Sensitivity of freshwater snails to aquatic contaminants: Survival and growth of 

endangered snail species and surrogates in 28-day exposures to copper, ammonia and 

pentachlorophenol 

John M. Besser, Douglas L. Hardesty, I. Eugene Greer, and Christopher G. Ingersoll 

U.S. Geological Survey, Columbia Environmental Research Center, Columbia, Missouri 

Administrative Report (CERC-8335-FY07-20-10) submitted to U.S. Environmental Protection 

Agency (USEPA) 

Project Number: 05-TOX-04, Basis 8335C2F, Task 3. 

Project Officer: Dr. David R. Mount 

USEPA/ORD/NHEERL 

Mid-Continent Ecology Division 

6201 Congdon Blvd, Duluth, MN 55804 USA 

April 13, 2009 
Abstract – Water quality degradation may be an important factor affecting declining populations 

of freshwater snails, many of which are listed as endangered or threatened under the U.S. 

Endangered Species Act. Toxicity data for snails used to develop U.S. national recommended 

water quality criteria include mainly results of acute tests with pulmonate (air-breathing) snails, 

rather than the non-pulmonate snail taxa that are more frequently endangered. Pulmonate pond 

snails (Lymnaea stagnalis) were obtained from established laboratory cultures and four taxa of 

non-pulmonate taxa from field collections. Field-collected snails included two endangered 

species from the Snake River valley of Idaho -- Idaho springsnail (Pyrgulopsis idahoensis; 

which has since been de-listed) and Bliss Rapids snail (Taylorconcha serpenticola) -- and two 

non-listed taxa, a pebblesnail (Fluminicola sp.) collected from the Snake River and Ozark 

springsnail (Fontigens aldrichi) from southern Missouri. Cultures were maintained using simple 

static-renewal systems, with adults removed periodically from aquaria to allow isolation of 

neonates for long-term toxicity testing. This method was successful for pond snails and Idaho 

springsnails, less successful with pebblesnails, and unsuccessful for Bliss Rapids snail or Ozark 

springsnail. Long-term (28-d) toxicity tests were conducted with copper, ammonia, and 

pentachlorophenol in hard reconstituted water (hardness 160-180 mg/L as CaCO3). Juvenile 

Idaho springsnails (5-7 weeks post-hatch) generally were more sensitive than neonate (<7 days 

post-hatch) pondsnails in survival and growth tests with copper, ammonia, and 

pentachlorophenol. In 28-d lethality tests with older snails (from field collections or laboratory 

cultures), copper sensitivity was similar among the four non-pulmonate taxa, but ammonia 

sensitivity differed widely, with Ozark springsnail the most sensitive taxon to ammonia. For all 

three chemicals, toxicity values for one or more taxa of non-pulmonate taxa were among the 

lowest reported in the literature, whereas toxicity values for pond snails were in the middle or 

2 
upper end of species sensitivity distributions. The ambient water quality criterion for copper, 

based on the Biotic Ligand Model, was protective for the snail taxa we tested, but one or more 

snail taxa had toxicity values less than criteria for pentachlorophenol or ammonia. These results 

indicate that inclusion of chronic toxicity data for freshwater snails during the development or 

revision of water quality criteria for protection of aquatic life can increase the ability of these 

criteria to protect aquatic taxa – including endangered and threatened snail taxa – from adverse 

effects of chemicals. 

Disclaimer: Any use of trade, product, or firm names is for descriptive purposes only and does 

not imply endorsement by the U.S. Government. 

3 
INTRODUCTION 

The suitability of U.S. Environmental Protection Agency (USEPA) ambient water quality 

criteria (WQC) for protection of aquatic species listed under the U.S. Endangered Species Act 

(ESA) has been the topic of ongoing consultations between USEPA, the U.S. Fish and Wildlife 

Service (USFWS) and states. Studies with ESA-listed freshwater fish species determined that 

these species were not consistently more sensitive to acute or sublethal effects of toxic chemicals 

than common surrogates (Dwyer et al. 2005; Besser et al. 2005), but recent studies indicate that 

freshwater mussels, including listed species, may be more sensitive than most fish and 

invertebrate species to toxic effects of some chemicals (e.g., ammonia and copper; Wang et al. 

2007; Augspurger et al. 2007). 

Many species of freshwater snails are ESA-listed, including six species from the Snake 

River region of Idaho (USFWS 1995), but there are relatively few data on the sensitivity of 

freshwater snails to toxic effects of contaminants. Most published toxicity tests with snails have 

been acute lethality tests conducted with field-collected snails, although greater sensitivity is 

likely in chronic tests with juvenile test organisms and sublethal endpoints (e.g., Gomot 1998, 

Duft et al. 2003, Grosell et al. 2006, Pounds et al. 2008). Air-breathing snails of the subclass 

Pulmonata (e.g., the families Physidae, Lymnaeidae, and Planorbidae) have been most widely 

used for laboratory toxicity tests, and their rapid growth, short generation times, and high 

reproductive output make them easy to use in toxicity tests, including chronic tests with 

sensitive, sublethal endpoints. Non-pulmonate snails (formerly included in subclass 

Prosobranchia) are more taxonomically diverse, and their physiology (inability to breathe 

atmospheric oxygen) and life history (slow growth and low reproductive rate) may make them 

both subject to endangerment and difficult to culture and test in the laboratory. 

4 
This report summarizes research conducted to help USEPA and USFWS to determine if 

WQC developed under the Clean Water Act are protective of ESA-listed snails in Idaho. 

Although some of the snail species and chemicals tested have special relevance to aquatic 

habitats in the Snake River valley of Idaho, the toxicity data generated during this study should 

prove to be useful for other consultations throughout the United States. 

The specific objectives of this study were: 

(1) to develop methods for laboratory culture and chronic toxicity testing (28-d survival and 

growth tests) with freshwater snails, including ESA-listed snails from Idaho and 

surrogate species; and 

(2) to conduct toxicity tests with several chemicals that may pose risks of toxicity in snail 

habitats and to compare chronic toxicity values for these chemicals among snail species 

and between snails and other aquatic taxa used to develop water quality criteria. 

METHODS 

Snail Collection and Culture 

Four species of non-pulmonate snails were collected from the field and shipped to the 

Columbia Environmental Research Center in Columbia Missouri for laboratory culture. Three 

species of non-pulmonate snails were collected by the USFWS from habitats in the Snake River 

valley of Idaho in fall 2005 and fall 2006: Idaho springsnail (SS; Pyrgulopsis idahoensis) and 

Bliss Rapids snail (BR; Taylorconcha serpenticola), and a species of pebblesnail (PS; 

Fluminicola sp.). Both BR and SS were listed under the Endangered Species Act at the time of 

these collections, although SS has since been de-listed, because it is now considered to be a 

5 
population of a species with wider distribution, the Jackson Lake springsnail (P. robusta). 

Another non-pulmonate species, Ozark springsnail (OZ; Fontigens aldrichi) was collected from 

southern Missouri in fall 2006. We also obtained a culture of the pulmonate pondsnail (Lymnaea 

stagnalis; LS) from Dr. Marie-Noële Croteau, (USGS, Menlo Park CA). 

Snails were held in 40-L aquaria with 35-L of well water (hardness 280 mg/L as CaCO3) 

at 18 oC. Aquaria were aerated and one-half of the water was replaced twice per week. 

Photoperiod was maintained at 16 hours light: 8 hours dark, with one-half of the tanks covered 

with shading material. Stainless steel tiles were offered as substrate. Snails were fed ad libitum a 

combination of Instant Algae and Shellfish Diet (Reed Mariculture, Campbell, CA). These algae 

suspensions were painted onto stainless steel substrates, which were air-dried before they were 

placed in the culture tanks. Additional foods offered to snail cultures included Wardley algae 

disks (Hartz Mountain Corp., Secaucus, NJ), and small portions of organically-grown carrots and 

iceberg lettuce. 

Even-aged cohorts of non-pulmonate snails were obtained by transferring adults to new 

tanks monthly and rearing neonates that hatched from deposited eggs. To collect neonates, 90% 

of the water was siphoned from the tank and the sides and the bottom of the tank were sprayed 

gently with well water to loosen snails and detritus. Water containing neonates and detritus was 

rinsed through a 150-μm stainless steel sieve and sieve contents were examined with a 3.5X 

dissecting microscope. Neonate snails were collected using a capillary tube syringe system. This 

syringe system consists of a glass capillary tube (1.17-mm inner diameter) connected by vinyl 

tubing (1.0-mm inner diameter) to a 2.5-cm, 1-mL syringe with 16-gauge needle (ASTM 2008a). 

After collection, the young snails were placed into a 4-L beaker containing 2 L of well water. 

These beakers were aerated gently , a small amount of food was offered, and the snails were 

6 
acclimated to the hard reconstituted test water (ASTM 2008b) over a 48-hr period. Only SS 

produced cohorts large enough for conducting toxicity tests with uniform-age groups, with more 

than 2500 offspring produced from an initial cohort of 300 snails between October 2005 and 

October 2006. Reproduction of this species predominantly occurred in winter and spring 2006, 

within nine months of collection. PS from the fall 2006 collection produced about 200 neonates 

between July and October 2006 and these animals were combined over several months to 

produce mixed-age groups for testing in summer 2007. BR and OZ did not reproduce in our 

cultures and were tested only in mixed-age field-collected groups. LS produced abundant egg 

masses, which were collected as needed to provide neonates (<7 d after hatching) for testing. 

Preliminary evaluation of test conditions 

Two preliminary studies were conducted to evaluate the performance of two snail 

species, SS and LS, under potential test conditions for 28-d toxicity tests. The first of these 

studies evaluated survival and growth of SS and LS fed three different food types during a 21-d 

period. Diets in this test included: an algae mixture (1 ml of Nannochloropsis and 2 ml Shellfish 

Diet in 1800 ml of well water; Wang et al. 2007) used for tests with juvenile freshwater mussels 

(ASTM 2008a); the standard yeast-cereal leaf-trout food (YCT) diet (1.8 mg/ml); and a 

suspension of Tetrafin® flake fish food (6 mg/ml). Each diet was fed at low and high feeding 

rates (1 ml and 2 ml per chamber). General test conditions are summarized in Table 1. Tests 

were conducted with four replicates of LS (<7 d post hatch; 10 snails per chamber) for each 

combination of diet and feeding rate. Because of the limited availability of SS, single replicates 

(10 snails per chamber) were tested with seven of eight treatment combinations, excluding the 

high feeding rate of the YCT diet. 

7 
The second preliminary study investigated possible causes for high mortality of SS in 

controls in the first two 28-d toxicity tests with copper (Test A) and PCP (Test B). This 28-d 

study compared survival and growth of SS and LS in treatment combinations that differed in 

water type (hard reconstituted water, described below, vs. well water), carrier solvent (hard 

reconstituted water with and without 0.26 ml/L triethylene glycol, the carrier solvent used in the 

PCP exposures), and feeding rate (flake food suspensions; low vs. high). Feeding rates in the 

‘low’ treatment were 0.5 ml/chamber on Monday-Wednesday-Friday for SS and 1.0 ml/chamber 

daily for LS. Feeding rates in the ’high’ treatment were increased after day 13 to 0.5 ml daily for 

SS and 2 ml daily for LS. Each of the eight treatment combinations was tested with both SS and 

LS (3 replicates per species with 10 animals in each replicate). 

Toxicity tests 

Test conditions for 28-d toxicity tests with freshwater snails were similar to methods for 

toxicity testing with freshwater mussels (ASTM 2008a; Wang et al. 2007). All tests were 

conducted in hard reconstituted water (170 mg/L hardness as CaCO3; ASTM 2008b) at 20 °C. 

Test chambers were 300 ml beakers with 200 ml water volume, with 3.75 volume additions per 

day. Test chambers for tests with LS were covered with screened caps (initially designed for tests 

with flying adult midges; USEPA 2000) to prevent these air-breathing snails from escaping from 

exposure chambers). Additional details of test conditions are presented in Table 1. 

Toxicity tests were conducted with copper (copper sulfate), pentachlorophenol (PCP; 

stock prepared in triethylene glycol), and ammonia (ammonium chloride). These chemicals were 

selected from the list of candidate chemicals with differing modes of toxic action suggested by 

Dwyer et al. (2005), based on their potential importance in snail habitats in the Snake River 

8 
valley (copper from aquaculture, ammonia from livestock) and sensitivity of snails in acute 

range-finding tests with copper, ammonia, PCP and carbaryl. In 96-hr range-finding tests, SS 

mortality was observed in exposures to copper (above 31 μg/L), pentachlorophenol (above 250 

μg/L) and ammonia (above 8 mg N/L), but not carbaryl (no mortality at 10 mg/L; all 

concentrations nominal). 

Automated proportional diluters prepared and delivered five test concentrations in a 50 

percent dilution series, plus a control. The control water for PCP tests included 0.26 ml/L of 

triethylene glycol at the same concentration of triethylene glycol in the highest PCP treatment. 

Stock solutions and test waters for ammonia tests were adjusted to pH 8.3. The pH of 

ammonium chloride stock solutions was increased by adding dilute sodium hydroxide and the 

pH of test water was decreased by titration with dilute hydrochloric acid using automatic pH 

controllers (Wang et al. 2008). 

Water samples were collected every two weeks for measurement of copper or PCP and 

weekly for analysis of ammonia. Samples for analysis of copper or PCP were filtered (a 

polypropylene syringe filter; 0.45-μm pore diameter) and samples were analyzed without 

concentration or cleanup steps. Copper concentrations were determined by inductively-coupled 

plasma-mass spectroscopy (May et al. 1997). PCP concentrations were analyzed by highperformance liquid chromatography using a C18 column, a mobile phase of acetonitrile/water, 

and UV detection (Orazio et al. 1983). Total ammonia concentrations were determined on 

unfiltered samples by ion-selective electrode. 

Different age/size groups of snails were used for toxicity tests, depending on the 

availability of various species (Table 2). All tests with LS were started with cohorts of neonates 

less than 7 d post-hatch. The first two 28-d toxicity tests with copper (Test A) and PCP (Test B) 

9 
were conducted with the youngest SS that could be handled routinely without mortality: (about 

3-6 weeks after egg deposition or 2-3 weeks post-hatch). Endpoints of these studies were 

survival (both species) and growth (wet weight; LS only). Because of high control mortality in 

these tests (5 to 60% survival at Day 28), subsequent survival and growth tests with all three 

chemicals (Tests 1-3) were conducted with juvenile SS (5-13 weeks post-hatch) and neonate LS 

(<7 d post-hatch), with three or four replicate groups per treatment (depending on availability of 

organisms). Additional survival tests (Tests 4 and 5) were conducted with mixed-age snails from 

field collections (BR and OZ) or with older, mixed-age snails from cultures (SS and PS), with 

two replicates per treatment. Field-collected snails were held for periods from four months (OZ) 

to six months (BR) -- in attempts to initiate laboratory cultures -- before they were used in 

toxicity tests. Older, laboratory-produced groups included animals of widely-varying ages: 

roughly 4-8 months old for SS and 6-12 months old for PS. All toxicity tests were conducted for 

28 d, with endpoints of survival (determined by observation of movement using microscopic 

examination) and growth. For preliminary tests A and B, growth of LS was determined by 

measurement of aggregate wet weights for each rep, and no growth data was collected for SS, 

which had problems with control survival. For subsequent survival and growth tests with SS and 

LS (Tests 1-3), growth of both species was evaluated by digital measurement of maximum shell 

diameter. Growth was not measured at the end of the survival tests conducted with the mixedage snails (Tests 4 and 5). 

Toxicity data were rank-transformed and analyzed by one-way ANOVA (Conover and 

Iman 1981), with significant differences from controls determined by Dunnett’s test (SAS/STAT 

software, version 9.1; SAS Institute, Cary NC). ANOVA results were used to determine the noobserved-effect concentration (NOEC), the highest exposure concentration that did not differ 

10 
significantly from the control, and the lowest-observed-effect concentration (LOEC), the lowest 

exposure concentration above the NOEC. Concentration-response curves were analyzed to 

determine median lethal concentrations (LC50s) and concentrations associated with 20 percent 

reductions in survival and growth (EC20), with concentrations log-transformed for all analyses. 

For tests with well-defined concentration-mortality curves (typically, tests with two or more 

treatments with partial mortalities), LC50s and EC20s for reduced survival were estimated by 

logistic regression using the Toxicity Response Analysis Program (TRAP, Version 1.0; R. 

Erickson, USEPA-MED, Duluth MN). For other tests, LC50s were estimated by the trimmed 

Spearman-Karber method (Hamilton et al. 1977) and EC20s for survival and growth were 

estimated using linear interpolation (Inhibition Concentration or ICp method; TOXSTAT 

software, version 3.5, WEST Inc., Cheyenne WY). For each species and chemical, the lowest 

EC20 value for single tests (or the geometric means of lowest EC20s for multiple tests) was used 

as the ‘species chronic value’ (SCV) for inter-species comparisons. 

RESULTS 

Evaluation of test conditions 

Results of the study of experimental diets indicated that LS had highest survival and 

growth when fed the flake diet (Figure 1), whereas SS had 100 percent survival in all diet 

treatments and had greater growth when fed either flake or YCT diets, compared to the algae 

diet. These results were the basis for selecting feeding rates of 0.5 ml/d of the flake diet (3 mg/d) 

for test chambers with neonate SS (5-13 weeks post-hatch) and 1 ml/d of the flake diet (6 mg/d) 

for chambers with neonate LS or with larger, mixed-age groups of other snail species. 

11 
Results of the second evaluation of test conditions indicated no consistent differences in 

survival (≥90% in all treatments) or growth (Figure 2) among water, carrier solvent, or feeding 

rate treatments. We concluded from this study that poor survival of SS in Test A and Test B was 

not caused by excessive feeding, inappropriate test water, or the presence of the carrier solvent. 

We concluded that problems with low SS survival in these tests was attributable to starting tests 

with very young snails (about 3-6 weeks after egg deposition). Subsequent toxicity tests were 

conducted with older SS (at least 5 weeks post-hatch; Table 2), with a reduced initial feeding rate 

(0.5 ml/d flake food on Monday, Wednesday, and Friday for the first 14 d of the 28-d exposures) 

and had no further problems with control survival. 

Exposure concentrations and water quality 

Exposure concentrations for toxicity tests generally were close to nominal concentrations. 

Measured copper concentrations generally were within 10% of nominal concentrations for all 

three tests (Table 3), except the highest exposure concentration in Test 1 averaged 146% of the 

nominal concentration, apparently caused by a short-term diluter malfunction. Measured copper 

concentrations in the lowest exposure levels were also slightly greater than nominal in all three 

tests, reflecting a low but consistent background copper concentration in our test systems 

(average for controls: 0.87 μg Cu/L). Measured concentrations of PCP in Test B were consistent 

with nominal concentrations except for losses, presumably attributable to sorption of PCP, at 

lower exposure levels (Table 4). In contrast, PCP concentrations in Test 2 averaged only about 

50% of nominal concentrations. The cause of this problem was never fully determined, but the 

data suggest a problem with the injection volume from the toxicant delivery system. Despite this 

12 
problem, means of five measured PCP concentrations for each exposure level were consistent 

within treatments, and standard deviations were less than 10 percent of means. Mean ammonia 

concentrations were close to nominal (Table 5), although some losses were evident in Test 5 

(i.e., measured concentrations as low as 83-84% of the nominal concentrations), probably 

indicating increased nitrification by the microbial community in the test system. 

Water quality of test waters remained within acceptable ranges throughout tests. Mean 

hardness (169 mg/L as CaCO3) and alkalinity (121 mg/L as CaCO3) measured in nine batches of 

test water delivered to the proportional diluters were within acceptable ranges (ASTM 2008a,b; 

data not shown). The mean pH of incoming test water (8.41) was above the specified range of 

7.8 to 8.0 for this water, but was within the normal range for freshly-prepared hard reconstituted 

waters prepared at CERC (e.g., Dwyer et al. 2005). Average major ion content of test waters 

(means of 7 analyses, in mg/L) were chloride, 4.9; sulfate 167; calcium, 30, magnesium, 22; 

potassium, 4.0; sodium, 50; and iron, 0.033 (Table 6). Dissolved organic carbon (DOC) was 

below the detection limit (<1.0 mg/L) for two analyses of incoming test water and subsequent 

analyses of ASTM hard water resulted in a measured DOC concentration of about 0.3 mg/L 

(Ning Wang, USGS, Columbia, MO, unpublished data). DOC also was below detection in seven 

of 14 analyses of water from test chambers (Table 6). Of the seven samples with detectable 

DOC, the four highest concentrations (59-100 mg/L) were collected during the PCP tests and 

reflect the presence of the triethylene glycol carrier solvent, which had a nominal maximum 

concentration of 137 mg/L in the high treatment and the solvent control. Three samples from 

tests without carrier solvent had low (<2 mg/L) but detectable concentrations. 

Concentrations of other water quality measured during tests (Table 7) were similar to 

those in the incoming water supply, except mean pH in these equilibrated samples fell within a 

13 
slightly lower range (8.0 - 8.4). Test waters from ammonia tests had mean pH within a narrow 

range (8.22-8.29) and had slightly lower alkalinity, reflecting the influence of additions of small 

volumes of acid by the automatic pH control system used in the ammonia exposures. Dissolved 

oxygen in test chambers remained above 5.4 mg/L in all samples. Total ammonia concentrations 

remained low in test chambers (except in ammonia tests), with mean concentrations of 1.2 mg 

N/L or lower. Mean temperatures of all tests remained within the nominal range (19-21 oC) 

although some individual measurements exceeded 22 oC, apparently reflecting warming of 

samples that were removed from test chambers. 

Toxicity data 

Snails generally adapted well to test conditions during toxicity tests. Snails in the controls 

and in the low-chemical treatments moved throughout the test beakers and fed actively (as 

evidenced by removal of food and biofilm). Snails in high-chemical treatments tended to be less 

active, often remaining on the bottoms of the test chambers, allowing food and biofilm to 

accumulate. Pondsnails (LS) in all treatments sometimes crawled above the water level; this 

behavior was most common in high-copper treatments. Control survival was acceptable (90­

100%) in all exposures except the first two SS exposures (Test A and Test B). In Test A (Table 

8), SS survival was only 60% in the control, but survival in copper treatments followed trends 

similar to subsequent copper tests. In Test B (Table 9), SS had low survival (0-25%) in all 

treatments. The failure of these two tests, and the results of the second test-conditions study 

(Figure 2), led to the decision to use older SS for subsequent tests at a lower initial feeding rate, 

which resulted in acceptable control survival of SS in all subsequent tests. Snails in the LS 

control groups in Tests 1, 2, and 3 (started with <7-d old neonates) grew rapidly and consistently, 

14 
with mean shell length increasing by more than 300 percent by day 28. Growth of juvenile SS in 

these tests (started with animals 5-12 weeks post-hatch) was slower and more variable among 

tests, with mean shell diameter increasing by 32 to 99 percent by the end of tests. Control groups 

of SS in Tests 4 and 5, which were started with older snails, had greater increases in mean shell 

length (85-86%). Control groups of mixed-age BR, OZ, and PS had little change in mean shell 

length (maximum increase of 30%) during Tests 4 and 5. 

Results of 28-d toxicity tests with copper, PCP, and ammonia are summarized in Tables 

10 through 14 and Figures 3 through 5. Statistical analyses of data indicated significant 

reductions in survival and growth, relative to controls (one-way rank ANOVA; P<0.05) for most 

tests and endpoints. Exceptions were the survival data for LS and growth data for SS in Test 3 

with ammonia, which had little variation among treatment means (Table 12), and survival data 

for PS in Test 5 with ammonia, which had a wide range of treatment means (0-100%), but also 

had extreme variation between replicates in two of the high-ammonia treatments (Table 14). 

Dunnett’s test indicated that one or more treatment means were significantly less than controls 

for all tests that had significant ANOVA results, allowing estimation of NOEC and LOEC. 

Chronic toxicity values (NOEC and LOEC) were determined for all endpoints with 

significant ANOVAs (Table 15). Median lethal concentrations (LC50s) were determined for all 

exposures, except two exposures that had survival greater than 50 percent in all treatments: LS 

exposure to PCP in test B (Table 9), and LS exposure to ammonia in Test 3 (Table 12. The LC50 

for PCP toxicity to LS in Test 2 was estimated by the inhibition concentration method (IC50) 

because this test did not have any treatments with zero survival (Table 11). 

Survival and growth EC20s (Table 15) were defined for all exposures except for 

ammonia exposures in Test 3 (Table 12). In this test, survival and growth of LS and growth of 

15 
SS did not have 20% reductions relative to controls (EC20 >8.0 mg N/L). In contrast, survival 

of SS had greater than 20% reductions in survival in all treatments except the control (EC20 < 

0.48 mg N/L). Species chronic values (SCV) were estimated as the lower of EC20s for survival 

or growth for each chemical in single tests for a species, or as the geometric mean of lowest 

EC20s from multiple tests with the same species. 

Differences in toxicity values among snail species 

Copper. Analysis of variance (ANOVA) demonstrated significant reductions in survival 

with increased copper concentrations for all eight exposures (Test A, Test 1, and Test 4; Figure 

3). LOECs for survival in copper exposures ranged from 13 μg/L, for all species in Test 4 (SS, 

BR, PS, and OZ), to 87 μg/L for LS in Test 1 (Tables 10 and 13). Median lethal concentrations 

(LC50s) in copper tests indicated a narrower range of sensitivity among species and between 

tests (Table 15). The ranges of LC50s for repeated tests with LS and SS overlapped, but survival 

EC20s for LS (16.9 and 22.4 μg/L) were greater than those for SS or any other non-pulmonate 

taxa (range: 8.58-16.8 μg/L). Analyses of the growth response for LS and SS were somewhat 

inconclusive, with LOECs and EC20s indicating somewhat different trends for Test A and Test 1 

(Tables 8, 10, 15). Overall, copper EC20s differed substantially among repeated tests and 

endpoints for LS (6.15-22.4 μg/L) and SS (7.4-13.4 μg/L), but SCVs for copper fell within a 

narrow range (9.55-16.8 μg/L) for all five snail taxa tested (Table 15). 

Pentachlorophenol. Responses of SS and LS to PCP exposure were quite similar in Test 2 

(Figure 4). Survival of SS and LS was reduced significantly only at the highest PCP 

concentrations tested (>200 μg/L), but growth of SS was affected significantly at much lower 

concentrations. Survival of LS was even less affected by PCP in Test B, but comparisons to SS 

16 
are not available for this test because of the failure of the SS exposure. In contrast, growth of 

both species was affected by PCP exposure – in Test B for LS and in Test 2 for SS (Table 15). 

The apparently greater sensitivity of LS growth in Test A compared to Test 2 suggests that the 

one-dimensional measure of shell diameter (Table 11) did not fully express changes that were 

evident in wet weight (Table 11). As a result of between-test differences in growth EC20, SCVs 

for PCP were lower for SS than for LS, even though the lowest growth EC20s for the two 

species were similar (Table 15).

 Ammonia. Sensitivity to ammonia toxicity varied widely among the snail taxa (Figure 5). 

LS was least sensitive to ammonia effects on survival, with no significant reductions at the 

highest concentrations tested (nominal 8.0 mg N/L as total ammonia), and OZ was most 

sensitive, with a LOEC 0.83 mg N/L (Tables 12 and 14). Although ANOVA indicated no 

significant differences among ammonia concentrations for PS in Test 5, due to wide variation 

between the two replicates in several treatments, the LC50 and survival EC20 for PS were the 

second-lowest of the species tested (Table 15). For SS, LC50s and survival EC20s for ammonia 

toxicity differed widely between Test 3 and Test 5, perhaps reflecting differences in sensitivity 

between juveniles and adults. Growth responses to ammonia exposure in Test 3 were not 

significant for SS, but reductions in growth were small but significant for LS. Although the 

growth LOEC for LS was low (1.8 mg N/L), the growth EC20 was not defined because the 

magnitude of growth reductions were small (from 6% at 1.8 mg N/L to 16% at 8.0 mg N/L). As 

a result, the SCV for ammonia toxicity to LS was much greater than those for the four nonpulmonate species. 

17 
DISCUSSION 

Sensitivity of snails relative to other aquatic taxa 

Copper. All five snail taxa tested were sensitive to copper exposure, relative to SCVs 

reported by USEPA (2007) and results from other recent studies at CERC (Figure 6a). After 

SCVs were adjusted to a water hardness of 85 mg/L (USEPA 2004), only five of 25 values – 

including three chronic values for freshwater mussels (Wang et al. 2007) – were lower than those 

for several snail taxa tested in this study (SS, PS, and LS). All five species tested were more 

sensitive than the only other snail species in the USEPA chronic copper toxicity database, the 

pulmonate species, Campeloma decisum (Arthur and Leonard 1970; Figure 6). The chronic 

WQC for copper derived using the Biotic Ligand Model (USEPA 2007; calculated for ASTM 

reconstituted water with hardness of 85 mg/L and DOC of 0.5 mg/L) appears to provide adequate 

protection for the snail taxa tested. These comparisons do not attempt to model the influence of 

DOC in various toxicity test waters, because DOC data were not reported for many of the studies 

reported by USEPA (2007). Most measured DOC concentrations in test chamber were below 

detection (<1.0 mg/L), with a few detectable concentrations as high as 1.8 mg/L (Table 6). 

Pentachlorophenol. The two snail species tested, LS and SS, had widely differing 

sensitivity to PCP (Figure 6b). The SCV for SS, based on an EC20 for growth of 16.6 μg/L, is 

the lowest chronic value reported for aquatic taxa exposed to PCP, and is lower than the current 

chronic water criterion (24 μg/L at pH 8.25). In contrast, the SCV for LS ranked near the middle 

of the species sensitivity distribution, and another pulmonate snail, Physa gyrina, was among the 

least sensitive aquatic taxa tested (Hedke et al. 1986; Figure 6). The fact that two of 11 available 

SCVs – for SS (this study) and the fountain darter (Besser et al. 2005) – fall at or below the 

18 
current chronic WQC suggests that this criterion may not adequately protect snails or other 

aquatic taxa from toxic effects of PCP. 

Ammonia. Tests with five taxa indicated that freshwater snails are among the aquatic taxa 

most sensitive to ammonia toxicity (Figure 6). Three of the five snail species we tested had 

SCVs (OZ, PS, and SS) close to or less than the lowest SCV reported by USEPA (1999). The 

SCV for OZ was lower than the current chronic WQC for ammonia, and was lower than chronic 

values for other aquatic taxa, except for two freshwater mussel taxa tested by Wang et al. (2007). 

For SS, both the survival EC20 and LC50 from Test 3 were even lower than the SCVs for OZ, 

although the SCV for SS was increased by the substantially higher survival EC20 from Test 5. 

Although the survival data for SS in Test 3 were highly variable, the contrast between 100 

percent control survival and mean survival of 60 percent or less in all ammonia treatments 

suggests that the juvenile SS in this test were substantially more sensitive than the cohort of older 

SS exposed to ammonia in Test 5. 

Relative sensitivity of snail taxa and endpoints 

Results of the chronic toxicity tests indicated that LS was less sensitive than the nonpulmonate snail species for two of the three chemicals tested. This trend is less evident in results 

of previous acute tests with these same chemicals, using a variety of pulmonate and nonpulmonate snail taxa (Table 16). For ammonia and PCP, 96-hr LC50s for pulmonate taxa 

(Lymnaea spp. and Physa spp.) overlapped broadly with those for non-pulmonate taxa 

(Potamopyrgus spp. and Gillia altilis). For copper, the 96-hr LC50 for Lymnaea luteola was 

lower than 96-hr LC50s reported for the non-pulmonate species, Potamopyrgus jenkinsi. 

19 
Evaluating differences in sensitivity among taxa is complicated by the influence of 

various test conditions. Acute toxicity of PCP under ambient conditions in Mississippi River 

water was substantially greater during summer than during winter (Hedke et al. 1986) and PCP 

was more acutely toxic in flow-through tests than in tests conducted under static conditions 

(Stuart and Robertson 1985). Acute LC50s for Potamopyrgus jenkinsi were lower for juveniles 

than adults for ammonia (by a factor of 2.6) and copper (by 50%; Watton and Hawkes 1984). 

Our 28-d tests with SS also indicated greater sensitivity of younger animals. In tests with both 

copper and ammonia, survival EC20s and LC50s for young SS (2-13 weeks old, in Test A, Test 

1, and Test 3) consistently were lower than values determined from tests with older, mixed-age 

cohorts (Test 4 and Test 5; Table 15) 

Comparison of chronic sensitivity of pulmonate and non-pulmonate snails also may be 

affected by differences in the endpoints that are feasible for laboratory chronic tests. Several 

chronic toxicity studies with Physa spp. and Lymnaea spp. have reported sensitive sublethal 

effects on reproduction (e.g., Hedke et al. 1986, Gomot 1998), and/or growth (e.g., Grosell et al. 

2006, De Schamphelaere et al. 2008) in these pulmonate taxa, which have rapid growth and short 

generation times. Fewer chronic studies have reported sensitive sublethal responses in nonpulmonate snails, but egg production was reported to be more sensitive than survival in 28-d 

water-only tests with Bithynia tentaculata (van Wijngaarden et al. 1998) and in spiked sediment 

tests with the New Zealand mudsnail, Potamopyrgus antipodarum (Duft et al. 2003). 

Characteristics of P. antipodarum, notably the occurrence of populations that are parthenogenic 

and ovoviviparous, have led to proposals for its use in reproductive toxicity studies (Duft et al. 

2007). Tests reporting reproductive endpoints of either pulmonate or non-pulmonate snails 

require long test duration (e.g., 5-6 weeks; Hedtke et al. 1986, Arthur and Leonard 1970) or tests 

20 
 

started with adult- or sub-adult snails. No reproduction of LS was observed in the 28-d tests, 

which started with early juveniles (<7 d post-hatch), but some egg production of PS in 28-d was 

observed in tests started with mixed-age groups (Test 5). There also were not consistent 

differences in sensitivity between growth and mortality endpoints in tests with LS or SS. Of 

seven tests with survival and growth endpoints, EC20s for growth were substantially lower in 

two tests (copper Test A for LS, PCP Test 2 for SS), the EC20 for survival was lower in one test 

(ammonia Test 3 for SS), and the two endpoints had similar EC20s in the remaining four tests 

(Table 15). 

Summary 

Objective 1: Develop methods for laboratory culture and chronic toxicity testing (28-d survival 

and growth tests) with freshwater snails, including ESA-listed snails from Idaho and surrogate 

species. 

Chronic (28-d) toxicity tests were completed successfully with two to five taxa of 

freshwater snails exposed to copper (7 tests), PCP (3 tests), and ammonia (6 tests). With 

the exception of the first two tests with SS, toxicity tests with five species of freshwater 

snails, conducted under routine flow-through test methods, had good control survival and 

produced reliable estimates of toxicity thresholds. Simple static culture methods 

produced neonates of two species (LS and SS) in numbers adequate for toxicity testing, 

and also produced lesser numbers of a third species (PS). Tests with laboratory-reared 

and field-collected snails produced useful data on snail survival in 28-d toxicity tests. 

Tests conducted with uniform-aged groups generated low-variability estimates of growth 

21 
based on digital measurement of shell diameter, and toxicity values for growth were 

lower than those for survival for two of seven exposures. 

Objective 2: Conduct toxicity tests with several chemicals that may pose risks of toxicity in snail 

habitats and to compare chronic toxicity values for these chemicals among snail species and 

between snails and other aquatic taxa used to develop water quality criteria. 

Sensitivity to the toxic effects of three test chemicals varied widely among the five snail 

species tested. The pulmonate pondsnail (LS) was less sensitive to PCP than the nonpulmonate Idaho springsnail (SS) and less sensitive to ammonia than all four nonpulmonate taxa. All five snail taxa had similar sensitivity to copper. The sensitivity of the 

two ESA-listed species, SS (which is no longer listed) and BR, to ammonia was similar 

to or less than that of the non-listed snail taxa. Overall, study results indicate that 

freshwater snails, especially non-pulmonate snails, are very sensitive to toxic effects of 

some aquatic contaminants. The WQC derived from datasets that do not represent snail 

taxa, such as the current ammonia criteria (USEPA 1999), may not adequately protect 

freshwater mollusks. The de-listing of SS and the limited testing conducted with the 

federally-endangered species, BR (survival data only, from tests with field-collected 

animals) leaves some uncertainty about the relative sensitivity of listed vs. unlisted snail 

species. Ongoing research in our laboratory is focused on development of improved 

culture methods for BR, with the goal of producing even-age cohorts that can be tested 

using the 28-d survival and growth test method presented here. 

22 
Acknowledgments 

Funding for this project was provided by USEPA through the Regional Applied Research 

Effort (RARE) program, but this report does not necessarily represent the views or policies of the 

USEPA. Burt Shepard of USEPA; Dave Hopper, Michael Morse, and Susan Burch of U.S. Fish 

and Wildlife Service; and Chris Mebane of USGS provided valuable guidance in planning this 

research. Dave Hopper collected Idaho springsnails, Bliss Rapids snails, and pebble snails and 

Ron Oesch assisted with collection of Ozark springsnails. David Whites assisted with laboratory 

culture of snails. Chris Ivey and James Kunz assisted with toxicity tests. Tom May and Mike 

Walther conducted copper analyses. Kevin Feltz conducted pentachlorophenol analyses. Dennis 

McIntyre and Scott Ireland provided peer review comments on the draft report. This report has 

been reviewed in accordance with U.S. Geological Survey peer review policy. 

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27 
Table 1. Summary of test conditions for toxicity studies with freshwater snails in accordance to 

ASTM (2008a,b,c). 

1. Test organisms See Table 2 

2. Test type Flow-through toxicity test 

3. Test Duration 28 d 

4. Toxicants 

Copper (copper sulfate) 

Pentachlorophenol (stock prepared in triethylene glycol) 

Ammonia (Ammonium chloride; pH of stock adjusted to 8.3 with 

sodium hydroxide) 

5. Dilution series Control and five concentrations in 50% dilution series (see Tables 3-5 

for nominal and measured concentrations) 

6. Temperature 20±1°C 

7. Lighting Ambient laboratory light (about 200 lux, 16 hr light:8 hr dark) 

8. Aeration None 

9. Feeding 

Homogenized flake fish food suspension (Tetrafin®, 6 mg/ml): 

Pondsnails (and adults of other species), 1 ml/beaker/d; 

Idaho springsnails, 0.5 ml /beaker on Monday, Wednesday, and Friday 

(daily starting on day 14). 

10. Test water 

Hard reconstituted water (hardness 160-180 mg/L as CaCO3; ASTM 

2008b). pH in ammonia tests maintained at 8.3 by automated titration 

with dilute HCl (Wang et al. 2008) 

11. Water addition 3.8 volume-additions/day (125 ml/chamber every 4 hr)) 

12. Test chamber 300 ml screened beaker (200 ml water). Beakers with pond snails had 

screened lids (midge emergence caps; USEPA 2000). 

13. Age of test organisms See Table 2 

14. Organisms/chamber 10 

15. Replication 2 to 4 chambers per exposure level (depending on snail availability) 

16. Water quality Temperature, dissolved oxygen, pH, alkalinity, hardness, ammonia 

(weekly); dissolved organic carbon (DOC) and major ions (day 14) 

17. Toxicant analyses Dissolved (filtered <0.45 μm) copper and PCP on days 1, 14, 27; total 

ammonia at least weekly 

18. Endpoints Survival, days 4, 7, 14, 21, and 28; growth (shell diameter), day 28 

28
 
2 

Table 2. Species, age, and size of snails used in 28-d toxicity tests. [SD=standard deviation, N=number of anima

B 

1 

2 

3 

3

Species (ID) Test Chemical 

Approximate 

age (weeks 

post-hatch) 

Starting sh

Mean 

Pondsnail, Lymnaea stagnalis (LS) A Cu <1 2.41 

PCP <1 1.33

Cu <1 1.65

PCP <1 1.83

3 NH3 <1 1.74 

Idaho springsnail, Pyrgulopsis idahoensis (SS) A Cu 2-3 0.463 

PCP 2-3 0.589

Cu 5-7 0.492

PCP 6-9 0.934

NH3 11-13 b 1.108 

 NH3 7-9 b 0.940 

4, 5 Cu, NH3 Mixed 2.000 

Bliss Rapids Snail, Taylorconcha serpenticola (BR) a 4, 5 Cu, NH3 Mixed 1.981 

Pebblesnail, Fluminicola sp. (PS) 4, 5 Cu, NH3 Mixed 2.685 Ozark springsnail, Fontigens aldrichi (OZ) 4, 5 Cu, NH3 Mixed 1.311 

B a E=Endangered species; b Larger cohort tested in replicates 1 and 2; smaller cohort tested in replicates 3 and 4. 

1 
Table 3. Nominal and measured concentrations of copper (Cu) in toxicity tests with freshwater 

snails. All samples filtered through polypropylene filter cartridges (0.45-μm pore diameter). [SD 

= standard deviation; N=number of analyses.] 

30 

Test 

 

Nominal 

Cu 

(μg/L) 

Measured Cu (μg/L) 

N Mean SD Minimum Maximum 

Percent of 

nominal 

A 

 

1 

 

4 

 

0 

1.9 

3.8 

7.5 

15 

30 

0 

3.8 

7.5 

15 

30 

60 

0 

3.8 

7.5 

15 

30 

60 

1 

1 

1 

1 

1 

1 

 

4 

4 

4 

4 

4 

4 

 

4 

4 

4 

4 

4 

4 

0.6 

2.6 

3.9 

6.1 

15 

33 

1.1 

4.6 

8.3 

17 

32 

87 

0.7 

4.3 

6.6 

13 

26 

62 

--

--

--

--

--

--

0.5 

0.6 

1.4 

3.1 

4.0 

9.3 

0.2 

0.7 

0.4 

1.2 

1.9 

9.2 

--

--

--

--

--

--

0.8 

4.0 

7.0 

14 

27 

75.6 

 

0.5 

3.4 

6.2 

12.2 

24.5 

54.9 

--

--

--

--

--

--

1.9 

5.2 

9.6 

20.5 

36.6 

97.1 

0.9 

5.0 

7.1 

14.7 

28.8 

75.0 

--

135

103

81

100

109

--

122 

111 

112 

107 

146 

--

113 

88 

88 

88 

103 
 

Table 4. Nominal and measured concentrations of pentachlorophenol (PCP) in toxicity tests with 

freshwater snails. All samples filtered through polypropylene filter cartridges (0.45-μm pore 

diameter). [SD = standard deviation; N=number of analyses.] 

Study Nominal 

PCP 

(μg/L) N Mean 

Measured PCP (μg/L) 

SD Minimum Maximum Percent of 

Nominal 

B 0 4 3 2 1 6 --

25 4 19 4 13 22 76 

50 4 41 5 37 47 83 

100 4 88 6 82 95 88 

200 4 178 5 170 182 89 

400 4 377 10 370 391 94 

2 0 5 0 0.3 0.03 0.8 --

25 5 11 2 9 13 43 

50 5 24 4 20 28 49 

100 5 50 5 45 54 50 

200 5 97 3 92 100 49 

400 5 224 22 200 240 56 

31 
Table 5. Nominal and measured concentrations of total ammonia (NH3) in toxicity tests with 

freshwater snails. SD [SD = standard deviation; N=number of analyses.] 

32 

Nominal Measured NH3 (mg N/L) 

Study 

 

NH3 

(mg N/L) N Mean SD Minimum Maximum 

Percent of 

nominal 

3 

 

5 

 

0 

0.5 

1 

2 

4 

8 

0 

0.5 

1 

2 

4 

8 

7 

7 

7 

7 

7 

7 

7 

7 

7 

7 

7 

6 

0.14 

0.48 

1.0 

1.8 

3.6 

8.0 

0.05 

0.45 

0.83 

1.7 

3.6 

7.9 

0.09 

0.08 

0.1 

0.3 

0.6 

0.8 

0.02 

0.09 

0.19 

0.3 

0.7 

0.5 

0.04 

0.36 

0.89 

1.4 

2.8 

6.6 

 

0.03 

0.27 

0.57 

1.1 

2.5 

7.3 

0.29 

0.61 

1.3 

2.3 

4.6 

9.3 

0.08 

0.55 

1.1 

2.1 

4.7 

8.4 

--

97 

101 

91 

91 

100 

--

90 

83 

84 

91 

99 
Table 6. Major ions and dissolved carbon in test waters during snail toxicity tests. [DOC=dissolved organic carb

inorganic carbon; feed=diluter water delivery line; -- indicates no data] 

Test Day Source Concentration (mg/L) DOC DIC Chloride Sulfate Calcium Magnesium Potassiu

A 

A 

A A 

A 

A 

B B B 

1 

1 

1 2 

2 2 4 

4 

4 

4 

4 5 

21 

21 

21 

28 

28 

28 28 28 

28 14 

14 

14 28 

28 28 21 

21 21 

21 

21 21 

Feed 

LS 

SS 

Feed 

LS 

SS 

LS 

SS 

Feed 

LS 

SS 

Feed 

LS 

SS 

Feed 

BR 

OZ 

PS 

SS 

Feed Feed 

--

1.8 

<1.0 

--

<1.0 

1.2 

100* 

59* 

--

<1.0 

1.1 

--

81* 

68* 

--

<1.0 

<1.0 

<1.0 

<1.0 

<1.0 <1.0 

--

24 

22 

--

25 

20 

----

--

--

--

--

--

--

----

--

--

--

----

6.2 

--

--

4.5 

--

--

----

7.9 

--

--

5.6 

--

--

5.2 

----

--

--

3.4 1.7 

188 

--

--

188 

--

--

----

189 

-­

--

136 

--

--

--

166 

-­

-­

--

--

--

148 

--

155 

31 

--

--

31 

--

--

----

30 

--

34 

----

28 

--

--

28 28 

21 

--

--

21 

--

--

----

23 

--

--

23 

----

23 

--

--

--

--

21.5 22.2 

4.3 

--

--

4.5 

--

--

----

4.4 

--

--

3.4 

----

4.2 

--

--

--

--

3.8 3.6 

*DOC measurements from PCP tests reflect presence of carrier solvent. 
Table 7. Water quality in exposure chambers during toxicity tests with freshwater snails. [SD = Standard deviatio

for feeding study, A1/A2=algae diet (1 or 2 ml/d), F1/F2=flake diet (1 or 2 ml/d), Y1/Y2=YCT diet (1 or 2 ml/d)

study, A=ASTM hard water, W=well water, C=control (no solvent), S=solvent (triethylene glycol), L/H=low/hig

Test Treat­ ment N Temperature (oC) 

Mean Max 

Dissolved 

oxygen (mg/L) 

Mean Min 

Ammonia 

(mg N/L) 

Mean SD Mean 

pH 

SD 

Hardness 

(mg/L) 

Mean SD 

A1 3 20.1 20.1 9.2 9.0 0.1 0.0 8.43 0.07 159 1 

A2 3 20.1 20.1 9.2 8.9 0.0 0.0 8.39 0.10 165 7 

Feeding F1 3 19.8 20.0 8.7 8.5 0.3 0.1 8.08 0.13 162 3 

study F2 3 19.8 20.0 7.6 6.6 0.3 0.1 8.03 0.07 161 1 

Y1 3 19.9 20.2 9.0 8.6 0.1 0.1 8.40 0.11 161 1 

Y2 3 20.1 20.1 8.9 8.6 0.2 0.1 8.40 0.17 160 14 

ACH 3 20.1 20.4 7.6 6.9 0.9 1.4 8.27 0.44 171 9 

ACL 1 19.6 19.6 8.4 8.4 0.1 -- 8.64 -- 180 -- 

Test 

conditions 

study 

ASH 

ASL 

WCH 

WCL 

WSH 

WSL 

3 

1 

3 

1 

3 

1 

20.0 

19.7 

19.4 

18.9 

19.7 

19.6 

20.2 

19.7 

19.7 

18.9 

19.8 

19.6 

7.6

8.2

8.08.2 8.37.8 7.68.3 8.3

 6.9 

 6.7 

 8.3 

0.2

0.1

0.2

--

0.10.1 0.2

--

0.1

 0.1 

 0.1 

 --

8.21 

8.70 

8.03 

8.44 

7.98 

8.10 

0.37 

--

0.11 

--

0.10 

--

174 

180 

239 

280 

280 

280 

7 

--

68 

--

2 

--

0 5 20.9 23.5 8.2 7.6 0.2 0.1 8.26 0.23 169 12 

Test A: 

Copper, 

(μg/L) 

2 4 

7.5 

15 30 

0 

5 5 

5 

5 5 4 

20.9 20.8 

20.8 

20.9 20.8 20.8 

23.5 23.5 

23.5 

23.5 23.5 21.4 

8.48.5

8.38.1 8.48.0 8.48.0 7.7

 8.0  7.9  7.3 

0.20.2

0.20.1 0.20.1 0.10.1 0.1

 0.1  0.1  0.1 

8.28 8.33 

8.30 

8.35 8.36 8.26 

0.14 0.14 

0.15 

0.15 0.13 0.14 

171 167 

171 

171 

173 166 

8 10 

10 

8 

9 5 

Test 1: 

Copper 

(μg/L) 

3.75 

7.5 

15 30 

60 

3 3 

4 3 

3 

20.4 20.5 

20.6 20.4 

20.5 

20.7 20.9 

21.2 20.6 

20.7 

7.47.2

7.36.9 7.16.4 6.96.7 

 7.3 

 6.4 

0.10.2

0.20.1 0.20.0 0.20.0 

 0.1 

 0.0 

8.20 8.17 

8.22 8.21 

8.22 

0.08 0.05 

0.14 0.12 

0.15 

167 172 

169 169 169 

7 7 

7 5 

5 
Table 7 (continued). 

Test Treat­ ment N Temperature (oC) Mean Max Dissolved oxygen (mg/L) Mean Min Ammonia (mg N/L) Mean SD 

pH 

Mean SD 

Hardness 

(mg/L) 

Mean S

0 5 20.2 20.8 7.9 5.9 0.2 0.2 8.39 0.11 168 8 

Test 4: 

Copper 

(μg/L) 

3.75

7.5

1530

 6 

5 

6 5 

20.5 20.4 

20.2 20.4 

21.5 21.5 

21.4 21.4 

7.7 7.7 

7.7 7.5 

6.0 6.8 

5.9 6.0 

0.2 0.4 

0.3 0.7 

0.1 0.5 

0.5 1.1 

8.38 8.35 

8.44 8.43 

0.10 0.14 

0.08 0.11 

171 169 

162 160 

6 8 

22

60 6 20.3 21.4 7.8 5.9 1.2 2.6 8.44 0.13 169 6 

0 5 21.0 22.3 7.9 6.1 0.2 0.1 8.42 0.30 170 7 

Test B: 

PCP 

(μg/L) 

2520

100200

 5 5 

5 5 

20.8 21.3 21.2 21.2 

21.7 22.3 22.1 22.2 

8.4 8.0 8.0 8.1 

6.5 5.4 7.3 6.7 

0.2 0.1 0.1 0.1 

0.1 0.0 0.1 0.1 

8.46 8.38 8.48 8.42 

0.17 0.20 0.10 0.16 

172 174 170 172 

2 4 7 4 

400 5 21.0 22.3 8.0 6.7 0.1 0.1 8.43 0.16 171 7 

0 4 20.8 23.4 8.1 7.5 0.2 0.1 8.47 0.12 191 1

25 4 20.5 21.2 8.1 7.4 0.1 0.1 8.47 0.13 190 8 

Test 2: PCP 50 4 20.1 20.8 7.8 6.5 0.1 0.1 8.39 0.21 189 7 

(μg/L) 100 4 20.2 20.8 7.6 5.9 0.1 0.1 8.37 0.26 187 1

200 4 20.4 21.0 7.9 7.0 0.1 0.1 8.42 0.15 189 8 

400 4 20.4 21.0 7.8 6.4 0.2 0.1 8.35 0.30 189 8 

0 8 20.3 21.6 7.7 6.0 8.26 0.04 170 5 

Test 3: 

Ammonia 

(mg N/L) 

0.5

1 2 4 

6 

7 7 7 

20.3 

20.0 20.0 19.8 

21.7 

21.3 21.1 21.1 

8.1 

7.8 7.7 7.6 

7.0 

5.9 6.0 6.1 

(See Table 5) 

8.27 

8.22 8.24 8.26 

0.06 

0.04 0.04 0.07 

168 

168 169 169 

6 

5 6 7 

8 7 20.0 21.0 7.9 5.9 8.25 0.04 170 8 

0 5 20.9 22.0 8.4 7.7 8.29 0.13 166 8 

Test 5: 0.5 5 21.0 22.3 8.3 7.8 8.23 0.07 166 6 

Ammonia 

(mg N/L) 

1 2 

5 5 

20.8 20.5 

21.9 21.8 

8.3 8.2 

7.6 7.5 (See Table 5) 8.28 8.27 0.04 0.06 166 166 7 8 

4 5 21.0 22.0 8.5 7.8 8.25 0.08 167 8 

8 5 20.8 22.5 8.4 7.8 8.25 0.06 167 8 
Table 8. Treatment means and analysis of variance for 28-d exposures of freshwater snails to copper (Test A). Fo

endpoint, p-values indicate significance of ANOVA for differences among treatments. Asterisks indicate means less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=Idaho springsnailerror.] 

Species 

LS 

Copper 

(μg/L) 

0.63 

N

4 

Mean 

92.5 

Survival (percent) 

SE Dunnett 

4.8 

p-value 

0.0186 

Copper 

(μg/L)

0.63 

N 

4 

Mean 

27.7 

Wet weight (m

SE Dunn

0.4 

LS 2.6 4 92.5 2.5 2.6 4 20.6 0.6 * 

LS LS 

LS 

LS 

3.9 6.1 

15 33 

4 4 

4 4 

87.5 92.5 

87.5 

0 

7.5 2.5 

2.5 

0 * 

3.9 6.1 

15 

33 

4 4 

4 0 

20.7 25.8 

6.8 

--

1.8 0.7 

1.0 

* 

* 

SS 0.63 4 60 10 <0.0001 

SS 2.6 4 80 8.2 

SS 3.9 4 82.5 8.5 

SS SS SS 

6.1 

15 33 

4 4 

4 

60 30 

0 

7.1 

10.8 

0 * 
Table 9. Treatment means and analysis of variance for 28-d exposures of freshwater snails to pentachlorophenol 

each species and endpoint, p-values indicate significance of ANOVA for differences among treatments. Asteriskare significantly less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=

= Standard error.] 

PCP Survival (percent) PCP Wet weight (mg) Species (μg/L) N Mean SE Dunnett p-value (μg/L) N Mean SE Dunne

LS 2.8 4 97.5 2.5 0.0029 2.8 4 44.3 2.1 

LS 19 4 102.5 2.5 19 4 35.4 1.7 LS 41 4 87.5 4.8 41 4 33.6 4.9 * 

LS 88 4 97.5 2.5 88 3 23.5 2.3 * LS 178 4 95 2.9 178 4 27.0 2.7 * 

LS 377 4 52.5 18.9 * 377 4 5.3 0.4 * 

SS 2.8 4 5 5 0.0007 SS 19 4 7.5 4.8 SS 41 4 25 6.5 SS 88 4 0 0 SS 178 4 0 0 SS 377 4 0 0 
Table 10. Treatment means and analysis of variance for 28-d exposures of freshwater snails to copper (Test 1). F

endpoint, p-values indicate significance of ANOVA for differences among treatments. Asterisks indicate means less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=Idaho springsnail

Species 

LS 

Copper. 

(μg/L) 

1.1 

N 

4 

Mean 

95.0 

Survival (percent) 

SE Dunnett 

5.0 

p-value 

0.0009 

Copper

(μg/L)

1.1 

N 

4 

Mean 

7.74 

Shell diamet

SE 

026 

LS 4.6 4 92.5 7.5 4.6 4 7.18 0.14 

LS LS LS 

LS 

8.3 

17 32 87 

4 4 4 4 

97.5 97.5 52.5 

0.0 

2.5 2.5 

18.9 

0.0 * 

8.3 

17 32 87 

4 4 

4 

7.58 7.49 

4.20 

--

0.16 0.19 

0.95 

SS 1.1 4 97.5 2.5 <0.0001 1.1 4 0.83 0.05 

SS SS 

SS 

SS SS 

4.6 

8.3 

17 

32 87 

4 

4 

4 4 4 

100.0 

100.0 

17.5 

0.0 0.0 

0.0 

0.0 

8.5 0.0 0.0 

* * * 

4.6 

8.3 

17 

32 87 

4 

4 

3 

1.14 

0.74 

0.62 

----

0.06 

0.02 

0.03 
Table 11. Treatment means and analysis of variance for 28-d exposures of freshwater snails to pentachloropheno

each species and endpoint, p-values indicate significance of ANOVA for differences among treatments. Asteriskare significantly less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=I

= standard error.] 

Survival (percent) Shell diameter 

PCP PCP Species (μg/L) N Mean SE. Dunnett p-value N Mean SE 

(μg/L)

LS 0.33 4 100.0 0.0 <0.0001 0.33 4 7.77 0.17 

LS 11 4 100.0 0.0 11 4 7.38 0.17 LS 24 4 100.0 0.0 24 4 7.21 0.13 LS 50 4 100.0 0.0 50 4 7.04 0.27 LS 97 4 100.0 0.0 97 4 6.95 0.36 

LS 224 4 25.0 18.5 * 224 3 3.88 0.68 

SS 0.33 4 100.0 0.0 0.0014 0.33 4 1.86 0.10 SS 11 3 100.0 0.0 11 3 1.56 0.08 SS 24 3 86.7 13.3 24 3 1.33 0.08 

SS 50 3 100.0 0.0 50 3 1.50 0.12 

SS 97 3 93.3 6.7 97 2 1.24 0.10 

SS 224 3 0.0 0.0 * 224 
 Table 12. Treatment means and analysis of variance for 28-d exposures of freshwater snails to ammonia (Test 3)

and endpoint, p-values indicate significance of ANOVA for differences among treatments. Asterisks indicate trea

significantly less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=Idah

standard error.] 

Survival (percent) Shell diamete

Species Ammonia Ammonia 

(mg N/L) N Mean SE Dunnett p-value (mg N/L) 

N Mean SE 

LS 0.14 4 97.5 2.5 0.8534 0.14 4 7.11 0.17 LS 0.48 4 100.0 0.0 0.48 4 6.87 0.05 

LS 1.0 4 95.0 2.9 1.0 

1.8 

4 6.93 0.10 

LS 1.8 4 90.0 10.0 4 6.67 0.08 

LS 3.6 4 95.0 5.0 3.6 4 6.38 0.24 

LS 8.0 4 97.5 2.5 8.0 4 5.98 0.06 

SS 0.14 4 100.0 0.0 0.0011 0.14 4 1.35 0.02 

SS 0.48 4 44.4 20.3 * 0.48 4 1.47 0.10 

SS 1.0 4 27.5 10.3 * 1.0 3 1.29 0.08 

SS 1.8 

3.6 

4 62.5 16.5 1.8 4 1.52 0.11 

SS 4 20.0 14.1 * 3.6 2 1.55 0.20 

SS 8.0 4 2.5 2.5 * 8.0 1 1.10
 

Table 13. Treatment means and analysis of variance for 28-d exposures of freshwater snails to 

copper (Test 4). For each species and endpoint, p-values indicate significance of ANOVA for 

differences among treatment means. Asterisks indicate means that are significantly less than 

control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [BR=Bliss Rapids snail; 

OZ=Ozark springsnail; PS=pebblesnail; SS=Idaho springsnail. SE = standard error.] 

41 

Copper Species (μg/L) N 

BR 0.70 2 

BR 4.3 2 

BR 6.6 2 

BR 13 2 

BR 26 2 

BR 62 2 

OZ 0.70 2 

OZ 4.3 2 

OZ 6.6 2 

OZ 13 2 

OZ 26 2 

OZ 62 2 

PS 0.70 2 

PS 4.3 2 

PS 6.6 2 

PS 13 2 

PS 26 2 

PS 62 2 

SS 0.70 2 

SS 4.3 2 

SS 6.6 2 

SS 13 2 

SS 26 2 

SS 62 2 

Mean 

100.0 

85.0 

100.0 

80.0 

0.0 

0.0 

100.0 

91.7 

100.0 

66.7 

58.3 

0.0 

90.0 

100.0 

100.0 

50.0 

0.0 

0.0 

100.0 

100.0 

100.0 

75.0 

50.0 

0.0 

Survival (percent)

SE Dunnett 

0.0 

5.0 * 

0.0 

0.0 * 

0.0 * 

0.0 * 

0.0 

8.3 

0.0 

0.0 * 

8.3 * 

0.0 * 

0.0 

0.0 

0.0 

10.0 * 

0.0 * 

0.0 * 

0.0 

0.0 

0.0 

0.0 * 

12.5 * 

0.0 * 

p-value 

<0.0001 

0.0008 

<0.0001 

<0.0001 
 

Table 14. Treatment means and analysis of variance for 28-d exposures of freshwater snails to 

ammonia (Test 5). For each species and endpoint, p-values indicate significance of ANOVA for 

differences among means. Asterisks indicate treatment means that are significantly less than 

control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [BR=Bliss Rapids snail; 

OZ=Ozark springsnail; PS=pebblesnail; SS=Idaho springsnail.SE = standard error.] 

42 

 

 

 

 

 

 

 

 

 

 

 

 

Species Survival (percent) 	 Ammonia 

(mg N/L) N Mean SE Dunnett p-value 

BR 0.046 2 100.0 0.0 0.0003 

BR 0.45 2 100.0 0.0 

BR 0.83 

1.7 

2 100.0 0.0 

BR 2 85.0 5.0 * 

BR 3.6 2 80.0 10.0 * 

BR 7.9 2 30.0 0.0 * 

OZ 0.046 2 100.0 0.0 <0.0001 

OZ 2 93.8 6.3 

OZ 2 50.0 25.0 * 

0.45 

0.83 

OZ 1.7 2 0.0 0.0 * 

OZ 3.6 2 0.0 0.0 * 

OZ 7.9 2 0.0 0.0 * 

PS 0.046 2 100.0 0.0 0.339 

PS 0.45 2 90.0 10.0 

PS 0.83 2 90.0 10.0 

PS 1.7 2 45.0 45.0 

PS 3.6 2 50.0 50.0 

PS 7.9 2 0.0 0.0 

SS 0.046 2 100.0 0.0 0.0209 

SS 0.45 2 100.0 0.0 

SS 0.83 2 100.0 0.0 

SS 1.7 2 100.0 0.0 

SS 3.6 2 75.0 25.0 

SS 7.9 2 12.5 12.5 * 
Table 15. Summary of 28-d toxicity values for freshwater snails. Species chronic value is lowest EC20 for a sing

mean of lowest survival or growth EC20s (bold) for multiple tests. Upper boundary value used for ‘less than’ va

J=juvenile; M=mixed. LC50=median lethal concentration; EC20=20% effect concentration; conf=95% confiden

Survival Growth 

Chemical Species Age Test 

EC20 (conf) LC50 (conf) EC20 (conf) 

Copper 

SS BR PCP 

OZ 

PS 

NH3 

SS 

SS 

*EC20 or L BR 

OZ 

**LC50 est PS 

LS 

LS 

LS 

C50 estim

imated by 

N 

N 

J J 

M 

M 

M M 

N 

N 

N 

N 

N 

J 

M 

M 

M 

M 

ated by T

linear int

A 

1 

A 

1 

4 

4 

4 

4 

B 

2 

B 

2 

3 

3 

5 

5 

5 5 

oxicity R

16.9 

22.4 

8.58* 

9.89 

13.7* 

16.8 

14.0* 

11.1 

>337 

121 

--

109 

>7.9 

<0.48

3.24* 

3.42* 

0.61 

1.02* 

esponse 

erpolation (IC50). 

(16-17) 21.7 (19-34) 36.2 

(1.7-43) 13.1* 

(9.7-10.3) 12.9 (7.1-26) 23.8* 

(16.8-16.8) 20.8 

(4.1-47) 25.6* 

(10.6-12.1) 16.9 

-- >337 

(115-152) 170** 

(96-114) 143 

-- >7.9 

 -- 0.609 

(4.4-5.3) 4.84* 

(2.0-5.7) 5.92* (0.52-0.87) 0.963 (0.12-8.6) 2.38* 

Analysis Program (TRA

(20-23) 

(30-44) 

(6.0-28) 

(12-14) 

(17-34) 

(18-24) 

(13-51) 

(14-20) 

--(154-191) 

(138-151) 

--(0.39-0.81) 

(4.4-5.3) (4.4-8.0) (0.78-1.18) (0.75-7.6) 

P, Version 1

6.15 

21.5 

7.4 

19.1 

118 

16.6 

>7.9 

>8.0 

.0; R. Eri

(6.1-6.6) 

(19-27) 

(6.8-8.5) 

(19-55) (100-135) 

(11-69) 

ckson, USEPA

-M
Table 16. Published toxicity values for toxicity of copper, pentachlorophenol, and ammonia to freshwater snails

S=survival, G=growth, R=reproduction. Metrics: LC50=median lethal concentration; NOEC=no observed effecLOEC=lowest observed effect concentration; ChV=geometric mean of NOEC and LOEC.] 

Species Duration Endpoint Metric Concentration Notes 

Copper (ug/L) 

Lymnaea luteola 96h S LC50 27 KhangaPotamopyrgus jenkinsi 96 h S LC50 54 juveniles Watton Potamopyrgus jenkinsi 96 h S LC50 77 adults Watton 

Potamopyrgus jenkinsi 96 h S LC50 79 senescent Watton Pomacea canaliculata 10 d G NOEC 68 juveniles Pena anPomacea canaliculata 50 d G NOEC >68 juveniles Pena anPhysa integra 6 wk SG LOEC 14.8 adults Arthur 

Campeloma decisum 6 wk SGR LOEC 14.8 adults Arthur 

Pentachlorophenol (ug/L) 

Lymnaea acuminata 96 h S LC50 160 Gupta a

Physa gyrina 96 h S LC50 220 summer Hedtke 

Physa gyrina 96 h S LC50 1380 winter Hedtke Physa gyrina 96 h S LC50 267 Lk. Superior Hedtke 

Gillia altilis 96 h S LC50 810 static Stuart aGillia altilis 96 h S LC50 300 flow-through Stuart aPhysa gyrina 96 h S ChV 150 Hedtke Physa gyrina 36 d G ChV 74 Hedtke 

Physa gyrina 21 d R ChV <26 Hedtke 
Table 16 (continued) 

Species Duration Endpoint Metric Concentration Notes 

Total Ammonia (mg N/L) 

Lymnaea stagnalis 96 h S LC50 0.825 William

Physa fontinalis 96 h S LC50 1.4 William

Potamopyrgus antipodarium 96 h S LC50 2.02 Alonso 

Potamopyrgus antipodarium 96 h S LC50 0.31 lowest Hickey 

Potamopyrgus antipodarium 96 h S LC50 0.44 highest Hickey Potamopyrgus jenkinsi 96 h S LC50 0.315 juveniles Watton 

Potamopyrgus jenkinsi 96 h S LC50 0.85 adults Watton 

Potamopyrgus jenkinsi 96 h S LC50 0.49 senescent Watton 
 Survival (%) 

0 

20 

40 

60 

80 

100 

no data 

Algae1 Algae2 Flake1 Flake2 YCT1 YCT2 

Wet weight (mg) 

0 

5 

10 

15 

20 

25 

LS 

SS 

no data 

Algae1 Algae2 Flake1 Flake2 YCT1 YCT2 

Figure 1. Survival and growth of pondsnail (LS) and Idaho springsnail (SS) in preliminary Study 

1 (21-d). Results for SS are from individual exposure groups; results for LS are means of 4 

replicates (with standard deviation). Labels that include ‘1’ and ‘2’ indicate low and high 

feeding rates, respectively, for each diet. 

46 
 

ASTM-L 

ASTM-H 

Well-L 

Well-H 

ASTM-L 

ASTM-H 

Well-L 

Well-H 

Shell diameter (mm) 

0 

2 

4 

6 

8 

10 

Water 

Solvent 

Springsnail (SS) Pondsnail (LS)
 

Figure 2. Growth of Idaho springsnail (SS) and pondsnail (LS) in preliminary Study 2. Bars are 

mean shell diameters for individual snails after 28 d, with standard deviation (n=9-10). 

Treatments are combinations of test water (ASTM hard vs. well water), solvent (control or 

triethylene glycol), and feeding rate (L=low; H=high). 

47 
 

Test A 

30 100
 

LS 

SS 

* 

* 

*

* 


 

80
 

Survival (%) 

20
 

60
 

40
 

10
 

20
 

0
 0 

Wet weight (mg) 

Test 1
 

1 10 100 

LS 

SS 

* * * 

1.5 8 100
 

80
 6
 

1.0 

60
 

* 

* 

1 10 100
 

Survival (%) 

4
 

40
 0.5 

20
 

0
 

2
 

0.0 0 

Test 4
 Copper (μg/L) 

Shell diameter, mm

(SS=left, LS=right) 

100
 

Copper (μg/L) 

1 10 100 

BR 

OZ 

PS 

SS 

* 

* 

* * * 

* 

* 

* 

* 

Survival (%) 

80
 

60
 

40
 

20
 

0
 

1 10 100 1 
 10 100

Figure 3. Toxicity of copper to freshwater snails. Survival and growth during 28-d exposures: 

means with standard errors (Test A and Test 1; n=4) or means only (Test 4; n=2). Asterisks 

indicate significant differences from controls (rank ANOVA with Dunnett’s test). 

48 
 

Test B Survival (%) 

0 

20 

40 

60 

80 

100 

LS 

SS * 

Wet weight (mg) 

0 

10 

20 

30 

40 

50 

* * 

* * 

* 

1 10 100 1 10 100

Test 2
 

2
 8 100
 

80
 6
 

LS 

SS 

* 

* 

1 10 100 
 

* * * * 

1 10 100

Shell diameter, mm

(Y-axis: SS=left, LS=right) 

Survival (%) 

60
 

40
 

20
 

0
 

1
 4
 

2
 

0 0 

PCP (μg/L) PCP (μg/L) 

Figure 4. Toxicity of pentachlorophenol (PCP) to freshwater snails. Survival and growth during 

28-d exposures (means with standard errors; n=4). Asterisks indicate significant differences from 

controls (rank ANOVA with Dunnett’s test). 

49 
 

60 

4 

6 

8 

Test 3 

0.1 1 10 

LS 

SS 

* 

* 

* 

* 

100
 

80
 

* * * 

0.1 1 10
 

Shell diameter (mm) 

Survival (%) 

40
 

20
 

0 

Total ammonia (mg N/L) 

0 

2 

Test 5
 

100
 

Total ammonia (mg N/L) 

0.1 1 10 

BR 

OZ 

PS 

SS * * 

* 

* 

* * 

* 

* 

Survival (%) 

80
 

60
 

40
 

20
 

0
 

Figure 5. Toxicity of ammonia to freshwater snails. Survival and growth during 28-d exposures: 

means with standard errors (Test 3; n=4) or means (Test 5; n=2). Asterisks indicate significant 

differences from controls (rank ANOVA with Dunnett’s test). 

50 
 

Copper (μg/L) 

0 

20 

40 

60 

80 

100 

Mussel 

Fish 

Snail 

Invert 

SS PS 

LS 

OZ 

BR 

Campeloma

1 10 100

10 100 500
 
 

PCP (μg/L) 

Percentile 

0 

20 

40 

60 

80 

100 

SS 

LS 

Physa

Ammonia (mg N/L) 

1 10

0 

20 

40 

60 

80 

100 

SS 

PS 

LS 

OZ 

BR 

0.2 20 

51 

Chronic value 

Figure 6. Species sensitivity distributions and chronic water quality criteria (dashed lines) for 

copper, PCP, and ammonia. Values were adjusted to common conditions for comparisons: 85 

mg/L hardness for copper (USEPA 19995, 2007); pH 7.8 for PCP (USEPA 1996); and pH 8.0 

and 25 oC for ammonia (USEPA 1999). Toxicity values from Tables 15 and 16.