Exposure-Related Effects of Formulated Pseudomonas 

fluorescens Strain CL145A to Glochidia from Seven 

Unionid Mussel Species 

By James A. Luoma, Kerry L. Weber, Todd J. Severson, Theresa M. Schreier, Denise A. Mayer, Douglas B. Aloisi 

and Nathan L. Eckert 

Open-File Report 2015–1094

U.S. Department of the Interior 

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

SALLY JEWELL, Secretary 

U.S. Geological Survey 

Suzette M. Kimball, Acting Director 

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

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

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

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

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

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

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

endorsement by the U.S. Government. 

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

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

must be secured from the copyright owner. 

Suggested citation: 

Luoma, J.A., Weber, K.L., Severson, T.J, Schreier ,T.M., Mayer, D.A., Aloisi, D.B, and Eckert N.L., 2015, 

Exposure-related effects of formulated Pseudomonas fluorescens strain CL145A to glochidia from seven unionid 

mussel species: U.S. Geological Survey Open-File Report 2015–1094, 474 p., 

http://dx.doi.org/10.3133/ofr20151094.

ISSN 2331-1258 (online) 
iii

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 thank Mark P. Gaikowski of the Upper Midwest Environmental 

Sciences Center for assisting with study design and statistical analysis as well as Jeremy K. Wise, 

Samuel M. Stafslien, Pajtshiab L. Moua, and Susan M. Schleis for technical assistance and data 

collection. 

v 

Contents 

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

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

Introduction ....................................................................................................................................................1

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

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

Test Article ..............................................................................................................................................3

Test system.............................................................................................................................................4

Animal Collection and Distribution..............................................................................................................7

Test Article Preparation and Application .....................................................................................................8

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

Glochidia Viability Assessments.................................................................................................................9

Data Analysis................................................................................................................................................ 10

Results and Discussion.................................................................................................................................. 10

Conclusions.................................................................................................................................................. 17

References Cited .......................................................................................................................................... 17

Appendix 1. Study Protocol, Amendments, and Datasheets.............................................................................. 19

Appendix 2. Deviations from the Study Protocol............................................................................................. 113

Appendix 3. Randomization Assignments...................................................................................................... 135

Appendix 4. Test Article Information.............................................................................................................. 237

Appendix 5. Test Animal Information............................................................................................................. 282

Appendix 6. Water Quality ........................................................................................................................... 325

Appendix 7. Glochidia Viability ..................................................................................................................... 353
vi

Figures

Figure 1. Photograph showing glochidia test system with individual test chambers......................................... 6

Figure 2. Photographs showing A, the extraction of glochidia from a gravid female mussel and, B, extracted 

glochidia.................................................................................................................................... 7

Figure 3. Photomicrographs showing A, glochidia before a drop of sodium chloride solution was added to 

induce valve closure in viable animals and, B, glochidia one minute after addition of the sodium 

chloride solution......................................................................................................................... 8

Figure 4. Photograph showing the distribution of glochidia to test chambers.................................................. 8

Tables

Table 1. Data for test animals, test articles and, exposure dates for studies of selected native freshwater 

mussel glochidia exposed to two formulations of Pseudomonas fluorescens, strain CL145A............. 4

Table 2. Data for initial glochidia viability by individual female mussel for each species................................. 5

Table 3. Estimated number of glochidia distributed to test chambers. .......................................................... 6

Table 4. Mean temperature, alkalinity, hardness, and conductivity of pre-exposure source water and pooledexposure termination-water samples.......................................................................................... 12

Table 5. Mean dissolved oxygen, mean temperature, and pH range of exposure water............................... 12

Table 6. Mean total ammonia nitrogen of exposure water by treatment group at 24 hours .......................... 14

Table 7. Mean un-adjusted glochidia viability from selected native 

 freshwater mussels at 6, 12 and 24 hours............................................................................... 15

Table 8. Mean adjusted glochidia viability from selected native freshwater mussels at 6, 12 and 24 hours.... 16
vii

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)

microliter (μL) 0.000033814 ounce, fluid (fl. oz)

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

Flow rate

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

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

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.

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).

Abbreviations

AI active ingredient

α alpha

CaCO3 calcium carbonate

DO dissolved oxygen

FDP freeze-dried powder

HD heat deactivated

p p-value

SDP spray-dried powder

TAN total ammonia nitrogen

USGS U.S. Geological Survey

1 

Exposure-Related Effects of Formulated Pseudomonas 

fluorescens Strain CL145A to Glochidia from Seven 

Unionid Mussel Species 

By James A. Luoma1, Kerry L. Weber1, Todd J. Severson1, Theresa M. Schreier1, Denise A. Mayer2, Douglas B. 

Aloisi3, and Nathan L. Eckert3

Abstract 

The study was completed to evaluate the exposure-related effects of a biopesticide for dreissenid 

mussel (Dreissena polymorpha, zebra mussel and Dreissena rostriformis bugensis, quagga mussel) 

control on glochidia from unionid mussels endemic to the Great Lakes and Upper Mississippi River 

Basins. The commercially prepared biopesticide was either a spray-dried powder (SDP) or freeze-dried 

powder (FDP) formulation of Pseudomonas fluorescens, strain CL145A. Glochidia of the unionid 

mussel species Lampsilis cardium, Lampsilis siliquoidea, Lampsilis higginsii, Ligumia recta, Obovaria 

olivaria, and Actinonaias ligamentina were exposed to SDP-formulated P. fluorescens and Lampsilis 

cardium and Megalonaias nervosa were exposed to FDP-formulated P. fluorescens. 

All exposures were static, 24 hours in duration, and included six treatment groups. The treatment 

groups included (1) an untreated control, (2) a positive control which received a nominal target active 

ingredient (AI) concentration of 300 milligrams per liter (mg/L) of heat-deactivated test article, and (3) 

treatments that received nominal target AI concentrations of 50, 100, 200, and 300 mg/L of test article. 

All treatment concentrations are reported based on active ingredient.

Glochidia viability was reduced in two of the six species exposed to 50 mg/L SDP and in four of 

the six species exposed to 100 mg/L SDP when compared to untreated control groups at 6, 12, and 

24 hours. Regardless of sample time, concentrations of 200 and 300 mg/L of SDP and 300 mg/L of 

heat-deactivated SDP (positive control) substantially reduced glochidia viability in all species except, L. 

higginsii. Glochidia viability was only reduced for L. cardium exposed to FDP at concentrations ≥

200 mg/L. After 24 hours of FDP exposure, differences in glochidia viability were only detected in M. 

nervosa that were exposed to 300 mg/L of heat-deactivated SDP. However, given the low viability in 

the control group, the results for M. nervosa should be interpreted with caution. 

Introduction 

North America has the greatest diversity of freshwater mussels in the World with historical 

evidence of approximately 297 taxa consisting of 281 species and 16 subspecies, but because of their 

sedentary nature, freshwater mussels are particularly vulnerable to anthropogenic influences, such as 

 

1

 U.S. Geological Survey 

2

 New York State Education Department 

3

 U.S. Fish and Wildlife Service 
2 

habitat alteration; habitat degradation; pollution and overharvest; and more recently, to impacts from 

dreissenid mussels (Dreissena polymorpha, zebra mussel and Dreissena rostriformis bugensis, quagga 

mussel) (Williams and others, 1993, Neves and others, 1997, Strayer and Malcom, 2007). Master and 

others (1998) listed: 67 percent of freshwater mussels in the United States as extinct or vulnerable to 

extinction; 95 species of North American freshwater bivalves were listed as vulnerable, endangered, or 

critically endangered; and 29 were listed as extinct or possibly extinct on the International Union for 

Conservation of Nature’s Red List (http://www.iucnredlist.org/, accessed March 6, 2015). Estimates 

indicate that 127 mussel species will become extinct in the next 100 years; however, this estimate may 

be low as it did not factor in impacts from dreissenid mussels (Ricciadi and Rasmussen, 1999). Native 

mussels in the southeastern United States are in particular danger with only 25 percent of the 

269 historical species listed as stable, 13 percent presumed extinct, and 60 percent reported as either 

endangered, threatened or of special concern (Neves and others, 1997). The negative impact of 

dreissenid mussels on unionid mussels is well documented in the scientific literature with reports as 

early as 1937 of extirpation of unionid mussels from European waters due to dreissenid mussel 

colonization (Sebestyen, 1937; cited in Burlakova and others, 2000). Severe declines in unionid 

abundance linked to dreissenid colonization in North American waters were reported approximately one 

decade after dreissenids invaded the United States (Haag and others, 1993; Nalepa, 1994; Ricciardi and 

others, 1996). 

The need to protect and recover imperiled species, such as freshwater mussels, was recognized 

in the 1973 Endangered Species Act (16 USC 1531-1544, 87 Stat. 884) and the U.S. Fish and Wildlife 

Service has developed recovery plans for many threatened and endangered unionid mussel species 

(U.S. Fish and Wildlife Service, 2013; http://www.fws.gov/endangered/species/recovery-plans.html, 

accessed April 1, 2015). Mitigating the severe impacts of dreissenid mussels on unionid species and the use 

of population enhancement and reintroduction into historical ranges with propagated animals are 

commonly cited tasks in unionid recovery plans (U.S. Fish and Wildlife Service, 2004). The U.S. Fish 

and Wildlife Service mussel propagation program uses wire-mesh rearing cages that are placed in 

natural waterways for approximately 18 months to rear unionid mussels (Brady and others, 2010). 

However, some areas previously used for rearing have been abandoned due to dreissenid mussel fouling 

of the rearing cages. A potential dreissenid mussel management tool for limited, high-value, open-water 

habitats, such as around unionid mussel rearing cages or mussel beds, is a commercially formulated 

biopesticide containing a specific strain (CL145A) of the common soil bacterium, Pseudomonas 

fluorescens as the active ingredient. Currently, the spray-dried powder (SDP) formulation of the 

biopesticide Zequanox® is manufactured by Marrone Bio Innovations, Inc. (Davis, California). It was 

registered by the U.S. Environmental Protection Agency (registration number 84059–15) for controlling 

dreissenid mussels in industrial water systems in 2012 and for open-water system use in 2014. The 

biopesticide containing P. fluorescens is readily ingested by dreissenid mussels, which causes necrosis 

of epithelial cells lining the digestive tract that results in the death of the dreissenid mussel (Molloy, 

Mayer, Gaylo, Morse, and others, 2013). The component of the P. fluorescens biopesticide that causes 

epithelial cell necrosis has been identified as a heat-liable natural metobilite associated with the 

bacterium’s cell wall (Molloy, Mayer, Gaylo, Burlakova, and others 2013). 

Freshwater mussels have a unique reproductive strategy that includes a short parasitic lifestage 

called glochidia. The ability of mature glochidia to attach to a suitable host (typically a fish) is a critical 

life-cycle step for most freshwater mussels (ASTM International, 2013). Therefore, evaluating the 

exposure-related effects of water-borne pesticides on freshwater mussel glochidia is prudent. 

The objective of this study was to evaluate the exposure-related effects of a commercially 

formulated P. fluorescens, strain CL145A biopesticide on glochidia from select unionid mussel species 
3 

present in the Great Lakes and Upper Mississippi River Basins. This report summarizes the methods 

and results of laboratory trials in which applications of SDP- or FDP-formulated P. fluorescens

biopesticides were administered for 24 hours to static test chambers containing glochidia extracted from 

female unionid mussels. During the course of this study, the test article manufacturer, Marrone Bio 

Innovations, Inc., requested a change to a freeze-dried powder (FDP) formulation. However, the FDP 

formulation was quickly discontinued; the resulting data for the two species exposed to FDP (Lampsilis 

cardium and Megalonaias nervosa) are included within the report. 

Materials and Methods 

The protocol and amendments for this study are presented in appendix 1 (items 1–6). All 

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

identified as deviations (appendix 2, items 1–12). Notes to file provide further documentation on the 

procedures used during the study (appendix 1, items 7 and 8). 

Experimental Design 

The study was completed at the U.S. Geological Survey’s Upper Midwest Environmental 

Sciences Center (La Crosse, Wisconsin). Seven species of unionid mussel glochidia (table 1) were 

exposed to either the SDP- (six species) or the FDP-formulation (two species) following procedures 

outlined in “Standard Guide for Conducting Laboratory Toxicity Tests with Freshwater Mussels” 

(Annex A1 of the ASTM International, 2013). The six species of unionid mussel glochidia exposed to 

the SDP-formulation were L. cardium, Lampsilis siliquoidea, Lampsilis higginsii, Ligumia recta, 

Obovaria olivaria, and Actinonaias ligamentina. The two species of unionid mussel glochidia exposed 

to the FDP-formulation were Lampsilis cardium and M. nervosa. For each species, glochidia were 

flushed from 3–4 gravid female mussels and viability was estimated to be over 80 percent before the 

glochidia were used in the study. Consistent with the Standard Guide for Conducting Laboratory 

Toxicity Tests with Freshwater Mussels, glochidia that closed their valves in response to sodium 

chloride exposure were considered viable (ASTM International, 2013). Photomicrographs were 

recorded before and after the addition of the sodium chloride solution and used for definitive 

determination of initial glochidia viability (table 2). Glochidia were pooled and distributed to test 

chambers according to a predetermined random distribution scheme (appendix 3, items 4–6). An 

estimated 843–4,602 glochidia were distributed to each test chamber (table 3). 

Each species was assigned to one of three test block locations and treatments were assigned to 

test chambers using randomized block designs (appendix 3, items 1–3 and 7–9, respectively). Six 

treatments (n = 3 per treatment) were tested and included (1) an untreated control, (2) nominal target 

concentrations of 50, 100, 200, and 300 milligrams per liter (mg/L) of test article based on active 

ingredient (AI), and (3) a nominal target concentration of 300 mg/L AI of heat-deactivated test article 

group (300 HD, positive control). Exposures were static and lasted 24 hours. Glochidia from each test 

chamber were assessed for viability at 6, 12 and 24 hours, with the exception of M. nervosa glochidia, 

which were only assessed at 24 hours. 

Test Article 

The test articles used in the study were commercially prepared SDP or FDP formulated P. 

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

fluorescens, strain CL145A cells), respectively. The test articles were produced by Marrone Bio 
4 

Innovations, Inc. and test article use was documented in log books (appendix 4, items 4–6). All test 

article concentrations are reported as active ingredient. Verification of biological activity (the ability of 

the test article to induce dreissenid mussel mortality) was determined for each test article lot after use in 

the study by the New York State Museum Field Research Laboratory (Cambridge, New York). 

Biological activity for each lot of test article was confirmed as indicated by mean zebra mussel mortality 

ranging from 85.3 to 94.7 percent in the test article treated groups compared to 0.0 to 1.3 percent in the 

untreated control groups (table 1; appendix 4, items 7–9). 

Table 1. Data for test animals, test articles and, exposure dates for studies of selected native freshwater mussel 

glochidia exposed to two formulations of Pseudomonas fluorescens, strain CL145A. 

[PPB, plain pocketbook; WAS, washboard; BLS, black sandshell; FAM, fatmucket; HGE, Higgins eye; HIC, hickorynut; 

MUC, mucket; FDP, freeze-dried powder; SDP, spray-dried powder; MBI, Marrone Bio Innovations, Inc.] 

 Test article 

Biological 

Scientific name Common name Abbreviation Formulation Lot number activity1 Exposure date 

(percent) 

Lampsilis Plain pocketbook PPB FDP 110607WB-FD-E 94.7 ± 6.1 October 18, 2011 cardium 

Megalonaias Washboard WAS FDP 110607WB-FD-E 94.7 ± 6.1 October 18, 2011 nervosa 

Ligumia Black sandshell BLS SDP MBI-401-SDP-4655-12-MIX 85.3 ± 11.5 January 17, 2012 recta 

Lampsilis Fatmucket FAM SDP MBI-401 110308AI-BD-3 86.7 ± 4.6 May 12, 2011 siliquoidea 

Lampsilis Higgins eye HGE SDP MBI-401-110308AI-BD-3 86.7 ± 4.6 May 12, 2011 higginsii 

Obovaria Hickorynut HIC SDP MBI-401-SDP-4655-12-MIX 85.3 ± 11.5 January 19, 2012 olivaria 

Actinonaias Mucket MUC SDP MBI-401-SDP-4655-12-MIX 85.3 ± 11.5 January 17, 2012 ligamentina 

Lampsilis Plain pocketbook PPB SDP MBI-401 110308AI-BD-3 86.7 ± 4.6 May 12, 2011 cardium 

1 Biological activity is the mean percent zebra mussel mortality in test article treated groups during a bioassay performed by the New York 

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

Test system 

The test system was a series of glass test chambers (80 × 40 millimeters [mm], width × height; 

catalog number 89000–286; VWR International, West Chester, Pennsylvania) arranged in three blocks. 

Each block contained 3 rows with 6 test chambers, for a total of 18 test chambers per species (fig. 1). 

Test chambers were labelled to allow for identification of treatment type and replicate number. During 

the exposure, light aeration (1–2 bubbles per second) was provided to each test chamber through a 

disposable glass pipet. Test water was well water conditioned to ambient temperature (≈20 °C) and 

dissolved-gas saturation by providing gentle aeration in 19-liter (L) glass jars for a minimum of 

24 hours before use. Indirect fluorescent lighting (mean of 108 to 536 lux) was provided on an 18 hours 

light to 6 hours dark cycle in accordance with ASTM International guidelines (ASTM International, 

2013). 
5 

Table 2. Data for initial glochidia viability by individual female mussel for each species. 

[Viability of glochidia by individual female mussel and species during exposures of selected native 

freshwater mussel glochidia to two formulations of Pseudomonas fluorescens, strain CL145A. SD, standard 

deviation—number in parentheses; SDP, spray-dried powder; FDP, freeze-dried powder]

Species Formulation Mussel 

number 

Total 

glochidia 

Nonviable 

glochidia1

Glochidia 

viability 

(percent)

Mean percent

glocihidia 

viability 

(SD) 

Lampsilis cardium

 

 FDP 1 

2 

212 

222 

16 

39 

92.5 

82.4 

84.7 (6.9) 

 3 150 31 79.3 

Megalonaias nervosa 

 

FDP 1 

2 

124 

155 

2 

1 

98.4 

99.4 

96.5 (5.5) 

 3 51 5 90.2 

 4 104 0 100.0 

Ligumia recta 

 

 

SDP 1 

2 

3 

312 

1,083 

405 

32 

250 

53 

89.7 

76.9 

86.9 

84.5 (6.7) 

Lampsilis siliquoidea

 

 SDP 1 

2 

548 

297 

70 

80 

87.2 

73.1 

80.3 (7.1) 

 3 456 89 80.5 

Lampsilis higginsii 

 

SDP 1 

2 

129 

168 

49 

20 

62.0 

88.1 

80.8 (16.5)

 3 357 27 92.4 

Obovaria olivaria 

 

SDP 1 

2 

366 

352 

54 

51 

85.2 

85.5 

86.0 (1.2) 

 3 309 39 87.4 

Actinonaias ligamentina 

 

SDP 1 

2 

161 

153 

14 

9 

91.3 

94.1 

92.7 (1.4) 

 3 287 21 92.7 

Lampsilis cardium 

 

SDP 1 

2 

467 

537 

36 

27 

92.3 

95.0 

93.8 (1.4) 

 3 927 55 94.1 

1

Nonviable glochidia are the sum of glochidia closed before the addition of, and open after the addition of, 

sodium chloride. 
6 

Table 3. Estimated number of glochidia distributed to test chambers

[Distributions to test chambers by species during exposures of selected 

native freshwater mussel glochidia to two formulations of Pseudomonas 

fluorescens, strain CL145A. SD, standard deviation—number in 

parentheses; SDP, spray-dried powder; FDP, freeze-dried powder] 

Estimated

Species Formulation number (SD) 

 of glochidia per test chamber 

Lampsilis cardium FDP 4,602 (1,225)

Megalonaias nervosa FDP 843 (382)

Ligumia recta SDP 2,021 (500)

Lampsilis siliquoidea SDP 2,702 (1,094)

Lampsilis higginsii SDP 3,427 (1,018)

Obovaria olivaria SDP 4,187 (2,503)

Actinonaias ligamentina SDP 2,200 (1,061)

Lampsilis cardium SDP 3,355 (1,077)

. 

Figure 1. Photograph showing glochidia test system with 

individual test chambers. 
7 

Animal Collection and Distribution 

Test animal information is presented in appendix 5, items 1-8. Gravid female mussels were 

collected from the Upper Mississippi, Chippewa, and St. Croix Rivers and identified to species as 

described in Cummings and Mayer (1992) and Watters and others (2009) by biologists from the Genoa 

National Fish Hatchery (Genoa, Wis.). Gravid mussels were maintained at the Genoa National Fish 

Hatchery on flowing pond water for up to 120 days before being transported to the Upper Midwest 

Environmental Sciences Center for glochidia extraction. Glochidia were extracted from the gravid 

female mussels by gently opening the valves and using a syringe to flush the marsupium with water 

(fig. 2). Glochidia from 6–10 marsupial tubes were flushed into a 500-milliliter (mL) beaker and 

viability of the glochidia was estimated to be > 80 percent by observation under a microscope before 

pooling. Glochidia viability was defined as valve closure in response to the addition of a drop of 

saturated sodium chloride solution (fig. 3). Glochidia from 3–4 mussels of each species were pooled in a 

500-mL beaker containing water. Definitive initial viability of glochidia extracted from each female 

mussel was determined from photomicrographs after exposures were completed (table 2). The beaker 

containing the pooled glochidia was gently swirled to position the glochidia in a concentrated group at 

the bottom of the beaker. Glochidia were then indiscriminately collected by placing a 1,000-microliter 

(μL) wide bore pipet tip (model T-1005-WB-C-R; Axygen Scientific, Union City, California) within the 

concentrated glochidia and withdrawing a 100-μL sample with an adjustable pipette (model research 

plus 100–1,000 μL; Eppendorf USA, Hauppauge, N.Y.). The glochidia were then placed into a 

randomly selected test chamber that contained 96.5 mL of water (fig. 4). After all test chambers had 

received glochidia, the distribution process was repeated until all test chambers received five 

independent glochidia distributions (appendix 3, items 4–6). Throughout the distribution process 

samples (100 μL, n =16 per species) containing glochidia were placed into a 35-mm petri dish and used 

to estimate the total number of glochidia distributed to the test chambers. Each treatment group 

contained three test chamber replicates with an estimated range of 843 to 4,602 glochidia per chamber 

(table 3). The volume of water within the test chambers after glochidia distribution and before test 

article administration was 97.0 mL. 

Figure 2. Photographs showing A, the extraction of glochidia from a gravid female mussel and, B, extracted 

glochidia. 
8 

Figure 3. Photomicrographs showing A, glochidia before a drop of sodium chloride solution was added to induce 

valve closure in viable animals and, B, glochidia one minute after addition of the sodium chloride solution.

Figure 4. Photograph showing the distribution of 

glochidia to test chambers. 

Test Article Preparation and Application 

For each species, a separate test article stock solution was prepared for the 50, 100, 200 and 

300 mg/L treatment groups immediately prior to application. A single heat-deactivated test article 

solution was prepared for each treatment day and used for all applications of heat-deactivated stock 

solution (positive control) that were completed on the same day. Untreated water was used as the test 

article stock solution for the untreated control treatment. The stock solutions were prepared by weighing 

a known amount of test article (1.0 grams FDP; 2.0 grams SDP) on an analytical balance and then 
9 

placing the test article into a 100-mL flask with water, which resulted in an AI stock solution of 

10,000 mg/L. The heat-deactivated stock was prepared by placing the test article into a 100-mL 

volumetric flask with approximately 50 mL of water, then the flask was placed into a 70 degrees Celsius 

(°C) water bath for 45 minutes to degrade the activity of the P. fluorescens according to methods 

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

York State Museum Field Research Laboratory, oral commun., 2010). After cooling to ambient 

temperature, the heat-deactivated stock solution was brought to a final 100 mL volume with water. 

Each test chamber received (1) 3.0 mL of untreated water (control), (2) 3.0 mL of heatdeactivated stock solution (positive control), (3) 3.0 mL of stock solution (300 mg/L treatment), or (4) 

3.0 mL of the appropriate combination of untreated water and stock solution (50, 100 and 200 mg/L 

treatments [appendix 1, items 20 and 24]). The final volume in each test chamber after application of the 

test article was 100 mL. 

Water Chemistry 

Prior to exposure, temperature, hardness, and alkalinity were measured on the source water for 

all trials. Conductivity was measured on the source water for all trials, excluding those completed in 

May 2011 (L. cardium, L. siliquoidea, and L .higginsii SDP exposures; table 1). For the trials completed 

in May 2011 the water chemistry parameters measured during the exposure included duplicate 

measurements of dissolved oxygen (DO), pH, and temperature on all control and high test article 

concentration (300 mg/L) test chambers and on one indiscriminately selected test chamber from the 

remaining treatment groups. For all other trials, the water chemistry parameters measured during the 

exposure included duplicate measurements of DO, pH, and temperature in all test chambers. At the 

termination of the exposure, the contents of each test chamber were pooled by treatment group and the 

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

on the pooled replicates. The TAN was measured using a YSI 9000 photometer (YSI, Inc., Yellow 

Springs, Ohio; L. cardium, L. siliquoidea, and L. higginsii SDP exposures) or using the automated 

phenate method (Standard Method 4500G in American Public Health Association and others, 2012; L. 

recta, O. olivaria, and A. ligamentina SDP exposures; L. cardium and M. nervosa FDP exposures). 

Glochidia Viability Assessments 

Definitive initial viability determinations for glochidia extracted from each female mussel and 

the viability of glochidia in 

photomicrographs using equation 1: 

where 

Viability	ሺpercent

N 

 NC

ሻ 	 ൌ

samples collected during the exposures were completed from 

൫N-ൣNCpre+NOpost൧൯

N

ൈ 100 (1) 

 is the total number of glochidia in the sample, 

pre is the number of glochidia closed before adding sodium chloride, and 

 NOpost is the number of glochidia open after adding sodium chloride. 

 During the exposure, glochidia from each test chamber were assessed for viability at 6, 12, and 

24 hours, with the exception of the M. nerovosa glochidia, which were assessed only at 24 hours. Water 

within each test chamber was gently swirled to position the glochidia in a concentrated group centered 

at the bottom exposure chamber. Then a 100-μL sample was withdrawn from each exposure chamber 

using a 1,000-μL wide-bore pipet tip attached to an adjustable pipette. For each species, excluding M. 
10 

nerovosa, the glochidia samples were placed into a 35-mm petri dish, viewed, and photomicrographed 

using a Nikon model SMZ 1500 compound stereo microscope fitted with a Nikon digital sight DS–Fi1 

camera controlled by Nikon Imaging Systems Elements-BR® software, version 3.10 (Nikon Imaging 

systems, 1991–2010). Photomicrographs were recorded before and after a drop of saturated sodium 

chloride solution was added to induce valve closure in viable glochidia. The second photomicrograph 

was recorded one minute after sodium chloride addition to provide a standard valve closure response 

time. Glochidia on each photomicrograph were enumerated by valve position (either open or closed) 

and the percentage of viable glochidia was calculated by using equation 1. Megalonaias nervosa 

glochidia are released in a mucoidal matrix, which precluded the ability to obtain an image of the 

glochidia on a single focal plane. Therefore, M. nervosa glochidia were immediately assessed for 

viability by placing the glochidia under a dissecting microscope (model SMZ745; Nikon Instruments) 

and enumerating, by valve position, before and after a after a drop of saturated sodium chloride solution 

was added to induce valve closure in viable glochidia. 

Data Analysis 

Water chemistry (DO, pH, temperature, hardness, alkalinity, conductivity, and TAN) data 

analyses were limited to simple descriptive statistics (appendix 6, items 1, 2, 4, and 7). In order to 

compensate for initial glochidia viabilities of less than 100 percent, the observed viabilities of glochidia 

during the exposure were adjusted with the initial viability of glochidia using the methods described by 

Wang and others (2007). Viability adjustments were completed using equation 2: 

Adjusted v ሺVO

I

ሻ (2)

where 

 V

iability	 ൌ ሺV ሻ ൈ 100

O is the observed (un-adjusted) viability of a treatment replicate (appendix 7, items 

5–7), and 

 VI is the initial mean viability observed for the species, in percent (table 3). 

For example, the mean initial viability of A. ligamentina glochidia was 92.7 percent. The observed 

(unadjusted) viability of A. ligamentina glochidia in a control group replicate, at 24 hours, was 90.0 

percent, therefore, the adjusted viability of A. ligamentina glochidia in the control group replicate, at 

24 hours, is 90/92.7 × 100 or 97.1 percent. 

 Statistical comparisons were completed with the observed (un-adjusted) glochidia viability of 

each treatment group replicate using SAS® software version 9.3 (SAS, 2010). Significance was declared 

at α ≤ 0.05. A generalized linear mixed model was used to analyze the viability of glochidia by 

treatment group and species (appendix 7, item 2). The proportion of mortalities (number of nonviable 

glochidia compared to the total number of glochidia present) were modeled with a binomial distribution 

and a logit link function. A scale parameter was added to the model using the “random_residual_” 

statement. At each sampling time, glochidia viability in each treatment group was individually 

compared to the viability in the untreated control group using a two-sided means comparison test. 

Results and Discussion 

Pre-exposure water chemistry parameters (temperature, alkalinity, hardness, and conductivity) of 

the source water are summarized in table 4 and are presented in appendix 6 (item 1). Pre-exposure 

temperature ranged from 18.4 to 19.9 °C; hardness ranged from 171 to 175 mg/L as calcium carbonate 

(CaCO3); alkalinity ranged from 122 to 130 mg/L as CaCO3; and conductivity ranged from 383 to 
11 

422 microsiemens per centimeter (μS/cm). Water chemistry parameters (DO, pH, and temperature) 

measured during the exposure are presented in table 5 and in appendix 6 (item 2). Dissolved oxygen 

remained well above the ASTM International criterion of 4 mg/L throughout the exposure period 

(ASTM International, 2013). The pH of exposure water ranged from 7.52 to 8.56 and the temperature 

ranged from 17.2 to 20.7 °C. The pooled replicate exposure water samples collected at exposure 

termination had mean temperatures from 18.2 to 20.3 °C, hardness from 180 to 210 mg/L as CaCO3,

alkalinity from 137 to 153 mg/L as CaCO3, and conductivity from 367 to 457 μS/cm (table 4; 

appendix 6, item 4). The mean TAN from samples collected at the termination of the exposure period are 

presented in table 6 and appendix 6 (items 5–7). The TAN remained below the 2013 

U.S. Environmental Protection Agency criterion for acute exposure (17 mg/L at pH 7 and 20 °C) throughout 

the entire exposure period for all species and treatment groups (U.S. Environmental Agency, 2013). 

The initial viability of glochidia extracted for each female mussel ranged from 62 to 100 percent, 

with glochidia extracted from four individuals below the 80 percent viability recommended in the 

“Standard Guide for Conducting Laboratory Toxicity Tests with Freshwater Mussels” (ASTM 

International, 2013). However, the mean viability of glochidia extracted from the female mussels for 

each species ranged from 80.3 to 96.5 percent (table 2, appendix 7, item 1). Statistical analyses of the 

un-adjusted glochidia viability are presented in appendix 7 (item 4) and the un-adjusted-mean glochidia 

viability of each treatment group are presented by species in table 7. The mean of the adjusted viability 

of each treatment group are presented by species in table 8 and in appendix 7 (item 5–7). 

 Regardless of species, the mean of the un-adjusted and adjusted control group viability during 

the SDP trials exceeded 66 and 82 percent, respectively, throughout the study. After 6 hours of SDP 

exposure at 50 mg/L, the un-adjusted-mean viabilities of L. recta and A. ligamentina glochidia were 

significantly lower (11.7 percent [p < 0.01 ] and 71.7 percent [p = 0.04], respectively) than the untreated 

control groups (85.7 and 88.6 percent, respectively). Concentrations of SDP ≥100 mg/L for 6 hours 

significantly lowered glochidia viability for all species, except for L. cardium and L. higginsii, which 

were impacted at concentrations ≥ 200 and 300 mg/L, respectively. The observed lower viability for 

O. olivaria at 6 hours in the 100 mg/L SDP-treated group is likely an outlier, given no differences were 

detected at 12 and 24 hours in the viability of glochidia in the 100 mg/L SDP-treated group compared to 

the control group (p = 0.16 and 0.15, respectively). 

The mean for the un-adjusted and adjusted control group viability for L. cardium during the FDP 

trial exceeded 81 and 96 percent, respectively. The un-adjusted- and adjusted-mean control group 

glochidia viability for M. nervosa at 24 hours was 52.1 and 53.9 percent, respectively. During the 

L. cardium FDP-trial, glochidia exposed to FDP concentrations ≥ 200 mg/L were significantly less viable 

than those in the untreated control group, regardless of sample time. After 24 hours of exposure, no 

difference was detected between the viability of M. nervosa glochidia in the FDP-treated groups 

compared to the control group, with the exception of the positive control group. However, given the low 

viability observed in the M. nervosa untreated controls (un-adjusted viability of 52.1 percent) the results 

should be interpreted with caution. 
12 

Table 4. Mean temperature, alkalinity, hardness, and conductivity of pre-exposure source water and pooledexposure termination-water samples. 

[Samples were collected before and during exposures of selected native freshwater mussel glochidia to two formulations of 

Pseudomonas fluorescens, strain CL145A. °C, degrees Celsius; mg/L, milligrams per liter; Cond., conductivity; μS/cm, 

microsiemens per centimeter at 25 degrees Celsius; SDP, spray-dried powder; FDP, freeze-dried powder; number in 

parentheses, standard deviation; --, no data collected or cannot calculate] 

 Preexposure Exposure termination 

Species Formulation Temperature 

(°C) 

Alkalinity Hardness

(mg/L) 1 (mg/L) 1

Cond. 

(μS/cm) 

 Temperature 

(°C) 

Alkalinity Hardness 

(mg/L) 1 (mg/L) 1

Cond. 

(μS/cm) 

Lampsilis 

cardium 

Megalonaias 

nervosa 

Ligumia 

recta

Lampsilis 

siliquoidea 

Lampsilis 

higginsii 

Obovaria 

olivaria

Actinonaias 

ligamentina

Lampsilis 

cardium

FDP 

FDP 

SDP 

SDP 

SDP 

SDP 

SDP 

SDP 

18.4 (0.0) 

18.4 (0.0) 

19.9 (0.0) 

19.3 (0.1) 

19.3 (0.1) 

19.9 (0.2) 

19.9 (0.0) 

19.3 (0.1) 

126 (1) 

126 (1) 

124 (1) 

130 (0) 

130 (0) 

122 (1) 

124 (1) 

130 (0) 

174 (0) 

174 (0) 

171 (1) 

171 (1) 

171 (1) 

175 (1) 

171 (1) 

171 (1) 

391 (--) 

391 (--) 

422 (9) 

-- 

-- 

383 (2) 

422 (9) 

-- 

18.2 (0.2) 

18.7 (0.3) 

20.3 (0.5) 

19.9 (0.1) 

19.5 (0.1) 

19.7 (0.7) 

20.0 (0.4) 

19.1 (0.1) 

144 (3) 

148 (5) 

153 (8) 

137 (2) 

142 (4) 

147 (7) 

153 (5) 

137 (3) 

196 (4) 

201 (2) 

199 (6) 

181 (2) 

185 (3) 

210 (4) 

198 (2) 

180 (1) 

431 (12) 

440 (18) 

433 (21) 

367 (13) 

387 (17) 

457 (27) 

440 (25) 

391 (14) 

1 Alkalinity and hardness reported as milligrams per liter of calcium carbonate. 

Table 5. Mean dissolved oxygen, mean temperature, and pH range of exposure water. 

[Values are by treatment group, during exposures of selected native freshwater mussel glochidia 

to two formulations of Pseudomonas fluorescens, strain CL145A. mg/L, milligrams per liter; 

pH, reported as standard units; °C, degrees Celsius; SDP, spray-dried powder; number in 

parentheses, standard deviation; HD, heat-deactivated test article, FDP, freeze-dried powder]

Treatment Dissolved oxygen Temperature pH group (mg/L) (°C) 

Lampsilis cardium, FDP formulation

Control 8.34 (0.51) 17.8 (0.4) 8.12–8.32 

50 8.13 (0.54) 17.2 (0.5) 8.26–8.31 

100 8.08 (0.81) 17.5 (0.3) 8.31–8.34 

200 7.92 (0.63) 17.4 (0.50 8.31–8.34 

300 7.81 (0.62) 17.6 (0.5) 8.29–8.33 

300 HD 7.19 (0.89) 17.7 (0.4) 8.31–8.36 

Megalonaias nervosa, FDP formulation

Control 7.43 (0.19) 18.6 (0.2) 8.39–8.41 

50 7.64 (0.64) 17.8 (0.4) 8.39–8.40 

100 8.39 (0.79) 18.3 (0.4) 8.39–8.41 

200 8.12 (0.69) 18.5 (0.3) 8.35–8.49 
13 

Treatment 

group 

Dissolved oxygen 

(mg/L) 

Temperature 

(°C) pH 

300 

300 HD 

7.87 (0.53) 

7.84 (0.68) 

18.1 (0.3) 

18.4 (0.2) 

8.35–8.39 

8.37–8.40 

Ligumia recta, SDP formulation

Control 9.17 (0.31) 20.4 (0.4) 8.27–8.56 

50 9.36 (0.48) 20.7 (0.7) 8.34–8.44 

100 9.59 (0.94) 20.5 (0.4) 8.32–8.38 

200 8.72 (0.57) 20.2 (0.3) 8.21–8.37 

300 8.75 (1.09) 20.6 (0.4) 8.15–8.31 

300 HD 9.29 (0.88) 19.6 (0.9) 8.27–8.50 

Lampsilis siliquoidea, SDP formulation

Control 8.59 (0.28) 19.4 (0.4) 8.39–8.48 

50 7.71 (0.57) 19.3 (0.4) 8.05–8.36 

100 7.58 (0.81) 19.5 (0.8) 8.09–8.35 

200 7.31 (1.82) 19.5 (0.6) 7.73–8.26 

300 6.92 (2.09) 19.7 (0.7) 7.52–8.37 

300 HD 6.78 (2.24) 19.1 (0.1) 7.68–8.31 

Lampsilis higginsii, SDP formulation

Control 8.05 (0.20) 19.3 (0.6) 8.38–8.46 

50 7.59 (0.26) 19.3 (0.2) 8.08–8.38 

100 8.02 (0.76) 19.0 (1.1) 8.00–8.29 

200 7.49 (0.68) 18.7 (0.9) 8.03–8.30 

300 7.21 (1.54) 19.3 (0.8) 7.58–8.41 

300 HD 7.02 (1.34) 19.5 (0.7) 7.85–8.34 

Obovaria olivaria, SDP formulation

Control 9.82 (0.78) 19.9 (0.2) 8.36–8.51 

50 10.41 (0.32) 19.7 (0.5) 8.32–8.48 

100 10.16 (0.42) 19.6 (0.5) 8.30–8.45 

200 9.67 (0.34) 19.7 (0.5) 8.10–8.35 

300 9.66 (0.93) 19.7 (0.2) 8.10–8.30 

300 HD 10.57 (0.30) 19.3 (1.0) 8.11–8.36 

Actinonaias ligamentina, SDP formulation

Control 8.40 (0.63) 20.1 (0.1) 8.34–8.53 

50 9.20 (0.47) 20.1 (0.1) 8.33–8.43 

100 8.65 (0.18) 19.8 (0.5) 8.31–8.43 

200 8.96 (0.50) 19.7 (0.2) 8.26–8.40 
14 

Treatment 

group 

Dissolved oxygen 

(mg/L) 

Temperature 

(°C) pH 

300 8.74 (0.25) 19.8 (0.3) 8.29–8.38 

300 HD 8.63 (0.31) 19.7 (0.6) 8.23–8.42 

Lampsilis cardium, SDP formulation

Control 8.50 (0.11) 19.5 (0.4) 8.23–8.42 

50 8.15 (0.11) 19.7 (0.7) 8.25–8.39 

100 8.04 (0.91) 19.3 (0.4) 8.03–8.39 

200 7.66 (0.87) 19.3 (0.4) 8.09–8.36 

300 7.21 (1.63) 19.6 (0.5) 7.74–8.29 

300 HD 7.46 (1.17) 19.6 (0.6) 8.07–8.37 

Table 6. Mean total ammonia nitrogen of exposure water by treatment group at 24 hours. 

 [Total ammonia nitrogen in milligrams per liter measured at the termination of 24 hours exposures of selected native 

freshwater mussel glochidia to two formulations of Pseudomonas fluorescens, strain CL145A. FDP, freeze-dried powder; 

SDP, spray-dried powder; --, cannot calculate data; number in parentheses, standard deviation; HD, heat-deactivated 

product] 

FDP formulation SDP formulation 

Treatment Lampsilis Megalonaias Ligumia Lampsilis Lampsilis Obovaria Actinonaias Lampsilis 

group cardium nervosa recta siliquoidea higginsii olivaria ligamentina cardium 

Control 0.058 0.026 0.080 0.10 0.09 0.066 0.066 0.06 

(0.016) (0.004) (0.002) (--) (--) (0.029) (0.039) (--) 

50 0.107 0.050 0.297 0.25 0.17 0.210 0.268 0.08 

(0.013) (0.010) (0.121) (--) (--) (0.097) (0.234) (--) 

100 0.137 0.056 0.714 0.24 0.31 0.367 0.445 0.13 

(0.011) (0.019) (0.014) (--) (--) (0.238) (0.241) (--) 

200 0.225 0.047 1.054 0.44 0.53 0.243 0.853 0.24 

(0.014) (0.013) (0.127) (--) (--) (0.027) (0.072) (--) 

300 0.296 0.123 1.452 0.63 0.45 0.302 1.047 0.44 

(0.012) (0.057) (0.242) (--) (--) (0.157) (0.116) (--) 

300 HD 0.193 0.086 0.230 0.55 0.50 0.489 0.581 0.51 

(0.012) (0.020) (0.028) (--) (--) (0.389) (0.197) (--) 
15 

Table 7. Mean un-adjusted glochidia viability from selected native freshwater mussels at 6, 12 and 24 hours. 

[Selected native freshwater mussel glochidia exposed for 6, 12 and 24 hours to two formulations of Pseudomonas 

fluorescens, strain CL145A. FDP, freeze dried powder; SDP, spray dried powder; number in parentheses, standard deviation; 

--, no data collected; HD, heat-deactivated test article] 

FDP formulation SDP Formulation 

Treatment 

group 

Lampsilis Megalonaias

cardium nervosa 

Ligumia

recta 

Lampsilis

siliquoidea 

Lampsilis Obovaria 

higginsii olivaria 

Actinonaias

ligamentina 

Lampsilis

cardium 

6 hours

Control 82.3 -- (4.7) 

85.7 

(1.1) 

72.3 

(4.0) 

72.3 

(9.5) 

80.0 

(11.5) 

88.6 

(4.4) 

92.3 

(1.0) 

50 69.6 -- (11.5) 

11.7a

(9.5) 

56.8 

(2.7) 

77.7 

(3.7) 

85.2 

(1.3) 

71.7a

(6.6) 

77.9 

(8.9) 

100 65.7 -- (3.4) 

0.5a

(0.8) 

29.0a

(14.8) 

63.2 

(1.0) 

61.0a

(13.8) 

72.2a

(9.7) 

75.3 

(6.1) 

200 48.7a

-- (11.4) 

1.7a

(1.6) 

19.3a

(17.1) 

56.7 

(5.5) 

40.6a

(11.5) 

40.5a

(16.3) 

52.2a

(21.2) 

300 40.7a

-- (14.7) 

1.1a

(1.6) 

11.9a

(16.7) 

29.1a

(21.7) 

15.7a

(12.7) 

11.8a

(7.3) 

9.9a

(10.4) 

300 HD 20.4a

-- (23.0) 

0.8a

(1.5) 

2.3a

(2.4) 

13.3a

(12.3) 

13.8a

(6.3) 

3.6a

(2.1) 

3.4a

(3.1) 

12 hours

Control 81.8 -- (3.4) 

89.5 

(0.5) 

66.6 

(6.5) 

68.7 

(6.1) 

86.4 

(6.3) 

94.2 

(1.5) 

86.1 

(2.0) 

50 76.7 -- (6.2) 

16.2a

(5.6) 

59.1 

(4.1) 

68.7 

(6.5) 

84.5 

(2.9) 

66.6a

(17.8) 

78.6 

(6.6) 

100 70.6 -- (8.4) 

8.8a

(3.3) 

43.6a

(3.4) 

65.5 

(3.1) 

77.2 

(12.4) 

64.4a

(17.7) 

52.7a

(45.6) 

200 34.9a

-- (30.6) 

1.2a

(0.3) 

19.6a

(13.2) 

46.2 

(17.9) 

50.3a

(16.5) 

40.0a

(4.5) 

46.7a

(22.3) 

300 9.1a

-- (8.8) 

0.0a

(0.0) 

4.5a

(1.9) 

38.8a

(8.1) 

39.2a

(3.0) 

10.2a

(2.5) 

6.5a

(4.2) 

300 HD 4.8a

-- (1.2) 

0.9a

(0.8) 

2.1a

(2.8) 

9.5a

(0.9) 

1.2a

(0.4) 

8.2a

(5.0) 

5.8a

(2.6) 

24 hours

Control 81.9 

(1.4) 

52.1 

(11.3) 

84.5 

(1.9) 

69.4 

(8.2) 

69.3 

(7.3) 

87.0 

(1.8) 

92.6 

(2.2) 

92.1 

(0.9) 

50 74.1 

(9.3) 

55.6 

(9.5) 

14.7a

(1.8) 

56.8 

(6.0) 

74.6 

(3.4) 

89.2 

(2.9) 

75.8a

(13.0) 

79.8 

(5.6) 

100 67.7 

(3.8) 

48.1 

(17.0) 

7.0a

(4.9) 

41.6a

(6.5) 

65.5 

(4.9) 

78.3 

(6.8) 

63.5a

(11.2) 

66.9a

(15.8) 

200 29.5a

(23.8) 

49.5 

(18.7) 

3.3a

(2.2) 

11.6a

(4.7) 

42.5a

(14.8) 

60.7a

(4.5) 

30.0a

(7.2) 

59.2a

(12.8) 

300 12.0a

(1.8) 

40.1 

(20.8) 

0.7a

(0.6) 

6.0a

(1.7) 

22.3a

(26.6) 

26.5a

(3.2) 

6.0a

(3.5) 

6.1a

(7.2) 

300 HD 4.9a

(2.3) 

26.8a

(10.6) 

0.7a

(0.8) 

1.7a

(0.9) 

13.9a

(10.4) 

0.5a

(0.3) 

5.2a

(3.4) 

4.7a

(1.3) 

a

At each sample time, the mean un-adjusted treatment group glochidia viability ( in percent) within the same column that have a letter superscript are 

significantly different (p > 0.05) from the control group. 
16 

Table 8. Mean adjusted glochidia viability from selected native freshwater mussels at 6, 12 and 24 hours. 

 [Selected native freshwater mussel glochidia exposed for 6, 12 and 24 hours to two formulations of Pseudomonas 

fluorescens, strain CL145A. FDP, freeze-dried powder; SDP, spray-dried powder; number in parentheses, standard 

deviation; --, no data collected; HD, heat-deactivated test article]

FDP formulation SDP Formulation 

Treatment 

group 

Lampsilis Megalonaias

cardium nervosa 

Ligumia

recta 

Lampsilis

siliquoidea 

Lampsilis Obovaria 

higginsii olivaria 

Actinonaias

ligamentina 

Lampsilis

cardium 

6 hours 

Control 

50 

97.1 -- (5.6) 

82.1 -- (13.6) 

101.4 

(1.3) 

13.9 

(11.3) 

90.1 

(5.0) 

70.7 

(3.4) 

89.5 

(11.8) 

96.2 

(4.6) 

93.0 

(13.4) 

99.1 

(1.5) 

95.6 

(4.8) 

77.3 

(7.1) 

98.4 

(1.1) 

83.1 

(9.5) 

100 

200 

77.6 -- (4.1) 

57.5 -- (13.5) 

0.6 

(1.0) 

2.1 

(1.8) 

36.1 

(18.4) 

24.0 

(21.4) 

78.2 

(1.3) 

70.1 

(6.8) 

70.9 

(16.1) 

47.3 

(13.3) 

77.9 

(10.5) 

43.7 

(17.6) 

80.2 

(6.5) 

55.7 

(22.6) 

300 48.0 -- (17.3) 

1.3 

(1.8) 

14.8 

(20.8) 

36.0 

(26.9) 

18.3 

(14.8) 

12.7 

(7.9) 

10.6 

(11.1) 

300 HD 24.1 -- (27.2) 

1.0 

(1.7) 

2.9 

(2.9) 

16.4 

(15.2) 

16.1 

(7.3) 

3.9 

(2.2) 

3.7 

(3.3) 

12 hours

Control 96.6 -- (4.0) 

105.9 

(0.6) 

82.9 

(8.0) 

85.0 

(7.6) 

100.5 

(7.4) 

101.7 

(1.6) 

91.8 

(2.2) 

50 

100 

200 

90.5 -- (7.4) 

83.3 -- (10.0) 

41.2 -- (36.1) 

19.1 

(6.6) 

10.5 

(3.9) 

1.4 

(0.4) 

73.6 

(5.1) 

54.3 

(4.2) 

24.4 

(16.4) 

85.0 

(8.0) 

81.1 

(3.9) 

57.2 

(22.2) 

98.2 

(3.4) 

89.7 

(14.5) 

58.4 

(19.2) 

71.8 

(19.1) 

69.4 

(19.1) 

43.2 

(4.9) 

83.8 

(7.0) 

56.1 

(48.7) 

49.8 

(23.8) 

300 10.8 -- (10.4) 

0.0 

(0.0) 

5.6 

(2.4) 

48.0 

(10.0) 

45.6 

(3.5) 

11.0 

(2.7) 

6.9 

(4.5) 

300 HD 5.7 -- (1.4) 

1.1 

(1.0) 

2.7 

(3.5) 

11.8 

(1.1) 

1.4 

(0.4) 

8.8 

(5.4) 

6.2 

(2.8) 

24 hours

Control 

50 

96.7 

(1.7) 

87.5 

(11.0) 

53.9 

(11.7) 

57.6 

(9.8) 

100.0 

(2.3) 

17.4 

(2.1) 

86.5 

(10.2) 

70.8 

(7.4) 

85.8 

(9.0) 

92.3 

(4.2) 

101.2 

(2.0) 

103.7 

(3.3) 

99.9 

(2.4) 

81.7 

(14.1) 

98.2 

(1.0) 

85.1 

(6.0) 

100 80.0 

(4.5) 

49.8 

(17.6) 

8.2 

(5.8) 

51.9 

(8.0) 

81.0 

(6.0) 

91.0 

(8.0) 

68.5 

(12.1) 

71.3 

(16.8) 

200 

300 

34.9 

(28.0) 

14.2 

(2.2) 

51.3 

(19.3) 

41.6 

(21.5) 

3.8 

(2.6) 

0.9 

(0.8) 

14.5 

(5.8) 

7.5 

(2.2) 

52.6 

(18.3) 

27.6 

(32.9) 

70.6 

(5.3) 

30.9 

(3.8) 

32.4 

(7.8) 

6.5 

(3.8) 

63.2 

(13.6) 

6.5 

(7.7) 

300 HD 5.8 

(2.7) 

27.7 

(11.0) 

0.9 

(0.9) 

2.1 

(1.1) 

17.2 

(12.9) 

0.6 

(0.4) 

5.6 

(3.7) 

5.1 

(3.3) 
17 

Conclusions 

The mean adjusted-viability of control group glochidia in trials with spray-dried powder (SDP) 

formulation of Pseudomonas fluorescens strain CL145A exceeded 82 percent. Differences in glochidia 

viability were detected in two of six species exposed to 50 milligrams per liter (mg/L) SDP and in four 

of six species exposed to 100 mg/L SDP at 6, 12, and 24 hours when compared to untreated controls. 

Regardless of sample time, viability of glochidia in the 200 and 300 mg/L SDP-treated groups and the 

300 mg/L heat-deactivated SDP positive control groups were significantly lower than glochidia in the 

untreated control for all species except Lampsilis higginsii. The significant impact of the SDP positive 

control treatments on glochidia viability indicate that the decreases in glochidia viability may not be 

caused by the same mode of action that causes dreissenid mussel (zebra mussel, Dreissena polymorpha 

and quagga mussel, Dreissena rostriformis bugensis) mortality. 

The mean adjusted-viability of control group glochidia in trials with freeze-dried powder (FDP) 

formulation of P. fluorescens (strain CL145A) exceeded 96 percent in the Lampsilis cardium trial and 

was 53.9 percent in the Megalonaias nervosa at 24 hours. Regardless of sample time, differences in 

glochidia viability were only detected in the L. cardium FDP-trial when the concentration was ≥

200 mg/L. In the M. nervosa FDP exposures, differences in glochidia viability were only detected 

between the 300 mg/L heat-deactivated FDP-treated group (positive control) and the untreated control 

group. 

However, given the low viability observed in the M. nervosa control group (52.1 percent unadjusted viability), the results for M. nervosa should be interpreted with caution. Similar to the SDPtrials, the impact of the FDP positive control treatments indicate that the observed decreases in glochidia 

viability may not be caused from the same mode of action that causes dreissenid mussel mortality. The 

results of these exposures indicate that applications of a biopesticide formulated with P. fluorescens as 

the active ingredient may impact the viability of native freshwater mussel glochidia if they are present in 

the water column during an application. However, freshwater mussel glochidia are only present in the 

water column for a relatively short period which could be avoided by timing P. fluorescens biopesticide 

applications. Additionally, applications of a P. fluorescens biopesticide for dreissenid mussel control 

would be of short duration and the biological activity of P. fluorescens degrades rapidly; further 

reducing the risk of exposure to P. fluorescens to native mussel glochidia. 

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. 

Cummings S.K. and Mayer C.A., 1992, Field guide to freshwater mussels of the Midwest, Illinois 

Natural History Survey Manual 5, 194 pp. 
18 

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. 

Master, L.L., Flack, S.R. and Stein, B.A., eds., 1998, Rivers of Life: Critical Watersheds for Protecting 

Freshwater Biodiversity. The Nature Conservancy, Arlington, Virginia. 

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

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

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

4, p. 71-79. 

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

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

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

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

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

Nikon Imaging System Elements-BR, 1991-2010, Version 3.10, Nikon Instruments Inc., Melville, New 

York, USA. 

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. 

SAS, 2010, Version 9.3, SAS Institute Inc., Cary, NC, USA. 

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

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

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

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

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

U.S. Fish and Wildlife Service, 2004, Higgins Eye Pearlymussel (Lampsilis higginsii) recovery plan: 

First Revision, Ft. Snelling, Minnesota, 126 p. 

U.S. Fish and Wildlife Service, 2013, ESA Basics 40 Years of Conserving Endangered Species, 2 p., 

accessed April 1, 2015, at http://www.fws.gov/endangered/esa-library/pdf/ESA_basics.pdf/. 

Wang, N., Augspurger, T., Barnhart, M.C., Bidwell, J. R., Cope, W. G., Dwyer, F. J., Geis S., Greer, E., 

Ingersoll, C.G., Kane, C.M, May, T.W., Neves, R.J., Newton T.J., Roberts, A.D., and Whites, D.W., 

2007, Intra‐and interlaboratory variability in acute toxicity tests with glochidia and juveniles of 

freshwater mussels (unionidae): Environmental Toxicology and Chemistry, v. 26, no. 10, 2029-2035. 

Watters, G.T., M.A. Hoggarth and D.H. Stansbery, 2009, The freshwater mussels of Ohio: Columbus, 

Ohio, Ohio State University Press, 421 p. 

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. 
19 

Appendix 1. Study Protocol, Amendments, and Datasheets

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Protocol: “Effects of Pseudomonas fluorescens(Pf-CL145A) to glochidia from seven unionid mussel 

species” 21 20

2 Amendment 1: Revision of Study Protocol, Study # AEH-11-PSEUDO-01 6 41

3 Amendment 2: Revision of Study Protocol, Study # AEH-11-PSEUDO-01 8 47

4 Amendment 3: Revision of Study Protocol, Study # AEH-11-PSEUDO-01 5 55

5 Amendment 4: Revision of Study Protocol, Study # AEH-11-PSEUDO-01 7 60

6 Amendment 5: Revision of Study Protocol, Study # AEH-11-PSEUDO-01 2 67

7 Note to File 1: Study # AEH-11-PSEUDO-01 6 69

8 Note to File 2: Study # AEH-11-PSEUDO-01 7 75

9 Version 1.1 “Donor Mussel UMESC Lot Number Assignment Form” Datasheet 1 82

10 Version 1.1 “Daily Care Worksheet Donor Mussel Holding” Datasheet 1 83

11 “Adult Mussel Holding Daily Algal Diet 1/13/2012” Datasheet 1 84

12 Version 1.1 “Glochidia Test Organism UMESC Lot Number Assignment Form” Datasheet 1 85

13 Version 1.1 “Glochidia Aliquot Distribution Form” Datasheet 1 86

14 Version 1.1 “Glochidia Photomicrograph Counting Record” Datasheet 1 87

15 Version 1.1 “Initial Viability and Concentration Determination of Glochidia” Datasheet 1 88

16 Version 1.1 “Water Quality – Form 1 Initial (Dilution water Hardness, Alkalinity, and Temperature)” 

Datasheet 1 89

17 Version 1.2 “Water Quality – Form 2 During Exposure (Dissolved Oxygen, pH, Temperature)” Datasheet 1 90

18 Version 1.2 “Water Quality – Form 3 Upon Termination (Hardness, Alkalinity, Conductivity, Temperature 

and pH)” Datasheet 1 91

19 Version 1.1 “Test Chemical Stock Preparation Data Form” Datasheet 1 92

20 Version 1.2 “Chemical Stock Solution Determination and Preparation” Datasheet 1 93

21 Version 1.1 “Pipette Calibration” Datasheet 1 94

22 OLD Versions of Datasheets 18 95
20 
21 
22 
23 
24 
25 
26 
27 
28 
29 
30 
31 
32 
33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
43 
44 
45 
46 
47 
48 
49 
50 
51 
52 
53 
54 
55 
56 
57 
58 
59 
60 
61 
62 
63 
64 
65 
66 
67 
68 
69 
70 
71 
72 
73 
74 
75 
76 
77 
78 
79 
80 
81 
82 
83 
84 
85 
86 
87 
88 
89 
90 
91 
92 
93 
94 
95 
96 
97 
98 
99 
100 
101 
102 
103 
104 
105 
106 
107 
108 
109 
110 
111 
112 
113 

Appendix 2. Deviations from the Study Protocol 

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Deviations #1 and #2 from Study Protocol, Study # AEH-11-PSEUDO-01 1 114

2 Deviation #3 from Study Protocol, Study # AEH-11-PSEUDO-01 1 115

3 Deviation #4 from Study Protocol, Study # AEH-11-PSEUDO-01 1 116

4 Deviation #5 from Study Protocol, Study # AEH-11-PSEUDO-01 1 117

5 Deviation #6 from Study Protocol, Study # AEH-11-PSEUDO-01 1 118

6 Deviation #7 from Study Protocol, Study # AEH-11-PSEUDO-01 2 119

7 Deviation #8 from Study Protocol, Study # AEH-11-PSEUDO-01 2 121

8 Deviation #9 from Study Protocol, Study # AEH-11-PSEUDO-01 1 123

9 Deviation #10 from Study Protocol, Study # AEH-11-PSEUDO-01 5 124

10 Deviation #11 from Study Protocol, Study # AEH-11-PSEUDO-01 2 129

11 Deviation #12 from Study Protocol, Study # AEH-11-PSEUDO-01 2 131

12 Deviation #13 from Study Protocol, Study # AEH-11-PSEUDO-01 2 133
114 
115 
116 
117 
118 
119 
120 
121 
122 
123 
124 
125 
126 
127 
128 
129 
130 
131 
132 
133 
134 
135 

Appendix 3. Randomization Assignments

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 SAS random assignment of mussel species to block for plain pocketbook, Higgins eye, and fatmucket 

(10-May-11) 4 136

2 SAS random assignment of mussel species to block for washboard and plain pocketbook (14-Oct-11) 4 140

3 SAS random assignment of mussel species to block for black sandshell, mucket, and hickory nut

(09-Jan-12) 5 144

4 SAS random assignment of glochidia to tank for plain pocketbook, Higgins eye, and fatmucket (12-May-11) 20 149

5 SAS random assignment of glochidia to tank for washboard and plain pocketbook (14-Oct-11) 13 169

6 SAS random assignment of glochidia to tank for black sandshell, mucket, and hickorynut (09-Jan-12) 20 182

7 SAS random assignment of treatment to tank for plain pocketbook, Higgins eye, and fatmucket (12-May-11) 13 202

8 SAS random assignment of treatment to tank for washboard and plain pocketbook (14-Oct-11) 9 215

9 SAS random assignment of treatment to tank for black sandshell, mucket, and hickorynut (09-Jan-12) 13 224
136 
137 
138 
139 
140 
141 
142 
143 
144 
145 
146 
147 
148 
149 
150 
151 
152 
153 
154 
155 
156 
157 
158 
159 
160 
161 
162 
163 
164 
165 
166 
167 
168 
169 
170 
171 
172 
173 
174 
175 
176 
177 
178 
179 
180 
181 
182 
183 
184 
185 
186 
187 
188 
189 
190 
191 
192 
193 
194 
195 
196 
197 
198 
199 
200 
201 
202 
203 
204 
205 
206 
207 
208 
209 
210 
211 
212 
213 
214 
215 
216 
217 
218 
219 
220 
221 
222 
223 
224 
225 
226 
227 
228 
229 
230 
231 
232 
233 
234 
235 
236 
237 

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 2 238

2 Material Safety Data Sheet: MBI-401 Freeze Dried Powder 2 240

3 MBI-401 FDP (lot # 110607WB-FD-E) Test Article: Certificate of Analysis 1 242

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

110308AI-BD-3 5 243

5 Copy of test article information from test article log book for MBI-401 FDP; lot number 110607WB-FD-E 4 248

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

4655-12-Mix 4 252

7 NYSM Post-Treatment Product Validation Assay (lot number MBI-401 SDP 110308AI-BD-3) 2 256

8 NYSM Post-Treatment Product Validation Assay (lot number MBI-401 FDP 110607WB-FD-E) 2 258

9 NYSM Post-Treatment Product Validation Assay (lot number MBI-401 SDP 4655-12-Mix) 2 260

10 Test Chemical Weights (12-May-11) 1 262

11 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (PPBSDP) 2 263

12 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (HGE SDP) 2 265

13 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (FAM SDP) 2 267

14 Test Chemical Stock Preparation Data Form (18-Oct-11) 1 269

15 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (PPB FDP) 2 270

16 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (WAS FDP) 2 272

17 Test Chemical Stock Preparation Data Form (17-Jan-12) 1 274

18 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (BLS SDP) 2 275

19 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (MUC SDP) 2 277

20 Test Chemical Stock Preparation Data Form (19-Jan-12) 1 279

21 Glochidia Exposure Dosing Form and Chemical Stock Solution Determination (HIC SDP) 2 280
238 
239 
240 
241 
242 
243 
244 
245 
246 
247 
248 
249 
250 
251 
252 
253 
254 
255 
256 
257 
258 
259 
260 
261 
262 
263 
264 
265 
266 
267 
268 
269 
270 
271 
272 
273 
274 
275 
276 
277 
278 
279 
280 
281 
282 

Appendix 5. Test Animal Information

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Test Organism Species List, Collection and Inclusion Criteria 1 283

2 Amended Test Organism Species List, Collection and Inclusion Criteria 2 284

3 Daily Care Worksheets (PPB, HGE, FAM, HIC, BLS, MUC) 6 286

4 Adult Mussel Holding Daily Algal Diet (1/13/2012) 1 292

5 Donor Mussel UMESC Lot Number Assignment Forms 8 293

6 USGS UMESC Fish/Animal History Forms (Donor Mussels) 8 301

7 Glochidia Test Organism UMESC Lot Number Assignment Forms 8 309

8 USGS UMESC Fish/Animal History Forms (Glochidia) 8 317
283 
284 
285 
286 
287 
288 
289 
290 
291 
292 
293 
294 
295 
296 
297 
298 
299 
300 
301 
302 
303 
304 
305 
306 
307 
308 
309 
310 
311 
312 
313 
314 
315 
316 
317 
318 
319 
320 
321 
322 
323 
324 
325 

Appendix 6. Water Quality

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Initial (Pre Exposure) Water Chemistry – Data Summary 2 326

2 Exposure Water Chemistry – Data Summary 9 328

3 Exposure Light Intensity – Data Summary 2 337

4 Termination Water Chemistry – Data Summary 2 339

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

November 9, 2011 1 341

6 Report of Analysis – Ammonia Report from Water Quality Laboratory at UMESC – Report Dated February 

3, 2012 1 342

7 Total Ammonia Nitrogen – Data Summary 10 343

 
326 
327 
328 
329 
330 
331 
332 
333 
334 
335 
336 
337 
338 
339 
340 
341 
342 
343 
344 
345 
346 
347 
348 
349 
350 
351 
352 
353 

Appendix 7. Glochidia Viability

Item 

number Item description

Number 

of 

pages

Report 

page 

number

1 Initial (Pre exposure) Glochidia Viability – Data Summary 2 354

2 SAS program for glochidia viability 2 356

3 SAS log for glochidia viability 4 358

4 SAS output for glochidia viability 81 362

5 Glochidia Viability Assessment – 6 hour – Data Summary 7 443

6 Glochidia Viability Assessment – 12 hour – Data Summary 7 450

7 Glochidia Viability Assessment – 24 hour – Data Summary 8 457

8 Glochidia Distribution Counts – Data Summary 10 465
354 
355 
356 
357 
358 
359 
360 
361 
362 
363 
364 
365 
366 
367 
368 
369 
370 
371 
372 
373 
374 
375 
376 
377 
378 
379 
380 
381 
382 
383 
384 
385 
386 
387 
388 
389 
390 
391 
392 
393 
394 
395 
396 
397 
398 
399 
400 
401 
402 
403 
404 
405 
406 
407 
408 
409 
410 
411 
412 
413 
414 
415 
416 
417 
418 
419 
420 
421 
422 
423 
424 
425 
426 
427 
428 
429 
430 
431 
432 
433 
434 
435 
436 
437 
438 
439 
440 
441 
442 
443 
444 
445 
446 
447 
448 
449 
450 
451 
452 
453 
454 
455 
456 
457 
458 
459 
460 
461 
462 
463 
464 
465 
466 
467 
468 
469 
470 
471 
472 
473 
474 
ISSN 2331-1258 (online) 

http://dx.doi.org/10.3133/ofr20151094

Luoma and others

—Exposure-Related Effects of Formulated Pseudomonas fluorescens Strain CL145A to Glochidia from Seven Unionid Mussel Species

—Open-File Report 2015–1094