Exposure-Related Effects of Pseudomonas fluorescens

(Pf-CL145A) on Juvenile Unionid Mussels

By Kerry L. Weber, James A. Luoma, Denise A. Mayer, Doug B. Aloisi, and Nathan L. Eckert

Open-File Report 2015–1066

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2015

For more information on the USGS—the Federal source for science about the Earth,

its natural and living resources, natural hazards, and the environment—visit

http://www.usgs.gov or call 1–888–ASK–USGS (1–888–275–8747)

For an overview of USGS information products, including maps, imagery, and publications,

visit http://www.usgs.gov/pubprod

To order this and other USGS information products, visit http://store.usgs.gov

Any use of trade, firm, or product names is for descriptive purposes only and does not imply

endorsement by the U.S. Government.

Although this information product, for the most part, is in the public domain, it also may

contain copyrighted materials as noted in the text. Permission to reproduce copyrighted items

must be secured from the copyright owner.

Suggested citation:

Weber, K.L., Luoma, J.A., Mayer, D.A., Aloisi, D.B., and Eckert, N.L. 2015, Exposure-related effects of 

Pseudomonas fluorescens (Pf-CL145A) on juvenile unionid mussels: U.S. Geological Survey Open-File Report 

2015–1066, 663 p., http://dx.doi.org/10.3133/ofr20151066. 

ISSN 2331-1258 (online)

ii
Acknowledgments

This study was funded through a combination of a U.S. Environmental Protection Agency Great 

Lakes Restoration Initiative grant and U.S. Geological Survey appropriated funds. The authors thank 

personnel from the Upper Midwest nvironmental Sciences Center, including Theresa M. Schreier, 

Maren Tuttle-Lau, Todd J. Severson, Jeremy K. Wise, Samuel M. Stafslien, Aaron R. Cupp, Pajtshiab 

L. Moua, Jonathan T. Vannata, and Daniel G. Burke, for assisting with data collection. We thank Mark 

Gaikowski, who assisted with study design and Brian Gray, who assisted with data analysis.

iii

Contents

Acknowledgments.......................................................................................................................................................iii

Abstract ...................................................................................................................................................................... 9

Introduction............................................................................................................................................................... 10

Materials and Methods ............................................................................................................................................. 11

Experimental Design................................................................................................................................................. 11

Test Article ............................................................................................................................................................ 12

Test System.......................................................................................................................................................... 12

Test Animals ......................................................................................................................................................... 15

Test Article Solutions ............................................................................................................................................ 16

Dosing................................................................................................................................................................... 16

Water Chemistry ................................................................................................................................................... 17

Survival Assessment............................................................................................................................................. 17

Data Analysis............................................................................................................................................................ 17

Results...................................................................................................................................................................... 18

Conclusion................................................................................................................................................................ 23

References Cited...................................................................................................................................................... 24

Appendix 1. Study Protocol With Data Forms........................................................................................................... 26

Appendix 2. Deviations From the Study Protocol.................................................................................................... 130

Appendix 3. Randomization Assignments............................................................................................................... 144

Appendix 4. Test Article Information ....................................................................................................................... 274

Appendix 5. Test Animal Information ...................................................................................................................... 341

Appendix 6. Water Quality and System Conditions ................................................................................................ 357

Appendix 7. Animal Feed Information..................................................................................................................... 539

Appendix 8. Juvenile Mussel Survival Summary and Statistical Analysis............................................................... 589

v
Figures

Figure 1. Test system used for dosing juvenile unionid mussels....................................................................... 13

Figure 2. Schematic of test system used for dosing juvenile unionid mussels. ................................................. 14

Figure 3. Example of three-section test chambers............................................................................................ 14

Figure 4. Three-section test chamber schematic: inflow section (left), center section containing juvenile unionid 

mussels (center), and outflow section (right). .......................................................................................................... 15

Figure 5. Representative juvenile unionid mussels displaying active foot movement. The photograph is of 

Lampsilis cardium juvenile unionid mussels at the end of the 7-day postexposure holding period.......................... 16

Tables

Table 1. Identification and characteristics of test animals and test articles formulations.................................. 11

Table 2. Mean (standard deviation) dissolved oxygen, temperature, and pH range by treatment group during 

the preexposure, exposure, and holding periods..................................................................................................... 19

Table 3. Mean (standard deviation) alkalinity, water hardness, and conductivity of preexposure and exposure 

termination water samples....................................................................................................................................... 21

Table 4. Mean (standard deviation) total ammonia nitrogen (TAN) and un-ionized ammonia (NH3) at 24 hour 

during the exposure period. ..................................................................................................................................... 22

Table 5. Mean (standard deviation) recovery and percent survival of unionid mussels exposed to spray-dried 

powder (SDP) or freeze-dried powder (FDP), by formulation type and treatment group.......................................... 23

vi
Conversion Factors

International System of Units to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

micrometer (μm) 3.937×10-5 inch (in.)

millimeter (mm) 0.03937 inch (in.)

Volume

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

milliliter (mL) 0.03382 ounce, fluid (fl. oz)

Flow rate

milliliter per minute (mL/min) 0.0002642 gallon per minute (gal/min)

Mass

milligram (mg) 3.527 ×10-5 ounce, avoirdupois (oz)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as °F = (1.8 × °C) + 32.

Conductivity is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 25 °C).

Concentrations of chemical constituents in water are given in milligrams per liter (mg/L).

vii
Abbreviations

AI active ingredient of Pseudomonas fluorescens

Alk. alkalinity as CaCO3

CaCO3 calcium carbonate

CL145A strain of Pseudomonas fluorescens

Cond. conductivity

DO dissolved oxygen

EPA U.S. Environmental Protection Agency

FDP freeze-dried powder

Hard. hardness as CaCO3

HD heat-deactivated Pseudomonas fluorescens

L × W × H length by width by height

NH3 un-ionized ammonia

NH3-N/L un-ionized ammonia nitrogen per liter

Pf-CL 145A Pseudomonas fluorescens strain CL 145A

SDP spray-dried powder

TAN total ammonia nitrogen

Temp temperature (in °C)

UMESC Upper Midwest Environmental Sciences Center

USGS U.S. Geological Survey

viii
Exposure-Related Effects of Pseudomonas fluorescens

(Pf-CL145A) on Juvenile Unionid Mussels 

By Kerry L. Weber,1 James A. Luoma,

1 Denise A. Mayer,2 Doug B. Aloisi,3 and Nathan L. Eckert3

Abstract

The exposure-related effects of a commercially prepared spray-dried powder (SDP) or 

freeze-dried powder (FDP) formulation of Pseudomonas fluorescens (strain CL145A) on the survival of 

seven species of newly metamorphosed (< 72 hours old) freshwater unionid mussels was evaluated. 

Forty unionid mussels of each species were randomly distributed to test chambers and each species 

independently exposed for 24 hours to a static dose of either SDP (four species: Lampsilis cardium, 

Lampsilis siliquoidea, Lampsilis higginsii, and Ligumia recta) or FDP (three species: Obovaria olivaria, 

Actinonaias ligamentina, and Megalonaias nervosa). 

Each test chamber was assigned to one of six treatment groups (n = four chambers per group) by 

using a randomized block design. The six treatment groups included (1) an untreated control group, 

(2) groups that received applications with nominal target active ingredient (AI) concentrations of 50, 

100, 200, and 300 milligrams per liter (mg/L), and (3) a group that received an application with a 

nominal target AI concentration of 300 mg/L of heat-deactivated test article (300 HD). After a 24-hour 

exposure period, water inflow to the test chambers was restored, and the unionid mussels were

maintained for an additional 7 days before they were assessed for survival.

Mean survival of four unionid mussels species exposed to SDP varied among species and 

treatment groups when compared to the untreated control groups. The results indicate that exposure to 

SDP-formulated P. fluorescens up to the maximum label concentration (100 mg/L AI) and up to three 

times the maximum label exposure duration (8 hours) is not likely to affect the survival of L. siliquoidea

and L. higginsii. Low mean survival in the L. recta control group (25.0 percent) indicates that results for 

L. recta should be interpreted with caution. Mean survival of the L. cardium was significantly lower in 

all treated groups (14.4 to 40.6 percent) compared to the control group (68.8 percent). These results 

indicate that further investigation on the impact of SDP-formulated P. fluorescens on L. recta and

L. cardium is warranted. 

Mean survival of three unionid mussels species exposed to FDP was not significantly different 

in the 50-, 100-, and 200-mg/L AI treatment groups and the 300 mg/L heat-deactivated treatment groups 

when compared to the control groups. Mean survival of O. olivaria and M. nervosa was significantly 

lower in the 300-mg/L AI treated groups (38.1 and 48.1 percent, respectively) compared to the control 

groups (71.9 and 88.1 percent, respectively). The results indicate that exposure to FDP-formulated 

P. fluorescens up to the maximum label concentration (100 mg/L AI) and up to three times the 

1

 U.S. Geological Survey. 

2 New York State Education Department.

3

 U.S. Fish and Wildlife Service. 

9 

 
maximum label exposure duration (8 hours) is not likely to affect the survival of O. olivaria, 

A. ligamentina, and M. nervosa. 

Introduction

Freshwater unionid mussel populations of North America were historically considered the most 

diverse in the world, but diversity has rapidly declined and continues to decrease in response to a variety 

of anthropogenic influences such as habitat degradation and alteration, pollution, and overharvest 

(Williams and others, 1993; Strayer and others, 2004). Out of the 250 species of unionid mussels in 

North America listed on the International Union for Conservation Red List, 39 species have a stable or 

increasing population, whereas 72 percent are currently threatened, imperiled, or candidates for listing,

and 12 percent are extinct (http://www.iucnredlist.org/, accessed January 7, 2015). According to 

Ricciardi and Rasmussen (1999), an estimated 127 unionid mussel species will be lost within the next 

100 years; however, this conservative 6.4 percent decadal extinction rate does not factor in extirpations 

related to invasions by dreissenid mussels (zebra, Dreissena polymorpha, and quagga, D. rostriformis 

bugensis). 

Because of their high reproductive capacity and their planktonic lifestage, dreissenid mussels 

can quickly disperse and inundate aquatic environments (Mackie, 1991; Birnbaum, 2011; Benson and 

others 2015). Since their introduction and establishment in the Great Lakes in the 1980s, zebra mussels 

have been documented in 680 lakes within 27 states (U.S. Geological Survey, 2014). Zebra mussels 

adhering to unionid mussels have been documented to contain > 10,000 individuals weighing from two

to five times as much as the unionid mussel (Hebert and others, 1991; Mackie, 1991). For example, in a 

power plant canal in Lake Erie, Schloesser and Kovalak (1991) observed in excess of 10,000 zebra 

mussels colonized on unionid mussels and a mean colonization estimate of 6,800 zebra mussels per 

unionid. 

Zebra mussel colonization may interfere with unionid mussel locomotion, feeding, reproduction, 

and respiration (Mackie, 1991; Schloesser and Kovalak, 1991), and it has been linked to the starvation 

of unionids through competition for food resources (Baker and Hornbach, 1997; Strayer and Malcom, 

2007). Heavily colonized unionid mussels may lack the energy reserves required to survive winter, and 

their ability to burrow into the sediment to avoid environmental stressors may be impeded (Nalepa, 

1994; Schloesser and Nalepa, 1994). Unionid mussel extirpations and population declines have been 

linked to zebra mussels (Burlakova and others, 2000); prediction models estimate that colonization by 

as few as 100 zebra mussels can result in unionid mussel mortality (Ricciardi and others, 1995).

Federal and state management agencies have implemented recovery and propagation programs 

for threatened and endangered unionid species that coincide with legislation and programs for control 

and removal of introduced and invasive species such as zebra mussels (Neves, 2004; Nalepa and 

Schloesser, 2014). There is a lack of safe and effective tools to control dreissenid mussels in open-water 

environments, and the impacts of zebra mussels on unionid mussels have made it clear that such tools 

are of utmost importance. One potential option for limited open-water control of dreissenid mussels is a 

commercially prepared formulation of a specific strain (CL145A) of the common soil bacterium 

Pseudomonas fluorescens (Molloy, Mayer, Gaylo, Morse, and others, 2013). Pseudomonas fluorescens

strain CL145A was discovered to be selectively toxic to dreissenid mussels by scientists at the New 

York State Museum Field Research Laboratory (Cambridge, New York). Ingestion of Pseudomonas 

fluorescens strain CL145A was found to cause cell necrosis in the epithelium lining of the dreissenid 

mussels’ digestive tract, which ultimately induces mortality (Molloy, Mayer, Gaylo, Burlakova, and 

others, 2013). 

10 
Newly metamorphosed unionid mussels are ecologically and physiologically different from adult 

unionid mussels (ASTM International, 2013). Therefore, it is critical to know the potential 

exposure-related effects of formulated P. fluorescens on the newly metamorphosed life stage of unionid 

mussels before P. fluorescens is applied to open-water environments containing unionid mussels. 

Recommendations in the standard guide for laboratory toxicity tests with freshwater unionid mussels 

include initiating tests on juvenile unionid mussels within 5 days of excysting from host fish and 

completing tests within 14 days because of high mortality rates observed 4 to 6 weeks after 

transformation (ASTM International, 2013). Consistent with these recommendations, the objective of 

this study was to determine the survival of newly metamorphosed unionid mussels after exposure to 

spray-dried powder (SDP) or freeze-dried powder (FDP) formulated P. fluorescens in a 24-hour 

exposure. 

Materials and Methods

The protocol for this study entitled “Effects of Pseudomonas fluorescens (Pf-CL145A) to newly 

metamorphosed juvenile unionid mussels from seven different unionid species” is presented in appendix 

1 (item 1). All methods and materials follow the written protocol except those instances that were 

identified in notes to file (appendix 1, items 2–3), study protocol amendments (appendix 1, items 4–12), 

and study deviations (appendix 2, items 1–12).

Experimental Design

Laboratory trials were completed at the U.S. Geological Survey’s Upper Midwest 

Environmental Sciences Center (UMESC) in La Crosse, Wisconsin, to assess the survival of newly 

metamorphosed unionid mussels following exposure to commercially prepared formulations of 

P. fluorescens, strain CL145A (SDP or FDP formulation). Seven species (table 1) endemic to the Great 

Lakes and (or) Mississippi River Basins were evaluated in the study trials. The four species of newly 

metamorphosed unionid mussels exposed to SDP were Lampsilis cardium, Lampsilis siliquoidea, 

Lampsilis higginsii, and Ligumia recta; the three species of newly metamorphosed unionid mussels 

exposed to FDP were Obovaria olivaria, Actinonaias ligamentina, and Megalonaias nervosa. Individual 

juvenile unionid mussels (< 72 hours old) were assessed for viability and randomly assigned to a test 

chamber in equal proportions (n = 960 per species; 40 per test chamber; appendix 3, items 7–12). 

Treatments were assigned to test chambers by using a randomized block design (appendix 3, 

items 13–18). Six treatment groups (n = 4 per treatment group) were tested and included (1) an 

untreated control, (2) nominal target treatment concentrations of 50, 100, 200, and 300 milligrams per 

liter (mg/L) based on active ingredient (AI), and (3) a nominal target treatment concentration of 

300 mg/L AI of heat-deactivated SDP or FDP test article (300 HD). The experimental unit for the trial 

was the individual test chamber.

At the conclusion of the 24-hour exposure period, the SDP- or FDP-treated water was drained

from each test chamber and each test chamber flushed with fresh water prior to resuming the chamber 

water inflow. The unionid mussels were maintained in the test chambers for an additional 7-day

postexposure holding period. At the conclusion of the 7-day postexposure holding period, each unionid 

mussel was individually assessed for survival as indicated by active foot movement. 

11 
Table 1. Identification and characteristics of test animals and test articles formulations.

Scientific 

name

Common name Abbreviation

Test article

Exposure date

Type Lot number Biological activity

(percent)

Obovaria 

olivaria Hickorynut HIC FDP 110510FD 98.7 ± 2.3 July, 12 2011

Actinonaias 

ligamentina Mucket MUC FDP 110510FD 98.7 ± 2.3 July 14, 2011

Megalonaias 

nervosa

Washboard WAS FDP 110928FD 96.0 ± 6.9 December 13, 2011

Lampsilis 

siliquoidea Fatmucket FAM SDP MBI-401 SDP 4655-12-Mix 85.3 ± 11.5 January 27, 2012 

Ligumia 

recta

Black sandshell BLS SDP TR4669-4-(7-8) 

2nd shipment 70.7 ± 2.3 April 17, 2012 

Lampsilis 

cardium Plain pocketbook PPB SDP TR4669-4-(5) 76.0 ± 8.0 May 16, 2012

Lampsilis 

higginsii Higgins’ eye HGE SDP TR4669-4-(5) 76.0 ± 6.9 May 26, 2012

Test Article

The test articles, produced by Marrone Bio Innovations, Inc. (Davis, California), were a 

commercially prepared SDP formulation and FDP formulation of P. fluorescens (strain CL145A) 

containing 50 and 100 percent AI (weight-to-weight ratio P. fluorescens, strain CL145A), respectively. 

Multiple formulations of test article were used during the course of this study because of the withdrawal

of support for the FDP formulation by Marrone Bio Innovations, Inc. Data derived with the FDP 

formulation are included within the report; however, the formulation is not currently being 

manufactured.

Test article concentrations are reported as nominal target concentrations of active ingredient. 

Test article use was documented in test chemical logbooks (appendix 4, items 24–28). Verification of 

biological activity was determined for each lot of test article after use in the study by the New York 

State Museum Field Research Laboratory (Cambridge, New York) using their standard dreissenid 

mussel bioassay (appendix 4, items 29–34). The bioassays verified retention of biological activity for 

each lot of test article with mean mortality rates of 70.7 to 98.7 percent for treated zebra mussels 

compared to 0.0 to 4.0 percent in the untreated groups (table 1; appendix 4, items 29–34). 

Test System

Three independent test systems were constructed, and each system consisted of a series of 

24 glass test chambers (17.8 × 5.1 × 8.3 millimeters [mm]; length by width by height [L × W × H]) 

separated into two blocks (n = 12 per block; figs. 1 and 2). Polycarbonate distribution boxes (41.0 ×

26.5 × 15.2 centimeters [cm]; L × W × H) were mounted above the test chambers and were used to 

deliver water and food solution to the test chambers. Polycarbonate head boxes (41.0 × 26.5 × 15.2 cm; 

L × W × H) were mounted above the distribution boxes and were used to mix the food solution (similar 

to Meinertz and others, 2011) with the water supply and then to supply a constant flow of food-enriched 

12 
water to the distribution boxes through glass standpipes (≈ 5 mm inner diameter). Food solutions were 

delivered to the inflow of each headbox via a peristaltic pump (Masterflex® Digi-staltic drive, model 

77310; Cole-Parmer, Vernon Hills, Illinois) fitted with Masterflex L/S 16 tubing (figs. 1 and 2). 

Test chamber inflows were controlled through 21-gauge luer-lock dispensing needles attached to 

3-milliliter (mL) syringe bodies. The syringe body tips were cut to a length of approximately 3 cm, 

fitted into silicone stoppers, and then positioned in 19-mm holes in the bottom of the distribution boxes. 

Well water (20 ± 2 degrees Celsius [°C]) inflow to each test chamber was maintained between 7.0 to 

10.0 milliliters per minute to provide a minimum of one test chamber exchange per hour. During the 

exposure period, water inflow was interrupted and supplemental aeration was supplied to the test 

chambers. Indirect fluorescent lighting (85 to 405 lux) was provided on an 18 hours light: 6 hours dark

cycle as in accordance with ASTM International guidelines (2013). 

The test chambers (fig. 3) contained three separate sections; the center section (7.0 × 4.5 × 

8.0 cm; L × W × H) was used to house the test animals, and the outer sections (4.5 × 4.5 × 8.0 cm; L × 

W × H) were used to isolate the water inflow and outflows (fig. 4). The center sections were separated 

from the outer sections by two glass partitions. Each partition consisted of two identical 0.32-cm-thick 

glass plates (4.5 × 8 cm; width by height) with two 1.4-cm holes positioned in a horizontal plane 

approximately 0.8 cm from the bottom (inflow partitions) or 2.5 cm from the bottom (outflow 

partitions). Nitex® mesh (150 micrometers [μm]) was placed between the partition plates before 

bonding them with silicone sealant. The Nitex mesh retained the test animals while allowing for water 

inflow/outflow. Test chambers were uniquely identified to allow for identification of treatment type and 

replicate.

13 

Figure 1. Test system used for dosing juvenile unionid mussels. 
Figure 2. Schematic of test system used for dosing juvenile unionid mussels. 

14 

Figure 3. Example of three-section test chambers. 
Figure 4. Three-section test chamber schematic: inflow section (left), center section containing juvenile unionid 

mussels (center), and outflow section (right).

Test Animals

Seven species of newly metamorphosed unionid mussels (excysted < 72 hours) were acquired 

from the U.S. Fish and Wildlife Service Genoa National Fish Hatchery and used as the test animals 

(table 1; appendix 5, items 1–15). The unionid mussels were acclimated to test dilution water and 

temperature through periodic water exchanges prior to transport and upon arrival at UMESC. Unionid 

mussels were observed under a dissecting microscope (model SMZ745; Nikon Instruments, Melville, 

New York), and those actively displaying foot movement (fig. 5; n = 960 per species [40 unionid 

mussels per replicate × 4 replicates × 6 treatment groups]) were randomly distributed to test chambers in 

groups of 20 until each chamber received a total of 40 unionid mussels. The unionid mussels were 

distributed to the test chambers ≤ 12 hours prior to exposure initiation by using a pipette with a widebore pipette tip to lessen the chances of injury to the animals. 

The newly metamorphosed unionid mussels were provided a diet consisting of Chlorella

sorokiniana, Tetraselmis, and Nannochloropsis at a nominal target concentration of 2.0 mg/L (algae dry 

weight). The diet used similar species and quantities that were to be successful for rearing newly 

metamorphosed juvenile mussels in a continuous flow-through system (Meinertz and others, 2011). A 

continuous supply of 20 ± 2 °C well water containing the algae food solution was provided to the test 

chambers during the acclimation and 7-day postexposure holding periods. Algae solutions (3.6 liters 

[L]) were prepared daily in a 4-L Erlenmeyer flasks. The algae solution contained a 1:1:1 (dry weight) 

mixture of live chlorella algae (Kentucky Department of Fish and Wildlife Resources, Center for 

Mollusk Conservation, Frankfort, Kentucky), Tetraselmis Instant Algae®, and Nannochloropsis Instant 

Algae®. Instant algae pastes were obtained from Reed Mariculture (Campbell, Calif.). The algae 

15 
solutions were continuously mixed on stir plates and delivered via a peristaltic pump directly into the 

water inflow stream for the headboxes at 2.0 ± 0.2 mL/minute. Airstones were placed in the headboxes

to assure thorough mixing of the algae solutions with the well-water inflow before delivery to the 

distribution boxes and test chambers. The resulting algae-enriched water delivered to the test chambers 

contained the nominal target concentration of 2.0 mg/L (algae dry weight). 

Figure 5. Representative juvenile unionid 

mussels displaying active foot movement. The 

photograph is of Lampsilis cardium juvenile 

unionid mussels at the end of the 7-day 

postexposure holding period. 

Test Article Solutions

For each species tested, a 10,000-mg/L AI treatment stock solution was prepared by placing 

either 10 grams SDP or 5 grams FDP in a 500-mL volumetric flask containing well water. A 5-L batch 

exposure solution was prepared immediately prior to exposure for each treatment group by adding the 

appropriate amount of the treatment stock solution to well water (appendix 4, items 17–23). The 

nominal target concentrations of the batch exposure solutions were 50, 100, 200, and 300 mg/L of active 

ingredient. 

On each exposure day, a 10,000-mg/L AI heat-deactivated treatment stock solution was prepared 

by placing either 10 grams SDP or 5 grams FDP in a 500-mL volumetric flask containing approximately 

300 mL of well water. The solution was placed in a 70 °C water bath for 45 minutes to degrade the 

active components of the P. fluorescens, according to the methods developed at the New York State 

Museum Field Research Laboratory (D. Mayer, Director of the New York State Museum Field Research 

Laboratory, oral commun., 2010). After cooling, the solution was brought to a final volume of 500 mL 

with well water. A 5-L batch deactivated exposure solution was prepared for each species by adding 

150 mL of the stock solution to 4,850 mL of well water (appendix 4, items 17–23). The nominal target 

concentration of the batch heat-deactivated exposure solutions was 300 mg/L of active ingredient. 

Dosing

Batch exposure solutions were prepared and immediately applied to test chamber replicates in a 

sequence. Exposures commenced with control treatments followed by the 300 mg/L heat-deactivated 

16 
(positive control) treatment, then the treatments of 50, 100, 200, and 300 mg/L. Treatments were 

initiated by interrupting water inflows and then removing the inflow and outflow standpipes to drain the 

water. After draining, residual water in the test chambers (≈25 mL) was replaced with exposure solution 

by flushing ≈300 mL of exposure solution through the test chambers. After test-chamber flushing, the 

standpipes were seated, and the test chambers were filled with exposure solution. Test chambers were 

supplied with supplemental aeration through disposable glass pipets placed near the bottom center of the 

test chamber. 

At the conclusion of the 24-hour exposure period, the standpipes were removed from the test 

chambers and the exposure water drained. The chambers were flushed with well water (20 ± 2 °C), and 

water inflow was restored.

Water Chemistry

Water hardness, alkalinity, conductivity, and total ammonia nitrogen (TAN) were measured 

prior to exposure on water samples collected from each distribution box. Dissolved oxygen (DO), pH, 

and temperature also were measured prior to exposure in each distribution box and in two

indiscriminately selected test chambers for each species. 

Dissolved oxygen, pH, and temperature were measured in the center section of each test 

chamber at 6, 12, and 24 hours after application of the test article. Immediately after measurement of the 

water chemistry parameters, water samples (≈5 mL) were collected from the center section of the test 

chambers for determination of TAN. The samples were filtered through a 0.45-μm syringe filter, 

acidified to pH ≤ 2.5 with 10 percent sulfuric acid, and stored at ≈4 °C until analyzed for TAN by using 

the automated phenate method (Standard Method 4500G in American Public Health Association and 

others, 2012). Upon exposure termination, water hardness, alkalinity, and conductivity were measured 

in pooled water samples obtained from the center sections of test chamber replicates for each species. 

Dissolved oxygen, pH, and temperature were measured daily throughout the 7-day postexposure 

holding period in two representative test chambers for each species. Water hardness, alkalinity, 

conductivity, and TAN were measured once during the 7-day postexposure holding period on a 

representative test chamber for each species. 

Survival Assessment

Seven days after exposure, the contents of each test chamber were drained and rinsed through a 

150-μm sieve with well water (20 ± 2 °C) to collect the unionid mussels. The contents retained on the 

sieve were backwashed into a petri dish (100 mm × 150 mm) and viewed under a dissecting microscope 

to ascertain survival of the mussels. Survival of the recovered unionid mussels was indicated by 

observation of foot movement. Unrecovered unionid mussels were treated as mortalities within the data 

analysis.

Data Analysis

Water chemistry (DO, pH, temperature, water alkalinity, water hardness, conductivity, and 

ammonia) data analyses were limited to simple descriptive statistics calculated by using SAS® software 

version 9.4 (SAS Institute, Inc., Cary, North Carolina). Un-ionized ammonia fractions were calculated 

by using the pH and temperature measured at the time of sample collection according to the formula of 

Emerson and others (1975). 

Statistical comparisons of zebra mussel survival also were completed by using SAS® software 

version 9.4. Significance was declared at α ≤ 0.05. A generalized linear mixed model was used to 

17 
analyze the survival of unionid mussels in each treatment group (appendix 8, item 3). The proportion of 

mortalities ([number of dead + number of unrecovered unionid mussels at the end of postexposure 

period] compared to the original number of unionid mussels present) was modeled with a binomial 

distribution and a logit link function. A scale parameter was added to the model by using the 

random_residual statement. Unionid mussel survival in each treatment group was individually compared 

to the survival in the untreated control group by using a two-sided means comparison test.

Results

Preexposure water chemistry parameters (DO, pH, and temperature) are summarized in table 2 

and in appendix 6 (items 1–8). Dissolved oxygen ranged from 8.33 to 8.79 mg/L, temperature ranged 

from 19.9 to 21.0 °C, pH ranged from 7.7 to 8.3, water hardness ranged from 172 to 180 mg/L as 

calcium carbonate (CaCO3), alkalinity ranged from 118 to 130 mg/L as CaCO3, and conductivity ranged 

from 370 to 396 microsiemens (μS)/cm. The TAN remained ≤ 0.18 mg as NH3-N/L, and un-ionized 

ammonia remained ≤ 0.01 mg as NH3/L. 

Exposure period water chemistry parameters (DO, pH, and temperature) are summarized in table

2 and in appendix 6 (items 1–4). Dissolved oxygen remained above the ASTM International (2013) 

criterion of 4 mg/L throughout the entire testing period. Water hardness, water alkalinity, and 

conductivity are summarized in table 3 and in appendix 6 (items 1–4). Water chemistry values for 

pooled exposure termination included water hardness ranges from 178 to 192 mg/L as CaCO3, alkalinity 

ranges from 141 to 149 mg/L as CaCO3, and conductivity ranges from 382 to 444 μS/cm. 

The TAN and un-ionized ammonia from samples collected during the exposure period are 

summarized in table 4 and in appendix 6 (items 5–8). The TAN remained below the 2013 U.S. 

Environmental Protection Agency (EPA) criterion of 17 mg/L TAN at pH 7 and 20 °C for acute 

exposure through 12 hours of the exposure period for all species and treatment groups and through the 

24-hour exposure period for all control animals (tables 5b and 6 in EPA, 2013). The TAN in the L. recta

300 mg/L heat-deactivated treatment group (4.99 mg/L as NH3/L; table 4) was the only SDP-treatment 

group that exceeded the acute criterion. The TAN observed in the O. olivaria and A. ligamentina 100, 

200, 300 mg/L FDP-treatment groups and 300 mg/L heat-deactivated FDP-treatment group (range from 

3.50 to 11.66 mg/L as NH3/L, table 4) exceeded the acute criterion at 24 hours. In addition, the TAN 

observed in the M. nervosa 200 and 300 mg/L FDP-treatment groups and 300 mg/L heat-deactivated 

FDP-treatment group (ranged from 5.39 to 7.09 mg/L as NH3/L; table 4) also exceeded the acute 

criterion. 

The 7-day postexposure holding period water chemistry parameters are summarized in tables 2 

and 3 and in appendix 6 (items 9–12). Dissolved oxygen ranged from 7.93 to 8.41 mg/L, temperature 

ranged from 19.6 to 20.4 °C, pH ranged from 8.0 to 8.2, water hardness ranged from 174 to 178 mg/L as 

CaCO3, alkalinity ranged from 121 to 131 mg/L as CaCO3, and conductivity ranged from 363 to 

402 μS/cm. The TAN remained ≤ 0.10 mg as NH3-N/L, and un-ionized ammonia remained ≤ 0.01 mg 

as NH3/L.

Unionid mussel recovery and survival are summarized in table 5 and in appendix 8 (items 1–4). 

The recovery of unionid mussels from individual test chambers ranged from 70 to 100 percent. Multiple 

factors may have been responsible for unionid mussel recoveries of < 100 percent. First, unionid 

mussels may have been injured in the initial handling and placement process, during which unionid 

mussels were handled with a disposable pipet at least twice. If the injury resulted in mortality, then the 

valves may have degraded to the point of being indiscernible during the survival assessment. Secondly, 

the valves of newly metamorphosed unionid mussels are very fragile, and they may have been damaged 

during the collection process when they were rinsed on a sieve before being rinsed into a petri dish. 

18 
Mean survival of the SDP-control groups ranged from 25.0 percent (L. recta) to 87.5 percent

(L. siliquoidea). Survival of the SDP-treated groups varied between species; however, exposures up to 

the maximum label concentration of SDP-formulated P. fluorescens (100 mg/L AI) for up to three times 

the labeled exposure duration (8 hours) resulted in significant differences in survival for only L. cardium

and L. recta. Significant differences in L. cardium survival were detected between the control group and 

all treatment groups. A significant difference in L. recta survival was detected between the control 

group and the 100-mg/L treatment group. Although no other differences were detected in mean survival 

of L. recta treated groups compared to the control group, the low survival of the control group 

(25.0 percent) indicates that the results should be interpreted with caution. Mean survival of 

L. siliquoidea in the 200 AI, 300 AI, and 300-mg/L heat-deactivated treatment groups was significantly 

lower than that of the control group. Differences were detected in the mean survival between the 200- 

and 300-mg/L SDP-treatment and control treatment groups for L. higginsii. 

Mean survival of the FDP control groups ranged from 71.9 percent (O. olivaria) to 88.1 percent 

(M. nervosa). With the exception of the O. olivaria and M. nervosa 300-mg/L FDP-treatment groups, 

mean survival in all of the M. nervosa, O. olivaria, and A. ligamentina FDP-treated groups did not 

significantly differ from the control groups. Mean survival of the O. olivaria 300-mg/L FDP-treated 

group was significantly lower (p = 0.01) than the control group. Mean survival in the O. olivaria 50-, 

100-, and 200-mg/L FDP-treatment groups and the 300-mg/L heat-deactivated FDP-treatment group did 

not differ from that of the control group. The mean survival of the M. nervosa 300-mg/L FDP-treated 

group was significantly lower (p < 0.01) than that of the control group. 

Table 2. Mean (standard deviation) dissolved oxygen, temperature, and pH range by treatment group 

during the preexposure, exposure, and holding periods. 

[mg/L, milligrams per liter; FDP, freeze-dried powder; SDP, spray-dried powder; HIC, hickorynut; MUC, mucket; 

WAS, washboard; PPB, plain pocketbook; FAM, fatmucket; HGE, Higgins’ eye; BLS, black sandshell; DO 

dissolved oxygen; Temp, temperature; °C, degrees Celsius; 300 HD, 300 mg/L of heat-deactivated product] 

Sampling 

period

Water 

quality 

parameter

Treatment 

group 

(mg/L) 

FDP formulation SDP formulation

HIC MUC WAS PPB FAM HGE BLS

Pre–

exposure DO (mg/L) 8.33 8.77 8.70 8.33 8.50 8.79 8.77

pH 8.21 8.24 8.20 8.10 7.68 8.22 8.32

Temp (°C) 21.0 20.6 20.1 20.7 20.8 20.5 19.9

Exposure DO (mg/L) Control

8.6

(0.2)

8.7

(0.3)

8.5

(0.2)

8.7

(0.1)

8.7

(0.1)

8.5

(0.1)

8.7

(0.2)

50

8.3

(0.3)

8.5

(0.1)

8.3

(0.3)

8.6

(0.2)

8.5

(0.2)

8.3

(0.2)

8.8

(0.1)

100

7.9

(0.9)

8.5

(0.2)

8.3

(0.2)

8.4

(0.5)

8.5

(0.1)

8.3

(0.3)

8.5

(0.4)

19 
Sampling 

period

Water 

quality 

parameter

Treatment 

group 

(mg/L) 

FDP formulation SDP formulation

HIC MUC WAS PPB FAM HGE BLS

200

8.0

(0.8)

8.4

(0.1)

8.1

(0.4)

8.0

(0.8)

8.2

(0.6)

7.9

(0.7)

8.1

(0.7)

300

7.4

(1.2)

8.2

(0.4)

7.8

(0.6)

8.0

(0.8)

7.8

(1.0)

7.7

(0.9)

7.8

(1.2)

300 HD

7.7

(1.0)

8.0

(0.4)

8.1

(0.6)

8.1

(0.8)

7.8

(1.0)

8.0

0.6)

8.0

1.0)

pH Control 8.20–8.35 8.12–8.358.05–8.27 8.28–8.387.98–8.35 8.13–8.40 8.02–8.43

50 8.19–8.27 8.14–8.267.96–8.23 8.21–8.367.99–8.28 8.10–8.28 8.04–8.33

100 8.06–8.20 8.09–8.307.99–8.20 8.00–8.347.98–8.23 8.15–8.30 7.99–8.32

200 8.08–8.21 8.04–8.237.93–8.08 7.96–8.317.92–8.17 7.91–8.11 7.95–8.07

300 7.95–8.11 7.98–8.127.85–7.99 7.97–8.347.81–8.03 7.81–8.13 7.84–8.05

300 HD 7.97–8.06 7.93–8.017.82–8.01 7.88–8.377.80–8.12 7.97–8.14 7.85–8.15

Temp (°C) Control

19.2

(0.9)

19.1

(0.8)

19.6

(0.3)

19.9

(0.2)

19.1

(0.2)

19.8

(0.3)

19.5

(0.2)

50

19.3

(0.9)

19.1

(0.8)

19.6

(0.2)

19.7

(0.1)

19.1

(0.1)

19.9

(0.2)

19.6

(0.2)

100

19.2

(0.9)

19.1

(0.7)

19.7

(0.2)

19.6

(0.1)

19.1

(0.1)

19.9

(0.2)

19.6

(0.3)

200

19.2

(0.9)

19.2

(0.6)

19.6

(0.2)

19.8

(0.2)

19.0

(0.3)

19.9

(0.2)

19.7

(0.3)

300

19.1

(0.8)

19.1

(0.6)

19.6

(0.3)

19.8

(0.2)

19.0

(0.3)

20.0

(0.3)

19.5

(0.2)

300 HD

19.3

(0.9)

19.0

(0.7)

19.6

(0.2)

19.7

(0.1)

19.0

(0.2)

20.0

(0.1)

19.6

(0.2)

Holding DO (mg/L) 7.93 7.93 8.19 8.28 8.09 8.16 8.41

pH 8.22 8.24 8.13 8.12 8.01 8.20 8.20

Temp (°C) 19.6 19.8 19.9 20.4 20.3 20.4 20.0

20 
Table 3. Mean (standard deviation) alkalinity, water hardness, and conductivity of 

preexposure and exposure termination water samples.

[Alk., alkalinity as CaCO3; Hard., hardness as CaCO3; CaCO3, calcium carbonate; Cond., 

conductivity; mg/L, milligrams per liter; μS/cm, microsiemens per centimeter at 25 degrees 

Celsius; HIC, hickorynut; MUC, mucket; WAS, washboard; PPB, plain pocketbook; FAM,

fatmucket; HGE, Higgins’ eye; BLS, black sandshell]

Species

Preexposure Exposure termination1 Holding period

Alk. 

(mg/L)

Hard. 

(mg/L)

Cond. 

(μS/cm)

Alk. 

(mg/L)

Hard. 

(mg/L)

Cond. 

(μS/cm)

Alk. 

(mg/L)

Hard. 

(mg/L)

Cond. 

(μS/cm)

HIC

118 172 374 147

(15)

180

(4)

427

(35)

129 178 386

MUC

130 179 371 149

(15)

179

(3)

444

(37)

131 178 390

WAS

125 178 387 148

(12)

189

(2)

429

(29)

121 178 402

PPB

128 172 372 143

(10)

181

(2)

382

(5)

126 174 364

FAM

126 180 396 148

(12)

192

(7)

430

(24)

123 175 383

HGE

128 174 370 141

(8)

178

(2)

390

(18)

121 178 363

BLS

125 180 376 146

(13)

183

(1)

409

(18)

121 176 378

1 Exposure termination parameters were measured on pooled water samples for each treatment group.

21 
Table 4. Mean (standard deviation) total ammonia nitrogen (TAN) and un-ionized ammonia 

(NH3) at 24 hour during the exposure period. 

[FDP, freeze-dried powder; SDP, spray-dried powder; HIC, hickorynut; MUC, mucket; WAS, washboard; 

PPB, plain pocketbook; FAM, fatmucket; HGE, Higgins’ eye; BLS, black sandshell; TAN, total ammonia 

nitrogen; < , less than; mg NH3-N/L, milligrams un-ionized ammonia nitrogen per liter; 300 HD, 300 

mg/L of heat-deactivated product; NH3, un-ionized ammonia; mg/L milligrams per liter] 

Water quality 

parameter

Treatment 

group 

(mg/L)

FDP formulation SDP formulation

HIC MUC WAS PPB FAM HGE BLS

TAN

(mg NH3-N/L)

0 

0.02

(0.01)

0.01

(<0.01)

0.03

(0.02)

0.08

(0.03)

0.08

(0.02)

0.06

(<0.01)

0.07

(0.01)

50

1.26

(0.08)

1.36

(0.11)

1.32

(0.38)

0.29

(0.10)

0.72

(0.15)

0.21

(0.10)

0.19

(0.02)

100

3.50a

(0.26)

3.71a

(0.82)

2.56

(0.19)

0.66

(0.35)

1.47

(0.12)

1.22

(0.54)

2.16

(1.21)

200

7.80a

(0.72)

8.30a

(1.47)

5.39a

(1.02)

1.06

(0.79)

3.15

(0.42)

2.22

(0.51)

3.13

(1.90)

300

10.50a

(1.12)

11.45a

(1.21)

6.81a

(1.32)

1.30

(0.93)

4.23

(0.72)

2.50

(0.82)

4.26

(0.51)

300 HD

11.66a

(0.26)

10.67a

(0.43)

7.09a

(2.04)

1.29

(0.96)

4.15

(0.68)

3.53

(0.44)

4.99a

(0.13)

NH3

(mg/L)

0 

<0.01

(<0.01)

<0.01

(<0.01)

<0.01

(<0.01)

0.01

(<0.01)

0.01

(<0.01)

0.01

(<0.01)

0.01

(<0.01)

50

0.07

(0.01)

0.08

(0.01)

0.07

(0.03)

0.02

(0.01)

0.04

(0.01)

0.01

(0.01)

0.01

(<0.01)

100

0.16

(0.02)

0.22

(0.04)

0.11

(0.03)

0.04

(0.02)

0.08

(0.01)

0.08

(0.04)

0.13

(0.08)

200

0.37

(0.07)

0.41

(0.10)

0.19

(0.05)

0.04

(0.03)

0.13

(0.04)

0.08

(0.03)

0.13

(0.08)

300

0.36

(0.07)

0.46

(0.09)

0.19

(0.03)

0.05

(0.03)

0.13

(0.05)

0.08

(0.05)

0.15

(0.04)

300 HD

0.40

(0.03)

0.35

(0.01)

0.22

(0.02)

0.06

(0.05)

0.14

(0.03)

0.15

(0.02)

0.21

(0.06)

a TAN values exceed the acute exposure criterion (tables 5b and 6, U.S. Environmental Protection Agency, 2013).

22 
Table 5. Mean (standard deviation) recovery and percent survival of unionid mussels 

exposed to spray-dried powder (SDP) or freeze-dried powder (FDP), by formulation type and 

treatment group.

[Survival treatment means within the same column with letters are significantly different (p > 0.05) 

from the control means; mg/L, milligrams per liter; FDP, freeze-dried powder; SDP, spray-dried 

powder; HIC, hickorynut; MUC, mucket; WAS, washboard; PPB, plain pocketbook; FAM, fatmucket; 

HGE, Higgins’ eye; BLS, black sandshell] 

Treatment 

group 

(mg/L)

FDP formulation SDP formulation

HIC MUC WAS PPB FAM HGE BLS

Recovery Control

88.1

(4.7)

89.4

(10.1)

96.9

(1.3)

91.3

(5.2)

91.3

(2.5)

93.1

(5.5)

93.1

(5.5)

50

88.8

(12.7)

93.8

(4.3)

92.5

(5.4)

90.6

(5.2)

91.3

(5.2)

88.1

(8.8)

93.1

(2.4)

100

90.0

(8.4)

90.0

(10.2)

94.4

(3.8)

91.3

(7.5)

96.3

(3.2)

92.5

(6.5)

92.5

(5.4)

200

90.6

(5.2)

94.4

(4.7)

96.3

(6.0)

91.9

(3.8)

91.9

(2.4)

86.3

(13.8)

93.8

(6.0)

300

94.4

(8.3)

93.8

(1.4)

96.3

(1.4)

90.0

(7.4)

91.9

(2.4)

87.5

(9.4)

93.1

(5.5)

300HD

88.1

(5.9)

94.4

(2.4)

94.4

(5.2)

90.0

(3.5)

91.3

(4.3)

94.4

(3.1)

95.0

(0.0)

Survival Control

71.9

(9.4)

78.8

(10.3)

88.1

(5.9)

68.8

(9.7)

87.5

(3.5)

88.1

(9.7)

25.0

(13.4)

50

65.0

(20.3)

78.1

(6.3)

77.5

(6.1)

14.4a

(7.7)

75.6

(13.8)

79.4

(9.0)

21.3

(18.1)

100

67.5

(15.9)

75.0

(11.5)

86.3

(6.6)

18.8a

(10.1)

86.3

(6.0)

78.8

(4.8)

6.3a

(2.5)

200

52.5

(23.0)

70.0

(10.2)

80.0

(9.6)

26.9a

(20.9)

50.6a

(21.2)

65.6a

(13.8)

8.1

(6.3)

300

38.1a

(14.0)

73.8

(10.3)

48.1a

(11.3)

18.8a

(14.2)

3.8a

(3.2)

61.9a

(6.6)

13.8

(4.8)

300HD

53.1

(11.8)

81.9

(3.1)

76.3

(10.3)

40.6a

(20.0)

30.0a

(35.5)

75.6

(12.3)

6.9

(12.1)

Conclusion

The study results indicate that exposure to spray-dried powder formulated Pseudomonas 

fluorescens at the maximum-labeled concentration (100 milligrams per liter [mg/L] active ingredient 

[AI]) and up to three times the maximum-labeled exposure duration (8 hours) is not likely to affect the 

23 
survival of newly metamorphosed Lampsilis siliquoidea and Lampsilis higginsii. The low mean survival 

of the Ligumia recta control group (25.0 percent) indicates that results for L. recta should be interpreted 

with caution. Significant differences were detected in the mean survival of the Lampsilis cardium

treated groups (14.4 to 40.6 percent) compared to the control group (68.8 percent). The results indicate 

that further investigation on the effects of spray-dried powder formulated P. fluorescens exposure on 

L. recta and L. cardium is warranted.

Mean survival of three unionid mussels species exposed to the freeze-dried powder formulated 

Pseudomonas fluorescens was not significantly different in the 50-, 100-, and 200-mg/L AI treatment 

groups and the 300-mg/L heat-deactivated treatment groups when compared to the control groups. The 

study results indicate that exposure to freeze-dried powder formulated Pseudomonas fluorescens up to

200 mg/L AI and up to 24 hours is not likely to impact the survival of newly metamorphosed Obovaria 

olivaria, Actinonaias ligamentina, and Megalonaias nervosa. 

References Cited

American Public Health Association, American Water Works Association, and Water Environment 

Federation, 2012, Standard methods for examination of water and wastewater (22d ed.): Washington, 

D.C., American Public Health Association, 1,360 p.

ASTM International, 2013, ASTM Standard E2455-06 (2013)—Standard guide for conducting 

laboratory toxicity tests with freshwater mussels: West Conshohocken, Pa., ASTM International, 52 p. 

Baker, S.M., and Hornbach, D.J., 1997, Acute physiological effects of zebra mussel (Dreissena 

polymorpha) infestation on two unionid mussels, Actinonaias ligamentina and Amblema plicata:

Canadian Journal of Fisheries and Aquatic Sciences, v. 54, p. 512–519. 

Benson, A.J., Raikow, D., Larson, J., Fusaro, A., and Bogdanoff, A.K., 2015, Dreissena polymorpha 

(fact sheet, revision date June 26, 2014): Gainesville, Fla., USGS Nonindigenous Aquatic Species 

Database, accessed February 25, 2015 at http://nas.er.usgs.gov/queries/factsheet.aspx?speciesid=5. 

Birnbaum, Christina, 2011, NOBANIS—Invasive alien species fact sheet—Dreissena polymorpha: 

Online Database of the European Network on Invasive Alien Species, accessed February 13, 2014, at 

http://www.nobanis.org/files/factsheets/Dreissena_polymorpha.pdf. 

Burlakova, L.E., Karatayev, A.Y., and Padilla, D.K., 2000, The impact of Dreissena polymorpha 

(PALLAS) invasion on unionid bivalves: International Review of Hydrobiology, v. 85, no. 5–6, p. 

529–541.

Emerson, K., Russo, R.C., Lund, R.E., and Thurston, R.V., 1975, Aqueous ammonia equilibrium 

calculations—Effect of pH and temperature: Journal of the Fisheries Research Board of Canada, 

v. 32, p. 2379–2383. 

Hebert, P.D.N., Wilson, C.C., Murdoch, M.H., and Lazar, R., 1991, Demography and ecological 

impacts of the invading mollusc Dreissena polymorpha: Canadian Journal of Zoology, v. 69, 

p. 405–409. 

Mackie, G.L., 1991, Biology of the exotic zebra mussel, Dreissena polymorpha, in relation to native 

bivalves and its potential impact in Lake St. Clair: Hydrobiologia, v. 219, p. 251–268. 

Meinertz, J.R., Schreier, T.M., Hess, K.R., and Bartsch, M.R., 2011, Survival and growth of newly 

transformed Lampsilis cardium and Lampsilis siliquoidea in a flow-through, continuous feeding test 

system: American Malacological Bulletin, v. 29, p. 69–75. 

Molloy, D.P., Mayer, D.A., Gaylo, M.J., Burlakova, L.E., Karatayev, A.Y., Presti, K.T., Sawyko, P.M., 

and Morse, J.T., 2013, Non-target trials with Pseudomonas fluorescens strain CL145A, a lethal 

control agent of dreissenid mussels (Bivalvia: Dreissenidae): Management of Biological Invasions, 

v. 4, p. 71–79. 

24 
Molloy, D.P., Mayer, D.A., Gaylo, M.J., Morse, J.T., Presti, K.T., Sawyko, P.M., Karatayev, A.Y., 

Burlakova, L.E., Laruelle, F., Nishikawa, K.C., and Griffin, B.H., 2013, Pseudomonas fluorescens

strain CL145A—A biopesticide for the control of zebra and quagga mussels (Bivalvia: Dreissenidae):

Journal for Invertebrate Pathology, v. 113, p. 104–114. 

Nalepa, T. F., 1994, Decline of native unionid bivalves in Lake St. Clair after infestation by the zebra 

mussel, Dreissena polymorpha: Canadian Journal of Fisheries and Aquatic Sciences, v. 51, 

p. 2227–2233. 

Nalepa, T.F., and Schloesser D.W., eds., 2014, Response, management, and mitigation, Part III of

Quagga and zebra mussels—Biology, impacts, and control (2d ed.): Boca Raton, Fla., CRC Press, 

Taylor & Francis Group, p. 116–284. 

Neves, R.J., 2004, Propagation of endangered freshwater mussels in North America: Journal of 

Conchology, Special Publication, 3, p. 69–80. 

Ricciardi, A., and Rasmussen, J.B., 1999, Extinction rates of North American freshwater fauna: 

Conservation Biology, v. 13, no. 5, p. 1220–1222. 

Ricciardi, A., Whoriskey, F.G., and Rasmussen, J.B., 1995, Predicting the intensity and impact of 

Dreissena infestation on native unionid bivalves from Dreissena field density: Canadian Journal of 

Fisheries and Aquatic Sciences, v. 52, p. 1449–1461. 

Schloesser, D.W., and Kovalak, W.P., 1991, Infestation of unionids by Dreissena polymorpha in a 

power plant canal in Lake Erie: Journal of Shellfish Research, v. 10, no. 2, p. 355–359. 

Schloesser, D.W., and Nalepa, T.F., 1994, Dramatic decline of unionid bivalves in offshore waters of 

western Lake Erie after infestation by the zebra mussel, Dreissena polymorpha: Canadian Journal of 

Fisheries and Aquatic Sciences, v. 51, p. 2234–2242. 

Strayer, D.L., and Malcom, H.M., 2007, Effects of zebra mussels (Dreissena polymorpha) on native 

bivalves—The beginning of the end or the end of the beginning?: Journal of the North American 

Benthological Society, v. 26, no. 1, p. 111–122. 

Strayer, D.L., Downing, J.A., Haag, W.R., King, T.L., Layzer, J.B., Newton, T.J., and Nichols, J.S.,

2004, Changing perspectives on pearly mussels, North America's most imperiled animals: 

BioScience, v. 54, no. 5, p. 429–439. 

U.S. Environmental Protection Agency, 2013, Ambient water quality criteria for ammonia—Freshwater 

2013: Washington, D.C., Office of Water, EPA 822–R–13–001, 242 p. 

U.S. Geological Survey, 2014, Zebra and quagga mussel distribution in U.S. lakes: USGS Web page,

accessed on March 27, 2014, at http://fl.biology.usgs.gov/Nonindigenous_Species/Zebra_mussel_ 

distribution/zebra_mussel_distribution.html.

Williams, J.D., Warren Jr., M.L., Cummings, K.S., Harris, J.L., and Neves, R.J., 1993, Conservation 

status of freshwater mussels of the United States and Canada: Fisheries, v. 18, no. 9, p. 6–22. 

25 
Appendix 1. Study Protocol With Data Forms

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Protocol: “Effects of Pseudomonas fluorescens (Pf–CL145A) to newly 

metamorphosed juvenile mussel from seven different unionid species” 22 28

2 Note to File 1: Clarification of pH and temperature data collected for un–ionized 

ammonia 1 50

3 Note to File 2: Clarify use of two Water Quality Form 2 datasheets for Hickorynut 

mussel test 1 51

4 Amendment 1: Initiating feed to exposure chambers during the pre–exposure period 2 52

5 Amendment 2: Alkalinity and hardness measurements collected during exposure 

period termination from each exposure concentration 2 54

6 Amendment 3: Reduces feeding rate during the holding period 2 56

7 Amendment 4: Changes software used to capture, record, and analyze 

photomicrographs to Nikon Elements BR® software 2 58

8 

Amendment 5: Changes ammonia sampling regime to include LTRMP using the 

automated phenate method of analysis to provide information regarding the 

accumulation rate of ammonia

2 60

9 Amendment 6: Alters pre–exposure feeding regime, removes use of substrate, and 

reduces holding period to 7 days 5 62

10 Amendment 7: Eliminates use of YSI 9000 Spectrophotometer for ammonia analysis 2 67

11 Amendment 8: Amends test material from freeze dried powder (FDP) to spray dried 

powder (SDP) 7 69

12 Amendment 9: Status of study changed to non–regulated 2 76

13 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form 1 78

14 Newly Metamorphosed Juvenile Mussel Distribution Form 1 79

15 Newly Metamorphosed Mussel Exposure Dosing Form 1 80

16 Test Chemical Stock Preparation Data Form 1 81

17 Chemical Stock Solution Determination and Preparation – Copy (original misplaced) 1 82

18 Water Quality – Form 1: Pre–exposure 1 83

19 Water Quality – Form 2: Exposure Period Dissolved Oxygen, pH, Temperature 1 84

20 Water Quality – Form 2a: Exposure Period pH and Temperature for Ammonia 

Analysis 1 85

21 Water Quality – Form 3: Exposure Termination Hardness, Alkalinity and Conductivity 1 86

22 Daily Water Quality – Form 4: Dissolved Oxygen, pH, and Temperature 1 87

23 Weekly Water Quality – Form 5: Hardness, Alkalinity and Conductivity 1 88

24 System Conditions – Form 1: Light Intensity 1 89

25 System Condition – Form 3: Daily Flow Rates (mL/minute) 1 90

26 Individual Algae Product Dry Weight Determination Datasheet 1 91

27 Algae Stock Solution Preparation 1 92

28 Algae Stock Solution Dry Weight Determination 1 93

26 
29 Juvenile Mussel Recovery Datasheet 1 94

30 Juvenile Mussel Micropictograph Datasheet 1 95

31 Juvenile Mussel Measurement Datasheet 1 96

32 Pipette Calibration 1 97

33 Revised Datasheets 32 98

27 
28 
29 
30 
31 
32 
33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
43 
44 
45 
46 
47 
48 
49 
50 
51 
52 
53 
54 
55 
56 
57 
58 
59 
60 
61 
62 
63 
64 
65 
66 
67 
68 
69 
70 
71 
72 
73 
74 
75 
76 
77 
78 
79 
80 
81 
82 
83 
84 
85 
86 
87 
88 
89 
90 
91 
92 
93 
94 
95 
96 
97 
98 
99 
100 
101 
102 
103 
104 
105 
106 
107 
108 
109 
110 
111 
112 
113 
114 
115 
116 
117 
118 
119 
120 
121 
122 
123 
124 
125 
126 
127 
128 
129 
Appendix 2. Deviations From the Study Protocol

Item 

number Item description

Number 

of 

pages

Report 

page

number

1 Deviation 1: Algae feed delivery line incorrectly inserted into pump; feed not supplied 

for ≈24 h 1 131

2 Deviation 2: Dry weight of chlorella not measured prior to use for feeding 1 132

3 Deviation 3: Algae feed delivery line obstructed; only ≈20% of feed rationed for ≈24 h 1 133

4 Deviation 4: Low water level in test chamber due to standpipe leak and water delivery 

line obstruction 1 134

5 Deviation 5: Power outage interrupted algae delivery pumps for ≈12 h 1 135

6 Deviation 6: Decreased number of preserved mussels for initial mussel valve length 2 136

7 Deviation 7: Microphotograph overwritten and restored 1 138

8 Deviation 8: Dosing stock solutions for 300 mg/L heat deactivated and 50 mg/L active 

solutions incorrectly prepared 1 139

9 Deviation 9: Conductivity meter incorrectly calibrated 1 140

10 Deviation 10: Algae feed delivery line obstructed; only ≈20% of feed rationed for 24 h 1 141

11 Deviation 11: Algae feed delivery line crimped causing clog; only ≈20% of feed 

rationed for ≈24 h – Dated April 27, 2012 1 142

12 Deviation 12: Low water level in test chamber due to standpipe leak 1 143

130 
131 
132 
133 
134 
135 
136 
137 
138 
139 
140 
141 
142 
143 
Appendix 3. Randomization Assignments

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 SAS generated random assignment of species to test system – dated July 7, 2011 4 145

2 SAS generated random assignment of species to test system – dated December 8, 2011 3 149

3 SAS generated random assignment of species to test system – dated January 23, 2012 4 152

4 SAS generated random assignment of species to test system – dated March 28, 2012 4 156

5 SAS generated random assignment of species to test system – dated May 2, 2012 4 160

6 SAS generated random assignment of species to test system – dated May 23, 2012 4 164

7 SAS generated random assignment of juvenile mussel distribution – dated July 7, 2011 7 168

8 SAS generated random assignment of juvenile mussel distribution – dated December 

8, 2011 7 175

9 SAS generated random assignment of juvenile mussel distribution – dated January 23, 

2012 13 182

10 SAS generated random assignment of juvenile mussel distribution – dated March 28, 

2012 6 195

11 SAS generated random assignment of juvenile mussel distribution – dated May 2, 

2012 for Lampsilis cardium 6 201

12 SAS generated random assignment of juvenile mussel distribution – dated May 23, 

2012 8 207

13 SAS generated random assignment of treatment to experimental tanks – dated July 7, 

2011 10 215

14 SAS generated random assignment of treatment to experimental tanks – dated 

December 8, 2011 9 225

15 SAS generated random assignment of treatment to experimental tanks – dated January 

23, 2012 18 234

16 SAS generated random assignment of treatment to experimental tanks – dated March 

28, 2012 7 252

17 SAS generated random assignment of treatment to experimental tanks – dated May 2, 

2012 7 259

18 SAS generated random assignment of treatment to experimental tanks – dated May 23, 

2012 8 266

144 
145 
146 
147 
148 
149 
150 
151 
152 
153 
154 
155 
156 
157 
158 
159 
160 
161 
162 
163 
164 
165 
166 
167 
168 
169 
170 
171 
172 
173 
174 
175 
176 
177 
178 
179 
180 
181 
182 
183 
184 
185 
186 
187 
188 
189 
190 
191 
192 
193 
194 
195 
196 
197 
198 
199 
200 
201 
202 
203 
204 
205 
206 
207 
208 
209 
210 
211 
212 
213 
214 
215 
216 
217 
218 
219 
220 
221 
222 
223 
224 
225 
226 
227 
228 
229 
230 
231 
232 
233 
234 
235 
236 
237 
238 
239 
240 
241 
242 
243 
244 
245 
246 
247 
248 
249 
250 
251 
252 
253 
254 
255 
256 
257 
258 
259 
260 
261 
262 
263 
264 
265 
266 
267 
268 
269 
270 
271 
272 
273 
Appendix 4. Test Article Information

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Material Safety Data Sheet: MBI–401 FDP 2 276

2 Material Safety Data Sheet: MBI–401 SDP 2 278

3 Test Chemical Stock Preparation Data Form – Stock preparation date July 12, 2011 1 280

4 Test Chemical Stock Preparation Data Form – Stock preparation date July 14, 2011 1 281

5 Test Chemical Stock Preparation Data Form – Stock preparation date December 13, 

2011 1 282

6 Test Chemical Stock Preparation Data Form – Stock preparation date January 27, 2012 1 283

7 Test Chemical Stock Preparation Data Form – Stock preparation date April 17, 2012 1 284

8 Test Chemical Stock Preparation Data Form – Stock preparation date May 16, 2011 1 285

9 Test Chemical Stock Preparation Data Form – Stock preparation date May 26, 2011 1 286

10 Newly Metamorphosed Mussel Exposure Dosing Form – dated July 12, 2011 1 287

11 Newly Metamorphosed Mussel Exposure Dosing Form – dated July 14, 2011 1 288

12 Newly Metamorphosed Mussel Exposure Dosing Form – dated December 13, 2011 1 289

13 Newly Metamorphosed Mussel Exposure Dosing Form – dated January 27, 2012 1 290

14 Newly Metamorphosed Mussel Exposure Dosing Form – dated April 17, 2012 1 291

15 Newly Metamorphosed Mussel Exposure Dosing Form – dated May 16, 2012 1 292

16 Newly Metamorphosed Mussel Exposure Dosing Form – dated May 26, 2012 1 293

17 Chemical Stock Solution Determination – Exposure termination date July 13, 2011 1 294

18 Chemical Stock Solution Determination – Exposure termination date July 15, 2011 1 295

19 Chemical Stock Solution Determination – Exposure termination date December 14, 

2011 1 296

20 Chemical Stock Solution Determination – Exposure termination date January 28, 2012 1 297

21 Chemical Stock Solution Determination – Exposure termination date April 18, 2012 1 298

22 Chemical Stock Solution Determination – Exposure termination date May 17, 2012 1 299

23 Chemical Stock Solution Determination – Exposure termination date May 27, 2012 1 300

24 Copy of test article information from test article logbook for MBI–401 FDP; lot 

number 110510FD – used for HIC and MUC exposures 6 301

25 Copy of test article information from test article logbook for MBI–401 FDP; lot 

number 110928FD – used for WAS exposure 5 307

26 Copy of test article information from test article logbook for MBI–401 SDP; lot 

number MBI–401 SDP 4655–12–Mix – used for FAM exposure 5 312

27 Copy of test article information from test article logbook for MBI–401 SDP; lot 

number TR4669–4–(7–8) 2nd shipment – used for BLS exposure 5 317

28 Copy of test article information from test article logbook for MBI–401 SDP; lot 

number TR 4669–4–(5) – used for PPB and HGE exposures 7 322

29 Post–treatment activity retention report from NYSM for lot MBI–401 FDP 110510FD 

– Start date of July 20, 2011 2 329

274 
30 Post–treatment activity retention report from NYSM for lot MBI–401 FDP 110928FD 

– Start date of December 29, 2011 2 331

31 Post–treatment activity retention report from NYSM for lot MBI–401 SDP 4655–12–

Mix – Start date of February 9, 2012 2 333

32 Post–treatment activity retention report from NYSM for lot MBI–401 SDP TR–4669–

4–(7–8) 2nd shipment – Start date of May 1, 2012 2 335

33 Post–treatment activity retention report from NYSM for lot MBI–401 SDP TR–4669–

4–(5) – Start date of May 29, 2012 2 337

34 Post–treatment activity retention report from NYSM for lot MBI–401 SDP TR–4669–

4–(5) – Start date of June 6, 2012 2 339

275 
276 
277 
278 
279 
280 
281 
282 
283 
284 
285 
286 
287 
288 
289 
290 
291 
292 
293 
294 
295 
296 
297 
298 
299 
300 
301 
302 
303 
304 
305 
306 
307 
308 
309 
310 
311 
312 
313 
314 
315 
316 
317 
318 
319 
320 
321 
322 
323 
324 
325 
326 
327 
328 
329 
330 
331 
332 
333 
334 
335 
336 
337 
338 
339 
340 
Appendix 5. Test Animal Information

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Test Organism Species List, Collection, and Inclusion Criteria – dated May 27, 2011 1 342

2 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Obovaria olivaria 1 343

3 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Actinonaias ligamentina 1 344

4 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Megalonaias nervosa 1 345

5 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Lampsilis siliquoidea 1 346

6 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Liguma recta 1 347

7 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Lampsilis cardium 1 348

8 Newly Metamorphosed Juvenile Mussel Test Organism UMESC Lot Number 

Assignment Form – Lampsilis higginsii 1 349

9 Newly Metamorphosed Juvenile Mussel Distribution Form – dated July 11, 2011 1 350

10 Newly Metamorphosed Juvenile Mussel Distribution Form – dated July 13, 2011 1 351

11 Newly Metamorphosed Juvenile Mussel Distribution Form – dated December 12, 2011 1 352

12 Newly Metamorphosed Juvenile Mussel Distribution Form – dated January 26, 2012 1 353

13 Newly Metamorphosed Juvenile Mussel Distribution Form – dated April 16, 2012 1 354

14 Newly Metamorphosed Juvenile Mussel Distribution Form – dated May 15, 2012 1 355

15 Newly Metamorphosed Juvenile Mussel Distribution Form – dated May 25, 2012 1 356

341 
342 
343 
344 
345 
346 
347 
348 
349 
350 
351 
352 
353 
354 
355 
356 
Appendix 6. Water Quality and System Conditions

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Exposure Period Water Chemistry Data Summary for SAS 9 358

2 SAS output for Statistical analysis of exposure period water chemistry 19 367

3 SAS program for Statistical analysis of exposure period water chemistry 2 386

4 SAS log for Statistical analysis of exposure period water chemistry 4 388

5 Total Ammonia Nitrogen Data Summary for SAS 13 392

6 SAS output for Statistical analysis of ammonia levels (TAN and un–ionized) 31 405

7 SAS program for Statistical analysis of ammonia levels (TAN and un–ionized) 2 436

8 SAS log for Statistical analysis of ammonia levels (TAN and un–ionized) 3 438

9 Holding Period Water Chemistry Data Summary for SAS 4 441

10 SAS output for Statistical analysis of holding period water chemistry 12 445

11 SAS program for Statistical analysis of holding period water chemistry 2 457

12 SAS log for Statistical analysis of holding period water chemistry 3 459

13 System Conditions – Flow Rates Data Summary for SAS – Certified November 21, 

2014 20 462

14 SAS output for Statistical Analysis of Holding Chamber Flow Rates – Dated May 7, 

2014 32 482

15 SAS program for Statistical Analysis of Holding Chamber Flow Rates – Dated May 7, 

2014 2 514

16 SAS log for Statistical Analysis of Holding Chamber Flow Rates – Dated May 7, 2014 3 516

17 System Conditions – Light Intensity Data Summary for SAS – Certified November 21, 

2014 3 519

18 SAS output for Statistical Analysis of Light Intensity – Dated May 7, 2014 3 522

19 SAS program for Statistical Analysis of Light Intensity – Dated May 7, 2014 1 525

20 SAS log for Statistical Analysis of Light Intensity – Dated May 7, 2014 2 526

21 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date August 23, 2011 5 528

22 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date January 9, 2012 1 533

23 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date February 21, 2012 1 534

24 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date April 24, 2012 1 535

25 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date May 8, 2012 1 536

26 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date May 24, 2012 1 537

27 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC –

Report date June 15, 2012 1 538

357 
358 
359 
360 
361 
362 
363 
364 
365 
366 
367 
368 
369 
370 
371 
372 
373 
374 
375 
376 
377 
378 
379 
380 
381 
382 
383 
384 
385 
386 
387 
388 
389 
390 
391 
392 
393 
394 
395 
396 
397 
398 
399 
400 
401 
402 
403 
404 
405 
406 
407 
408 
409 
410 
411 
412 
413 
414 
415 
416 
417 
418 
419 
420 
421 
422 
423 
424 
425 
426 
427 
428 
429 
430 
431 
432 
433 
434 
435 
436 
437 
438 
439 
440 
441 
442 
443 
444 
445 
446 
447 
448 
449 
450 
451 
452 
453 
454 
455 
456 
457 
458 
459 
460 
461 
462 
463 
464 
465 
466 
467 
468 
469 
470 
471 
472 
473 
474 
475 
476 
477 
478 
479 
480 
481 
482 
483 
484 
485 
486 
487 
488 
489 
490 
491 
492 
493 
494 
495 
496 
497 
498 
499 
500 
501 
502 
503 
504 
505 
506 
507 
508 
509 
510 
511 
512 
513 
514 
515 
516 
517 
518 
519 
520 
521 
522 
523 
524 
525 
526 
527 
528 
529 
530 
531 
532 
533 
534 
535 
536 
537 
538 
Appendix 7. Animal Feed Information

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Chlorella Algae: FedEx Standard Overnight Label (shipped from Monte McGregor) –

dated May 31, 2011 1 541

2 Chlorella Algae: FedEx Standard Overnight Label (shipped from Monte McGregor) –

dated July 11, 2011 1 542

3 Chlorella Algae: FedEx Priority Overnight Label (shipped from Monte McGregor) –

dated May 7, 2011 1 543

4 Nanno–3600 and Shelfish Diet 1800 Algae – Order Invoice from Reed Mariculture –

dated May 11, 2011 1 544

5 Tet 3600 and Nanno–3600 Algae – Order Invoice from Reed Mariculture – dated 

March 20, 2012 1 545

6 Note from Monte McGregor regarding chlorella algae shipment – Harvest date May 

10, 2011 1 546

7 Note from Monte McGregor regarding chlorella algae shipment – Harvest date June 24 1 547

8 Reed Mariculture literature on Shellfish Diet, Nannochloropsis, and Tetraselmis –

Internet printout 6 548

9 Algae Stock Dry Weight Determination Procedure 1 554

10 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date May 26, 

2011 3 555

11 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date June 5, 

2011 1 558

12 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date June 25, 

2011 1 559

13 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date July 7, 

2011 1 560

14 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date 

December 8, 2011 3 561

15 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date January 

26, 2012 1 564

16 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date March 

31, 2012 2 565

17 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date April 13, 

2012 1 567

18 Algae Stock Dry Weight Determination Datasheet – 48 hour dry weight date May 12,

2012 1 568

19 Algae Stock Preparation Chart – Effective date June 3, 2011 2 569

20 Algae Stock Preparation Chart – Effective date June 6, 2011 2 571

21 Algae Stock Preparation Chart – Effective date June 17, 2011 2 573

22 Algae Stock Preparation Chart – Effective date June 27, 2011 2 575

23 Algae Stock Preparation Chart – Effective date July 7, 2011 2 577

24 Algae Stock Preparation Chart – Effective date December 8, 2011 2 579

25 Algae Stock Preparation Chart – Effective date January 25, 2012 2 581

539 
26 Algae Stock Preparation Chart – Effective date January 27, 2012 2 583

27 Algae Stock Preparation Chart – Effective date April 14, 2012 2 585

28 Algae Stock Preparation Chart – Effective date May 14, 2012 2 587

540 
541 
542 
543 
544 
545 
546 
547 
548 
549 
550 
551 
552 
553 
554 
555 
556 
557 
558 
559 
560 
561 
562 
563 
564 
565 
566 
567 
568 
569 
570 
571 
572 
573 
574 
575 
576 
577 
578 
579 
580 
581 
582 
583 
584 
585 
586 
587 
588 
Appendix 8. Juvenile Mussel Survival Summary and Statistical 

Analysis

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Juvenile Mussel Survival Data Summary for SAS 5 590

2 SAS output for All Juvenile Mussel Survival Data 59 595

3 SAS program for All Juvenile Mussel Survival Data 3 654

4 SAS log for All Juvenile Mussel Survival Data 7 657

589 
590 
591 
592 
593 
594 
595 
596 
597 
598 
599 
600 
601 
602 
603 
604 
605 
606 
607 
608 
609 
610 
611 
612 
613 
614 
615 
616 
617 
618 
619 
620 
621 
622 
623 
624 
625 
626 
627 
628 
629 
630 
631 
632 
633 
634 
635 
636 
637 
638 
639 
640 
641 
642 
643 
644 
645 
646 
647 
648 
649 
650 
651 
652 
653 
654 
655 
656 
657 
658 
659 
660 
661 
662 
663 
Weber and others—Exposure-Related Effects of Pseudomonas fluorescens (Pf-CL145A) on Juvenile Unionid Mussels—Open-File Report 2015–1066

ISSN 2331-1258 (online)

http://dx.doi.org/10.3133/ofr20151066