump, gravity drain and control tanks.
A likely explanation as to why fish eggs survived gravity draining and pumping is that
their small size (approximately 1 millimeter diameter) allows them to enter the impeller cavity of
the pump without significant contact with the blades or the impeller cavity walls or experiencing
excessive shear or cavitational stress.
Conclusion
Based on this bench-scale study, it appears that ballast water pumps have measurable
mortality for larger organisms such as fish, but that mortality is not measurable for fish eggs.
Although the data from this study indicate roughly one-half of adult minnows and essentially no
minnow eggs will be killed when passing through a centrifugal pump at unit flow rates
comparable to the ballast water flows on a Laker, these data are not definitive of ballast water
pump mortality due, in part, to differences in pump design. For example, ballast pump data
provided by Phil Moore, Interlake Steamship Company (Moore, 2014) show that main ballast
pumps on some of their vessels have 30 inch diameter impellers with a 30° blade angle, a 2.5
inch gap between the outer edge of the impeller and the cavity walls, and operate at speeds of
690 revolutions per minute (RPM). Note that the ballast pump gap is larger than the length of the
largest fish used for this testing, and much larger than the diameter of the fish eggs. In contrast,
the impeller diameter on the trash pump used for this study was 5 inches and had a 0.008 to
0.014 inch (0.20 to 0.36 mm) gap between the outer edge of the impeller and the cavity walls.3
The angle of the blades and the speed of the impeller used for this study were unknown. Note
also that the trash pump gap is much smaller than the length/width of the fish used for this testing
and is somewhat smaller than the diameter of the fish eggs. Because of these significant
differences in physical size of the pumps, EPA can make only generalized conclusions based on
the mortality caused by the centrifugal pump used in this study and the mortality that would be
expected by main ballast pumps on Lakers. Future research evaluating ballast pump mortality
could involve a similar study conducted on-board a Laker using actual main ballast pumps. Such
a study would also account for other significant differences in physical size and configuration
between our simulation and actual ballast water systems that could affect organism mortality,
such as vessel ballast water piping and tankage.
1 Wacker-Neuson 2" diameter centrifugal trash pump powered by a 4.8 horsepower Honda gasoline engine.
3-5
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 4 - Data Quality
SECTION 4
DATA QUALITY
Quality assurance/quality control (QA/QC) procedures applicable to this sampling
episode are outlined in the QAPP for this program, approved by EPA on May 16, 2014, and its
amendment dated August 29, 2014. This section describes the quality control practices used to
assess the precision and accuracy of the analytical data presented in Section 3.0. Quality control
(QC) practices used for this sampling episode include the duplicate mortality counts and
laboratory quality control checks.
4.1 ANALYTICAL QUALITY CONTROL
ERG verified that laboratory performance was acceptable by verifying that all samples
received by the laboratory were analyzed within the method-specific holding times and that the
quality checks of the mortality data, as specified by the QAPP, were conducted. Data review
biologists from GLEC prepared a written data review narrative (see Appendix E) describing any
qualifications of the data. The laboratory quality control measures for mortality analysis of
fathead minnows and fathead minnow eggs are described below.
4.1.1 Completeness
Completeness is defined in terms of the percentage of data that were collected and
deemed to be acceptable for use in this study. The goals for this study were a minimum of 80%
sampling completeness and 90% analytical completeness resulting in a minimum overall
completeness of 72% (determined by multiplying sampling and analytical completeness goals).
For the fathead minnow tests, all 15 of the targeted samples were collected and analyzed
resulting in a sampling and analytical completeness of 100%. For the fathead minnow eggs, ERG
had originally planned to complete 4 replicates, but due to a lack of eggs, only 3 replicates were
completed, resulting in sampling completeness of 75%. All 9 fathead minnow egg samples from
the 3 replicates were analyzed and the data deemed acceptable resulting in an analytical
completeness of 100%. The overall completeness for the study was calculated to be 89% (24 of
27 samples collected, and 24 of 24 samples analyzed).
4.1.2 Precision
Precision is a measure of the agreement among repeated measurements and is
quantitatively assessed by calculating the relative percent difference (RPD) between duplicate
sample results. For this study, precision was measured by comparing the counts of living/dead
organisms for every 10th subsample4 to counts from the subsequent subsample (i.e., every 11th
subsample). Per the requirements of the QAPP, duplicate recounts were conducted at a frequency
4 For minnows, a subsample consisted of collecting a fraction of the total number of minnows in each sample and
analyzing the sample for live/dead organisms. The number of subsamples collected from the minnows samples
received in the laboratory ranged between 3 and 10. For fathead minnow eggs, a subsample consisted of a small
aliquot (20 to 30 milliliters) of water obtained from the primary sample bottle and analyzed for live/dead organisms.
The number of subsamples collected from the egg samples received in the laboratory ranged between 7 and 13.
4-1
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 4 - Data Quality
of 10%, and the RPD between the duplicate counts samples was calculated to determine if the
target RPD of + 10 % was achieved. Table 4-1 and Table 4-2 show the results of the duplicate
mortality counts and the calculated RPDs for minnows and eggs, respectively.
For dead minnows, only 1 of the 33 duplicate quality assurance subsamples exceeded the
target RPD of 10%. For live minnows, only 3 of the 33 duplicate quality assurance subsamples
exceeded the target RPD of 10%. The elevated RPD of 66.7% for one live minnow subsample is
a result of the low number of minnows in the sample. For dead fathead minnow eggs, only 1 of
the 10 duplicate quality assurance subsamples exceeded the target RPD, and for live fathead
minnow eggs, none of the quality assurance subsamples exceeded the target RPD.
Table 4-1. Duplicate Sample Results and Calculated RPDs for Fathead Minnows
Treatment
Type
Control
Control
Control
Gravity
Gravity
Pump
Pump
Pump
Control
Control
Gravity
Pump
Pump
Pump
Control
Control
Gravity
Pump
Pump
Control
Gravity
Control
Pump
Pump
Gravity
Pump
Control
Control
Treatment
Replicate
1
1
1
1
1
1
1
1
2
2
2
2
2
2
o
J
3
3
3
3
4
4
4
4
4
4
4
5
5
Sub-
Sample
Number
2
3
4
1
6
2
2
2
2
4
1
1
3
2
1
1
1
1
1
1
2
4
1
1
12
3
3
3
Original
Sample
Result
(Dead)
1
0
0
0
0
11
8
12
0
0
0
13
13
7
0
0
0
2
4
0
0
0
9
10
0
2
0
0
Duplicate
Sample
Result
(Dead)
1
0
0
0
0
12
8
13
0
0
0
17
14
7
0
0
0
2
4
0
0
0
9
11
0
2
0
0
RPD
0.0%
NC
NC
NC
NC
8.7%
0.0%
8.0%
NC
NC
NC
26.7%
7.4%
0.0%
NC
NC
NC
0.0%
0.0%
NC
NC
NC
0.0%
9.5%
NC
0.0%
NC
NC
Original
Sample
Result
(Alive)
8
7
9
14
6
2
o
J
1
8
11
13
12
5
4
15
17
23
9
11
14
12
14
1
16
8
9
12
11
Duplicate
Sample
Result
(Alive)
8
7
9
14
6
2
o
J
1
8
11
13
15
4
4
15
17
22
9
11
14
11
14
1
17
8
9
12
11
RPD
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
22.2%
22.2%
0.0%
0.0%
0.0%
4.4%
0.0%
0.0%
0.0%
8.7%
0.0%
0.0%
6.1%
0.0%
0.0%
0.0%
0.0%
4-2
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 4 - Data Quality
Table 4-1. Duplicate Sample Results and Calculated RPDs for Fathead Minnows
Treatment
Type
Gravity
Pump
Pump
Pump
Gravity
Treatment
Replicate
5
5
5
5
5
Sub-
Sample
Number
3
1
1
6
16
Original
Sample
Result
(Dead)
0
4
14
o
J
0
Duplicate
Sample
Result
(Dead)
0
4
13
3
0
RPD
NC
0.0%
7.4%
0.0%
NC
Original
Sample
Result
(Alive)
9
9
1
12
11
Duplicate
Sample
Result
(Alive)
9
9
2
12
11
RPD
0.0%
0.0%
66.7%
0.0%
0.0%
NC = Not calculated.
Table 4-2. Duplicate Sample Results and Calculated RPDs for Fathead Minnow Eggs
Treatment
Type
Control
Gravity
Gravity
Pump
Control
Gravity
Pump
Control
Pump
Gravity
Treatment
Replicate
1
1
1
1
2
2
2
2
3
3
Sub-
Sample
Number
1
1
3
4
3
3
3
12
1
3
Original
Sample
Result
(Dead)
0
0
0
1
0
0
2
0
0
0
Duplicate
Sample
Result
(Dead)
0
0
0
1
0
0
1
0
0
0
RPD
NC
NC
NC
0.0%
NC
NC
66.7%
NC
NC
NC
Original
Sample
Result
(Alive)
19
4
7
32
8
6
15
1
16
12
Duplicate
Sample
Result
(Alive)
19
4
7
32
8
6
15
1
16
12
RPD
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
NC = Not calculated.
4.1.3 Accuracy
Accuracy is a measure of the agreement between a measured value and a reference of
"true" value. In this study, the values in question are whether or not each organism type is alive
or dead. Live organisms will exhibit either movement (with or without a stimulus) for small fish,
or a heartbeat and movement within the egg for fish eggs. Possible accuracy errors are
movements created by water currents that make a dead organism appear alive and non-
responsive organisms that appear dead but are actually alive. To determine the accuracy of the
live or dead analysis, a second GLEC analyst observed every 10th subsample and independently
assessed mortality. This accuracy check was conducted by the second analyst immediately
following counting by the first analyst to guard against organism death due to prolonged
exposure to microscope lights. Accuracy is then measured by duplicate live/dead counts of the
same subsample. The target difference between the duplicate counts of the same subsample
should be less than 10 percent, resulting in 90 percent accuracy.
4-3
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 4 - Data Quality
Accuracy data are provided in Table 4-3 and Table 4-4 for fathead minnows and fathead
minnow eggs, respectively. For fathead minnows, only 1 of the 33 quality assurance subsamples
for live and dead analysis had accuracy less than 90 percent. For fathead minnow eggs, only one
of the 10 quality assurance subsamples had accuracy less than 90% and this was likely caused by
the very low number of organisms in the sample.
Table 4-3. Accuracy Data for Fathead Minnow Quality Assurance Subsamples
Treatment
Type
Control
Control
Control
Gravity
Gravity
Pump
Pump
Pump
Control
Control
Gravity
Pump
Pump
Pump
Control
Control
Gravity
Pump
Pump
Control
Gravity
Control
Pump
Pump
Gravity
Pump
Control
Control
Gravity
Pump
Pump
Pump
Gravity
Treatment
Replicate
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
5
5
Sub-
Sample
Number
2
3
4
1
6
2
2
2
2
4
1
1
o
J
2
1
1
1
1
1
1
2
4
1
1
12
3
3
o
J
o
J
1
1
6
16
Original
Sample
Result
(Dead)
1
0
0
0
0
11
8
12
0
0
0
13
13
7
0
0
0
2
4
0
0
0
9
10
0
2
0
0
0
4
14
o
J
0
Duplicate
Sample
Result
(Dead)
1
0
0
0
0
12
8
13
0
0
0
17
14
7
0
0
0
2
4
0
0
0
9
11
0
2
0
0
0
4
13
3
0
Dead
Organism
Accuracy
100%
100%
100%
100%
100%
92%
100%
92%
100%
100%
100%
76%
93%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
91%
100%
100%
100%
100%
100%
100%
108%
100%
100%
Original
Sample
Result
(Alive)
8
7
9
14
6
2
3
1
8
11
13
12
5
4
15
17
23
9
11
14
12
14
1
16
8
9
12
11
9
9
1
12
11
Duplicate
Sample
Result
(Alive)
8
7
9
14
6
2
3
1
8
11
13
15
4
4
15
17
22
9
11
14
11
14
1
17
8
9
12
11
9
9
2
12
11
Live
Organism
Accuracy
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
80%
80%
100%
100%
100%
96%
100%
100%
100%
109%
100%
100%
94%
100%
100%
100%
100%
100%
100%
50%
100%
100%
4-4
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 4 - Data Quality
Table 4-4. Accuracy Data for Fathead Minnow Eggs Quality Assurance Subsamples
Treatment
Type
Control
Gravity
Gravity
Pump
Control
Gravity
Pump
Control
Pump
Gravity
Treatment
Replicate
1
1
1
1
2
2
2
2
3
3
Sub-
Sample
Number
1
1
o
J
4
o
J
o
J
o
J
12
1
o
J
Original
Sample
Result
(Dead)
0
0
0
1
0
0
2
0
0
0
Duplicate
Sample
Result
(Dead)
0
0
0
1
0
0
1
0
0
0
Dead
Organism
Accuracy
100%
100%
100%
100%
100%
100%
50%
100%
100%
100%
Original
Sample
Result
(Alive)
19
4
7
32
8
6
15
1
16
12
Duplicate
Sample
Result
(Alive)
19
4
7
32
8
6
15
1
16
12
Live
Organism
Accuracy
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
4.2 FIELD QUALITY CONTROL
Field quality control was evaluated by first verifying that the organisms used in the study
were alive prior to testing and then evaluating the variability between replicate tests. The
following subsections describe each of these field quality controls.
4.2.1 Integrity of Stock Organisms
One indicator of field quality control was verification that the fathead minnows and
fathead minnow eggs were alive prior to testing. To verify the fathead minnows were alive prior
to each test replicate, the physical condition of the minnows was observed and any that were
floating or appeared dead were removed. To verify the eggs were alive prior to testing, GLEC
collected 10 subsamples of eggs from the stock and analyzed the eggs under the microscope. The
data, provided in Table 4-5, indicate that in 8 of the 10 subsamples, all the eggs were alive prior
to testing. Only two subsamples had any eggs that were dead. These results verify that the
fathead minnow eggs received from MED were alive prior to testing.
Table 4-5. Mortality Analysis Results for Fathead Minnow Eggs Prior to Testing
Sample
1
2
o
J
4
5
6
7
8
Live
5
7
7
7
14
18
8
13
Dead
0
0
4
0
0
1
0
0
Indeterminate
2
1
1
0
1
2
1
0
% Mortality
0.0%
0.0%
36.4%
0.0%
0.0%
5.3%
0.0%
0.0%
4-5
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 4 - Data Quality
Table 4-5. Mortality Analysis Results for Fathead Minnow Eggs Prior to Testing
Sample
9
10
Live
9
5
Dead
0
0
Indeterminate
1
2
% Mortality
0.0%
0.0%
4.2.2 Variability
Another field quality control measure is the variability among replicate tests for each
organism and tank type (pump, gravity drain, and control). For fathead minnows, the average
was estimated for each of the 5 replicates in the control tank; then an average of these 5 averages
was taken and the 95 percent confidence interval around this average was estimated. The same
procedure was conducted for the gravity drain and pump tanks. Similarly, for fathead minnow
eggs, an average was estimated for each of the 3 replicates and then an average of these 3
averages was taken and the 95 percent confidence interval calculated.
As indicated in Table 4-6, the lower bound of the 95 percent confidence interval on
percent mortality for the fathead minnow pump tank is 38.0% (44.8% minus 6.8%) and the upper
bound mortality rate is 55.6% (44.8% plus 6.8%). Four of the five replicates are within this
range, which indicates that the methodology is fairly replicable and consistent. The 95 percent
confidence interval on percent mortality for the fathead minnow control tank is 0% (0.3% minus
0.8%) through 1.1% (0.3% plus 0.8%). Replicate 1 has a mean of 1.7%, which is outside this
confidence interval. This implies there is some variability across replicates for the control tank.
The other four point estimates are all zero and thus fall within the confidence interval. The 95
percent confidence interval for the gravity tank is 0 to 0.7% (0.2% minus and plus 0.5%). Once
again the four zero values fall within this range but the average for the replicate where a single
fish died is outside this range. These two samples lying outside the confidence intervals do not
necessarily invalidate the results; when evaluating the incidence of rare events (i.e., low
probability events, such as a fish dying in the control tank) the traditional measures of deviation
often do not perform well. For instance, these intervals both include negative values which are
not feasible outcomes (there cannot be negative dead fish), and thus the lower bounds are
reported as zero. This results in a compressed confidence interval, which makes it less likely for
a value to lie within this range.
The three replicates for fathead minnow eggs in the pump tank average 1.4% mortality
with a confidence interval from 0 to 3.2% (1.4% minus and plus 1.8%). Two of the three sample
averages fall within this range. For the control tank the interval is 0 to 1.4% (0.5% minus and
plus 0.9%), and once again two of the three samples are within this interval. For the gravity tank
the interval is 0 to 3.7% (1.8% minus and plus 1.9%), and all three samples are within this
specified range.
4-6
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study
Section 4 - Data Quality
Table 4-6. Averages of the Mean Percent Mortality Rates and 95% Confidence Intervals
Replicate
Control Tank
Gravity Drain Tank
Pumped Tank
Fathead Minnows
1
2
3
4
5
Average
95% Confidence Interval
1.7%
0.0%
0.0%
0.0%
0.0%
0.3%
+ 0.8%
0.0%
0.0%
1.1%
0.0%
0.0%
0.2%
+ 0.5%
53.7%
43.2%
34.8%
46.9%
45.6%
44.8%
+ 6.8%
Fathead Minnow Egg
1
2
3
Average
95% Confidence Interval
1.6%
0.0%
0.0%
0.5%
+ 0.9%
3.7%
0.0%
1.8%
1.8%
+ 1.9%
0.8%
3.5%
0.0%
1.4%
+ 1.8%
4-7
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 5 - References
SECTION 5
REFERENCES
1. Briski, E., Wiley, C.J., & Bailey, S.A. (2012), Role of Domestic Shipping in the
Introduction or Secondary Spread of Non-indigenous Species: Biological Invasions
within the Laurentian Great Lakes. Journal of Applied Ecology, 49(5), 1124-1130.
2. Cangelosi, A., Schwerdt, T., Mangan, T., Mays, N., and Prihoda, K., A Ballast Discharge
Monitoring System for Great Lakes Relevant Ships: A Guidebook for Researchers, Ship
Owners, and Agency Officials, Table 1, November 2011.
3. Dahberg, M.D., A Review of Survival Rates of Fish Eggs and Larvae in Relationship to
Impact Assessments, Marine Fisheries Review, March 1979.
4. ERG, Sampling and Analysis Plan for the Vessel General Permitting Program Pump
Mortality Study, August 2014.
5. ERG, Quality Assurance Project Plan Addendum for Technical Support for the Vessel
General Permitting Program - Pump Mortality Study, August 2014.
6. Moore, P., Main ballast pump design data provided by Phil Moore at Interlake Steamship
Company to Mark Briggs at ERG via email on July 11, 2014.
7. Ruiz, G., Reid, D. Current state of understanding about the effectiveness of ballast water
exchange (BWE) in reducing aquatic nonindigenous species (ANS) introductions to the
Great Lakes Basin and Chesapeake Bay, USA: synthesis and analysis of existing
information. NOAA Technical Memorandum GLERL-142. Ann Arbor, MI. 2007.
8. Rup, M. P., Bailey, S. A., Wiley, C. I, Minton, M. S., Miller, A. W., Ruiz, G. M., &
Maclsaac, H. J. (2010). Domestic Ballast Operations on the Great Lakes: Potential
Importance of Lakers as a Vector for Introduction and Spread of Non-indigenous
Species. Canadian Journal of Fisheries and Aquatic Sciences, 67(2), 256-268.
9. USCG Acquisition Directorate, Asian Carp Survivability Experiments and Water
Transport Surveys in the Illinois River, Volume 1. Report No. CG-D-01-13, January
2013a.
10. USCG, Acquisition Directorate, Ballast Water Treatment, U.S. Great Lakes Bulk Carrier
Engineering and Cost Study, Volume 1, Report No. CG-D-12-13, November 2013b.
11. USEPA, Environmental Technology Verification (ETV) Program, Generic Protocol for
the Verification of Ballast Water Treatment Technology, September 2010.
12. USEPA, Vessel General Permit For Discharges Incidental To The Normal Operation Of
Vessels (VGP), December 2013.
5-1
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Section 5 - References
13. Wilcoxin, F., Individual Comparisons by Ranking Methods. Biometrics Bulletin, Vol. 1,
No. 6. (Dec., 1945), pp. 80-83.
5-2
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Appendix A
Appendix A:
SAMPLING AND ANALYSIS PLAN
-------
feERG
Sampling and Analysis Plan for the
Vessel General Permitting Program
Pump Mortality Study
Prepared for:
U.S. Environmental Protection Agency
Office of Wastewater Management
Office of Water
1200 Pennsylvania Avenue, NW
Washington, D.C. 20460
Prepared by:
Eastern Research Group, Inc.
14555 Avion Parkway
Suite 200
Chantilly, VA20151
August 29, 2014
EPA Contract No. EP-C-12-021
Work Assignment 1-53
-------
Revision No. 1
Date: August 29, 2014
DISCLAIMER
Neither the United States Government nor any of its employees, contractors,
subcontractors, or their employees make any warrant, expressed or implied, or assume any legal
liability or responsibility for any third party's use of, or the results of, such use of any
information, apparatus, product, or process discussed in this report, or represents that its use by
such party would not infringe on privately owned rights.
The primary contact regarding questions or comments on this document is:
Dr. Ryan Albert
U.S. Environmental Protection Agency
Office of Wastewater Management
Mail Code: 4203M
1200 Pennsylvania Ave., N.W.
Washington DC 20460
(202) 564-0763
albert.ryan@epa.gov
-------
Revision No. 1
Date: August 29, 2014
CONTENTS
Page
1. INTRODUCTION 1-1
1.1 Background 1-1
1.2 Objectives and General Approach 1-1
1.3 Bench-Scale Study Location 1-2
2. MORTALITY TESTING 2-1
2.1 Bench-Scale Testing Apparatus 2-1
2.2 Fish Egg Mortality Testing Procedure 2-5
2.3 Fish Mortality Testing Procedure 2-5
3. SAMPLE COLLECTION AND ANALYSIS METHODS 3-1
3.1 Organism Sampling Procedures 3-1
3.1.1 Fish Egg Sampling Procedure 3-1
3.1.2 Fish Sampling Procedure 3-1
3.2 Organism Analysis Procedures 3-1
3.2.1 Fish Egg Mortality Assessment 3-2
3.2.2 Fish Mortality Assessment 3-2
3.3 Sample Labeling 3-3
3.4 Chain of Custody 3-3
4. QUALITY ASSURANCE FOR FIELD SAMPLE ANALYSIS 4-1
4.1 Documentation of Sample Custody 4-1
4.2 Field Replicates 4-1
4.3 Laboratory Duplicates 4-1
5. TESTING ACTIVITIES 5-1
5.1 Study Team Organization 5-1
5.2 Pre-Visit Preparation 5-1
5.3 Field Testing Schedule 5-1
5.4 Logistics 5-3
5.4.1 Field Study Team Contacts 5-4
5.4.2 Site Contact 5-4
5.4.3 EPA Contacts 5-4
5.4.4 ERG Contact 5-5
6. DATA ANALYSIS 6-1
7. RELATED BIBLIOGRAPHY 7-1
Appendix A: Standard Operating Procedures for Laboratory-Based Viability Analysis of Fish
Embryos and Larvae for Toxicity Testing and Other Special Environmental
Studies and Purposes
-------
Revision No. 1
Date: August 29, 2014
LIST OF TABLES
Page
3-1 Summary Number of Samples, Sample Bottles, Preservation, and Holding Time
Requirements 3-2
5-1 Fish (Minnows) Daily Testing Schedule 5-2
5-2 Fish Egg Daily Testing Schedule 5-3
LIST OF FIGURES
Page
2-1 Pump System Testing Apparatus 2-1
2-2 Gravity Drain System Testing Apparatus 2-2
2-3 Control System Testing Apparatus 2-2
2-4 Layout of Testing System 2-3
6-1 GLEC Field Laboratory Mortality Data Sheet - Fish Eggs 6-2
6-2 GLEC Field Laboratory Mortality Data Sheet - Minnows 6-3
in
-------
Revision No. 1
Date: August 29, 2014
1. INTRODUCTION
The U.S. Environmental Protection Agency (EPA) is conducting a bench-scale study to
determine fish egg and fish mortality caused by ballast water pumps on Great Lakes bulk carriers
(Lakers)1. This study plan provides the approach that will be used to simulate ballast water
discharges from Lakers, the methodology for preparing the challenge water for testing, and the
procedures for collecting and analyzing living organisms for the test. The bench-scale study will
be performed by EPA and EPA's technical contractor Eastern Research Group, Inc. (ERG) and
ERG's subcontractor, Great Lakes Environmental Center, Inc. (GLEC). This document, in
combination with the Quality Assurance Project Plan (QAPP), is intended to serve as a guide for
study personnel, as well as a study review mechanism for EPA personnel.
1.1 Background
Due to a 2006 court order, EPA began permitting incidental vessel discharges from many
vessels on February 6, 2009. The 2013 Vessel General Permit (VGP) (USEPA, 2013) regulates
discharges incidental to the normal operation of vessels operating in a capacity as a means of
transportation. The VGP includes general effluent limits applicable to all discharges; general
effluent limits applicable to 27 specific discharge streams; narrative water-quality based effluent
limits; inspection, monitoring, recordkeeping, and reporting requirements; and additional
requirements applicable to certain vessel types.
Literature has shown that during ballast water intake, a diverse community of live
organisms present in both the water column and the lake sediments is entrained into the ballast
tanks (Ruiz et al, 2007). When Lakers ballast in a Great Lakes port which has been colonized by
an aquatic nuisance species (ANS), and then discharge their ballast in another Great Lakes port,
they have the potential to spread ANS within the Great Lakes. In Part 2.2.3.3 of the 2013 VGP,
EPA included several best management practices (BMPs) for ballast water management for
Lakers to reduce the likelihood of those vessels dispersing and spreading aquatic invasive
species. One of those BMPs is for vessels to use their ballast pumps to empty their ballast tanks,
rather than gravity draining, to produce both sheer and cavitational stresses on these organisms,
theoretically resulting in higher mortality. Although pumping ballast water rather than gravity
draining should result in additional organism mortality, only one study has been conducted
(USCG, 2013) to support the BMP, and this study examined only larval fish and did not
investigate other organisms such as zooplankton or fish eggs that can also be drawn into ballast
tanks. As such, EPA is actively gathering data on the mortality caused by pumps on other types
of organisms.
1.2 Objectives and General Approach
The objective of this bench-scale study is to determine if emptying ballast tanks by
pumping creates greater mortality for fish eggs and fish (minnows) than emptying ballast tanks
"Laker" is the common name for the large and uniquely designed and constructed dry bulk vessels (or carriers)
used to transport bulk material commodities throughout the Great Lakes system. U. S. flag Lakers usually only
transport goods on the four upper Great Lakes and connecting channels, as most are limited by their size from
transiting the Welland Canal. The primary commodities transported by the Lakers include iron ore pellets, coal,
grain, limestone, cement, sand, and salt.
-------
Revision No. 1
Date: August 29, 2014
by gravity draining. Based on the results of the bench-scale testing, additional pilot or full scale
testing may be conducted under future work assignments.
The general approach of this bench-scale study will include:
• Collect fish eggs and small minnows from a laboratory-raised culture;
• Place the organisms into two process feed tanks and a control tank;
• Gravity drain one feed tank into a collection net and count the number of live and
dead organisms following gravity draining;
• Pump the second feed tank into a collection net and count the number of live and
dead organisms following pumping. The pumping rate will be adjusted to
simulate the ballast pumping rate on a Laker;
• Determine the test handling mortality by analyzing live and dead organisms in the
control tank;
• Using stastical analysis, determine the differences in mortality between the
control, gravity draining and pumping for each organism type.
1.3 Bench-Scale Study Location
ERG and GLEC will conduct the bench-scale study at the Lake Superior National
Estuarine Research Reserve (LSNERR) located in Superior, Wisconsin in early September,
2014. This facility was selected for the study due to its location on the shore of Lake Superior's
St. Louis River estuary (a major shipping port) and the availability of Lake Superior estuary
water for maintaining the organisms before and after testing. In addition, the facility can provide
laboratory and dock space needed for both the test tanks.
1-2
-------
Revision No. 1
Date: August 29, 2014
2.
MORTALITY TESTING
This section provides the detailed procedure that will be used to conduct the bench-scale
organism mortality tests at LSNERR. The bench-scale testing procedure will be divided into
three phases that include: (1) constructing the test apparatus including the tanks, piping, pumps,
and post treatment organism collection nets; (2) obtaining fish eggs (fathead minnow eggs) and
conducting the gravity drain, pump and control testing and live/dead sample analysis; and (3)
obtaining small fish (fathead minnows) and conducting the gravity drain, pump and control
testing and live/dead sample analysis. Testing for each organism group (fish eggs and minnows)
will be conducted separately. All tests will have multiple replicates. The following subsections
describe each of these three phases in greater detail.
2.1 Bench-Scale Testing Apparatus
Figure 2-1, Figure 2-2, and Figure 2-3 are diagrams depicting the pump, gravity drain,
and control system testing apparatus, respectively. Figure 2-4 is a site layout showing the
arrangement of the test tanks and the sample receiving areas.
Live organisms
55-gallontesttank
2" transfer hose
Pump
4" transfer hose
Net Support
Structure
4'
30" diameter plankton net
X
Lake Bed
Figure 2-1. Pump System Testing Apparatus
2-1
-------
Revision No. 1
Date: August 29, 2014
Live organisms
55-gallor test tank
2" transfer hose
Dock
4" transfer hose
Net Support
Structure
4'
30" diameter plankton net
X
Lake Bed
Figure 2-2. Gravity Drain System Testing Apparatus
Live organisms
Dock
55-galion test tank in estuarv
30" diameter plankton net inside test tank
X
Lake Bed
Figure 2-3. Control System Testing Apparatus
2-2
-------
Revision No. 1
Date: August 29, 2014
Dock
Pump Test
Gravity Test
/ Control Test Tank i
in
Plankton Net Inside Control
Plankton
Plankton
Figure 2-4. Layout of Testing System
The bench-scale testing system will consist of three 55-gallon open top plastic test tanks
placed on wood stands. Each test tank has a diameter of 30 inches and a height of 35 inches. The
pump and gravity drain tanks will be fitted with a 2" diameter valve beneath the tank for draining
or pumping. The third tank, which will be used as a control to measure mortality caused by
handling of the organisms will not be drained and therefore will not require a valve. The pump
and gravity drain tanks will be filled with water directly from the St. Louis River estuary using a
pump. The water will be filtered through 35 micron mesh before it enters each tank. Each
organism group (fish eggs or minnows) will be placed in the water within the test tanks
immediately prior to testing. The pump and gravity drain test tanks will be directed to the
plankton nets placed in approximately 4' of water in the estuary. The plankton nets have a 35
micron screen size2, are 30" diameter and will have a 1-liter cod-end cup on the bottom to
capture the organisms rinsed from the nets.
The pump test tank will discharge below the water line into the plankton net to simulate a
ballast discharge through the sea chest. The gravity drain test tank will also discharge into the
plankton net below the water line to simulate gravity draining by Lakers.3 ERG decided to place
the plankton nets in the estuary rather than another receiving tank to buffer the force of the water
and minimize the potential mortality that could be caused by the organisms contacting plankton
2 U.S. Environmental Protection Agency Environmental Technology Verification (ETV) Program. Generic Protocol for the
Verification of Ballast Water Treatment Technology. Section 5.4.6.4 requires the use of a 35 micron screen size for capture of
zooplankton.
3 According to Mr. Mr. Jason Toast with The Interlake Steamship Company and Mr. Jim Weakly at the Lake
Carriers Association, American Lakers discharge ballast water through either their low or high sea chests below the
water line.
2-3
-------
Revision No. 1
Date: August 29, 2014
nets during discharge. In addition, to further reduce mortality caused by organisms being forced
into the plankton nets by water pressure, the pipe diameter on the pump discharge will be
increased from 2" to 4". This will reduce the pressure and force with which test water will be
discharged into the plankton net to help counteract the force imposed by the pump. The gravity
drain tank will also include a pipe size increase from 2" to 4" near the middle of the pipe run in
to ensure consistency between the pump and gravity test systems. To ensure against
"contamination" of the test net with organisms in the water (and escape of live organisms inside
the test nets), net supports will be constructed to hold nets 12 inches above the water level. Once
the pump and gravity drain test tanks are empty, a 1-hour recovery period will occur4, after
which the plankton nets will be gently lifted out of the receiving areas and the organisms will be
collected for analysis by GLEC.
To evaluate organism mortality caused by handling, the control tank (3rd tank) will be
submerged in the estuary adjacent to the pump and gravity drain collection nets. The control tank
will be lined with a 35 micron plankton net. The control tank will be submerged rather than
drained to reduce possible organism mortality that could be caused by the change in elevation
from the dock to the sample receiving area in the estuary. Water in the control tank will also be
filtered through 35 micron mesh, the same as water in the gravity and pump test tanks. Test
organisms will be introduced into the control tank and its plankton net at the same time that test
organisms are introduced into the test tanks on the dock. This net will also be supported 12
inches above the water level to guard against organism escape or entrainment of additional
organisms from estuary water. The control tank plankton net will be raised at the same time as
the nets from the gravity drain and pump test nets and the mortality of the organisms collected in
the control tank net will be evaluated.
To simulate ballast pump conditions from a Laker, ERG will use a Honda trash pump
with 2" diameter hose connected to the valve on the bottom of the pump test tank. Based on data
provided in the literature, ballast flow through the piping of a Laker is 10 ft/second; therefore,
flow from the pump should range between 1,380 and 2,450 gal/hour with an average of 1,920
gal/hr (32 gal/min).5 Because the power of the water pressure coming out of a 2" diameter hose
from the pump would be high enough to cause mortality in test organisms by forcing them at
high velocity into the plankton net, the diameter of the discharge hose will be increased to 4",
thus reducing the pressure by a factor of 2 and reducing undesirable mortality caused by the
sampling procedure rather than the pump.
2.2 Fish Egg Mortality Testing Procedure
If live fathead minnow eggs are available from EPA's Mid-Continent Ecological
Division (MED) laboratory in Duluth for testing in early September, mortality testing with this
organism will be conducted. Fathead minnows were selected for testing because they are either
native to Lake Superior and the St. Louis River. Eggs will be acclimated to estuary water by
bringing them to test water temperature at a rate of no more than 5°C per hour. Hatchery water
4 Great Ships Initiative Standard Operating Procedure GSI/SOP/LB/RA/SA/2 Procedure for Zooplankton Sample
Analysis, July 2009.
5 Cangelosi, A., Schwerdt, T., Mangan, T., Mays, N., and Prihoda, K., A Ballast Discharge Monitoring System for
Great Lakes Relevant Ships: A Guidebook for Researchers, Ship Owners, and Agency Officials, Table 1, November
2011.
-------
Revision No. 1
Date: August 29, 2014
hardness will also be obtained and compared to estuary water to ascertain whether test water will
need to have its hardness increased with well water or mineral water to ensure the osmotic
integrity of the eggs.
Each of the three test tanks will be filled with filtered (35 micron) St. Louis River estuary
water and dosed with enough eggs to provide a concentration of approximately three eggs per
gallon. For three 55-gallon test tanks and four replicates, approximately 2,000 fathead minnow
eggs will be needed to complete the study. Four replicates rather than 5 will be conducted for
fish eggs due to their limited availability from MED. For each test, the eggs will be introduced
into the three tanks (gravity drain, pump and control test tanks) at the same time. Because eggs
will already be at test-water temperature, no acclimation period is necessary or desirable because
the test tanks on the dock will heat up in the September sun, increasing stress on the eggs. Once
the eggs pass from the gravity drain and pump test tanks into the plankton nets suspended in the
sample receiving areas established in the estuary, the one hour recovery period will being.
Following this, the plankton nets will be raised to capture the eggs in the cod-end collection
container for analysis of mortality. Mortality assessment will be conducted under dissecting
microscopes and will be determined by the presence of a heartbeat or larval movement in each
egg. Egg condition will be noted as appropriate.
2.3 Fish Mortality Testing Procedure
Small fathead minnows or other similar bait fish will be obtained from a local bait
supplier in Wisconsin and used to test mortality of small fish passing through a pump. Fathead
minnows or other bait fish were selected for testing because they are either native to Lake
Superior and the St. Louis River, or their populations have already been established. These
minnows also tend to be relatively hardy, helping to ensure that there will be little handling and
control mortality. Minnows will be acclimated to estuary water by bringing them to test water
temperature at a rate of no more than 5°C per hour. Culture water hardness will also be obtained
and compared to estuary water to ascertain whether test water will need to have its hardness
increased with well water or mineral water to ensure no undue osmotic stress on the minnows.
Each 55-gallon test tank will be filled with St. Louis River estuary water and dosed with
approximately 100 minnows. For three test tanks and five replicate tests, approximately 1,500
minnows will be required for the study. Minnows will be introduced into the three test tanks at
the same time and allowed five minutes to acclimate (a long acclimatization period is not
desirable due to the difficulty of keeping test tank water from heating up; minnows will already
acclimated to test water temperature by the time the test starts). Test tanks will then be pumped
or drained into the plankton nets established in the sample receiving areas in the estuary. Once
minnows are pumped or gravity drained from the test tanks into the sample receiving area
plankton nets, the one hour recovery period will begin. Following this, field personnel will raise
the nets and collect the organisms to evaluate mortality. Mortality assessment will be conducted
by GLEC personnel either under the microscope or with the naked eye by assessing body or gill
movement depending on fish size. Notes will be made on fish body condition as appropriate.
2-5
-------
Revision No. 1
Date: August 29, 2014
3. SAMPLE COLLECTION AND ANALYSIS METHODS
This section describes the sampling procedures and analytical methods that will be used
by the study team to determine organism mortality in the control, gravity drain, and pump test
tanks.
3.1 Organism Sampling Procedures
The following sampling procedures will be used to raise the plankton nets from the
sample receiving areas in the estuary and collect the organisms for mortality analysis.
3.1.1 Fish Egg Sampling Procedure
Plankton nets containing fish eggs will be individually raised using their tow harnesses
by personnel standing on the dock. Because all water from the tests will be released into or
contained within the nets, all eggs used in each test will be contained within the net for each test
(control, pump, or gravity drain). As each net is raised, personnel in the water will gently wash
down the sides of the net to wash eggs down into the collection container. Once all eggs have
been washed into the collection container of each net, the containers will be removed and carried
to the lab area for microscopic analysis. In the lab area, the containers will be placed into a
cooler with water, which will be maintained at estuary water temperature using ice.
3.1.2 Fish Sampling Procedure
Plankton nets containing minnows will be individually raised using their tow harnesses
by personnel standing on the dock. Because all water from the tests and control will be released
into or contained within the nets, all fish used in each test will be contained with the net for each
test (control, pump, or gravity drain). As each net is raised, personnel in the water will wash
down the sides of the net as needed to wash minnows down into the collection container
(depending on fish size, several containers may be used to avoid overcrowding the fish). Once all
fish have been washed into collection containers from each net, the containers will be carried to
the lab area for microscopic analysis. In the lab area, containers from each test will be decanted
into 10-gallon aquaria (one aquarium for each test: control, pump, and gravity drain). Aquaria
will be maintained at estuary water temperature using ice; dissolved oxygen levels will be
maintained using aerators.
3.2 Organism Analysis Procedures
Samples collected in the 1-liter containers from the cod end of the plankton nets will be
immediately analyzed in the LSNERR on-site laboratory by GLEC laboratory staff. Table 3-1
shows the total number of samples that will be collected and analyzed for each organism type.
Standard operating procedures (SOPs) that will be used by field laboratory personnel for
organism mortality assessments are provided in Appendix A. Samples will be hand-carried from
the sample receiving areas directly to the laboratory. Containers will be capped with plankton
netting and placed in coolers containing estuary water that is aerated with bubblers and whose
temperature is regulated using ice to maintain estuary water temperatures. This will minimize
mortality due to crowded holding conditions before mortality assessment can be completed.
3-1
-------
Revision No. 1
Date: August 29, 2014
Table 3-1. Summary Number of Samples, Sample Bottles,
Preservation, and Holding Time Requirements
Parameter
Fish Eggs
Fish
(minnows)
Estimated Number
of Samples
12
15
Sample Bottle and Volume
1 -liter container from cod end of
plankton net.
1 -liter container from cod end of
plankton net.
Preservation
Maintain temperature
and DO levels.
Maintain temperature
and DO levels.
Holding
Time
Immediate
Immediate
The following subsections provide additional details on how samples will be analyzed in
the on-site laboratory for live/dead fish eggs and minnows.
3.2.1 Fish Egg Mortality Assessment
Eggs will be removed from each container for microscopic examination by gently
swirling each container to re-suspend any eggs that have settled to the bottom and then using a
large pipette to place an approximately 20 milliliter subsamples into a counting chamber. This
process will be repeated 30 - 50 times (600 - 1000 mL from a 1000 mL container) until 60
minutes have elapsed while the nets and tanks are cleaned and re-set for the next replicate test
run. With four personnel concurrently examining eggs under microscopes, about eighty percent
of the eggs from each net (130 eggs) should be able to be assessed for mortality between test
replicates.
Egg mortality will be assessed under dissecting microscopes by either viewing a
heartbeat or by seeing larval movement inside each egg, depending on the age of the egg. If the
egg is so undeveloped that it does not have a visible heart, it will be recorded as "indeterminate".
If the egg contains a visible larva that is not moving, the egg will be gently prodded with very
fine forceps in an attempt to elicit a response. Egg larvae that do not respond to prodding will be
recorded as dead. Thus, data collection will consists of counts of live, dead, and "indeterminate"
eggs. Percent mortality calculations will use only live and dead counts. All eggs will be
preserved separately from each net so that a total count of eggs can be generated for each
replicate.
3.2.2 Fish Mortality Assessment
Minnows will be held in 10-gallon, temperature-monitored, aerated aquaria (one for each
test type: pump, gravity drain, control) to insure against post-test mortality. Minnows will be
gently removed from each aquarium using a fine-mesh aquarium net and placed in an
appropriately-sized dish for examination (whether examination is by the naked eye, a magnifying
glass, or a microscope will depend on minnow size). With three personnel examining minnows,
GLEC personnel should be able to examine nearly all the minnows used in each test.
Mortality assessment will be based on movement or, if none, gill movement. Intact but
non-moving minnows will be gently prodded. Notes will be made on body condition of the
minnows as appropriate (e.g., if individuals are noticeably abraded or only pieces of minnows
are found). If minnows have been chopped apart by the pump, only heads will be counted for the
mortality count. Data collection will consist of counts of live and dead minnows. A separate
3-2
-------
Revision No. 1
Date: August 29, 2014
category of "injured" minnows may also be counted, but this will be a completely separate count,
so that injured/damaged minnows are also counted as either alive or dead. Percent mortality
calculations will use only live and dead counts; counts of injured/damaged minnows will be
reported separately (but not used in the percent mortality calculations) to help assess cause of
death by pump propeller. Percent mortality will be calculated as a percentage of dead individuals
out of the total count in each test replicate.
3.3 Sample Labeling
Each sample container will be coded with a unique sample number and labeled at the
time of collection. Samples will be labeled with the replicate number (1, 2, or 3) and if the
sample is from the pump, gravity drain or control test tank discharge. For example, a sample for
fish eggs collected from the pump discharge during the second replicate test would be labeled
"Egg Pump Rep 2, sample 1 of 1" (if only one container per replicate).
3.4 Chain of Custody
Due to the extremely short holding times for the organism samples, individual Chain of
Custody reports (CCRs) will not be prepared prior to each sample being delivered to the
laboratory. Instead, the person delivering the sample to the laboratory will log the sample
information on a CCRs being maintained in the laboratory. A CCR will be developed for each
replicate test for each organism group. The CCR will remain in the laboratory and the individual
delivering the sample will be responsible for logging the sample information including the
sample number, analysis to be performed (e.g., live/dead fish eggs), time and date of sample
collection, and the initials of the person delivering the sample to the laboratory.
3-3
-------
Revision No. 1
Date: August 29, 2014
4. QUALITY ASSURANCE FOR FIELD SAMPLE ANALYSIS
Quality assurance/quality control (QA/QC) procedures applicable to this study are
outlined in the QAPP (ERG, 2014). The QA/QC program includes the components discussed in
the following subsections.
4.1 Documentation of Sample Custody
All samples will be delivered to the on-site laboratory by a member of the study team.
While samples are being collected, samples and sampling equipment will be maintained in the
physical possession or view of at least one member of the sampling crew. To maintain a record
of sample custody, the sampling crew will complete a CCR form for each study replicate. These
CCR forms will be used to document sample custody transfer from the field to the on-site
laboratory.
4.2 Field Replicates
A total of 5 mortality tests (pump, gravity drain, and control) for fish eggs and fish
(fathead minnows) will be conducted. The average percent mortality in the control for each
replicate will be compared to the average percent mortality of the pumped and gravity drain tests
to determine if the gravity and pumped tests have greater mortality than the control. Variability
between the control replicates for each organism will be measured as a standard deviation that
will be used to determine an upper bound control mortality. Average mortality in the pumped
and gravity tests, along with variability measured as a standard deviation between replicates, will
be used to determine a lower bound control mortality. The average and upper bound control
mortality will be compared to the average and lower bound control mortality of the pump and
gravity tests to determine if the pump and gravity tests caused more mortality than the control.
4.3 Laboratory Duplicates
GLEC anticipates analyzing half or more of all organisms in each net for each test
replicate. For eggs, this will entail counting 30 - 50 twenty milliliter subsamples in the counting
chamber for analysis of live/dead organisms. Live/dead analysis will be made by counting
organisms under the dissecting microscope (or magnifying glass or naked eye for fathead
minnows). During analysis, ten percent (3 to 5) of the subsamples will be re-counted by a second
observer. The second live/dead count designated for duplicate analysis will be conducted
immediately following the initial count. Results from both counts will be recorded and the values
used to calculate a relative percent difference.
4-1
-------
Revision No. 1
Date: August 29, 2014
5. TESTING ACTIVITIES
This section of the plan summarizes the ERG and GLEC sampling team organization,
pre-visit preparation, field sampling activities, and logistics including site contacts and site
location.
5.1 Study Team Organization
Mr. Mark Briggs will serve as ERG's on-site project manager. He will be assisted by Ms.
Kathleen Wu, also with ERG. ERG will be responsible for procuring test tanks and plankton
nets, renting the pumps and associated transfer hoses, and procuring the supplies (extra cod-end
containers for the plankton nets, lumber to build support structures, fish tanks with aerators, etc.)
to establish the collection areas in the St. Louis River Estuary. ERG is also responsible for
procuring fathead minnow eggs and small minnows for testing, and for procuring ice to maintain
test tank temperatures. During the field study, ERG will be responsible for calibrating the pump
speed and/or flow rate, filling the test tanks with estuary water prior testing, adding organisms to
the test tanks, and pumping or gravity draining the test tanks to the plankton nets.
Mr. Chris Turner will serve as GLEC's Principal Investigator (PI). He will be assisted by
two additional GLEC field and microscopy lab technicians who will work primarily on mortality
assessment. Mr. Turner will oversee and assist several aspects of the mortality assessment and
mortality detection limit assessments. GLEC will provide microscopes, forceps, sub-sampling
gear, and counters.
5.2 Pre-Visit Preparation
Prior to conducting the field study, the ERG crew chief and the GLEC PI will distribute
this Sampling and Analysis Plan (SAP), the QAPP, and the Health and Safety Plan (HASP) to
each team member and ensure they are completely familiar with the sampling, quality, and health
and safety requirements. The ERG crew chief will also provide LSNERR site personnel copies
of the SAP and any site-specific supplemental information prior to the start of sampling.
The crew chief will also coordinate the procurement and shipment of all necessary
sampling and health and safety equipment.
5.3 Field Testing Schedule
The field study is tentatively scheduled for early September 2014. Prior to equipment
setup and testing, the crew chief will notify ERG's Health and Safety Coordinator of any revised
activities along with recommended revisions to the proposed health and safety procedures.
Together, they will review the proposed health and safety procedures, incorporate any site-
specific changes indicated by the Health and Safely Coordinator, and obtain approval for
sampling from the Health and Safely Coordinator before proceeding with testing activities.
Table 5-1 and Table 5-2 show potential timelines for the fish (minnows) and fish eggs
test days. Equipment will be setup and tested the day prior to beginning the study (Monday).
Setup will include establishing the laboratory area where mortality observations will be made,
placing the test tanks and pump on the dock, constructing the plankton net support structures,
-------
Revision No. 1
Date: August 29, 2014
plumbing the system, and testing the system using ambient water. At the end of the setup day,
equipment will be cleaned and readied for minnow testing which will begin the next day.
Fish (minnow) testing is expected to be conducted the first day of the study (Tuesday),
followed by fish egg testing on the second day (Wednesday). At the end of testing, the tanks,
pumps, piping, plankton nets, and laboratory equipment will be removed from LSNERR and
returned.
Table 5-1. Fish (Minnows) Daily Testing Schedule
Time
0730
0800
0830
0835
0935
1030
1035
1135
1300
1305
1405
1500
1505
1605
1700
1705
1805
Fish Testing (Day 1)
ERG arrives at LSNERR and readies tanks and establishes receiving nets in the water.
ERG arrives at LSNERR with fathead minnows from the Wisconsin bait supplier and places minnows
in aerated, temperature-monitored storage tank. Minnows are acclimated to test temperature water at a
rate of no more than 5°C per hour. GLEC arrives and collects a water sample to verify water hardness,
conductivity, temperature and dissolved oxygen levels are appropriate for the minnows.
ERG fills tanks with filtered estuary water, adds approximately 100 fathead minnows to each test tank,
and allows to acclimate for 5 minutes.
Replicate 1 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 1 sample
containers delivered to on-site laboratory for mortality assessment.
Plankton nets cleaned and reestablished for Replicate 2. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water and 100 fathead minnows added to each test tank and
allowed to acclimate for 5 minutes.
Replicate 2 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 2 sample
containers delivered to on-site laboratory for mortality assessment.
Plankton nets cleaned and reestablished for Replicate 3. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water and 100 fathead minnows added to each test tank and
allowed to acclimate for 5 minutes.
Replicate 3 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 3 sample
containers delivered to on-site laboratory for mortality assessment.
Plankton nets cleaned and reestablished for Replicate 4. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water and 100 fathead minnows added to each test tank and
allowed to acclimate for 5 minutes.
Replicate 4 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 4 sample
containers delivered to on-site laboratory for mortality assessment.
Plankton nets cleaned and reestablished for Replicate 5. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water and 100 fathead minnows added to each test tank and
allowed to acclimate for 5 minutes,
Replicate 5 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 5 sample
containers delivered to on-site laboratory for mortality assessment.
5-2
-------
Revision No. 1
Date: August 29, 2014
Table 5-1. Fish (Minnows) Daily Testing Schedule
Time
Fish Testing (Day 1)
1930
Fish testing complete and plankton nets cleaned and reestablished for Day 2 testing with fish eggs.
Table 5-2. Fish Egg Daily Testing Schedule
Time
0800
0830
0930
0935
1035
1130
1135
1235
1330
1335
1435
1530
1535
1635
1800
Fish Egg Testing (Day 2)
ERG arrives at LSNERR and readies tanks and establishes receiving nets in the water.
GLEC crew arrives at LSNERR. ERG arrives at LSNERR with fathead minnow eggs from EPA-MED
and places eggs in aerated, temperature-monitored storage tank. Eggs are acclimated to test
temperature water at a rate of no more than 5°C per hour. GLEC collects a water sample for analysis of
ambient water parameters (hardness, conductivity, temperature and DO).
ERG fills tanks with estuary water, adds approximately 165 fathead minnow eggs to each test tank,
and allows to acclimate for 5 minutes.
Replicate 1 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 1 sample
containers delivered to on-site laboratory for mortality assessment.
Plankton nets cleaned and reestablished for Replicate 2. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water. ERG places approximately 165 fathead minnow eggs
in test tanks for acclimation for Replicate 2.
Replicate 2 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 2 sample
containers delivered to on-site laboratory for fish egg mortality assessment.
Plankton nets cleaned and reestablished for Replicate 3. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water. ERG places approximately 165 fathead minnow eggs
in test tanks for acclimation for Replicate 3
Replicate 3 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 3 sample
containers delivered to on-site laboratory for mortality assessment.
Plankton nets cleaned and reestablished for Replicate 4. Pump, gravity drain, and control test tanks
cleaned and refilled with filtered estuary water. ERG places approximately 165 fathead minnow eggs
in test tanks for acclimation for Replicate 3.
Replicate 4 - pump, gravity drain and control test tanks discharged into plankton nets and 1 hr
recovery period begins.
Plankton nets lifted for the pump, gravity drain and control test tank discharges and Replicate 4 sample
containers delivered to on-site laboratory for fish egg mortality assessment.
Plankton nets cleaned and stowed. Fish egg testing complete.
5.4 Logistics
This subsection summarizes the field study team personnel, site contacts, EPA contacts
and address, and ERG project management contact and address.
5-3
-------
Revision No. 1
Date: August 29, 2014
5.4.1 Field Study Team Contacts
Mark Briggs (Crew Chief)
ERG
3400 Jack Morris Drive
West Branch, MI 48661
Office: (989)345-7595
Cell: (989)701-5850
mark.briggs@erg.com
Kathleen Wu
ERG
14555 Avion Parkway, Suite 200
Chantilly, VA20151
Office: (703)633-1625
Cell: (703)581-7390
kathleen.wu@erg.com
Mr. Chris Turner (PI)
Great Lakes Environmental Center, Inc.
739 Hastings Street
Traverse City, MI 49686
Office: (231)941-2230
cturner@glec.com
5.4.2 Site Contact
Shon Schooler
Lake Superior National Estuarine Research Reserve
14 Marine Drive
Superior, WI 54880
Office: (715)392-3141
sschoole@uwsuper.edu
5.4.3 EPA Contacts
Dr. Ryan Albert
Mail Code: 4203M
1200 Pennsylvania Ave., NW
Washington DC 20460
Office: (202)564-0763
albert.ryan@epa.gov
Kathryn Kelley
Mail Code: 4203M
1200 Pennsylvania Ave., NW
Washington DC 20460
-------
Revision No. 1
Date: August 29, 2014
Office: (202)564-7004
kellev.kathryn@epa.gov
5.4.4 ERG Contact
Debra Falatko (Work Assignment Manager)
ERG
14555 Avion Parkway, Suite 200
Chantilly, VA20151
(703)633-1607
debra.falatko@erg.com
5-5
-------
Revision No. 1
Date: August 29, 2014
6. DATA ANALYSIS
Mortality analysis data will be recorded on the datasheets for each of the organism types.
Data sheets for fish eggs and minnows are provided in Figures 6-1 and 6-2. Following
completion of testing and data quality analysis, the qualified data will be transferred from the
datasheets to Excel spreadsheets. A separate Excel workbook containing mortality data will be
established for fish eggs and minnows. Within each workbook, tabs will be created for the
control, gravity drain, and pump mortality data. Data will include the number of live and dead
organisms counted in each plankton net container collected from the individual plankton nets
retrieved. The spreadsheet will calculate an average count of live and dead organisms from each
plankton net retrieved for each test replicate. Duplicate QC count results and relative percent
differences will also be calculated and reported. ERG and GLEC will analyze the data by
comparing organism mortality in the control to organism mortality caused by gravity draining
and pumping for each organism type. Ultimately, percent mortality will be statistically tested for
significant differences among pump, gravity drain, and control data for each organism type.
6-1
-------
Revision No. 1
Date: August 29, 2014
Counter
Replicate
Date
Test Type: Control Pump Gravity
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Figure 6-1. GLEC Field Laboratory Mortality Data Sheet - Fish Eggs
6-2
-------
Revision No. 1
Date: August 29, 2014
Counter
Replicate
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Date
Test Type: Control Pump Gravity
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Replicate
Dead
Alive
Subl
Sub 2
Sub 3
Sub 4
Sub 5
Sub 6
Sub?
Sub 8
Sub 9
Sub 10
QA Check
Total
Damage Notes:
Figure 6-2. GLEC Field Laboratory Mortality Data Sheet - Minnows
6-3
-------
Revision No. 1
Date: August 29, 2014
7. RELATED BIBLIOGRAPHY
1. Bailey, S., Duggan, I, VanOverdijk, Johengen, T., Reid, D., and Maclsaac, H., Salinity
Tolerance of Diapausing Eggs of Freshwater Zooplankton. Freshwater Biology, 49, 286-
295, 2004.
2. Cangelosi, A., Schwerdt, T., Mangan, T., Mays, N., and Prihoda, K., A Ballast Discharge
Monitoring System for Great Lakes Relevant Ships: A Guidebook for Researchers, Ship
Owners, and Agency Officials, November 2011.
3. Ruiz, G., and Reid, D., Current State of Understanding About the Effectiveness of Ballast
Water Exchange in Reducing Aquatic Nonindigenous Species Introductions to the Great
Lakes Basin and Chesapeake Bay. Synthesis and Analysis of Existing Information,
NOAA Technical Memorandum GLERL-142, September 2007.
4. U.S. Coast Guard Acquisition Directorate Research and Development Center. Asian Carp
Survivability Experiments and Water Transport Surveys in the Illinois River, Volume 1,
Report CG-D-01-13, January 2013.
5. U.S. Coast Guard, Ballast Water Treatment, U.S. Great Lakes Bulk Carrier Engineering
and Cost Study. Volume II Analysis of On-Board Treatment Methods, Alternative Ballast
Water Management Practices, and Implementation Costs. Report Number CG-D-12-13.
November 2013.
6. U.S. Coast Guard Acquisition Directorate Research and Development Center. Ballast
Water Treatment, U.S. Great Lakes Bulk Carrier Engineering and Cost Study, Volume 1,
Report CG-D-12-13, November 2013.
7. U.S. Environmental Protection Agency Environmental Technology Verification (ETV)
Program. Generic Protocol for the Verification of Ballast Water Treatment Technology.
EPA/600/R-10/146, September 2010.
8. U.S. Environmental Protection Agency. U.S. Final Issuance of National Pollutant
Discharge Elimination System (NPDES) Vessel General Permit (VGP) for Discharges
Incidental to the Normal Operation of Vessels.
9. U.S. Environmental Protection Agency. Quality Assurance Project Plan for Technical
Support for the Vessel General Permitting Program-Pump Mortality Sampling.
7-1
-------
Revision No. 1
Date: August 29, 2014
Appendix A:
STANDARD OPERATING PROCEDURES FOR LABORATORY-BASED VIABILITY
ANALAYSIS OF FISH EMBRYOS AND LARVAE FOR TOXICITY TESTING AND
OTHER SPECIAL ENVIRONMENTAL STUDIES AND PURPOSES
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 1 of 16
PROCEDURE FOR CONDUCTING LABORATORY-BASED ANALYSIS OF FISH
EMBRYOS AND LARVAE VIABILITY FOR TOXICITY TESTING AND OTHER
SPECIAL ENVIRONMENTAL STUDIES
Method Reference: ASTM E 1241-98.1999. Standard Guide for Conducting Early Life-
Stage Toxicity Tests with Fishes. ASTM, Volume 11.05.
September 8, 2014
Great Lakes Environmental Center, Inc.
(GLEC)
Tyler Linton
Technical Author
~/
9/8/14
Chris Turner
User Reviewer
Date
.Mick DeGraeve
GLEC Management
Date
C.
JenruSr Hansen
GLEC Quality Assurance Officer
Date
Training Statement:
I have read, understand, and agree to follow this SOP.
Signature
Date
Printed Name
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 2 of 16
TABLE OF CONTENTS
Page No.
I. SCOPE/PURPOSE 3
II. SUMMARY OF METHOD 3
III. DEFINITIONS 4
IV. INTERFERENCES AND CAUTIONS 4
V. HEALTH AND SAFETY 4
VI. EQUIPMENT AND SUPPLIES 5
VII. REAGENTS AND STANDARDS 5
VIII. SAMPLE COLLECTION, PRESERVATION, AND STORAGE 6
IX. QUALITY CONTROL 9
X. CALIBRATION 10
XL PROCEDURE 10
XII. DATA ANALYSIS AND CALCULATIONS 14
XIII. INSTRUMENT MAINTENANCE 15
XIV. QUALITY ASSURANCE 15
XV. WASTE MANAGEMENT/POLLUTION PREVENTION 15
XVI. DEVIATIONS 15
XVII. REFERENCES 16
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page3 of 16
I. SCOPE/PURPOSE
1.1 This procedure describes the methodology for conducting viability (live/dead)
analysis offish embryos and larvae used for aquatic toxicity testing and other
environmental studies, such as special fish embryo-larval studies or entrainment
studies. The emphasis is on fathead minnows (Pimephalespromelas), but the
method is adaptable to other related minnow or small fish species.
1.1.1 During early-life stage chronic toxicity testing and certain other tests and
studies fish embryos are continuously exposed for days or weeks to
selected concentrations of the test material, with observations of
embryonic development, hatching, survival and growth being recorded.
1.1.2 Tests are typically initiated with embryos between 2 and 24-hrs old, and
generally must be less than a defined age, e.g., 48-hrs old for early-life
stage toxicity testing.
1.1.3 Minnow species such as P. promelas are available from commercial
sources or in-house laboratory cultures.
1.1.4 The development, hatching, growth and survival of fertilized eggs and
larvae is used to determine chronic toxic effect concentrations and other
unacceptable or adverse impact levels reflective of chemical effects or
other types of specific biological and physical effects such as tissue
contaminant loading an entrainment.
1.2 Experience with fish embryos and larvae or training with someone with such
experience is required before using this SOP with analytical samples.
II. SUMMARY OF METHOD
2.1 The procedure to observe the embryos for viability consists of dividing them in
sub-samples, taking each of them out of the exposure or other temporary
container, and examining them under a microscope for viability status and/or
other irregularities.
2.2 The procedure to observe larvae or small fish for viability consists of visually
verifying that each individual in the test, whether in an exposure treatment
replicate of an aquatic toxicity test or in a different container representing a
unique treatment/test group from a special study, is viable and/or otherwise free
of physical abnormalities or irregularities in appearance.
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 4 of 16
III. DEFINITIONS
3.1 Embryo - a fertilized egg.
3.2 Entrainment - to pull or draw in and transport.
3.3 Fungused - organisms (usually embryos) infected with waterborne fungus.
3.4 Operculum - the hard bony flap covering and protecting the gills of a fish.
3.5 Viability - alive based the observance of heartbeat, opercular movement, and the
ability to respond to stimulus (a gentle touch with a laboratory pipette tip).
IV. INTERFERENCES AND CAUTIONS
4.1 Removal of fungused embryos should be done quickly, and the remaining viable
embryos returned to the incubation tanks as quickly as possible so that they are
not damaged by desiccation.
4.2 It is recommended to maintain organisms in the incubation/holding tanks at the
same environmental conditions at which embryos/fish have been exposed during
spawning, or while being reared.
4.3 The water temperature in the rearing tanks is allowed to follow ambient
laboratory temperatures of 20-25°C, but sudden, extreme, variations in
temperature must be avoided.
4.4 To prevent unnecessary osmotic stress it is recommended that while larval and
other small-sized fish should not be subjected to more than a 50 mg/L (as CaCOs)
change in water hardness in any one 24-h period; acclimation to more than a 100
mg/L in total is acceptable.
4.5 Equipment used to handle embryos must be sterilized (soap and hot water and
well rinsed at a minimum), and hands should be washed before and after
handling.
V. HEALTH AND SAFETY
5.1 Regard each chemical/reagent as a potential health hazard, and read the MSDS for
each before starting work.
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 5 of 16
VI. EQUIPMENT AND SUPPLIES
6.1 Reflected-light (dissecting) stereo microscope;
6.2 Illuminator box(es);
6.3 Watch glass(es);
6.4 Petri dish(es);
6.5 Magnifying glass(es);
6.6 Forceps;
6.7 Blunt probe;
6.8 Pipette(s) with bulb(s);
6.9 Plastic squirt bottle(s);
6.10 Plastic sample and other containers (as required);
6.11 Meter to check hardness; and
6.12 Thermocouple thermometer to monitor temperature during incubation.
VII. REAGENTS AND STANDARDS
7.1 Gases - Not Applicable. No gases are used in this procedure.
7.2 Reagent Water - Not Applicable. No reagent water is used in this procedure.
7.2.1 Deionized (DI) or Dechlorinated water for rinsing specimens
7.3 Reagents - Not Applicable. No reagents are used in this procedure.
7.3.1 70% Ethanol (when preserving specimens is required).
7.4 Standard Solutions - Not Applicable. No standard solutions are used in this
procedure.
7.5 Biological Specimens - The only biological specimens are those used in this
procedure to determine effects (i.e., the analytical samples).
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 6 of 16
VIII. SAMPLE COLLECTION PRESERVATION AND STORAGE
8.1 Sources
8.1.1 Commercial Sources. Small fish (such as the fathead minnow), juveniles
and adults are available from commercial biological supply houses or
laboratory culture facilities. Fish obtained from outside sources for use
in toxicity or other tests may not always be of suitable age and quality.
Fish provided by supply houses should be guaranteed to be: (1) the
correct species, (2) disease free and in good condition, and (3) in the
requested age range. The latter can be ascertained by obtaining a record
of the date on which the eggs were deposited.
8.1.2 In-house Culture. Suitability of fish for use in toxicity testing and for
other scientific testing purposes can be assured by developing an in-
house culture. Fathead minnows are particularly suited to in-house
culture because they are: 1) common and widely distributed, and
therefore adults for brood stock are easy to obtain; 2) their life history is
well known, and they are relatively hardy and easy to reproduce and
maintain in good condition in the laboratory or culture facility; and 3)
embryos can be available throughout the year and are less likely to be
diseased.
Note: Because the quality of embryos and/or fish represents a crucial
factor for hatching and study success and for production of test
organisms of good quality, any stress to embryos and fish should be
avoided, such as: physical shock during transport, acclimation, and/or
incubation; thermal or osmotic shock. It is recommended that organisms
are maintained in the incubation/holding tanks at the same
environmental conditions at which embryos/fish have been exposed
during spawning or while being reared.
8.2 Embryo Incubation, Acclimation and Handling
8.2.1 Incubation. There are three primary methods for incubating fathead
minnow (and other minnow) embryos obtained from outside or internal
sources: on substrates, in a separately funnel, or in embryo incubation
cups (see USEPA, 2002). After fertilization, fathead minnow embryos
are approximately 1.2 mm to 1.6 mm in diameter. The incubation time
depends on temperature, and is 4.5 to 6 days at 25°C.
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 7 of 16
8.2.2 Incubation on substrates. Several (2-4) tile substrates are placed on end
in a circular pattern (with the embryos on the inner side) in 10 cm of
water in a tray. The tray is then placed in a constant temperature water
bath, and the embryos are aerated with a 2.5 cm air stone placed in the
center of the circle. The embryos are examined daily, and the dead and
fungused embryos are counted, recorded, and removed with forceps. At
an incubation temperature of 25°C, 75-100% the embryos hatch in five
days. At 22°C, embryos incubated on aerated tiles require seven days for
50% hatch.
8.2.3 For additional details regarding spawning, fertilization and embryo
incubation on substrates, see GLEC SOP TOX 0006 Culturing
Pimephales promelas.
8.2.4 Incubation a in a separatory funnel. The embryos are removed from the
substrates with a gentle circular rolling action of the index finger
("rolled off) (Gast and Brungs, 1973), their total volume is measured,
and the number of embryos is calculated using a conversion factor (for
fathead minnows) of approximately 430 embryos/mL. The embryos
(approximately 1500 to 2000) are incubated in about 1.5 L of water in a 2
L separatory funnel maintained in a water bath. The embryos are stirred
in the separatory funnel by bubbling air from the tip of a plastic micro-
pipette placed at the bottom, inside the separatory funnel. During the
first two days, the embryos are taken from the funnel daily, those that
are dead and fungused are removed, and those that are alive are returned
to the separatory funnel in clean water. The embryos hatch in four days
at a temperature of 25°C. However, usually on day three the eyed
embryos are removed from the separatory funnel and placed in water in
a plastic tray and gently aerated with an air stone. Using this method, the
embryos hatch in five days.
Note: With this incubation method, hatching time is greatly influenced
by the amount of agitation of the embryos and the incubation
temperature. If on day three the embryos are transferred from the
separatory funnel to a static, un-aerated container, 50% of the embryos
will hatch in six days (instead of five), and a 100% will hatch in 7 days.
8.2.5 Incubation in incubation cups. The embryos are "rolled off the
substrates, and the total number is estimated by determining the volume.
The embryos are then placed in incubation cups attached to a rocker arm
assembly (Mount, 1968). Both flow-through and static renewal
incubation can be used. On day one, the embryos are removed from the
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 8 of 16
cups and those that are dead and fungused are removed. After day one,
only dead embryos are removed from the cups. Most of the embryos will
hatch in five days if incubated at 25°C.
8.2.6 Acclimation. In general, embryos should be standing in dilution water
within 3°C of the desired test temperature. To acclimate, embryos should
not be subjected to more than a 3°C change in water temperature in any
one 12-h period, and preferably not more than 3°C in 72 hr (ASTM,
2013). The concentration of dissolved oxygen should be maintained
between 60 and 100% saturation. To prevent unnecessary osmotic
stress, embryos should not be subjected to more than a 50 mg/L (as
CaCOs) change in water hardness in any one 24-h period, and preferably
not more than a 100 mg/L hardness change in total.
Note: during the incubation and acclimation period, the embryos are
examined daily for viability and fungal growth, until they hatch.
Unfertilized eggs and embryos that have become infected by fungus
should be removed with forceps using a table top magnifier-illuminator
(see Microscopic Viability Observations and Viability Analysis of
Embryos, below). Non-viable eggs become milky and opaque, and are
easily recognized. The non-viable eggs are very susceptible to fungal
infection, which may then spread throughout the egg mass. Removal of
fungused embryos should be done quickly, and the remaining good
embryos returned to the incubation tanks as quickly as possible so that
they are not damaged by desiccation.
8.2.7 Handling. Embryos should be handled as little as possible. When
handling is necessary, it must be done gently, carefully and quickly so
that the organisms are not unnecessarily stressed. Smooth bore glass
tubes are best for handling and transporting embryos.
8.2.8 Equipment used to handle embryos must be sterilized (soap and hot
water and well rinsed at a minimum), and hands should be washed
before and after handling.
8.3 Larval and Small Fish Rearing/Holding and Acclimation and Handling
8.3.1 Rearing/Holding. Newly-hatched larvae are transferred daily from the
egg incubation apparatus to small rearing tanks, using a large bore
pipette, until the hatch is complete. New rearing tanks are set up on a
daily basis to separate fish by age group. Up to 1500 newly hatched
larvae can be placed in a 60 L (15 gal) or 76 L (20 gal) all-glass
aquarium for 30 days. A density of 150 fry per liter is suitable for the
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 9 of 16
first four weeks. The water temperature in the rearing tanks should
follow ambient laboratory temperatures of 20-25°C, but sudden,
extreme, variations in temperature must be avoided.
8.3.2 When larval and other small-sized fish are acquired from a commercial
source, they should be isolated for a short time before use to acclimate to
test conditions (see below), and to ensure they are healthy and disease-
free. Unhealthy or diseased fish should not be used. In general, if
greater than 20% of the fish die within the holding and acclimation
period, the fish should not be used.
8.3.3 Acclimation. Like embryos, larval and other small-sized fish should be
standing in dilution water within 3°C of the desired test temperature. To
acclimate, fish should not be subjected to more than a 3°C change in
water temperature in any one 12-h period, and preferably not more than
3°C in 72 hr (ASTM, 2013). The concentration of dissolved oxygen
should be maintained between 60 and 100% saturation. To prevent
unnecessary osmotic stress, however, it is recommended that while
larval and other small-sized fish should not be subjected to more than a
50 mg/L (as CaCOs) change in water hardness in any one 24-h period;
acclimation to more than a 100 mg/L in total hardness is acceptable.
8.3.4 Handling. Larval and other small-sized fish should also be handled as
little as possible. When handling is necessary, it must be done gently,
carefully and quickly so that the organisms are not unnecessarily
stressed. In general, small dip nets are best for handling fish that weigh
over 0.5g each (for any size less than that use a smooth-bore pipette).
Equipment used to handle fish must be sterilized (soap and hot water
and well rinsed at a minimum), and hands should be washed before and
after handling.
IX. QUALITY CONTROL
9.1 A reliable quality control usually requires 20-30 eggs or fish placed under a
microscope or viewed at the macroscopic scale using the same process but
analyzed separately by a second analyst immediately following counting by the
first analyst, to guard against delayed effects of sample processing.
9.2 The number of quality control samples is project-specific, but should generally
reflect a 10-20% effort, and increased based on the number and frequency of
errors detected. Note that for small fish, the 10-20% quality control sample effort
could amount to re-examination of the appropriate number offish set aside in a
separate container to be analyzed by a second analyst.
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 10 of 16
9.3 Any deviations from quality control procedures must be recorded - see under
Deviations below.
9.4 Chain-of-custody forms must be completed in cases where samples change hands
from one party to another, or to a third party. Completion of chain of custody
forms as per GLEC SOP LAB 1014.
X. CALIBRATION
10.1 Calibrate meters and probes in the morning (or before use) and again mid-day, or
about every three hours when in use, and whenever the meter produces erratic
results.
10.2 Follow calibration and maintenance procedures in the parameter/equipment
specific SOP.
XL PROCEDURE
111 Microscope and Microscope Use (General)
11.1.1 Microscopes. Depending on the size of the specimen, appropriate
microscopes are a reflected-light (dissecting) stereo microscope. For
large specimens, a simple table top magnifier-illuminator unit may be
acceptable.
11.1.2 General Microscope Use. Ensure the microscope is located on a flat and
sturdy surface. Turn on the illumination source (if applicable) and place
the slide or other device holding the specimen (watch glass or petri dish)
on the stage. The proper position for the sample is directly over the light
source. Set the microscope to the lowest objective powered lens and use
the coarse and fine focus knobs to bring the image into focus. Always
look into the ocular lens with both eyes open, resisting the urge to close
one eye and squint with the other which will increase the strain and
tension on your eyes. Adjust the diaphragm as needed (and as
applicable) to allow more or reduce the amount of light let in. Switch to
higher magnification powers as needed, refocusing each time if needed.
Wipe the lenses with lens paper if dirty then cover the microscope once
finished.
11.2 Microscopic Viability Observations and Viability Analysis of Embryos
11.2.1 The procedure to observe the embryos for viability consists of dividing
them in sub-samples, taking each of them out of the exposure or other
temporary container, and examining them under a microscope for
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 11 of 16
viability status and/or other irregularities. Eggs can be preserved in
ethanol for archive and for obtaining total counts of eggs observed, or,
returned to the exposure tank or other temporary container.
11.2.1.1 With a large, smooth-bore pipette, carefully, gently and
quickly transfer embryos from the temporary container to a
watch-glass or Petri dish, making sure that the embryos form
a single layer.
11.2.1.2 Adjust the microscope as described above.
11.2.1.3 Check for the following embryo viability characteristics,
depending on the scope of the work:
• At a minimum (and assuming all eggs are fertilized), look
for:
o Presence/absence of heartbeat and/or larval
movement and/or opaque discoloration in species
whose embryos are normally translucent (depending
on stage of development); and
o Movement after gentle prodding with a pipette.
• On a project-specific basis, look for irregularities such as:
o irregular rounded shape and size (diameter in fathead
minnow approximately 1.2 to 1.6 mm);
o irregular yolk formation (e.g., a decrease in the
amount of vitellogenic/yolk material that is deposited
in the developing oocyte); and
o irregular transparency (no superficial spots and dark
areas).
11.2.1.4 Record observations. If the embryo is so undeveloped such
that live/dead status cannot be ascertained (via gentle
prodding with fine forceps or blunt probe to elicit a response,
when necessary), the viability status of the embryo should be
recorded as "indeterminate."
Note: An early estimate of irregular or aborted eggs can be
made at this stage. Any irregular egg will soon develop into
an abortive embryo or an abnormal larva. Spots on the
external chorion account for physical or bacterial damages.
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 12 of 16
As a general rule, good egg batches have usually less than
10% abnormal eggs. Any batch containing more than 20%
abnormal eggs should be discarded or is sign of adverse
effect/impact.
11.2.1.5 Sub-samples are taken by gently swirling containers to re-
suspend any eggs that have settled to the bottom and using a
large, smooth-bore pipette to place a volume (e.g., 10 - 20
milliliters or mL) into a counting chamber (watch glass or
petri dish). Volume of sub-sample is driven by number of
eggs in sub-sample volume, and should generally not exceed
more than 1 egg per 2 mLs.
11.2.1.6 The sub-sampling process is repeated as many times as
needed over a specified period of time in order to ensure a
certain percentage of embryos is examined within a specified
time frame. For example, repeating the sub-sampling process
using a 20 mL sub-sample volume from a 1000 mL container
25 times in one hour allows for one technician to evaluate
half (500 mLs) the volume of the 1 L container containing
embryos. If the estimated number of embryos in the 1 L
container is 200 eggs, that researcher will examine
approximately 100 eggs in the time allotted.
11.2.1.7 The sub-sampling process, the amount sub-sampled, and the
timeframe for sampling is project-specific, but should
generally not exceed 100 - 130 eggs per researcher per hour,
pending experience.
11.2.1.8 Surviving embryos are returned to exposure tanks/containers,
euthanized, or, can be preserved for later analysis.
11.2.1.9 Uncontaminated dead embryos are bagged and discarded
according to laboratory specifications, or stored frozen for
later analysis and disposal.
11.3 Microscopic and Macroscopic Observations and Viability Analysis of Larval
and Small Fish
11.3.1 The procedure to observe larvae or small-sized fish for viability consists
of visually verifying that each individual in the test, whether in an
exposure treatment replicate of an aquatic toxicity test or in a different
container representing a unique treatment/test group from a special
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 13 of 16
study, is viable and/or otherwise free of physical abnormalities or
irregularities in appearance. Fish can be preserved in ethanol for archive
and obtaining total counts observed, or, returned to the exposure tank or
other temporary container.
11.3.1.1 With a small dip net (for fish weighing 0.5 g or greater)
carefully, gently and quickly transfer the fish from the
temporary container to an appropriately-sized vessel from
which to make microscopic or macroscopic observations,
depending on the size of the fish.
11.3.1.2 Adjust (dissecting) microscope as described above if it is
necessary to use a microscope, i.e., for very small newly-
hatched larvae, or, place vessel holding specimen(s) on
illuminator box and use visual inspection or inspection with
magnifying glass, as needed. The vessel used for inspection
can contain one or more fish, so long as the research
technician making the observations can distinguish amongst
individuals for accurate viability counts. Note, it is not
recommended that more than 10 fish be evaluated at any one
time in any vessel, and it is preferable to limit the maximum
number offish to 5 at any one time.
11.3.1.3 Check for the following fish viability characteristics,
depending on the scope of the work:
• At a minimum (and assuming all fish are intact):
o Presence/absence of voluntary body movement and/or
respiratory (gill) movement and/or heartbeat and/or
presence/absence of movement to stimulus (gentle
prodding with blunt probe/instrument).
• And, on a project-specific basis, other physical and
behavioral irregularities/abnormalities:
o Uncoordinated swimming and/or skin damage or
abrasion (indication of physical damage);
o Skin damage or abrasion and/or bleeding (indication
of physical damage);
o Improper pigmentation (physical or other damage);
and
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 14 of 16
o Hyperventilation or surfacing (could be a severe
stress response due to handling and not treatment, but
could be due to internal damage).
11.3.2 Record observations. Note that in the case of small fish entrainment and
other special studies, whole, intact fish may not be available for
live/dead counting. In such a case, the fish heads of dead animals can be
counted. Additionally, injured/damaged fish, while alive, can be counted
separately for viability analysis (presumably, per the definition of
viability, injury or damage precludes "ability to survive.").
11.3.3 Sub-sampling for fish is generally constrained by how many fish can be
observed accurately at one time, as per above. For most studies, and
unlike embryo analysis, all fish tested/treated are assessed for viability,
unless sample numbers are excessive for number of technicians to
process (usually a limitation in the field, not in the laboratory or via a
bench-scale test).
11.3.4 The sub-sampling process is repeated as many times as needed over a
specified period of time in order to ensure all (or a certain percentage) of
fish are examined within a specified time frame.
11.3.5 The sub-sampling process, the amount sub-sampled, and the timeframe
for sampling is project specific.
11.3.6 Surviving fish are returned to exposure tanks/containers, euthanized, or,
can be preserved for later analysis.
11.3.7 Uncontaminated dead animals are bagged and discarded according to
laboratory specifications, or stored frozen for later analysis and disposal.
XII. DATA ANALYSIS AND CALCULATIONS
12.1 Data analysis and calculations will be project specific. Any marked activities for
the project (Project Name, Date, Scope of Work, Location, Sample Type, Sample
Numbers, etc.) and any notes or calculations taken/made during the event will be
documented in a project-specific log book.
12.2 Dedicated raw data sheets will be generated prior to the study/analysis, and data
recorded by hand in hardcopy. Raw data in hardcopy form can be transferred to
electronic spreadsheet form, as needed. A 100% QC of data transferred to
hardcopy is required. Alternatively, data can be hand-entered in electronic form
on-site using a portable computer, but values must be recorded in hardcopy on
raw data sheets to ensure transparency and for QA/QC purposes.
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 15 of 16
12.3 Other forms of data reporting are project-specific, and are subject to requests and
agreement by the project manager/leader prior to project initiation.
XIII. INSTRUMENT MAINTENANCE
13.1 Maintain equipment and meters and thermometers according to the
parameter/instrument specific SOP.
13.2 Cover microscopes when not in use, and replace light bulbs as needed.
XIV. QUALITY ASSURANCE
14.1 Each part of the raw data, data transcription, and final report is reviewed by the
primary generator of the information, and then a 100% QC is conducted by a peer
familiar with the test. These reviews consist of checking any mathematical
computations performed, and the accuracy and traceability of the data, sample IDs
and completeness/clarity of the data sheets. The final report is then reviewed by
an upper level staff member or a management designee for scientific soundness
and assessment of any usual results.
XV. WASTE MANAGEMENT/POLLUTION PREVENTION
15.1 This method will be conducted with active pollution prevention as an objective
by: modifying processes to reduce or eliminate waste, promoting the use of non-
toxic or less-toxic substances, implementing conservation techniques, and re-
using materials rather than putting them into the waste stream.
15.2 Pour liquid waste from this procedure (ethanol) down a sink drain, connected to a
municipal sewer system, with the cold water faucet fully open. Allow the faucet
water to flow for ~2 minutes.
XVI. DEVIATIONS
16.1 Any change in protocol from an approved study plan must be signed off by the
GLEC Quality Assurance Officer after notifying and gaining approval from the
client.
16.2 All deviations must be recorded in the project-specific log book, and hardcopies
of specific deviations should be provided to the GLEC QAO for record keeping
purposes.
16.3 Acclimation of embryos and fish. The recommended magnitude and time rate of
change associated with acclimation of embryos and fish noted above are specific
to use of organisms for aquatic toxicity testing purposes. In special studies, both
-------
Great Lakes Environmental Center, Inc.
GLEC SOP Number: LAB 1054
Date of Previous Revision: N/A
Revision Date: September 8, 2014
Page 16 of 16
magnitude and time rate of change may be altered for specific purposes, but
should not exceed thermal or osmotic maxima based on species or taxa-specific
guidance, if such guidance exists. Any deviations from the recommended
magnitude and time rate of change must be noted in the project-specific log book
along with the justification for the deviation.
XVII. REFERENCES (NEEDS COMPLETED AS NECESSARY)
17.1 ASTM £1241-05(2013). Standard Guide for Conducting Early Life-Stage
Toxicity Tests with Fishes. ASTM, Volume 11.06.
17.2 Gast, M.H. and Brungs, W.A. 1973. A Procedure for Separating Eggs of the
Fathead Minnow. Prog. Fish. Cult. 35:54.
17.3 GLEC SOP LAB 1014. Chain of Custody.
17.4 GLEC SOP TOX 0006. Culturing Pimephalespromelas.
17.5 Mount, D.I. 1968. Chronic toxicity of copper to fathead minnows (Pimephales
promelas Rafinesque). Water Research. 2:214-223.
17.6 U.S. Environmental Protection Agency (USEPA). 2002. Methods for Measuring
the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine
Organisms, Fifth Edition. October 2002. EPA-821-R-02-012. USEPA Office of
Water, Washington, DC. Appendix A.
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Appendix B
Appendix B:
FISH AND FISH EGG STOCKING PERMITS
-------
state of Wisconsin Fish Stocking Permit
Department of Natural Resources Form g40Q 61 Rey 2_Q »
Box 7921
Madison, Wl 53707
TO WHOM IT MAY CONCERN
This is to certify that the below named is hereby permitted pursuant to Section 29.745 of the Wisconsin
Statutes to import into the state and/or stock fish in the waters specified below. Personally identifiable
information found on this form is not intended to be used for any other purpose.
Permittee: Permit ID# 6812
Mark Briggs Permit Issue Date: 07/29/2014
3400 Jack Morris Drive . .. .. __ .... ..
Application Modifications:
West Branch, Ml 48661 ™
989 345 7595
Organization: Eastern Reseach Group, Inc.
Stocking Site
County: Waterbody Name WBIC:
DOUGLAS LAKE SUPERIOR 2751220
Status of Water
Public Waters - Public Access
Species to be Stocked Avg Length
Fish Farm Information Species Age (Inches) Number Pounds
Urbank Live Bait, Reg. No.: LIS #35 FATHEAD MINNOW ADULT 2 1,500 1
To be Obtained From
Supplier Name: HAYWARD BAIT AND TACKLE
THIS PERMIT IS ISSUED SUBJECT TO THE FOLLOWING CONDITIONS:
1. This permit is effective from 08/18/2014 until 10/18/2014.
2. A Receipt of Fish for Planting, Form 3600-16, for all fish stocked in public waters is attached to this Permit. The Receipt
must be completed and returned to the Fish Biologist listed on this Permit and Receipt when stocking is complete.
3. Paul Piszczek, telephone number (715) Ur392-7990 , must be notified of the time of stocking at least 3 days
in advance of stocking
4. The fish being stocked under this permit must have been certified by a qualified inspector, and must meet fish health
standards and requirements promulgated under Wisconsin Stats. 95.60(4s)(b) and Wisconsin Administrative Code s.
ATCPCh. 10.60.
5. A valid signed DATCP Fish Health Certificate (FHC) must accompany the stocking permit during all stocking operations.
6. To prevent or control the spread of the Viral Hemorrhagic Septicemia (VHS) virus, fish may not be moved or stocked
into non-VHS infected waters from VHS infected waters, including fish farms that are directly connected to any VHS
infected water. VHS infected waters are defined to be Lake Michigan, Lake Superior, Lake Winnebago System and
their tributaries upstream to the first dam or barrier impassable to fish.
7. The department reserves the right to inspect all loads of fish to be stocked under this permit, including but not limited
to: verify species, count, measure, weigh, assess water quality, or collect tissue samples
8. No fish may be stocked under the authority of this permit if the fish have been transported to their final destination for
stocking in any container which also contains any fish not authorized for stocking under this permit. Other fish may be
transported on the same truck as long as they are in separate compartments
Biologist Signature: Paul Piszczek (e-signature) Date Signed. 07/29/2014
-------
Return Completed Stocking Receipt to:
Paul Piszczek
Wisconsin Department of Natural Resources
1701 N4THST
Superior, Wl 54880
-------
AH-AQ-200Z (rev. 12/2008)
Wisconsin Department of Agriculture,
Trade and Consumer Protection
Division of Animal Health
P OBox891i
Madison, WI 53708-8911
Phone: 60&-224-4887 Fax; 608-224-4871
FISH IMPORT PERMIT APPLICATION
ATCP 10.62, Wfe, Adm. Code, and Sec, 95.60 (Wis. Stats.)
OFFICE USE ONLY
Import Permit Number;
Date:
Veterinarian's Signature
Exp
-Ite
DN^ Permit Required?
or No
INSTRUCTIONS: Complete all fields, sign, date, and make a copy for your records. If paying with check or money order,
submit fees (payable to WI DATCP), the application and copies of the FISH HEALTH CERTIFICATE to the address listed
above. Legible fax copies are welcome. A copy of the Issued Import permit will be faxed, and Jne original will be malted.
Upon receipt of the approved import permit, forward a copy to the fish source. Ensure that the hauler receives copies of
the Import permit and fish health certificate because they must accompany each shipment Into Wisconsin.
I. IMPORTER INFORMATION
(Person/Business that owns fish/fish eggs when the shipment enters Wisconsin.)
Fish Farm Registration Number;
(and) Livestock Premises Code:
Legal Name „
df/ayr?'"*/ /?#$•&• ret*. Crr&^f>
Mailing Address
3 yao ~\f^c,tc ^,w> t>i2-
Telephone Number
•96$ ~^$' 9$ 95
(or) WI DNR Stocking Permit Number:
6*8 1&
Contact Name (
StfcsX &tw s
City / State / Zip ' ' , ,
u*s>T 0,1^^4 »*»^ yg^
Facsimile Number
Stocking Location (if different than mailing address) Including City / State /Zip
/f>/^>t?f ^#s~ UiJ'JL SY&OO
Applicant Signature/ y / sj ^
M6/f&v*JL
Date of Application „ ^^.^$, y
n. FISH
(Fish/fish egg owner outside Wisconsin)
Legal Name
05 £7*7
Bua'ness Address ai . a
£10} C^^K^'V VLiAX,
Telephone Number ^ ^ __ ^ ^ ^0 a
Location of Fish (if other than business address)
including City /State /Zip
Contact Name _ i~ . \
/^^/\ CiJkf /&/*« ^i/ •
City /State /Zip
t>wtt>fu tvt//
Facsimile Number
Out of State Registration or License Number
(and) Livestock Premises Code (If any)
REQUIRED; Attach a copy of the Fish Health Certificate (FHC) and all laboratory tests that cover the fish/fish
eggs for this application. The Import permit cannot be processed until the original FHC is accepted at the
Division of Animal Health.
IH. PAYMENT METHOD [$90.00 fee per ATCP 10.62(4)(c)j
Payment Method d Check Q Money Order D Visa $U~ Master Card
(To maintain confidentiality, section III (Payment Method) will be shredded after collection of fees.)
Credit Card Number
Card Holder Address v
Card Holder, Signature & Printed Name
Expiration Date (mm/yy)
City /State /Zip
UJ&$/~~ Mfl'V'Yzifi y*?
E-mail address (for receipt)
/*?<£-* A*< J-v *&> e f.
&/&
j^ Y $ ' & &/
'^^- C O^L^_
-------
IV. INFORMATION
Species Name ^ , *
Species Name
Species Name
Species Name
Species Name
Species Name
D Fish or JET Eggs
O Fish or O Eggs
D Fish or D Eggs
O Fish or JU Eggs
O Fish or D Eggs
D Fish or D Eggs
Size
Size
Size
Size
Size
Size
V. RICIPIENT INFORMATION (If other than importer in Section I)
For multiple recipients, copy this page and complete this section for each recipient
Recipient Number One
Fish Farm Registration Number:
(and) Livestock Premises Code:
Legal Name
Mailing Address
Telephone Number
(or) WI DNR Stocking Permit Number;
(or) WI DNR Bait Dealer License Number:
Contact N$me
City/ State /Zip
Receiving Location (if different than mailing address)
Recipient Number Two
Fish Farm Registration Number:
(and) Livestock Premises Code:
Legal Name
Mailing Address
Telephone Number
(or) WI DNR Stocking Permit Number:
(or) WI DNR Bait Dealer License Numbers
Contact Name
City /State /Zip
Receiving Location (If different than mailing address)
VI. HAULER INFORMATION
Legal Name _
Registration or License Number
Contact Name
Mailing Address
„
3
Telephone Number
' '
City /State /Zip
Jhe department shall grant or deny an application under ATCP 10,62 within 30 days after receipt of a
complete application, If the department denies the application, the department shall issue the denial notice in
writing and shall state the reasons for the denial.
Personal information you provide may be used for purposes other than that for which It was originally collected -
sec,15,04(i)(m), Wis* Stats. However, any information that Jdentffes the type or number of fish or fish eggs, the.
supplier, or purchaser will be kept CONFIDENTIAL by the department as required |Jy law [s, 95,60(7), Wis. Stats,]
-------
AH-AQ-2803 (rev. 02/2010) PREVIOUS VERSIONS SHOULD BE DESTROYED
SUBMIT ORIGINAL WITHIN SEVEN DAYS AFTER ISSUE TO;
Wisconsin Department of Agriculture,
Trade and Consumer Protection
Division of Animal Health
PO Box 8911, Madison, Wl 53708-8911
Phone: 608-224-4872 Fax; 6Q8-224-4871
OFFICE USE ONLY
ATCP 10,65, Wis, Adm, Code
n .
?7^5~
3f be Issuedi
INSPECTION DATE
\L\
ISSUE DATE
(A fish health certificate CANNOT be Issued un
ite are obtained.)
FISH FARM REGISTRATION
LIVESTOCK PREMISES CODE (if any)
FISH FARM
A *
6 1
OWNER / MANAGER NAMi
FISH FARM ADDRESS / CITY / STATE /ZIP
BUSINESS ADDRESS / CITY / STATE / ZIP
u i
TYPEOF WATER SUPPLY (Check all that apply)
& Lake Q Stream D Spring D Weil
D Qther_i[speclfy) ___^__
TYPE OF FISH HEALTH CERTIFICATE (FHC)
D Annual (Expires 12 months from date Issued)
13" Lot (Expires 30 days from date issued)
List fish species present on date of inspection,
p.gTt
Hca
•LLQUAUIFIEP B8H HEAyTH INSpCljjJRlMEQRMA^IQN (4)
A-'. LM(M^ py INTACT TELEPHONE -
PRINTED NAME
- tj 2- "
LICENSE NUpER
'ADDRES
CITY/ STATE /ZIP
\ certify that fish for any required laboratory tests have bsen sampled and inspected by lot or facility according to the current version of
the Inspection Section of the AFS-FHS Blue Book or the DIE Manual and Code, 1 have also visually Inspected a minimum of 60 fish
per species (or 1 00% of the population for populations of 60 fish or less) and certify that the fish have no gross el Weal signs of
contagious or infectious diseases except as noted on this form, All laboratory test results are summarized in the table below and the •
laboratory's report Is appended to this document.
(4) & t ,, f . . /
&- X^'*^^1 &VM ~7 1 ^ / / V
CorriVnenWon viisfoie signs of contagious or Infectious disease (5),
t_ _
e 2 fpr-jacd4pfe^Jes^§|ilpi
-------
state or Wisconsin Fish Stocking Permit
Department of Natural Resources F M00.61 R 2,0f
Box 7921
Madison, Wl 53707
TO WHOM IT MAY CONCERN
This is to certify that the below named is hereby permitted pursuant to Section 29.745 of the Wisconsin
Statutes to import into the state and/or stock fish In the waters specified below. Personally identifiable
information found on this form is not intended to be used for any other purpose,
Permittee: Permit \0# 6816
Mark Briggs Permit Issue Date: 07/29/2014
3400 Jack Morris Drive . „ .. ... _,.,. ,.
, . . . . App cat on Modifications:
West Branch, Mi 48661
989 345 7595
Organization: Eastern Research Group, Inc.
Stocking Site
County: Waterbody Name WBIC:
DOUGLAS LAKE SUPERIOR • 2751220
Status of Water
Public Waters - Public Access
Species to be Stocked ^ Avg, Length
Fish Farm Information Species Age (2^ (Inches) Number Pounds
|USEPA Mid Continentlcological Division] FATHEAD MINNOW [ FJ#f £6*$ | O.l"~~"f~2.000 | 1
To be Obtained From
Supplier Name; MID-CONTINENT ECOLOGY DIVISION - EPA
THIS PERMIT IS ISSUED SUBJECT TO THE FOLLOWING CONDITIONS:
1, This permit Is effective from 08/20/2014 until 10/19/2014,
2, A Receipt of Fish for Planting, Form 3600-16, for ail fish stocked In public waters is attached to this Permit. The Receipt
must be completed and returned to the Fish Biologist listed on this Permit and Receipt when stocking !s complete.
3, Paul Piszczek, telephone number (715) 392-7990 , must be notified of the time of stocking at least 3 days
In advance of stocking
4, The fish being stocked under this permit must have been certified by a qualified inspector, and must meet fish health
standards and requirements promulgated under Wisconsin Stats. 95,60(4s){b) and Wisconsin Administrative Code s,
ATCPCh. 10.60,
5. A valid signed DATCP Fish Health Certificate (FHC) must accompany the stocking permit during all stocking operations.
6. To prevent or control the spread of the Viral Hemorrhaglc Septicemia (VHS) virus, fish may not be moved or stocked
into non-VHS infected waters from VHS infected waters, Including fish farms that are directly connected to any VHS
Infected water, VHS infected waters are defined to be Lake Michigan, Lake Superior, Lake Winnebago System and
their tributaries upstream to the first dam or barrier impassable to fish.
7. The department reserves the right to inspect all loads of fish to be stocked under this permit, including but not limited
to; verity species, count, measure, weigh, assess water quality, or collect tissue samples
8; No fish may be stocked under the authority of this permit if the fish have been transported to their final destination for
stocking In any container which also contains any fish not authorized for stocking under this permit. Other fish may be
transported on the same truck as long as they are in separate compartments
Biologist Signature: Paul Piszczek (e-signature) Dgte s|gned; 07/29/2014
-------
State of Wisconsin - Animal Health E-payment Services Page I of I
- "-Sii&^s
Exit
You must click the "Continue" button below in order to return to the state agency's website,
Please keep a record of your Confirmation Number, or print this page for your records.
Confirmation Number WISAHE009551968
Payment Details
Description WI Animal Health
DATCP Animal Health E-payment Services
http://www.datcp.state.wi.us/
Payment Amount $90.00
Payment Date 08/20/2014
Status PROCESSED
Payment Method
Payer Name Mark Briggs
Card Number *Q459
Card Type Master Card
Approval Code T7380B
Confirmation Email Caroi.PauIs@wi.gov
Billing Address
Address 1 3400 Jack Morris Dr.
City West Branch,
State MI
Zip Code 48661
ht^s://epayment.epymtservicexon^main/pay^ 8/20/2014
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Appendix C
Appendix C:
PHOTOGRAPHS
-------
Appendix C: Photographs
Day 1: Fathead Minnow Test
Photograph 1: Control & Gravity Nets
Photograph 2: Setting Up Gravity Net
4
^^^m a^
Photograph 3: Setting Up Control Net
Photograph 4: Control Net
C-l
-------
Photograph 5: View of Dock (left)
Photograph 6: View of Dock (right)
Photograph 7: Net Set-Up
Photograph 8: Security Gate
Photograph 10: Empty Dock
Photograph 9: Effluent Fish Tanks
C-2
-------
Photograph 11: Equipment Set-Up Area
Photograph 12: Filling Drums
Photograph 13: Filling Drums 2
Photograph 14: Fish Tank Set-up
Photograph 15: Fish Tank Set-up
Photograph 16: Control Net on Pulley
C-3
-------
Photograph 17: GLEC Analysis Area
Photograph 18: Gravity Fish Tank
Photograph 19: Influent Fish Tanks
Photograph 20: Influent Tanks with Aerators and Thermometers
Photograph 21: Influent Fish Tank
Photograph 22: Influent Fish Tanks
C-4
-------
Photograph 23: Inside View of Drum
Photograph 24: Indoor Set-up
Photograph 25: NERR Building
Photograph 26: Net Set-up With Hoses
Photograph 28: Net Set-up
Photograph 27: Net Set-up With Hoses
C-5
-------
Photograph 29: Pulling the Gravity Net
Photograph 30: Pulling the Gravity Net
Photograph 31: Pulling the Control Net
Photograph 32: Pulling the Control Net
C-6
-------
Photograph 33: Pulling the Pump Net
Photograph 34: Pulling the Pump Net
Photograph 36: Pump Effluent Replicate 1
Photograph 35: Pump Effluent Replicate 1
C-7
-------
Photograph 37: Pump Effluent Replicate 1
Photograph 38: Pump Efflent Replicate 2
Photograph 39: Pump Effluent in Netting
Photograph 40: Pump Effluent in Netting
C-8
-------
Day 2: Fathead Minnow Eggs Test
Photograph 41: Collecting Control Sample
Photograph 42: Collecting Gravity Sample
Photograph 43: Counting Egg Mortality
Photograph 45: GLEC Analysis Area
Photograph 44: Counting Egg Mortality
Photograph 46: GLEC Analysis Area
C-9
-------
Photograph 47: Dock Set-up
Photograph 49: Effluet Egg Samples
Photograph 51: Three Sample Collecting Area
Photograph 48: Emptying Drum
Photograph 50: Influent Eggs
Photograph 52: Pump Set-up
C-10
-------
Photograph 53: Pump Valve Set-up
Photograph 55: Fish Egg Under Scope
Photograph 54: Trash Pump
C-ll
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Appendix D
Appendix D:
LABORATORY MORTALITY DATA
-------
Fish/Egg Mortality Tests - GLEC log
Fish/Egg
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Treatment
Control
Gravity
Pump
Control
Gravity
Pump
Control
Gravity
Pump
Rep#
1
1
1
2
2
2
3
3
3
Date
9/10/14
9/10/14
9/10/14
9/10/14
9/10/14
9/10/14
9/10/14
9/10/14
9/10/14
Time In
10:43
10:54
11:00
12:00
12:06
12:11
13:12
13:16
13:20
Temp
Start
17.0
17.0
17.0
16.5
16.5
16.5
16.5
16.5
16.0
Temp
11:27
18.5
17.5
17.5
Temp
11:50
17.0
17.0
17.0
Temp
12:22
17.0
17.0
15.5
Temp
13:50
17.0
17.0
16.0
Temp
Time
Finished
11:52
12:06
12:00
13:12
13:16
13:20
14:15
14:25
14:20
Comments
127 processed
100% examined
126 processed
100% examined
100% examined
100% examined
100% examined
100% examined
100% examined
-------
Technician:
Replicate _1
David Rosier
Mortality Data Sheet - Fish
Test Type: D Control [7] Pump D Gravity
Date:
9/9/2014
Dead
Alive
Injured*
QC?
Subl
5
3
0
U
Sub 2
11
2
0
bd CT
Sub 3
10
9
0
U
Sub 4
1
11
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
27
25
0
Damage Notes:
Replicate _3
Many fish macerated by pump and killed
Dead
Alive
Injured*
QC?
Subl
4
11
0
Ld CT
Sub 2
3
10
1
U
Sub 3
4
11
3
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
11
32
4
Damage Notes:
Replicate _4
Sub 2 - slight hemorrhaging at mouth
Sub 3 - Sever damage to head on one, cut in half, one slightly hemorrhaging
Dead
Alive
Injured*
QC?
Subl
9
1
0
Ld CT
Sub 2
8
10
2
U
Sub 3
6
4
1
U
Sub 4
3
0
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
26
15
3
Damage Notes:
Replicate _5_
Sub 2 - Damage to head/eyes
Sub 3 - Small cut on flank
Dead
Alive
Injured*
QC?
Subl
4
9
0
Ld CT
Sub 2
2
8
1
U
Sub 3
6
10
2
U
Sub 4
2
12
0
U
Sub 5
11
4
1
U
Sub 6
3
12
3
Ld CT
Sub?
0
10
4
U
Sub
U
Sub
U
Sub
U
Total
28
65
11
Damage notes:
* Fish which are
Sub 2 - Hemmorging on face Sub 5 - Dorsal hemmoraging and caudal half missing
Sub 3 - Eyes missing, damage to head Sub 6 and 7 - Hemmorrhaging on dorsal and mouth/head
alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
Eliot Hoitsma
Replicate _1
Mortality Data Sheet - Fish
Test Type: d Control [7J Pump Q Gravity
Date:
9/9/2014
Dead
Alive
Injured*
QC?
Subl
0
10
5
U
Sub 2
12
1
1
bd CT
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
12
11
6
Damage Notes:
Replicate _2_
Tail fins chopped off
Dead
Alive
Injured*
QC?
Subl
5
3
1
U
Sub 2
4
16
2
U
Sub 3
13
5
2
Ld CT
Sub 4
1
13
2
U
Sub 5
6
1
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
29
38
7
Damage Notes:
Replicate _3_
Tail fin chopped off, bleeding from gills
Dead
Alive
Injured*
QC?
Subl
2
9
0
Ld CT
Sub 2
6
5
1
U
Sub 3
7
2
1
U
Sub 4
5
10
1
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
20
26
3
Damage Notes:
Replicate _5_
missing half of body, bent body
Dead
Alive
Injured*
QC?
Subl
14
1
0
bd CT
Sub 2
8
o
6
i
U
Sub 3
5
11
0
U
Sub 4
14
1
0
U
Sub 5
o
J
5
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
44
21
1
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
Tracey Ladder
Replicate _1
Mortality Data Sheet - Fish Date:
Test Type: L~H Control [7] Pump Qj" Gravity
9/9/2014
Dead
Alive
Injured*
QC?
Subl
14
3
1
u
Sub 2
8
3
1
Ld CT
Sub 3
2
11
0
U
Sub 4
2
3
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
26
20
2
Damage Notes:
Replicate _2
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate _4
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate
Dead
Alive
Injured*
QC?
Sub 1 - cut in half
Sub 2 - cut in half
Subl
13
12
0
Ld CT
Subl
10
16
2
Ld CT
Sub 2
7
4
0
Ld CT
Sub 2
6
5
1
U
Sub 3
2
13
0
U
Sub 3
2
9
1
bd CT
Sub 1 - eye damage
Sub 2 - eye damage
Sub
U
Sub
U
Sub
U
Sub
U
Sub 4
1
6
1
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
22
29
0
Total
19
36
5
Sub 3 - mouth injury
Sub 4 - mouth injury
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
0
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
David Rosier
Mortality Data Sheet - Fish
Date:
9/9/2014
Replicate _2
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate
Dead
Alive
Injured*
QC?
Test Type: d Control Q Pump [7] Gravity
Subl
0
13
1
Ld CT
Sub 2
0
12
0
U
Sub 3
0
13
0
U
Sub 4
0
13
0
U
Sub 5
0
13
1
U
Sub 6
0
13
0
U
Sub 1 - Nearly unresponsive, minimal movement
Sub 5 - Injured with bloody mouth
Sub
u
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub?
0
13
0
U
Sub 8
0
12
0
U
Sub 9-2 minor injuries,
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub 9
0
13
2
U
bloody fins
Sub
U
Sub
U
Sub
U
Sub 10
0
6
0
U
Sub
U
Sub
U
Sub
U
Total
0
121
4
Total
0
0
0
Total
0
0
0
Total
0
0
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
Eliot Hoitsma
Mortality Data Sheet - Fish
Test Type: L~H Control QPump [7] Gravity
Date: 9/9/2014
Replicate _1
Dead
Alive
Injured*
QC?
Subl
0
14
0
Ld CT
Sub 2
0
6
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
20
0
Damage Notes:
Replicate _4_
Dead
Alive
Injured*
QC?
Subl
0
12
0
U
Sub 2
0
12
0
Ld CT
Sub 3
0
11
0
U
Sub 4
0
10
0
U
Sub 5
0
13
0
U
Sub 6
0
17
0
U
Sub?
0
9
0
U
Sub 8
0
9
0
U
Sub 9
0
11
0
U
Sub 10
0
8
0
U
Total
0
112
0
Damage Notes:
Replicate _4_
Dead
Alive
Injured*
QC?
Sub 11
0
7
0
U
Sub 12
0
8
0
Ld CT
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
15
0
Damage Notes:
Replicate
Dead
Alive
Injured*
QC?
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
0
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
Tracey Ladder
Replicate _1
Mortality Data Sheet - Fish
Test Type: d Control Qj Pump [7] Gravity
Date:
9/9/2014
Dead
Alive
Injured*
QC?
Subl
0
22
0
U
Sub 2
0
23
0
U
Sub 3
0
10
0
U
Sub 4
0
20
0
U
Sub 5
0
8
0
U
Sub 6
0
6
0
Ld CT
Sub?
0
4
0
U
Sub
U
Sub
U
Sub
U
Total
0
93
0
Damage Notes:
Replicate _3_
Dead
Alive
Injured*
QC?
Subl
0
23
0
Ld CT
Sub 2
0
15
0
U
Sub 3
0
16
0
U
Sub 4
0
8
1
U
Sub 5
0
10
0
U
Sub 6
0
13
0
U
Sub?
1
9
0
U
Sub
U
Sub
U
Sub
U
Total
1
94
1
Damage Notes:
Replicate _5_
Sub 4 - Bloody lip
Dead
Alive
Injured*
QC?
Subl
0
11
0
U
Sub 2
0
17
0
U
Sub 3
0
9
0
Ld CT
Sub 4
0
12
0
U
Sub 5
0
16
0
U
Sub 6
0
11
0
U
Sub?
0
10
0
U
Sub 8
0
14
0
U
Sub 9
0
7
0
U
Sub 10
0
11
0
U
Total
0
118
0
Damage Notes:
Replicate _5_
Dead
Alive
Injured*
QC?
Sub 11
0
9
0
U
Sub 12
0
13
0
U
Sub 13
0
12
0
U
Sub 14
0
7
0
U
Sub 15
0
6
0
U
Sub 16
0
11
0
Ld CT
Subl?
0
9
0
U
Sub 18
0
10
0
U
Sub
U
Sub
U
Total
0
77
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
David Rosier
Replicate _1
Mortality Data Sheet - Fish Date:
Test Type: 0 Control Qj Pump Q Gravity
9/9/2014
Dead
Alive
Injured*
QC?
Subl
0
10
1
u
Sub 2
0
9
0
U
Sub 3
0
10
0
u
Sub 4
0
9
0
Ld CT
Sub 5
0
9
0
U
Sub 6
0
3
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
50
1
Damage Notes:
Replicate _3_
Dead
Alive
Injured*
QC?
Subl
0
15
0
Ld CT
Sub 2
0
14
0
U
Sub 3
0
16
0
U
Sub 4
0
15
0
U
Sub 5
0
10
0
u
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
70
0
Damage Notes:
Replicate _4_
Dead
Alive
Injured*
QC?
Subl
0
14
0
L£J CT
Sub 2
0
14
0
U
Sub 3
0
13
0
U
Sub 4
0
12
0
U
Sub 5
0
11
0
U
Sub 6
0
14
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
78
0
Damage Notes:
Replicate _5_
Dead
Alive
Injured*
QC?
Subl
0
13
0
U
Sub 2
0
12
0
U
Sub 3
0
12
0
Ld CT
Sub 4
0
15
0
U
Sub 5
0
15
0
U
Sub 6
0
11
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
78
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
Eliot Hoitsma
Replicate _1
Mortality Data Sheet - Fish Date:
Test Type: 0 Control Q Pump Q Gravity
9/9/2014
Dead
Alive
Injured*
QC?
Subl
0
11
0
U
Sub 2
0
9
0
U
Sub 3
0
7
1
U CT
Sub 4
0
7
0
U
Sub 5
0
7
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
41
1
Damage Notes:
Replicate _2_
Bloody face
Dead
Alive
Injured*
QC?
Subl
0
9
0
U
Sub 2
0
8
0
Ld CT
Sub 3
0
11
0
U
Sub 4
0
8
0
U
Sub 5
0
8
0
U
Sub 6
0
6
0
U
Sub 7
0
10
0
U
Sub
U
Sub
U
Sub
U
Total
0
60
0
Damage Notes:
Replicate _3_
Dead
Alive
Injured*
QC?
Subl
0
17
0
L£J CT
Sub 2
0
6
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
23
0
Damage Notes:
Replicate _5_
Dead
Alive
Injured*
QC?
Subl
0
13
0
U
Sub 2
0
10
0
U
Sub 3
0
11
0
L_l CT
Sub 4
0
14
0
U
Sub 5
0
13
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
61
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
Technician:
Replicate _1
Tracey Ladder
Mortality Data Sheet - Fish
Test Type: 0 Control D Pump D Gravity
Date:
9/9/2014
Dead
Alive
Injured*
QC?
Subl
1
4
0
U
Sub 2
1
8
0
Ld CT
Sub 3
0
12
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
2
24
0
Damage Notes:
Replicate _2
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate _4
Dead
Alive
Injured*
QC?
Damage Notes:
Replicate
Dead
Alive
Injured*
QC?
Subl
0
16
0
U
Subl
0
20
0
U
Sub
U
Sub 2
0
9
0
U
Sub 2
0
9
0
U
Sub
U
Sub 3
0
8
0
U
Sub 3
0
11
0
U
Sub
U
Sub 4
0
11
0
Ld CT
Sub 4
0
14
0
Ld CT
Sub
U
Sub 5
0
8
0
U
Sub 5
0
11
0
U
Sub
U
Sub 6
0
9
0
U
Sub 6
0
11
0
U
Sub
U
Sub 7
0
8
0
U
Sub
U
Sub
U
Sub 8
0
8
0
U
Sub
U
Sub
U
Sub 9
0
8
0
U
Sub
U
Sub
U
Sub 10
0
7
0
U
Sub
U
Sub
U
Total
0
92
0
Total
0
76
0
Total
0
0
0
Damage notes:
* Fish which are alive but have a noticeable injury. NOTE, these are a subset of alive and all fish should be tallied as either live or dead.
-------
ERG Ballast Fish Data
Pump
Dead
Live
TOTAL
% Mort
RepJ
65
56
121
53.7%
Rep 2
51
67
118
43.2%
Rep 3
31
58
89
34.8%
Rep 4
45
51
96
46.9%
Rep 5
72
86
158
45.6%
TOTAL
264
318
582
45.4%
Gravity
Dead
Live
TOTAL
% Mort
RepJ
0
113
113
0.0%
Rep 2
0
121
121
0.0%
Rep 3
1
94
95
1.1%
Rep 4
0
127
127
0.0%
Rep 5
0
195
195
0.0%
TOTAL
1
650
651
0.2%
Control
Dead
Live
TOTAL
% Mort
RepJ
2
115
117
1 .7%
Rep 2
0
152
152
0.0%
Rep 3
0
93
93
0.0%
Rep 4
0
154
154
0.0%
Rep 5
0
139
139
0.0%
TOTAL
2
653
655
0.3%
-------
Technician:
David Rosier
Replicate _1
Mortality Data Sheet - Eggs
Test Type: D Control [7] Pump D Gravity
Date:
9/10/2014
Dead
Alive
Indeterminate*
QC?
Subl
0
10
0
u
Sub 2
0
4
0
U
Sub 3
0
48
1
U
Sub 4
1
32
0
LJ CT
Sub 5
0
1
I
U
Sub 6
0
6
0
U
Sub?
0
17
2
U
Sub_
U
Sub_
U
Sub_
U
Total
1
124
4
Damage Notes:
Replicate _2
Dead
Alive
Indeterminate*
QC?
Subl
0
14
0
U
Sub 2
0
5
0
U
Sub 3
2
15
4
Ld CT
Sub 4
0
4
0
U
Sub 5
0
8
1
U
Sub 6
0
3
0
U
Sub?
0
3
0
U
Sub 8
0
1
0
U
Sub 9
0
1
0
U
Sub 10
0
1
0
U
Total
2
55
5
Damage Notes:
Replicate _3
Dead
Alive
Indeterminate*
QC?
Subl
0
16
0
Ld CT
Sub 2
0
1
0
U
Sub 3
0
11
2
U
Sub 4
0
2
1
U
Sub 5
0
4
0
U
Sub 6
0
1
0
U
Sub?
0
2
0
U
Sub 8
0
1
0
U
Sub
U
Sub
U
Total
0
38
3
Damage Notes:
Replicate
Dead
Alive
Indeterminate*
QC?
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
0
0
Damage notes:
* If the egg is so undeveloped that it does not have a visible heart, it will be recorded as "indeterminate"
-------
Technician:
Eliot Hoitsma
Replicate _1
Mortality Data Sheet - Eggs
Test Type: D Control DPump [7] Gravity
Date:
9/10/2014
Dead
Alive
Indeterminate*
QC?
Subl
0
4
0
Ld CT
Sub 2
0
4
0
U
Sub 3
0
1
2
Ld CT
Sub 4
1
1
1
U
Sub 5
0
1
0
U
Sub 6
0
3
0
U
Sub?
0
2
0
U
Sub 8
0
4
0
U
Sub_
U
Sub_
U
Total
1
26
3
Damage Notes:
Replicate _2
Dead
Alive
Indeterminate*
QC?
Subl
0
4
0
U
Sub 2
0
1
1
U
Sub 3
0
6
1
Ld CT
Sub 4
0
4
0
U
Sub 5
0
1
0
U
Sub 6
0
2
0
U
Sub?
0
3
0
U
Sub 8
0
4
0
U
Sub_
U
Sub_
U
Total
0
25
2
Damage Notes:
Replicate _3
Dead
Alive
Indeterminate*
QC?
Subl
0
2
0
U
Sub 2
0
1
0
U
Sub 3
0
12
0
Ld CT
Sub 4
0
4
1
U
Sub 5
1
18
0
U
Sub 6
0
4
1
U
Sub?
0
1
0
U
Sub 8
0
4
0
U
Sub 9
0
10
0
U
Sub
U
Total
1
56
2
Damage Notes:
Replicate
Dead
Alive
Indeterminate*
QC?
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
0
0
Damage notes:
* If the egg is so undeveloped that it does not have a visible heart, it will be recorded as "indeterminate"
-------
Technician:
Tracey Ladder
Mortality Data Sheet - Eggs
Test Type: 0 Control DPump D Gravity
Date:
9/10/2014
Replicate _1
Dead
Alive
Indeterminate*
QC?
Subl
0
19
2
Ld CT
Sub 2
0
6
2
u
Sub 3
0
60
4
U
Sub 4
2
34
1
U
Sub 5
0
8
0
U
Sub
U
Sub
U
Sub
U
Sub_
U
Sub
U
Total
2
127
9
Damage Notes:
Replicate _2
Dead
Alive
Indeterminate*
QC?
Subl
0
2
0
U
Sub 2
0
1
0
U
Sub 3
0
8
3
Ld CT
Sub 4
0
1
0
U
Sub 5
0
2
1
U
Sub 6
0
3
0
U
Sub?
0
2
0
U
Sub 8
0
0
1
U
Sub 9
0
7
0
U
Sub 10
0
0
1
U
Total
0
26
6
Damage Notes:
Replicate _2 (Cont)_
Dead
Alive
Indeterminate*
QC?
Sub 11
0
1
0
U
Sub 12
0
1
0
Ld CT
Sub 13
0
12
0
Ld CT
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
14
0
Damage Notes:
Replicate _3_
Dead
Alive
Indeterminate*
QC?
Subl
0
0
1
u
Sub 2
0
0
1
U
Sub 3
0
1
0
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Sub
U
Total
0
1
2
Damage notes: NOTE, NO QC WAS DONE DUE TO THE LOW COUNTS, PROCESSING WAS OVER TOO SOON.
* If the egg is so undeveloped that it does not have a visible heart, it will be recorded as "indeterminate"
-------
ERG Ballast Egg Data
Pump
Dead
Live
TOTAL
% Mort
RepJ
1
124
125
0.8%
Rep 2
2
55
57
3.5%
Rep 3
0
38
38
0.0%
TOTAL
3
217
220
1.4%
Gravity
Dead
Live
TOTAL
% Mort
RepJ
1
26
27
3.7%
Rep 2
0
25
25
0.0%
Rep 3
1
56
57
1 .8%
TOTAL
2
107
109
1.8%
Control
Dead
Live
TOTAL
% Mort
RepJ
2
127
129
1 .6%
Rep 2
0
40
40
0.0%
Rep 3
0
1
1
0.0%
TOTAL
2
168
170
1.2%
-------
Mortality QC Data Sheet
QA Analyst: Chris Turner
Date:
9/9/2014
Technician
Ledder
Hoitsma
Rosier
Hoitsma
Ledder
Rosier
Ledder
Hoitsma
Hoitsma
Ledder
Rosier
Ledder
Hoitsma
Ledder
Rosier
Hoitsma
Ledder
Hoitsma
Rosier
Rosier
Hoitsma
Ledder
Rosier
Ledder
Hoitsma
Ledder
Rosier
Hoitsma
Fish/Egg
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Treatment
Control
Control
Control
Gravity
Gravity
Pump
Pump
Pump
Control
Control
Gravity
Pump
Pump
Pump
Control
Control
Gravity
Pump
Pump
Control
Gravity
Control
Pump
Pump
Gravity
Pump
Control
Control
Rep#
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
4
4
5
5
Sub#
2
3
4
1
6
2
2
2
2
4
1
1
3
2
1
1
1
1
1
1
2
4
1
1
12
3
3
3
Dead
Orig.
1
0
0
0
0
11
8
12
0
0
0
13
13
7
0
0
0
2
4
0
0
0
9
10
0
2
0
0
QC
1
0
0
0
0
12
8
13
0
0
0
17
14
7
0
0
0
2
4
0
0
0
9
11
0
2
0
0
RPD
0.0%
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!
8.7%
0.0%
8.0%
#DIV/0!
#DIV/0!
#DIV/0!
26.7%
7.4%
0.0%
#DIV/0!
#DIV/0!
#DIV/0!
0.0%
0.0%
#DIV/0!
#DIV/0!
#DIV/0!
0.0%
9.5%
#DIV/0!
0.0%
#DIV/0!
#DIV/0!
Alive
Orig.
8
7
9
14
6
2
3
1
8
11
13
12
5
4
15
17
23
9
11
14
12
14
1
16
8
9
12
11
QC
8
7
9
14
6
2
3
1
8
11
13
15
4
4
15
17
22
9
11
14
11
14
1
17
8
9
12
11
RPD
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
22.2%
22.2%
0.0%
0.0%
0.0%
4.4%
0.0%
0.0%
0.0%
8.7%
0.0%
0.0%
6.1%
0.0%
0.0%
0.0%
0.0%
Accuracy:
Dead
I Alive
1
100%
100%
100%
100%
100%
92%
100%
92%
100%
100%
100%
76%
93%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
91%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
80%
80%
100%
100%
100%
96%
100%
100%
100%
109%
100%
100%
94%
100%
100%
100%
100%
-------
Mortality QC Data Sheet
QA Analyst: Chris Turner
Date:
9/9/2014
Technician
Ledder
Rosier
Hoitsma
Rosier
Ledder
Fish/Egg
Fish
Fish
Fish
Fish
Fish
Treatment
Gravity
Pump
Pump
Pump
Gravity
Rep#
5
5
5
5
5
Sub#
3
1
1
6
16
Dead
Orig.
0
4
14
3
0
QC
0
4
13
3
0
RPD
#DIV/0!
0.0%
7.4%
0.0%
#DIV/0!
Alive
Orig.
9
9
1
12
11
QC
9
9
2
12
11
RPD
0.0%
0.0%
66.7%
0.0%
0.0%
Accuracy:
Dead
| Alive
100%
100%
108%
100%
100%
100%
100%
50%
100%
100%
Accuracy:
Dead counts - Only 1 of 33 QC'd
subsamples where accuracy was
less than target of 90%
Live counts - Only 1 of 33 QC'd
subsamples where accuracy was
less than target of 90%;
difference is excessively low
(50%) in the one instance
because of very low sample
numbers
Precision:
Dead counts - Only 1 of 33 QC'd subsamples where RPD exceeded target of 10%
Live counts - Only 3 of 33 QC'd subsamples where RPD exceeded 10%; RPD is excessively high (66.7%) in the one instance because of very low sample numbers
No perceived data use limitations
-------
Mortality QC Data Sheet
QA Analyst: Chris Turner
Date:
9/10/2014
Technician
Ledder
Hoitsma
Hoitsma
Rosier
Ledder
Hoitsma
Rosier
Ledder
Rosier
Hoitsma
Fish/Egg
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Eggs
Treatment
Control
Gravity
Gravity
Pump
Control
Gravity
Pump
Control
Pump
Gravity
Rep#
1
1
1
1
2
2
2
2
3
3
Sub#
1
1
3
4
3
3
3
12
1
3
Dead
Orig.
0
0
0
1
0
0
2
0
0
0
QC
0
0
0
1
0
0
1
0
0
0
RPD
#DIV/0!
#DIV/0!
#DIV/0!
0.0%
#DIV/0!
#DIV/0!
66.7%
#DIV/0!
#DIV/0!
#DIV/0!
Alive
Orig.
19
4
7
32
8
6
15
1
16
12
QC
19
4
7
32
8
6
15
1
16
12
RPD
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
Indeterminate
Orig.
2
0
2
0
3
1
4
0
0
0
QC
2
0
2
0
3
1
5
0
0
0
RPD
0.0%
#DIV/0!
0.0%
#DIV/0!
0.0%
0.0%
22.2%
#DIV/0!
#DIV/0!
#DIV/0!
Precision:
Dead counts - Only 1 of 10 QC'd subsamples where RPD exceeded target of 10%; RPD is excessively high (66.7%) in the one instance because of very low
sample numbers
Live counts - 0 of 10 QC'd subsamples where RPD exceeded target of 10%
Indeterminate counts - Only 1 of 10 QC'd subsamples where RPD exceeded target of 10%; RPD is excessively high (22.2%) in the one instance because of
low sample numbers
Accuracy:
JDead
100%
100%
100%
100%
100%
100%
50%
100%
100%
100%
JAIive
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
[indeterminate
100%
100%
100%
100%
100%
100%
80%
100%
100%
100%
Accuracy:
Dead counts - Only 1 of 10 QC'd subsamples where
accuracy was less than 100%; difference is excessively low
(50%) in the one instance because of very low sample
numbers
Live counts - 0 of 10 QC'd subsamples where accuracy was
less than 100%
Indeterminate counts - Only 1 of 10 QC'd subsamples
where accuracy was less than 100%; difference is low
(80%) in the one instance because of low sample numbers
No perceived data use limitations
No perceived data use limitations
-------
Fish/Egg Mortality Tests - GLEC log
Fish/Egg
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Treatment
Control
Gravity
Pump
Control
Gravity
Pump
Control
Gravity
Pump
Rep#
1
1
1
2
2
2
3
3
3
Date
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
Time In
10:22
10:27
10:33
11:36
11:41
11:46
12:48
12:51
12:54
Temp
Start
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Temp
10:40
20.0
20.0
20.0
Temp
10:51
20.0
20.0
20.0
Temp
11:48
20.0
20.0
20.0
Temp
12:01
20.0
20.5
21.0
Temp
13:10
20.0
20.0
20.0
Time
Finished
10:45
10:51
10:51
11:48
11:57
12:03
12:58
13:05
13:05
Comments
preserved
Specimens
preserved
Specimens
preserved
Specimens
Fish/Egg
Fish
Fish
Fish
Fish
Fish
Fish
Treatment
Control
Gravity
Pump
Control
Gravity
Pump
Rep#
4
4
4
5
5
5
Date
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
9/9/14
Time In
13:58
14:00
14:04
15:05
15:06
15:08
Temp
Start
20.0
20.0
20.0
20.0
20.5
20.5
Temp
14:15
20.0
20.0
20.0
Temp
14:23
20.0
20.0
20.0
Temp
15:20
20.5
20.5
20.5
Temp
Temp
Time
Finished
14:08
14:16
14:23
15:14
15:30
15:33
Comments
Preserved
Specimens
-------
Initial Examination of Eggs prior to testing
Live
5
7
7
7
14
18
8
13
9
88
Dead
0
0
4
0
0
1
0
0
0
5
Indeterminate
2
1
1
0
1
2
1
0
1
9
% Mortality
0.0%
0.0%
36.4%
0.0%
0.0%
5.3%
0.0%
0.0%
0.0%
5.4%
QC'd
s
n
n
0
n
n
n
0
n
-------
Sampling Report for the Vessel General Permitting Program
Pump Mortality Study Appendix E
Appendix E:
LABORATORY QUALITY ASSURANCE DATA
-------
ERG Superior Ballast Water - Quality Assurance Summary and Statement Regarding Data Use
Primary Data collected by:
GLEC Senior Tech Dave Rosier
GLECTech Eliot Hoitsma
LSNERRTech Tracey Ledder
Field QC Analyst: Chris Turner
QA/QC effort included:
100% QC of raw data against data entered into Excel spreadsheet by Deb Turner (GLEC).
Additional 10 to 20% QA of comparison of raw data against data entered into spreadsheet, including all
calculations by Tyler Linton (GLEC).
100% QA of information from chain of custody against data spreadsheet by Tyler Linton (GLEC).
Results:
No errors found in QA effort
Egg Analysis:
Analytical Completeness:
QC goal was at least 1 of every 10 samples checked by a second analyst; also at minimum one of every
subsample per replicate checked by a second analyst. Goal was met.
10 of 68 subsamples QC'd = Duplicate QC of every 6.8 subsamples observed; Chris Turner (CT)
Precision:
Dead counts - Only 1 of 10 QC'd subsamples where RPD exceeded target of 10%; RPD is excessively
high (66.7%) in the one instance because of very low sample numbers
Live counts - 0 of 10 QC'd subsamples where RPD exceeded target of 10%
Indeterminate counts - Only 1 of 10 QC'd subsamples where RPD exceeded target of 10%; RPD is
excessively high (22.2%) in the one instance because of low sample numbers
Accuracy:
Dead counts - Only 1 of 10 QC'd subsamples where accuracy was less than 100%; difference is
excessively low (50%) in the one instance because of very low sample numbers
Live counts - 0 of 10 QC'd subsamples where accuracy was less than 100%
Indeterminate counts - Only 1 of 10 QC'd subsamples where accuracy was less than 100%; difference
is low (80%) in the one instance because of low sample numbers
Findings -
-------
No perceived data use limitations based on completeness, precision or accuracy
Fish Analysis:
Analytical Completeness:
33 of 163 subsamples QC'd = Duplicate QC of every 4.9 subsamples; Chris Turner (CT)
Precision:
Dead counts - Only 1 of 33 QC'd subsamples where RPD exceeded target of 10%
Live counts - Only 3 of 33 QC'd subsamples where RPD exceeded 10%; RPD is excessively high
(66.7%) in the one instance because of very low sample numbers
Accuracy:
Dead counts - Only 1 of 33 QC'd subsamples where accuracy was less than target of 90%
Live counts - Only 1 of 33 QC'd subsamples where accuracy was less than target of 90%; difference is
excessively low (50%) in the one instance because of very low sample numbers
Findings -
No perceived data use limitations based on completeness, precision or accuracy
------- |