### Determining the Appropriate Duration of Toxicity Tests with Glochidia of Native Freshwater Mussels

Final Completion Report for the period August 1, 2010 – July 31, 2012 Submitted to Edward Hammer U.S. Environmental Protection Agency 77 West Jackson Blvd. Chicago, IL 60604-3590 by Robert B. Bringolf, Ph.D. The University of Georgia Warnell School of Forestry & Natural Resources 180 East Green St., Athens, GA 30602-2152 Telephone: 706-542-1477 E-mail: bringo@uga.edu
M. Christopher Barnhart, Ph.D. Biology Department Missouri State University 901 South National Ave. Springfield, MO 65897 Telephone: 417-836-5166 Email: chrisbarnhart@missouristate.edu and
W. Gregory Cope, Ph.D. North Carolina State University Department of Environmen tal & Molecular Toxicology Box 7633, Raleigh, NC 27695-7633 Telephone: 919-515-5296 E-mail: greg_cope@ncsu.edu

Submitted May 8, 2013

1Executive Summary The overall goal of this project was to provide information for determining the appropriate duration of toxicity tests with the larval stage (glo chidia) of freshwater mussels belonging to the family Unionidae. The projec t was developed to prov ide insight into the ecological relevance of using the glochidia closing response to salt as an endpoint for toxicity tests and as a measure of condition in determin ing the appropriate dura tion of standardized toxicity tests with glochidia. Specific objectiv es were to determine: 1) if the duration of glochidia viability, defined as the ability to close in response to salt (NaCl) solution, is equivalent to the duration of in fectivity- the abili ty to attach to a host fish and metamorphose successfully into the juvenile stage; 2) if the duration of infectivity of glochidia deposited in natural stream sediment is sim ilar to the duration observed in water-only exposures; and 3) the probability that glochidia deposited onto the sediment surface will in fect host fish, using representative mussel-host species pairs.
During the first phase of the project, we tested glochidia viability a nd infectivity of six mussel species that differ in their host-infection strategies. Three of these species use mantle lures and host extraction ( Lampsilis siliquoidea, Lampsilis cardium, Villosa lienosa), two species broadcast glochidia into the water column ( Utterbackia imbecillis and Amblema plicata ), and one species releases glochidia in conglutinates ( Ptychobranchus occidentalis) . Our results indicate that within the first 24 h after release from the female mussel, glochidia viability was indicative of infectivity, as l ong as viability was >90%. After 24 h, viability remained relatively high (>60%) in many species tested, but infectivity of glochidia free in water sometimes declined rapidly after 24 h. Infectivity was high at all times when viability was >90%. The current American Society for Testing and Materials (AST M) guideline for conducti ng toxicity tests with

2glochidia of freshwater mussels requires >90% vi ability for control groups at test termination (commonly 24 h). Our results indicated that viab ility is an adequate proxy for infectivity of glochidia free in water when viab ility is >90%, and that infectivity decreased markedly in some species when viability dropped below 90%. Our resu lts also indicated that glochidia released in conglutinates remained viable and infective for longer periods than glochi dia released free into the water. We also noted for species that brood for several months (long term brooders), glochidia collected from females toward the end of the brooding season were viable for a shorter duration than those collected ea rlier in the brooding season. Ba sed on these data, we recommend that the current ASTM guideline for conducting toxicity tests with glochidia be retained with a maximum test duration of 24 h for all mussel species. Additionally, we recommend that glochidia from long term brooders be collected before the e nd of the brooding season. For species with previously defined duration of viability of ≥ 24 h, we recommend a 24 h toxicity test; however, for species with little or no data on duration of viability, we recommend that viability be assessed at an intermediate time poi nt(s) (e.g., 6, 12, 18, 24 h) during toxicity tests in the event that control viability is <9 0% by 24 h (e.g. at test termination). In the second phase of the project, we comp ared the duration of glochidia viability and infectivity 1) in natural water a nd sediment versus reconstituted wa ter, and 2) in free glochidia versus glochidia within conglutinates. These experiments were performed with Ptychobranchus
occidentalis glochidia and rainbow darters ( Etheostoma caeruleum ). Infectivity of free glochidia of Ptychobranchus declined to near zero within 24 h, more ra pidly than the other species tested. Viability declined more rapidly in natural wa ter and sediment than in reconstituted water, perhaps because of microorganisms that attack the glochidia. In contrast to free glochidia, glochidia in conglutinates remain ed viable and infective for the duration of the study (96 h). The

3apparent protective effects of the conglutinate suggest that 24-h toxicity te st duration with intact conglutinates would be conservative. In a third phase of the proj ect, we evaluated infectivity for glochidia that had been exposed to NaCl or copper (Cu) for 24 hr. We used glochidia of two mussel species, Lampsilis
cardium and Lampsilis dolabraeformis, and largemouth bass for this study. Two experiments were performed with glochidia of L. cardium , one with glochidia colle cted early in the brooding season (January) and a second with glochidia collected near the e nd of the brooding season (June) to determine if maternal brooding time influe nced sensitivity to the toxicant, viability, or infectivity. Following the 24-h exposure to a ra nge of concentrations of NaCl or Cu, we observed predictable results that viability decrea sed with increasing toxi cant concentration. The remaining viable glochidia in each treatment were placed on largemouth bass to determine if they were also able to attach and metamorphose on the fish. In each of the 5 trials, we observed that the viable glochidia were indeed able to metamorphose at a rate similar to those in the control. These results suggest that the glochi dia viability endpoint is a proxy for metamorphosis success and is therefore ecologically relevant for 24 h toxicity tests. The data also demonstrated that glochidia collected early in the brooding season were signif icantly less sensitive to the toxicant, had higher metamorphosis success, an d remained viable (>90%) longer than those collected late in the brooding season. These re sults have important imp lications for timing of glochidia collection for toxicity test with long-term brooding mussel species (e.g., members of the tribe Lampsilini). In summary, the results of this study provide d data that support retaining the current ASTM guideline for toxicity testing with glochi dia using viability as the endpoint, but with recommended maximum test duration of 24 h. We r ecommend that glochidia be collected before

4the end of the brooding season for toxicity tests to ensure health and viability of glochidia. We recommend that glochidia viabilit y be assessed in toxicity test s at a time point between 0 and 24 h (e.g., 6, 12, 18 h) for species for which duration of viability has not been defined in the event that viability of control mussels of a given spec ies decreases to <90% by the 24 h time point.

Formatted for submission to: Environmental Toxicology and Chemistry

5

### Determining the appropriate duration of t oxicity tests with glochidia of native freshwater mussels

Andrea K. Fritts 1 , M. Christopher Barnhart 2 , Megan Bradley 2 , Na Liu 2 , W. Gregory Cope 3 , and Robert B. Bringolf 1,4

1 University of Georgia, Warnell School of Forestry & Natural Resources, Athens, Georgia 30602

2 Missouri State University, Department of Biology, Springfield, Missouri 65897

3 North Carolina State University, Department of Environmental and Molecular Toxicology,

Raleigh, North Carolina 27695

4 Corresponding author: bringo@uga.edu

6Abstract Freshwater mussels (Unionidae) have a remarkable life cycle in which larvae (glochidia) are brooded by the female parent and then released into the water where they must attach to a fish host to metamorphose into a juven ile mussel. Glochidia attach by closing the shell valves on the host. Glochidia also close in response to salt so lution, and this closing res ponse to salt is widely used as a measure of viability to determine if glochidia are suitable for host fish suitability trials, as an endpoint for standard toxicity tests, and fo r a variety of other applications. The lifespan of glochidia in water is limited. We tested the a ssumption that glochidia viability, measured by the closing response to salt, is indicative of infectivity, the ability of glochidia to attach to a host fish and successfully metamorphose to the juvenile stag e. We compared the durations of glochidia viability and infectivity in water for seven mussel species ( Lampsilis siliquoidea, Lampsilis cardium, Lampsilis dolabraeformis, Villosa lienosa, Ptychobranchus occidentalis, Amblema plicata, and Utterbackia imbecillis ). Viability was indicative of infectivity between 0 and 24 hours. However, often infectivity of glochidia in water decreased after 24 h even when viability remained relatively high, especially for glochidi a collected near the e nd of the brooding season. The duration of viability and infectivity for glochidia of P. occidentalis was shorter in river water with sediment compared to reconstituted water. Ptychobranchus occidentalis releases membrane-bound packets (conglutinates) containing th e glochidia. Glochidia in conglutinates remained infective longer indicating that conglutin ates may provide protection from the external environment for at least four days. We also compared the viability and metamorphosis success of two mussel species after 24 h exposure to a toxicant (sodium chloride or copper). Metamorphosis success of remaining viable gloc hidia did not differ among the concentrations of

7toxicants, suggesting that viability is indicative of metamorpho sis success and that the decrease in glochidia reproductive potential after exposure to a toxicant is at tributed to a smaller number of glochidia that are open and able to attach to a host, rather th an a decrease in metamorphosis. Our results indicate that glochidia viability is a valid proxy for infectivity and an ecologically relevant endpoint for standard toxicity tests wi th freshwater mussels when control viability is
≥ 90%.

Keywords: Unionidae, larvae, viability, metamorphosis, toxicity

Introduction Freshwater mussels (Order Unionida) compri se six families and more than 850 species worldwide (Graf and Cummings 2007). North America is an epicenter of unionid diversity with approximately 300 species currently recognized (Parmalee and Bogan 1998). The study of freshwater mussels has increased markedly in recent years, spurred by the recognition that many mussel taxa have become extinct and many more are threatened by anth ropogenic perturbations (Ricciardi and Rasmussen 1999, Lydeard et al . 2004, Strayer et al. 2004). The extinction or extirpation of species can have cascading, ecosyst em-level effects because freshwater mussels provide essential ecosystem services such as wa ter filtration, nutrient se questration and cycling, and habitat for other aquatic organisms (V aughn and Spooner 2006, Strayer 2008, Vaughn et al. 2008). The life cycle of freshwater mussels include s a parasitic stage in which the larvae (glochidia) must attach to a host (usually a fish) to transform into the juvenile stage. To infect the

8appropriate host fish species, adult female mussels use a variety of strate gies, including mantle lures, conglutinates, and broadcast of free glochi dia into the water (Bar nhart et al. 2008, Haag 2012). In each case, the glochidia must be rele ased from the female marsupium and from egg or conglutinate membranes before they are able to c ontact the host and attac h. Attachment occurs by clamping the valves shut on the host gills or skin. Depending on the host infection strategy, glochidia may contact the host almost immediatel y after entering the water or they may remain free in the water for varying lengths of time before encountering a host. Lefevre and Curtis (1912) repor ted that glochidia closed in response to various salt solutions or to blood, and Coker et al. (1921) reco mmended that the viability of glochidia could be assessed by observing the closing respons e. This approach remains popular today for assessment of glochidia condition before propagati on or host testing. Gloc hidia that are: a) initially open and b) able to close in response to NaCl are considered to be “viable” and assumed to be able to infect a host fish (Ingersoll et al. 2007). The closi ng response has also been widely used as an endpoint in toxico logical studies (e.g., Keller and Ru essler 1997, Bri ngolf et al. 2007, Wang et al. 2007, Cope et al. 2008). In this context, glochidia may either close in response to the toxicant or become unable to clos e in response to salt. Either condition prevents completion of the lifecycle. Few quantitative studies have investigated th e correlation of the ability of glochidia to close in response to salt (viability ) with the ability to attach to a host fish and metamorphose to the juvenile life stage (infectivity). Zimmerman and Neves (2 002) assessed effects of different holding temperatures (0°, 10°, 25° C) on glochidia viability and i noculated fish with glochidia from the 0° and 10°C treatments at 7 and 14 days post extraction from the females. Metamorphosis to the juvenile stage occurre d, but the metamorphosis success (proportion of

9glochidia that metamorphosed to the juvenile st age) was not reported. Fisher and Dimock (2000) held glochidia of Utterbackia imbecillis in reconstituted water and f ound that the ability of the glochidia to metamorphose in ce ll culture media declined more rapidly than the closing response to potassium chloride. To date, it appears that no other studies have quantitatively investigated the relationship between viability and metamorphosis success. With the increasing interest in use of glochidia for standard toxicity tests for derivation of water qua lity criteria, assessing whether the closing response is indicative of the more ecologically relevant ab ility of glochidia to successfully attach and metamor phose on a host fish is necessar y. A related question is whether the duration of glochidia infec tivity in laboratory conditions is similar to that in natural conditions. For example, survival might be shor tened by microorganisms present in natural water and sediment but absent from reconstituted water used in laboratory tests. Survival might be lengthened in species whose glochidia are normally protected by conglutinat es. Lastly, there is a need to evaluate the effect on metamorphosis of glochidial exposure to a toxicant. Such data would allow researchers to assess if the decrease in reproductive potential is primarily attributed to a smaller number of glochidia that are open and ab le to attach to a host, or rather a decrease in metamorphosis success. Our objectives for this study were to determine: 1) if the duration of glochidia viability (determined by a shell closing response to NaCl) is equivalent to the duration of infectivity--the ability to attach to a host fish and metamorphos e successfully into the ju venile stage, 2) the protective effect of conglutinat es, 3) the effect of natural water and sediment on glochidia viability and infectivity, and 4) the effect of exposure to a toxicant on metamorphosis success. These findings will provide insight into the ecolog ical relevance of laboratory tests of glochidia viability, infectivity, and the appr opriate duration for standard t oxicity tests with glochidia.

10Methods Test organisms We used snorkeling, scuba divi ng, and tactile searches to co llect gravid female mussels from four different states over the course of three years (Table 1). These seven mussel species use the three main infect ion strategies found in Un ionidae: mantle lures ( Lampsilis siliquoidea, Villosa lienosa, Lampsilis cardium, Lampsilis dolabraeformis ), conglutinates ( Ptychobranchus occidentalis), and glochidia broadcasters ( Amblema plicata , Utterbackia imbecillis ).

Duration of viability vs. duration of infectivity We extracted glochidia from 3-8 female mu ssels per species by gently opening their valves and using a syringe and a gentle stream of water to flush the glochidia from the marsupial gills. Viability of glochidia from individual fe males was measured by uniformly suspending the glochidia in a known volume of water, rem oving ten 200-μL subsamples, and placing the subsamples into a 10-cm Petri dish. Each subsam ple contained ~15-30 glochidia, for a total of approximately 150-300 glochidia tested for viabil ity. A stereomicroscope at 10x magnification was used to count the open and closed glochi dia in each of the ten subsamples. A drop of saturated NaCl solution was then added to each 200-μL subsample, and the number of glochidia that remained open 1-min after the addition of the saline solu tion was counted. Glochidia that were open initially and closed in response to NaCl were considered viable, those that were closed initially were considered functionally dead because they no longer had the ability to attach to a host fish, and those that remained open after the addition of NaCl were considered definitively dead. The viability of a batch of glochidia was calculated by subtracting the number of glochidia

11that did not close after the addi tion of NaCl from the number that were originally open, then dividing by the total number of glochi dia (initially open and closed). Glochidia from female mussels (n= 3-8 with initial viability >90%) were pooled and randomly divided into replicate (n= 5) glass b eakers with 2 L of aerat ed, moderately hard reconstituted water (ASTM 2006a) where they were maintained at 20°C (± 1 o C) with a 16L: 8D photoperiod. At the beginning of the experiment a nd every 24 h thereafter, the viability of each replicate was calculated followi ng the method described above. Subsampling continued daily until mean viability for the five replicates wa s <10%. At each time point, a second subsample of glochidia from each replicate was tested for in fectivity on known host fish (two fish per replicate) for a total of 10 fish pe r time point. Host fish, largemouth bass ( Micropterus salmoides ) (8-18 cm) and rainbow darters ( Etheostoma caeruleum ) (5-8 cm) were obtained from hatcheries, which insured that the fish had not been previously expos ed to glochidial infections. Host fish were exposed to 4000 vi able glochidia per liter of wate r for 15 min; glochidia were kept in suspension with a large-bul b pipette and vigor ous aeration. Attachment and metamorphosis success we re assessed in multiunit recirculating aquarium systems (Aquatic Habitats, Apopka, Florid a) as described by Dodd et al. (2005). Fish infested with glochidia were rins ed, then placed individually in unit tanks (1-3 L) fitted with 153­
μ m filters to collect all sloughed glochidia a nd metamorphosed juveniles. Juveniles were distinguished from glochidia by possessing a foot and valve movement. Cont ents of the filters were examined under a stereomicroscope every 1-2 days for a minimum of 14 days and until no glochidia or juveniles were coll ected for at least three consecu tive days. Percent metamorphosis was calculated by dividing the tota l number of juveniles by the total number of glochidia and juveniles recovered from each fish. The aquari um systems were maintained at 20-22°C. All

12water-chemistry variables were measured da ily throughout the study a nd ranged from 7.5 to8.2 mg/L dissolved oxygen, pH: 6.8 to7.6, and total ammonia nitrogen <0.1 mg/L (Table 2).

Effects of conglutin ates and sediment The durations of glochidia vi ability and infectivity in conglutinates and free in water were compared in Ptychobranchus occidentalis . These durations in reconstituted water and in natural water and sediment were also compared. Hosts were rainbow darter ( Etheostoma
caeruleum , 5-8 cm total length). Conglutinates were dissected from the marsupial demibranchs of gravid female mussels with No. 0 insect pi ns. Conglutinates from six females were pooled, and each conglutinate was examined carefully unde r magnification to determine if it was intact. Only unbroken conglutinates were used for conglu tinate exposures. Free glochidia were obtained by using pins to gently tear open the outer c onglutinate membrane. The ruptured conglutinate was then gently drawn in and out of a 3-mL transfer pipette to di slodge the glochidia. Both free glochidia and intact conglutinates we re exposed to reconstituted hard water or to river water with fine sediment. Reconstitute d water was similar in formula to that of Smith et al. (1997), but concentrations of all solutes we re doubled. River water and wet sediment were collected from the James River (Greene Count y, Missouri). Approximately 15L of water and 250-ml of sediment were combined and filtered to 32 μm with Nitex filter cloth. Water and sediment were homogenized by stirring with a pa int stirrer while being portioned in 200-ml aliquots to each of the test chambers (250-ml beakers). A layer of sediment about 1 mm deep settled in each beaker. Treatments (three repli cates per treatment x exposure time; 60 beakers total) were: 1) reconstituted water with free gloc hidia, 2) reconstituted water with conglutinates, 3) river water and sediment with free glochi dia, and 4) river water and sediment with

13conglutinates. Each beaker recei ved 1000 glochidia or 3 conglutinat es containing a similar total number of glochidia. At time zero, and then each 24-h interval for 96 h, viability and infectivity were assessed. In conglutinate treatments, the conglutinates were opened and the glochidia freed. Two darters were placed in each beaker fo r 15 min, while gently stirring the water with a pipette, to achieve infestation. Methods for calcu lating viability and infectivity were similar to those previously described.

Effects of glochidial exposure to toxicants We extracted glochidia from 3-8 female mu ssels per species by gently opening their valves and using a syringe and a gentle stream of water to flush the glochidia from the marsupial gills. Viability of glochidia from individual fema les was measured in the same manner as that described above. According to ASTM guide lines (ASTM 2006b), glochidia from female mussels (n= 3-8 with initial viability >90 %) were pooled and randomly divided into six concentrations of NaCl (0, 0.5, 1.0, 1.5, 2.0, 2.5 ppt) or four concentrations of copper (0, 15, 30, 60 ppm) dissolved in moderately hard recons tituted water (ASTM 2006a). In all glochidia exposures temperature was 20 o C ±1 o C, pH ranged from 7.1 to 7.5, dissolved oxygen was >80% of saturation, hardness was 75-83 mg/L as CaCO 3 , and alkalinity was 71-85 mg/L as CaCO 3 (Table 3, Table 4). Glochidia from each concentration were then split into replicate (n= 3) glass beakers where they were maintained at 20°C with a 16L: 8D photoperiod. After 24 h of exposure to the respective toxicants, the viability of each replicate was calculated following the method described above. A second subsample of glochidia from each replicate was tested for infectivity on largemouth bass (two fish per replicate) fo r a total of six fish per concentration. The inoculation procedure follows the methods descri bed above. In each replicate, the inoculation

14suspension was adjusted to provi de 4000 viable glochidia per lite r. The aquarium systems were maintained at 18-22°C. All water-chemistry vari ables were measured daily throughout the study and ranged from 7.5 to 8.6 mg/L dissolved oxygen, pH: 6.8 to7.7, and tota l ammonia nitrogen
<0.1 mg/L. Concentrations of NaCl were verified daily in one repl icate of each treatment with a handheld salinity meter (YSI 30, Yellow Springs Instruments, Yellow Springs, OH) and were within 0.1 ppt of nominal at all times. Water samp les were collected and pr eserved to pH <2 for Cu analysis prior to analysis by inductively c oupled plasma mass spectrometry. All measured Cu concentrations (n=10) were 80-120% of nominal and duplicate samples (n=4) varied by <5%. All statistical analyses were base d on nominal concentrations.

Statistical analysis Data were tested for normality using th e Kolmogorov-Smirnov test (SAS, version 9.3; SAS Institute, Cary, North Carolina). To achie ve normality and equal variance, viability and metamorphosis data were transformed with the arcs ine of the square root. Transformed data were analyzed with a one-way ANOVA, followed by a post-hoc Dunnett’s test to assess for differences between the control (i .e., 0 h in the aging exposures) and each time point or toxicant concentration. Attachment data (the number of glochidia attached per fish) were analyzed with a one-way ANOVA, followed by a post-hoc Tukey’s test. Level of significance for all tests was

set at α = 0.05. A 24-h EC50 was calculated for each 24-h glochidia viability test with the Trimmed Spearman-Karber Method (ToxStat, WEST, Inc.). We considered EC50s significantly different based on non-overlapping 95% conf idence intervals (Bri ngolf et al. 2007).

15Results Duration of viability vs. duration of infectivity The initial (0 h) glochidia viability met th e current American Society for Testing and Materials (ASTM) guideline of >90% (ASTM 2006b) for all seven mussel species tested; however, the trial with L. siliquoidea collected in June had an in itial viability of only 80%. Viability and metamorphosis re mained higher for longer with L. siliquoidea glochidia collected in January (Fig. 1A) compared to those collected in June (Fig. 1B), which is the end of the brooding season for many members of the tribe Lamps ilini. At the 24-h time point, viability was <90% for L. cardium, U. imbecillis , and L. siliquoidea (June) (Fig. 1). According to the ASTM guideline for conducting laborator y toxicity tests with freshw ater mussels (ASTM 2006b), data from these time points would not be valid for to xicological testing purposes because the viability was <90%. Five species ( L. siliquoidea (Jan), L. dolabraeformis, V. lienosa, A. plicata, and P. occidentalis ) had >90% viability at 24 h (Fig. 1). For these species, metamorphosis success did not differ between 0 and 24 h (Fig. 1, Table 4). The exception was P. occidentalis free glochidia, which had viability >90% through 72 h but had low (<5%) metamorphosis by 24 h (Fig. 2A). For all but one species, metamorphosis success decreased rapidly as viability dropped below 90%, although viability remained near or above 60% for 3 d with four of the seven species. Overall, metamorphosis success ranged from 19 to 78% (mean 54%) at time zero for the species tested, typical of rates achieved in musse l propagation trials with known host fish-mussel pairs, at common temperatures, and with naïve test fish (Dodd et al. 2005). Total glochidia attachment did not differ among treatments for any of the mantle lure or conglutinate species tested ( p = 0.08-0.64), but the two broadcasting species ( U. imbecillis and A. plicata ) had significantly greater gl ochidia attachment ( p = <0.001) at the final i noculation time point (mean

16attachment ± 95% CI) ( U. imbecillis : 0 to24 h = 353 ± 61, 48 h = 1306 ± 208; A. plicata : 0 to 48 h = 365 ± 44, 72 h = 745 ± 88).

Effects of conglutin ates and sediment Initial (0 h) glochidia viabil ity was > 90% in all treatments . Viability of free glochidia exposed to sediment and river water decreased rapi dly (52% viable by 48 h) whereas viability of free glochidia in reconstituted water (no sediment ) remained >90% until 72 h (Fig. 2A & 2C). Sediment also reduced the duration of viability for glochidia in c onglutinates but viability did not decrease to <90% until 72 h of exposure (Fig. 2B & 2D). Vi ability of conglutinates in reconstituted water was >90% for at least 96 h, the time at which th e experiment was terminated. Metamorphosis success of free glochidia declin ed more rapidly than that of glochidia within conglutinates and declined similarly in both river water and reconstituted water (Fig. 2, Table 2). Metamorphosis success was high (45% ± 13) for glochidia placed on fish immediately after removal from the mussel, but decreased ra pidly to <5% for free glochidia aged 24 h or more, even though viability remained high (>90%). In contrast, glochidia in conglutinates remained infective for at least 96 h. Total gloc hidia attachment (mean ± 95% CI) did not differ
among P. occidentalis conglutinate treatments nor free glochidia in reconstituted water (38 ± 6; p

= 0.11-0.61 ), but free glochidia exposed to river water and sediment had significantly less glochidia attachment ( p = 0.0113) during all inoculation time poi nts after the init ial inoculation (0 h = 45 ± 20; 24-96 h = 12 ± 3).

Effects of glochidial exposure to toxicants

17In each toxicant test, viability decrease d (ranged from 95% to 25%) as toxicant concentration increased (Fig. 3). However, for th e remaining viable glochidia in each treatment, metamorphosis success was similar among treatment s regardless of viability (Fig. 3, Table 5). Lampsilis cardium glochidia collected in December (early brooding period; Fig. 3A) maintained high viability longer than those of the same speci es collected in June (late brooding period; Fig. 3B) and also had consistently hi gher (55-70%) metamorphosis rates than those collected in June (18-23%). Glochidia collected early in the brooding period (December) were significantly less sensitive (based on non-overlapping 95% confidence in tervals) to NaCl than those collected late in the brooding period (June). The 24-h EC50 (± 95% CI) for December glochidia was 2.04 ±
0.11 and for June glochidia was 0.80 ± 0.16 (Fig. 3D). Total gloc hidia attachment (mean ± 95% CI) did not differ among L. cardium or L. dolabraeformis trials ( p = 0.16-0.58).

Discussion Our results indicate that glochidia viability (as measured by the shell closing response to NaCl exposure) was indicative of th e ability to metamorphose into th e juvenile life stage, as long as viability was >90%. Timing of glochidia collection also appear s to be critical to glochidia health; viability of glochidia collected late in the brooding season declined rapidly compared to those collected earlier in the brooding season. The current ASTM guideline for conducting toxicity tests with glochidia of freshwater mussels (ASTM 2006b) requires >90% viability for control groups at test termina tion (commonly 24 h) and our data support the continued use of this criteria. Additionally, glochidia that remained viable following exposure to a toxicant for 24 h were able to successfully metamorphose, thus the viability endpoint ap pears to have direct ecological relevance. Based on these data, we recommend that the curre nt ASTM guideline for

18glochidia toxicity testing be retained with a minimum control viability of 90% throughout the duration of the test for all mussel species. Addi tionally, we recommend that glochidia from long term brooders be collected before the end of th e brooding season because those collected late in the season appeared less healthy based on our results that showed glochidia collected late in the brooding season were more sensitive to toxicants , had lower initial viability which declined quickly, and had poorer metamorphosi s success. For species with previously defined duration of viability of ≥ 24 h, we recommend a 24 h toxicity test; how ever, for species with little or no data on duration of viability, we recomme nd that viability be assessed at an intermediate time point(s) (e.g., 6, 12, 18, 24 h) during toxicity te sts in the event that control viability is <90% by 24 h (e.g. at test termination). Shipping time must also be a consideration if glochidia cannot be obtained locally. Further, for species with previously defined viability of ≥ 90% for ≥ 24h, tests of shorter duration (because of low c ontrol survival) should not be considered acceptable. The duration of viability for free glochidia of P. occidentalis was shorter in river water and sediment than in reconstituted water, but th e effect was much less pronounced for glochidia in conglutinates. We suspect that the relatively rapid decl ine of free glochidia in river water with sediment was the result of microorganisms that compromised glochidia survival and ability to attach to a host fish. In a preliminary test of this hypothesis, a group of free glochidia was tested in 0.4 μ filter-sterilized river water and they remain ed >80% viable at 72 h. This result indicated that microorganism removal positively affected viability. The subsequent infectivity of these glochidia was not tested, however. Wher eas infectivity of free glochidia of P. occidentalis decreased rapidly in both treatments, infectivity of glochidi a in conglutinates remained high for the duration of the study (96 h). Th e apparent protective effects of the conglutinate suggest that a 24-h toxicity test duration would be conserva tive for species that release glochidia in

19conglutinates. Conglutinates pr ovide protection from some toxi cants (e.g., copper) and thereby reduce toxicity (increase LC50, EC50 values; Gillis et al. 2008). Our results suggest that th e conglutinate packet of P. occidentalis provides protection from the external environment for at least 4 d. Even greater protection may be afforded by the marsupial gill of the brooding female, where glochidia of long-term brooders develop and survive for many months (Haag 2012). Once released from the prot ection of the conglutinate or the marsupia of the female mussel, the infectivity of free glochidia appears to decrease after 24 h, depending on the species. Our data indicate th at metamorphosis success decreases before viability decreases; at 48 h post extraction, L. siliquoidea (Jan) and V. lienosa exhibited 80% viability, but the corresponding metamorphosis success was reduced by 50% (Fig. 1A &1B). Importantly, the reduced infectivity was not a result of lower attachment to host fish, which remained consistent across inoculation days. The copper and NaCl toxicant e xposure results suggest that the decrease in glochidia reproductive potential after exposure to a toxicant is attributed to a smalle r number of glochidia that are open and able to attach to a host, rather than a decrease in metamorphosis success. Since the inoculation suspensions were adjusted to a unif orm number of viable gl ochidia, the consistent metamorphosis success supports th e use of the viability test as a proxy for metamorphosis. These results were consistent with 2 mussel spec ies and two toxicants wi th different modes of action. For mussel species that are long-term brooders , the length of time that glochidia have aged within the female mussel also may aff ect glochidia function and condition. Our data suggest that older glochidia may be in poorer physiological condition (e.g., less energy reserves) and are unable to remain viable for as long afte r the glochidia are removed from the female. In

20our study, L. siliquoidea glochidia early in the brooding pe riod (January) registered 80% viability 48-h after being removed from the female, whereas glochidia from the same species collected late in the brooding period (June) on ly had 20% viability at 48 h post extraction. Additionally, Lampsilis cardium glochidia tested late in the brooding period (June) also showed a rapid decline in viability compared to L. cardium tested early in the brooding period (December). Dodd et al. 2006 reported that Lampsilis reeveiana glochidia aged 241-360 d had significantly less metamorphosis success (67.5%) compared to glochidia aged 0-120 d (87.5%) and 121-240 d, (88.7%). Cope et al. (2008) reported longer du ration of viability (>90%) for some of the species used in the present study; ho wever, timing of collection of the glochidia was not reported. The implications for the results of this study are multifaceted. For the purposes of toxicological testing, our recommen dation is to retain the current ASTM guideline for toxicity testing with glochidia, but specify a maximum test duration of 24 h when us ing viability (i.e., the valve closure response to NaCl) as the endpoint. Ideally toxicity tests sh ould be conducted with glochidia immediately following removal from the female mussel, thus shipping of brooding females rather than free glochidi a may be considered. However, sh ipping of adult mussels is less practical and may create logistic al and regulatory (e.g., interbasin transfer of species or threatened or endangered status) problems, so sh ipping of free glochidia may be necessary. Our results indicate that this time frame may be suitable (i.e., viability and infectivity can remain
≥ 90% for 48 h) but we recommend that viability be assessed at one or more time points between 0 and 24 h (e.g., 6, 12, 18 h) in the event that viabili ty of controls decreas es to <90% by the 24 h time point of the toxicity test. This approach is especially im portant when a species is being used for which no viability duration information has been previously reported. These

21recommendations are supported by Cope et al. ( 2008), who showed that viability (determined with the NaCl method) of glochi dia from 20 species of mussels, three of which were tested in this study ( L. cardium , L. siliquoidea , U. imbecillis ), was suitable ( ≥ 90%) for 24 h or less. This time period corresponds with the ecol ogically relevant endpoint of in fectivity determined in this study. The higher toxicant sensit ivity, lower initial viability and lower metamorphosis success of glochidia collected late in th e brooding season suggest s they are not appropriate for use in toxicity tests. Use of glochidia from late in the brooding period (for long-term brooders) may result in toxicity test results that are substantia lly different that if gloc hidia were collected early in the brooding season. More resear ch is required to better define the period when glochidia fitness begins to decline to the point that to xicant sensitivity is increased. Furthermore, implications for freshwater mussel host suitability research include the possibility of obtaining a false-negative result, if the glochidia have been outside of the female for more than 24 h. The decrease in metamorphosis success should be noted by those who conduct propagation efforts with imperiled mussels, and every effort should be made to place glochidia onto host fish as soon as practical after the glochidi a are removed from the female mussel. Future propagation and culture research evaluating the infectivity of the glochidia life stage of freshwater mussels would benefit from accurate calculation and reporting of viability data. In summary, we found that glochidia viability of ≥ 90% was indicative of infectivity; however, after 24 h infectivity ofte n declined more rapidly than vi ability. Glochidia of long-term brooders collected late in the br ooding season had low fitness comp ared to those collected early in the brooding season. Glochidia in contact with sediment ma y have a reduced duration of viability and infectivity, but gl ochidia in conglutinates may remain viable and infective for extended periods after release from brooding fema les. Glochidia viability is, therefore, an

22ecologically relevant endpoint in standard toxic ity tests when viability is high and the current ASTM criteria for control animal viability (90%) is appropriate.

Acknowledgements Funding for this research was provided by the U. S. Environmental Protection Agency Region 5 Great Lakes Restoration Init iative Program through Work Order no. 104 from the U.S. Geological Survey. We thank Charles Delos, Ed Hammer, Linda Holst, Lisa Huff, Dave Mount, Charles Stephan, and Brian Thompson of the U.S. EPA for their valuable insight and assistance on project need and experimental design. We thank J. Creamer, M. Fritts, R. Ratajczak, A. Tuck, and J. Wisniewski for a ssistance in the laboratory and field, and B. Sietman and T. Newton for providing mussels. Largemouth bass were donated by Owens and Williams Fish Hatchery (Hawkinsville, Georgi a) and rainbow darter s were provided by Cons ervation Fisheries (Knoxville, Tennessee). The Georgia Cooperative Fi sh and Wildlife Research Unit is jointly supported by the University of Georgia, Geor gia Department of Natural Resources, U.S. Geological Survey, U.S. Fish and Wildlife Service, and Wildlife Management Institute. Any use of trade, product, or firm names is for descri ptive purposes only and does not imply endorsement by the U.S. Government.

23Literature cited ASTM (American Society for Testing and Ma terials). 2006a. Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians. E729-96. pp 79–100 in Annual Book of ASTM Standards , Vol 11.06. Philadelphia, PA. ASTM (American Society for Testing and Ma terials). 2006b. Standard guide for conducting laboratory toxicity tests with freshwater mussels E2455-06. pp 1393–1444 in Annual Book of ASTM Standards , Vol 11.06. Philadelphia, PA. Barnhart, M. C., W. R. Haag, and W. N. Rost on. 2008. Adaptations to host infection and larval parasitism in Unionoida. Journal of the North Amer ican Benthological Society 27:370­
394. Bringolf, R. B., W. G. Cope, M. C. Barnhart, S. Mosher, P. R. Lazaro, and D. Shea. 2007. Acute and chronic toxicity of pesticide fo rmulations (atrazine, chlorpyrifos and permethrin) to glochidia and juveniles of Lampsilis siliquoidea (Unionidae). Environmental Toxicology and Chemistry 26:2101–2107. Coker, R. E., A. F. Shira, H. W. Clark, and A. D. Howard. 1921. Natura l history and propagation of freshwater mussels. Bulletin of the U.S. Bureau of Fisheries 37:75–182. Cope, W. G., R. B. Bringolf, D. B. Buchwalter, T. J. Newton, C. G. Ingersoll, N. Wang, T. Augspurger, F. J. Dwyer, M. C. Barnhart, R. J. Neves, and E. Hammer. 2008. Differential exposure, duration, and sensitivity of unionoid ean bivalve life stages to environmental contaminants. Journal of the North American Benthological Society 27:451-462. Dodd, B. J., Barnhart, M.C., Rogers-Lowery, C.L ., Fobian, T.B. & R.V. Dimock Jr. 2005. Cross resistance of largemouth bass to glochidia of unionid mussels. Journal of Parasitology
91:1064-1072.

24Dodd, B. J., M. C. Barnhart, C. L. Rogers-L owery, T. B. Fabian, and R. V. Dimock. 2006. Persistence of host response to glochidia larvae in Micropterous salmoides

. Fish and Shellfish Immunology 21:473–484. Fisher, G. R. and R. V. Dimoc k. 2000. Viability of glochidia of Utterbackia imbeccilus (Bivalvia: Unionidae) following their rem oval from the parent mussel. Pages 185-188 in R. A. Tankersley, D. I. Warmolts, G. T. Watters, B. J. Armitage, P. D. Johnson, and R. S. Butler (editors). Freshwater Mollusk Symposia Proceedings . Ohio Biological Survey, Columbus, Ohio. 241 pp. Graf, D. L. and K. S. Cummings. 2007. Review of the systematics and global diversity of freshwater mussel species (Bivalvia : Unionoida). Journal of Molluscan Studies 73:291­
314. Gillis, P. L., R. J. Mitchell, A. N. Schwalb, K. A. McNichols, G. L. Mackie, C. M. Wood, and J. D. Ackerman. 2008. Sensitivity of the glochidia (l arvae) of freshwater mussels to copper: assessing the effect of water hardness and dissolved organic carbon on the sensitivity of endangered species. Aquatic Toxicology 88(2):137-145. Haag, W. R. 2012. North American Freshwater Mussels : Natural History, Ecology, and
Conservation . Cambridge University Press, Cambridge, United Kingdom. 536 pp. Ingersoll, C. G., N. J. Kernaghan, T. S. Gr oss, C. D. Bishop, N. Wang, and A. Roberts. 2007. Laboratory toxicity testing with freshwater mussels. Pages 95-134 in J. L. Farris and J. H. Van Hassel (editors). Freshwater bivalve ecotoxicology . CRC Press, Boca Raton, Florida, and SETAC Press, Pensacola, Florida. 321 pp.

25Keller, A. E. and D. S. Ruesller. 1997. The toxi city of malathion to unionid mussels: relationship to expected environmental concentrations. Environmental Toxicology and Chemisty 16(5):1028-1033. Lefevre, G. and W. C. Curtis. 1912. Studies on the reproductive and artificial propagation of fresh-water mussels. Bulletin of the Bureau of Fisheries 30:105-201+12 plates. Lydeard, C., R. H. Cowie, W. F. Ponder, A. E. Bogan, P. Bouchet, S. A. Clark, K. S. Cummings,
T. J. Frest, O. Gargominy, D. G. Herbert, R. Hershler, K. E. Perez, B. Roth, M. Seddon,
E. E. Strong, and F. G. Thompson. 2004. The global decline of nonmarine mollusks.
Bioscience 54:321-330. Parmalee, P. W. and A. E. Bogan. 1998. The Freshwater Mussels of Tennessee . University of Tennessee Press, Knoxville, TN. 328 pp. Ricciardi, A. and J. B. Rasmussen. 1999. Extinction rates of North American freshwater fauna. Conservation Biology 13:1220-1222. Smith, M. E., J. M. Lazorchak, L. E. Herri n, S. Brewer-Swartz, and W. T. Thoeny. 1997. A reformulated, reconstituted water for testi ng the freshwater amphipod, Hyalella azteca. Environmental Toxicology and Chemistry 16(6):1229-1233. Strayer, D. L., J. A. Downing, W. R. Haag, T. L. King, J. B. Layzer, T. J. Newton, and S. J. Nichols. 2004. Changing perspectives on pearly mussels, North America's most imperiled animals. Bioscience 54:429-439. Strayer, D. L. 2008. Freshwater Mussel Ecology: A Multifactor Approach to Distribution and
Abundance . University of California Pre ss, Berkeley, California. 204 pp.

26Vaughn, C. C., and D. E. Spooner. 2006. Unioni d mussels influence macroinvertebrate assemblage structure in streams. Journal of the North American Benthological Society
25:691–700. Vaughn, C. C., S. J. Nichols, and D. E. Spooner. 2008. Community and foodweb ecology of freshwater mussels. Journal of North American Benthological Society 27:409–423. Wang, N., C. G. Ingersoll, D. K. Hardesty, C. D. Ivey, J. L. Kunz, T. W. May, F. J. Dwyer, A. D. Roberts, T. Augspurger, C. M. Kane, R. J. Neves, and M. C. Barnhart. 2007. Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae). Environmental Toxicology and Chemistry 26:2036–2047. Zimmerman, L. L. and R. J. Neves. 2002. Effect s of temperature on duration of viability for glochidia of freshwater musse ls (Bivalvia: Unionidae). American Malacological Bulletin
17:31-35.

27Table1: Dates and locations of freshwater mussel collections. Species Water body County Date Amblema plicata Sac River Cedar, MO Jul 2011 Villosa lienosa Cooleewahee Creek Baker, GA Oct 2010 Lampsilis cardium Mississippi River La Crosse, WI Jun 2011 L. siliquoidea Silver Fork of Perche Creek Boone, MO Jan 2011 L. siliquoidea Red Lake River Pennington, MN Jun 2012 L. dolabraeformis Oconee River Montgomery, GA Oct 2012 Utterbackia imbecillis Lake Oconee Morgan, GA Jul 2011 Ptychobranchus occidentalis St. Francis River Wayne, MO Feb 2011

Table 2. Water quality parameters (mean ± SD) for ASTM moderately hard reconstituted water used for glochidia viability and infectivity stud ies in which the glochidia were aged before inoculation on fish. Hardness and alkalinity (mg CaCO 3 /L) were measured once at the beginning of each study, other parameters were meas ured at least 10 times throughout each study.

Study pH Dissolved Oxygen (mg/L) Temp ( o C) Hardness (mg CaCO3/L) Alkalinity (mg CaCO3/L) Amblema plicata Villosa lienosa Lampsilis cardium L. siliquoidea (Jan) L. siliquoidea (June) L. dolabraeformis Utterbackia imbecillis 7.1 ± 0.2 7.7 ± 0.2 7.5 ± 0.2 7.6 ± 0.1 7.6 ± 0.2 7.7 ± 0.1 7.2 ± 0.1 7.7 ± 0.9 8.1 ± 1.0 7.6 ± 0.9 7.8 ± 1.0 8.1 ± 0.9 7.9 ± 0.8 7.1 ± 1.0 20.5 ± 0.2 19.7 ± 0.1 20.4 ± 0.1 19.4 ± 0.2 20.3 ± 0.1 19.9 ± 0.1 19.4 ± 0.1 75 85 82 80 82 84 78 55 62 64 62 60 65 56

28Table 3. Water quality parameters for ASTM mo derately hard reconstituted water used for glochidia viability and infectivity studies in whic h the glochidia were exposed to a gradient of NaCl for 24 hours, after which time fish were inoculated. Hardness and alkalinity (mg CaCO 3 /L) was measured at the beginning of the exposure an d all other parameters were measured at the end of the 24 hour exposure.

Study NaCl (ppt) 0 0.5 1.0 1.5 2.0 Lampsilis cardium (Dec) pH DO (mg/L) Temp (°C) Hardness Alkalinity L. cardium (June) pH DO (mg/L) Temp (°C) Hardness Alkalinity L. dolabraeformis pH DO (mg/L) Temp (°C) Hardness Alkalinity 7.3 6.3 18.4 86 62
7.0 8.1 19.4 88 64
7.6 8.4 20.9

80 60 7.9 6.0 18.6 86 62
7.3 8.3 19.3 88 64

7.7 8.2 20.8

80 60 7.9 6.3 18.5 86 62
7.3 5.7 19.4 88 64
7.6 7.8 20.8

80 60 7.9 6.2 18.6 86 62
7.3 5.4 19.4 88 64

7.7 8.1 20.9

80 60 7.9 6.4 18.7 86 62
7.3 6.6 19.7 88 64
7.8 7.4 20.8

80 60
Table 4. Water quality parameters for ASTM moderately hard reconstituted water during Lampsilis dolabraeformis glochidia viability and infectivity test in which the glochidia were exposed to a gradient of copper (Cu) for 24 hours, after which time fish were inoculated. Hardness and alkalinity (mg CaCO 3 /L) was measured at the beginning of the exposure and all other parameters were measured at the end of the 24 hour exposure.

Parameter Cu (ppm) 0 15 30 60 pH 7.7 7.7 7.5 7.6 DO (mg/L) 8.7 7.1 8.2 7.9 Temp (°C) 20.9 20.9 20.8 20.7 Hardness 80 80 80 80 Alkalinity 60 60 60 60

29Table 5. Results of one-way ANOVA comparing the change in glochidia viability or metamorphosis over time for seven freshwater musse l species. Degrees of freedom (df), test statistic (F value) and P values are presented. Level of significance was α = 0.05.

30 Species df F value P value Viability Amblema plicata Villosa lienosa Lampsilis cardium L. siliquoidea , (Jan) L. siliquoidea , (June) L. dolabraeformis Utterbackia imbecillis Ptychobranchus occidentalis , glochidia/water P. occidentalis , conglutinate/water P. occidentalis , glochidia/sediment P. occidentalis , conglutinate/sediment Metamorphosis A. plicata V. lienosa L. cardium L. siliquoidea , (Jan) L. siliquoidea , (June) L. dolabraeformis U. imbecillis P. occidentalis , glochidia/water P. occidentalis , conglutinate/water P. occidentalis , glochidia/sediment P. occidentalis , conglutinate/sediment 4, 20 5, 24 3, 16 5, 24 2, 12 4, 20 3, 16 4, 10 4, 10 4, 10 4, 10 3, 16 3, 16 2, 12 4, 20

1, 8 3, 16 2, 12 4, 10 4, 10 4, 10 4, 10 45.23 59.83 211.85

47.13 31.69 75.39 286.73

13.18

0.83 35.17

4.00

7.75 9.25 49.65 86.85

2.68 20.41 105.09

32.69

6.68 6.77 1.37 <0.0001 <0.0001 <0.0001 <0.0001

0.0005 <0.0001 <0.0001

0.0005 0.5372 <0.0001

0.0344
0.0020 0.0009 <0.0001 <0.0001

0.1404 <0.0001 <0.0001 <0.0001

0.0070 0.0066 0.3118Table 6. Results of one-way ANOVA comparing the change in glochidia viability or metamorphosis for glochidia exposed to increasing concentrations of sodium chloride (NaCl) or copper (Cu). Degrees of freedom (d f), test statistic (F value) and P values are presented. Level of significance was α = 0.05. Species df F value P value Viability L.cardium (Dec), NaCl 5, 12 45.65 <0.0001 L.cardium (Jun), NaCl 3, 8 165.67 <0.0001 U.imbecillis , NaCl 2, 6 58.49 0.0001 L.dolabraeformis , NaCl 5, 12 92.31 <0.0001 L.dolabraeformis , Cu 3, 8 188.06 <0.0001 Metamorphosis L.cardium (Dec), NaCl 5, 12 0.82 0.5595 L.cardium (Jun), NaCl 3, 8 2.67 0.1187 U.imbecillis , NaCl 2, 6 3.73 0.0886 L.dolabraeformis , NaCl 5, 12 10.21 0.0005 L.dolabraeformis , Cu 3, 8 3.13 0.0875

31Figure Legends Fig. 1. Comparison of percent viability (bars) and percent metamorphosis success (lines; ± 95% CI) for glochidia following release from female mussels of A) Lampsilis siliquoidea (Jan), B) Lampsilis siliquoidea (Jun), C) Lampsilis cardium, D) Lampsilis dolabraeformis, E) Amblema plicata , F) Villosa lienosa, and G) Utterbackia imbecillis . Viability was determined with approximately 200 glochidia per replicate (n=5 ) and metamorphosis success was assessed on largemouth bass (n=10 fish, two per replicate). Pr esence of a black asterisk (*) indicates a significant difference in viability, and a white as terisk indicates a sign ificant difference in metamorphosis success. Fig. 2. Comparison of percent viability (bars) and percent metamorphosis success (lines; ± 95% CI) for glochidia of Ptychobranchus occidentalis : A) free glochidia in reconstituted water, B) glochidia in conglutinates in r econstituted water, C) free glochi dia exposed to river water and sediment, and D) glochidia in conglutinates e xposed to river water and sediment. Viability was determined with approximately 200 glochidia per replicate (n=3) and metamorphosis success was assessed on rainbow darter (n=6 fish, two per replicate). Presence of a black asterisk (*) indicates a significant difference in viability, and a white aste risk indicates a significant difference in metamorphosis success. Fig. 3. Comparison of percent viability (bars) and percent metamorphosis success (lines; ± 95% CI) for glochidia exposed to incr easing concentrations of sodium chloride (NaCl) or copper (Cu) for 24 h: A) Lampsilis cardium (Dec) NaCl exposure, B) Lampsilis cardium (Jun) NaCl exposure, C) Lampsilis dolabraeformis exposed to Cu. Viability was determined with approximately 200 glochidia per replicate (n=3 ) and metamorphosis success was assessed on largemouth bass (n=6 fish, two per replicate). Presence of a black asterisk (*) indicates a significant difference in viability, and a white as terisk indicates a sign ificant difference in metamorphosis success. Fig. 4. Comparison of NaCl 24-h EC50 (± 95% confidence interval) for Lampsilis cardium glochidia collected early in the brooding pe riod (December) and late in the brooding period (June). Asterisk indicates si gnificant difference in EC50s ( non-overlapping 95% confidence intervals).

32Fig. 1. 

## A 100Percent (%) 

## 80

60 40 20

0 

## * * *

* *

* *

0 24 48 72 96 120 Exposure (h) 

# B 100Percent (%) 

## 80

60 40 20

0 

## *

*

0 24 48 Exposure (h)

100

80



## * *

* *

*Percent (%) 

## 60 40 20

0 0 24 48 72 Exposure (h) 

# C

33

# D 100Percent (%) 

## *
*

* * 80 60 40 20

0 0 24 48 72 96 Exposure (h)Percent (%) 

# E

* *

* *

*

* 100

80 60 40 20

0 0 24 48 72 96 120 Exposure (h)

34Percent (%) 

# F 100



## * *

* *

* 80 60 40 20

0 0 24 48 72 96 Exposure (h)

## 0 20 40 60 80 100Percent (%)



## * * *

* * 

# G

0 24 48 72 Exposure (h)

35Fig. 2. 

# A 100Percent (%) 

## 80

60 40 20

0 

## * * *

* *

* 0 24 48 72 96 Exposure (h) 

# B 100Percent (%) 

## 80

60 40 20

0 

## * *

0 24 48 72 96 Exposure (h)

36 

# C 100Percent (%) 

## 80

60 40 20

0 

## * * *

* *

*

* 0 24 48 72 96 Exposure (h)

## 0 20 40 60 80 100Percent (%) 

## * 

# D

0 24 48 72 96 Exposure (h)

37Fig. 3. 

# A 100

80 

## * *

*Percent (%) 

## 60 40 20

0 0.0 0.5 1.0 1.5 2.0 NaCl (ppt ) 

# B

0 20 40 60 80 100Percent (%) 

## * * *

*

0.0 0.5 1.0 1.5 2.0 NaCl (ppt )

38 

## * 

# C 100 

## * *
* *Percent (%) 

## 80

60 40 20

0 0 15 30 60 Copper (ppm)Fig. 4.



## 0.0 0.5 1.0 1.5 2.0 2.524-h EC50 (g/L)



## *

Dec June

39