ABSTRACT POPP, ANAKELA. Advancing

ABSTRACT

POPP, ANAKELA. Advancing the Tools of Freshwater Mussel Conservation: Determining the Relative Chemical Sensitivity of In Vivo and In Vitro Propagated Juvenile Mussels (Under the direction of William Cope).

Freshwater mussels of the family Unionidae are ecologically important and globally imperiled, and research is urgently needed to guide their protection and conservation.

Identifying and mitigating chemical stressors is an important part of the process, as is assessing mussel-specific sensitivity to pollutants to establish protective concentrations (e.g., establishing suitable habitat condition, effluent permit limits, state water quality standards, and water quality criteria). The newly transformed juvenile life stage has been shown to be sensitive to certain toxicants and is often used in toxicity testing. A ready supply of recently transformed juveniles are needed to conduct laboratory-based toxicity studies. Over the past several decades, host-fish (in vivo) propagation techniques have significantly advanced, as have long-term growth and maintenance of propagated mussels. Alternative media-based (in vitro) culture methods have made laboratory rearing of juveniles more efficient and cost- effective. However, ASTM International guidelines caution against using in vitro propagated juveniles in toxicity tests unless their relative chemical sensitivity to in vivo juveniles is described. The first objective of this study was to evaluate the relative sensitivity of juvenile mussels produced from both propagation methods to selected chemical toxicants. We conducted 96-hour acute toxicity tests according to the ASTM International guidelines with three species (Lampsilis cardium, L. abrupta, and Utterbackia imbecillis) and six chemicals: chloride, nickel, ammonia, copper (as copper sulfate), and aquatic herbicides Clearigate and

Nautique. We calculated the median effective concentration (EC50) for each species- chemical combination and compared the EC50s of in vitro and in vivo juveniles. Statistically significant differences in EC50 between in vitro and in vivo propagated juveniles were observed in 8 of the 17 trials. In 7 of the 8 statistically different tests, in vitro juveniles were more sensitive than in vivo juveniles. There was also a significant effect of the interaction between propagation method and concentration on survival in 6 of the 17 tests. Among all species, EC50s varied between in vitro and in vivo juveniles by a factor of 1.6 for ammonia,

1.1 for chloride, 1.4 for Clearigate, 2.1 for copper, 1.2 for Nautique, and 1.0 for nickel. All of these statistical differences were within the variation for between-laboratory comparisons for a given chemical demonstrated in a recently published evaluation of results of mussel toxicity tests, and therefore, indicate that in vitro propagated juvenile mussels may be appropriate for use in ASTM-based toxicity testing. The second objective of this study was to examine the effect of age on relative chemical sensitivity for one toxicant (copper). Mussels from each of the three species previously mentioned were tested during the first, second, and third week post-transformation and the EC50s of in vitro and in vivo juveniles were compared for each age group. Of the 8 EC50 comparisons, 4 statistically significant differences between in vitro and in vivo juveniles were observed, and in all of these instances, in vitro juveniles were more sensitive than in vivo juveniles. However, these differences were evenly distributed across all ages, and further analysis revealed the lack of a significant relationship between age and relative chemical sensitivity, indicating in vitro juveniles may be appropriate for use in toxicity testing regardless of age.

by Anakela Popp

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science

Fisheries, Wildlife, and Conservation Biology

Raleigh, North Carolina 2017

APPROVED BY:

______William Cope Thomas Augspurger Committee Chair

______Thomas Kwak Jay Levine

DEDICATION

To my parents and wonderful family: for loving me well, for supporting my endeavors (academic and otherwise), and for taking me camping so often that I was shocked when I found out people stayed in hotels for vacation. I love you.

And to the wonderful mentors who have taught, inspired, and encouraged me: Sandy

Stowe, who used her days off to impart scientific knowledge to middle school minds; Bill and Kelli Schuyler, whose passion for their students and the sciences still inspires me to keep studying this amazing world we live in; Robert Bringolf, Jay Shelton, Susan Wilde, and

Andrea Fritts, for sharing their love and knowledge of aquatic resources; Brett Albanese,

Jason Wisniewski, and Deb Weiler, for showing me the amazing diversity of my home state and for training me as a field biologist; and Greg Cope, for introducing me to the world of ecotoxicology and training me as a researcher. Thank you for investing in me.

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BIOGRAPHY

Anakela Popp was born and raised in the suburbs of Atlanta, Georgia. Though their day-to-day lives were centered in suburbia, her family made a point to spend time outdoors.

Family vacations were spent camping, hiking, canoeing, and fishing along various rivers, mountains, and beaches throughout the Southeast. Science and the outdoors have always been her passions, and Anakela was thrilled to discover that the two combined could become a career. She attended the University of Georgia, earning a Bachelor of Science in Forest

Resources with dual concentrations in Wildlife and Aquatic Sciences. Upon graduation in

2013, she worked as a Nongame Aquatics Technician for the Georgia Department of Natural

Resources before beginning her graduate career in 2014.

ACKNOWLEDGEMENTS

I would first like to thank my adviser, Dr. Greg Cope, and the rest of my committee,

Drs. Tom Augspurger, Tom Kwak, and Jay Levine, for their guidance, support, insights, and advice throughout this process. Your mentorship as I begin my professional career has been invaluable. Thanks to my lab mates, Jennifer Archambault and Sean Buczek, for showing me the ropes and offering endless support along the way. Joseph McIver and Mary Silliman were my excellent technicians—thank you for your diligent work and patience, even when mussels kept dying. Thanks also to Emilee Wooster, Mike Walter, Spencer Gardner, and Dylan

Owensby for additional laboratory assistance. Additional gratitude is for Casey Greishaber,

Tiffany Penland, Megan Thoemmes, Mary Henson, and the rest of DCL 258 for support, encouragement, and laughter along the way. Special thanks to Dr. Monte McGregor and the rest of the group at the Kentucky Center for Mollusk Conservation for producing the mussels for these experiments and for providing insight at various stages through the project. Thanks to Dr. Mac Law for offering time and expertise on options for potential histological analysis.

Masaki Miyazawa provided technical expertise and laboratory space for biomarker assays.

My gratitude goes out to Chris Ingersoll and Ning Wang for chemical advice, and to West

Bishop for additional chemical consultation and analysis of copper samples.

Funding for this research was provided by the U.S. Geological Survey (USGS) and

U.S. Fish and Wildlife Service through the Science Support Partnership Program via

Research Work Order No. 211, administered through the USGS North Carolina Cooperative

Fish and Wildlife Research Unit. In addition to Drs. Tom Kwak, Greg Cope, Chris Ingersoll,

Ning Wang, and Monte McGregor, I would like to thank Dr. Damian Shea, Dr. Christopher

Owen, and Anthony Velasco for submitting the initial research proposal.

TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………….vii LIST OF FIGURES…………………………………………………………..……………..ix Chapter 1: A Comparison of the Chemical Sensitivities of In vitro and In vivo Propagated Juvenile Freshwater Mussels: Implications for Standard Toxicity Testing Guidelines………………………………………………………………………………….1 Abstract……………...………………………………………………………………………..2 Introduction……….………………………………………………………………………….3 Methods………….……………………………………………………………………………8 Results…………….…………………………………………………………………………13 Discussion……………….…………………………………………………………………..15 Acknowledgements……………...………………………………………………………….20 References……………………...……………………………………………………………21 Tables………………………………………………………………………………………..25 Figures……………………………………………………………………………………….29 Chapter 2: Influence of Age on Juvenile Freshwater Mussel Chemical Sensitivity: A Comparison of Progeny from Two Propagation Methods……………………….……….30 Abstract……………………………………………..…………………………………....….31 Introduction……………………………….………………………………………………...32 Methods…………………………………….………………………………………………..35 Results…………………………………….…………………………………………………39 Discussion……………………………………….…………………………………………..40 Acknowledgements…………………………………...…………………………………….44 References…………………………………………...………………………………………46 Tables………………………………………………………………………………………..49 Figures……………………………………………………………………………………….50 Appendix A………………………………………………………………………………….54 Appendix B………………………………………………………………………………….60 Appendix C………………………………………………………………………………….61 Appendix D………………………………………………………………………………….62 Appendix E……………………………………………………………………….…………68

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LIST OF TABLES

Chapter 1 Table 1. Toxicant and associated treatment concentrations used in the acute toxicity tests with in vitro and in vivo propagated juvenile mussels for the three species tested………...……..…25 Table 2. Median effective concentration (EC50) causing 50% mortality (with 95% confidence intervals) in in vitro and in vivo propagated juvenile mussels at 48 and 96 hours……………..26 Table 3. Results from the Kruskal-Wallis two-way Analysis of Variance, with propagation (prop), concentration (conc) and their interaction as the main effects………………..……….27 Table 4. Inter-species ratio of variance between EC50s of in vitro and in vivo propagated juvenile mussels among three species for each chemical tested for this study………..………28

Chapter 2 Table 1. Median effective concentration (EC50) of copper causing 50% mortality (with 95% confidence intervals) in juvenile mussels at 96 h………………………………………..……49

Appendix A Table 1. Water chemistry data for tests using sodium chloride………………….…………..54 Table 2. Water chemistry data for tests using nickel chloride……………………………….55 Table 3. Water chemistry data for tests with copper sulfate…………………………………56 Table 4. Water chemistry data for tests with Clearigate®…………………………………..57 Table 5. Water chemistry data for tests with Nautique®…………………………………….58 Table 6. Water chemistry data for tests with ammonium chloride…………………………..59

Appendix B Table 1. Nominal and measured concentrations of toxicants…………...……………………60

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Appendix C Table 1. Effective concentration causing immobility or mortality for 5% of test organisms (EC05) at 96 hours……………………………………………………………………………61

Appendix D Table 1. Juvenile mussel survival in tests with ammonium chloride…………………………62 Table 2. Juvenile mussel survival in tests with sodium chloride……………………………..63 Table 3. Juvenile mussel survival in tests with Clearigate®……………………………….…64 Table 4. Juvenile mussel survival in tests with copper sulfate………………………….…….65 Table 5. Juvenile mussel survival in tests with Nautique®…………………………….…….66 Table 6. Juvenile mussel survival in tests with nickel chloride……..………………………..67

Appendix E Table 1. Survival data for L. abrupta in vitro juvenile mussels. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University………………...…………68 Table 2. Survival data for L. abrupta in vivo juvenile mussels. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University……………….………………….69 Table 3. Survival data for L. cardium in vitro juvenile mussels. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University…………………..……….70 Table 4. Survival data for Lampsilis cardium in vitro juvenile mussels, age 17-19 days….….71 Table 5. Survival data for Lampsilis cardium in vivo juvenile mussels, age 17-19 days...... …72 Table 6. Survival data for Utterbackia imbecillis in vitro juvenile mussels, age 3 and 17 days. …………………………………………………………………………………………..……73 Table 7. Survival data for Utterbackia imbecillis in vitro and in vivo juvenile mussels, age 27 and 3 days………………………………………………………………………………….....74 Table 8. Survival data for Utterbackia imbecillis in vitro and in vivo juvenile mussels, age 27 and 3 days………………………………………………………………………………….…75

LIST OF FIGURES

Chapter 1 Figure 1. Representative survival responses of in vitro and in vivo juveniles……………...…29

Chapter 2 Figure 1. Ratio of variation between in vitro and in vivo propagated juvenile mussel median effective concentration (EC50) by age………………………………………………….……50 Figure 2. Protein concentration of in vitro and in vivo juvenile mussels with age, normalized by length in mm…………………………………………………………………………..…..51 Figure 3. Adenosine triphosphate (ATP) concentration of in vitro and in vivo propagated juvenile mussels by age ……………………………………….……………………………..52

Chapter 1 A Comparison of the Chemical Sensitivities of In vitro and In vivo Propagated Juvenile Freshwater Mussels: Implications for Standard Toxicity Testing Guidelines

Abstract

Freshwater mussels of the family Unionidae are ecologically important and globally imperiled. Research is urgently needed to guide their protection and conservation.

Identifying and mitigating environmental chemical stressors is an important part of the process, as is assessing mussel-specific sensitivity to pollutants to establish water quality criteria. The newly transformed juvenile life stage has been shown to be sensitive to certain toxicants and is often used in toxicity testing. Thus, there is a need to transform mussel larvae (glochidia) into juveniles within a laboratory setting. Over the past several decades, conservation aquaculture has significantly advanced propagation techniques and long-term growth and maintenance of propagated mussels. Improving standard host-fish (in vivo) infection techniques has contributed to this success, but recently, in vitro culture methods have made it more efficient and cost-effective to raise juvenile mussels in the laboratory.

However, the international standards organization ASTM International cautions against using in vitro propagated juveniles in toxicity tests unless their relative chemical sensitivity to in vivo juveniles is described. The objective of this study was to evaluate the relative sensitivity of juvenile mussels produced from both propagation methods to selected chemical toxicants.

We conducted 96-hour acute toxicity tests according to the ASTM International guidelines with three species (Lampsilis cardium, L. abrupta, and Utterbackia imbecillis) and six chemicals: chloride, nickel, ammonia, copper, and aquatic herbicides Clearigate and

Nautique. We calculated the median effective concentration (EC50) for each species- chemical combination and compared the EC50s of the in vitro and in vivo juveniles.

Statistically significant differences in EC50 between in vitro and in vivo propagated juveniles

were observed in 8 of the 17 trials, and in vitro juveniles were more sensitive in 7 of the 8 significant differences. Six of the 8 statistically different tests for a given chemical were within the intra-laboratory variation demonstrated in a recently published evaluation of mussel toxicity tests (a factor of 2). All of these statistical differences were within the variation for inter-laboratory comparisons for a given chemical in the same study. Moreover, a Kruskal-Wallis Analysis of Variance revealed a significant effect of either propagation or the interaction of propagation and concentration in 7 tests. These findings indicate that in vitro propagated juvenile mussels may be appropriate for use in ASTM-based toxicity testing with reasonable precision.

Introduction

Freshwater mussels (family Unionidae) play an important ecological role in aquatic systems, yet are a critically imperiled taxonomic group. As filter- and suspension-feeders, unionids remove significant amounts of particulate matter—such as bacteria, plankton, and suspended sediments—from the water column (Vaughn et al. 2004, 2008; Howard and

Cuffey 2006). Items not assimilated after filtration are deposited onto the sediment, providing a link between surface water and sediment (Howard and Cuffey 2006). Despite their critical function, freshwater mussels are experiencing concerning rates of decline. In 1993, Williams et al. estimated that approximately 70% of the nearly 300 species in North America were declining in at least some part of their range. A number of factors have contributed to this decline, including habitat alteration, stress from introduced species, and chemical pollution of waterways (Bogan 1993, Strayer et al. 2004, Augspurger et al. 2007).

Freshwater mussels are sensitive to chemical contaminants and can be used as sentinels for aquatic systems. Unionid mussels live at the sediment-water interface, so they are susceptible to multiple routes of toxicant exposure, in particular, surface water, sediment, and pore water (Cope et al. 2008). The concept of using mussels as bioindicators originated in the early 20th century. In 1909, Ortmann observed that mussels were the first faunal group to die off in streams affected by coal mining and would not persist when reintroduced to an affected area. However, freshwater mussels were not included in criteria developed using the first United States Environmental Protection Agency (US EPA) water quality derivation guidelines because little toxicity information was available (Stephan et al. 1985; Keller et al.

2006). In recent decades, toxicity testing with freshwater mussels has become more common

(e.g., Bringolf et al 2007; Wang et al. 2007a), due in large part to the establishment of accepted testing guidelines for mussel early life stages (ASTM International 2006). From this proliferation of testing, it is known that mussels are sensitive to many chemicals, and have been found to be more acutely sensitive than many traditionally tested laboratory species

(e.g., daphnids, fathead minnows, rainbow trout) to certain chemical contaminants, including copper and ammonia (Augspurger et al. 2003; March et al. 2007; Wang et al. 2017). The U.S.

EPA currently considers unionid sensitivity data from tests meeting the ASTM International data quality objectives when setting national water quality criteria (U.S. EPA 2013).

Water quality criteria are established to protect the entire life history of a species. For many species of freshwater mussels, the larval (glochidia) and early juvenile life stages are typically the most sensitive, though can vary by species and contaminant (Dimock and

Wright 1993; Wang et al. 2007a). The most effective way to determine protective chemical

thresholds is to test the chemical sensitivity at the most sensitive life stages. These tolerance thresholds are most often determined through toxicity testing with standard methods (e.g.,

ASTM International 2006) in a controlled laboratory setting, thus, there is need for a consistent and high-quality supply of young juvenile mussels for testing.

Unionid mussels have a unique life history. For most mussel species, the glochidia are obligate fish parasites (Lefevre and Curtis 1912; Kat 1983; Fritts et al. 2013). The male mussel releases sperm conglutinates, which are then filtered from the water by the female.

The female then broods the fertilized glochidia in a modified gill structure (known as the marsupium) until they are mature. To ensure optimal transfer of mature glochidia onto a host fish, female mussels use an array of infection strategies to attract the proper host and maximize reproductive success (Barnhart et al. 2008). The host fish supplies the nutritional needs of the glochidia until transformation into the juvenile stage is complete, usually 14 to

21 days, depending on temperature and other environmental conditions (Barnhart et al. 2008;

Fritts et al. 2013). The relatively small size of these juveniles (<500 µm) and the quantity required for testing (approximately 200 per each acute toxicity test) necessitates that they are produced in the laboratory with captive propagation techniques to provide mussels for use in toxicity testing.

Traditional captive propagation methods involve extracting the glochidia from a female mussel and artificially infecting a host fish (Barnhart et al 2008). The fish are held in aquaculture-type conditions until the glochidia complete the transformation into juveniles. In addition to providing a means to study mussel-host transformation efficiency and other

interactions (Fritts et al. 2013; Douda 2015), this method typically produces abundant, high- quality juveniles, an important prerequisite for use in toxicity testing. However, for this method to be most efficient, the host fish must be known. Furthermore, this is a labor- and cost-intensive process requiring daily care for the host fish and monitoring and removal of transformed juveniles. There are also possibilities of brood failure, disease, parasite infestation, or other events that can result in poor juvenile transformation (Lima et al. 2012).

An alternative method of mussel propagation has been refined in recent decades that mitigates some of the problems associated with traditional host fish (in vivo) mussel propagation. This is an in vitro culture method, in which glochidia are placed into a culture media consisting of animal serum (usually rabbit or horse). The glochidia complete the transformation process in the culture media, where they derive protein and other nutrients rather than from the host fish tissues (Isom and Hudson 1982; Dimock and Wright 1993;

Lima et al. 2012). This technique was developed in 1926, when Ellis and Ellis first described the transformation of glochidia in a culture medium. However, this technique remained largely unknown and unused until the 1980s, when Hudson and Isom (1982) successfully transformed several mussel species in a culture medium. Since the resurrection of the method, techniques have greatly improved (Owen et al. 2010) and over 40 species have been successfully transformed in culture media (Lima et al. 2012).

In vitro mussel propagation holds several advantages over traditional host-fish methods. Namely, glochidia-to-juvenile transformation rates are greater with in vitro than in vivo propagation, typically above 90%, and the removal of the host fish eliminates much of

the unpredictability associated with mussel culture (Lima et al. 2012). The in vitro technique is also a more economical method, both in terms of financial cost and efficient use of glochidia.

Although there are many benefits to in vitro culture, there are also several disadvantages. For example, specialized cell culture equipment and supplies are needed, and there is a risk of fungal or bacterial outbreaks in the culture media (Lima et al. 2012).

Additionally, there are few data available on the relative health and chemical sensitivity of in vitro juveniles compared to in vivo juveniles (ASTM International 2006, 2013). In vitro juveniles tend develop anatomically and physiologically at slower rates than in vivo juveniles of the same cohort and exhibit greater mortality post-transformation (Fisher and Dimock

2006; Fox 2014). However, Summers (1999) did not observe statistically significant differences in sensitivity to copper between in vitro and in in vivo propagated juveniles, although the in vitro juveniles generally died at lower concentrations of copper than their in vivo counterparts. Likewise, a review of copper toxicity studies on mussels by March et al.

(2007) did not observe a consistent relationship in chemical sensitivity between in vitro and in vivo juveniles. The first ASTM International (2006) guidelines for conducting toxicity tests with juvenile mussels did not recommend using in vitro propagated juveniles until their relative chemical sensitivities were described, and no comprehensive data have been generated to advise more recent revisions (ASTM International 2013). The aim of this research was to provide a comparison of the relative chemical sensitivities of in vitro and in vivo cultured juvenile mussels through a series of standard acute toxicity tests with compounds representing different chemical classes and modes of action.

Methods

Test organisms

Three species of mussels were selected for testing in this study based on availability, native range, and conservation status. Lampsilis cardium, the Plain Pocketbook (Rafinesque,

1820), is a relatively common species from the subfamily Ambleminae, tribe Lampsilini, native to the Interior and Great Lakes drainage basins (Parmalee and Bogan 1998). Lampsilis abrupta, the Pink Mucket (Say, 1831), co-occurs with L. cardium in the Interior basin, but is listed as federally endangered. Utterbackia imbecillis, the Paper Pondshell (Say, 1829), is in the subfamily Unioninae, tribe Anodontini. It is relatively common throughout the Interior and Atlantic slope drainages and is often used in toxicity testing (Parmalee and Bogan 1998;

Summers 1999).

Propagation

All juvenile mussels were propagated at the Kentucky Center for Mollusk

Conservation (Kentucky Department of Fish and Wildlife Resources, Frankfort, Kentucky) using traditional host-fish (in vivo) and media culture (in vitro) propagation methods (Coker et al. 1921; Isom and Hudson 1982; Hudson and Isom 1984; Owen et al. 2010; Fritts et al.

2013). For each species, gravid female mussels were collected from wild sustaining populations and held in hatchery conditions to serve as broodstock. Lampsilis abrupta were collected in the lower Tennessee River (Tennessee drainage), L. cardium were collected from the lower Licking River (Ohio River drainage), and U. imbecillis were collected from small streams in the Cedar Creek watershed (Kentucky River drainage). Glochidia from three

females were used for each test batch. Half the glochidia were extracted from each female, mixed with glochidia from the other females, and used to inoculate the appropriate fish for the given species. The other half were extracted, mixed, and placed in a culture media mixture of rabbit serum, antibiotics, amino acids, and lipids (Owen et al. 2010). After transformation, mussels were held at the Kentucky Center for Mollusk Conservation under their standard rearing protocols. At the time of testing, juvenile mussels were shipped by overnight courier in successive batches, one per week, to the Aquatic Toxicology Laboratory at NC State University (Raleigh, North Carolina). During shipping, approximately 150-250 mussels of a given species were placed into a 50 mL centrifuge tube, which was then placed into a 1.2 L Thermos® containing water from the culture facility to provide a thermal buffer.

The Thermos was then placed into a Styrofoam® cooler with warm or cool packs, depending on the ambient temperature of the shipping season.

Juvenile assessment and acclimation

Upon arrival at the laboratory, juvenile mussels were assessed for initial viability by looking for foot movement (ASTM 2006). Initial arrival water temperature was measured, and mussels were acclimated to test temperature (20°C) at the rate of 2.5°C per day.

Additionally, mussels were acclimated to ASTM International hard water, used in testing

(ASTM 2007) at a rate of 25% volume exchange per hour. After acclimation to 100% test water and target temperature, mussels were held for a minimum of 24 h to ensure complete acclimation of test organisms. Prior to test initiation, viability was assessed again (considered viable upon detection of foot movement inside or outside of the shell or the presence of a

heart beat) and when overall viability was >90%, the mussels were allocated to test chambers. Tests were 96 hour non-aerated static tests with 100% water renewal at 48 hours, conducted according to ASTM International (2006) guidelines for juvenile mussel testing.

Viability was assessed at 48 and 96 hours.

Photographs were taken for a subset of mussels at the time of arrival and the individual mussels were measured to the nearest micrometer using a Leica EZ4 D stereo microscope with integral digital camera and Leica Application Suite EZ digital photographic software (Leica Microsystems, Ltd., Switzerland). Individuals used in toxicity tests ranged in age from 0 to 8 weeks post-transformation. Average shell lengths were 853 µm (+/- 430 µm) and 464 µm (+/- 368 µm) for in vivo and in vitro cultured Lampsilis abrupta, respectively;

973 µm (+/- 476 µm) and 860 µm (+/- 364 µm) for L. cardium; and 913 µm (+/- 467 µm) and 653 µm (+/- 264 µm) for Utterbackia imbecillis.

Chemical sensitivity

To determine the relative chemical sensitivity of in vitro and in vivo propagated mussels, juveniles from each propagation method were exposed to a range of chemical concentrations for a suite of six toxicants. Each test had a control (no toxicant) and six treatment concentrations, each conducted in triplicate with ten mussels per replicate in 250 mL evaporating dishes (Table 1). Mussels were assessed for survival at 48 and 96 hours, and the median effective concentration (EC50) causing immobility or mortality, and effective concentration for 5% of the population (EC05) were calculated for each species-toxicant combination.

Six chemicals with three modes of toxic action (MOA) were selected based on known toxicity to mollusks and environmental relevance. Chloride (as sodium chloride: Sigma-

Aldrich, St. Louis, MO, 99% purity) is a well-described mussel toxicant (MOA: respiratory stress) (Wang et al. 2007a). Copper (as copper sulfate: Sigma-Aldrich, St. Louis, MO, 99% purity) is another well-known mussel toxicant (MOA: metallic stress) (Wang et al. 2007a).

Clearigate® (Applied Biochemists, Germantown, WI, 3.82% elemental copper) and Nautique

® (SePRO Corporation, Carmel, IN, 9.1% metallic copper), both chelated copper aquatic herbicides, were also chosen. Nickel (as nickel chloride: Sigma-Aldrich, St. Louis, MO, 99% purity, MOA: metallic stress) and ammonia (as ammonium chloride: Fisher Scientific,

Waltham, MA, 99% purity, MOA: narcosis) are also well-described mussel toxicants selected for use in the comparison tests (Wang et al. 2007a). Toxicity test concentrations were based on published EC50 or median lethal concentration (LC50) values (Wang et al.

2007a) or by pilot range-finding tests.

Quality assurance

All tests were conducted according to the ASTM International Standard Guide for

Conducting Laboratory Toxicity Tests with Freshwater Mussels (2006). All tests, except for three, met the recommended >90% control survival at test termination (ASTM 2006); three tests that had between 80-90% control viability (L. cardium in vivo chloride, L. cardium in vitro nickel, and U. imbecillis ammonia) were still included in analysis. Water quality conditions of temperature, conductivity, dissolved oxygen, and pH were measured with a calibrated meter (YSI model 566 MPS multi-probe, Yellow Springs Instrument Co., Yellow

Springs, Ohio) from a composite water sample at each treatment concentration (Appendix

A). Alkalinity and hardness were measured using standard titrimetric procedures (APHA

2005).

Toxicant exposure concentrations were verified with standard analytical chemistry methods; RTI International (Research Triangle Park, NC) for copper and nickel; NC State

University Center for Applied Aquatic Ecology (Raleigh, NC) for ammonia; and SePRO

Corporation (Whitakers, NC) for Nautique, Clearigate, and additional copper samples

(Appendix B). Ammonia samples were verified at 0, 24, and 48 hours of the tests to ensure concentrations remained stable over time. In tests containing a copper-based toxicant, all glassware was pre-treated with the appropriate test concentration 24 hours prior to test initiation to minimize adsorptive loss of copper ions to the glass. Chloride concentrations were verified in the laboratory using a YSI conductivity probe. Chloride concentration was expressed as the volume of chloride ions (g/L) in addition to the salts required to make the

ASTM International hard water, which reflects a background conductivity of ~550 µS/cm.

Statistical analysis

The median effective concentration, i.e., the concentration of the toxicant producing an immobilizing or lethal effect to 50% of the test population (EC50) and the effective concentration to 5% of the population (EC05), were estimated with the Comprehensive

Environmental Toxicity Information Software package (CETIS) (v1.8.0.12, Tidepool

Scientific, LLC, McKinleyville, California). Confidence intervals (95%) were used to determine significant differences between EC05s and EC50s. ECs with overlapping

confidence intervals were considered similar, whereas ECs with non-overlapping confidence intervals were considered statistically different (Archambault et al. 2013).

Survival data from each species-chemical combination were analyzed separately in

JMP (SAS Institute Inc., 2015), with a Kruskal-Wallis 2-way Analysis of Variance

(ANOVA) on ranks. This included the main effects of concentration, propagation method, and their interaction. Significance level was set to α = 0.05.

An inter-species in vitro:in vivo ratio was determined as in Raimondo et al. (2016).

First, the ratio of the in vitro to in vivo EC50s for each species-toxicant combination was calculated (i.e., dividing one EC50 into the other). Ratios for each species were then averaged within a chemical to calculate the inter-species variation.

Results

Juvenile mussel acute toxicity tests

Tests were conducted on three mussel species with each propagation method (in vitro and in vivo) with 6 chemicals, with the exception of U. imbecillis and Clearigate due to limited availability of mussels. In total, 17 pairs of tests were conducted. Additionally, in vitro and in vivo tests (three replicates of 10 mussels) for L. abrupta were not able to be conducted concurrently due to constraints with mussel production, thus the in vitro and in vivo juveniles for this species were on average, one week different in age at the time of testing. To assess this potential age influence and overall test accuracy with this species, an in vivo test with one replicate of 10 mussels (“Test A” or “L. abrupta-A”) was conducted

alongside each in vitro test. The in vivo L. abrupta test with three replicates of ten mussels is referred to as “Test B” or L. abrupta-B.

Non-overlapping 95% confidence intervals (a statistically significant difference) for the 96 hour EC50 between in vitro and in vivo propagated juveniles were observed in 8 of the

17 trials (Table 2). EC50s for ammonia ranged from 3.3-10.1 mg TAN/L, and the L. cardium test produced significant differences between in vitro and in vivo juveniles, as did the L. abrupta Test A in vivo and in vitro test. Chloride EC50s ranged from 1.7-3.0 g Cl/L. EC50s for Clearigate ranged from 176-508 µg Cu/L. For both chloride and Clearigate, the difference between Test A and Test B for in vivo propagated juveniles was significant, as was the difference between Test B and in vitro propagated juveniles. The difference between in vitro and in vivo L. cardium for chloride and Clearigate was also significant. The EC50s for copper sulfate ranged from 53.7-178.1 µg Cu/L, and in vitro and in vivo juveniles were significantly different for all species. Nautique EC50s ranged from 720.1-5,656.9 µg Cu/L. While there was no difference between the in vitro juveniles and both Test A and Test B in vivo tests for

L. abrupta, Test A and Test B were significantly different from each other. EC50s for nickel ranged from 701.0 to greater than 1,500 µg/L Ni. There was no difference between in vitro and in vivo juveniles in any Ni test, except for L. abrupta in vivo Test A, which could not be calculated because the EC50 exceeded the test concentration range. Of the nine pairs of

EC05s calculated, four produced significant differences between in vitro and in vivo propagated juveniles (Appendix C). EC05s could not be calculated for the remainder of the tests due to limited amount of partial mussel mortality in treatment replicates below the EC50

and will not be discussed further in this document. Raw survival data for all tests is available in Appendix D.

Effect of propagation method on survival

The Kruskal-Wallis two-way ANOVA revealed a significant effect of concentration on survival in all tests, a significant effect of propagation in 5 tests (29%), and a significant interaction between propagation and concentration in 6 tests (35%) (Table 3). Differences in the main effect of propagation were in the L. abrupta copper test; the L. cardium ammonia,

Clearigate, and copper tests; and the U. imbecillis Nautique test. Differences in the interaction effect were in the L. abrupta copper test; the L. cardium ammonia, Clearigate, copper, and Nautique tests; and the U. imbecillis ammonia test.

Inter-species variation ratios

An interspecies ratio was calculated for each chemical. Interspecies ratios greater than

1 indicate in vivo mussels were less sensitive than in vitro mussels, ratios less than 1 indicate in vitro mussels were less sensitive, and ratios equal to 1 indicate equal sensitivity. The inter- species ratio of EC50 was 1.6 for ammonia, 1.1 for chloride, 1.4 for Clearigate, 2.1 for copper sulfate, 1.2 for Nautique, and 1.0 for nickel (Table 4). All values were greater than 1, indicating that in vitro mussels were generally more sensitive than in vivo mussels.

Discussion

In vitro propagated juveniles were statistically more sensitive about half the time (8 of 17 trials), and overall, generally more sensitive than their in vivo counterparts to chemical

toxicants. Thus, if precise toxicity values for a toxicant are required, it would be more appropriate to use in vivo propagated juveniles rather than in vitro propagated individuals.

However, although these findings indicate a difference in chemical sensitivity between in vitro and in vivo propagated mussels, the variation observed was not greater than that in the published literature with juvenile unionids, where age, mussel species, toxicant or formulation, and test conditions all contribute to variation (Wang et al 2007b, 2017;

Raimondo et al. 2016). Several recent studies have examined the variation in in vivo juvenile mussel toxicity within and among laboratories across multiple mussel species and chemicals.

For example, Raimondo et al. (2016) performed a meta-analysis of available mussel toxicity data and calculated the average inter-species ratio of within and among laboratory variation for a number of chemicals. The overall variation ratio, among all chemicals, was 1.9 within labs and 3.6 among labs. Wang et al. (2007b) performed a collaborative series of chemical tests among five independent laboratories, each using mussels from the same propagation batch and same test water for each test. They found a within lab variation ratio of 1.2, and an among lab variation ratio of 1.5. In a recent study comparing the toxicity of newly transformed juveniles to a number of chemicals, including ammonia, chloride, copper, and nickel; Wang et al. (2017) reported a factor of <= 2 for chloride and <= 5 for ammonia, copper, and nickel. The toxicity results for mussels from the two propagation types and test chemicals in this study were within the 3.6 among laboratory ratio found in Raimondo et al.

(2016), and all of the chemical-specific EC50 ratios are comparable to both Raimondo et al.

(2016) and Wang et al. (2007b) ratios (Table 4).

The main effect of concentration was significant in all tests (Table 3). This is not particularly informative, as concentration ranged from low to high, with the intention of generating a gradient of mortality response. The interaction between propagation method and chemical was significant in 6 of 17 (35%) of cases. This means that survival at each concentration was influenced by propagation in those tests where the interaction was significant (and vice versa). These differences occurred in all species, but mostly occurred in test with copper-based compounds (4 of 6 tests), indicating that there may be a mechanistic component to in vitro vs. in vivo chemical sensitivity. In 4 of the 6 tests where the interaction of propagation method and concentration was significant, the main effect of propagation was also significant. The one test where only the main effect of propagation method was significant was U. imbecillis with Nautique.

Unionid chemical sensitivity can vary with age (Klaine et al. 1997; Wang et al.

2007a). If in vitro mussels are developmentally delayed relative to in vivo mussels (Dimock and Wright 1993; Fox 2014), their relative chemical sensitivities may depend on the time since their transformation or the developmental stage. Summers (1999) did not find statistically significant differences between in vitro and in vivo produced juveniles at 2-3 days post-transformation, but the in vitro juveniles in that study were generally more sensitive to copper. This study did not directly examine the effect of age on chemical sensitivity, but tests with younger L. cardium juveniles resulted in higher EC50 ratios than tests with older juveniles. The possible effect of age or developmental stage on relative toxicity warrants future examination.

Although this study prudently used mussel species from different subfamilies, tribes, and conservation statuses in testing, only three species were assessed, and future research would benefit from testing mussels of the two propagation methods with additional genera.

However, approximately 70% of mussel species that have been used in toxicity testing vary from two commonly used surrogate species (Lampsilis siliquoidea and Utterbackia imbecillis) by a factor of ≤ 2 (Raimondo et al. 2016). Additionally, as both types of propagation and culture methods continue to improve, any potential developmental gaps between in vitro and in vivo juvenile mussels should continue to be reduced.

Overall, EC50s from both in vitro and in vivo mussels in this study are comparable to those published in the literature. Median effective concentrations for ammonia in this study ranged from 6.2 to 10.1 mg TAN/L for in vivo juveniles, and from 3.3 to 7.9 mg TAN/L for in vitro juveniles. Both are comparable to the results found in Wang et al. (2007), which ranged from 2.3 to 11.1 mg TAN/L. Chloride values from this study (in vivo: 1.7-2.9 g Cl/L; in vitro: 1.7-2.95) were also comparable to those in the literature (e.g., 1.71-5.23 g Cl/L from

Raimondo et al. 2016, supplemental data). Copper values determined in this study

(normalized to total water hardness of 100 ppm); in vivo: 116.9-178.1 µg Cu/L; in vitro 53.7-

84.9 µg Cu/L) were greater than those in the literature (e.g., 6.8-60.0 µg Cu/L from Wang et al. 2007a). Copper sulfate was also the only toxicant that produced significant differences between in vitro and in vivo mussel EC50s among all species. However, Raimondo et al.

(2016) found a between-lab variation ratio of 4.8 for copper, so the apparent variation in the results of this study is not beyond typical variation in toxicity tests. Nickel values in the literature were less (e.g., 96-377 µg Ni/L from Raimondo et al. 20l6, supplemental data) than

those generated in this study (when normalized to a hardness of 50 mg/L; in vivo: 262- >560

µg Ni/L; in vivo: 271-470 µg Ni/L).

Both in vitro and in vivo juveniles from all species were more sensitive to copper sulfate than to Clearigate or Nautique. Juvenile L. abrupta and L. cardium were more sensitive to Clearigate than to Nautique. Clearigate is a chelated copper aquatic herbicide with a surfactant, whereas Nautique is an aquatic herbicide with two forms of chelated copper without a surfactant. The lowest recommended application rate for both herbicides

(Clearigate: 100 µg Cu/L [0.9 gal Clearigate/acre*foot], Nautique: 500 µg Cu/L [1.5 gal

Nautique/ acre*foot]) are below the EC50s found in this study. However, an EC50 from an acute, water-only test may not be the most valuable measure in assessing whether these application rates would be protective of juvenile mussels. For example, the Clearigate EC05s for L. abrupta and L. cardium (Appendix C) ranged from 82 to 312 µg Cu/L, encompassing the lowest recommended application rate. However, these water-only tests do not account for copper uptake and binding by plants and sediment, and likely represent the worst-case scenario (Mastin and Rodgers 2000). A mesocosm-type experiment incorporating potential copper binding may be more informative in a risk assessment for these herbicides.

Overall, there was minimal variation in the results of toxicity tests between juvenile mussels produced by in vitro or in vivo propagation methods, and that the variation observed was within that commonly encountered in data sets generated within and among laboratories

(Wang et al. 2007b; Raimondo et al. 2016). Therefore, the current ASTM International

(2013) guide for conducting toxicity tests with juvenile mussels may be revised in the future

to state that juvenile mussels cultured in vitro may be used to conduct toxicity tests, because this study demonstrates that the sensitivity of the juvenile mussels cultured in vitro is similar to the sensitivity of juvenile mussels cultured in vivo. Large numbers of mussels can be produced relatively inexpensively using in vitro propagation techniques. The use of in vitro propagated juvenile mussels in standard toxicity tests broadens opportunities for a multitude of private, state, and federal laboratories to generate data for mussel species that are of local conservation concern and require toxicity data. Despite the potential remaining uncertainties about delayed growth and development in the very early stages of in vitro juveniles compared to in vivo juveniles (Fisher and Dimock 2006; Fox 2014), it appears that in vitro propagated mussels can be used as reasonable surrogates for in vivo juveniles in standard toxicity testing.

Acknowledgements

Funding for this research was provided by the U.S. Geological Survey (USGS) and

U.S. Fish and Wildlife Service through the Science Support Partnership Program via

Research Work Order No. 211, administered through the USGS North Carolina Cooperative

Fish and Wildlife Research Unit. We thank Jennifer Archambault, Sean Buczek, Joseph

McIver, Mary Silliman, Mike Walter, and Emilee Wooster for laboratory assistance, and

West Bishop, Chris Ingersoll, and Ning Wang for providing chemical expertise. Special thanks to Dr. Monte McGregor and the rest of the group at the Kentucky Center for Mollusk

Conservation for producing the mussels for this experiment and for providing insight at various stages through the project.

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Tables Table 1: Toxicant and associated treatment concentrations used in acute toxicity tests with in vitro and in vivo propagated juvenile mussels for the three species tested.

Mussel Species Toxicant Lampsilis abrupta Lampsilis cardium Utterbackia imbecillis Ammonia (NH Cl), 4 0, 1, 2 ,4, 8, 16, 32 0, 1, 2 ,4, 8, 16, 32 0, 1, 2 ,4, 8, 16, 32 mg TAN/L Clearigate ®, 0, 50, 100, 250, 500, 750, 0, 25, 50, 100, 250, 500, 750 -- µg Cu/L 1000, 1500 Chloride (NaCl), 0, 0.3, 0.6, 1.2, 2.4, 3.6, 4.8 0, 0.3, 0.6, 1.2, 2.4, 3.6, 4.8 0, 0.3, 0.6, 1.2, 2.4, 3.6, 4.8 g Cl/L Copper (CuSO ), 0, 12.5, 25, 50, 100, 200, 4 0, 25, 50, 100, 200, 300, 400 0, 12.5, 25, 50, 100, 200, 300 µg Cu/L 300 Nautique ®, 0, 250, 500, 1000, 2000, 0, 250, 500, 1000, 2000, 0, 12.5, 25, 50, 100, 200, 300 µg Cu/L 4000, 8000 4000, 8000 Nickel (NiCl), 0, 100, 250, 500, 750, 1000, 0, 100, 250, 500, 750, 1000, 0, 100, 250, 500, 750, 1000, 1500 µg Ni/L 1500 1500

Table 2: Median effective concentration (EC50) causing 50% immobilization or mortality (with 95% confidence intervals) in in vitro and in vivo propagated juvenile mussels at 48 and 96 hours. In vitro and in vivo EC50 pairs in bold type are significantly different at 96 h. L. abrupta–A= Test with one replicate of ten mussels. L. abrupta–B= Test with three replicates of 10 mussels. N/A= not able to calculate EC50 due to insufficient mortality. * = No test run for Utterbackia imbecillis with Clearigate®.

48h EC50 96h EC50 Species Toxicant Units In vivo In vitro In vivo In vitro L. abrupta-A Ammonia mg NH4/L 22.6 (16.0-32.0) 10.6 (7.7-14.7) 7.5 (5.8-9.8) 5.7 (4.6-6.9) L. abrupta-B Ammonia mg NH4/L 17.5 (15.4-20.0) 6.2 (4.4-7.1) L. cardium Ammonia mg NH4/L 16.2 (13.9-18.8) 7.1 (6.3-8.1) 7.7 (6.8-8.8) 3.3 (3.0-3.7) U. imbecillis Ammonia mg NH4/L 26.9 (22.8-31.7) >32 (N/A) 10.1 (9.0-11.3) 7.9 (6.5-9.7)

L. abrupta-A Chloride g NaCl/L 4.2 (3.6-4.9) 3.3 (3.1-3.6) 2.9 (2.8-3.0) 3.0 (2.9-3.1) L. abrupta-B Chloride g NaCl/L 2.7 (2.5-2.9) 1.9 (1.7-2.1) L. cardium Chloride g NaCl/L 3.1 (2.5-3.0) 2.7 (2.5-3.0) 2.7 (2.5-3.0) 1.8 (1.6-1.9) U. imbecillis Chloride g NaCl/L 1.9 (1.6-2.0) 2.3 (2.1-2.5) 1.7 (1.6-1.8) 1.7 (1.6-1.8)

L. abrupta-A Clearigate® µg Cu/L >1,000 (N/A) 800.3 (693.9-922.9) 508.9 (405.6-638.6) 480.1 (364.7-632.1) L. abrupta-B Clearigate µg Cu/L 296.3 (249.4-351.6) 176.0 (149.4-207.4) L. cardium Clearigate µg Cu/L 722.9 (601.0-868.0) 487.9 (423.2-562.5) 480.1 (400.7-575.3) 230.2 (189.5-279.7) U. imbecillis Clearigate µg Cu/L * * * *

L. abrupta-A Copper µg Cu/L 251.5 (191.-330.0) 128.1 (106.8-153.9) 178.1 (122.0-260.2) 53.7 (42.0-68.7) L. abrupta-B Copper µg Cu/L >300 (N/A) 130.0 (110.8-152.6) L. cardium Copper µg Cu/L 232.7 (207.7-261.0) 129.3 (109.8-152.4) 160.8 (147.7-175.0) 84.9 (70.3-102.5) U. imbecillis Copper µg Cu/L 669.4 (451.2-993.3) 691.5 (464.1-1030.2) 116.9 (96.2-142.1) 76.9 (63.5-93.2)

L. abrupta-A Nautique® µg Cu/L >8,000 (N/A) 5901.5 (5449.78-6390.5) 4924.6 (4132.5-5868.5) 3968.5 (3374.5-4667.0) L. abrupta-B Nautique µg Cu/L 5656.9 (4000-8000) 3406.3 (2954.3-3927.5) L. cardium Nautique µg Cu/L >8,000 (N/A) >8,000 (N/A) 5656.9 (4000-8000) 5157.5 (4732.3-5620.9) U. imbecillis Nautique µg Cu/L >1,000 (N/A) >1,000 (N/A) >1,000 (N/A) 720.1 (339.2-1528.6)

L. abrupta-A Nickel µg Ni/L >1,500 (N/A) 1383.7 (1226.7-1560.7) >1,500 (N/A) 1160.3 (1095.2-1229.3) L. abrupta-B Nickel µg Ni/L 1375.7 (1253.0-1510.0) 1228.2 (1182.3-1275.8) L. cardium Nickel µg Ni/L 1246.9 (1136.1-1368.7) 1264.6 (1183.46-1351.3) 701.0 (629.1-781.2) 725.7 (668.1-788.4) U. imbecillis Nickel µg Ni/L >1,500 (N/A) >1,500 (N/A) 1291.7 (1214.7-1373.5) 1257.6 (1201.3-1316.4)

Table 3. Results from the Kruskal-Wallis two-way Analysis of Variance, with propagation (prop), concentration (conc) and their interaction as the main effects. Concentration always has a significant effect on survival, as concentrations were selected to produce a range of low to high mortality.

Species Chemical Effect d. f. F-stat p-value L. abrupta Chloride Prop 1 1.11 0.298 Conc 6 37.71 <0.0001 Prop*Conc 6 1.44 0.218 L. cardium Chloride Prop 1 0.0008 0.9782 Conc 6 24.98 <0.0001 Prop*Conc 6 1.06 0.4098 U. imbecillis Chloride Prop 1 0 1 Conc 6 111.33 <0.001 Prop*Conc 6 0.583 0.7405 L. abrupta Ammonia Prop 1 0.308 0.5823 Conc 6 90.804 <0.0001 Prop*Conc 6 2.331 0.0535 L. cardium Ammonia Prop 1 47.08 <0.0001 Conc 6 138.43 <0.0001 Prop*Conc 6 11.133 <0.0001 U. imbecillis Ammonia Prop 1 0.0037 0.9517 Conc 6 30.136 <0.0001 Prop*Conc 6 3.623 0.0087 L. abrupta Clearigate Prop 1 -- -- Conc 7 79.536 <0.0001 Prop*Conc 7 0.887 0.457 L. cardium Clearigate Prop 1 30.94 <0.0001 Conc 6 48.98 <0.0001 Prop*Conc 6 2.94 0.0235 L. abrupta Copper Prop 0 71.022 <0.0001 Conc 3 129.3 <0.0001 Prop*Conc 3 8.835 <0.0001 L. cardium Copper Prop 1 60.12 <0.0001 Conc 6 221.67 <0.0001 Prop*Conc 6 13.112 <0.0001 U. imbecillis Copper Prop 1 0.1 0.753 Conc 6 29.076 <0.0001 Prop*Conc 6 1.47 0.225 L. abrupta Nickel Prop 1 2.67 0.1112 Conc 6 10.922 <0.0001 Prop*Conc 6 0.802 0.575 L. cardium Nickel Prop 1 0.0594 0.8092 Conc 6 32.405 <0.0001 Prop*Conc 6 1.0842 0.396 U. imbecillis Nickel Prop 1 0.003 0.9565 Conc 6 13.022 <0.0001 Prop*Conc 6 0.518 0.79 L. abrupta Nautique Prop 1 0.0353 0.852 Conc 6 36.133 <0.0001 Prop*Conc 6 0.585 0.74 L. cardium Nautique Prop 1 3.99 0.055 Conc 6 52.767 <0.0001 Prop*Conc 6 3.991 0.0052 U. imbecillis Nautique Prop 1 5.8 0.0229 Conc 7 8.143 <0.0001 Prop*Conc 7 2.0131 0.0863

Table 4: Inter-species ratio of variance between EC50s of in vitro and in vivo propagated juvenile mussels among three species for each chemical tested for this study. Ratios greater than 1 indicate in vivo mussels were less sensitive than in vitro mussels, ratios less than 1 indicate in vitro mussels were less sensitive. Ratios from this study are compared with inter-species ratios calculated in Wang et al. (2007) and Raimondo et al. (2016) for within- and between-lab acute EC50 variation of tests with in vivo propagated juveniles. ND= no ratio available.

Mean Toxicity Value Ratio

Within Lab Variation Between Lab Variation In vivo vs Wang et al. Raimondo et al. Wang et al. Raimondo et al. Toxicant In vitro 2007 2016 2007 2016 Ammonia 1.6 ND 2.0 ND 4.8 Chloride 1.1 ND 1.5 ND 1.8 Clearigate 1.4 1.2 2.1 1.5 4.8 Copper 2.1 1.2 2.1 1.5 4.8 Nautique 1.2 1.2 2.1 1.5 4.8 Nickel 1.0 ND 2.0 ND 1.6

1 A 1 B 0.8 0.8 0.6 0.6

0.4 0.4

0.2 0.2

0 0

0 100 200 300 400 0 100 200 300 400 Survival(proportion) Concentration (µg/L Cu) Survival(proportion) Concentration (µg/L Cu)

In vivo In vitro In vivo EC50 In vitro EC50

Figure 1: Representative survival responses of in vitro and in vivo juveniles. Panel A: Lampsilis cardium to copper, where median effective concentrations (EC50s) between in vivo and in vitro juveniles were significantly different. Panel B: Utterbackia imbecillis to copper, where EC50s between in vivo and in vitro juveniles were significantly different.

Chapter 2 Influence of Age on Juvenile Freshwater Mussel Chemical Sensitivity: A Comparison of Progeny from Two Propagation Methods

Abstract Freshwater mussels (Unionidae) are a rapidly declining faunal group. Freshwater mussels are sensitive to chemical toxicants, and much of their decline can be contributed to chemical contamination of aquatic habitats. Toxicity threshold data for freshwater mussels are increasingly being used to calculate and establish water quality criteria, most often from tests using newly transformed juveniles, which, for most contaminants, is the most sensitive life stage. Unionid mussels are parasitic on host fish as larvae (glochidia) until they transform into juveniles and fall to the sediment, where they begin their life as benthic adults.

Traditional host-fish propagation (in vivo propagation) involves artificially infecting host fish and monitoring larval transformation. Although in vivo propagation techniques have greatly improved in recent years, challenges and uncertainties associated with host fish health and successful transformation of glochidia remain. In vitro propagation bypasses the need for a host fish by using a culture medium; however, the relative fitness and chemical sensitivity of in vitro juveniles compared to in vivo juveniles is not known. Because these potential differences have not been described, the ASTM International guideline for toxicity testing with juvenile mussels does not recommend using in vitro propagated juveniles until their relationship to their in vivo counterparts is defined. Additionally, chemical sensitivity of juvenile mussels can decrease with age, so potential differences between in vitro and in vivo juveniles may become more convergent as the mussels age, or decreased fitness of in vitro juveniles could increase differences with age. The aim of this study was to examine the relative chemical sensitivities between in vitro and in vivo propagated juveniles along a

developmental timeline. Acute toxicity tests (96 h) with copper sulfate were conducted for

Lampsilis abrupta, L. cardium, and Utterbackia imbecillis juveniles from each propagation method. The median effective concentration (EC50) for each test was calculated and the

EC50 for in vitro and in vivo juveniles were compared at each age tested. Of the 8 EC50 comparisons, 4 statistically significant differences between in vitro and in vivo juveniles were observed. However, these differences were evenly distributed across all ages, and further analysis revealed no significant relationship between age and relative chemical sensitivity.

Additionally, no significant differences were found in protein and ATP concentrations between in vitro and in vivo juveniles along the developmental gradient, indicating a similar physiological condition. These findings demonstrate that in vitro juveniles may be appropriate for use in toxicity testing regardless of age.

Introduction

Freshwater mussels (Family Unionidae) can be useful indicators of ecosystem heath and are often one of the most sensitive faunal groups to chemical contaminants in aquatic systems (Augspurger et al. 2003; March et al. 2007). Toxicity data from freshwater mussels can be used to set water quality criteria (U.S. EPA 2013). For many toxicants, the newly transformed juvenile is the life stage most sensitive to chemical contaminants, and is thus used in laboratory toxicity testing (Wang et al. 2007a). Because unionid mussels have an obligate parasitic life stage on fish (Lefevre and Curtis 1912; Kat 1983; Fritts et al. 2013), rearing mussels in a laboratory requires maintaining appropriate host fish for the duration of the transformation from larvae (glochidia) to juvenile. Rearing mussels using a host fish (in

vivo propagation) presents several challenges, including limited transformation success, care for and uncertainty associated with the health and condition of host fish, and effectively monitoring the drop-off of juveniles (Lima et al. 2012).

The in vitro propagation method bypasses some of the challenges associated with traditional mussel propagation. This method was first developed in the early 20th century, and has been greatly improved in recent decades (Ellis and Ellis 1926; Hudson and Isom 1982;

Isom and Hudson 1982; Owen et al. 2010; Lima et al. 2012). In vitro propagation involves placing glochidia in a culture medium composed of animal serum, amino acids, lipids, and antibiotics until the transformation process is complete (Hudson and Isom 1982; Owen et al.

2010). The in vitro method removes uncertainty associated with the host fish, results in greater larval to juvenile transformation success, and is more cost-effective than traditional, host-fish propagation. However, in vitro propagation requires specialized cell culture equipment, and there is risk of fungal or bacterial outbreaks in the culture media (Owen et al.

2010; Lima et al. 2012). Additionally, the relative health of in vitro juveniles is not well described compared to their in vivo counterparts, particularly with regards to chemical sensitivity (Fisher and Dimock 2006).

The relative chemical sensitivity of juvenile mussels from the two propagation types may depend upon the time since transformation. Younger juveniles are generally more sensitive to toxicants than older juveniles, though this can vary by chemical (Klaine et al.

1997; Wang et al. 2007a). In vitro juveniles have higher initial mortality rates and develop more slowly than in vivo juveniles of the same age, though mortality and growth rates

become comparable after 30 days post-transformation (Fisher and Dimock 2006; Fox 2014).

In previous toxicity tests with copper sulfate, in vitro juveniles were generally more sensitive than in vivo juveniles (Summers 1999). This finding, however, was not observed in more recent studies or reviews, where no clear relationship between in vitro and in vivo juveniles was observed (Valenti et al. 2006; March et al. 2007).

An understanding of the initial relative health of juveniles may provide insight into potential differences in chemical sensitivity of in vitro and in vivo propagated juvenile mussels. Protein and adenosine triphosphate (ATP) concentrations can be used as indicators of general health and condition by reflecting comparative energy available between in vivo and in vitro juveniles (Vetter and Hodson 1982; Fisher and Dimock 2006). Fisher and

Dimock (2006) determined that relative protein concentration is an appropriate measure of general condition in juvenile mussels. Protein content prior to a toxicity test could indicate differences in physiological condition of in vitro and in vivo cultured juveniles, either stress from the non-sterile environment of the host fish (in vivo juveniles) or from missing nutritional requirements (in vitro juveniles).

Currently, ASTM International does not recommend using in vitro propagated juveniles in toxicity testing unless their relative chemical sensitivity to in vivo transformed juveniles is described (ASTM International 2013). Because younger juveniles tend to be more sensitive to certain chemicals, and because no study has yet been able to consistently describe the relationship between in vivo and in vitro juveniles, it seems plausible that the relative chemical sensitivities may change with age. The aim of this study was to examine

potential differences between in vitro and in vivo propagated juvenile mussels to a single toxicant based on post-transformation age.

Methods

Test organisms

Three species native to the United States were selected for use in this study based on availability for production, range distribution, and conservation status. Lampsilis abrupta, the

Pink Mucket (Say, 1831), is a federally endangered species from the subfamily Ambleminae, tribe Lampsilini, native to Interior drainage basins (Parmalee and Bogan 1998). Lampsilis cardium, the Plain Pocketbook (Rafinesque, 1820), is a common species co-occurring with L. abrupta in the Interior basin, but is also found in the Great Lakes drainage. Utterbackia imbecillis, the Paper Pondshell (Say, 1829), is in the subfamily Unioninae, tribe Anodontini.

It is relatively common throughout the Interior and Atlantic Slope drainages and is often used in toxicity testing (Parmalee and Bogan 1998; Summers 1999).

Propagation

Juvenile mussels were propagated at the Kentucky Center for Mollusk Conservation

(Frankfort, Kentucky) using traditional host-fish (in vivo) and media culture (in vitro) propagation methods (Coker et al. 1921; Isom and Hudson 1982; Hudson and Isom 1984;

Owen et al. 2010; Fritts et al. 2013). Gravid female mussels of each species were collected from wild populations to serve as broodstock. Lampsilis abrupta were collected in the lower

Tennessee River (Tennessee drainage), L. cardium were collected from the lower Licking

River (Ohio River drainage), and U. imbecillis were collected from small streams in the

Cedar Creek watershed (Kentucky River drainage). Glochidia from three females were extracted and mixed. Half were used to inoculate the appropriate host fish for the given species and half were placed in a culture media mixture of rabbit serum and gentamicin.

After transformation, mussels were held at the Kentucky Center for Mollusk Conservation under their standard rearing protocols until required for testing. Mussels were then shipped overnight in successive weekly batches to NC State University (Raleigh, North Carolina) for use in toxicity testing. For transport, approximately 250 mussels were placed into a 50 mL centrifuge tube, which was then placed into a Thermos® containing water from the culture facility for added temperature stability. The Thermos was then placed in a Styrofoam® cooler with hot or cold packs, depending on the ambient temperature.

Juvenile assessment and acclimation

Juvenile mussels were assessed for viability according to the ASTM International guideline upon arrival at the laboratory (ASTM 2013). After initial water temperature was measured, mussels were acclimated to the test temperature of 20°C at a rate of 2.5°C per day.

Additionally, mussels were acclimated to the test water (ASTM International Hard Water,

ASTM 2007) at a rate of 25% volume exchange per hour. After 100% test water concentration and target temperature were reached, mussels were held for a minimum 24 h acclimation period. Mussels were again assessed for viability (using foot movement) at the start of the test, if acceptable (>80%), mussels were distributed to test chambers. Tests were

96 h non-aerated static tests with 100% water renewal at 48 h, conducted according to ASTM

International guidelines for juvenile mussels (2006). Viability was assessed at 48 and 96 h.

Digital photographs were taken of the mussels upon arrival at the laboratory. Length for a subset of mussels (~30 per batch) was measured to the nearest micrometer using a Leica

EZ4 D stereo microscope with integral digital camera and Leica Application Suite EZ digital photographic software (Leica Microsystems, Ltd., Switzerland).

Chemical sensitivity

To determine the influence of age (as a proxy for development) on the relative chemical sensitivity of in vitro and in vivo juvenile mussels, juveniles from each propagation method were exposed to copper (as copper sulfate: Sigma-Aldrich, St. Louis, MO, 99% purity). Test concentrations were 0, 12.5, 25, 50, 100, 200, and 300 µg Cu/L. Mussels were assessed for survival at 48 and 96 h, and the median effective concentration (EC50) resulting in immobility or mortality was calculated. To assess the magnitude of any potential difference in survival between in vitro and in vivo mussels for a particular age, the ratio of the two EC50s in each age pairing was calculated. The EC50 variation ratio was calculated by comparing in vitro:in vivo EC50s in an age pair. All juvenile mussel ages are reported as post-transformation age at the start of the test, and age is expressed as “X days in vivo-Y days in vitro.” Additionally, the variation ratio was calculated within all in vivo tests and all in vitro tests for a species.

Protein and ATP quantification

Immediately after test initiation, 20 mussels from each test batch (when available) were placed in mdRIPA lysis buffer [25 mM Tris (pH 7.4), 1% NP-40, 0.5% sodium deoxycholate, 15 mM NaCl] and frozen in an ultracold freezer until analysis. Samples were homogenized using a pestle in a 1.5 mL microcentrifuge tube, then read on a BioRad

SmartSpec3000 spectrophotometer following the manufacturer protocol for the protein assay kit (Bio-Rad, Life Science Research, Hercules, California). After analysis, protein samples were diluted to a uniform concentration for use in ATP measurements. ATP production was then measured using an Enliten® ATP assay kit and associated standard protocols and read on a GLOMAX 20/20 Luminometer (Promega Corporation, Madison, Wisconsin, USA).

Statistical analysis

The median effective concentration, i.e., the concentration of a toxicant causing immobility or mortality to 50% of the population (EC50), was computed with the

Comprehensive Environmental Toxicity Information Software package (CETIS) (v1.8.0.12,

Tidepool Scientific, LLC, McKinleyville, California). The associated 95% confidence intervals were used to determine significant differences between EC50s. If the 95% confidence intervals for in vitro and in vivo tests of the same age did not overlap, tests were considered to be significantly different (as in Archambault et al. 2013). Tests from all species were pooled for additional statistical analysis, then split into two groups based on the age of juveniles used: ‘younger’ (<10 d) and ‘older’ (>10 d). An Analysis of Variance (ANOVA)

was run on the combined EC50s from all species to examine the effect of age (‘younger’ vs

‘older’) and propagation method (in vitro vs in vivo) and the potential interaction between the two. Protein and ATP concentrations were analyzed separately with multiple linear regression (JMP: SAS Institute Inc., 2015), looking at the main effects of age and propagation method on protein and ATP concentration, respectively.

Results

Eight age-paired and five unpaired (four in vitro and one in vivo) tests were conducted (Table 1). Control survival was >80% in all but one test (Appendix E), and all but

6 tests were above the 90% ASTM survival threshold (ASTM 2013). Juveniles in paired tests came from the same broodstock, except for the L. cardium 9-10 day test. Four of the eight age-paired tests resulted in statistically significant differences in EC50 between in vitro and in vivo juveniles: L. abrupta at 12-12 days and 22-20 days, and L. cardium at 3-4 and 9-10 days. Age-paired variation ratios in vivo:in vitro averaged 1.7 and ranged from 0.8 to 4.3

(Figure 1). Lampsilis abrupta pairs had ratios of 4.3 (12-12 days), 1.9 (18-20 days), and 2.4

(22-20 days). Lampsilis cardium had ratios of 1.4 (3-4 days), 0.9 (3-5 days), 1.6 (7-4 days), and 1.0 (7-5 days). Variation ratios for Utterbackia imbecillis were 0.8 (3-3 days), 1.5 (6-6 days), and 1.2 (17-17 days). The variation between all in vivo tests was 1.1 for L. abrupta,

2.0 for L. cardium, and 1.9 for U. imbecillis. In vitro variation was 2.0 for L. abrupta, 1.3 for

L. cardium, and 1.2 for U. imbecillis.

There was no interaction effect between age grouping of juveniles (‘younger’=<10 days or ‘older’ =10> days old) and propagation method on EC50 (p=0.601). However, the main effect of propagation method on EC50 was significant (p=0.031).

Due to limited number of mussels, protein and ATP samples were taken from the following tests: L. abrupta in vivo at 12, 18, and 22 d and in vitro at 6, 12, and 16 d; L. cardium in vivo at 8, and 12 d and in vitro at 5, 9, and 19 d; and U. imbecillis in vivo at 6 and

21 d and in vitro at 18 and 16 d. Generally, in vitro juvenile mussels had higher levels of protein than their in vivo counterparts (Figure 2). However, when protein content was normalized for length, there was no significant interaction effect between the age of juveniles

(in days) and propagation method on the protein content of juveniles (p=0.523), and the effects of propagation and age were not significant (p=0.073 and p=0.054, respectively).

Additionally, age and propagation method did not have a significant interaction effect on

ATP of juveniles (Figure 3) (p=0.902), and propagation and age were not significant

(p=0.223 and p=0.89, respectively).

Discussion

In vitro juveniles were significantly more sensitive than in vivo juveniles in four of eight tests, and overall generally more sensitive. The only significantly different EC50s occurred within the genus Lampsilis. Significant differences for L. cardium occurred at 3-4 and 9-10 days, but not in the test at 5-7 days. For L. abrupta, these differences were at 12-12 days and 20-22 days. Significant differences in EC50 were evenly distributed across ages and

did not indicate a difference in chemical sensitivity associated with age. The ANOVA confirmed this finding, as the interaction between age groups (<10 d old and >10 d old) and propagation method on EC50 was not significant (p=0.601). However, the main effect of propagation method on EC50 was significant (p=0.031).

Variation between the EC50s ranged from a factor of 0.8 (U. imbecillis at 3 d) to 4.3

(L. cardium at 12 d), averaging 1.7 across all species and age-pairs. There did not seem to be an effect of age on the magnitude of variation between EC50s. This is comparable to rates of variation that have previously been published for in vivo juvenile toxicity tests using copper

(Wang et al. 2007b, Raimondo et al. 2016). Four age-pairs were within the 1.2 within-lab variation found by Wang et al. (2007b), and eight were within the within-lab variation of 2.1 reported by Raimondo et al. (2016). Six age-pairs were within the 1.5 between-lab variation found by Wang et al. (2007b), and all eight age-pairs were within the 4.8 between-lab variation described by Raimondo et al. (2016). Additionally, the within- in vivo and within– in vitro variations for each species were all equal to or below the within-lab variation of 2.1

(Raimondo et al. 2016).

We determined that in vitro and in vivo juvenile mussels are physiologically similar in terms of protein and ATP concentrations. The difference in protein concentration between in vitro and in vivo juveniles was not significant, though in vitro juveniles generally had greater levels of protein than in vivo juveniles of the same age. Perhaps in vitro juveniles had higher protein concentrations because they were not growing as rapidly as in vivo juveniles

(Fisher and Dimock 2006; Fox 2014) and had not yet completely metabolized lipid reserves,

necessitating the metabolism of protein. Protein from culture media may be more concentrated or readily available than protein from the fish host plasma. However, in a study conducted by Fisher and Dimock (2006), in vivo propagated juveniles had higher levels of protein, cholesterol, and glycogen than in vitro juveniles at 1 d post-transformation, and higher levels of protein and cholesterol at 7 d post-transformation. The in vitro juveniles they studied had levels comparable to glochidia for all three physiological indicators. Their study was conducted in 2006, and culture and feeding methods have continued to improve over the past decade (e.g., Owen et al. 2010; Fox 2014), potentially influencing the early health and condition of the mussels. In our study, energy expenditure (in terms of ATP production) was not descriptive of differences in health or condition at the initiation of a toxicity test.

Although this study robustly examined potential differences in EC50 for age-paired tests with juvenile mussels from the two propagation methods, there are several ways it could be improved for future experiments. First, our studies were conducted during the first three weeks of development, but no age-paired tests with individuals older than 20 d were conducted due to limited availability of juveniles. Moreover, no species used in this study had tests conducted at every time interval (1, 2, and 3 weeks), which limited statistical and analytical chemistry comparisons. All samples for protein and ATP measurements were taken pre-exposure. It would be valuable to collect post-exposure samples to compare relative stress placed on the juveniles throughout the toxicant exposure—this was not possible in the present study due to limited numbers of juveniles.

This study demonstrated that variation in EC50s from toxicity tests conducted with in vitro propagated mussels is acceptable when compared to the variation from the more routinely performed toxicity tests with in vivo propagated juveniles, however, opportunities remain to examine the health of in vivo juvenile mussels in future studies. For example, it would be valuable to measure additional biomarkers that indicate general physiological condition as well as exposure to, and effect of toxicants, such as oxidative stress, reactive oxygen species, and lipid peroxidation, each of which would provide a measure of relative health or condition (Ringwood et al. 1999; Lushchak 2011). In addition to evaluating biomarker responses, studying the effect of the culture media recipe on the growth and condition of in vitro propagated mussels and its potential influence on EC50 variation in toxicity tests may be warranted. Such studies could examine potential health differences between in vitro juveniles raised on culture media compared to those raised on a host fish.

Additionally, it would be beneficial to perform chronic tests (e.g., 21-28d in length) to examine differences in chemical sensitivity at lower toxicant concentrations over a longer time period.

In toxicity testing with freshwater mussels, several species (<10) are most commonly used (Raimondo et al. 2010, 2016) due to their ease of propagation, availability of gravid females, and propensity for long-term culture in the laboratory; the rarer species of conservation importance tend to be less well-represented. In freshwater mussel toxicity testing, Lampsilis siliquiodea and Utterbackia imbecillis are commonly used as surrogates for other mussel species (Raimondo et al. 2016). Raimondo et al. (2016) found that

approximately 70% of tested mussel species varied from L. siliquiodea and U. imbecillis by no more than a factor of 2, which infers adequate representation. The average variation between in vitro and in vivo juveniles in this study was 1.7, below the variation associated with the aforementioned surrogate species.

In conclusion, we found that in vitro propagated juveniles were more sensitive to a chemical toxicant than their in vivo counterparts, and the differences between EC50s were significant approximately 50% of the time. However, even with statistically significant differences between in vitro and in vivo juvenile chemical sensitivities, variation in chemical sensitivity was no greater than the among-lab difference observed in published results from in vivo propagated juveniles used in toxicity testing. Therefore, in vitro propagated juvenile mussels can be considered functionally similar to in vivo propagated mussels. More research is needed to further define the health and condition relationship between in vitro and in vivo propagated mussels, however, this study indicates it may be appropriate to use in vitro juveniles in freshwater mussel toxicity testing with reasonable precision.

Acknowledgements

Funding for this research was provided by the U.S. Geological Survey (USGS) and

U.S. Fish and Wildlife Service through the Science Support Partnership Program via

Research Work Order No. 211, administered through the USGS North Carolina Cooperative

Fish and Wildlife Research Unit. We thank Jennifer Archambault, Sean Buczek, Spencer

Gardner, and Joseph McIver for laboratory assistance, and Masaki Miyazawa for assistance

with biomarker processing. Special thanks to Dr. Monte McGregor and the rest of the group at the Kentucky Center for Mollusk Conservation for producing the mussels for this experiment and for providing insight at various stages through the project.

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Table 1: Median effective concentration (EC50) of copper causing 50% immobility or mortality (with 95% confidence intervals) in juvenile mussels at 96 h. Significantly different EC50s are distinguished in bold type. Age is reported as ‘days in vivo, days in vitro’, unless in vivo and in vitro ages were the same. ‘--‘ = no test run.

EC50 Species Age (days) In vivo In vitro L. abrupta 6 -- -- 20.7 (14.5-29.5) L. abrupta 12 115.7 (95.2-140.6) 26.6 (20.8-33.9) L. abrupta 18 100.9 (84.2-120.9) -- -- L. abrupta 22, 20 130.0 (110.8-152.6) 53.7 (42.0-68.7) L. cardium 3,4 60.1 (52.8-68.4) 44.0 (37.3-52.0) L. cardium 7,5 70.0 (60.8-80.7) 67.0 (55.3-81.2) L. cardium 9, 10 160.8 (147.7-175.0) 52.4 (45.8-59.9) L. cardium 16 -- -- 84.9 (70.3-102.5) L. cardium 17 -- -- 89.6 (76.2-105.3) U. imbecillis 3 33.7 (26.5-42.8) 41.0 (34.6-48.6) U. imbecillis 6 116.9 (96.2-142.1) 76.9 (63.5-93.2) U. imbecillis 17 62.1 (51.3-75.1) 52.6 (42.6-64.5) U. imbecillis 27 -- -- 63.0 (51.3-77.2)

′

† ′ ′ † † # # # † † †

Figure 1. Ratio of variation between in vitro and in vivo propagated juvenile mussel median effective concentration (EC50) by age. Horizontal line represents a ratio of 1:1. Here, in vitro juvenile EC50s are divided into in vivo EC50s for mussels of the same age and species. Ratios greater than 1 indicate in vivo mussels were less sensitive than in vitro mussels, ratios of less than 1 indicate in vivo mussels were more sensitive than in vitro mussels. # = Utterbackia imbecillis. † = Lampsilis cardium. ′ = Lampsilis abrupta.

0.12

0.10

0.08

0.06

0.04 In vivo

0.02 In vitro

Protein, mg/mL/mm oflength mg/mL/mm Protein, 0.00 0 5 10 15 20 25 Age (days)

Figure 2. Protein concentration of in vitro and in vivo juvenile mussels with age, normalized by length in mm. Neither propagation method (p=0.073), age (p=0.054), or their interaction (p=0.523) were significant.

1.6E-14 1.4E-14 In vivo 1.2E-14 1E-14 In vitro 8E-15 6E-15 4E-15 2E-15 Moles of ATP/8 mg protein mg ATP/8 of Moles 0 0 5 10 15 20 25 Age (days)

Figure 3. Adenosine triphosphate (ATP) concentration of in vitro and in vivo propagated juvenile mussels by age. Neither propagation method (p=0.223), age (p=0.890), or their interaction (p=0.902) were significant.

APPENDICES

Appendix A Table 1. Water chemistry data for tests using sodium chloride.

Prop. Conc. Temp. Conductivity DO DO Alkalinity Hardness Species Method (g Cl/L) (ᵒC) (µS/cm) (%) (mg/L) pH (mEq/L) (ppm) L. cardium in vitro 0 20.95 541 96.9 8.64 8.01 114 154 L. cardium in vitro 0.3 20.92 1499 97 8.6 8.09 L. cardium in vitro 0.6 20.96 2423 97.1 8.62 8.17 L. cardium in vitro 1.2 20.9 4241 96 8.51 8.15 L. cardium in vitro 2.4 20.97 7787 97.2 8.5 8.07 L. cardium in vitro 3.6 20.98 11267 96 8.28 8.17 L. cardium in vitro 4.8 21.03 14741 97.9 8.31 8.11 L. cardium in vivo 0 20.71 652 98.2 8.8 8.22 114 154 L. cardium in vivo 0.3 20.62 1571 98.2 8.78 8.24 L. cardium in vivo 0.6 20.9 2461 98.2 8.72 8.28 L. cardium in vivo 1.2 20.97 4351 98.5 8.67 8.16 L. cardium in vivo 2.4 20.94 8006 98.9 8.63 8.23 L. cardium in vivo 3.6 20.93 11473 96.6 8.29 8.17 L. cardium in vivo 4.8 20.04 15406 97.2 8.23 8.16 U. imbecillis in vitro 0 19.55 593 95.4 8.46 8.25 110 162 U. imbecillis in vitro 0.3 19.48 1588 92.9 8.49 8.4 U. imbecillis in vitro 0.6 19.3 2594 93.6 8.57 8.38 U. imbecillis in vitro 1.2 19.07 45.14 95.3 8.71 8.39 U. imbecillis in vitro 2.4 19.01 81.26 95 8.57 8.32 U. imbecillis in vitro 3.6 19.24 11768 94.5 8.36 8.29 U. imbecillis in vitro 4.8 19.33 15021 93.6 8.19 8.25 U. imbecillis in vivo 0 19.37 587 94.4 8.68 8.33 110 162 U. imbecillis in vivo 0.3 19.53 1596 92.9 8.5 8.38 U. imbecillis in vivo 0.6 19.49 2600 92 8.4 8.39 U. imbecillis in vivo 1.2 19.33 4481 96.4 8.75 8.36 U. imbecillis in vivo 2.4 19.15 8205 94.1 8.47 8.33 U. imbecillis in vivo 3.6 19.22 11725 94.3 8.35 8.27 U. imbecillis in vivo 4.8 19.36 15181 94.2 8.22 8.27 L. abrupta in vitro 0 21.89 496 96.4 8.42 8.32 110 152 L. abrupta in vitro 0.3 21.58 1427 92.2 8.13 8.29 L. abrupta in vitro 0.6 21.54 2339 95.9 8.4 8.2 L. abrupta in vitro 1.2 21.45 4176 960 8.36 8.22 L. abrupta in vitro 2.4 21.46 7575 95.4 8.23 8.2 L. abrupta in vitro 3.6 21.38 11078 96.2 8.2 8.22 L. abrupta in vitro 4.8 21.25 14245 95.4 8.05 8.19 L. abrupta-B in vivo 0 20.7 492 95.4 8.38 8.29 102 128 L. abrupta-B in vivo 0.3 21.68 1420 93.9 8.27 8.3 L. abrupta-B in vivo 0.6 21.47 2363 95.7 8.4 8.29 L. abrupta-B in vivo 1.2 21.43 4142 95.3 8.32 8.3 L. abrupta-B in vivo 2.4 21.36 9858 95.3 8.18 8.2 L. abrupta-B in vivo 3.6 21.27 11052 94.7 8.11 8.2 L. abrupta-B in vivo 4.8 21.35 14204 95 8.02 8.21 L. abrupta-A in vivo 0 20.74 500 99.3 8.87 8.43 110 152 L. abrupta-A in vivo 0.3 20.66 1357 99 8.84 8.4 L. abrupta-A in vivo 0.6 20.65 2233 99.4 8.87 8.38 L. abrupta-A in vivo 1.2 20.66 3894 99.1 8.79 8.35 L. abrupta-A in vivo 2.4 20.67 7080 98 8.61 8.31 L. abrupta-A in vivo 3.6 20.71 10201 99 8.58 8.28 L. abrupta-A in vivo 4.8 20.72 13242 99 8.49 8.25

Table 2. Water chemistry data for tests using nickel chloride.

Prop Conc. Temp. Conductivity DO DO Alkalinity Hardness Species Method (µg Ni/L) (ᵒC) (µS/cm) (%) (mg/L) pH (mEq/L) (ppm) L. cardium in vitro 0 20.74 550 93.8 8.39 8.39 118 166 L. cardium in vitro 100 20.63 551 95.4 8.55 8.41 L. cardium in vitro 250 19.66 557 94.0 8.60 8.42 L. cardium in vitro 500 19.68 568 94.5 8.46 8.43 L. cardium in vitro 750 19.58 558 93.9 8.59 8.44 L. cardium in vitro 1,000 19.72 562 94.0 8.59 8.46 L. cardium in vitro 1,500 19.83 572 94.2 8.60 8.45 L. cardium in vivo 0 21.08 551 95.5 8.47 8.36 118 166 L. cardium in vivo 100 20.55 552 95.3 8.55 8.38 L. cardium in vivo 250 20.47 552 94.9 8.51 8.43 L. cardium in vivo 500 19.80 555 95.2 8.69 8.42 L. cardium in vivo 750 18.72 558 95.3 8.70 8.44 L. cardium in vivo 1,000 19.57 557 94.6 8.66 8.45 L. cardium in vivo 1,500 19.73 560 93.9 8.59 8.45 U. imbecillis in vitro 0 20.12 536 96 8.7 8.31 108 156 U. imbecillis in vitro 100 20.02 537 95.5 8.66 8.39 U. imbecillis in vitro 250 20.03 536 94.9 8.62 8.36 U. imbecillis in vitro 500 19.98 538 95.1 8.65 8.39 U. imbecillis in vitro 750 20.21 538 94.8 8.52 8.39 U. imbecillis in vitro 1,000 20.11 539 95.3 8.63 8.40 U. imbecillis in vitro 1,500 20.28 539 96.8 8.73 8.39 U. imbecillis in vivo 0 20.06 538 95.2 8.64 8.16 108 156 U. imbecillis in vivo 100 19.98 537 95.5 8.67 8.36 U. imbecillis in vivo 250 19.92 536 94.9 8.63 8.39 U. imbecillis in vivo 500 19.96 536 94.9 8.63 8.4 U. imbecillis in vivo 750 20.03 537 94.3 8.57 8.4 U. imbecillis in vivo 1,000 20.09 538 94.7 8.62 8.4 U. imbecillis in vivo 1,500 20.2 540 95.5 8.62 8.4 L. abrupta in vitro 0 21.57 548 95.1 8.36 8.32 108 164 L. abrupta in vitro 100 21.52 550 94.6 8.37 8.38 L. abrupta in vitro 250 21.35 557 96.1 8.48 8.42 L. abrupta in vitro 500 21.34 553 95.9 8.48 8.43 L. abrupta in vitro 750 21.31 554 96.0 8.48 8.44 L. abrupta in vitro 1,000 21.28 555 96.0 8.49 8.44 L. abrupta in vitro 1,500 21.25 560 96.2 8.52 8.43 L. abrupta-B in vivo 0 21.81 552 94.7 8.31 8.41 108 164 L. abrupta-B in vivo 100 21.76 554 95.2 8.35 8.45 L. abrupta-B in vivo 250 21.64 553 95.9 8.43 8.46 L. abrupta-B in vivo 500 21.50 555 96.1 8.76 8.46 L. abrupta-B in vivo 750 21.37 555 96.4 8.51 8.45 L. abrupta-B in vivo 1,000 21.24 557 96.6 8.56 8.45 L. abrupta-B in vivo 1,500 21.26 557 96.3 8.53 8.44 L. abrupta-A in vivo 0 19.65 527 91.8 8.41 8.27 118 166 L. abrupta-A in vivo 100 19.44 531 91.3 8.40 8.26 L. abrupta-A in vivo 250 19.33 530 92.8 8.55 8.31 L. abrupta-A in vivo 500 19.33 531 92.9 8.55 8.32 L. abrupta-A in vivo 750 19.27 531 92.8 8.55 8.31 L. abrupta-A in vivo 1,000 19.33 531 92.4 8.49 8.32 L. abrupta-A in vivo 1,500 19.36 533 92.5 8.51 8.30

Table 3. Water chemistry data for tests with copper sulfate.

Prop Conc. Temp. Conductivity DO DO Alkalinity Hardness Species Method (µg Cu/L) (ᵒC) (µS/cm) (%) (mg/L) pH (mEq/L) (ppm) U. imbecillis in vitro 0 19.60 565 97.5 8.92 8.47 100 154 U. imbecillis in vitro 12.5 19.42 553 97.8 8.98 8.46 U. imbecillis in vitro 25 19.38 554 97.8 8.99 8.45 U. imbecillis in vitro 50 19.33 556 97.3 8.95 8.44 U. imbecillis in vitro 100 19.33 554 98.3 9.05 8.44 U. imbecillis in vitro 200 19.38 553 98.1 9.03 8.44 U. imbecillis in vitro 300 19.40 555 98.0 9.00 8.44 U. imbecillis in vivo 0 19.48 553 97.4 8.93 8.46 100 154 U. imbecillis in vivo 12.5 19.39 553 98.7 9.01 8.45 U. imbecillis in vivo 25 19.41 553 98.4 9.02 8.45 U. imbecillis in vivo 50 19.39 554 98.3 9.03 8.44 U. imbecillis in vivo 100 19.38 554 98.5 9.05 8.44 U. imbecillis in vivo 200 19.39 554 98.2 9.02 8.44 U. imbecillis in vivo 300 19.42 553 98.2 9.01 8.44 L. cardium in vitro 0 22.69 494 90.9 7.84 8.39 92 140 L. cardium in vitro 25 22.53 489 91.9 7.95 8.38 L. cardium in vitro 50 22.39 488 92.2 7.99 8.37 L. cardium in vitro 100 22.43 489 91.6 7.91 8.42 L. cardium in vitro 200 22.31 488 92.1 7.99 8.35 L. cardium in vitro 300 22.34 489 91.9 7.97 8.35 L. cardium in vitro 400 22.49 493 91.7 7.93 8.34 L. cardium in vivo 0 22.82 494 91.2 7.84 8.39 92 140 L. cardium in vivo 25 22.51 491 91.6 7.93 9.38 L. cardium in vivo 50 22.34 490 91.3 7.93 8.38 L. cardium in vivo 100 22.33 490 92.2 8.00 8.37 L. cardium in vivo 200 22.28 490 91.3 7.93 8.36 L. cardium in vivo 300 22.30 490 91.2 7.93 8.35 L. cardium in vivo 400 22.42 490 92.2 7.97 8.35 L. abrupta in vitro 0 21.52 552 98.0 8.63 8.37 108 164 L. abrupta in vitro 12.5 21.55 559 96.3 8.48 8.43 L. abrupta in vitro 25 21.90 552 96.5 8.49 8.44 L. abrupta in vitro 50 21.55 558 96.5 8.50 8.44 L. abrupta in vitro 100 21.59 558 96.0 8.44 8.44 L. abrupta in vitro 200 21.69 554 95.2 8.36 8.44 L. abrupta in vitro 300 21.76 552 95.3 8.36 8.44 L. abrupta in vitro 1,000 21.45 553 96.4 8.5 8.41 L. abrupta-A in vivo 0 21.53 550 93.0 8.20 8.27 108 164 L. abrupta-A in vivo 12.5 21.48 552 95.5 8.42 8.39 L. abrupta-A in vivo 25 21.36 551 95.8 8.48 8.43 L. abrupta-A in vivo 50 21.58 552 96.1 8.46 8.43 L. abrupta-A in vivo 100 21.68 552 95.4 8.38 8.44 L. abrupta-A in vivo 200 21.66 558 95.1 8.36 8.44 L. abrupta-A in vivo 300 21.73 553 95.8 8.46 8.44 L. abrupta-A in vivo 1,000 21.53 553 95.8 8.46 8.42 L. abrupta-B in vivo 0 19.11 554 93.7 8.66 8.36 106 156 L. abrupta-B in vivo 12.5 18.94 559 93.7 8.68 8.36 L. abrupta-B in vivo 25 18.93 558 94.2 8.72 8.35 L. abrupta-B in vivo 50 18.93 555 93.5 8.67 8.37 L. abrupta-B in vivo 100 18.93 556 93.7 8.7 8.37 L. abrupta-B in vivo 200 18.92 556 93.9 8.71 8.36 L. abrupta-B in vivo 300 18.99 556 93.6 8.67 8.36

Table 4. Water chemistry data for tests with Clearigate®.

Prop Conc. Temp. Conductivity DO DO Alkalinity Hardness Species Method (µg Cu/L) (ᵒC) (µS/cm) (%) (mg/L) pH (mEq/L) (ppm) L. cardium in vitro 0 20.65 520 97.2 8.67 8.41 108 164 L. cardium in vitro 25 20.49 519 96.4 8.66 8.41 L. cardium in vitro 50 20.42 520 96.1 8.63 8.42 L. cardium in vitro 100 20.45 519 96.4 8.64 8.42 L. cardium in vitro 250 20.64 522 96.5 8.66 8.42 L. cardium in vitro 500 20.77 519 96.3 8.6 8.43 L. cardium in vitro 750 20.92 522 96.3 8.59 8.43 L. cardium in vivo 0 20.79 521 97.1 8.67 8.41 108 164 L. cardium in vivo 25 20.75 520 96.6 8.64 8.42 L. cardium in vivo 50 20.63 522 96.4 8.64 8.42 L. cardium in vivo 100 20.68 521 96.3 8.63 8.43 L. cardium in vivo 250 20.78 521 96.7 8.64 8.43 L. cardium in vivo 500 20.97 518 96.2 8.57 8.42 L. cardium in vivo 750 21.05 524 96 8.53 8.42 L. abrupta-A in vivo 0 18.89 550 92.8 8.61 8.35 106 156 L. abrupta-A in vivo 50 18.83 556 92.5 8.6 8.36 L. abrupta-A in vivo 100 18.83 562 93.2 8.66 8.31 L. abrupta-A in vivo 250 18.85 556 93 8.64 8.32 L. abrupta-A in vivo 500 18.86 557 93.6 8.7 8.34 L. abrupta-A in vivo 750 18.83 558 93.2 8.66 8.35 L. abrupta-A in vivo 1,000 18.88 558 92.3 8.58 8.35 L. abrupta-A in vivo 1,500 18.9 561 92.5 8.58 8.37 L. abrupta in vitro 0 21.94 560 93.1 8.13 8.33 110 158 L. abrupta in vitro 100 21.91 556 91.7 8.03 8.38 L. abrupta in vitro 500 21.87 556 92.2 8.05 8.43 L. abrupta in vitro 1,000 21.86 556 92.1 8.07 8.42 L. abrupta-B in vivo 0 22.22 557 92.5 8.05 8.33 110 158 L. abrupta-B in vivo 100 21.93 555 91.6 8.01 8.39 L. abrupta-B in vivo 500 21.9 556 90.9 7.95 8.38 L. abrupta-B in vivo 1,000 21.94 558 91 7.8 8.4

Table 5. Water chemistry data for tests with Nautique®

Prop Conc. Temp. Conductivity DO DO Alkalinity Hardness Species Method (µg Cu/L) (ᵒC) (µS/cm) (%) (mg/L) pH (mEq/L) (ppm) U. imbecillis in vitro 0 19.65 565 96.7 8.85 8.3 112 156 U. imbecillis in vitro 12.5 19.65 561 97.2 8.89 8.39 U. imbecillis in vitro 25 19.68 554 97.4 8.91 8.4 U. imbecillis in vitro 50 19.66 558 97.2 8.89 8.42 U. imbecillis in vitro 100 19.68 557 97.2 8.89 8.42 U. imbecillis in vitro 200 19.69 557 97 8.86 8.43 U. imbecillis in vitro 300 19.64 558 97.1 8.88 8.43 U. imbecillis in vitro 1,000 19.44 560 97.1 8.92 8.44 U. imbecillis in vivo 0 19.69 702 97.3 8.89 8.05 112 156 U. imbecillis in vivo 12.5 19.58 561 97 8.89 8.36 U. imbecillis in vivo 25 19.64 559 97 8.88 8.41 U. imbecillis in vivo 50 19.63 561 97.1 8.89 8.41 U. imbecillis in vivo 100 19.64 557 97 8.88 8.42 U. imbecillis in vivo 200 19.66 558 96.8 8.86 8.43 U. imbecillis in vivo 300 19.68 558 97.3 8.9 8.43 U. imbecillis in vivo 1,000 19.58 558 97 8.89 8.44 L. cardium in vitro 0 20.7 559 92.4 8.27 8.47 110 162 L. cardium in vitro 250 20.69 559 92.3 8.27 8.46 L. cardium in vitro 500 20.59 560 92.3 8.23 8.46 L. cardium in vitro 1,000 20.67 563 91.5 8.19 8.47 L. cardium in vitro 2,000 20.56 564 92 8.27 8.47 L. cardium in vitro 4,000 20.5 570 92.1 8.28 8.48 L. cardium in vitro 8,000 20.44 576 92.5 8.3 8.49 L. cardium in vivo 0 20.67 555 90 8.13 8.4 110 162 L. cardium in vivo 250 20.68 560 92.5 8.27 8.45 L. cardium in vivo 500 20.65 560 91.3 8.18 8.46 L. cardium in vivo 1,000 20.62 566 91.7 8.23 8.47 L. cardium in vivo 2,000 20.55 564 91.5 8.21 8.58 L. cardium in vivo 4,000 20.49 569 92.1 8.27 8.48 L. cardium in vivo 8,000 20.28 583 92 8.3 8.53 L. abrupta in vitro 0 20.29 577 94.5 8.52 8.76 132 158 L. abrupta in vitro 250 20.16 573 93.3 8.45 8.55 L. abrupta in vitro 500 20.42 558 93.1 8.4 8.41 L. abrupta in vitro 1,000 20.48 563 93.3 8.38 8.38 L. abrupta in vitro 2,000 20.56 559 92.1 8.26 8.39 L. abrupta in vitro 4,000 20.63 581 93 8.34 8.42 L. abrupta in vitro 8,000 20.73 584 93.6 8.38 8.42 L. abrupta-A in vivo 0 20.58 579 95 8.52 9 132 158 L. abrupta-A in vivo 250 20.34 606 94.8 8.53 8.61 L. abrupta-A in vivo 500 20.3 587 93.4 8.43 8.46 L. abrupta-A in vivo 1,000 20.52 558 92 8.28 8.41 L. abrupta-A in vivo 2,000 50.53 561 93.5 8.35 8.4 L. abrupta-A in vivo 4,000 20.6 565 93.3 8.34 8.4 L. abrupta-A in vivo 8,000 20.73 567 93.2 8.34 8.41 L. abrupta-B in vivo 0 21.7 541 96.9 8.51 8.28 108 162 L. abrupta-B in vivo 250 21.64 548 97.3 8.54 8.35 L. abrupta-B in vivo 500 21.71 549 97.4 8.55 8.39 L. abrupta-B in vivo 1,000 21.66 551 97.7 8.57 8.41 L. abrupta-B in vivo 2,000 21.79 553 97.5 8.54 8.41 L. abrupta-B in vivo 4,000 21.82 560 97.2 8.52 8.34 L. abrupta-B in vivo 8,000 21.89 565 96.2 8.42 8.46

Table 6. Water chemistry data for tests with ammonium chloride.

Prop Conc. Temp. Conductivity DO DO Alkalinity Hardness Species Method (mg TAN/L) (ᵒC) (µS/cm) (%) (mg/L) pH (mEq/L) (ppm) U. imbecillis in vitro 0 19.37 672 92.5 8.51 8.00 102 158 U. imbecillis in vitro 1 19.31 565 93.1 8.57 8.34 U. imbecillis in vitro 2 19.35 568 93.2 8.58 8.39 U. imbecillis in vitro 4 19.35 571 93.5 8.6 8.40 U. imbecillis in vitro 8 19.4 582 93.2 8.57 8.40 U. imbecillis in vitro 16 19.46 600 92.8 8.52 8.39 U. imbecillis in vitro 32 19.49 642 92.9 8.53 8.38 U. imbecillis in vivo 0 19.34 567 92.2 8.49 8.27 102 158 U. imbecillis in vivo 1 19.34 566 92.7 8.53 8.37 U. imbecillis in vivo 2 19.36 568 93.1 8.55 8.39 U. imbecillis in vivo 4 19.38 571 93 8.55 8.4 U. imbecillis in vivo 8 19.43 581 93.4 8.57 8.4 U. imbecillis in vivo 16 19.49 601 92.6 8.49 8.39 U. imbecillis in vivo 32 19.48 712 93.1 8.54 8.36 L. cardium in vitro 0 20.62 545 92.4 8.29 8.27 112 166 L. cardium in vitro 1 20.33 556 93.5 8.43 8.35 L. cardium in vitro 2 20.35 558 93.8 8.45 8.34 L. cardium in vitro 4 20.27 577 93.9 8.48 8.32 L. cardium in vitro 8 20.26 612 94 8.49 8.33 L. cardium in vitro 16 20.27 681 93.5 8.45 8.29 L. cardium in vitro 32 20.35 818 94 8.46 8.25 L. cardium in vivo 0 20.44 549 93.4 8.4 8.31 112 166 L. cardium in vivo 1 20.29 553 93.3 8.43 8.33 L. cardium in vivo 2 20.16 561 94 8.5 8.34 L. cardium in vivo 4 20.11 577 94 8.52 8.33 L. cardium in vivo 8 19.97 608 94.2 8.56 8.36 L. cardium in vivo 16 20.12 678 93.9 8.5 8.3 L. cardium in vivo 32 20.27 811 93.4 8.42 8.25 L. abrupta in vitro 0 21.28 554 92.8 8.22 8.38 132 158 L. abrupta in vitro 1 21.07 584 94.1 8.34 8.37 L. abrupta in vitro 2 21.16 575 93.3 8.29 8.37 L. abrupta in vitro 4 21.01 612 94.4 8.39 8.34 L. abrupta in vitro 8 21.01 648 91.5 8.19 8.32 L. abrupta in vitro 16 21.05 730 92 8.15 8.28 L. abrupta in vitro 32 20.98 905 92.3 8.2 8.24 L. abrupta-B in vivo 0 21.36 571 91.7 8.13 8.34 132 158 L. abrupta-B in vivo 1 21.16 595 95.4 8.47 8.37 L. abrupta-B in vivo 2 21.16 575 93.5 8.27 8.37 L. abrupta-B in vivo 4 21.12 597 93.4 8.29 8.36 L. abrupta-B in vivo 8 21.09 638 92.7 8.23 8.34 L. abrupta-B in vivo 16 21.09 725 92.6 8.22 8.35 L. abrupta-B in vivo 32 21.15 898 92 8.16 8.24 L. abrupta-A in vivo 0 21.76 550 97.8 8.57 8.39 L. abrupta-A in vivo 1 21.6 558 97.2 8.55 8.4 L. abrupta-A in vivo 2 21.56 567 97.3 8.56 8.4 L. abrupta-A in vivo 4 21.52 582 97 8.53 8.39 L. abrupta-A in vivo 8 21.55 616 97.1 8.55 8.36 L. abrupta-A in vivo 16 21.62 688 95.3 8.34 8.31 L. abrupta-A in vivo 32 21.79 836 96.8 8.48 8.24

Appendix B Table 1: Nominal and measured concentrations of toxicants. †=Background (non-chlorine) salinity for all chemicals is ~0.3 ppt.

Nominal Measured % Nominal Measured % Chemical Conc. Conc. Recovery Chemical Conc. Conc. Recovery Ammonia 0 0.012 n/a Clearigate 100 67.1 67.1 (mg TAN/L) (µg Cu/L) Ammonia 1 0.834 83.4 Clearigate 500 375 75 (mg TAN/L) (µg Cu/L) Ammonia 2 2.017 100.9 Clearigate 1,000 758 75.8 (mg TAN/L) (µg Cu/L) Ammonia 4 3.087 77.2 Copper 0 0 n/a (mg TAN/L) (µg Cu/L) Ammonia 8 6.914 86.4 Copper 12.5 4.84 97 (mg TAN/L) (µg Cu/L) Ammonia 16 13.155 82.2 Copper 25 9.57 96 (mg TAN/L) (µg Cu/L) Ammonia 32 25.04 78.25 Copper 50 25.38 102 (mg TAN/L) (µg Cu/L) Chloride † 0 0.33 n/a Copper 100 49.61 99 (ppt) (µg Cu/L) Chloride † 0.5 0.84 168.0 Copper 200 103.79 104 (ppt) (µg Cu/L) Chloride † 1 1.28 128.0 Copper 300 197.01 99 (ppt) (µg Cu/L) Chloride † 2 2.26 113.0 Copper 500 503.75 101 (ppt) (µg Cu/L) Chloride † 4 4.27 106.8 Copper 1,000 996.06 100 (ppt) (µg Cu/L) Chloride † 6 6.32 105.3 Nautique 250 0.225 90 (ppt) (µg Cu/L) Chloride † 8 8.43 105.4 Nautique 500 0.475 95 (ppt) (µg Cu/L) Nickel 0 0.01 n/a Nautique 1,000 0.966 96.6 (µg Ni/L) (µg Cu/L) Nickel 100 96.85 97.0 Nautique 2,000 1.96 98 (µg Ni/L) (µg Cu/L) Nickel 200 196.59 98.0 Nautique 4,000 3.95 98.75 (µg Ni/L) (µg Cu/L) Nickel 300 304.25 101.0 Nautique 8,000 7.66 95.75 (µg Ni/L) (µg Cu/L) Nickel 500 489.44 98.0 (µg Ni/L) Nickel 1,000 1017.90 102.0 (µg Ni/L) Nickel 1,500 1500.10 100.0 (µg Ni/L)

Appendix C Table 1: Effective concentration causing immobility or mortality for 5% of test organisms (EC05) at 96 hours. N/A = EC05s could not be calculated due to constraints in the mortality response. * = No test run for Utterbackia imbecillis with Clearigate®.

96 h EC05

Species Toxicant In vivo In vitro Lampsilis abrupta Ammonia 3.6 (2.6-2.3) 2.5 (1.6-3.4) N/A† Lampsilis cardium Ammonia N/A N/A Utterbackia imbecillis Ammonia N/A N/A Lampsilis abrupta Chloride 1.2 (1.1-1.4) N/A N/A Lampsilis cardium Chloride N/A 1.2 (0.8-1.6) Utterbackia imbecillis Chloride N/A N/A Lampsilis abrupta Clearigate® 82.4 (56.8-103.9) 311.9 (139.1-407.1) N/A Lampsilis cardium Clearigate 254.3 (166.5-316.2) 91.4 (51.0-127.2) Utterbackia imbecillis Clearigate * * Lampsilis abrupta Copper 61.5 (42.6-76.9) 15.7 (9.0-21.2) 63.9 (14.9-101.2) Lampsilis cardium Copper N/A 29.7 (19.9-38.7) Utterbackia imbecillis Copper 49.0 (20.1-73.0) 25.3 (13.1-36.7) Lampsilis abrupta Nautique® 2050.5 (1314.2-2515.9) 2420.2 (1105.1-3062.7) N/A† Lampsilis cardium Nautique N/A N/A Utterbackia imbecillis Nautique N/A 106.6 (50.9-156.6) Lampsilis abrupta Nickel 961.3 (802.1-1059.8) N/A N/A† Lampsilis cardium Nickel 455.5 (329.6-538.4) 525.0 (359.2-611.3) Utterbackia imbecillis Nickel 987.0 (788.7-1099.3) 892.4 (744.8-988.9)

Appendix D Table 1. Juvenile mussel survival in tests with ammonium chloride.

Prop. Conc. (mg 48h 96h Prop. Conc. (mg 48h 96h Species Method TAN/L) Rep. survival survival Species Method TAN/L) Rep. survival survival L. abrupta in vitro 0 1 10/10 10/10 L. cardium in vivo 0 1 11/11 10/10 L. abrupta in vitro 0 2 10/10 10/10 L. cardium in vivo 0 2 11/11 11/11 L. abrupta in vitro 0 3 10/10 10/10 L. cardium in vivo 0 3 10/10 10/10 L. abrupta in vitro 1 1 7/7 7/7 L. cardium in vivo 1 1 9/9 9/9 L. abrupta in vitro 1 2 8/8 8/8 L. cardium in vivo 1 2 10/10 10/10 L. abrupta in vitro 1 3 7/7 7/7 L. cardium in vivo 1 3 10/10 10/10 L. abrupta in vitro 2 1 8/8 8/8 L. cardium in vivo 2 1 10/10 10/10 L. abrupta in vitro 2 2 8/8 8/8 L. cardium in vivo 2 2 10/10 10/10 L. abrupta in vitro 2 3 7/7 7/7 L. cardium in vivo 2 3 10/10 10/10 L. abrupta in vitro 4 1 7/7 5/7 L. cardium in vivo 4 1 10/10 10/10 L. abrupta in vitro 4 2 7/7 6/7 L. cardium in vivo 4 2 10/10 10/10 L. abrupta in vitro 4 3 6/7 3/7 L. cardium in vivo 4 3 10/10 10/10 L. abrupta in vitro 8 1 4/7 2/7 L. cardium in vivo 8 1 8/10 6/10 L. abrupta in vitro 8 2 5/7 2/7 L. cardium in vivo 8 2 7/10 3/10 L. abrupta in vitro 8 3 4/7 3/7 L. cardium in vivo 8 3 8/11 5/11 L. abrupta in vitro 16 1 3/7 0/7 L. cardium in vivo 16 1 7/10 0/10 L. abrupta in vitro 16 2 1/7 0/7 L. cardium in vivo 16 2 8/10 0/10 L. abrupta in vitro 16 3 2/7 0/7 L. cardium in vivo 16 3 9/10 0/10 L. abrupta in vitro 32 1 0/7 0/7 L. cardium in vivo 32 1 0/10 0/10 L. abrupta in vitro 32 2 2/7 0/7 L. cardium in vivo 32 2 0/10 0/10 L. abrupta in vitro 32 3 2/7 0/7 L. cardium in vivo 32 3 0/10 0/10 L. abrupta-A in vivo 0 1 11/11 10/11 U. imbecillis in vitro 0 1 10/10 9/9 L. abrupta-A in vivo 1 1 10/10 10/10 U. imbecillis in vitro 0 2 10/10 8/9 L. abrupta-A in vivo 2 1 11/11 9/10 U. imbecillis in vitro 0 3 10/10 8/9 L. abrupta-A in vivo 4 1 11/11 9/10 U. imbecillis in vitro 1 1 9/9 8/9 L. abrupta-A in vivo 8 1 10/10 5/10 U. imbecillis in vitro 1 2 9/9 8/9 L. abrupta-A in vivo 16 1 10/10 0/10 U. imbecillis in vitro 1 3 9/9 9/9 L. abrupta-A in vivo 32 1 0/10 0/10 U. imbecillis in vitro 2 1 9/9 8/9 L. abrupta-B in vivo 0 1 10/10 10/10 U. imbecillis in vitro 2 2 9/9 8/9 L. abrupta-B in vivo 0 2 10/10 10/10 U. imbecillis in vitro 2 3 9/9 9/9 L. abrupta-B in vivo 0 3 10/10 10/10 U. imbecillis in vitro 4 1 9/9 7/9 L. abrupta-B in vivo 1 1 10/10 10/10 U. imbecillis in vitro 4 2 9/9 5/9 L. abrupta-B in vivo 1 2 10/10 10/10 U. imbecillis in vitro 4 3 9/9 7/9 L. abrupta-B in vivo 1 3 10/10 9/10 U. imbecillis in vitro 8 1 8/9 7/9 L. abrupta-B in vivo 2 1 10/10 10/10 U. imbecillis in vitro 8 2 9/9 6/9 L. abrupta-B in vivo 2 2 10/10 10/10 U. imbecillis in vitro 8 3 9/9 7/9 L. abrupta-B in vivo 2 3 9/10 9/10 U. imbecillis in vitro 16 1 9/9 0/9 L. abrupta-B in vivo 4 1 10/10 9/10 U. imbecillis in vitro 16 2 9/9 0/9 L. abrupta-B in vivo 4 2 10/10 10/10 U. imbecillis in vitro 16 3 8/9 0/9 L. abrupta-B in vivo 4 3 10/10 9/10 U. imbecillis in vitro 32 1 8/9 0/9 L. abrupta-B in vivo 8 1 9/10 2/10 U. imbecillis in vitro 32 2 9/9 0/9 L. abrupta-B in vivo 8 2 10/10 3/10 U. imbecillis in vitro 32 3 7/9 0/9 L. abrupta-B in vivo 8 3 9/10 2/10 U. imbecillis in vivo 0 1 9/10 8/10 L. abrupta-B in vivo 16 1 9/10 0/10 U. imbecillis in vivo 0 2 9/10 9/10 L. abrupta-B in vivo 16 2 7/10 0/10 U. imbecillis in vivo 0 3 9/10 9/10 L. abrupta-B in vivo 16 3 5/10 0/10 U. imbecillis in vivo 1 1 9/9 7/9 L. abrupta-B in vivo 32 1 0/10 0/10 U. imbecillis in vivo 1 2 9/9 7/9 L. abrupta-B in vivo 32 2 0/10 0/10 U. imbecillis in vivo 1 3 7/9 7/9 L. abrupta-B in vivo 32 3 0/10 0/10 U. imbecillis in vivo 2 1 9/9 7/9 L. cardium in vitro 0 1 10/10 10/10 U. imbecillis in vivo 2 2 9/9 9/9 L. cardium in vitro 0 2 11/11 9/10 U. imbecillis in vivo 2 3 9/9 9/9 L. cardium in vitro 0 3 10/10 10/10 U. imbecillis in vivo 4 1 9/9 9/9 L. cardium in vitro 1 1 10/10 10/10 U. imbecillis in vivo 4 2 8/9 7/9 L. cardium in vitro 1 2 10/10 10/10 U. imbecillis in vivo 4 3 8/9 9/9 L. cardium in vitro 1 3 8/9 8/9 U. imbecillis in vivo 8 1 9/9 7/9 L. cardium in vitro 2 1 11/11 11/11 U. imbecillis in vivo 8 2 9/9 6/9 L. cardium in vitro 2 2 10/10 10/10 U. imbecillis in vivo 8 3 8/9 7/9 L. cardium in vitro 2 3 10/10 10/10 U. imbecillis in vivo 16 1 8/9 0/9 L. cardium in vitro 4 1 10/10 2/10 U. imbecillis in vivo 16 2 8/9 0/9 L. cardium in vitro 4 2 10/10 3/10 U. imbecillis in vivo 16 3 8/9 0/9 L. cardium in vitro 4 3 9/9 2/10 U. imbecillis in vivo 32 1 2/9 0/9 L. cardium in vitro 8 1 3/12 0/12 U. imbecillis in vivo 32 2 3/9 0/9 L. cardium in vitro 8 2 3/9 0/9 U. imbecillis in vivo 32 3 4/9 0/9 L. cardium in vitro 8 3 3/10 0/10 L. cardium in vitro 16 1 0/11 0/11 L. cardium in vitro 16 2 0/10 0/10 L. cardium in vitro 16 3 2/10 0/10 L. cardium in vitro 32 1 0/13 0/13 L. cardium in vitro 32 2 0/10 0/10 L. cardium in vitro 32 3 0/11 0/11

Table 2. Juvenile mussel survival in tests with sodium chloride.

Prop. Conc. (g 48h 96h Prop. Conc. (g 48h 96h Species Method NaCl/L) Rep survival survival Species Method NaCl/L) Rep survival survival L. abrupta in vitro 0 1 10/10 10/10 L. cardium in vitro 0 1 10/10 9/9 L. abrupta in vitro 0 2 10/10 9/10 L. cardium in vitro 0 2 10/10 9/10 L. abrupta in vitro 0 3 10/10 10/10 L. cardium in vitro 0 3 10/10 10/10 L. abrupta in vitro 0.5 1 10/10 10/10 L. cardium in vitro 0.5 1 10/10 10/10 L. abrupta in vitro 0.5 2 10/10 10/10 L. cardium in vitro 0.5 2 10/10 10/10 L. abrupta in vitro 0.5 3 10/10 10/10 L. cardium in vitro 0.5 3 10/10 7/10 L. abrupta in vitro 1 1 10/10 10/10 L. cardium in vitro 1 1 10/10 10/10 L. abrupta in vitro 1 2 10/10 10/10 L. cardium in vitro 1 2 10/10 10/10 L. abrupta in vitro 1 3 10/10 9/10 L. cardium in vitro 1 3 10/10 10/10 L. abrupta in vitro 2 1 10/10 10/10 L. cardium in vitro 2 1 10/10 8/10 L. abrupta in vitro 2 2 10/10 10/10 L. cardium in vitro 2 2 10/10 9/10 L. abrupta in vitro 2 3 10/10 10/10 L. cardium in vitro 2 3 10/10 10/10 L. abrupta in vitro 4 1 10/10 10/10 L. cardium in vitro 4 1 9/10 1/10 L. abrupta in vitro 4 2 9/9 9/9 L. cardium in vitro 4 2 8/10 2/10 L. abrupta in vitro 4 3 9/10 9/10 L. cardium in vitro 4 3 7/10 1/10 L. abrupta in vitro 6 1 3/10 0/10 L. cardium in vitro 6 1 2/10 0/10 L. abrupta in vitro 6 2 4/10 0/10 L. cardium in vitro 6 2 0/10 0/10 L. abrupta in vitro 6 3 5/10 0/10 L. cardium in vitro 6 3 1/10 0/10 L. abrupta in vitro 8 1 0/10 0/10 L. cardium in vitro 8 1 0/10 0/10 L. abrupta in vitro 8 2 0/10 0/10 L. cardium in vitro 8 2 0/10 0/10 L. abrupta in vitro 8 3 0/10 0/10 L. cardium in vitro 8 3 0/10 0/10 L. abrupta-A in vivo 0 1 10/10 10/10 L. cardium in vivo 0 1 10/10 8/10 L. abrupta-A in vivo 0 2 10/10 10/10 L. cardium in vivo 0 2 10/10 8/10 L. abrupta-A in vivo 0 3 8/9 8/9 L. cardium in vivo 0 3 10/10 10/10 L. abrupta-A in vivo 0.5 1 10/10 10/10 L. cardium in vivo 0.5 1 10/10 9/10 L. abrupta-A in vivo 0.5 2 10/10 10/10 L. cardium in vivo 0.5 2 10/10 10/10 L. abrupta-A in vivo 0.5 3 10/10 10/10 L. cardium in vivo 0.5 3 10/10 9/10 L. abrupta-A in vivo 1 1 10/10 10/10 L. cardium in vivo 1 1 10/10 9/10 L. abrupta-A in vivo 1 2 11/11 11/11 L. cardium in vivo 1 2 10/10 10/10 L. abrupta-A in vivo 1 3 10/10 9/9 L. cardium in vivo 1 3 10/10 10/10 L. abrupta-A in vivo 2 1 11/11 11/11 L. cardium in vivo 2 1 10/10 9/10 L. abrupta-A in vivo 2 2 10/10 10/10 L. cardium in vivo 2 2 10/10 10/10 L. abrupta-A in vivo 2 3 10/10 10/10 L. cardium in vivo 2 3 10/10 10/10 L. abrupta-A in vivo 4 2 10/10 9/10 L. cardium in vivo 4 1 10/10 8/10 L. abrupta-A in vivo 4 3 10/10 10/10 L. cardium in vivo 4 2 10/10 7/10 L. abrupta-A in vivo 6 1 10/10 0/10 L. cardium in vivo 4 3 10/10 9/10 L. abrupta-A in vivo 6 2 10/10 0/10 L. cardium in vivo 6 1 1/10 0/10 L. abrupta-A in vivo 6 3 10/10 0/10 L. cardium in vivo 6 2 2/10 0/10 L. abrupta-A in vivo 8 1 0/10 0/10 L. cardium in vivo 6 3 0/10 0/10 L. abrupta-A in vivo 8 2 0/10 0/10 L. cardium in vivo 8 1 0/10 0/10 L. abrupta-A in vivo 8 3 0/10 0/10 L. cardium in vivo 8 2 0/10 0/10 L. abrupta-B in vivo 0 1 10/10 10/10 L. cardium in vivo 8 3 0/10 0/10 L. abrupta-B in vivo 0 2 9/10 9/10 U. imbecillis in vitro 0 1 9/9 10/10 L. abrupta-B in vivo 0 3 10/10 10/10 U. imbecillis in vitro 0 2 10/10 10/10 L. abrupta-B in vivo 0.5 1 10/10 10/10 U. imbecillis in vitro 0 3 10/10 10/10 L. abrupta-B in vivo 0.5 2 10/10 10/10 U. imbecillis in vitro 0.5 1 11/11 11/11 L. abrupta-B in vivo 0.5 3 10/10 10/10 U. imbecillis in vitro 0.5 2 10/10 10/10 L. abrupta-B in vivo 1 1 9/10 10/10 U. imbecillis in vitro 0.5 3 10/10 9/10 L. abrupta-B in vivo 1 2 10/10 10/10 U. imbecillis in vitro 1 1 10/10 9/10 L. abrupta-B in vivo 1 3 10/10 10/10 U. imbecillis in vitro 1 2 10/10 10/10 L. abrupta-B in vivo 2 1 9/9 7/9 U. imbecillis in vitro 1 3 10/10 10/10 L. abrupta-B in vivo 2 2 10/10 9/10 U. imbecillis in vitro 2 1 10/10 10/10 L. abrupta-B in vivo 2 3 9/9 9/9 U. imbecillis in vitro 2 2 10/10 10/10 L. abrupta-B in vivo 4 1 9/10 3/10 U. imbecillis in vitro 2 3 10/10 10/10 L. abrupta-B in vivo 4 2 7/10 4/10 U. imbecillis in vitro 4 1 7/10 0/10 L. abrupta-B in vivo 4 3 9/10 2/10 U. imbecillis in vitro 4 2 4/10 0/10 L. abrupta-B in vivo 6 1 0/10 0/10 U. imbecillis in vitro 4 3 5/10 0/10 L. abrupta-B in vivo 6 2 0/10 0/10 U. imbecillis in vitro 6 1 0/10 0/10 L. abrupta-B in vivo 6 3 0/10 0/10 U. imbecillis in vitro 6 2 0/10 0/10 L. abrupta-B in vivo 8 1 0/10 0/10 U. imbecillis in vitro 6 3 0/10 0/10 L. abrupta-B in vivo 8 2 0/10 0/10 U. imbecillis in vitro 8 1 0/10 0/10 L. abrupta-B in vivo 8 3 0/10 0/10 U. imbecillis in vitro 8 2 0/10 0/10 U. imbecillis in vitro 8 3 0/10 0/10 U. imbecillis in vivo 0 1 10/10 10/10 U. imbecillis in vivo 0 2 10/10 10/10 U. imbecillis in vivo 0 3 10/10 10/10 U. imbecillis in vivo 0.5 1 10/10 9/10 U. imbecillis in vivo 0.5 2 10/10 10/10 U. imbecillis in vivo 0.5 3 10/10 9/9 U. imbecillis in vivo 1 1 10/10 10/10 U. imbecillis in vivo 1 2 10/10 10/10 U. imbecillis in vivo 1 3 10/10 10/10 U. imbecillis in vivo 6 1 0/10 0/10 U. imbecillis in vivo 2 1 10/10 10/10 U. imbecillis in vivo 6 2 0/10 0/10 U. imbecillis in vivo 2 2 10/10 9/10 U. imbecillis in vivo 6 3 0/10 0/10 U. imbecillis in vivo 2 3 10/10 10/10 U. imbecillis in vivo 8 1 0/10 0/10 U. imbecillis in vivo 4 1 1/10 0/10 U. imbecillis in vivo 8 2 0/10 0/10 U. imbecillis in vivo 4 2 3/10 0/10 U. imbecillis in vivo 8 3 0/10 0/10 U. imbecillis in vivo 4 3 1/10 0/10

Table 3. Juvenile mussel survival in tests with Clearigate®.

Conc. Prop. Conc. (µg 48h 96h Prop. (µg 48h 96h Species Method Cu/L) Rep. survival survival Species Method Cu/L) Rep. survival survival L. abrupta in vitro 0 1 10/10 10/10 L. cardium in vitro 0 1 10/10 10/10 L. abrupta in vitro 0 2 10/10 10/10 L. cardium in vitro 0 2 10/10 10/10 L. abrupta in vitro 0 3 10/10 10/10 L. cardium in vitro 0 3 10/10 10/10 L. abrupta in vitro 100 1 7/7 7/7 L. cardium in vitro 25 1 10/10 10/10 L. abrupta in vitro 100 2 7/7 7/7 L. cardium in vitro 25 2 10/10 10/10 L. abrupta in vitro 100 3 7/7 7/7 L. cardium in vitro 25 3 10/10 10/10 L. abrupta in vitro 500 1 7/7 5/7 L. cardium in vitro 50 1 10/10 9/10 L. abrupta in vitro 500 2 7/7 4/7 L. cardium in vitro 50 2 10/10 9/10 L. abrupta in vitro 500 3 6/7 4/7 L. cardium in vitro 50 3 10/10 10/10 L. abrupta in vitro 1000 1 2/7 0/7 L. cardium in vitro 100 1 10/10 10/10 L. abrupta in vitro 1000 2 3/7 0/7 L. cardium in vitro 100 2 9/10 9/10 L. abrupta in vitro 1000 3 1/7 1/7 L. cardium in vitro 100 3 10/10 8/10 L. abrupta-A in vivo 0 1 10/10 10/10 L. cardium in vitro 250 1 10/10 8/10 L. abrupta-A in vivo 0 2 10/10 10/10 L. cardium in vitro 250 2 8/10 4/10 L. abrupta-A in vivo 0 3 10/10 10/10 L. cardium in vitro 250 3 10/10 4/10 L. abrupta-A in vivo 100 1 7/7 7/7 L. cardium in vitro 500 1 6/10 2/10 L. abrupta-A in vivo 100 2 7/7 7/7 L. cardium in vitro 500 2 5/10 1/10 L. abrupta-A in vivo 100 3 7/7 7/7 L. cardium in vitro 500 3 5/10 1/10 L. abrupta-A in vivo 500 1 7/7 6/7 L. cardium in vitro 750 1 2/10 0/10 L. abrupta-A in vivo 500 2 7/7 5/7 L. cardium in vitro 750 2 1/12 0/10 L. abrupta-A in vivo 500 3 7/7 4/7 L. cardium in vitro 750 3 1/10 0/10 L. abrupta-A in vivo 1000 1 5/7 0/7 L. cardium in vivo 0 1 10/10 10/10 L. abrupta-A in vivo 1000 2 6/7 0/7 L. cardium in vivo 0 2 10/10 10/10 L. abrupta-A in vivo 1000 3 3/7 0/7 L. cardium in vivo 0 3 10/10 10/10 L. abrupta-B in vivo 0 1 10/10 10/10 L. cardium in vivo 25 1 10/10 10/10 L. abrupta-B in vivo 0 2 10/10 10/10 L. cardium in vivo 25 2 10/10 10/10 L. abrupta-B in vivo 0 3 10/10 10/10 L. cardium in vivo 25 3 10/10 10/10 L. abrupta-B in vivo 50 1 10/10 10/10 L. cardium in vivo 50 1 10/10 10/10 L. abrupta-B in vivo 50 2 10/10 10/10 L. cardium in vivo 50 2 10/10 10/10 L. abrupta-B in vivo 50 3 10/10 10/10 L. cardium in vivo 50 3 10/10 10/10 L. abrupta-B in vivo 100 1 8/10 8/10 L. cardium in vivo 100 1 10/10 11/11 L. abrupta-B in vivo 100 2 9/10 9/10 L. cardium in vivo 100 2 10/10 10/10 L. abrupta-B in vivo 100 3 9/10 9/10 L. cardium in vivo 100 3 10/10 10/10 L. abrupta-B in vivo 250 1 7/10 4/10 L. cardium in vivo 250 1 9/9 9/9 L. abrupta-B in vivo 250 2 8/10 1/10 L. cardium in vivo 250 2 10/10 9/10 L. abrupta-B in vivo 250 3 9/10 3/10 L. cardium in vivo 250 3 10/10 10/10 L. abrupta-B in vivo 500 1 0/10 0/10 L. cardium in vivo 500 1 9/10 5/10 L. abrupta-B in vivo 500 2 3/10 0/10 L. cardium in vivo 500 2 8/10 5/10 L. abrupta-B in vivo 500 3 2/10 0/10 L. cardium in vivo 500 3 8/10 4/10 L. abrupta-B in vivo 750 1 0/10 0/10 L. cardium in vivo 750 1 4/10 2/10 L. abrupta-B in vivo 750 2 0/10 0/10 L. cardium in vivo 750 2 5/10 2/11 L. abrupta-B in vivo 750 3 0/10 0/10 L. cardium in vivo 750 3 5/10 2/10 L. abrupta-B in vivo 1000 1 0/10 0/10 L. abrupta-B in vivo 1000 2 0/10 0/10 L. abrupta-B in vivo 1000 3 0/10 0/10 L. abrupta-B in vivo 1500 1 0/10 0/10 L. abrupta-B in vivo 1500 2 0/10 0/10 L. abrupta-B in vivo 1500 3 0/10 0/10

Table 4. Juvenile mussel survival in tests with copper sulfate.

Prop. Conc. (µg 48h 96h Prop. Conc. (µg 48h 96h Species Method Cu/L) Rep. survival survival Species Method Cu/L) Rep. survival survival L. abrupta in vitro 0 1 7/7 7/7 L. cardium in vivo 0 1 10/10 10/10 L. abrupta in vitro 0 2 10/10 9/9 L. cardium in vivo 0 2 10/10 10/10 L. abrupta in vitro 0 3 10/10 10/10 L. cardium in vivo 0 3 10/10 10/10 L. abrupta in vitro 12.5 1 7/7 7/7 L. cardium in vivo 25 1 10/10 10/10 L. abrupta in vitro 12.5 2 7/7 6/7 L. cardium in vivo 25 2 10/10 10/10 L. abrupta in vitro 12.5 3 7/7 10/10 L. cardium in vivo 25 3 10/10 10/10 L. abrupta in vitro 25 1 7/7 7/7 L. cardium in vivo 50 1 10/10 10/10 L. abrupta in vitro 25 2 7/7 7/7 L. cardium in vivo 50 2 10/10 10/10 L. abrupta in vitro 25 3 7/7 5/7 L. cardium in vivo 50 3 10/10 10/10 L. abrupta in vitro 50 1 7/7 4/7 L. cardium in vivo 100 1 10/10 10/10 L. abrupta in vitro 50 2 7/7 2/7 L. cardium in vivo 100 2 10/10 10/10 L. abrupta in vitro 50 3 8/8 3/8 L. cardium in vivo 100 3 10/10 10/10 L. abrupta in vitro 100 1 6/10 3/10 L. cardium in vivo 200 1 6/10 3/10 L. abrupta in vitro 100 2 3/7 2/7 L. cardium in vivo 200 2 6/10 2/10 L. abrupta in vitro 100 3 3/8 1/8 L. cardium in vivo 200 3 7/10 2/10 L. abrupta in vitro 200 1 4/7 0/7 L. cardium in vivo 300 1 4/10 0/10 L. abrupta in vitro 200 2 3/7 0/7 L. cardium in vivo 300 2 5/10 0/10 L. abrupta in vitro 200 3 3/7 0/7 L. cardium in vivo 300 3 4/10 0/10 L. abrupta in vitro 300 1 0/9 0/9 L. cardium in vivo 400 1 0/10 0/10 L. abrupta in vitro 300 2 0/7 0/7 L. cardium in vivo 400 2 0/10 0/10 L. abrupta in vitro 300 3 0/9 0/9 L. cardium in vivo 400 3 0/10 0/10 L. abrupta-A in vivo 0 1 11/11 11/11 U. imbecillis in vitro 0 1 9/10 9/10 L. abrupta-A in vivo 12.5 1 7/7 7/7 U. imbecillis in vitro 0 2 10/10 10/10 L. abrupta-A in vivo 25 1 7/7 7/7 U. imbecillis in vitro 0 3 10/10 10/10 L. abrupta-A in vivo 50 1 7/7 7/7 U. imbecillis in vitro 12.5 1 9/10 9/10 L. abrupta-A in vivo 100 1 6/7 5/7 U. imbecillis in vitro 12.5 2 9/10 9/10 L. abrupta-A in vivo 200 1 7/7 4/7 U. imbecillis in vitro 12.5 3 10/10 10/10 L. abrupta-A in vivo 300 1 1/7 1/7 U. imbecillis in vitro 25 1 9/10 9/10 L. abrupta-B in vivo 0 1 10/10 10/10 U. imbecillis in vitro 25 2 9/10 9/10 L. abrupta-B in vivo 0 2 10/10 10/10 U. imbecillis in vitro 25 3 10/10 10/10 L. abrupta-B in vivo 0 3 10/10 10/10 U. imbecillis in vitro 50 1 9/10 7/10 L. abrupta-B in vivo 12.5 1 10/10 10/10 U. imbecillis in vitro 50 2 10/10 6/10 L. abrupta-B in vivo 12.5 2 10/10 10/10 U. imbecillis in vitro 50 3 10/10 8/10 L. abrupta-B in vivo 12.5 3 10/10 10/10 U. imbecillis in vitro 100 1 10/10 2/10 L. abrupta-B in vivo 25 1 10/10 10/10 U. imbecillis in vitro 100 2 10/10 3/10 L. abrupta-B in vivo 25 2 10/10 10/10 U. imbecillis in vitro 100 3 10/10 4/10 L. abrupta-B in vivo 25 3 10/10 10/10 U. imbecillis in vitro 200 1 9/10 2/10 L. abrupta-B in vivo 50 1 10/10 10/10 U. imbecillis in vitro 200 2 8/10 1/9 L. abrupta-B in vivo 50 2 10/10 10/10 U. imbecillis in vitro 200 3 9/10 1/10 L. abrupta-B in vivo 50 3 10/10 10/10 U. imbecillis in vitro 300 1 10/10 0/10 L. abrupta-B in vivo 100 1 10/10 7/10 U. imbecillis in vitro 300 2 8/10 0/10 L. abrupta-B in vivo 100 2 10/10 6/10 U. imbecillis in vitro 300 3 9/10 0/10 L. abrupta-B in vivo 100 3 10/10 7/10 U. imbecillis in vivo 0 1 10/10 8/10 L. abrupta-B in vivo 200 1 9/10 3/9 U. imbecillis in vivo 0 2 10/10 9/10 L. abrupta-B in vivo 200 2 9/9 2/9 U. imbecillis in vivo 0 3 10/10 10/10 L. abrupta-B in vivo 200 3 10/10 2/10 U. imbecillis in vivo 12.5 1 10/10 9/10 L. abrupta-B in vivo 300 1 8/10 1/10 U. imbecillis in vivo 12.5 2 7/9 7/9 L. abrupta-B in vivo 300 2 7/10 0/10 U. imbecillis in vivo 12.5 3 10/10 10/10 L. abrupta-B in vivo 300 3 7/10 0/10 U. imbecillis in vivo 25 1 10/10 6/10 L. cardium in vitro 0 1 10/10 10/10 U. imbecillis in vivo 25 2 9/10 8/10 L. cardium in vitro 0 2 9/9 10/10 U. imbecillis in vivo 25 3 10/10 10/10 L. cardium in vitro 0 3 9/9 9/9 U. imbecillis in vivo 50 1 10/10 8/10 L. cardium in vitro 25 1 10/10 10/10 U. imbecillis in vivo 50 2 9/10 8/10 L. cardium in vitro 25 2 10/10 9/10 U. imbecillis in vivo 50 3 10/10 10/10 L. cardium in vitro 25 3 10/10 10/10 U. imbecillis in vivo 100 1 9/9 7/9 L. cardium in vitro 50 1 10/10 8/10 U. imbecillis in vivo 100 2 10/10 5/10 L. cardium in vitro 50 2 10/10 8/10 U. imbecillis in vivo 100 3 10/10 4/10 L. cardium in vitro 50 3 10/10 7/10 U. imbecillis in vivo 200 1 10/10 3/10 L. cardium in vitro 100 1 5/10 5/10 U. imbecillis in vivo 200 2 10/10 1/10 L. cardium in vitro 100 2 4/10 4/10 U. imbecillis in vivo 200 3 10/10 2/10 L. cardium in vitro 100 3 7/10 4/10 U. imbecillis in vivo 300 1 10/10 2/10 L. cardium in vitro 200 1 1/10 1/10 U. imbecillis in vivo 300 2 8/10 0/10 L. cardium in vitro 200 2 5/10 2/10 U. imbecillis in vivo 300 3 9/10 0/10 L. cardium in vitro 200 3 3/10 0/10 L. cardium in vitro 300 1 1/10 0/10 L. cardium in vitro 300 2 2/10 0/10 L. cardium in vitro 300 3 3/10 0/10 L. cardium in vitro 400 1 0/10 0/10 L. cardium in vitro 400 2 0/10 0/10 L. cardium in vitro 400 3 0/10 0/10

Table 5. Juvenile mussel survival in tests with Nautique®.

Conc. Prop. Conc. (µg 48h 96h Prop. (µg 48h 96h Species Method Cu/L) Rep. survival survival Species Method Cu/L) Rep. survival survival L. abrupta in vitro 0 1 10/10 9/10 L. cardium in vivo 0 1 10/10 10/10 L. abrupta in vitro 0 2 10/10 10/10 L. cardium in vivo 0 2 10/10 10/10 L. abrupta in vitro 0 3 10/10 10/10 L. cardium in vivo 0 3 10/10 10/10 L. abrupta in vitro 250 1 7/7 7/7 L. cardium in vivo 250 1 10/10 10/10 L. abrupta in vitro 250 2 8/8 8/8 L. cardium in vivo 250 2 10/10 10/10 L. abrupta in vitro 250 3 7/7 6/6 L. cardium in vivo 250 3 10/10 10/10 L. abrupta in vitro 500 1 7/7 7/7 L. cardium in vivo 500 1 10/10 10/10 L. abrupta in vitro 500 2 7/7 7/7 L. cardium in vivo 500 2 10/10 10/10 L. abrupta in vitro 500 3 6/6 7/7 L. cardium in vivo 500 3 10/10 10/10 L. abrupta in vitro 1000 1 7/7 7/7 L. cardium in vivo 1000 1 10/10 10/10 L. abrupta in vitro 1000 2 7/7 7/7 L. cardium in vivo 1000 2 10/10 10/10 L. abrupta in vitro 1000 3 7/7 7/7 L. cardium in vivo 1000 3 10/10 10/10 L. abrupta in vitro 2000 1 7/7 7/7 L. cardium in vivo 2000 1 10/10 10/10 L. abrupta in vitro 2000 2 7/7 6/7 L. cardium in vivo 2000 2 10/10 10/10 L. abrupta in vitro 2000 3 7/7 7/7 L. cardium in vivo 2000 3 10/10 10/10 L. abrupta in vitro 4000 1 7/8 5/7 L. cardium in vivo 4000 1 10/10 10/10 L. abrupta in vitro 4000 2 8/8 4/7 L. cardium in vivo 4000 2 10/10 10/10 L. abrupta in vitro 4000 3 7/7 2/7 L. cardium in vivo 4000 3 10/10 10/10 L. abrupta in vitro 8000 1 0/7 0/7 L. cardium in vivo 8000 1 11/11 0/11 L. abrupta in vitro 8000 2 1/7 0/7 L. cardium in vivo 8000 2 10/10 0/10 L. abrupta in vitro 8000 3 2/7 0/7 L. cardium in vivo 8000 3 10/10 0/10 L. abrupta-A in vivo 0 1 10/10 10/10 U. imbecillis in vitro 0 1 10/10 10/10 L. abrupta-A in vivo 250 1 10/10 10/10 U. imbecillis in vitro 0 2 10/10 10/10 L. abrupta-A in vivo 500 1 10/10 10/10 U. imbecillis in vitro 0 3 10/10 10/10 L. abrupta-A in vivo 1000 1 10/10 10/10 U. imbecillis in vitro 12.5 1 10/10 10/10 L. abrupta-A in vivo 2000 1 10/10 10/10 U. imbecillis in vitro 12.5 2 10/10 10/10 L. abrupta-A in vivo 4000 1 10/10 8/10 U. imbecillis in vitro 12.5 3 10/10 10/10 L. abrupta-A in vivo 8000 1 9/10 0/10 U. imbecillis in vitro 25 1 10/10 10/10 L. abrupta-B in vivo 0 1 10/10 10/10 U. imbecillis in vitro 25 2 10/10 10/10 L. abrupta-B in vivo 0 2 10/10 10/10 U. imbecillis in vitro 25 3 10/10 10/10 L. abrupta-B in vivo 0 3 10/10 10/10 U. imbecillis in vitro 50 1 10/10 10/10 L. abrupta-B in vivo 250 1 10/10 10/10 U. imbecillis in vitro 50 2 10/10 10/10 L. abrupta-B in vivo 250 2 10/10 8/10 U. imbecillis in vitro 50 3 10/10 10/10 L. abrupta-B in vivo 250 3 10/10 10/10 U. imbecillis in vitro 100 1 9/10 9/10 L. abrupta-B in vivo 500 1 10/10 10/10 U. imbecillis in vitro 100 2 10/10 9/10 L. abrupta-B in vivo 500 2 10/10 10/10 U. imbecillis in vitro 100 3 10/10 10/10 L. abrupta-B in vivo 500 3 10/10 10/10 U. imbecillis in vitro 200 1 10/10 9/10 L. abrupta-B in vivo 1000 1 10/10 10/10 U. imbecillis in vitro 200 2 10/10 10/10 L. abrupta-B in vivo 1000 2 10/10 10/10 U. imbecillis in vitro 200 3 10/10 7/10 L. abrupta-B in vivo 1000 3 10/10 10/10 U. imbecillis in vitro 300 1 10/10 8/10 L. abrupta-B in vivo 2000 1 10/10 9/10 U. imbecillis in vitro 300 2 10/10 8/10 L. abrupta-B in vivo 2000 2 10/10 9/10 U. imbecillis in vitro 300 3 10/10 7/10 L. abrupta-B in vivo 2000 3 10/10 10/10 U. imbecillis in vitro 1000 1 8/10 4/10 L. abrupta-B in vivo 4000 1 10/10 2/10 U. imbecillis in vivo 0 1 10/10 9/10 L. abrupta-B in vivo 4000 2 10/10 2/10 U. imbecillis in vivo 0 2 10/10 10/10 L. abrupta-B in vivo 4000 3 10/10 6/10 U. imbecillis in vivo 0 3 10/10 10/10 L. abrupta-B in vivo 8000 1 0/10 0/10 U. imbecillis in vivo 12.5 1 10/10 10/10 L. abrupta-B in vivo 8000 2 0/10 0/10 U. imbecillis in vivo 12.5 2 9/9 9/9 L. abrupta-B in vivo 8000 3 0/10 0/10 U. imbecillis in vivo 12.5 3 10/10 10/10 L. cardium in vitro 0 1 10/10 10/10 U. imbecillis in vivo 25 1 10/10 10/10 L. cardium in vitro 0 2 10/10 10/10 U. imbecillis in vivo 25 2 10/10 10/10 L. cardium in vitro 0 3 10/10 10/10 U. imbecillis in vivo 25 3 10/10 9/9 L. cardium in vitro 250 1 10/10 10/10 U. imbecillis in vivo 50 1 10/10 10/10 L. cardium in vitro 250 2 10/10 10/10 U. imbecillis in vivo 50 2 10/10 10/10 L. cardium in vitro 250 3 10/10 10/10 U. imbecillis in vivo 50 3 10/10 10/10 L. cardium in vitro 500 1 10/10 10/10 U. imbecillis in vivo 100 1 10/10 9/9 L. cardium in vitro 500 2 10/10 10/10 U. imbecillis in vivo 100 2 10/10 10/10 L. cardium in vitro 500 3 10/10 10/10 U. imbecillis in vivo 100 3 10/10 10/10 L. cardium in vitro 1000 1 10/10 10/10 U. imbecillis in vivo 200 1 10/10 10/10 L. cardium in vitro 1000 2 10/10 10/10 U. imbecillis in vivo 200 2 10/10 10/10 L. cardium in vitro 1000 3 10/10 10/10 U. imbecillis in vivo 200 3 10/10 10/10 L. cardium in vitro 2000 1 10/10 10/10 U. imbecillis in vivo 300 1 9/9 8/10 L. cardium in vitro 2000 2 10/10 10/10 U. imbecillis in vivo 300 2 10/10 9/10 L. cardium in vitro 2000 3 10/10 10/10 U. imbecillis in vivo 300 3 10/10 10/10 L. cardium in vitro 4000 1 9/10 7/10 U. imbecillis in vivo 1000 1 10/10 9/10 L. cardium in vitro 4000 2 10/10 10/10 L. cardium in vitro 4000 3 10/10 9/10 L. cardium in vitro 8000 1 10/11 0/11 L. cardium in vitro 8000 2 8/10 0/10 L. cardium in vitro 8000 3 9/10 0/10

Table 6. Juvenile mussel survival in tests with nickel chloride

Prop. Conc. (µg 48h 96h Prop. Conc. (µg 48h 96h Species Method Ni/L) Rep. survival survival Species Method Ni/L) Rep. survival survival L. abrupta in vitro 0 1 10/10 9/10 L. cardium in vivo 0 1 10/10 10/10 L. abrupta in vitro 0 2 9/9 9/9 L. cardium in vivo 0 2 9/10 9/10 L. abrupta in vitro 0 3 9/10 7/10 L. cardium in vivo 0 3 9/10 9/10 L. abrupta in vitro 100 1 7/7 7/7 L. cardium in vivo 100 1 10/10 10/10 L. abrupta in vitro 100 2 7/7 7/7 L. cardium in vivo 100 2 10/10 10/10 L. abrupta in vitro 100 3 7/7 7/7 L. cardium in vivo 100 3 10/10 10/10 L. abrupta in vitro 250 1 7/7 7/7 L. cardium in vivo 250 1 9/10 9/10 L. abrupta in vitro 250 2 7/7 7/7 L. cardium in vivo 250 2 10/10 10/10 L. abrupta in vitro 250 3 7/7 7/7 L. cardium in vivo 250 3 9/10 9/10 L. abrupta in vitro 500 1 6/7 6/7 L. cardium in vivo 500 1 10/10 9/10 L. abrupta in vitro 500 2 7/7 7/7 L. cardium in vivo 500 2 10/10 9/10 L. abrupta in vitro 500 3 7/7 7/7 L. cardium in vivo 500 3 9/10 9/10 L. abrupta in vitro 750 1 7/7 7/7 L. cardium in vivo 750 1 9/10 4/10 L. abrupta in vitro 750 2 6/7 5/7 L. cardium in vivo 750 2 8/10 4/10 L. abrupta in vitro 750 3 5/6 6/7 L. cardium in vivo 750 3 9/10 3/10 L. abrupta in vitro 1000 1 7/7 7/7 L. cardium in vivo 1000 1 9/11 3/11 L. abrupta in vitro 1000 2 7/7 6/7 L. cardium in vivo 1000 2 7/10 0/10 L. abrupta in vitro 1000 3 7/7 7/7 L. cardium in vivo 1000 3 7/10 3/10 L. abrupta in vitro 1500 1 3/7 0/7 L. cardium in vivo 1500 1 1/10 0/10 L. abrupta in vitro 1500 2 1/7 0/7 L. cardium in vivo 1500 2 3/10 0/10 L. abrupta in vitro 1500 3 4/7 0/7 L. cardium in vivo 1500 3 4/10 0/10 L. abrupta-A in vivo 0 1 10/10 10/10 U. imbecillis in vitro 0 1 10/10 10/10 L. abrupta-A in vivo 100 1 7/7 7/7 U. imbecillis in vitro 0 2 10/10 10/10 L. abrupta-A in vivo 250 1 7/7 7/7 U. imbecillis in vitro 0 3 10/10 10/10 L. abrupta-A in vivo 500 1 7/7 7/7 U. imbecillis in vitro 100 1 10/10 10/10 L. abrupta-A in vivo 750 1 7/7 7/7 U. imbecillis in vitro 100 2 10/10 10/10 L. abrupta-A in vivo 1000 1 7/7 7/7 U. imbecillis in vitro 100 3 10/10 10/10 L. abrupta-A in vivo 1500 1 4/7 3/7 U. imbecillis in vitro 250 1 10/10 10/10 L. abrupta-B in vivo 0 1 10/10 10/10 U. imbecillis in vitro 250 2 10/10 10/10 L. abrupta-B in vivo 0 2 10/10 10/10 U. imbecillis in vitro 250 3 10/10 10/10 L. abrupta-B in vivo 0 3 9/10 9/10 U. imbecillis in vitro 500 1 10/10 10/10 L. abrupta-B in vivo 100 1 10/10 10/10 U. imbecillis in vitro 500 2 10/10 10/10 L. abrupta-B in vivo 100 2 9/9 9/9 U. imbecillis in vitro 500 3 10/10 10/10 L. abrupta-B in vivo 100 3 10/10 10/10 U. imbecillis in vitro 750 1 10/10 9/10 L. abrupta-B in vivo 250 1 10/10 10/10 U. imbecillis in vitro 750 2 10/10 10/10 L. abrupta-B in vivo 250 2 9/9 9/9 U. imbecillis in vitro 750 3 10/10 10/10 L. abrupta-B in vivo 250 3 10/10 10/10 U. imbecillis in vitro 1000 1 10/10 10/10 L. abrupta-B in vivo 500 1 8/9 9/9 U. imbecillis in vitro 1000 2 10/10 10/10 L. abrupta-B in vivo 500 2 10/10 10/10 U. imbecillis in vitro 1000 3 10/10 8/10 L. abrupta-B in vivo 500 3 10/10 9/9 U. imbecillis in vitro 1500 1 8/10 2/10 L. abrupta-B in vivo 750 1 10/10 11/11 U. imbecillis in vitro 1500 2 7/10 0/10 L. abrupta-B in vivo 750 2 10/10 10/10 U. imbecillis in vitro 1500 3 10/10 3/10 L. abrupta-B in vivo 750 3 10/10 9/10 U. imbecillis in vivo 0 1 10/10 8/9 L. abrupta-B in vivo 1000 1 9/10 10/10 U. imbecillis in vivo 0 2 10/10 10/10 L. abrupta-B in vivo 1000 2 9/9 8/10 U. imbecillis in vivo 0 3 10/10 10/10 L. abrupta-B in vivo 1000 3 10/10 9/10 U. imbecillis in vivo 100 1 10/10 10/10 L. abrupta-B in vivo 1500 1 3/10 3/10 U. imbecillis in vivo 100 2 10/10 9/9 L. abrupta-B in vivo 1500 2 4/10 0/10 U. imbecillis in vivo 100 3 10/10 10/10 L. abrupta-B in vivo 1500 3 4/10 0/10 U. imbecillis in vivo 250 1 10/10 10/10 L. cardium in vitro 0 1 10/10 9/10 U. imbecillis in vivo 250 2 10/10 10/10 L. cardium in vitro 0 2 10/10 10/10 U. imbecillis in vivo 250 3 10/10 10/10 L. cardium in vitro 0 3 10/10 9/10 U. imbecillis in vivo 500 1 10/10 10/10 L. cardium in vitro 100 1 9/10 9/10 U. imbecillis in vivo 500 2 10/10 10/10 L. cardium in vitro 100 2 10/10 10/10 U. imbecillis in vivo 500 3 10/10 10/10 L. cardium in vitro 100 3 9/10 9/10 U. imbecillis in vivo 750 1 10/10 10/10 L. cardium in vitro 250 1 10/10 10/10 U. imbecillis in vivo 750 2 10/10 10/10 L. cardium in vitro 250 2 9/10 9/10 U. imbecillis in vivo 750 3 10/10 10/10 L. cardium in vitro 250 3 10/10 10/10 U. imbecillis in vivo 1000 1 10/10 10/10 L. cardium in vitro 500 1 10/10 10/10 U. imbecillis in vivo 1000 2 10/10 8/10 L. cardium in vitro 500 2 10/10 10/10 U. imbecillis in vivo 1000 3 10/10 10/10 L. cardium in vitro 500 3 10/10 7/10 U. imbecillis in vivo 1500 1 7/9 3/10 L. cardium in vitro 750 1 9/10 5/10 U. imbecillis in vivo 1500 2 8/10 4/10 L. cardium in vitro 750 2 7/10 4/10 U. imbecillis in vivo 1500 3 3/10 0/10 L. cardium in vitro 750 3 8/10 5/10 L. cardium in vitro 1000 1 10/10 1/10 L. cardium in vitro 1000 2 9/10 1/10 L. cardium in vitro 1000 3 9/10 0/10 L. cardium in vitro 1500 1 2/10 0/10 L. cardium in vitro 1500 2 3/10 0/10 L. cardium in vitro 1500 3 2/10 0/10

Appendix E Table 1. Survival data for L. abrupta in vitro juvenile mussels. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival L. abrupta 6 in vitro 0 1 9/10 8/10 L. abrupta 6 in vitro 0 2 9/10 6/7 L. abrupta 6 in vitro 0 3 9/10 9/10 L. abrupta 6 in vitro 12.5 1 7/12 6/12 L. abrupta 6 in vitro 12.5 2 11/11 9/10 L. abrupta 6 in vitro 12.5 3 6/9 3/9 L. abrupta 6 in vitro 25 1 7/10 5/10 L. abrupta 6 in vitro 25 2 8/10 3/10 L. abrupta 6 in vitro 25 3 6/10 4/10 L. abrupta 6 in vitro 50 1 8/10 0/10 L. abrupta 6 in vitro 50 2 11/12 2/11 L. abrupta 6 in vitro 50 3 10/10 0/7 L. abrupta 6 in vitro 100 1 2/10 0/10 L. abrupta 6 in vitro 100 2 6/10 0/10 L. abrupta 6 in vitro 100 3 4/10 0/10 L. abrupta 6 in vitro 200 1 4/10 0/10 L. abrupta 6 in vitro 200 2 2/12 0/10 L. abrupta 6 in vitro 200 3 7/10 0/10 L. abrupta 6 in vitro 300 1 1/10 0/10 L. abrupta 6 in vitro 300 2 1/12 0/10 L. abrupta 6 in vitro 300 3 1/10 0/10 L. abrupta 12 in vitro 0 1 8/10 8/10 L. abrupta 12 in vitro 0 2 10/10 10/10 L. abrupta 12 in vitro 0 3 9/10 9/10 L. abrupta 12 in vitro 12.5 1 10/10 7/10 L. abrupta 12 in vitro 12.5 2 10/10 8/10 L. abrupta 12 in vitro 12.5 3 8/10 7/8 L. abrupta 12 in vitro 25 1 8/10 4/10 L. abrupta 12 in vitro 25 2 4/9 4/8 L. abrupta 12 in vitro 25 3 9/10 6/10 L. abrupta 12 in vitro 50 1 5/8 1/8 L. abrupta 12 in vitro 50 2 5/10 3/10 L. abrupta 12 in vitro 50 3 6/10 2/10 L. abrupta 12 in vitro 100 1 1/10 0/10 L. abrupta 12 in vitro 100 2 0/10 0/10 L. abrupta 12 in vitro 100 3 2/11 0/11 L. abrupta 12 in vitro 200 1 0/10 0/10 L. abrupta 12 in vitro 200 2 0/9 0/9 L. abrupta 12 in vitro 200 3 0/10 0/10 L. abrupta 12 in vitro 300 1 0/10 0/10 L. abrupta 12 in vitro 300 2 0/10 0/10 L. abrupta 12 in vitro 300 3 0/11 0/11

Table 2. Survival data for L. abrupta in vivo juvenile mussels. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival L. abrupta 12 in vivo 0 1 7/7 7/7 L. abrupta 12 in vivo 0 2 9/9 9/9 L. abrupta 12 in vivo 0 3 10/10 9/10 L. abrupta 12 in vivo 12.5 1 9/10 9/10 L. abrupta 12 in vivo 12.5 2 10/10 10/10 L. abrupta 12 in vivo 12.5 3 10/10 9/10 L. abrupta 12 in vivo 25 1 9/10 9/10 L. abrupta 12 in vivo 25 2 10/10 10/10 L. abrupta 12 in vivo 25 3 9/10 9/10 L. abrupta 12 in vivo 50 1 9/10 8/10 L. abrupta 12 in vivo 50 2 9/9 6/9 L. abrupta 12 in vivo 50 3 9/10 8/10 L. abrupta 12 in vivo 100 1 8/10 6/10 L. abrupta 12 in vivo 100 2 8/10 6/10 L. abrupta 12 in vivo 100 3 8/10 5/10 L. abrupta 12 in vivo 200 1 8/10 4/10 L. abrupta 12 in vivo 200 2 8/10 3/10 L. abrupta 12 in vivo 200 3 6/10 4/10 L. abrupta 12 in vivo 300 1 4/10 0/10 L. abrupta 12 in vivo 300 2 8/10 0/10 L. abrupta 12 in vivo 300 3 6/10 0/10 L. abrupta 18 in vivo 0 1 10/10 10/10 L. abrupta 18 in vivo 0 2 10/10 10/10 L. abrupta 18 in vivo 0 3 11/11 10/11 L. abrupta 18 in vivo 12.5 1 10/10 8/10 L. abrupta 18 in vivo 12.5 2 10/10 7/10 L. abrupta 18 in vivo 12.5 3 10/10 7/9 L. abrupta 18 in vivo 25 1 10/10 10/10 L. abrupta 18 in vivo 25 2 10/10 10/10 L. abrupta 18 in vivo 25 3 9/10 8/10 L. abrupta 18 in vivo 50 1 10/10 10/10 L. abrupta 18 in vivo 50 2 10/10 8/10 L. abrupta 18 in vivo 50 3 9/10 8/10 L. abrupta 18 in vivo 100 1 8/10 8/10 L. abrupta 18 in vivo 100 2 8/10 3/10 L. abrupta 18 in vivo 100 3 8/10 3/10 L. abrupta 18 in vivo 200 1 4/10 2/10 L. abrupta 18 in vivo 200 2 3/10 0/10 L. abrupta 18 in vivo 200 3 4/10 2/10 L. abrupta 18 in vivo 300 1 2/10 0/10 L. abrupta 18 in vivo 300 2 0/10 0/10 L. abrupta 18 in vivo 300 3 3/10 0/10

Table 3. Survival data for L. cardium in vitro juvenile mussels. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival L. cardium 4 in vitro 0 1 10/10 10/10 L. cardium 4 in vitro 0 2 10/10 10/10 L. cardium 4 in vitro 0 3 10/10 10/10 L. cardium 4 in vitro 12.5 1 10/10 10/10 L. cardium 4 in vitro 12.5 2 10/10 0/10 L. cardium 4 in vitro 12.5 3 10/10 10/10 L. cardium 4 in vitro 25 1 9/10 8/10 L. cardium 4 in vitro 25 2 10/10 9/10 L. cardium 4 in vitro 25 3 10/10 8/10 L. cardium 4 in vitro 50 1 11/11 4/11 L. cardium 4 in vitro 50 2 10/10 6/10 L. cardium 4 in vitro 50 3 9/10 3/10 L. cardium 4 in vitro 100 1 5/11 1/11 L. cardium 4 in vitro 100 2 4/10 0/10 L. cardium 4 in vitro 100 3 5/10 1/10 L. cardium 4 in vitro 200 1 2/10 0/10 L. cardium 4 in vitro 200 2 3/10 0/10 L. cardium 4 in vitro 200 3 2/10 0/10 L. cardium 4 in vitro 300 1 0/10 0/10 L. cardium 4 in vitro 300 2 0/10 0/10 L. cardium 4 in vitro 300 3 0/10 0/10 L. cardium 5 in vitro 0 1 10/10 9/9 L. cardium 5 in vitro 0 2 10/10 10/10 L. cardium 5 in vitro 0 3 10/10 10/10 L. cardium 5 in vitro 12.5 1 9/9 9/9 L. cardium 5 in vitro 12.5 2 9/9 9/9 L. cardium 5 in vitro 12.5 3 10/10 9/10 L. cardium 5 in vitro 25 1 9/10 9/10 L. cardium 5 in vitro 25 2 10/11 9/11 L. cardium 5 in vitro 25 3 9/9 8/9 L. cardium 5 in vitro 50 1 10/10 7/10 L. cardium 5 in vitro 50 2 10/11 9/11 L. cardium 5 in vitro 50 3 9/10 5/10 L. cardium 5 in vitro 100 1 10/10 3/10 L. cardium 5 in vitro 100 2 9/10 4/10 L. cardium 5 in vitro 100 3 9/10 2/10 L. cardium 5 in vitro 200 1 9/10 0/10 L. cardium 5 in vitro 200 2 10/10 0/10 L. cardium 5 in vitro 200 3 8/10 0/10 L. cardium 5 in vitro 300 1 0/10 0/10 L. cardium 5 in vitro 300 2 1/10 0/10 L. cardium 5 in vitro 300 3 0/10 0/10

Table 4. Survival data for Lampsilis cardium in vitro juvenile mussels, age 17-19 days. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival L. cardium 17 in vitro 0 1 10/10 6/10 L. cardium 17 in vitro 0 2 9/9 9/9 L. cardium 17 in vitro 0 3 10/10 9/10 L. cardium 17 in vitro 12.5 1 10/10 10/10 L. cardium 17 in vitro 12.5 2 10/10 10/10 L. cardium 17 in vitro 12.5 3 10/10 10/10 L. cardium 17 in vitro 25 1 10/10 10/10 L. cardium 17 in vitro 25 2 10/10 9/10 L. cardium 17 in vitro 25 3 10/10 10/10 L. cardium 17 in vitro 50 1 10/10 9/10 L. cardium 17 in vitro 50 2 11/11 5/10 L. cardium 17 in vitro 50 3 10/10 8/10 L. cardium 17 in vitro 100 1 10/10 6/10 L. cardium 17 in vitro 100 2 10/10 5/10 L. cardium 17 in vitro 100 3 11/11 5/11 L. cardium 17 in vitro 200 1 6/11 0/10 L. cardium 17 in vitro 200 2 4/10 0/10 L. cardium 17 in vitro 200 3 4/10 0/10 L. cardium 17 in vitro 300 1 2/11 0/11 L. cardium 17 in vitro 300 2 3/10 0/10 L. cardium 17 in vitro 300 3 1/10 0/10 L. cardium 19 in vitro 0 1 10/10 10/10 L. cardium 19 in vitro 0 2 10/10 9/9 L. cardium 19 in vitro 0 3 10/10 10/10 L. cardium 19 in vitro 12.5 1 11/11 11/11 L. cardium 19 in vitro 12.5 2 10/10 10/10 L. cardium 19 in vitro 12.5 3 11/11 11/11 L. cardium 19 in vitro 25 1 11/11 10/10 L. cardium 19 in vitro 25 2 10/10 10/10 L. cardium 19 in vitro 25 3 10/10 10/10 L. cardium 19 in vitro 50 1 10/10 5/10 L. cardium 19 in vitro 50 2 10/10 6/10 L. cardium 19 in vitro 50 3 10/10 5/10 L. cardium 19 in vitro 100 1 11/11 0/10 L. cardium 19 in vitro 100 2 9/9 1/9 L. cardium 19 in vitro 100 3 11/11 0/10 L. cardium 19 in vitro 200 1 3/10 0/10 L. cardium 19 in vitro 200 2 4/10 0/10 L. cardium 19 in vitro 200 3 3/11 0/10 L. cardium 19 in vitro 300 1 1/10 0/10 L. cardium 19 in vitro 300 2 0/10 0/10 L. cardium 19 in vitro 300 3 0/10 0/10

Table 5. Survival data for Lampsilis cardium in vivo juvenile mussels, age 17-19 days. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival L. cardium 3 in vivo 0 1 10/10 10/10 L. cardium 3 in vivo 0 2 10/10 10/10 L. cardium 3 in vivo 0 3 10/10 10/10 L. cardium 3 in vivo 12.5 1 10/10 10/10 L. cardium 3 in vivo 12.5 2 11/11 11/11 L. cardium 3 in vivo 12.5 3 10/10 10/10 L. cardium 3 in vivo 25 1 10/10 9/10 L. cardium 3 in vivo 25 2 10/10 9/10 L. cardium 3 in vivo 25 3 10/10 9/10 L. cardium 3 in vivo 50 1 9/10 8/10 L. cardium 3 in vivo 50 2 10/10 9/10 L. cardium 3 in vivo 50 3 10/10 8/10 L. cardium 3 in vivo 100 1 9/11 0/11 L. cardium 3 in vivo 100 2 9/10 1/10 L. cardium 3 in vivo 100 3 8/10 0/10 L. cardium 3 in vivo 200 1 3/10 0/10 L. cardium 3 in vivo 200 2 2/10 0/10 L. cardium 3 in vivo 200 3 3/10 0/10 L. cardium 3 in vivo 300 1 0/10 0/12 L. cardium 3 in vivo 300 2 0/10 0/11 L. cardium 3 in vivo 300 3 0/11 0/11 L. cardium 7 in vivo 0 1 10/10 10/10 L. cardium 7 in vivo 0 2 9/10 8/10 L. cardium 7 in vivo 0 3 10/11 9/11 L. cardium 7 in vivo 12.5 1 9/10 9/10 L. cardium 7 in vivo 12.5 2 9/10 8/10 L. cardium 7 in vivo 12.5 3 9/10 9/11 L. cardium 7 in vivo 25 1 10/10 9/10 L. cardium 7 in vivo 25 2 8/10 7/10 L. cardium 7 in vivo 25 3 10/10 10/10 L. cardium 7 in vivo 50 1 9/10 7/10 L. cardium 7 in vivo 50 2 10/10 8/10 L. cardium 7 in vivo 50 3 10/10 6/10 L. cardium 7 in vivo 100 1 10/10 2/10 L. cardium 7 in vivo 100 2 10/10 2/10 L. cardium 7 in vivo 100 3 9/11 1/11 L. cardium 7 in vivo 200 1 9/11 0/11 L. cardium 7 in vivo 200 2 8/10 0/10 L. cardium 7 in vivo 200 3 8/11 0/10 L. cardium 7 in vivo 300 1 2/10 0/10 L. cardium 7 in vivo 300 2 0/10 0/10 L. cardium 7 in vivo 300 3 1/10 0/10

Table 6. Survival data for Utterbackia imbecillis in vitro juvenile mussels, age 3 and 17 days. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival U. imbecillis 3 in vitro 0 1 10/10 10/10 U. imbecillis 3 in vitro 0 2 9/10 7/10 U. imbecillis 3 in vitro 0 3 10/10 9/10 U. imbecillis 3 in vitro 12.5 1 9/10 9/10 U. imbecillis 3 in vitro 12.5 2 7/10 7/10 U. imbecillis 3 in vitro 12.5 3 10/10 8/10 U. imbecillis 3 in vitro 25 1 8/10 5/10 U. imbecillis 3 in vitro 25 2 8/10 7/10 U. imbecillis 3 in vitro 25 3 10/10 4/10 U. imbecillis 3 in vitro 50 1 8/11 4/11 U. imbecillis 3 in vitro 50 2 9/10 5/11 U. imbecillis 3 in vitro 50 3 10/10 5/10 U. imbecillis 3 in vitro 100 1 6/10 0/10 U. imbecillis 3 in vitro 100 2 2/10 0/10 U. imbecillis 3 in vitro 100 3 4/10 0/10 U. imbecillis 3 in vitro 200 1 1/10 0/10 U. imbecillis 3 in vitro 200 2 0/10 0/10 U. imbecillis 3 in vitro 200 3 0/10 0/10 U. imbecillis 3 in vitro 300 1 0/10 0/10 U. imbecillis 3 in vitro 300 2 0/10 0/10 U. imbecillis 3 in vitro 300 3 0/10 0/10 U. imbecillis 17 in vitro 0 1 10/10 10/10 U. imbecillis 17 in vitro 0 2 9/10 9/10 U. imbecillis 17 in vitro 0 3 10/10 10/10 U. imbecillis 17 in vitro 12.5 1 10/10 9/10 U. imbecillis 17 in vitro 12.5 2 10/10 9/10 U. imbecillis 17 in vitro 12.5 3 10/10 10/10 U. imbecillis 17 in vitro 25 1 9/9 8/10 U. imbecillis 17 in vitro 25 2 9/9 6/10 U. imbecillis 17 in vitro 25 3 9/9 7/10 U. imbecillis 17 in vitro 50 1 7/10 4/10 U. imbecillis 17 in vitro 50 2 8/10 5/10 U. imbecillis 17 in vitro 50 3 7/10 7/10 U. imbecillis 17 in vitro 100 1 7/10 2/10 U. imbecillis 17 in vitro 100 2 7/10 4/10 U. imbecillis 17 in vitro 100 3 6/10 2/10 U. imbecillis 17 in vitro 200 1 4/11 0/11 U. imbecillis 17 in vitro 200 2 5/10 0/10 U. imbecillis 17 in vitro 200 3 3/10 0/10 U. imbecillis 17 in vitro 300 1 0/11 0/11 U. imbecillis 17 in vitro 300 2 0/10 0/10 U. imbecillis 17 in vitro 300 3 0/11 0/11

Table 7. Survival data for Utterbackia imbecillis in vitro and in vivo juvenile mussels, age 27 and 3 days. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival U. imbecillis 27 in vitro 0 1 10/10 8/10 U. imbecillis 27 in vitro 0 2 9/10 8/10 U. imbecillis 27 in vitro 0 3 9/9 9/9 U. imbecillis 27 in vitro 12.5 1 9/10 9/10 U. imbecillis 27 in vitro 12.5 2 9/10 9/10 U. imbecillis 27 in vitro 12.5 3 9/10 8/10 U. imbecillis 27 in vitro 25 1 9/10 4/10 U. imbecillis 27 in vitro 25 2 10/10 10/10 U. imbecillis 27 in vitro 25 3 9/10 8/10 U. imbecillis 27 in vitro 50 1 9/10 8/10 U. imbecillis 27 in vitro 50 2 8/10 7/10 U. imbecillis 27 in vitro 50 3 8/10 4/10 U. imbecillis 27 in vitro 100 1 6/10 2/10 U. imbecillis 27 in vitro 100 2 6/10 3/10 U. imbecillis 27 in vitro 100 3 7/10 3/10 U. imbecillis 27 in vitro 200 1 6/10 0/10 U. imbecillis 27 in vitro 200 2 3/10 0/10 U. imbecillis 27 in vitro 200 3 3/10 0/10 U. imbecillis 27 in vitro 300 1 2/10 0/10 U. imbecillis 27 in vitro 300 2 2/10 0/10 U. imbecillis 27 in vitro 300 3 3/10 0/10 U. imbecillis 3 in vivo 0 1 9/10 9/10 U. imbecillis 3 in vivo 0 2 10/10 10/10 U. imbecillis 3 in vivo 0 3 10/10 9/10 U. imbecillis 3 in vivo 12.5 1 9/10 7/10 U. imbecillis 3 in vivo 12.5 2 10/10 9/10 U. imbecillis 3 in vivo 12.5 3 10/10 8/10 U. imbecillis 3 in vivo 25 1 7/10 7/10 U. imbecillis 3 in vivo 25 2 9/10 7/10 U. imbecillis 3 in vivo 25 3 7/10 5/10 U. imbecillis 3 in vivo 50 1 7/10 3/10 U. imbecillis 3 in vivo 50 2 6/10 4/10 U. imbecillis 3 in vivo 50 3 5/10 1/10 U. imbecillis 3 in vivo 100 1 5/10 2/10 U. imbecillis 3 in vivo 100 2 4/10 1/10 U. imbecillis 3 in vivo 100 3 0/10 0/10 U. imbecillis 3 in vivo 200 1 0/10 0/10 U. imbecillis 3 in vivo 200 2 0/10 0/10 U. imbecillis 3 in vivo 200 3 1/10 0/10 U. imbecillis 3 in vivo 300 1 0/10 0/10 U. imbecillis 3 in vivo 300 2 0/10 0/10 U. imbecillis 3 in vivo 300 3 0/10 0/10

Table 8. Survival data for Utterbackia imbecillis in vitro and in vivo juvenile mussels, age 27 and 3 days. Mussels were exposed to copper sulfate. * = Age is at time of arrival at NC State University.

Age Prop. Conc. 48h 96h Species (days)* Method (µg Cu/L) Rep. survival survival U. imbecillis 6 in vivo 0 1 10/10 10/10 U. imbecillis 6 in vivo 0 2 10/10 9/10 U. imbecillis 6 in vivo 0 3 5/10 3/10 U. imbecillis 6 in vivo 12.5 1 10/10 7/10 U. imbecillis 6 in vivo 12.5 2 10/10 6/10 U. imbecillis 6 in vivo 12.5 3 10/10 9/10 U. imbecillis 6 in vivo 25 1 9/10 7/10 U. imbecillis 6 in vivo 25 2 6/9 5/9 U. imbecillis 6 in vivo 25 3 7/10 4/10 U. imbecillis 6 in vivo 50 1 7/10 4/10 U. imbecillis 6 in vivo 50 2 7/10 5/10 U. imbecillis 6 in vivo 50 3 6/10 3/10 U. imbecillis 6 in vivo 100 1 1/10 2/10 U. imbecillis 6 in vivo 100 2 6/10 2/10 U. imbecillis 6 in vivo 100 3 5/11 2/10 U. imbecillis 6 in vivo 200 1 3/11 0/10 U. imbecillis 6 in vivo 200 2 3/9 0/10 U. imbecillis 6 in vivo 200 3 3/12 0/10 U. imbecillis 6 in vivo 300 1 0/10 0/10 U. imbecillis 6 in vivo 300 2 0/9 0/10 U. imbecillis 6 in vivo 300 3 1/10 0/10 U. imbecillis 17 in vivo 0 1 10/10 9/10 U. imbecillis 17 in vivo 0 2 10/10 10/10 U. imbecillis 17 in vivo 0 3 9/10 10/11 U. imbecillis 17 in vivo 12.5 1 10/10 11/11 U. imbecillis 17 in vivo 12.5 2 9/10 9/10 U. imbecillis 17 in vivo 12.5 3 10/10 9/10 U. imbecillis 17 in vivo 25 1 8/10 8/10 U. imbecillis 17 in vivo 25 2 11/12 11/13 U. imbecillis 17 in vivo 25 3 9/10 7/10 U. imbecillis 17 in vivo 50 1 10/10 9/10 U. imbecillis 17 in vivo 50 2 7/10 4/10 U. imbecillis 17 in vivo 50 3 9/10 7/10 U. imbecillis 17 in vivo 100 1 5/10 1/10 U. imbecillis 17 in vivo 100 2 8/10 4/10 U. imbecillis 17 in vivo 100 3 8/11 2/11 U. imbecillis 17 in vivo 200 1 4/10 1/10 U. imbecillis 17 in vivo 200 2 3/10 0/10 U. imbecillis 17 in vivo 200 3 2/10 0/10 U. imbecillis 17 in vivo 300 1 3/10 0/10 U. imbecillis 17 in vivo 300 2 2/11 0/11 U. imbecillis 17 in vivo 300 3 3/10 0/10