Efficacy of Pseudomonas fluorescens Strain CL145A 

Spray Dried Powder for Controlling Zebra Mussels 

Adhering to Native Unionid Mussels Within Field 

Enclosures

By James A. Luoma, Kerry L. Weber, Todd J. Severson, and Denise A. Mayer

Open-File Report 2015-1051

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,

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must be secured from the copyright owner.

Suggested citation:

Luoma, J.A., Weber, K.L., Severson, T.J., and Mayer, D.A., 2015, Efficacy of Pseudomonas fluorescens strain 

CL145A spray dried powder for controlling zebra mussels adhering to native unionid mussels within field 

enclosures: U.S. Geological Survey Open-File Report 2015–1051, 301 p., http://dx.doi.org/10.3133/ofr20151051. 

ISSN 2331-1258 (online)
iii

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. Michelle R. Bartsch, 

Jeremy K. Wise, and Hugh E. McMath (Upper Midwest Environmental Sciences Center) assisted with 

data collection. Mark Gaikowski (Upper Midwest Environmental Sciences Center) assisted in study

design, data analysis, and report preparation. Joe Eisterhold and Mark Ranweiler (Minnesota 

Department of Natural Resources) assisted with field logistics and project permitting. Special thanks are 

extended to Carl Towley and Mike Bump for logistical assistance and for allowing us to use their 

private property for equipment storage and project operations. 
iv
v 

Contents

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

Abstract ...................................................................................................................................................................... 1

Introduction................................................................................................................................................................. 2

Materials and Methods ............................................................................................................................................... 3

Experimental Design................................................................................................................................................... 3

Test Enclosure Area ................................................................................................................................................... 3

Test Article.................................................................................................................................................................. 5

Test Animals............................................................................................................................................................... 5

Reduction of Adhering Zebra Mussels .................................................................................................................... 6

Estimation of Initial Number of Adhering Zebra Mussels (Group 1)..................................................................... 6

Estimation of the Number of Adhering Zebra Mussels (Group 2)........................................................................ 7

Calculation of Zebra Mussel Body Weight Burden (Group 2).............................................................................. 7

Calculation of Adhering Zebra Mussels Reduction (Group 2).............................................................................. 7

Calculation of SDP Reduction Coefficient and Efficiency (Group 2).................................................................... 8

Unionid Mussel Survival.......................................................................................................................................... 8

Stock-Solution Preparation and Dosing ...................................................................................................................... 8

Test Enclosure Water Sample Collection................................................................................................................ 8

Concentration Verification ................................................................................................................................... 8

Water Chemistry.................................................................................................................................................. 9

Data Analysis.............................................................................................................................................................. 9

Reduction of Adhering Zebra Mussels .................................................................................................................... 9

Unionid Mussel Survival........................................................................................................................................ 10

Exposure Concentration........................................................................................................................................ 10

Results...................................................................................................................................................................... 10

Adverse Events..................................................................................................................................................... 10

Reduction of Adhering Zebra Mussels .................................................................................................................. 10

Unionid Mussel Survival........................................................................................................................................ 11

Dose Verification................................................................................................................................................... 12

Water Chemistry ................................................................................................................................................... 12

Conclusions .............................................................................................................................................................. 13

References ............................................................................................................................................................... 14

Appendix 1.Study Protocol With Data Forms............................................................................................................ 16

Appendix 2.Deviations From the Study Protocol....................................................................................................... 75

Appendix 3. Randomization Assignments................................................................................................................. 87

Appendix 4.Test Article Information.......................................................................................................................... 98

Appendix 5.Test Animal Information ....................................................................................................................... 126

Appendix 6.Water Quality ....................................................................................................................................... 168

Appendix 7.Spectrophotometric Summary and SAS Output, Program, and Log .................................................... 181

Appendix 8.Zebra Mussel Density Association Summary Data Analysis................................................................ 201

Appendix 9.Unionid Mussel Survival Assessment Summary.................................................................................. 217

Appendix 10.Statistical Analysis, Including SAS Programs, Outputs, and Logs for Unionid Mussel Survival, Zebra 

Mussel Colonization Density Associations, and Zebra Mussel Test Animal Lengths.............................................. 223
vi 

Figures

Figure 1. Test enclosure site and test animal collection locations......................................................................... 4

Figure 2. Example of 1-square-meter (m2) expanded metal mussel retention barrier (left) and 

2.25-m2 impermeable test enclosure (right)............................................................................................................... 4

Figure 3. Representative unionid mussel with adhering zebra mussels preexposure (top left), 

postexposure (top right) and with zebra mussels removed (bottom).......................................................................... 6

Tables

Table 1. Universal Transverse Mercator (UTM) centroid location of each test enclosure. ................................... 5

Table 2. Mean (standard deviation) number of adhering zebra mussels per unionid mussel 

before and after exposure, percent change of zebra mussel reduction, zebra mussel burden 

(as percent unionid body weight), and spray dried powder (SDP) reduction coefficient for number 

of adhering zebra mussels for each treatment group............................................................................................... 11

Table 3. Mean (standard deviation; number measured) length of adhering zebra mussels (live) 

by treatment group................................................................................................................................................... 11

Table 4. Mean (standard deviation) percent survival of Group 1 and 2 unionid mussels. .................................. 12

Table 5. Mean (standard deviation) spray dried powder exposure concentration 

in surface water samples collected from test enclosures during the exposure period. ............................................ 12

Table 6. Mean (standard deviation) water chemistry (dissolved oxygen, temperature) and 

pH range of surface water samples collected from each treatment group during the study period.......................... 13

Table 7. Mean (standard deviation) alkalinity, hardness, conductivity, and ammonia content 

of surface water samples collected from each treatment group during the study period.......................................... 13
vii 

Conversion Factors

International System of Units to Inch/Pound

Multiply By To obtain

Length

micrometer (μm) 3.937 × 10-5 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft) 

nanometer (nm) 3.937 × 10-8 inch (in.)

Area

square meter (m2) 10.76 square foot (ft2)

Volume

liter (L) 0.2642 gallon (gal)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

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).

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

Abbreviations

AI active ingredient

CaCO3 calcium carbonate

DO dissolved oxygen 

MBI Marrone Bio Innovations

mE meters East

mN meters North

NH3 un-ionized ammonia

OR odds ratio

SAS Statistical Analysis System

SD standard deviation

SDP spray dried powder

TAN total ammonia nitrogen

Pf-CL145A Pseudomonas fluorescens strain CL145A

USGS U.S. Geological Survey

UTM Universal Transverse Mercator

w/w weight to weight ratio
1 

Efficacy of Pseudomonas fluorescens Strain CL145A 

Spray Dried Powder for Controlling Zebra Mussels 

Adhering to Native Unionid Mussels Within Field 

Enclosures

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

1 Todd J. Severson,1 and Denise A. Mayer2

1 U.S. Geological Survey.

2 New York State Education Department.

Abstract

The efficacy of a commercially prepared spray dried powder (SDP) formulation of Pseudomonas 

fluorescens (strain CL145A) was evaluated for removing zebra mussels (Dreissena polymorpha) 

adhering to a population of unionid mussels in Lake Darling (Alexandria, Minnesota). Two groups of 

unionid mussels were used in the study. Unionid mussels were collected near the test area, weighed, 

photographed, individually tagged, and randomly allocated to one of nine test enclosures in equal 

proportions and then divided into two groups. The first group of unionid mussels (Group 1, n = 5 per 

test enclosure) were indiscriminately selected from each test enclosure and used to estimate the number 

of zebra mussels adhering to unionid mussels prior to exposure. The second group of unionid mussels 

(Group 2, n = 22 per test enclosure) were used to evaluate the efficacy of SDP for removal of adhering 

zebra mussels. Both Group 1 and Group 2 mussels were used to evaluate the effects of SDP exposure on 

unionid mussel survival. 

Treatment was assigned to each test enclosure by using a randomized block design. The three 

treatment groups were tested in triplicate and included an untreated control group and groups that

received a single application of 50 or 100 milligrams per liter (mg/L) of SDP based on active ingredient. 

All treatment concentrations are reported as active ingredient of SDP. Test enclosures were removed at 

the 8-hour exposure termination. Both Group 1 and Group 2 mussels remained in their assigned 

exposure location during the postexposure holding period. The number of zebra mussels adhering to 

Group 2 mussels (live and dead) was assessed 18 to 20 days postexposure in addition to assessing the 

survival of Group 1 and Group 2 unionid mussels. 

SDP, administered as a single treatment, significantly (p < 0.01) reduced the number of adhering 

zebra mussels when compared to the untreated controls. The number of zebra mussels adhering to 

unionid mussels (Group 2) was reduced 53 percent in the 50-mg/L treatment group and 68 percent in the 

100-mg/L treatment group. The number of adhering zebra mussels did not differ (p = 0.79) between the 

50- and 100-mg/L treatment groups after exposure. When standardized to the amount of SDP applied 

per square meter, each gram (g) of SDP applied in the 50-mg/L treatment reduced the number of 

adhering zebra mussel 59.8 percent more than the 100-mg/L treatment group.

 
2 

Group 1 mussel survival did not differ between treatment groups (p > 0.05); however, a 

difference was detected (p < 0.01) in the survival of Group 2 mussels. The survival of Group 2 mussels 

did not differ (p > 0.23) between control and treated groups. A difference in Group 2 mussel survival 

was detected (p = 0.03; odds ratio [OR] = 0.290) between the 50- and 100-mg/L treatment groups (that 

is, the survival was highest in the 50-mg/L treatment group and lowest in the 100-mg/L treatment 

group), however, the biological significance of the difference is indeterminate. 

Introduction

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

in the world, but diversity is declining rapidly in response to a variety of anthropogenic influences such 

as habitat degradation and alteration, pollution, and overharvest (Williams and others, 1993; Neves and 

others, 1997). Master (1990) found that 55 percent of North American mussel species were either 

extinct or imperiled. At least 127 imperiled mussel species are predicted to reach extinction within the 

next 100 years; however, the 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) 

(Ricciardi and Rasmussen, 1999). 

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

can quickly disperse and inundate aquatic environments (Mackie, 1991; Birnbaum, 2011). Since their 

introduction into the United States and establishment in the Great Lakes in the 1980s, zebra mussels 

have been identified in 680 lakes, not including impoundments and connected waterways, within 

27 states (Benson and others, 2015). In a power plant canal in Lake Erie, Schloesser and Kovalak (1991) 

estimated zebra mussel colonization as high as approximately 10,700 zebra mussels per unionid mussel, 

with a mean estimated density of approximately 6,800 zebra mussels per unionid mussel. Adhering 

zebra mussel colonies may contain in excess of 10,000 individuals, which could weigh two to five times 

as much as the unionid mussel (Hebert and others, 1991; Mackie, 1991). Prediction models estimate that 

a colonization density of as few as 100 zebra mussels per unionid can result in unionid mussel mortality 

(Ricciardi and others, 1995). 

Colonization by zebra mussels may interfere with unionid mussel locomotion, feeding, 

reproduction, and respiration (Mackie, 1991; Schloesser and Kovalak, 1991). Heavy zebra mussel 

colonization may contribute to starvation of the unionid through reduced intake of food and increased 

metabolic costs (Baker and Hornbach, 1997; Strayer and Malcom, 2007). Heavily colonized unionid 

mussels may lack the energy stores required to survive winter, and their ability to burrow into the 

sediment to avoid winter or other environmental stressors is impeded (Nalepa, 1994; Schloesser and 

Nalepa, 1994). Colonization by zebra mussels causes declines in unionid mussel populations and is 

dependent upon zebra mussel density, biomass, time since invasion, and substrate type (Burlakova and 

others, 2000). 

Federal and state management agencies have implemented recovery and propagation programs 

for threatened and endangered unionid species coinciding with legislation and programs for control and 

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

2014). However, there is currently a lack of access to environmentally safe and effective tools to control 

dreissenid mussel populations in open-water environments. A potential tool for limited open-water 

control of dreissenid mussels is a commercially formulated spray dried powder (SDP) product, which 

contains the killed cells of a specific strain (CL145A) of the common soil bacterium Pseudomonas 

fluorescens. The goal of this study was to determine the potential use of SDP in unionid mussel 

recovery and restoration efforts by controlling zebra mussel populations. The study objectives were 

(1) to determine the efficacy of SDP to control zebra mussels adhering to unionid mussels, and (2) to 
3 

evaluate the effects of SDP exposure on survival of unionid mussels compromised by zebra mussel 

colonization. This report summarizes a single field trial completed from July 18, 2013, to November 18, 

2013. The exposures were completed on July 26, 2013, and the postexposure assessments were 

completed August 13–15, 2013. 

Materials and Methods

The protocol for this study entitled “Efficacy of Pseudomonas fluorescens (Pf-CL145A) SDP for 

controlling zebra mussels within field enclosures” is contained in appendix 1, item 1. All methods and 

materials follow the written protocol except those instances that were identified in a note to file 

(appendix 1, item 2) and in study deviations (appendix 2, items 1–5).

Experimental Design

The study was done in Lake Darling (Alexandria, Minnesota) and consisted of a twofold 

assessment to evaluate the reduction in the number of zebra mussels adhering to unionid mussels and to 

evaluate unionid mussel survival following exposure to SDP. Zebra mussel reduction was assessed by 

comparing the estimated number of zebra mussels adhering to unionid mussels prior to SDP exposure to 

the enumerated number of zebra mussels adhering to mussels after SDP exposure. Unionid mussel 

survival was assessed after SDP exposure by observing foot movement or resistance to valve pressure. 

Unionid mussels were collected, individually tagged, and randomly assigned (appendix 3, 

item 4) to test enclosures in equal proportions (n = 243; 27 per test enclosure). A group of unionid 

mussels (Group 1; n = 45; 5 per test enclosure) were removed and used to estimate the number of 

adhering zebra mussels prior to SDP exposure (that is, adhering zebra mussels were removed and 

enumerated). After zebra mussel removal, Group 1 unionid mussels were returned to their assigned 

treatment enclosure for the remainder of the study. The undisturbed unionid mussels (Group 2; n = 198; 

22 per test enclosure) remained in the assigned test enclosure area for the entire study duration and were 

used to evaluate the efficacy of SDP for removal of adhering zebra mussels. Both groups were exposed 

concurrently in the test enclosures. 

Treatments were administered in triplicate as a single SDP exposure according to a randomized 

block design (appendix 3, item 1). The treatment levels were (1) an untreated control group, (2) a group

that received an application of 50 milligrams per liter (mg/L) of SDP active ingredient (AI) for 8 hours, 

and (3) a group that received an application of 100 mg/L of SDP AI for 8 hours. The experimental unit 

for the trial was the individual test enclosure.

Test Enclosure Area

Nine test enclosure areas were identified near the north shore of Lake Darling (fig. 1), and the 

Universal Transverse Mercator (UTM) coordinates (test area centroid) were recorded (table 1). Each test 

area was characterized as predominately sandy substrate with minimal algae and macrophyte growth. 

The areas selected were approximately 1.4 meter (m) deep and were spaced at least 6 m apart (by center 

point). Unionid mussel retention barriers (fig. 2) were placed in each test enclosure area approximately 

48 hours prior to exposure. The expanded metal 1-square-meter (m2) retention barriers were used to 

confine the unionid mussels to the test enclosure area for the duration of the study. After unionid mussel 

allocation, a 2.25-m2 test enclosure (fig. 2) was placed around each retention barrier and secured to 

fenceposts driven into the lakebed. The bottom sealing flaps of each test enclosure were secured to the 

lakebed with sandbags. 
4 

Figure 1. Test enclosure site and test animal collection locations.

Figure 2. Example of 1-square-meter (m2) expanded metal mussel retention barrier (left) and 

2.25-m2 impermeable test enclosure (right).
5 

Table 1. Universal Transverse Mercator (UTM) centroid 

location of each test enclosure.

[mg/L, milligrams per liter; mE, meters East; mN, meters North]

Enclosure

Treatment 

group 

(mg/L)

Zone Row Easting 

(mE)

Northing 

(mN)

1 100 15 T 315318 5089217

2 50 15 T 315305 5089224

3 0 15 T 315292 5089230

4 50 15 T 315281 5089231

5 50 15 T 315279 5089221

6 100 15 T 315290 5089218

7 0 15 T 315294 5089215

8 0 15 T 315302 5089211

9 100 15 T 315307 5089205

Test Article

The test article was a commercially prepared SDP formulation of Pseudomonas fluorescens, 

strain CL145A containing 50 percent AI (weight to weight ratio [w/w] P. fluorescens, strain CL145A). 

The test article (lot number TR 4669-4-3) was obtained from Marrone Bio Innovations, Inc. (MBI; 

Davis, California; Certificate of Analysis, appendix 4, item 2). Test article use was documented in the 

test chemical logbooks (appendix 4, items 5 and 6). Verification of test article biological activity was 

determined on a sample of test article by the New York State Museum Field Research Laboratory 

(Cambridge, New York), using their standard dreissenid mussel bioassay (appendix 4, item 7).

Biological activity was confirmed as demonstrated by a mean zebra mussel mortality in the treated 

group of 96.0 ± 6.9 percent compared to 1.3 ± 2.2 percent in the untreated group. 

Test Animals

Adult fatmucket unionid mussels (Lampsilis siliquoidea) with adhering zebra mussels (fig. 3) 

were collected 3 days prior to exposure from an existing population located within 300 m of the test 

enclosures. An incidental number of fragile papershell unionid mussels (Leptodea fragilis, n = 0–3 per 

enclosure) were inadvertently included. The L. fragilis were treated and analyzed the same as L. 

siliquoidea. Unionid mussels were confined within a 1-m2 retention barrier in approximately 0.75 m of 

water until distributed to the test enclosures. A Hallprint® shellfish tag with a unique alphanumeric code 

was fixed to each mussel shell with cyanoacrylate glue, and an initial wet weight with adhering zebra 

mussels was obtained for each unionid mussel. 

Groups of unionid mussels (n = 4–5) with adhering zebra mussels were randomly allocated to 

each test enclosure (appendix 3, item 4) and placed within a retention barrier until each test 

enclosure/retention barrier received a total of 27 unionid mussels. 

At exposure termination, the test enclosures were removed and both Group 1 and Group 2 

unionid mussels remained in the retention barriers at the treatment location for a postexposure holding 

period. At 18–20 days postexposure, the unionid mussels were removed from the retention barriers, and 
6 

wet weights were measured before and after removal of adhering zebra mussels. Additionally, each 

unionid mussel was photographed, assessed for survival, and measured for shell length. Unionid 

mussels were euthanized according to permit requirements (appendix 5, items 1–3). The unionid mussel 

shells were retained and used for confirmative species identification. All zebra mussels removed were 

sorted into categories of dead or alive and were enumerated; a subsample from each group was 

measured for shell length.

Figure 3. Representative unionid mussel with adhering zebra mussels preexposure (top 

left), postexposure (top right) and with zebra mussels removed (bottom).

Reduction of Adhering Zebra Mussels

Estimation of Initial Number of Adhering Zebra Mussels (Group 1)

After distribution, five unionid mussels (Group 1) were indiscriminately removed from each test 

enclosure and used to estimate the initial preexposure biomass of adhering zebra mussels for each test 

enclosure (that is, the initial number of adhering zebra mussels per gram of zebra mussels [equation 1]): 

 ee = nzz

[W1− W2]

 (1) 

where en is the estimated number of adhering zebra mussels per gram of zebra mussels, 

nzm is the number of live adhering zebra mussels removed from Group 1 mussels 

(preexposure), 

 W1 isthe group 1 mussel wet weight with adhering zebra mussels, in grams (g), and 
7 

 W2 is the group 1 mussel wet weight after zebra mussel removal (g). 

The average number of adhering zebra mussels, per gram, from each test enclosure was used to estimate 

the initial number of adhering zebra mussels on Group 2 mussels (equation 2). 

Estimation of the Number of Adhering Zebra Mussels (Group 2)

For each test enclosure, the number of zebra mussel adhering to Group 2 mussels prior to 

exposure was estimated (equation 2) by using the mean estimated number (x ̄e ) of adhering zebra 

mussels, per gram, of Group 1 mussels (derived from equation 1). Initial Group 2 mussel wet weight 

with adhering zebra mussels before exposure and Group 2 mussel wet weight after zebra mussel 

removal were used to determine the weight of adhering zebra mussels (equation 2); 

 ee2 = [W1 − W2] × x ̄e (2) 

where en2 is the estimated number of zebra mussels adhering to Group 2 mussel prior to 

exposure, 

W1 is the preexposure wet weight of Group 2 mussel with adhering zebra mussels (g), 

W2 is the postexposure wet weight Group 2 mussel after zebra mussel removal (g), and 

x ̄e is the mean estimated number of adhering zebra mussels per gram of zebra mussels 

for each test enclosure (derived from equation 1). 

Calculation of Zebra Mussel Body Weight Burden (Group 2)

The percent body weight burden of adhering zebra mussels was calculated for each individual 

unionid mussel by comparing the estimated weight of adhering zebra mussels to the weight of the 

unionid mussel without zebra mussels (equation 3). A mean percent body weight burden was calculated 

for each treatment group: 

 ZZ BBBB (percent) = (W1/ W2) × 100 (3) 

where 

ZM Burden is the adhering zebra mussel burden expressed as percent of unionid mussel body 

weight, 

W1 is the estimated preexposure wet weight of zebra mussels (g), and

W2 is the postexposure wet weight unionid mussel without zebra mussels (g). 

Calculation of Adhering Zebra Mussels Reduction (Group 2)

For each test enclosure, the reduction in the number of zebra mussels adhering to Group 2 

mussels was estimated by using the mean estimated number (x ̄e 2

) of adhering zebra mussels (derived 

from equation 2) and the number of adhering zebra mussels postexposure (equation 4). A mean percent 

reduction was calculated for each treatment group: 

 P P P Δ = ���x ̄ ee2

−nzz}

x ̄ ee2

� × 100� (4) 

where Δ is the percent reduction in the number of adhering zebra mussels on Group 2 

unionids, 

x ̄e 2 is the mean estimated number of zebra mussels adhering to Group 2 mussel prior to 

exposure (derived from equation 2), 

nzm is the number of live zebra mussels removed from Group 2 mussels (postexposure). 
8 

Calculation of SDP Reduction Coefficient and Efficiency (Group 2)

A reduction coefficient of SDP was calculated for each test enclosure by comparing the 

reduction in the number of adhering zebra mussels to the amount of SDP applied to the test enclosure 

area (equation 5). A mean reduction coefficient was calculated for each treatment group: 

 RR = � ∆

�P

A� �

� (5) 

where RC (reduction coefficient) is the percent reduction of adhering zebra mussels per gram of 

SDP applied, 

Δ is the percent reduction in the number of adhering zebra mussels on Group 2 unionids 

(derived from equation 4), 

P is the amount of SDP applied (g), and 

A is the area treated (=2.25 m2). 

Unionid Mussel Survival

Each unionid mussels was assessed for survival 18–20 days after SDP exposure. Survival was 

defined as foot or valve movement in response to tactile stimuli or resistance to valve pressure by 

adductor muscle contraction. Unrecovered unionid mussels were treated as mortalities within the data 

analysis.

Stock-Solution Preparation and Dosing

A separate SDP stock solution was prepared and immediately applied to each test enclosure. The

appropriate amount of SDP required to treat each test enclosure (appendix 4, item 4) was added to 

approximately 10 liters (L) of unfiltered lake water and mixed. Immediately after mixing, the stock was 

poured through a strainer, and clumps of test article were macerated with a pestle and rinsed into the 

stock solution with unfiltered lake water, bringing the final stock volume to approximately 15 L. 

The prepared stock solution was transported in a 19.4-L screw-top bucket to the assigned test 

enclosure. Treatments were individually applied in the following sequence: control, 50-, and 100-mg/L

SDP. The 50- and 100-mg/L SDP treatments were applied by adding the stock solution to four areas of 

the test enclosure. The water and stock solution within the test enclosure was then mixed by using a boat 

paddle. The control treatments involved the same application technique used for the SDP-treated 

groups, except that untreated Lake Darling water was applied in place of SDP stock solution. 

Test Enclosure Water Sample Collection

Water samples were collected by submersing a 1-L screw-top container below the surface of 

each test enclosure. The water samples were used to verify exposure concentration and to measure 

water-quality parameters (that is, hardness, alkalinity, conductivity, dissolved oxygen, temperature, pH, 

and ammonia). 

Concentration Verification

Exposure water SDP concentrations were determined at 1, 4, and 8 hours by comparing surface 

water samples collected from each test enclosure to a linear regression curve created from known 

concentrations (25, 50, 100 and 200 mg/L) and absorbance of the test article. Sample absorbance was 

measured on a Barnstead-Turner SP-830 Plus (model SM110215) spectrophotometer at 660 nanometers 

(nm). Linear regression equations were fit by using the SAS® software Proc Reg procedure (SAS®
9 

version 9.3; SAS Institute Inc., Cary, North Carolina), and the exposure sample concentrations were 

predicted from the regression analysis (appendix 7, items 2 and 3). 

Water Chemistry

Hardness, alkalinity, and conductivity were measured in surface water samples collected from 

each test enclosure before administering the test article. Dissolved oxygen, pH, and temperature were 

measured in surface water samples collected from each test enclosure prior to exposure as well as 4 and 

8 hours after exposure initiation. Immediately before exposure period termination, water samples were 

collected, filtered (0.45 micrometer [μm]), acidified with 10 percent sulfuric acid to ≤ pH 2.5, and 

stored at approximately 4 degrees Celsius (°C) until analyzed for total ammonia nitrogen (TAN) content 

by using the automated phenate method (Standard Method 4500G; American Public Health Association

and others, 2012). The un-ionized ammonia fractions were calculated by using the sample pH and 

temperatures measured at the time of sample collection according to the formula identified by Emerson 

and others (1975). Temperature loggers (Onset, Bourne, Massachusetts, HOBO® Pendent 

Temperature/Light Data Logger) were attached to the retention barriers and used to measure water 

temperature every 3 hours during the postexposure period. Dissolved oxygen, pH, and temperature of 

the lake water were measured daily during the assessment period (18–20 days postexposure) near the 

retention barriers.

Data Analysis

Water chemistry data analyses were limited to simple descriptive statistics; comparative 

statistics were not generated. Statistical comparisons of zebra mussel density, zebra mussel length, and 

unionid mussel survival were performed by using SAS® software version 9.3 (SAS Institute, Inc., Cary, 

N.C.). Significance for all analyses was declared at p ≤ 0.05. Exposure concentrations were determined 

by using SAS® software version 9.3. 

Reduction of Adhering Zebra Mussels

A general linear model created with the SAS® software Proc GLM procedure was used to 

analyze the number of zebra mussels adhering to unionid mussels before and after exposure 

(appendix 10, item 5). The mean number of zebra mussels adhering to unionid mussels in each 

treatment group before and after exposure was modeled with the “no intercept” and “solution” options 

specified. The assumptions of normal distribution and homogeneity of variance were assessed by using 

the univariate procedure with the “normal” option specified and the Bartlett’s test. The mean number of 

zebra mussels adhering to unionid mussels of each treatment group (before and after exposure) was 

individually compared to the number of zebra mussels adhering to unionid mussels of the untreated 

control group by using a two-sided means comparison test. 

By using the procedures previously described (SAS® software Proc GLM; appendix 10, item 8), 

the mean length of zebra mussels removed from unionid mussels collected from each treatment replicate 

before (Group 1) and after (Group 2) SDP exposure was analyzed to determine whether a correlation 

existed between zebra mussel length and zebra mussel survival within each treatment assignment. The 

analysis of test animal length was limited to adhering live zebra mussels because dead zebra mussel

shells could not be retained. 
10 

Unionid Mussel Survival

A generalized linear mixed model (SAS® software Proc GLIMMIX) was used to analyze the 

survival of unionid mussels in each treatment (appendix 10, item 2). The proportion of mortalities 

(number of dead unionid mussels compared to the total number unionid mussels) was modeled with a 

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

“random_residual” statement. Unionid mussel survival of each treatment group was individually 

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

with a Tukey post hoc adjustment. 

Exposure Concentration

The mean exposure concentrations were determined for individual test enclosures, for each 

sampling time, and for each treatment group using the SAS® software Proc Means statement (appendix 

7, item 3).

Results

Adverse Events

There were no observable adverse effects observed in the unionid mussels or zebra mussels 

within the test enclosures. There was a significant algal bloom within Lake Darling (dominated by 

Mougeotia and Spirogyra species) during the postexposure period. The study was terminated 

approximately 10 days earlier than planned because of concerns that the algae bloom may impact native 

or zebra mussel survival (appendix 2, items 1 and 6). 

Reduction of Adhering Zebra Mussels

Statistical analysis output can be found in appendix 10 (item 4), and the data summary can be 

found in appendix 8 (item 1). The number of zebra mussels adhering to unionid mussels before and after 

treatment is summarized in table 2. No difference (p > 0.74) was detected in the number of zebra 

mussels adhering to unionid mussels in each treatment group prior to exposure. The observed percent 

body weight burden of adhering zebra mussels from each treatment group ranged from 125.9 to 130.0 

percent of unionid mussel body weight. The mean number of adhering zebra mussels before and after 

exposure differed in the 50- and 100-mg/L treatment groups (160 versus 74 [p < 0.01] and 180 versus

59 [p < 0.01], respectively). The number of adhering zebra mussels was reduced on average by 53 and 

68 percent in the 50- and 100-mg/L treatment groups, respectively. The number of adhering zebra 

mussels did not differ (p = 0.79) between the 50- and 100-mg/L treatment groups after exposure. When 

standardized to the amount of SDP applied per square meter, the 50-mg/L treatment was more efficient 

than the 100-mg/L treatment at reducing the number of adhering zebra mussels (0.44 percent reduction 

per gram applied versus 0.28 percent reduction per gram applied, respectively). 

Statistical analysis output can be found in appendix 10 (item 7), and the data summaries can be 

found in appendix 5 (items 6–7). Mean length of adhering zebra mussels is summarized in table 3. Mean 

length did not differ (p > 0.89), indicating that zebra mussel length was not a predictor of mortality.
11 

Table 2. Mean (standard deviation) number of adhering zebra mussels per unionid mussel before and after 

exposure, percent change of zebra mussel reduction, zebra mussel burden (as percent unionid body weight), and 

spray dried powder (SDP) reduction coefficient for number of adhering zebra mussels for each treatment group. 

[mg/L, milligrams per liter; ZM, zebra mussel; SDP, spray dried powder; NA, not applicable. Means with the same letter are 

not significantly different (p > 0.05).]

Treatment group 

(mg/L)

Zebra mussels per unionid mussel

ZM burden1 SDP reduction coefficient2

(percent) Preexposure Postexposure Colonization change 

(percent)

Control (0) 165a (15) 179a (9) 9 (9) 130.0 (4.4) NA

50 160a (11) 74b (9) -53 (8) 125.9 

(12.5) 0.441 (0.043)

100 180a (13) 59b (23) -68 (10) 127.1 

(11.7) 0.276 (0.033)

1

Expressed as a percentage of unionid mussel body weight. 

2

Expressed as percent reduction in the number of adhering zebra mussels per gram of SDP applied. 

Table 3. Mean (standard deviation; number measured) length of adhering zebra mussels (live) by treatment 

group.

[mg/L, milligrams per liter; mm, millimeters. Means with the same letter are not significantly different (p > 0.05)]

Treatment group (mg/L) Length (mm)

Preexposure Postexposure

Control (0) 12.0a (0.1; 1,453) 11.2a (2.3; 1,366)

50 12.1a (0.5; 1,266) 11.8a (0.5; 590)

100 11.2a (0.4; 1,359) 12.0a (0.5; 584)

Unionid Mussel Survival

Statistical analysis output can be found in appendix 10 (item 1), and the data summary can be 

found in appendix 9 (item 1). Survival of unionid mussels is summarized in table 4. Treatment did not 

significantly affect Group 1 mussel survival (p > 0.05) but did significantly affect Group 2 mussel 

survival (p < 0.01) and Group 1 and Group 2 combined mussel survival (p < 0.01). No difference 

(p > 0.23) was detected in survival between the Group 2 control group and the 50- or 100-mg/L 

treatment groups. A difference (p = 0.03) was detected in the mean survival between the 50- and the 

100-mg/L treatment groups (96.8 and 85.1 percent, respectively), however, the biological significance 

of the difference is indeterminate. The log odds of unionid mussel survival in the 100-mg/L treatment 

was 0.290 times the odds of survival of unionid mussels assigned to the 50-mg/L treatment.
12 

Table 4. Mean (standard deviation) percent 

survival of Group 1 and 2 unionid mussels.

[mg/L, milligrams per liter. Means within columns with 

the same letter are not significantly different (p > 0.05)]

Treatment group (mg/L) Survival (percent)

Group 1 Group 2

Control (0) 100.0a (0.0) 89.4ab (6.9)

50 100.0a (0.0) 96.8a (3.9)

100 80.0a (20.0) 85.1b (13.8)

Dose Verification

The linear regression, exposure concentrations, and data summary can be found in appendix 7 

(items 1–2). Exposure concentrations for each treatment group are summarized in table 5. The measured 

exposure concentrations were lower than the target concentration. The mean SDP exposure 

concentrations measured throughout the exposure period were 35.3 ± 3.3 mg/L and 74.2 ± 3.4 mg/L for 

the 50- and 100-mg/L treatment groups, respectively. 

Table 5. Mean (standard deviation) spray dried powder 

exposure concentration in surface water samples collected from 

test enclosures during the exposure period.

[mg/L, milligrams per liter; ND, not detectable/below detection limit]

Treatment group (mg/L) Time (hours)

1 4 8

Control (0) ND ND ND

50 38.6 (1.7) 35.5 (2.7) 31.9 (1.0)

100 76.9 (1.3) 75.5 (2.4) 70.3 (1.3)

Water Chemistry

The water-chemistry data summaries are presented in appendix 6 (items 1–5). Water-chemistry 

parameters (dissolved oxygen, pH, and temperature) in the test enclosures are summarized in table 6. 

Dissolved oxygen concentration remained above the minimum level recommended (4 mg/L) in the 

ASTM International guide for conducting laboratory tests with freshwater mussels (ASTM 

International, 2013). Hardness, alkalinity, conductivity, and ammonia are summarized in table 7. The 

alkalinity ranged from 174 to 176 mg/L as calcium carbonate (CaCO3), hardness from 189 to 193 mg/L 

as CaCO3, and conductivity from 322 to 324 microsiemens per centimeter (μS/cm) (automatic 

temperature corrected to 25 °C); un-ionized ammonia was ≤ 0.01 mg/L. Data collected by the 

temperature data loggers during the postexposure period indicated a water temperature range of 22.3 °C 

to 23.9 °C. Mean (standard deviation [SD]) water-chemistry parameters measured 18–20 days

postexposure were dissolved oxygen, 8.60 mg/L (0.40); temperature, 21.9 °C (0.3); and pH range, 8.54–

8.63. 
13 

Table 6. Mean (standard deviation) water chemistry (dissolved 

oxygen, temperature) and pH range of surface water samples collected 

from each treatment group during the study period.

[mg/L, milligrams per liter; °C; degrees Celsius]

Sample time pH Dissolved oxygen (mg/L) Temperature (°C)

Control (0 mg/L)

Preexposure 8.45 – 8.58 7.03 (0.12) 20.6 (0.5)

4 hours 8.45 – 8.50 6.49 (0.12) 22.9 (0.2)

8 hours 8.40 – 8.49 6.36 (0.39) 23.0 (0.0)

50 mg/L

Preexposure 8.47 – 8.56 7.22 (0.48) 20.4 (0.2)

4 hours 8.35 – 8.38 5.95 (0.17) 23.1 (0.1)

8 hours 8.25 – 8.36 5.63 (0.06) 23.1 (0.1)

100 mg/L

Preexposure 8.53 – 8.56 6.86 (0.24) 19.6 (0.6)

4 hours 8.16 – 8.28 5.85 (0.19) 23.0 (0.2)

8 hours 8.18 – 8.23 5.36 (0.17) 23.1 (0.2)

Table 7. Mean (standard deviation) alkalinity, hardness, conductivity, and ammonia content of surface water 

samples collected from each treatment group during the study period.

[mg/L, milligrams per liter; μS/cm, microsiemens per centimeter; TAN, total ammonia nitrogen; mg NH3-N/L, milligrams unionized ammonia nitrogen per liter; NH3, un-ionized ammonia; <, less-than; CaCO3, calcium carbonate; °C, degrees Celsius]

Treatment group 

(mg/L)

Preexposure Exposure termination

Alkalinity 

(mg/L)1

Hardness 

(mg/L)1

Conductivity 

(μS/cm)2

TAN (mg NH3-

N/L) NH3 (mg/L)

Control (0) 175 (0) 189 (1) 323 (5) 0.04 (0.02) <0.01 (<0.01)

50 174 (2) 190 (2) 322 (3) 0.10 (0.02) <0.01 (<0.01)

100 176 (3) 193 (4) 324 (4) 0.13 (0.01) 0.01 (<0.01)

1

Reported as milligrams per liter CaCO3. 

2

Temperature compensated to 25 °C.

Conclusions

The application of a single dose of spray dried powder (SDP) formulation of Pseudomonas 

fluorescens (strain CL145A, 50 or 100 milligrams per liter [mg/L]) for 8 hours to open-water test 

enclosures significantly reduced the number of zebra mussels adhering to unionid mussels. The number 

of adhering zebra mussels after exposure did not differ (p = 0.79) between the 50-mg/L and the 

100-mg/L treatment groups. When standardized to the amount of SDP applied, the 50-mg/L treatment 
14 

was more efficient than the 100-mg/L treatment at reducing the number of zebra mussels adhering to 

zebra mussels (0.44 percent reduction per gram of SDP applied versus 0.28 percent reduction per gram 

of SDP applied, respectively). Multiple 50-mg/L applications may be more efficient at reducing zebra 

mussels than a single 100-mg/L application of SDP. The use of a subsurface injection application 

technique to deliver SDP only to the benthic zone may further reduce the amount required to achieve 

control. Treatment success was not dependent upon zebra mussel length (p > 0.89). No difference was 

detected in unionid mussel survival between the treated and control groups. A difference (p = 0.03; OR 

= 0.290) in unionid mussel survival was detected between the Group 2 50-mg/L and Group 2 100-mg/L 

treatment groups. Though statistically different, the biological significance of the slight difference in 

observed mortality between the treatment groups is indeterminate. Unionid mussel mortality may have 

been related to poor initial condition, as indicated by the high initial burden of adhering zebra mussels 

(> 125 percent of unionid mussel body weight). 

References

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

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

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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 27, 2015 at http://nas.er.usgs.gov/queries/FactSheet.aspx?speciesID=5. 

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

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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. 

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

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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–

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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. 

Master, L., 1990, The imperiled status of North American aquatic animals: Biodiversity Network News, 

v. 3, no. 3, p. 5–8. 

Nalepa, T.F., 1994, Decline of native unionids 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. 
15 

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

Conchology, special publication 3, p. 69–80. 

Neves, R.J., Bogan, A.E., Williams, J.D., Ahlstedt, S.A., and Hartfield, P.W., 1997, Status of aquatic 

mollusks in the southeastern United States—A downward spiral of diversity, in Benz, G.W., and 

Collins, D.E., eds., Aquatic fauna in peril—The southeastern perspective: Decatur, Ga., Southeastern 

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

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

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

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16 

Appendix 1. Study Protocol With Data Forms 

Item 

number Item description

Number 

of 

pages

Report 

Page 

number

1 Protocol: “Efficacy of Pseudomonas fluorescens (Pf-CL145A) SDP for controlling 

zebra mussels within field enclosures” 32 17

2 Note to File 1: Foot biopsy collection and resulting data use 1 49

3 Test Chemical Stock Preparation 1 50

4 Mussel Species Identification 9 51

5 Mussel Distribution Data 1 60

6 Water Quality 1 61

7 Alkalinity 1 62

8 Conductivity and Hardness 1 63

9 HOBO Data 1 64

10 Spectrophotometric Data 2 65

11 Initial Assessment of Native and Zebra Mussels 1 67

12 Mussel Initial Assessment 1 68

13 Mussel Weight and Length Assessment 1 69

14 Mussel Survival Assessment 1 70

15 Mussel Final Survival Assessment 1 71

16 Zebra Mussel Lengths 1 72

17 Assessment Zebra Mussel Lengths 1 73

18 Mussel Volume Displacement Assessment – Not Used in Study 1 74
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Appendix 2. Deviations From the Study Protocol 

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Deviation 1: Early termination of holding period 1 76

2 Deviation 2: Unrecovered unionid mussels during post-exposure assessment 1 77

3 Deviation 3: Mussel #C331 assessed twice 1 78

4 Deviation 4: Discontinuation of volume displacement 1 79

5 Deviation 5: Unequal ratios of Fragile Papershell (Leptodea fragilis) mussels in the 

study 2 80

6 Filamentous algae report from John Manier 5 82
76 
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79 
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81 
82 
83 
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85 
86 
87 

Appendix 3. Randomization Assignments

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 SAS output for random assignment of treatment to experimental enclosures 1 88

2 SAS program for random assignment of treatment to experimental enclosures 2 89

3 SAS log for random assignment of treatment to experimental enclosures 2 91

4 SAS output for random assignment of mussels to enclosures 2 93

5 SAS program for random assignment of mussels to enclosures 1 95

6 SAS log for random assignment of mussels to enclosures 2 96
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Appendix 4. Test Article Information 

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Material Safety Data Sheet: MBI-401 Spray Dried Powder 2 99

2 MBI-401 SDP (lot # TR-4669-4-3) Test Article: Certificate of Analysis 1 101

3 Test Material Requirements for Enclosure treatments 1 102

4 Test Chemical Stock Preparation 1 103

5 

Copy of test article information from test article log book for MBI-401 SDP; lot 

number TR4669-4-3; Container 1 of 2 – Used for enclosure treatment at Lake 

Darling

6 104

6 

Copy of test article information from test article log book for MBI-401 SDP; lot 

number TR4669-4-3; Container 2 of 2 – Used for enclosure treatment at Lake 

Darling

6 110

7 Post-treatment product validation assay for test article (lot # TR4669-4-3) 2 116

8 MBI-401 SDP (lot # 401P120724C) Test Article: Certificate of Analysis – Not Used 

for Treatment 1 118

9 Test Chemical Stock Preparation – Not Used for Treatment 1 119

10 Pre-treatment product validation assay for test article (lot # 401P120724C) – FAILED 2 120

11

Copy of test article information from test article log book for MBI-401 SDP; lot 

number 401P120724C; Container 1 of 1 – Not used in dosing (FAILED pre-test 

validation)

4 122
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Appendix 5. Test Animal Information

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Special permit (No. 18804) for taking and possession of mussels 2 127

2 Prohibited Invasive Species Permit (No. 290) 1 129

3 Collection Report for Special Permit 18804 2 130

4 Mussels Species Identification datasheet 9 132

5 Unionid Mussel Weight and Length – Data Summary 6 141

6 Zebra Mussel Lengths Pre-exposure Assessment – Data Summary 10 147

7 Zebra Mussel Lengths Post-exposure Assessment – Data Summary 11 157
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Appendix 6. Water Quality

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Pre-Exposure Period Water Chemistry – Data Summary 2 169

2 Exposure Period Water Chemistry – Data Summary 2 171

3 Ammonia – Data Summary 3 173

4 Temperature Data Loggers (HOBOs) – Data Summary 4 176

5 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC 1 180

 
169 
170 
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174 
175 
176 
177 
178 
179 
180 
181 

Appendix 7. Spectrophotometric Summary and SAS Output, 

Program, and Log 

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Spectrophotometric Data – Data Summary 2 182

2 SAS output for Standard Curve Linear Regression and sample concentrations 11 184

3 SAS program for Standard Curve Linear Regression and sample concentrations 2 195

4 SAS log for Standard Curve Linear Regression and sample concentrations 4 197
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Appendix 8. Zebra Mussel Density Association Summary Data 

Analysis

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Zebra Mussel Density Associations – Data Summary 15 202
202 
203 
204 
205 
206 
207 
208 
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210 
211 
212 
213 
214 
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217 

Appendix 9. Unionid Mussel Survival Assessment Summary 

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Unionid Mussel Survival – Data Summary 5 218
218 
219 
220 
221 
222 
223 

Appendix 10. Statistical Analysis, Including SAS Programs, 

Outputs, and Logs for Unionid Mussel Survival, Zebra Mussel 

Colonization Density Associations, and Zebra Mussel Test 

Animal Lengths 

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 SAS output for unionid mussel survival 11 224

2 SAS program for unionid mussel survival 3 235

3 SAS log for unionid mussel survival 6 238

4 SAS output for zebra mussel density 23 244

5 SAS program for zebra mussel density 2 267

6 SAS log for zebra mussel density 4 269

7 SAS output for zebra mussel lengths 23 273

8 SAS program for zebra mussel lengths 2 296

9 SAS log for zebra mussel lengths 4 298
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ISSN 2331-1258 (online) 

http://dx.doi.org/10.3133/2015-1051