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 Environmental & Molecular Toxicology 

Box 7633, Raleigh, NC 27695-7633 

Telephone: 919-515-5296 

E-mail: greg_cope@ncsu.edu
 

Submitted May 8, 2013 

1
 
Executive Summary 

The overall goal of this project was to provide information for determining the 

appropriate duration of toxicity tests with the larval stage (glochidia) of freshwater mussels 

belonging to the family Unionidae. The project was developed to provide 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 determining the appropriate duration of standardized 

toxicity tests with glochidia. Specific objectives 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 infectivity- the ability 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 similar to the duration observed in water-only exposures; and 3) the 

probability that glochidia deposited onto the sediment surface will infect host fish, using 

representative mussel-host species pairs. 

During the first phase of the project, we tested glochidia viability and 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 long 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 (ASTM) guideline for conducting toxicity tests with 

2
 
glochidia of freshwater mussels requires >90% viability for control groups at test termination 

(commonly 24 h). Our results indicated that viability is an adequate proxy for infectivity of 

glochidia free in water when viability is >90%, and that infectivity decreased markedly in some 

species when viability dropped below 90%. Our results also indicated that glochidia released in 

conglutinates remained viable and infective for longer periods than glochidia 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 earlier in the brooding season. Based 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 end 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 point(s) (e.g., 6, 12, 18, 24 h) during toxicity tests in 

the event that control viability is <90% by 24 h (e.g. at test termination). 

In the second phase of the project, we compared the duration of glochidia viability and 

infectivity 1) in natural water and sediment versus reconstituted water, 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 rapidly than the other species tested. 

Viability declined more rapidly in natural water and sediment than in reconstituted water, 

perhaps because of microorganisms that attack the glochidia. In contrast to free glochidia, 

glochidia in conglutinates remained viable and infective for the duration of the study (96 h). The 

3
 
apparent protective effects of the conglutinate suggest that 24-h toxicity test duration with intact 

conglutinates would be conservative. 

In a third phase of the project, 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 collected early in the brooding 

season (January) and a second with glochidia collected near the end of the brooding season 

(June) to determine if maternal brooding time influenced sensitivity to the toxicant, viability, or 

infectivity. Following the 24-h exposure to a range of concentrations of NaCl or Cu, we 

observed predictable results that viability decreased with increasing toxicant 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 glochidia 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 significantly less sensitive to the 

toxicant, had higher metamorphosis success, and remained viable (>90%) longer than those 

collected late in the brooding season. These results have important implications 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 provided data that support retaining the current 

ASTM guideline for toxicity testing with glochidia using viability as the endpoint, but with 

recommended maximum test duration of 24 h. We recommend that glochidia be collected before 

4
 
the end of the brooding season for toxicity tests to ensure health and viability of glochidia. We 

recommend that glochidia viability be assessed in toxicity tests 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 species decreases to <90% by the 24 h time point. 

Formatted for submission to: Environmental Toxicology and Chemistry 

5
 
Determining the appropriate duration of toxicity tests with glochidia of native 

freshwater mussels 

Andrea K. Fritts1, M. Christopher Barnhart2, Megan Bradley2, Na Liu2, W. Gregory Cope3, and 

Robert B. Bringolf1,4 

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 

6
 
Abstract 

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 juvenile mussel. Glochidia attach by closing the shell valves on the 

host. Glochidia also close in response to salt solution, and this closing response 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 for a variety of other applications. The lifespan of 

glochidia in water is limited. We tested the assumption 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 stage. 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 glochidia collected near the end 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 the glochidia. Glochidia in conglutinates 

remained infective longer indicating that conglutinates 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 glochidia did not differ among the concentrations of 

7
 
toxicants, suggesting that viability is indicative of metamorphosis success and that the decrease 

in glochidia reproductive potential after exposure to a toxicant is attributed to a smaller number 

of glochidia that are open and able to attach to a host, rather than 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 with freshwater mussels when control viability is 

≥90%. 

Keywords: Unionidae, larvae, viability, metamorphosis, toxicity 

Introduction 

Freshwater mussels (Order Unionida) comprise 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 anthropogenic perturbations 

(Ricciardi and Rasmussen 1999, Lydeard et al. 2004, Strayer et al. 2004). The extinction or 

extirpation of species can have cascading, ecosystem-level effects because freshwater mussels 

provide essential ecosystem services such as water filtration, nutrient sequestration and cycling, 

and habitat for other aquatic organisms (Vaughn and Spooner 2006, Strayer 2008, Vaughn et al. 

2008). 

The life cycle of freshwater mussels includes 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 

8
 
appropriate host fish species, adult female mussels use a variety of strategies, including mantle 

lures, conglutinates, and broadcast of free glochidia into the water (Barnhart et al. 2008, Haag 

2012). In each case, the glochidia must be released from the female marsupium and from egg or 

conglutinate membranes before they are able to contact the host and attach. 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 immediately 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) reported that glochidia closed in response to various salt 

solutions or to blood, and Coker et al. (1921) recommended that the viability of glochidia could 

be assessed by observing the closing response. This approach remains popular today for 

assessment of glochidia condition before propagation or host testing. Glochidia 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 closing response has also been widely 

used as an endpoint in toxicological studies (e.g., Keller and Ruessler 1997, Bringolf 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 close in response to salt. Either condition prevents completion of 

the lifecycle. 

Few quantitative studies have investigated the 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 (2002) assessed effects of different 

holding temperatures (0°, 10°, 25°C) on glochidia viability and inoculated 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 occurred, but the metamorphosis success (proportion of 

9
 
glochidia that metamorphosed to the juvenile stage) was not reported. Fisher and Dimock (2000) 

held glochidia of Utterbackia imbecillis in reconstituted water and found that the ability of the 

glochidia to metamorphose in cell 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 quality criteria, assessing whether 

the closing response is indicative of the more ecologically relevant ability of glochidia to 

successfully attach and metamorphose on a host fish is necessary. A related question is whether 

the duration of glochidia infectivity in laboratory conditions is similar to that in natural 

conditions. For example, survival might be shortened 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 conglutinates. 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 able 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 metamorphose successfully into the juvenile stage, 2) the 

protective effect of conglutinates, 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 ecological relevance of laboratory tests of glochidia 

viability, infectivity, and the appropriate duration for standard toxicity tests with glochidia. 

10
 
Methods 

Test organisms 

We used snorkeling, scuba diving, and tactile searches to collect gravid female mussels 

from four different states over the course of three years (Table 1). These seven mussel species 

use the three main infection strategies found in Unionidae: 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 mussels 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 females was measured by uniformly suspending the 

glochidia in a known volume of water, removing ten 200-μL subsamples, and placing the 

subsamples into a 10-cm Petri dish. Each subsample contained ~15-30 glochidia, for a total of 

approximately 150-300 glochidia tested for viability. A stereomicroscope at 10x magnification 

was used to count the open and closed glochidia 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 solution 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 

11
 
that did not close after the addition of NaCl from the number that were originally open, then 

dividing by the total number of glochidia (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 beakers with 2 L of aerated, moderately hard 

reconstituted water (ASTM 2006a) where they were maintained at 20°C (± 1oC) with a 16L: 8D 

photoperiod. At the beginning of the experiment and every 24 h thereafter, the viability of each 

replicate was calculated following the method described above. Subsampling continued daily 

until mean viability for the five replicates was <10%. At each time point, a second subsample of 

glochidia from each replicate was tested for infectivity on known host fish (two fish per 

replicate) for a total of 10 fish per 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 exposed to glochidial infections. 

Host fish were exposed to 4000 viable glochidia per liter of water for 15 min; glochidia were 

kept in suspension with a large-bulb pipette and vigorous aeration. 

Attachment and metamorphosis success were assessed in multiunit recirculating 

aquarium systems (Aquatic Habitats, Apopka, Florida) as described by Dodd et al. (2005). Fish 

infested with glochidia were rinsed, then placed individually in unit tanks (1-3 L) fitted with 153­

μm filters to collect all sloughed glochidia and metamorphosed juveniles. Juveniles were 

distinguished from glochidia by possessing a foot and valve movement. Contents 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 collected for at least three consecutive days. Percent metamorphosis 

was calculated by dividing the total number of juveniles by the total number of glochidia and 

juveniles recovered from each fish. The aquarium systems were maintained at 20-22°C. All 

12
 
water-chemistry variables were measured daily throughout the study and 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 conglutinates and sediment 

The durations of glochidia viability 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 pins. Conglutinates from six females were pooled, 

and each conglutinate was examined carefully under magnification to determine if it was intact. 

Only unbroken conglutinates were used for conglutinate exposures. Free glochidia were obtained 

by using pins to gently tear open the outer conglutinate membrane. The ruptured conglutinate 

was then gently drawn in and out of a 3-mL transfer pipette to dislodge the glochidia. 

Both free glochidia and intact conglutinates were exposed to reconstituted hard water or 

to river water with fine sediment. Reconstituted water was similar in formula to that of Smith et 

al. (1997), but concentrations of all solutes were doubled. River water and wet sediment were 

collected from the James River (Greene County, 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 paint 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 replicates per treatment x exposure time; 60 beakers 

total) were: 1) reconstituted water with free glochidia, 2) reconstituted water with conglutinates, 

3) river water and sediment with free glochidia, and 4) river water and sediment with 

13
 
conglutinates. Each beaker received 1000 glochidia or 3 conglutinates 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 for 15 min, while gently stirring the water with a 

pipette, to achieve infestation. Methods for calculating viability and infectivity were similar to 

those previously described. 

Effects of glochidial exposure to toxicants 

We extracted glochidia from 3-8 female mussels 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 females was measured in the same manner as that 

described above. According to ASTM guidelines (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 reconstituted water (ASTM 2006a). In all glochidia 

exposures temperature was 20o

C ±1oC, pH ranged from 7.1 to 7.5, dissolved oxygen was >80% 

of saturation, hardness was 75-83 mg/L as CaCO3, and alkalinity was 71-85 mg/L as CaCO3 

(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) for a total of six fish per concentration. The 

inoculation procedure follows the methods described above. In each replicate, the inoculation 

14
 
suspension was adjusted to provide 4000 viable glochidia per liter. The aquarium systems were 

maintained at 18-22°C. All water-chemistry variables were measured daily throughout the study 

and ranged from 7.5 to 8.6 mg/L dissolved oxygen, pH: 6.8 to7.7, and total ammonia nitrogen 

<0.1 mg/L. Concentrations of NaCl were verified daily in one replicate 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 samples were collected and preserved to pH <2 for 

Cu analysis prior to analysis by inductively coupled 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 based on nominal concentrations. 

Statistical analysis 

Data were tested for normality using the Kolmogorov-Smirnov test (SAS, version 9.3; 

SAS Institute, Cary, North Carolina). To achieve normality and equal variance, viability and 

metamorphosis data were transformed with the arcsine 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% confidence intervals (Bringolf et al. 2007). 

15
 
 

Results 

Duration of viability vs. duration of infectivity 

The initial (0 h) glochidia viability met the 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 initial viability of only 80%. 

Viability and metamorphosis remained 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 Lampsilini. 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 laboratory toxicity tests with freshwater mussels (ASTM 2006b), data 

from these time points would not be valid for toxicological 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 mussel 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 glochidia attachment (p = <0.001) at the final inoculation time point (mean 

16
 
attachment ± 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 conglutinates and sediment 

Initial (0 h) glochidia viability was > 90% in all treatments. Viability of free glochidia 

exposed to sediment and river water decreased rapidly (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 conglutinates but viability did not 

decrease to <90% until 72 h of exposure (Fig. 2B & 2D). Viability of conglutinates in 

reconstituted water was >90% for at least 96 h, the time at which the experiment was terminated. 

Metamorphosis success of free glochidia declined 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 rapidly 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 glochidia 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 points after the initial inoculation 

(0 h = 45 ± 20; 24-96 h = 12 ± 3). 

Effects of glochidial exposure to toxicants 

17
 
In each toxicant test, viability decreased (ranged from 95% to 25%) as toxicant 

concentration increased (Fig. 3). However, for the remaining viable glochidia in each treatment, 

metamorphosis success was similar among treatments 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 species collected in June (late brooding period; Fig. 

3B) and also had consistently higher (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 intervals) 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 glochidia 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 the ability to metamorphose into the juvenile life stage, as long 

as viability was >90%. Timing of glochidia collection also appears 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 termination (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 appears to have direct 

ecological relevance. Based on these data, we recommend that the current ASTM guideline for 

18
 
glochidia toxicity testing be retained with a minimum control viability of 90% throughout the 

duration of the test for all mussel species. Additionally, we recommend that glochidia from long 

term brooders be collected before the end of the 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 metamorphosis success. 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 point(s) 

(e.g., 6, 12, 18, 24 h) during toxicity tests 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 control 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 the effect was much less pronounced for glochidia 

in conglutinates. We suspect that the relatively rapid decline 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 remained >80% viable at 72 h. This result indicated 

that microorganism removal positively affected viability. The subsequent infectivity of these 

glochidia was not tested, however. Whereas infectivity of free glochidia of P. occidentalis 

decreased rapidly in both treatments, infectivity of glochidia in conglutinates remained high for 

the duration of the study (96 h). The apparent protective effects of the conglutinate suggest that a 

24-h toxicity test duration would be conservative for species that release glochidia in 

19
 
conglutinates. Conglutinates provide protection from some toxicants (e.g., copper) and thereby 

reduce toxicity (increase LC50, EC50 values; Gillis et al. 2008). 

Our results suggest that the 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 protection 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 that 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 exposure results suggest that the decrease in glochidia 

reproductive potential after exposure to a toxicant is attributed to a smaller 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 uniform number of viable glochidia, the consistent 

metamorphosis success supports the use of the viability test as a proxy for metamorphosis. 

These results were consistent with 2 mussel species and two toxicants with 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 affect 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 after the glochidia are removed from the female. In 

20
 
our study, L. siliquoidea glochidia early in the brooding period (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) only 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 duration of viability (>90%) for 

some of the species used in the present study; however, 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 recommendation is to retain the current ASTM guideline for toxicity 

testing with glochidia, but specify a maximum test duration of 24 h when using viability (i.e., the 

valve closure response to NaCl) as the endpoint. Ideally toxicity tests should be conducted with 

glochidia immediately following removal from the female mussel, thus shipping of brooding 

females rather than free glochidia may be considered. However, shipping of adult mussels is less 

practical and may create logistical and regulatory (e.g., interbasin transfer of species or 

threatened or endangered status) problems, so shipping 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 viability of controls decreases to <90% by the 24 h 

time point of the toxicity test. This approach is especially important when a species is being 

used for which no viability duration information has been previously reported. These 

21
 
recommendations are supported by Cope et al. (2008), who showed that viability (determined 

with the NaCl method) of glochidia 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 ecologically relevant endpoint of infectivity determined in this 

study. The higher toxicant sensitivity, lower initial viability and lower metamorphosis success of 

glochidia collected late in the brooding season suggests 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 substantially different that if glochidia were collected early 

in the brooding season. More research is required to better define the period when glochidia 

fitness begins to decline to the point that toxicant 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 glochidia 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 often declined more rapidly than viability. Glochidia of long-term 

brooders collected late in the brooding season had low fitness compared to those collected early 

in the brooding season. Glochidia in contact with sediment may have a reduced duration of 

viability and infectivity, but glochidia in conglutinates may remain viable and infective for 

extended periods after release from brooding females. Glochidia viability is, therefore, an 

22
 
ecologically relevant endpoint in standard toxicity 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 Initiative 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 assistance 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, Georgia) and rainbow darters were provided by Conservation Fisheries 

(Knoxville, Tennessee). The Georgia Cooperative Fish and Wildlife Research Unit is jointly 

supported by the University of Georgia, Georgia 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 descriptive purposes only and does not imply endorsement 

by the U.S. Government. 

23
 
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27
 
Table1: 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 studies in which the glochidia were aged before 

inoculation on fish. Hardness and alkalinity (mg CaCO3/L) were measured once at the beginning 

of each study, other parameters were measured 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 

 

28
 
Table 3. Water quality parameters for ASTM moderately hard reconstituted water used for 

glochidia viability and infectivity studies in which the glochidia were exposed to a gradient of 

NaCl for 24 hours, after which time fish were inoculated. Hardness and alkalinity (mg CaCO3/L) 

was measured at the beginning of the exposure and 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 CaCO3/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 

 

29
 
Table 5. Results of one-way ANOVA comparing the change in glochidia viability or 

metamorphosis over time for seven freshwater mussel 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 

0.3118 

 
 

 

 

Table 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 (df), 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 

31
 
 

Figure 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). Presence of a black asterisk (*) indicates a 

significant difference in viability, and a white asterisk indicates a significant 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 reconstituted water, C) free glochidia exposed to river water and 

sediment, and D) glochidia in conglutinates exposed 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 asterisk 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 increasing 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 asterisk indicates a significant difference in 

metamorphosis success. 

Fig. 4. Comparison of NaCl 24-h EC50 (± 95% confidence interval) for Lampsilis cardium 

glochidia collected early in the brooding period (December) and late in the brooding period 

(June). Asterisk indicates significant difference in EC50s (non-overlapping 95% confidence 

intervals). 

32
 
Fig. 1. 

A

100 Percent (%)80 

60 

40 

20 

0 

*

*

*

*

*

*

*

0 24 48 72 96 120 

Exposure (h) 

B 100 Percent (%)

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

100 Percent (%)

*

*

* *

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) 

34

Percent (%)

F

100 

*

*

*

*

*

80 

60 

40 

20 

0 

0 24 48 72 96 

Exposure (h) 
0 

20 

40 

60 

80 

100 Percent (%)

*

*

*

*

*

G

0 24 48 72

Exposure (h)
 

35
 
Fig. 2. 

A

100 

Percent (%)80 

60 

40 

20 

0 

* *

*

*

*

*

0 24 48 72 96 

Exposure (h) 

B

100 

Percent (%)80 

60 

40 

20 

0 

*

*

0 24 48 72 96 
 

Exposure (h) 

36

C

100 

Percent (%)80 

60 

40 

20 

0 

* *

*

*

*

*

*

0 24 48 72 96 

Exposure (h) 
0 

20 

40 

60 

80 

100 

Percent (%)

*

D

0 24 48 72 96 
 

Exposure (h)
 

37
 
Fig. 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 

100 Percent (%)

*

*

*

*

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 

2.5 

24-h EC50 (g/L)

*

Dec June
 

39