## Sensitivity of freshwater snails to aqua tic 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 Chri stopher G. Ingersoll

U.S. Geological Survey, Colu mbia 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 impor tant factor affectin g declining populations

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

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

water quality criteria include mainly results of ac ute 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 labo ratory cultures and four taxa of

non-pulmonate taxa from field collections. Fi eld-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 pe riodically 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, an d unsuccessful for Bliss Rapids snail or Ozark

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

pentachlorophenol in hard reconstituted wa ter (hardness 160- 180 mg/L as CaCO 3 

## ). Juvenile

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

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

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

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

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

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

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

2

## upper end of species sensitivity distributions. Th e 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 chroni c 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 aqua tic taxa – including endangered and th reatened 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 Protec tion 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. Stud ies with ESA-listed freshwater fish species determined that

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

than common surrogates (Dwyer et al. 2005; Besser et al. 2005), but recen t 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-lis ted, 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-br eathing snails of the subclass

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

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

reproductive output make them easy to use in to xicity tests, includi ng chronic tests with

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

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

atmospheric oxygen) and life hist ory (slow growth and low repr oductive 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-liste d 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, th e toxicity data generate d during this study should

prove to be useful for other consulta tions throughout the United States.

The specific objectives of this study were:

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

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

surrogate species; and

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

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

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

METHODS

Snail Collection and Culture

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

Columbia Environmental Research Center in Co lumbia 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 distri bution, 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 obtai ned a culture of the pulmonate pondsnail ( Lymnaea

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

Snails were held in 40-L aquaria with 35-L of well water (hardness 280 mg/L as CaCO 3 

## )

at 18 oC. Aquaria were aerated and one-half of the water was repl aced 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 o ffered 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 stai nless steel substrates, which were air-dried before they were

placed in the culture tanks. Additional foods o ffered 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 we re 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 detr itus. Water containing neon ates and detritus was

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

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

syringe system consists of a glass capillary tube (1.17-mm in ner 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 we re placed into a 4-L beaker containing 2 L of well water.

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

6

## acclimated to the hard recons tituted 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 pred ominantly 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 animal s were combined over several months to

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

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

masses, which were collected as needed to prov ide neonates (<7 d after ha tching) 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 include d: an algae mixture (1 ml of Nannochloropsis and 2 ml Shellfish

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

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

suspension of Tetrafin® flake fish food (6 mg/m l). 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 seve n 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 t oxicity 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 solv ent used in the

PCP exposures), and feeding rate (flake food su spensions; 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’ treatm ent were increased after day 13 to 0.5 ml daily for

SS and 2 ml daily for LS. Each of the eight treatm ent 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 fr eshwater snails were similar to methods for

toxicity testing with freshwater mussels (A STM 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 preven t these air-breathing snails from escaping from

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

Toxicity tests were conducte d with copper (copper sulfat e), pentachlorophenol (PCP;

stock prepared in triethylene gl ycol), and ammonia (ammonium ch loride). 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 impo rtance in snail habita ts in the Snake River

8

## valley (copper from aquaculture, ammonia from livestock) and sens itivity of snails in acute

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

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

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

concentrations nominal).

Automated proportional diluters prepared and de livered 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 c oncentration of triethylene glycol in the highest PCP treatment.

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

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

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

controllers (Wang et al. 2008).

Water samples were collected every two w eeks 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 diamet er) and samples were analyzed without

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

plasma-mass spectroscopy (May et al. 1997). PCP concentrations were analyzed by highperformance liquid chromatography using a C18 co lumn, 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 (T able 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 with out mortality: (about

3-6 weeks after egg deposition or 2-3 weeks pos t-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), subseq uent survival and growth tests with all three

chemicals (Tests 1-3) were conducted with juve nile 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, mi xed-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, labora tory-produced groups included an imals of widely-varying ages:

roughly 4-8 months old for SS and 6-12 months old for PS. All toxi city 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, grow th 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 s ubsequent survival and gr owth tests with SS and

LS (Tests 1-3), growth of both species was ev aluated by digital measur ement of maximum shell

diameter. Growth was not measured at the end ofthe 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 co ntrols determined by D unnett’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, a nd the lowest-observed-effect c oncentration (LOEC), the lowest

exposure concentration above the NOEC. Concen tration-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-transf ormed for all analyses.

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

treatments with partial mortalities), LC50s a nd 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 inter polation (Inhibition Concentra tion 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 experi mental diets indicated that LS had highest survival and

growth when fed the flake diet (Figure 1), wh ereas 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 feed ing 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 m l/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 consis tent differences in

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

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

not caused by excessive feeding, ina ppropriate test water, or the pr esence of the carrier solvent.

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

with very young snails (about 3-6 weeks after egg deposition). S ubsequent 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 Frid ay 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 toxi city tests generally were clos e 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, a pparently caused by a short-term diluter malfunction. Measured copper

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

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

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

with nominal concentrations except for losses, pr esumably 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 toxican t delivery system. Despite this

12

## problem, means of five measured PCP concentra tions 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 nitrifi cation by the microbial commun ity in the test system.

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

hardness (169 mg/L as CaCO 3 

## ) and alkalinity (121 mg/L as CaCO 3 

## ) measured in nine batches of

test water delivered to the proportional dilute rs 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 norm al 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; calci um, 30, magnesium, 22;

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

below the detection limit (<1.0 mg/L) for two anal yses 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 wate r 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 fr om 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 th e 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 ammo nia 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 treatm ents 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. Contro l survival was acceptable (90­

100%) in all exposures except the first two SS expos ures (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, a nd the results of the second test-conditions study

(Figure 2), led to the decision to use older SS for subsequent tests at a lo wer 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 da y 28. Growth of juvenile SS in

these tests (started with anim als 5-12 weeks post-hatch) was sl ower 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 ol der 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 ra nk ANOVA; P<0.05) for most

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

with ammonia, which had little variation among tr eatment 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 re plicates in two of the high-a mmonia treatments (Table 14).

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

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

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

significant ANOVAs (Table 15). Medi an 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 ammoni a 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 treatm ents with zero survival (Table 11).

Survival and growth EC20s (Table 15) we re 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% reduc tions 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 va lues among snail species

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

with increased copper concentratio ns 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 indicat ed 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 gr owth response for LS and SS were somewhat

inconclusive, with LOECs and EC20 s indicating somewhat different trends for Test A and Test 1

(Tables 8, 10, 15). Overall, copper EC20s di ffered 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 expos ure were quite similar in Test 2

(Figure 4). Survival of SS and LS was redu ced 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 affe cted by PCP in Test B, but comparisons to SS

16

## are not available for this test because of the fa ilure of the SS exposure. In contrast, growth of

both species was affected by PCP exposure – in Te st 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 signifi cant 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 (T ables 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 reflec ting 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 ex posure, relative to SCVs

reported by USEPA (2007) and resu lts 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 fo r freshwater mussels (Wang et al . 2007) – were lower than those

for several snail taxa tested in this study (SS, PS, and LS). All five sp ecies 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 Li gand 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 compar isons 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 hi gh 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 SC V for LS ranked near the middle

of the species sensitivity distribut ion, 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 dart er (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 indi cated 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 reporte d 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 fres hwater mussel taxa test ed 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 subs tantially higher survival EC20 from Test 5.

Although the survival data for SS in Test 3 we re highly variable, th e contrast between 100

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

suggests that the juvenile SS in th is 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 ammoni a 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 t oxicity of PCP under ambient c onditions 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 se nsitivity 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 valu es determined from test s with older, mixed-age

cohorts (Test 4 and Te st 5; Table 15)

Comparison of chronic sensitivity of pulmona te 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 pulmona te taxa, which have rapid growth and short

generation times. Fewer chronic studies have re ported sensitive subl ethal 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 popul ations that are parthenogenic

and ovoviviparous, have led to proposals for its us e 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, Art hur and Leonard 1970) or tests

20

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

which started with early juveni les (<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 mo rtality endpoints in test s 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 ha d similar EC20s in the remaining four tests

(Table 15).

Summary

Objective 1: Develop methods fo r laboratory culture and chronic to xicity testing (28-d survival

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

species.

Chronic (28-d) toxicity tests were complete d 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, toxi city tests with five species of freshwater

snails, conducted under routin e flow-through test methods, ha d good control survival and

produced reliable estimates of toxicity th resholds. 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 produ ced useful data on snail surviv al in 28-d toxicity tests.

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

21

## based on digital measurement of shell diamet er, and toxicity values for growth were

lower than those for survival for two of seven exposures.

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

habitats and to compare chroni c toxicity values for these ch emicals 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 simila r sensitivity to copper. The sensitivity of the

two ESA-listed species, SS (whi ch 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 sn ails, are very sensitiv e 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 da ta only, from tests with field-collected

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

species. Ongoing research in our laborato ry 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 grow th test method presented here.

22

## Acknowledgments

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

Effort (RARE) program, but this report does not necessari ly 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 spri ngsnails. David Whites assisted with laboratory

culture of snails. Chris Ivey and James Kunz a ssisted with toxicity te sts. Tom May and Mike

Walther conducted copper analyses. Kevin Feltz conducted pentachloroph enol analyses. Dennis

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

been reviewed in accordance with U.S. Ge ological 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-thr ough toxicity test

3. Test Duration 28 d

4. Toxicants Copper (copper sulfate) Pentachlorophenol (stock prepar ed 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 othe r 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 CaCO 3 

## ; 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 wa ter). Beakers with pond snails had screened lids (midge emer gence 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

282Table 2. Species, age, and size of snails used in 28-d toxicity tests. [SD=standarddeviation, N=number of animals measured.]B123329Species (ID)TestChemicalApproximateage (weekspost-hatch)Starting shell diameter (mm)Mean SD NPondsnail,Lymnaea stagnalis(LS) ACu<12.41PCP<1 1.33Cu<1 1.65PCP <1 1.833 NH3 <1 1.74Idaho springsnail,Pyrgulopsis idahoensis(SS)A Cu 2-3 0.463PCP2-3 0.589Cu5-7 0.492PCP 6-9 0.934NH3 11-13b1.108NH3 7-9b0.9404, 5 Cu, NH3 Mixed 2.000Bliss Rapids Snail,Taylorconcha serpenticola(BR)a4, 5 Cu, NH3 Mixed 1.981Pebblesnail,Fluminicolasp. (PS)4, 5 Cu, NH3 Mixed 2.685Ozark springsnail,Fontigens aldrichi(OZ)4, 5 Cu, NH3 Mixed 1.3110.460.580.170.260.250.0400.0890.1010.1020.3060.1230.6600.3480.8610.28822261011102327131081015321212BaE=Endangered species;bLarger cohort tested in replicates 1 and 2; smaller cohort tested in replicates 3 and 4.1

## Table 3. Nominal and measured co ncentrations 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 No minal

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 co ncentrations of pentachlorophenol (PCP) in toxicity tests with

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

diameter). [SD = standard devi ation; 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 con centrations of total ammonia (NH 3 

## ) in toxicity tests with

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

32 No minal Measured NH 3 

## (mg N/L) Study NH 3 

## (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

99Table 6. Major ions and dissolved carbon intest waters during snail toxicity tests.[DOC=dissolved organic carbon; DIC=dissolvedinorganic carbon; feed=diluter waterdelivery line; -- indicates no data]TestDaySourceConcentration (mg/L)DOCDICChlorideSulfate CalciumMagnesiumPotassiumSodiumIronAAAAAABBB111222444445212121282828282828141414282828212121212121FeedLSSSFeedLSSSLSSSFeedLSSSFeedLSSSFeedBROZPSSSFeedFeed--1.8<1.0--<1.01.2100*59*--<1.01.1--81*68*--<1.0<1.0<1.0<1.0<1.0<1.0--2422--2520------------------------------6.2----4.5--------7.9----5.6----5.2--------3.41.7188----188--------189-­--136------166-­-­------148--15531----31--------30--34----28----282821----21--------23----23----23--------21.522.24.3----4.5--------4.4----3.4----4.2--------3.83.647----51--------48----51----57--------49480.03----0.01--------0.06----<.01----ND------------*DOC measurements from PCP tests reflect presence of carrier solvent.33Table 7. Water quality in exposure chambersduring toxicity tests with freshwater snails. [SD = Standard deviation. Treatment codes: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); for test conditionsstudy, A=ASTM hard water, W=well water, C=control (no solvent),S=solvent (triethylene glycol),L/H=low/high feeding rates.]34TestTreat­mentNTemperature(oC)Mean MaxDissolvedoxygen(mg/L)Mean MinAmmonia(mg N/L)Mean SDMeanpHSDHardness(mg/L)Mean SDAlkalinity(mg/L)Mean SDA1320.120.19.29.00.10.08.430.0715911208A2320.120.19.28.90.00.08.390.1016571208FeedingF1319.820.08.78.50.30.18.080.1316231200studyF2319.820.07.66.60.30.18.030.0716111223Y1319.920.29.08.60.10.18.400.1116111217Y2320.120.18.98.60.20.18.400.17160141134ACH320.120.47.66.90.91.48.270.4417191236ACL119.619.68.48.40.1--8.64--180--130--TestconditionsstudyASHASLWCHWCLWSHWSL31313120.019.719.418.919.719.620.219.719.718.919.819.67.68.28.08.28.37.87.68.38.36.96.78.30.20.10.2--0.10.10.2--0.10.10.1--8.218.708.038.447.988.100.37--0.11--0.10--1741802392802802807--68--2--12712424423023723412--8--6--0520.923.58.27.60.20.18.260.23169121226Test A:Copper,(μg/L)247.51530055555420.920.820.820.920.820.823.523.523.523.523.521.48.48.58.38.18.48.08.48.07.78.07.97.30.20.20.20.10.20.10.10.10.10.10.10.18.288.338.308.358.368.260.140.140.150.150.130.1417116717117117316681010895122123124124124122455434Test 1:Copper(μg/L)3.757.51530603343320.420.520.620.420.520.720.921.220.620.77.47.27.36.97.16.46.96.77.36.40.10.20.20.10.20.00.20.00.10.08.208.178.228.218.220.080.050.140.120.151671721691691697775512112312412512522344Table 7 (continued).TestTreat­mentNTemperature(oC)Mean MaxDissolvedoxygen (mg/L)Mean MinAmmonia(mg N/L)Mean SDpHMeanSDHardness(mg/L)Mean SDAlkalinity(mg/L)Mean SD0520.220.87.95.90.20.28.390.1116881213Test 4:Copper(μg/L)3.757.51530656520.520.420.220.421.521.521.421.47.77.77.77.56.06.85.96.00.20.40.30.70.10.50.51.18.388.358.448.430.100.140.080.11171169162160682123121121122120333460620.321.47.85.91.22.68.440.13169612330521.022.37.96.10.20.18.420.3017071273Test B:PCP(μg/L)2520100200555520.821.321.221.221.722.322.122.28.48.08.08.16.55.47.36.70.20.10.10.10.10.00.10.18.468.388.488.420.170.200.100.1617217417017224741261271271265445400521.022.38.06.70.10.18.430.16171712860420.823.48.17.50.20.18.470.1219113127325420.521.28.17.40.10.18.470.1319081261Test 2: PCP50420.120.87.86.50.10.18.390.2118971232(μg/L)100420.220.87.65.90.10.18.370.26187101289200420.421.07.97.00.10.18.420.1518981286400420.421.07.86.40.20.18.350.30189812640820.321.67.76.08.260.0417051176Test 3:Ammonia(mg N/L)0.5124677720.320.020.019.821.721.321.121.18.17.87.77.67.05.96.06.1(See Table 5)8.278.228.248.260.060.040.040.07168168169169656711911911712043638720.021.07.95.98.250.04170811650520.922.08.47.78.290.1316681196Test 5:0.5521.022.38.37.88.230.0716661207Ammonia(mg N/L)125520.820.521.921.88.38.27.67.5(See Table 5)8.288.270.040.0616616678119119444521.022.08.57.88.250.08167811958520.822.58.47.88.250.061678119535Table 8. Treatment means and analysis of variance for 28-d exposuresof freshwater snails to copper (Test A). For each speciesandendpoint, p-values indicate significance of ANOVA for differences among treatments. Asterisks indicate means that are significantlyless than control (Dunnett’s test). Cell bordersindicate NOEC and LOEC. [LS=pondsnail, SS=Idaho springsnail. SE = Standarderror.]36SpeciesLSCopper(μg/L)0.63N4Mean92.5Survival (percent)SEDunnett4.8p-value0.0186Copper(μg/L)0.63N4Mean27.7Wet weight (mg)SE Dunnett0.4p-value<0.0001LS2.6492.52.52.6420.60.6*LSLSLSLS3.96.11533444487.592.587.507.52.52.50*3.96.11533444020.725.86.8--1.80.71.0**SS0.6346010<0.0001SS2.64808.2SS3.9482.58.5SSSSSS6.11533444603007.110.80*Table 9. Treatment means and analysis of variance for 28-d exposuresof freshwater snails to pentachlorophenol (PCP; Test B). Foreach species and endpoint, p-values indicatesignificance of ANOVA for differences among treatments. Asterisks indicate means thatare significantly less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=Idaho springsnail. SE= Standard error.]37PCPSurvival (percent)PCPWet weight (mg)Species(μg/L)N Mean SE Dunnett p-value(μg/L)N Mean SE Dunnett p-valueLS 2.8 4 97.5 2.50.0029 2.8 4 44.3 2.1<0.0001LS 19 4 102.5 2.519 4 35.4 1.7LS 41 4 87.5 4.841 4 33.6 4.9 *LS 88 4 97.5 2.588 3 23.5 2.3 *LS 178 4 95 2.9178 4 27.0 2.7 *LS 377 4 52.5 18.9 *377 4 5.3 0.4 *SS 2.8 4 5 50.0007SS 19 4 7.5 4.8SS 41 4 25 6.5SS 88 4 0 0SS 178 4 0 0SS 377 4 0 0Table 10. Treatment means and analysis of variance for 28-d exposures of freshwater snails to copper (Test 1). For each speciesandendpoint, p-values indicate significance of ANOVA for differences among treatments. Asterisks indicate means that are significantlyless than control (Dunnett’s test). Cell bordersindicate NOEC and LOEC. [LS=pondsnail, SS=Idaho springsnail. SE=standard error.]38SpeciesLSCopper.(μg/L)1.1N4Mean95.0Survival (percent)SEDunnett5.0p-value0.0009Copper(μg/L)1.1N4Mean7.74Shell diameter (mm)SE Dunnett026p-value0.0058LS4.6492.57.54.647.180.14LSLSLSLS8.3173287444497.597.552.50.02.52.518.90.0*8.31732874447.587.494.20--0.160.190.95*SS1.1497.52.5<0.00011.140.830.05<0.0001SSSSSSSSSS4.68.317328744444100.0100.017.50.00.00.00.08.50.00.0***4.68.31732874431.140.740.62----0.060.020.03*Table 11. Treatment means and analysis of variance for 28-d exposures of freshwater snails to pentachlorophenol (PCP; Test 2).Foreach species and endpoint, p-values indicatesignificance of ANOVA for differences among treatments. Asterisks indicate means thatare significantly less than control (Dunnett’s test). Cell borders indicate NOEC and LOEC. [LS=pondsnail, SS=Idaho springsnail.SE= standard error.]39Survival (percent)Shell diameter (mm)PCPPCPSpecies(μg/L)(μg/L)N Mean SE. Dunnett p-value N Mean SE Dunnett p-valueLS 0.33 4 100.0 0.0<0.0001 0.33 4 7.77 0.170.0115LS 11 4 100.0 0.011 4 7.38 0.17LS 24 4 100.0 0.024 4 7.21 0.13LS 50 4 100.0 0.050 4 7.04 0.27LS 97 4 100.0 0.097 4 6.95 0.36LS 224 4 25.0 18.5 *224 3 3.88 0.68 *SS 0.33 4 100.0 0.00.0014 0.33 4 1.86 0.100.0023SS 11 3 100.0 0.011 3 1.56 0.08SS 24 3 86.7 13.324 3 1.33 0.08 *SS 50 3 100.0 0.050 3 1.50 0.12 *SS 97 3 93.3 6.797 2 1.24 0.10 *SS 224 3 0.0 0.0 *224Table 12. Treatment means and analysis of variance for 28-d exposures of freshwater snails toammonia (Test 3). For each speciesand endpoint, p-values indicate significanceof ANOVA for differences among treatments.Asterisks indicate treatment means thataresignificantly less than control (Dunnett’s test).Cell borders indicate NOEC and LOEC.[LS=pondsnail, SS=Idaho springsnail.SE=standard error.]40Survival (percent)Shell diameter (mm)SpeciesAmmoniaAmmonia(mg N/L)(mg N/L)N Mean SE Dunnett p-valueNMean SE Dunnett p-valueLS 0.14 4 97.5 2.5 0.8534 0.14 4 7.11 0.17LS 0.48 4 100.0 0.00.48 4 6.87 0.05LS 1.0 4 95.0 2.91.01.84 6.93 0.10LS 1.8 4 90.0 10.04 6.67 0.08 *LS 3.6 4 95.0 5.03.6 4 6.38 0.24 *LS 8.0 4 97.5 2.58.0 4 5.98 0.06 *SS 0.14 4 100.0 0.0 0.0011 0.14 4 1.35 0.02SS 0.48 4 44.4 20.3 * 0.48 4 1.47 0.10SS 1.0 4 27.5 10.3 * 1.0 3 1.29 0.08SS1.83.64 62.5 16.51.8 4 1.52 0.11SS 4 20.0 14.1 *3.6 2 1.55 0.20SS 8.0 4 2.5 2.5 *8.0 1 1.100.00070.3691

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

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

differences among treatment means. Asterisks in dicate 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=Idah o 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 varian ce 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. Asterisk s 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=Id aho springsnail.SE = standard error.]

42 Survival (percent) Species 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 valuesfor freshwater snails. Species chronic valueis lowest EC20 for a single test, or geometricmean of lowest survival or growth EC20s (bold) for multiple tests. Upper boundary valueused for ‘less than’ values. [N=neonate,J=juvenile; M=mixed. LC50=median lethal concentration; EC20=20% effect concentration; conf=95% confidence interval.]43SurvivalGrowthSpeciesChemicalSpeciesAgeTestEC20(conf)LC50(conf)EC20(conf)chronicvalueCopperSSBRPCPOZPSNH3SSSSBR*EC20 or LOZ**LC50 estPSLSLSLSC50 estimimated byNNJJMMMMNNNNNJMMMMated by Tlinear intA1A14444B2B2335555oxicity R16.922.48.58*9.8913.7*16.814.0*11.1>337121--109>7.9<0.483.24*3.42*0.611.02*esponseerpolation (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 16.1521.57.419.111816.6>7.9>8.0.0; R. Eri(6.1-6.6)(19-27)(6.8-8.5)(19-55)(100-135)(11-69)ckson, USEPA11.59.5516.814.011.147.516.6>7.91.253.420.611.02-MED, Duluth MN)..Table 16. Published toxicity values for toxicity of copper, pentachlorophenol, andammonia to freshwatersnails. [Endpoints:S=survival, G=growth, R=reproduction. Metrics: LC50=median lethal concentration;NOEC=no observed effect concentration;LOEC=lowest observed effect concentration; ChV=geometricmean of NOEC and LOEC.]44SpeciesDurationEndpointMetricConcentrationNotesReferenceCopper (ug/L)Lymnaea luteola96hS LC50 27Khangarot et al. 1988Potamopyrgus jenkinsi96 hS LC50 54juvenilesWatton and Hawkes 1984Potamopyrgus jenkinsi96 hS LC50 77adultsWatton and Hawkes 1984Potamopyrgus jenkinsi96 hS LC50 79senescentWatton and Hawkes 1984Pomacea canaliculata10 dG NOEC 68juvenilesPena and Pocsidio 2007Pomacea canaliculata50 dG NOEC >68juvenilesPena and Pocsidio 2007Physa integra6 wkSG LOEC 14.8adultsArthur and Leonard 1970Campeloma decisum6 wkSGR LOEC 14.8adultsArthur and Leonard 1970Pentachlorophenol (ug/L)Lymnaea acuminata96 hS LC50 160Gupta and Rao 1982Physa gyrina96 hS LC50 220summerHedtke et al. 1986Physa gyrina96 hS LC50 1380winterHedtke et al. 1986Physa gyrina96 hS LC50 267Lk. SuperiorHedtke et al. 1986Gillia altilis96 hS LC50 810staticStuart and Robertson 1985Gillia altilis96 hS LC50 300flow-throughStuart and Robertson 1985Physa gyrina96 hS ChV 150Hedtke et al. 1986Physa gyrina36 dG ChV 74Hedtke et al. 1986Physa gyrina21 dR ChV <26Hedtke et al. 1986Table 16 (continued)45Species Duration Endpoint MetricConcentration Notes ReferenceTotal Ammonia (mg N/L)Lymnaea stagnalis96 h S LC50 0.825Williams et al. 1985Physa fontinalis96 h S LC50 1.4Williams et al. 1985Potamopyrgus antipodarium96 h S LC50 2.02Alonso and Camargo 2003Potamopyrgus antipodarium96 h S LC50 0.31 lowest Hickey and Vickers 1994Potamopyrgus antipodarium96 h S LC50 0.44 highest Hickey and Vickers 1994Potamopyrgus jenkinsi96 h S LC50 0.315 juveniles Watton and Hawkes 1984Potamopyrgus jenkinsi96 h S LC50 0.85 adults Watton and Hawkes 1984Potamopyrgus jenkinsi96 h S LC50 0.49 senescent Watton and Hawkes 1984Survival (%)



## 0 20 40 60 80 100no data



## Algae1 Algae2 Flake1 Flake2 YCT1 YCT2Wet weight (mg)



## 0 5 10 15 20 25



# LS

SSno 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 e xposure 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.

46ASTM-L ASTM - H Well- L Well -H

A STM- L

A ST M

-H Wel l-L Wel l -HShell 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 af ter 28 d, with standard deviation (n=9-10).

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

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

47

# Test A

30 100



### LS

SS 

# * * * * 80Survival (%) 20 60

40
10

20

0 0Wet weight (mg)



# Test 1

1 1 0 1 0 0 

### LS

SS 

# * * * 1.5 8 100

80 6

1.0
60



# * *

1 10 100Survival (%)

4

40 0.5

20

0 2

0.0 0



# Test 4 Copper ( μ g/L)Shell diameter, mm(SS=left, LS=right)

100



### Copper ( μ g/L) 1 1 0 1 0 0 

### BR

OZ

PS

SS 

# * * * * * *

* *

*Survival (%) 80

60

40

20

0 1 1 0 1 0 0 

### 1 10 100



## Figure 3. Toxicity of copper to freshwater sna ils. 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 c ontrols (rank ANOVA with Dunnett’s test).

48

# Test BSurvival (%)



### 0 20 40 60 80 100

LS

SS 

# *Wet weight (mg)



### 0 10 20 30 40 50



# * *

* *

*

1 1 0 1 0 0 1 1 0 1 0 0



# Test 2

2 8 

### 100

80 6



### LS

SS 

# * *

1 1 0 1 0 0 

# *

* * *

1 1 0 1 0 0Shell diameter, mm(Y-axis: SS=left, LS=right)Survival (%)



### 60

40

20

0 1 4

2

0 0



### PCP ( μ g/L) PCP ( μ g/L)



## Figure 4. Toxicity of pentachlor ophenol (PCP) to freshwater sna ils. Survival and growth during

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

controls (rank ANOVA w ith Dunnett’s test).

49

### 60 4 6 8 

# Test 3

0.1 1 10 

### LS

SS 

# *

* * * 100

80 

# * * *

0.1 1 10Shell 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 sn ails. Survival and grow th during 28-d exposures:

m eans with standard errors (Test 3; n=4) or m eans (Test 5; n=2). Asteri sks indicate significant

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

50

## Copper ( μ g/L)

0 20 40 60 80 100

Mussel

Fish

Snail

Invert SS PS

LS OZ BR Campeloma



### 1 1 0 1 0 0

500 10 100 

## PCP ( μ g/L)Percentile



### 0 20 40 60 80 100

SS LS Physa



## Ammonia (mg N/L)

1 1 0 0 20 40 60 80 100

SS PS LS

OZ BR



### 0.2 20



## 51 

# Chronic value

Figure 6. Species sensitivity dist ributions and chronic water quali ty criteria (dashed lines) for

copper, PCP, and ammonia. Values were adju sted 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). T oxicity values from Tables 15 and 16.