Safety of Spray-Dried Powder Formulated Pseudomonas 

fluorescens Strain CL145A Exposure to Subadult/Adult 

Unionid Mussels During Simulated Open-Water 

Treatments

By James A. Luoma, Kerry L. Weber, Diane L. Waller, Jeremy K. Wise, Denise A. Mayer, and Douglas B. Aloisi

Open-File Report 2015–1064

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 

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Suggested citation:

Luoma, J.A., Weber, K.L., Waller, D.L., Wise, J.K., Mayer, D.A., and Aloisi, D.B., 2015, Safety of spray-dried 

powder formulated Pseudomonas fluorescens strain CL145A exposure to subadult/adult unionid mussels during 

simulated open-water treatments: U.S. Geological Survey Open-File Report 2015–1064, 248 p., 

http://dx.doi.org/10.3133/ofr20151064. 

ISSN 2331-1258 (online)

ii
Acknowledgments

This study was funded through a grant from the U.S. Environmental Protection Agency Great 

Lakes Restoration Initiative awarded to the New York State Education Department and U.S. Geological 

Survey appropriated funds. 

The authors would like to thank the staff of the Genoa National Fish Hatchery for the many 

hours dedicated to propagating the seven species of mussels and for their expert advice, which was 

integral to the study design and execution. The Wisconsin Department of Natural Resources assisted in 

the permitting required for the study, and the La Crosse Regional Airport (La Crosse, Wisconsin) 

allowed us to conduct study operations on their property. 

The authors thank Mr. Hugh McMath (USGS), who assisted in study conduct as well as data and 

sample collection. We also thank Mr. Mark Gaikowski (USGS) for his assistance in study design and

data analysis. 

iii
Contents

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

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

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

Materials and Methods ............................................................................................................................................... 4

Experimental Design............................................................................................................................................... 4

Test Article .......................................................................................................................................................... 4

Test System ........................................................................................................................................................ 5

Test Animals........................................................................................................................................................ 6

Postexposure Handling.................................................................................................................................... 6

Survival Assessment ....................................................................................................................................... 6

Dosing and Dose Verification .............................................................................................................................. 7

Water Chemistry.................................................................................................................................................. 8

Data Analysis .......................................................................................................................................................... 8

Results........................................................................................................................................................................ 8

Conclusions .............................................................................................................................................................. 11

References Cited...................................................................................................................................................... 12

Appendix 1. Study Protocol with Data Forms............................................................................................................ 13

Appendix 2. Deviations from the Study Protocol..................................................................................................... 100

Appendix 3. Randomization Assignments............................................................................................................... 104

Appendix 4. Test Article Information ....................................................................................................................... 123

Appendix 5. Test Animal Information ...................................................................................................................... 137

Appendix 6. Water Quality ...................................................................................................................................... 154

Appendix 7. Spectrophotometric Summary, SAS Program, Output and Log .......................................................... 167

Appendix 8. Statistical Analysis including SAS Programs, Outputs and Logs for Survival and Treatment 

Concentration ......................................................................................................................................................... 193

Appendix 9. Survival Assessment Summary .......................................................................................................... 237

iv
Figures

Figure 1. Exposure and postexposure test animal holding location. ................................................................... 3

Figure 2. Test tanks positioned in the mobile bioassay laboratory (left) and containment baskets positioned in 

test tank (right)........................................................................................................................................................... 5

Figure 3. Example specimens of the seven unionid mussel species tested (left) and alphanumeric-tagged 

mussels (right). .......................................................................................................................................................... 6

Figure 4. Example wire mesh cage used to hold mussels during the postexposure period. ............................... 7

Tables

Table 1. Scientific and common name, source, lifestage, mean (standard deviation) length, and total number 

of test animals used in the study................................................................................................................................ 4

Table 2. Mean (standard deviation) percent survival of mussels exposed to SDP by treatment group and 

exposure duration...................................................................................................................................................... 9

Table 3. Mean (standard deviation) dissolved oxygen, temperature and pH range observed for each treatment 

group during the study period. ................................................................................................................................. 10

Table 4. Mean (standard deviation) alkalinity, hardness, conductivity, total ammonia nitrogen (TAN), and 

un-ionized ammonia (NH3) observed during the exposure period............................................................................ 10

Table 5. Mean (standard deviation) exposure concentration for each treatment group throughout the exposure 

period. ......... ........................................................................................................................................................... 11

v 
Conversion Factors

International System of Units to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

micrometer (μm) 3.937x10-5 inch (in.)

millimeter (mm) 0.03937 inch (in.)

Volume

liter (L) 1.057 quart (qt)

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 x10-5 ounce, avoirdupois (oz)

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

Specific conductance 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).

Abbreviations

AEH Aquatic Ecosystem Health Branch

CaCO3 calcium carbonate

DO dissolved oxygen

ID inner diameter

NH3 un-ionized ammonia

SDP spray dried powder

SDP/L spray dried powder per liter

TAN total ammonia nitrogen

TEMP temperature

Pf Pseudomonas fluorescens

μS/cm microsiemens per centimeter

vi 
Safety of Spray-Dried Powder Formulated Pseudomonas 

fluorescens Strain CL145A Exposure to Subadult/Adult 

Unionid Mussels During Simulated Open-Water 

Treatments

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

1 Diane L. Waller,1 Jeremy K. Wise,1 Denise A. Mayer,2 and Douglas B. 

Aloisi3

Abstract

The exposure effects of a commercially prepared spray dried powder (SDP) formulation of 

Pseudomonas fluorescens (strain CL145A) on the survival of seven species of unionid mussels endemic 

to the Great Lakes and Mississippi River basins was evaluated in this study. The study exposures were

completed within replicated 350-liter test tanks contained within a mobile bioassay laboratory sited on 

the shores of the Black River near La Crosse, Wisconsin. The test tanks were supplied with flowing, 

filtered river water which was interrupted during the exposure period. 

Two groups of seven species of mussels were used in equal proportions in the study. The first 

group was exposed to SDP for 8 hours, and the second group was exposed to SDP for 24 hours. 

Individually tagged mussels were randomly allocated to test tanks until all test tanks contained 8 to 

10 mussels of each species (dependent upon the number available for testing). 

The experimental unit for the trial was the individual test tank, and treatment group was assigned 

using a randomized block design. The treatment groups for each exposure duration consisted of (1) an 

untreated control group, (2) a group that received an application of 50 milligrams SDP per liter (mg 

SDP/L), and (3) a group that received an application of 100 mg SDP/L. All mussel species and both 

exposure duration groups were exposed concurrently (that is, one-half of the mussels were removed 

after 8 hours of SDP exposure and the remaining mussels were removed after 24 hours of SDP 

exposure). All treatment concentrations are reported as active ingredient. 

After exposure, the mussels were consolidated into wire mesh cages and placed in the Black 

River for a 27-28 day postexposure period, after which time survival of mussels was assessed. Of the 

1,170 mussels tested in the study, 3 were confirmed dead and 5 were not recovered and treated as 

mortalities in the analysis. The effect and interactions of species, SDP exposure concentration, and SDP 

exposure duration were analyzed and did not affect mussel survival (p > 0.98). The results from this 

study indicate that SDP exposure at the maximum approved open-water concentration of 100 mg/L for 

up to 3 times the maximum approved open-water exposure duration of 8 hours (in other words for 

24 hours of exposure) is unlikely to reduce survival of subadult or adult mussels. 

1 U.S. Geological Survey. 2 New York State Education Department.

3 U.S. Fish and Wildlife Service.

1 

 
Introduction

Native freshwater mussels of North America were historically considered the most diverse in the 

world, with about 297 recognized taxa consisting of 281 species and 16 subspecies (Williams and 

others, 1993). Mussels are largely sedentary in nature, relying on movement of host fish during 

glochidial attachment as means of transport. Thus, they are particularly vulnerable to a variety of 

anthropogenic influences, including habitat degradation and alteration, pollution, and overharvest. 

Neves (2004) reported that 70 species of freshwater mussels in the United States are listed as threatened

or endangered, and 40 are candidates for possible listing. The International Union for Conservation of 

Nature’s Red List has 95 species of North American freshwater bivalves currently listed as vulnerable, 

endangered, or critically endangered and 29 listed as extinct or possibly extinct 

(http://www.iucnredlist.org/, accessed February, 2014). Ricciardi and Rasmussen (1999) suggested that 

127 mussel species will become extinct in the next 100 years; this is claimed to be a conservative 

extinction rate of 6.4 percent per decade given that it did not take into account extirpations caused by 

invasive dreissenid mussels (zebra mussel, Dreissena polymorpha, and quagga mussel, D. bugensis). 

Concerns for native mussels in the Southeast are even greater given that only 25 percent of the 269 

species historically present are reported as stable compared to the 13 percent presumed extinct and the 

28, 14, and 18 percent listed, respectively, as endangered, threatened, or of special concern (Neves and 

others, 1997). 

Many mussels endemic to North America were imperiled prior to epizoic colonization by zebra 

and quagga mussels. The introduction of these dreissenids, however, has dramatically heightened 

concerns for the continued survival of native mussel species. Zebra mussels were reported to be 

responsible for the extirpation of unionid mussels from waters in Europe as early as 1937 (Sebestyen, 

1937; cited in Burlakova and others, 2000). Severe declines in unionid abundance in Europe (Karatayev 

and Burlakova, 1995; and Burlakova, 1998; cited in Burlakova and others, 2000) and North America 

(Haag and others, 1993; Nalepa, 1994; Ricciardi and others, 1996; Neves, 2004) have since been well 

documented in the literature. 

The 1973 Endangered Species Act brought forth the need to recognize, protect, and recover rare 

mussels in the United States. The U.S. Fish and Wildlife Service is responsible for developing recovery 

plans for threatened and endangered species. Recovery plans can include restoring and acquiring critical 

habitat, removing introduced or invasive species, and captive propagation and release into historic 

ranges.

One potential tool for limited, open-water control of dreissenid mussels in critical, high-value 

habitats is a commercially prepared spray dried powder (SDP) formulation of a specific strain 

(CL145A) of the common soil bacterium Pseudomonas fluorescens. The SDP formulation is produced 

by Marrone Bio Innovations, Inc. (Davis, California) and it was registered by the U.S. Environmental 

Protection Agency (Zequanox®; registration # 84059-15) for controlling dreissenid mussels in industrial 

water conveyance systems (for example, cooling and service water systems) in 2012 and for controlling 

dreissenid mussels in open-water systems in 2014. Evaluation of SDP exposure effects on nontarget 

animals, particularly sedentary filter feeding animals such as native unionid mussels, is a critical step 

before SDP should be applied to control dreissenid mussels in open-water environments.

The study objective was to determine the survival of unionid mussels after exposure to SDP up 

to the maximum approved open-water application concentration (100 milligrams per liter [mg/L]) and 

up to 3 times the maximum approved open-water exposure duration (8 hours). The SDP exposures were 

completed within a mobile bioassay laboratory sited along the Black River (La Crosse, Wisconsin) and 

after the exposures the test animals were held in the Black River adjacent to the exposure location 

(fig. 1). 

2 
Figure 1. Exposure and postexposure test animal holding location. 

3 
Materials and Methods

The protocol and amendments for this study are contained in appendix 1 (items 1–2). All 

methods and materials followed the written protocol except those instances that were identified as 

deviations (appendix 2, items 1–3).

Experimental Design

The study was designed to assess the survival of unionid mussels after exposure to target 

concentrations of 50 and 100 mg/L of SDP for 8 and 24 hours. Five species of subadult and two species 

of adult unionid mussels were evaluated (table 1). A total of 1,170 mussels were individually tagged, 

measured, and randomly assigned (appendix 3, item 2) to test tanks in equal proportions. Each test tank 

contained two containment baskets, one to hold mussels for the 8-hour exposure group and one for the 

24-hour exposure group. Each exposure group consisted of 8–10 mussels of each species. 

Treatment was assigned to each test tank by using a randomized block design (appendix 3, item

1). Three treatment groups were tested in triplicate and included (1) an untreated control group, (2) a 

group that received an application of 50 milligrams SPD per liter (mg SDP/L), and (3) a group that 

received an application of 100 mg SDP/L. The experimental unit for the trial was the individual test 

tank. At the conclusion of the exposure period, the mussels were consolidated into wire mesh cages and 

placed in the Black River (La Crosse, Wis.) for a 27–28 day postexposure period, after which time 

survival was assessed.

Table 1. Scientific and common name, source, lifestage, mean (standard deviation) length, and total number 

of test animals used in the study.

[mm, millimeter]

Scientific name Common name Source Lifestage Mean length 

(mm)

Total number

used

Megalonaias nervosa Washboard Propagated Subadult 44.0 (3.8) 180

Lampsilis cardium Plain pocketbook Propagated Subadult 39.2 (4.2) 162

Obovaria olivaria Hickorynut Propagated Subadult 30.9 (2.2) 180

Lampsilis siliquoidea Fatmucket Propagated Subadult 57.0 (4.8) 180

Lampsilis higginsii Higgins eye Propagated Subadult 23.8 (2.3) 144

Amblema plicata Threeridge Wild Adult 63.2 (8.3) 180

Fusconaia flava Wabash pigtoe Wild Adult 51.1 (8.0) 144

Test Article

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

strain CL145A (Zequanox®) containing 50 percent active ingredient (weight to weight ratio [w/w] 

P. fluorescens, strain CL145A); provided by Marrone Bio Innovations as a mixed lot [401P12163C and 

401P12164C]; Certificates of Analysis, appendix 4, items 2 and 3). Test article use was documented in 

the test chemical log books (appendix 4, item 7). Concentrations of test article are reported as active 

4 
ingredient. Retention of test article biological activity was assessed after the study by the New York 

State Museum Field Research Laboratory, using their standard dreissenid mussel bioassay. Results of 

the test article biological activity verification bioassay demonstrated the retention of test article 

biological activity as indicated by a mean zebra mussel mortality of 98.7 ± 2.3 percent in the SDPtreated group compared with mean mortality of 0.0 ± 0.0 percent in the untreated control (appendix 4, 

item 6). 

Test System

The test system was a series of nine independent circular test tanks (76.2 x 95.3 centimeters

[cm], diameter × high; 350-L capacity [fig. 2]) contained within a mobile bioassay laboratory. The test 

system utilized filtered Black River water for the static exposures, containment baskets to confine the 

test animals, and substrate to allow for test animal positioning. River water was supplied to the test 

system using a 3-horsepower submersible pump (ITT Goulds Pumps, Seneca Falls, New York, model 

18GS30). Water was filtered (200 micrometer) using an automatic backwashing microscreen filtration 

system (Forsta Filter Inc., Los Angeles, Calif.; model M2-90). Filtered water was delivered to a headbox 

(55.9 x 114.3 x 30.5 cm, width x length x height) positioned over each row of test tanks and gravity fed 

to each test tank at ≈6 liters per minute for approximately one tank-exchange per hour. Water flow was 

interrupted during the exposure period. Water during the preexposure period was discharged to the 

river; SDP treated exposure water was retained in portable frame tanks, collected by a state-licensed 

septic hauler, and discharged to the La Crosse, Wis., sanitary treatment system. 

Two semi-rigid plastic mesh containment baskets were positioned within each test tank (fig. 2). 

The baskets were secured to a frame constructed with 1.9 and 2.5-cm wide × 0.3-cm-thick welded 

aluminum that had an attached aluminum rod handle (0.95 x 76.0 cm, diameter × length). The 

containment baskets were used to confine the test animals during the exposure period and to facilitate 

removal after exposure. Approximately 2.5 cm (≈11.4 kg) of washed sand substrate (River Run 

Products, Inc. Marathon, Wis.) was placed in the test tanks after the placement of the containment 

baskets. The substrate facilitated test animal positioning and normal behavior (that is, siphoning). 

Figure 2. Test tanks positioned in the mobile bioassay laboratory (left) and containment baskets 

positioned in test tank (right).

5 
Test Animals

A total of seven species of unionid mussels, composed of five species of subadult mussels and 

two species of adult mussels, were used as the test animals (table 1; fig. 3). Subadult mussels were 

propagated animals, ≤ 3 years of age, obtained from the U.S. Fish and Wildlife Service’s Genoa 

National Fish Hatchery (NFH). The adult mussels were wild collected from the Upper Mississippi River 

at river mile 670.5 by Genoa NFH biologists. A Hallprint® shellfish tag with a unique alphanumeric 

code was fixed to each mussel shell with cyanoacrylate glue (fig. 3), and individual shell length was 

measured parallel to the hinge. The mussels were maintained at the Genoa NFH in flowing pond water 

then transported to the test location. Prior to distribution, the mussels were acclimated to Black River 

water by periodic additions of Black River water to the transportation coolers. 

Figure 3. Example specimens of the seven unionid mussel species tested (left) and alphanumeric-tagged 

mussels (right). 

Postexposure Handling

At exposure termination (8 and 24 hours), the assigned containment baskets were removed and 

the mussels were consolidated into one of six wire mesh cages, constructed according to Brady and 

others (2010; fig. 4), for the postexposure period. Mussels from one test tank replicate of each treatment 

group and exposure duration were placed into a single wire mesh (that is, mussels removed at the same 

exposure termination from a control, a 50 mg/L replicate and a 100 mg/L replicate were placed into the 

same wire mesh cage). The wire mesh cages were placed in ≈3 meters of water in the Black River 

(15 T 064099mE 4858943mN, fig. 1) for the postexposure period. 

Survival Assessment

Survival of mussels was assessed at 27 or 28 days after exposure. Survival was defined as valve 

or foot movement in response to tactile stimuli or resistance to valve pressure by adductor muscle 

contraction. The mussels recovered from each wire mesh cage were sorted by species and individually 

assessed for survivorship. 

6 
Figure 4. Example wire mesh cage used to hold mussels during the postexposure period.

Dosing and Dose Verification

One of three treatments was assigned to each test tank according to a randomized design scheme 

(appendix 3, item 1). Three treatment groups were assigned in triplicate and consisted of (1) an 

untreated control group, (2) a 50-mg SDP/L group, and (3) a 100-mg SDP/L group. A dosing stock 

solution was prepared from the test article and immediately applied to each test tank. Depending on 

treatment assignment, a premeasured amount of SDP (0 grams [g] for control; 35 g for 

50-mg SDP/L treatment; 70 g for 100-mg SDP/L treatment) was added to ≈12 L of test tank water and 

mechanically mixed for 3–5 minutes with a paint mixer attached to an electric drill. Immediately after 

mixing, the stock solution was poured through a strainer, and clumps of test article were macerated with 

a pestle and rinsed into the stock solution. The resulting stock solutions for each test tank contained the 

amount of SDP required to achieve the desired concentration. The SDP treatments were administered to 

the test tanks by gently pouring the stock solution into the test tank. The stock solution and water within 

the test tank were gently mixed by hand, using a polyvinyl chloride pipe. Care was used to minimize 

substrate disturbance. 

Water samples were collected to verify SDP exposure and total ammonia nitrogen (TAN) 

concentrations. Water samples were collected by submersing a 50-mL beaker below the water surface of 

each test tank. The SDP exposure concentrations were determined by comparison to a linear regression 

curve created from known concentrations (0, 25, 50, 100 and 200 mg/L [as SDP active ingredient]) and 

absorbance of the test article. Sample absorbance was measured on a Beckman-Coulter model DU-800 

spectrophotometer at 660 nanometers. A linear regression equation (appendix 7, items 1–2) was fit 

using the Statistical Analysis Software Proc Reg procedure (SAS® Version 9.3, SAS Institute, Inc., 

7 
Cary, North Carolina), and the exposure concentrations were determined from the linear regression 

(appendix 7, item 1). Exposure concentrations are reported as active ingredient.

Water Chemistry

Dissolved oxygen, pH, and temperature were measured in each test tank before treatment and 

≈1, 6, 12, 18 and 24 hours thereafter. Water hardness, alkalinity, and conductivity were measured prior 

to treatment on samples collected from each headbox and from water samples collected from each test 

tank ≈3 hours after administering the treatment. Immediately before the exposure period was 

terminated, water samples were collected and analyzed for total ammonia nitrogen content by the 

UMESC water quality laboratory, 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 temperature 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 each wire mesh cage 

and recorded water temperature every 6 hours during the postexposure period. 

Data Analysis

Statistical comparisons of mussel survival were completed using Statistical Analysis Software 

(SAS® Version 9.3, SAS Institute, Inc., Cary, N.C.). Significance for all analyses was declared at 

α ≤ 0.05. A generalized linear mixed model was used to analyze the effect and interactions of species, 

SDP exposure concentration, and exposure duration on the survival of mussels. The proportion of 

mortalities ([number of dead + unrecovered mussels]/total number of mussels in the sample) were 

modeled by using the SAS Proc GLIMMIX procedure with a binomal distribution and a logit link 

function (appendix 8, item 1). A scale parameter was added to the model by using the 

“random_residual_” statement. Mussel survival in each active treatment group was individually 

compared to the mussel survival of the untreated control group by using a two-sided means comparison 

test. 

Data analyses for water chemistry were limited to simple descriptive statistics. The SAS 

Software Proc Means procedure was used to determine the mean exposure concentration by individual 

test tank and by treatment group (appendix 7, items 1–2). All concentrations are reported as active 

ingredient. 

Results

Statistical analyses for survival output are presented in appendix 8 (item 3), and the summarized 

data are presented in appendix 9 (items 1–8). Survival of mussels in each treatment group is 

summarized in table 2. Mean survival of all mussel species in both the 8- and the 24-hour exposure 

duration groups exceeded 95 percent for all treatment groups, and mussel survival exceeded 87 percent

for all test replicates, regardless of species, treatment group, or exposure duration. Of the 1,170 mussels 

used in the study, there were a total of 3 confirmed mortalities and 5 test animals that were not 

recovered. The unrecovered mussels were treated as mortalities in the data analyses. No difference in 

mussel survival was detected (p > 0.98) when comparing the effects and interactions of species, 

exposure concentration, and exposure duration. 

The water chemistry data are presented in appendix 6 (items 1-4). Water chemistry parameters 

(dissolved oxygen, pH, and temperature) observed during the study period are summarized in table 3. 

Dissolved oxygen in each test tank remained above the minimum level recommended (4.0 mg/L) for 

8 
laboratory tests with freshwater mussels (ASTM International, 2013) throughout the first 12 hours of 

exposure. The mean dissolved oxygen concentration of the untreated control tanks at 18 and 24 hours 

was 3.3 and 2.7 mg/L, respectively. The mean dissolved oxygen concentration of the treated tanks at 

18 and 24 hours was < 1.0 mg/L. The test water hardness, alkalinity, conductivity, and ammonia 

observed during the study period are summarized in table 4. The mean alkalinity ranged from 42 to 

45 mg/L as calcium carbonate (CaCO3), hardness from 52 to 57 mg/L as CaCO3, conductivity from 

103 to 115 microsiemens per centimeter, pH from 6.05 to 7.19; and un-ionized ammonia concentrations 

were < 0.01 mg/L. The mean water temperature during the preexposure and exposure period ranged 

from 16.2 to 18.7 degrees Celsius (°C). The mean daily water temperature during the postexposure 

period ranged from 16.9 to 25.0 °C. 

Table 2. Mean (standard deviation) percent survival of mussels exposed to SDP by treatment 

group and exposure duration. 

[Means with the same letter are not significantly different (α > 0.05); mg/L, milligram per liter]

Species

8-hour exposure 24-hour exposure

Control 50 mg/L 100 mg/L Control 50 mg/L 100 mg/L

Megalonaias nervosa

100a

(0)

96.7a

(5.8)

100a

(0)

100a

(0)

100.0a

(0.0)

100a

(0)

Lampsilis cardium

100a

(0)

100a

(0)

100a

(0)

100a

(0)

96.3a

(6.4)

96.3a

(6.4)

Obovaria olivaria

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

96.7a

(5.8)

Lampsilis siliquoidea

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

Lampsilis higginsii

95.8a

(7.2)

100a

(0)

100a

(0)

95.8a

(7.2)

95.8a

(7.2)

95.8a

(7.2)

Amblema plicata

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

Fusconaia flava

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

100a

(0)

The individual test tank SDP exposure concentrations and group means are presented in 

appendix 7 (items 1–2); the exposure concentrations for each treatment group are summarized in table 5. 

The mean SDP concentrations measured throughout the 24-hour exposure period were within 5 percent

of the target concentrations of 50 and 100 mg SDP/L with the exception of the initial sample (≈1 hour 

after SDP application) and the 12-hour sample. The mean initial SDP exposure concentrations were 

72.6 mg/L and 127.3 mg/L in the 50- and 100-mg/L treatment groups, respectively. The mean 12-hour 

exposure concentrations were 72.5 and 104.9 mg/L in the 50- and 100-mg/L treatment groups, 

respectively. The elevated concentrations were most likely caused by sediment disturbance that 

occurred (1) during initial mixing of the SDP stock solution with the test tank water and (2) when the 

9 
containment baskets were removed from the test tanks at the conclusion of the 8-hour exposure period. 

Sediment disturbance increased the turbidity which interfered (increased spectrophotometer readings) 

with the absorbance measurements. 

Table 3. Mean (standard deviation) dissolved oxygen, temperature and pH range observed for each 

treatment group during the study period.

[mg/L, milligram per liter; °C, degree Celsius; pre, preexposure]

Time

Control 50 mg/L 100 mg/L

pH

DO Temp pHDO Temp pHDO Temp

(mg/L) (°C) (mg/L) (°C) (mg/L) (°C)

Pre 7.15-7.19

6.59

(0.06)

16.2

(0.0)

7.16-7.18

6.64

(0.03)

16.2

(0.0)

7.06-7.18

6.69

(0.09)

16.2

(0.0)

1 7.07-7.08

6.32

(0.01)

16.8

(0.0)

7.04-7.06

6.51

(0.04)

16.8

(0.0)

6.42-6.61

6.54

(0.09)

16.9

(0.0)

6 6.98-7.00

5.38

(0.09)

17.7

(0.1)

6.99-7.00

5.83

(0.03)

17.8

(0.2)

6.97-6.99

5.76

(0.04)

17.9

(0.1)

8 6.94-6.97

4.85

(0.14)

18.1

(0.0)

6.92-6.94

5.48

(0.04)

18.0

(0.1)

6.93

5.42

(0.07)

18.2

(0.1)

12 6.45-6.71

4.18

(0.08)

18.4

(0.2)

6.72-6.82

4.38

(0.14)

18.4

(0.2)

6.83-6.86

4.15

(0.09)

18.4

(0.1)

18 6.99-7.02

3.27

(0.13)

18.5

(0.1)

6.77-6.80

0.63

(0.14)

18.5

(0.1)

6.63-6.66

0.16

(0.01)

18.6

(0.1)

24 6.90-6.99

2.71

(0.19)

18.6

(0.1)

6.39-6.42

0.04

(0.00)

18.7

(0.1)

6.05-6.13

0.03

(0.00)

18.7

(0.1)

Table 4. Mean (standard deviation) alkalinity, hardness, conductivity, total ammonia 

nitrogen (TAN), and un-ionized ammonia (NH3) observed during the exposure period.

[mg/L, milligram per liter; μS/cm, microsiemens per centimeter at 25 degrees Celsius] 

Treatment 

group

Alkalinity

(mg/L)1

Hardness

(mg/L)1

Conductivity

(μS/cm)

TAN

(mg NH3-N/L)

NH3

(mg/L)

Control 42

(0)

52

(2)

103

(2)

0.25

(0.01)

<0.01

(<0.01)

50 mg/L 43

(1)

53

(1)

111

(0)

0.07

(0.01)

<0.01

(<0.01)

100 mg/L 44

(1)

53

(1)

115

(1)

0.07

(0.02)

<0.01

(<0.01)

1 Reported as calcium carbonate (CaCO3). 

10 
Table 5. Mean (standard deviation) exposure concentration for each treatment group 

throughout the exposure period.

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

Treatment

group

Time (hours)

1 6 12 18 24

Control ND ND

5.0

(5.1)

ND ND

50 mg/L

72.6

(3.7)

49.0

(4.1)

72.5

(12.6)

51.9

(2.9)

51.2

(3.7)

100 mg/L

127.3

(11.6)

99.3

(3.1)

104.9

(7.9)

95.0

(3.1)

95.7

(4.6)

Conclusions

Regardless of species, the application of Zequanox up to the maximum approved open-water 

application concentration of 100 mg/L (as active ingredient) for up three times the maximum approved 

open-water exposure duration (24 hours; the maximum approved open-water exposure duration is 

8 hours) did not reduce the survival of the unionid mussels (p > 0.98). The results of this study indicate 

that exposure of up to 100 mg of SDP active ingredient/L for up to 24 hours is unlikely to reduce the 

survival of subadult or adult unionid mussels. Dissolved oxygen suppression was observed in treated 

tanks after 12 hours of exposure; however, the maximum approved exposure duration for open-water 

application is 8 hours. 

11 
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. 

Brady, T.R., Aloisi, D., Gordon, D and Wege G., 2010, A method for Culturing Mussels Using In-River 

Cages: Journal of Fish and Wildlife Management, v. 2, no. 1, p.85–89. 

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. 

Haag, W.R., Berg, D.L., Garton, D.W., and Farris, J.L., 1993, Reduced survival and fitness in native 

bivalves in response to fouling by the introduced zebra mussel (Dreissena polymorpha) in western 

Lake Erie: Canadian Journal of Fisheries and Aquatic Sciences, v. 50, no. 1, p. 13–19. 

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. 

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 

Aquatic Research Institute, Lenz Design and Communications, p. 43–85. 

Ricciardi, A., Whoriskey F.G., and Rasmussen J.B., 1996, Impact of the Dreissena invasion on native 

unionid bivalves in the upper St. Lawerence River, Canadian Journal of Fisheries and Aquatic 

Sciences, v. 53, p. 1434–1444. 

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

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

Williams, J.D., Warren, M.L., Jr., 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. 

12 
Appendix 1. Study Protocol With Data Forms

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 

Protocol: “Safety of Spray Dried Powder (SDP) Formulated Pseudomonas fluorescens

strain CL 145A (Zequanox) Exposure to Sub-Adult Unionid Mussels During 

Simulated Open Water Treatments”

32 14 

2 Amendment 1 Revision of Study Protocol, Study # AEH-13-PSEUDO-06 12 46 

3 Test Chemical Stock Preparation Datasheet 1 58 

4 Alkalinity Datasheet 1 59

5 Conductivity and Hardness Datasheet 1 60 

6 Water Quality Datasheet 1 61 

7 Mussel Histology Samples Datasheet 1 62 

8 Plain Pocketbook Survival Assessment 5 63 

9 Hickorynut Survival Assessment 5 68 

10 Washboard Survival Assessment 5 73 

11 Higgins Eye Survival Assessment 5 78 

12 Fatmucket Survival Assessment 5 83 

13 Threeridge Survival Assessment 5 88 

14 Wabash Pigtoe Survival Assessment 5 93 

15 Native Mussel Lengths Datasheet (version 1) 1 98 

16 Native Mussel Lengths Datasheet (version 2; revised) 1 99

13 
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99 
Appendix 2. Deviations From the Study Protocol

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 Deviation #1 – Native mussels recovered from tanks/equipment after the termination 

of the assigned treatment period 1 101 

2 Deviation #2 – Native mussels not recovered after 30 day holding period 1 102 

3 Deviation #3 – Native mussel used in exposure >3 years of age 1 103 

100 
101 
102 
103 
Appendix 3. Randomization Assignments

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 SAS generated random assignment of treatment to experimental tanks. 5 105 

2 SAS generated random assignment of mussels to test tanks/time sections. 6 110 

3 Hickorynut assignment to treatment tanks 1 116 

4 Washboard assignment to treatment tanks 1 117 

5 Higgins eye assignment to treatment tanks 1 118 

6 Fatmucket assignment to treatment tanks 1 129 

7 Threeridge assignment to treatment tanks 1 120 

8 Wabash Pigtoe assignment to treatment tanks 1 121 

9 Plain Pocketbook assignment to treatment tanks 1 122 

104 
105 
106 
107 
108 
109 
110 
111 
112 
113 
114 
115 
116 
117 
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119 
120 
121 
122 
Appendix 4. Test Article Information

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 Material Safety Data Sheet: Zequanox® 2 124 

2 MBI-401 SDP (lot # 401P12163C) Test Article: Certificate of Analysis 1 126 

3 MBI-401 SDP (lot # 401P12164C) Test Article: Certificate of Analysis 1 127 

4 MBI-401 SDP (lots # 401P12163C and 401P121164C) Test Article: Packing List 

USPS (shipped to James Luoma 08/06/2012) 1 128 

5 Test Article Stock Preparation 1 129

6 New York State Museum Post-Exposure Product Validation Assay 2 130 

7 Copy of test article log book [MBI-401 SDP]; lot number 401P12163C and 

401P12164C (Received mixed in Containers) Container 5 of 6 5 132 

123 
124 
125 
126 
127 
128 
129 
130 
131 
132 
133 
134 
135 
136 
Appendix 5. Test Animal Information

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 Email from GNFH: Origin of Test Organisms 2 138 

2 Native Mussel Lengths – Data Summary 1 140 

3 Plain Pocketbook (L. cardium) Length Summary 1 141 

4 Hickorynut (O. olivaria) Length Summary 1 142 

5 Washboard (M. nervosa) Length Summary 1 143 

6 Higgins Eye (L. higginsii) Length Summary 1 144 

7 Fatmucket (L. siliquoidea) Length Summary 1 145 

8 Threeridge (A. plicata) Length Summary 1 146 

9 Wabash Pigtoe (F. flava) Length Summary 1 147 

10 Histological Examination of native mussel test organisms – Summary 6 148 

137 
138 
139 
140 
141 
142 
143 
144 
145 
146 
147 
148 
149 
150 
151 
152 
153 
Appendix 6. Water Quality

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 Water Chemistry – Data Summary (Preexposure and exposure) 4 155 

2 Ammonia – Data Summary 3 159

3 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC 1 162 

4 Data Logger (HOBO) 30 day Temperature Summary – Data Summary 4 163 

154 
155 
156 
157 
158 
159 
160 
161 
162 
163 
164 
165 
166 
Appendix 7. Spectrophotometric Summary, SAS Program, 

Output and Log

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 Spectrophotometric Data – Data Summary with SAS output 18 168 

2 SAS program; Exposure Concentration Analysis 2 186 

3 SAS log; Exposure Concentration Analysis 5 188 

167 
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173 
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175 
176 
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184 
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192 
Appendix 8. Statistical Analysis Including SAS Programs, 

Outputs and Logs for Survival and Treatment Concentration

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 SAS program for native mussel survival 2 194 

2 SAS log for native mussel survival 3 196 

3 SAS output for native mussel survival 38 199

193 
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Appendix 9. Survival Assessment Summary

Item 

Number Item Description

Number 

of 

Pages

Report 

Page 

Number

1 Mortality Data 4 238 

2 Plain Pocketbook (L. cardium) Mortality Summary 1 242 

3 Hickorynut (O. olivaria) Mortality Summary 1 243 

4 Washboard (M. nervosa) Mortality Summary 1 244 

5 Higgins Eye (L. higginsii) Mortality Summary 1 245 

6 Fatmucket (L. siliquoidea) Mortality Summary 1 246 

7 Threeridge (A. plicata) Mortality Summary 1 247 

8 Wabash Pigtoe (F. flava) Mortality Summary 1 248 

237 
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248 
Weber and others— Safety of Spray-Dried Powder Formulated Pseudomonas fluorescens Strain CL145A Exposure to 

 Subadult/Adult Unionid Mussels During Simulated Open-Water Treatments

—Open-File Report 2015–1064

ISSN 2331-1258 (online)

http://dx.doi.org/10.3133/ofr20151064