28

- DAY AMMONIA TOXICITY TESTING OF NON

- PULMONATE SNAILS

EPA Contract Number: EP

- C

- 09

- 001 Work Assignment 2

- 31, Task 1, Subtask 1

Final Report

PREPARED FOR THE U.S. Environmental Protection Agency

By

Great Lakes Environmental Center

1295 King A ve. Columbus, OH 43212 Phone: (614) 487

- 1040 Facsimile: (614) 487

- 1920

May 27 , 2011ii TABLE OF CONTENTS

EXECUTIVE SUMMARY

................................ ................................ ................................ ........... vi

INTRODUCTIO N

................................ ................................ ................................ .......................... 1

OBJECTIVE

................................ ................................ ................................ ................................ ... 2

METHODS

................................ ................................ ................................ ................................ ..... 3

Experimental Facility

................................ ................................ ................................ .................. 3

Dilution Water

................................ ................................ ................................ ............................. 3

Test Material

................................ ................................ ................................ ............................... 4

Test Organisms

................................ ................................ ................................ ............................ 4

Toxicity Range Finding Test

................................ ................................ ................................ ....... 4

28

- Day Toxicity Test

................................ ................................ ................................ ................. 5

Data Analysis

................................ ................................ ................................ .............................. 6

RESULTS AND DISCUSSION

................................ ................................ ................................ ..... 6

Toxicity Range Finding Test

................................ ................................ ................................ ....... 6

28

- Day Toxicity Test

– Water Quality

................................ ................................ ........................ 7

28

- Day Toxicity Test

- TAN Measurement and Concentrations

................................ ................ 8

Determination of a 96

- h EC50 in the 28

- day Exposure

................................ .............................. 9

Determination of EC20s

................................ ................................ ................................ ............ 10

TEST UNCERTAINTY

................................ ................................ ................................ ................ 10

Variation in TAN concentrations and select water quality p arameters

................................ ..... 11

Data interpretation

................................ ................................ ................................ ..................... 12

SUMMARY AND CONCLUSIONS

................................ ................................ ........................... 13

REFEREN CES

................................ ................................ ................................ ............................. 14iii LIST OF TABLES

Table 1. Water quality summary statistics for 96

- hr ammonia toxicity range

- finding test with

mature, mixed

- age P. canaliculata

. ................................ ................................ .............................. 15

Table 2. Results of the 96

- hr range

- finding test with mature, mixed

- age P. canaliculata (N=6
snails per treatment).

................................ ................................ ................................ ..................... 16

Table 3. Overall water qu ality summary statistics for 28

- day ammonia toxicity test with mature,

mixed

- age P. canaliculata . Values represent summary statistics derived using all measured
values within a treatment (nominal) for a given parameter.

................................ ......................... 17

Table 4. Daily average measured total ammonia concentrations (mg TAN/L) by nominal
treatment level. Number in parentheses following the coefficient of variation (CV) is the number

of available TAN measurements at that concent ration on that day, i.e., N = 1 to 4 based on
number of replicates sampled per treatment. A value of (0) indicates that the TAN concentration
for that day was estimated by interpolating the average concentrations from the closest days for

which measuremen ts were taken (see text).

................................ ................................ .................. 18

Table 5. Daily average calculated unionized ammonia concentration (mg UIA/L) by nominal
treatment level. Number in parentheses following the coefficient of variatio n (CV) is the number
of available measurements at that concentration on that day, i.e., N = 1 to 4 based on number of
replicates sampled per treatment. A value of (0) indicates that the UIA concentration for that day
was estimated by interpolating the ave rage concentrations from the closest days for which
measurements were taken (see text).

................................ ................................ ............................ 19

Table 6. Concentration

- response results for P. canaliculata 28 day survival in response to TAN,

with an d without replicates from nominal 1.9 mg TAN/L treatment. Toxicity predictions

obtained from a 3

- parameter logistic model using the EPA TRAP (v. 1.21) analysis program.

.. 20

LIST OF FIGURES

Figure 1. TRAP logistic equation results of 96

- hr range

- finding test at pH 8.19 and 24.7°C.
Ninety

- six hour EC50 normalized to pH 8.0 and 25°C is 13.56 (9.91

- 18.53) mg TAN/L. TAN
concentration represents average concentration cal culated from the measurement of samples

taken over the 96

- hr test period. Statistics below figure represent output from TRAP where “initial” represents the input value required as a first guess to generate a model and where “final”
represents actual stati stic given by the fitted model equation.

................................ ...................... 21iv

Figure 2. Graphical display of the variability in measured water temperatures over the 28

- day
test period amongst replicate chambers within the variou s test treatments (based on nominal
TAN concentrations).

................................ ................................ ................................ .................... 22

Figure 3. Graphical display of the variability in measured pH of test water over the 28

- day test
period amongst replicate chambers wit hin the various test treatments (based on nominal TAN
concentrations)

................................ ................................ ................................ .............................. 23

Figure 4. Graphical display of the variability in measured dissolved oxygen concentrations over

the 28

- day test period am ongst replicate chambers within the various test treatments (based on
nominal TAN concentrations).

................................ ................................ ................................ ...... 24

Figure 5. Graphical display of the variability in measured TAN concentrations over the 28

- da y test period amongst replicate chambers within the various test treatments (based on nominal
TAN concentrations). Line represents the daily average TAN from all replicates within a
treatment when measured, and interpolation between the daily average conce ntrations when

measurements were not made.

................................ ................................ ................................ ...... 25

Figure 6. Graphical display of the variability in calculated unionized ammonia (UIA)

concentrations over the 28

- day test period amongst replicate ch ambers within the various test
treatments (based on nominal TAN concentrations). Line represents the daily average UIA from
all replicates within a treatment when measured, and interpolation between the daily average

concentrations when measurements were n ot made.

................................ ................................ .... 26

Figure 7. Twenty eight day survival of P. canaliculata across an ammonia concentration
gradient with replicate values averaged. P. canaliculata in the control treatment were

mainta ined for an additional 14 days after test completion to further assess controls.

................ 27

Figure 8. Twenty

- eight day survival of P. canaliculata across an ammonia concentration gradient
by replicate c hamber. Note the anomalous early mortality in replicates 3 and 4 of the nominal
1.9 mg TAN/L treatment.

................................ ................................ ................................ .............. 28

Figure 9. Graphical display of concentration

- response results for P. canaliculata afte r 28 days in
response to TAN, (a) all replicates from all treatments included, and (b) minus the four replicates
from the second lowest (nominal 1.9 mg TAN/L) treatment. TRAP logistic equation results are
as those presented in Table 5.

................................ ................................ ................................ ....... 29

LIST OF APPENDICES

APPENDIX 1. Justification for selection of test species and species distribution.

..................... 30v

APPENDIX 2. S ummary of acquisition, acclimation, maintenance, and health of test snail
species.

................................ ................................ ................................ ................................ .......... 35

APPENDIX 3. Metals characterization of dilution water from East Arm Grand Traverse Bay,
Lake Michigan.

................................ ................................ ................................ ............................. 42

APPENDIX 4. Cross validation of archived samples using new standard curve.

....................... 43

APPENDIX 5A. Water quality measurements.

................................ ................................ ........... 44

APPENDIX 6. TAN measurements.

................................ ................................ ............................ 50

APPENDIX 7. Trimatrix duplicate TAN analysis using Phenate Method.

................................ . 51

APPENDIX 8A. P. canaliculata survival.

................................ ................................ ................... 52

APPENDIX 9. P. canaliculata growth measurements.

................................ ............................... 54vi

EXECUTIVE SUMMARY

The purpose of the st udy outlined in this report was t o independently test and confirm previous

28

- d ay toxicity test results indicating that specialized and/or Federally

- Listed non

- pulmonate, gill

- bearing snails such as the Idaho springsnail, Bliss Rapids snail and pebblesnail are potentially both : 1) extra sensitive to prolonged ammonia exposure , and 2) as sensitive as

ammonia

- sensitive freshwater unionid mussel species are to such exposure . To do so, EPA sponsored a study to conduct additional 28

- day toxicit y tests using mor e widely

- distribute d non

pulmonate snail species. The candidate non

- pulmonate snail species targeted for testing were

mature , mixed

- age globe siltsnail ( Birgella subglobosus ), pointed campeloma ( Campeloma

decisum ), and silty hornsnail ( Pleurocerca canalic ulata ), each represent ative of three widely

distributed families within the continental United States; Hydrobiidae, Viviparidae, and Pleuroceridae, respectively

. Of the three wild

- caught snail species collected for testing, EPA’s contractor (Great Lakes E nvironmental Center, Inc., i.e., GLEC ) was only able to successfully hold, maintain, and test, via a 28

- day , measured flow

- through ammonia toxicity test, silty

hornsnail ( P. canaliculata ) . The 96 h acute EC50 estimated from a toxicity range finding test

w ith the species was 1 3.56 mg total ammonia nitrogen or TAN/L, after normalizing to 25°C and pH 8.0. The corresponding EC20 estimated from t he 28

- day survival test was as low as 0.55 mg
TAN/L after normalizing to 25 ° C and pH 8.0 ; however , because of the un certainty in the test, the

recommended 28

- d ay ammonia survival effects concentration for the species is <2.2 8 mg TAN/L

at 25 ° C and pH 8.0 . This upper limit concentration is not recommended for FCV calculation as originally intended, but instead, should be considered as contributing to the weight of evidence indicating the unique sensitivity of gill

- bearing snails to extended ammonia exposure in general.

An ACR estimated by dividing the normalized 96 h EC50 estim ate from the range finding test by

the normal ized and recommended 28

- day ammonia survival effects concentration i s calculated to

be >5.9

.

Based on the results reported herein , GLEC concludes that for P. canaliculata , the 28

- d ay ammonia survival effects concentration of <2.2 8 mg TAN/L at 25°C and pH 8.0 confirm s that :

1) The sensitivity of m ature, mixed age P. canaliculata to ammonia toxicity via a 28

- d ay test approaches that of their more specialized and/or Federally

- Listed counterparts

, e.g., Idaho springsnail ( Pyrgulopsis idahoensis ) , Bliss Rapids ( Taylorconcha serpenticola ) and pebblesnails ( Fluminicola sp. )

– see Besser et al. 2009 and Besser (2011) ; and, 2) May also approach that of the highly ammonia

- sensitive freshwater unionid mussel species : wavy

- rayed lamp mussel, Lampsilis fasciola ; fatmucket, Lampsilis siliquoidea ; and rainbow mussel, Villosa iris

– see Wang et al. (2007a, b) and Wang et al. (2008)

.

A dditional 28

- d ay toxici ty tests are recommended for this and other non

- pulmonate snails to

further substant iate the preliminary findings obtained in this and other similar stud ies with freshwater gill

- bearing snails

.1

INTRODUCTION

The U.S. EPA, Office of Water is charged with protecting aquatic life, wildlife, and human health from adverse anthropogenic and water

- mediated effects under the purview of the Clean Water Act (CWA). In support of this mission, o n December 30, 2009 , U.S. EPA’s Health and Ecological Criteria Division (HECD), within the Office of Science and Technology (OST) of EPA, published the “Draft 2009 Update Aquatic Life Ambient Wate r Quality Criteria for

Ammonia

– Freshwater” in the Federal Register (FR). Included in the 2009 draft ammonia criteria update are several 28

- day EC20 values (concentrations associated with a 20% effect) from a series of toxicity tests with various freshwa ter snail species from a United States Geological Survey (USGS) study (Besser et al. 2009) . The Besser et al. (2009) study involved

the conduct of 28

- day flow

- through survival and growth toxicity tests with five species of snails:

four non

- pulmonate or gi ll

- bearing snails ( Pyrgulopsis idahoensis

, Fontigens aldrichi

, Taylorconcha serpenticola , and Fluminicola sp.), and one commonly tested pulmonate (capable

of air

- breathing) snail ( Lymnaea stagnalis) . Two of the non

- pulmonate species were Federally

Listed species at the time of testing, i.e., P. idahoensis and T. serpenticola , and all of the non

pulmonate snails tested require specialized habitats and exhibit limited distribution within the

continental United States.

All snail tests performed in the Besser et al. (2009) study were conducted in ASTM hard water (mean hardness and alkalinity of 169 and 121 mg/L as CaCO 3 , respectively) which had a pH range of 8.20

- 8.29 and a temperature range of 19

- 21 o C during testing. The pH (8.0) and temperature (25 o C) norma lized 28

- day EC20 (based on survival and expressed in terms of total ammonia nitrogen (TAN) ) for the five snail species ranged from a low of <0.52 mg TA N/L for juvenile Idaho springsnail ( P. idahoensis ) to >8.5 mg TA N/L for less than 1 week old post

- hatch pond snails ( L. stagnalis ). The 28

- day EC20 for the other three non

- pulmonate snail species was

lower than the 28

- day EC20 for L. stagnalis , i.e., the 28

- day EC20 for the mixed age Ozark springsnail ( F. aldrichi ) was 0.71 mg TA N/L; 3.98 mg TA N/L for the m ixed age Bliss Rapids snail ( T. serpenticola ); 3.77 mg TA N/L for the adult Idaho springsnail ( P. idahoensis ); and <7.9

mg TA N/L for the mixed age pebblesnail ( Fluminicola sp.). Thus, it appears in this instance that gill

- bearing, non

- pulmonate snail speci es may be slightly to substantially more sensitive to 28

- day ammonia exposure than at least one non

- gill bearing, pulmonate snail species, L. stagnalis

. Moreover, the comparatively low survival

- based 28

- day EC20 for the juvenile Idaho springsnail

coincide s with the Species Mean Chronic Values (SMCVs) reported in the 2009 draft ammonia criteria update for several ammonia

- sensitive freshwater mussel species (e.g., wavy

- rayed lamp mussel, Lampsilis fasciola ; fatmucket, Lampsilis siliquoidea ; and rainbow musse l, Villosa iris )

– see Wang et al. ( 2007a, b ) and Wang et al. ( 2 008) . Therefore, EPA commissioned GLEC to

conduct a dditional 28

- day toxicity tests targeting more widely

- distributed non

- pulmonate snail species to evaluate whether this group of snail specie s is generally: 1) as sensitive to 28

- day ammonia toxicity as their more specialized and/or Federally

- Listed counterparts (e.g., springsnail, Bliss Rapids snail and pebblesnail), and 2) as sensitive as ammonia

- sensitive freshwater mussel species.2

OBJECTIV E

The specific objective of this study was to generate 28

- day toxicity test data to determine the

concentration of TAN that causes 20 percent lethality and/or immobilization to one or more widely

- distributed snail species within the continental United Stat es. Candidate non

- pulmonate snail species were identified using species distributions from NatureServe © Explorer. GLEC, working in cooperation with U.S. EPA, selected three c andidate snail species based on the following factors: 1) distribution within th e conterminous United States, 2) degree of habitat specialization, 3) representativeness of the taxonomic group as a whole, and 4) availability for collection (see Appendix 1 ). Of the three species collected for testing: siltsnail ( Birgella subglobosus ), p ointed campeloma ( Campeloma decisum ), and silty hornsnail ( Pleurocerca

canaliculata ), only the latter species was successfully acclimated and maintained at a level of health necessary for 28

- day ammonia toxicity testing (see Appendix 2 for additional detai ls regarding the acquisition and maintenance of all t hree snail species targeted for testing, and for a short summary of the health status achieved during their acclimation and holding in the laboratory).

The 28

- day ammonia toxicity test data for P. cana liculat a descri b ed herein are to be used to inform the final revision of EPA’s update of the freshwater ambient water quality criteria for the protection of aquatic life (ALC) for ammonia. The 28

- day toxicity tests w ere performed generally following appli cable guidance outlined in ASTM E 1241

- 05 , Standard Guide for Conducting Early Life

- Stage Toxicity Tests with Fishes (ASTM 2010a ), and supplemented with any applicable methods and protocol established in ASTM E2455

- 06, Standard Guide for Conducting Laborat ory Toxicity Tests with Freshwater Mussels (ASTM 20 10b ). As aquatic toxicity tests utilizing non

- pulmonate snails are extremely limited, acclimation and holding procedures we re also based on other laboratory experience (e.g., the Besser et al. (2009) stud y conducted at the USGS facility in Columbia, MO) gained from 28

- day snail toxicity testing, and following recommendations from snail culture experts such as Paul Johnson at Alabama Aquatic Biodiversity Center. It is important to recognize th e current kno wledge of snail culturing,

holdi ng and feeding is very limited and not completely applicable to aquatic toxicity testing. Due to certain limitations such as the inability to culture the selected snail species to obtain test organisms of a certain life sta ge (e.g., young juveniles), the primary focus for this study w as on testing survival of mature, mixed age snails. And because mature, mixed age snails were targeted for use , growth over the 28

- day test period wa s expected to be slow and thus, this endpoin t was not expected to provide useful concentration

- response information. Instead, the study conducted by GLEC focu s ed on survival/immobility as the endpoint for 28

- day point

estimat ion/effect determination

.

To provide additional ancillary information p otentially useful for ALC development for ammonia, the test protocol included enhanced observations of live/dead counts and collection of samples for chemical analysis to support calculation of a 96

- hr EC50. Prior to testing, a 96

- hr acute toxicity range

- finding test was used to establish the test concentrations for the 28

- day toxicity tests. The acute EC50 estimated from the 96

- hr range

- finding test and 28

- day ammonia3

survival effects concentration 1 w ere subsequently used to estimate an Acute

- Chronic Rati o (ACR) for P. canaliculata

.

METHODS

Experimental Facility

The 28

- day toxicity test was conducted using a modified Benoit semi

- continuous flow mini

dilutor system at GLEC’s environmental toxicology laboratory in Traverse City, Michigan. The dilutor syst em was located in an enclosed facility with a dedicated water source and electricity. Dilution water used in this study was collected from Lake Michigan, delivered to an external

temperature

- controlled storage tank that in turn delivered water to a single internal glass headbox within the enclosed facility. Dilution water from the glass headbox was t he n gravity fed to the

mini

- dilut or system. All construction materials for this system consist ed of conditioned glass, PVC, or TYGONTM, and 100% clear silica gel was used as the adhesive material.

Dilution water in both the internal headbox and external storage tank was continuously aerated and mixed. Water temperature within the external storage tank was maintained at 25±1°C with

an electronic heater/chiller unit. Water within the internal headbox was maintained at 25°C through a combination of temperature control within the facility coupled with immersion heaters as required. The facility is equipped with an array of lights that were maintained at 150

- 500 lux under a photoperiod of 16 hours light and 8 hours dark.

An ammonia stock solution was continuously maintained throughout the study, and this stock solution was continuously distributed to a mixing cell within the mini

- dilutor that continuously

recei ved a constant supply of Lake Michigan dilution water and ammonia stock solution via a metered pump (Fluid Metering, Inc.). The resulting solution was then distributed to the first treatment mixing cell of the system, representing the highest ammonia test concentration. A portion of this test solution was then used to create subsequent two

- fold serial dilutions for the four remaining treatment mixing cells resulting in five ammonia concentrations with an approximate dilution factor of 0.5. The test solut ion within each treatment level mixing cell was then evenly distributed to the four replicate 2.5 L test chambers for the corresponding treatment

level via flow

- splitting boxes . The mini

- dilutor system was monitored daily and cleaned and

cal ibrated as req uired to maintain a flow rate of approximately 32 mL/min from the outlet s of mixing cell s that were positioned in

- line with and directly above the six flow

- splitting boxes resulting in a volume of water sufficient to provide at least 3.5 complete test solu tion renewals per replicate test chamber per day.

Dilution Water The laboratory dilution water for the acclimation period and toxicity test was Lake Michigan water that was collected directly from the West Arm of Grand Traverse Bay and delive red to

1 Note: GLEC recommends use of the 28

- day ammonia survival effects concentration for this test instead of an

EC20 point estimate because of the uncertainty in the 28

- day test due to variable ammonia exposure, particularly in
the first 18 days of the test, and varying response within critical treatments

– see additional explanation under Test
Uncertainty below.4

GLEC ’s laboratory. Similar dilution water has previously been characterized for: pH, total suspended solids, total organic carbon (TOC), organophosphorus pesticides, total residual chlorine, total PCBs, chlorinated phenoxy herbicides, ammonia, cyanide, sulfid e, bromide, fluoride, iodide, nitrate, phosphate, sulfate, calcium, magnesium, potassium, aluminum, arsenic, beryllium, boron, cadmium, chromium, cobalt, copper, iron, lead, manganese, mercury, molybdenum, nickel, selenium, silver, zinc, hardness, alkalini ty, conductivity, sodium and chloride. The characterization (shown for metals and other select constituents only) is summarized in Appendix 3

. In every case , the concentration of the analyzed components in the

dilution water is less than the concentratio n shown to adversely affect freshwater aquatic

invertebrate species based on current 304(a) ambient water quality criteria and datasets , or is

below the minimum detection limit.

The dilution water was pH adjusted and controlled to maintain a prescribed t est pH using an automated pH controller (Bar nantTM) in the internal headbox and adjusting the pH of the dilution

water with the addition of dilute HCl.

Test Material A mixture of reagent grade ammonium chloride (NH 4 Cl: BDH) and ammonium hydroxide (NH 4 OH: E M Science) was used in this 28

- day toxicity test to more effectively maintain a constant pH in the test solutions

. Stock solutions to be delivered to the dilutor system were initially prepared as aqueous solutions in de

- ionized water. Prior to test initi ation, the appropriate mixture of NH 4 Cl and NH 4 OH to maintain a target pH of 8.0 was identified.

Test Organisms Mixed age adult Pleurocera canaliculata (Family Pleuroceridae) were collected from the Ohio River on August 11, 2010 (see Appendix 2 for additi onal details regarding location, water quality conditions, acclimation procedures, etc.) . Snails were transported from the collection site to the Traverse City, MI experimental facility in cool, oxygenated Ohio River water. Upon arrival at the experiment al facility the following morning, snails were transferred to Lake Michigan test

dilution water (see Appendix 2 for further detail) and held until September 22, 2010 (approximately six weeks total) at approximately 25°C and pH=8.1 to acclimate snails to te st water and conditions as well as to ensure wild caught organisms did not show any signs of stress, physical damage, disease, external parasites, or any other condition that would contribute to mortality.

Snails were fed a diet of periphyton ad libitum on unglazed PVC tiles introduced initially and after approximately two weeks into each test chamber during the study, and were also observed supplementing that diet by consuming periphyton biofilms that accrued on test chamber surfaces, particularly durin g the latter stages of the experiment. Periphyton was colonized in a separate flow through system receiving Lake Michigan dilution water by allowing periphyton biofilms to colonize and develop on the unglazed PVC tiles

.

96

- hr Toxicity Range

- Finding Test

Prior to the initiation of the 28

- day toxic ity test, a 96

- hr toxicity range

- finding test was conducted to determine a suitable range of test concentrations for use in the 28

- day toxicity test

. The range

- finding test ran from September 14

- 18, 2010, and con sisted of five treatment levels5

and a control, with three adult snails per replicate and two r eplicates per treatment (N=6 snails total per treatment) . Nominal treatment concentrations for the range

- finding test were 3, 6, 12,

25 and 50 mg TA N/L. Target test temperature and pH were 25°C and 8.0, respectivel y. The endpoint for this range

- finding test was death and/or immobilization, which was measure d daily

and used to calculate a 96

- hr EC50 for the test. Individuals presumed dead were removed from

the t est chamber and held in a separate beaker containing overlying water for up to 30 minutes, and if observations were confirmed, dead individuals were discarded. If death was not confirmed, then individuals were returned to their respective test chamber.

2 8

- Day Toxicity Test Following the completion o f the range

- finding test, a 28

- day toxicity test using mature, mixed

age Pleurocera canaliculata was conducted from September 22

– October 20, 2010. The test design consisted of five ammonia treatment levels and one control treatment. Each treatment consisted of four replicate chambers with six snails (N=24 snails total per treatment)

. Note: for this study, only 280 P. canaliculata were collected from the Ohio River, 46 of which were smaller in size (range: 0.5 to 0.8 cm total shell length) and subsequently not used for 28

- day toxicity testing. Because of the expedited timeline for this study, and also because of the length

of time chosen a priori to acclimate and hold the wild

- caught snails prior to testing to ensure adequate health (approximately six weeks), sample si ze could not be increased to the desired number of ten without causing a delay in testing. Nominal treatment concentrations were 0.9, 1.9, 3.8, 7.5, and 15 mg TAN/L. The target test temperatu re and pH were 25°C and 8.0, respectivel y. The endpoint for the 28

- day toxicity test was death and/or immobilization, which was measured daily and used to attempt calculation of a definitive EC50 for the first 96 hours of

the test and an EC20 for the 28 d ay test period 2

. Except for the first 96 hours of the 28

- day test where observations were made daily, d ead and/or immobile snails were evaluated on Mondays, Wednesdays and Fridays of each week, as described above for the range

- finding test.

GLEC experien ced some difficulty maintaining the desired target water quality conditions throughout the test largely due to problems with the maintenance and operation of the dilutor

system under the difficult test conditions (i.e., high test temperature and use of nat ural, unfiltered lake water) selected for testing (see additional details provided in the Test Uncertainty section provided below). Dissolved oxygen, temperature and pH were measured daily in each replicate chamber on nearly every day of the testing perio d using bench top and hand held meters : dissolved oxygen [YSI Model 57 or 52]; pH [Orion Model 710A or 250A]; temperature [VWR

Traceable Thermometer

- d igital]). Specific conductivity was measured weekly in each replicate on nearly every Monday of the testi ng period using a bench top meter (YSI Model 35). The minimum acceptable dissolved oxygen concentration in any test chamber was 60% saturation, and any time dissolved oxygen fell below 70% saturation, the replicate chamber in question was further investig ated and the problem remedied , primarily through increased attention and

maintenance of the mini

- dilutor system

. Likewise, the pH of the test water within a given test chamber was maintained to within 0.2 standard units of the target pH of 8.0, and temper ature to
within ± 1 °C of the target temperature of 25°C . I f any individual measured pH or temperature in any test chamber was more than 1.5 pH units or 3 o C (respectively) different from the mean of the

2 Agai n, due to uncertainty in the test, GLEC recommends an alternate 28

- day ammonia survival effects
concentration for the test, as described in more detail in the Test Uncertainty section of this report.6

time

- weighted average measured temperature or pH f or the individual test chambers, then the replicate chamber in question was further investigated and the problem remedied

, again, primarily through increased attention and maintenance of the mini

- dilutor system . If all snails within a particular test chamber died, then dissolved oxygen, temperature, and pH measurements for that chamber were no longer taken.

TAN measurements were measured at GLEC with an Orion Research Microprocessor pH

mil l ivolt meter 811 with an Orion 9512 ion specific ammonia probe through out the study and within replicate test chambers following U

. S

. EPA method 350.3, Standard Methods for the Examination of Water and Wastewater, 21st edition, 2005, Method 4500

- NH3 (D) and corresponding GLEC Standard Operating Procedures (SOPs). The report ing limit for this method was 0.5 mg TAN/L. Ammonia measurements were made in all chambers on days 1, 2, 5, 16, 19, and 21

- 28 of the test , the latter (days 21

- 28 of the test) only in test chambers with surviving snails . On the remaining test days, ammoni a concentrations were measured in a minimum of two replicate chambers per treatment every other day. In addition to the metered measurements, a subsample of 35 measurements from water samples collected between days 20 and 28 were analyzed for TAN by a sec ond laboratory (Trimatrix) using the Phenate M ethod (Standard Methods for the Examination of Water and Wastewater, 21st edition, 2005, Method

4500

- NH3 (G)). Subsamples of ten archived samples were measured after the study at GLEC

but with a re

- calculated standard curve with a lower concentration range (see Appendix 4 ).

One day prior to test initiation, the metering system and flow rates upstream and out of the flow

splitting boxes were checked to assure the system was functioning properly. The meter syst em and flow rates were subsequently checked dail y throughout the remainder of the 28

- day test period. Water from all replicate chambers was siphoned and chambers were allowed to fill overnight with test water. A total of six adult P. canaliculata were ran domly assigned to each test chamber from a single holding cha mber. Snails used in the range

- finding test were not used

in the 28

- day test. Water qua lity measurements and observations of survival were made throughout the study as previously described, wit h sufficient measurements made during the first

96 hours to determine a 96

- h r EC50. After the completion of the 28

- day test, all surviving snails from the control group were held for an additional 14 days in control water and monitored for

survival or imm obilization to further demonstrate the health of the snails used in this test.

Data Analysis All effect concentration (EC20 and EC50) measurements were made using three parameter logistic equation functions within EPA’s Toxicity Relationship Analysis Prog ram (TRAP, Version 1.21, October 30, 2010). T he survival NOEC and LOEC of the 28

- day toxicity test were

not calculated for the test for reasons explained later in the report. S ummary statistics for all water quality parameters (including TAN concentrat i o ns) were calculated in MS Excel.

RESULTS AND DISCUSSION

96

- hr Toxicity Range

- Find ing Test Average temperature, pH, and dissolved oxygen throughout the toxicity range

- finding test were similar across treatments, with temperature ranging from 24.3

- 25.1°C, pH ranging from 8.07

- 8.23, and dissolved oxyg en ranging from 7.3

- 7.6 mg/L (Table 1). The ov erall average7

temperature (24.7 °C) was similar to the target temperature of 25°C, while the overall average pH (8.19) was slightly higher than the target value of 8 .0. M ean m easured TAN concentrations were on average 36 percent lower than nominal concentrations across nearly the entire concentration

range except for the lowest concentration (Table 1). Overall, measured TAN increased over the

course of the range

- fin ding test

, possibly due to improved performanc e and operation of the dilutor and ammonia delivery s ystem

.

Prior to the initiation of the 28

- day toxicity test

, a 96

- h r EC50 estimate was calculated from the results of the range finder test using the EPA P robit Analysis Program (see Table 2) . Because the control concentrations were below detection level, they were not included in the initial analysis. The resulting 96

- hr EC50 at measured test conditions was 9.06 mg TAN/L, with a 95%

confidence interval of 6.00

- 15.0 mg TAN/L (Table 2) . After normalizing to pH 8.0 and 25°C,

the normalized 96

- hr EC50 was 12.7 1 mg TAN/L, with a 95% confidence interval of 8.4 2

- 21.1 mg TAN/L.

The 96

- hr EC50 was recalculated in TRAP using a logistic equation model after assigni ng a concentration of 0.06 mg TAN/L to the controls (see text below under 28

- Day Toxicity T est

– TAN Measurement and Concentrations ) . Based on measured test conditions (pH

- 8.19, Temp

24.7°C) the resulting 96

- hr EC50 was 9.66 mg TAN/L, with a 95% confidenc e interval of 7.06

- 13.2 mg TAN/L (Figure 1). After normalizing to pH 8.0 and 25°C, the 96

- hr EC50 was

estimated to be 13.56 mg TAN/L, with a 95% confidence int erval of 9.91

- 18. 53 mg TAN/L , but because of low sampl e size (N=6 per concentration ) in the rang e

- finding test, this particular 96

- hr EC50 should be considered preliminary for this species.

28

- Day Toxicity Test

– Water Quality Temperature, pH, and dissolved oxygen levels fluctuated throughout the study

, particularly over

the 4

- day period from Friday , October 1, 2010 to Monday O ctober 4, 2010, test days 9

- 12 (see Figures 2, 3 and 4, respectively; corresponding raw data for each treatment is included in Appendix 5)

. During this period

, several measured values for water t emperature , pH, and

dissolved o xygen were reduced relative to the remainder of the test period. The lowest temperatures during this period fell just below the acceptable range (<22 o C) for this test (Figure 2) . Similarly, the lowest pH values observed were just below 7.5 (Figure 3) , an d dissolved

oxygen fell below the acceptable range ( 60% saturation ) in a total of five test chambers (Figure 4) . Further explanation for the likely decrease in these key water quality parameters is provided in the Test Uncertainty section below.

Overall, the average water temperature, pH, and di ssolved oxygen levels were 24.7 o C , 8.15, and 7.3 mg/L, respectively. Average w ater temperature for a giv en treatment in general fell just below the target temperature of 25 o C throughout the entire test concentrati on ( range of 24.5 to

24.8 o C ) , while pH was slightly higher overall (average difference of approximately 0.07 s.u.) at the lower end of the ammonia concentration gradient (including the control treatment) than at the high end, possibl y as a result of the re lative ratios of NH 4 Cl to NH 4 OH across treatments (Table 3). Mean dissolved oxygen concentrations ranged from a low of 7.1 mg/L in the 1.9 and 3.8 mg TAN/L ( nominal) treatments to an average high o f 7.7 mg/L in the control treat ment (Table 3). Specific c onductivity was relatively stable throughout the study within a given ammonia treatment level; however, specific conductivity increased from 307

- 415 μS/cm across the8

concentration gradient, most likely the result of the additional chloride from the test ma terial (Table 3 ).

28

- Day Toxicity Test

- TAN Measurement and Concentrations

T he temporal distribution of TAN measurements taken during the test was uneven, with more measurements taken during test days 1

- 5 (to determine a definitive 96

- hr EC5) and 19

- 28 ( to
assess the effectiveness of the remedial actions taken to address the problems GLEC encountered trying to maintain stable TAN concentrations within each treatment)

, respectively, and fewer

m easurements taken between days 6

- 18 (Figure 5; Appendix 6). Be cause there was a high degree of temporal variab ility in observed TAN concentrations, average TAN for a given

treatment cannot be accurately estimated as a simple average of all measurements within a particular treatment . This is because the average TAN c oncentration for a particular treatment will be biased towards the more data rich sampling periods , which do not encompass the full

range of temporal variability during the 28

- day study . In order to remove this potential source of

bias from the average TA N measurements, the following procedure was performed. For every day during the test, the average daily TAN concentration was calculated for each treatment level (Table 4). By collapsing the replicate measurements into a single treatment level average on a daily basis , much of the problem associated with the paucity of measurement data during days 6

- 18 is overcome. While TAN may not have be en measured in a single replicate chamber for up to a week (or longer, in those chamber s that had already experience d 100% snail mortality) , TAN was measured in at least one, and usually at least two chambers

, on most test days ( see Appendix

6 ). For those days where no measurements were taken in any replicate chamber for a particular treatment level, the daily average was estimated through interpolation of the nearest temporal measurements available. For example, in the nominal 1.9 mg TAN/L t reatment, there were no measurements in any of the replicates on test day 6. The estimated concentration of 0.53 mg

TAN/L is the average of the day 5 concentration of 0.50 mg TAN/L and the day 7 concentration of 0.56 mg TAN/L

. It is worth noting that t he average daily concentrations estimated using this method are generally lower than the concentrations one would obtain from avera ging all of the available measurements, because for all but the highest TAN treatment level, the data are most depauperate during the period when they are also the lowest (Figure 5). T his approach allows for the cumulative average TAN concentration s to be calcu la ted for the entire 28

- day test period

which are used to calculate the 28

- day EC20s described later in this report

.

Based on the above, the 28

- day cumulative average TAN concentrations (i.e., treatment mean TAN concentrations, or x

 ̄ in T able 4) were below nominal concentrations f or all treatment levels (Table 4 ). Mean measured TAN as a percentage of nominal TAN increased across the concentration gradient, rising from 38 % at the lowest test concentration to 80 % at the highest test

concent ration. TAN concentrations were temporally variable in all test concentrations, with the ratio of highest to lowest measured TAN concentration for single measurements within each

treatment all exceed ing 1.5 ( Figure 5; also see Appendix 6 ). The relative t emporal fluctuations in TAN measurements were highest in the two lowest test concentrations, peaking in the second

l owest test concentration (nominal = 1.9 mg TAN/L ; m easured = 0.81 mg TAN/L ), then

decreas ing with increasing test concentration for the thre e highest treatment levels ( Figure 5,

Table 4 ). Note that within the three test concentrations representing the m iddle of the concentration gradient (e.g., measured 0.81, 1.86 and 4.50 mg TAN/L), the lowest measured

TAN concentration coincided with test d ays 9

- 12

, w hi ch was the timeframe w hen temperature,9

pH and dissolved oxygen also dropped below target test specifications (compare Figure 5 against Figures 2 through 4 ). Also note that the highest measured TAN concentration for all but the highest treatme nt level generally occurred o n the first day of the 28

- day test. The se within

treatment fluctuations in TAN measurements amongst replicate test chambers explain a significant portion of the variability in exposure for the test, and thus, are a major s ourc e of uncertainty precluding accurate estimation of the 28

- day EC20

, as discussed in greater detail

below.

For convenience to the reader, the temporal distribution of calculated unionized ammonia (UIA)

concentrations was summarized in table form and graph ically as for TAN (see Table 5 and Figure 6). The pattern of temporal variability observed for TAN is similarly evident for UIA, but even more pronounced

.

Due to the observed variability in measured TAN concentrations, and a s an attempt to not only

verif y the accuracy and precision of the metered TAN measurements but also to check the validity of the TAN measurements , as many as 35 additional water samples were collected

be tween days 20

- 28 for TAN measurement and a corresponding split sample was sent to a
second laborat ory and analyzed for TAN using the phenate method ( Method 4500

- NH3 (G) ). The phenate method was more sensitive by an order of magnitude than the meter method (reporting limit 0.05 mg TAN/L). The seven sub

- samples from the experimental cont rol were all below the phenate method reporting limit of 0.05 mg TA N/L. Concentrations of the non

- control samples measured by the phenat e method were approximately 1.3 times higher than corresponding metered measurements, and that relative difference was similar across the test concentration gradient (Appendix 7 ).

A s part of the remedial action taken above, a n additional ten sub

- samples were also collected during days 20

- 28 and archived by GLEC until the completion of the study. After the completion of t he study, the sub

- samples were re

- measured by the same meter used for the study, but using a refined standard curve equation with additional standards at the low end of the concentration gradient. Using th e revised standard curve, the cross

- validated cont rol concentration was 0.06 mg TAN/L (Appen dix 4 )

.

As can be seen in Table 4 and corresponding appendix (A ppendix 6 ) , and i n order to facilitate

calculation of 28

- day EC20 s using TRAP, all non

- detects were assigned the value of 0.06 mg

TAN/L obtained from re

- measuring archived samples using the revised standard curve as described above. The value of 0.06 mg TAN/L was likely a conservative estimate, based on the

results of the seven control sub

- samples measured using the phenate method, all of which were

be low the method rep orting limit of 0.05 mg TAN/L.

Determination of a 96

- h r EC50 in the 28

- D ay Exposure After 96 hours, P. canaliculata survival was greater than 50 percent for all test concentrations in

the 28

- day exposure . As a result, a 96

- hr EC50 coul d not be calculated from the data gen erated during the 28

- day exposure using either TRAP or the Probit Analysis Program. Sim ilarly, ANOVA analysis of 96

- h r survival revealed no statistically significant differences between treatments (P=0.3), so no post h oc analysis was performed. T he 96

- h r acute EC50 based on the

f irst four days of th e 28

- day toxicity test was >9.59 mg TAN/L after normalizing the highest10

TAN treatment test concentr ation measured after 96

- h r ( i.e., cumulative average of 9.83 mg TAN/L at p H 8.02 and temperature 24.2 o C ) to 25 o C and pH 8.0. This is in contrast to the re sult from the acute range

- finding test where a 96

- h r EC50 of 13.56 mg TAN/L normalized to 25 o C and pH 8.0 was determined for th e species.

Determination of 28

- Day EC20 s Viewed in the aggregate, snail mortality increased with increasing TAN concentration, and the initiation of snail mortality generally occurred sooner at higher TAN concentrations compared to lower concentrations ( see Figure s 7 and 8 ; and corresponding raw surviv al data provided in Appendix 8 A and B). When individual replicates within each treatment are considered

, however, it is apparent that replicates 3 and 4 within the second l owest treatment concentration (nominal

– 1.9 mg TAN/L; m easured

– 0.81 mg TAN/L) ex perienced very rapid mortality

, with only one surviving snail in each of these replicate s after three days, and with replicate 3 experiencing complete mortality after four days (Figure 8 ). The onset of mortality for the snails in these two replicate chamb ers occurred earlier than for snails in any of the other test concentrations, and was likely the result of some unmeasured factor aside from ammonia toxicity. Water quality measurements in those chambers during that time period provide little

additional e vidence of why the rapid die

- off may have occurred (see additional details in the Test Uncertainty section below)

. Moreover, in contrast to the early onset of mortality for snails in replicates 3 and 4 of this treatm ent , all six snails survived in replica te 1 (Figure 8)

. Because of

the obvious incongruity in response amongst replicates within this treatment (nominal

- 1.9 mg TAN/L; measured

- 0.81 mg TAN/L), dose

- response analysis was performed in TRAP on the 28

- day survival response of snails with (Figur e 9a) and without (Figure 9b) the four replicates from this potentially anomalous treatment

.

The 28

- day EC20 estimate based on the response of snails in all replicates of every treatment at test conditions (pH 8.15 and 24.7°C) was 0.4475 mg TAN/L , with a 95% confide nce interval of

0.1657

- 1.208 mg TAN/L . Normalized to pH 8.0 and 25°C, the EC20 was 0.5508 mg TAN/L

, with a 95% confidence i nterval of 0.2039

- 1.487 mg TAN/L (Table 6 ). In contrast, t he 2 8

- day

EC20 estimate without the four replicates from the s econd lowest treatment at test conditions (pH 8.15 and 24.7°C) was 0. 6841 mg TAN/L , with a 95% confidence interval of 0. 2017

- 2.320 mg TAN/L . Normalized to pH 8.0 an d 25°C, th is EC20 was 0. 8584 mg TAN/L , with a 95%

confidenc e interval of 0. 2531

- 2.911 mg TA N/L (Table 6 ).

T here were no readily apparent differences amongst treatment groups based on measurements of growth taken on surviving snails at the termination of the test on day 28 ( see Appendix 9), and therefore, growth data we re not used for 28

- day po int estimation.

TEST UNCERTAINTY

As is clear from above, a measure of uncertainty exists with this test owing to both the variable ammonia exposure s and the unequal response s observed within and among treatments

, such that the accuracy of the 28

- day EC 20 for the test is questionable . The following provides additional explanation r egarding interpretation of the se test results, as well as a recommendation on how these results might best be considered given this variability in exposures and responses

.11

Va riation in TAN concentrations and select water quality parameters

A s discussed in the preceding section

, much of the temporal variability in TAN conce ntrations may be attributable to the very high TAN concentrations measured on test day 1 and very low

TAN concentrations measured on test day 12. T here is no obvious explanation for the high and

low TAN measurements based upon review of test record s and observations, with the exception

of measured TAN stock concentrations . Meter calibration logs, recorded di luter flow rates measured in diluter cells upstream of the splitter boxes, ammonia stock preparation and procedure logs, etc., do not reveal any extraordinary circumstances over the particular timeframe in question (or throughout the entire 28

- day test per iod).

A general decline in stock TAN concentration may parti ally explain some of the temporal

variation in TAN c oncentration among treatments (see Table 4 and Appendix 6) . However

, records indicate that recipe and stock preparation procedures were impleme nted similarly throughout the exposure ; the exception being the small, likely inconsequential, addition of

NH 4 OH (approximately 200 ml per 4 L) used to raise the pH of the stock solution to near 8.0

. During the test, the measured TAN of freshly

- prepared s tock solution s declined over the first 12 days by approximately 30 percent ( 422 to 295 mg TAN/L ) , after which ammonia stock

concentrations increased to a maximum of 467 mg TAN/L on day 16 before stabilizing at a measured average concentration of 450 mg TAN /L for the remainder of the test ( N ote : fresh ammonia stock solutions were prepared daily or near daily from test days 21

- 28, but less were prepared every 2 to 5 days until then).

Corrective action to the problem of the declining (and variable) measured T AN concentrations among replicates and treatments included increased delivery system maintenance (e.g., scrubbing

of flow

- splitting boxes and adjustment and mai n te n ance of siphons to ensure equal flows of test

solution from flow

- splitting boxes), as well a s increased sampling for TAN measurement in every replicate chamber on every day over the final 10 days of the test. In addition, several duplicate samples were collected and sent to a third

- party contract laboratory ( Trimatrix ) for analytical determinati on of ammonia using the Phenate Method (see text and Appendix 7). Also, additional samples taken from select replicates within each treatment and on select days were

co llected and measured for TAN throughout the day (i.e., to assess diurnal variability

– note: as these measured values were found to be consistent, the values were averaged for the purpose of reporting).

In addition to the 30% decline in TAN stock concentrations over the first 12 days of the experiment

, snails withi n select replicates of all treatments did experience low water temperature, pH or dissolved oxygen (or any combination of the three) between test days 9 through 12 (see Fi gures 2

- 4 ). Of the se , only low dissolved oxygen could be consid ered to directly affect snail survival, but upo n further review of these data

, one cannot definitively

conclude that the drop in dissolved oxygen resulted in decreased snail survival . For example, dissolved oxygen concentrations decreased to as low as 2.1 and 1.9 mg/L over the day 9 to 12

timeframe in r eplicates 2 and 4 in the nominal 0.9 and 3.8 mg TAN/L treatments, respectively (see Figure 4)

, but these decreases did not result in subsequent decreases in snail survival (see Figure 8 and Appendix 8 )

.12

GLEC suspect s the drop in temperature, pH, and di ssolved oxygen between days 9

- 12, as well as some of the temporal variability in TAN concentrations between days 1

- 18, were the result of

blocked flow from the diluter splitter boxes to select replicates (via line plugging by algae and other biological deb ris )

. Observations by labo ratory technicians confirm such circumstance on

several occasions , although any observations of no or reduced flow to test chambers was immediately rectified upon recognition by increased attention and proper maintenance (e.g.,

s crubbing or physical manipulation to remove blockages/de bris or adjustment of siphons in flow

splitting boxes to re

- establish equal flow to all test chambers)

. Nevertheless, with such low flo w rates ( only 3.5 turnovers per day) restriction of flow for eve n a short period of time could lead to large reductions in temperature, pH, dissolved oxygen and TAN concentration via ambient heat

transfer (in the case of temperature) and bacterial and periphyton uptake and utilization (in the case of pH, dissolved oxyg en and TAN). Note : the amount of periphyton and biofilm on the sides of individual exposure cha mbers was moderate to substantial and largely permitted (i.e.,

individual test chambers were only scrubbed on a weekly as opposed to a daily basis)

, as such

mat erial proved a supplemental food source to the snails during the test and was assumed to positively contribute to their overall health

. This latter practice, in addition to the low turnover rates within each test chamber, was likely problematic as t he dil ution water used for testing was natural, unfiltered Lake Michigan water

, and the test temperature was a high 25 o C , thus promoting substantial periphyton growth within the test system.

Data interpretation

The variable TAN (and consequently, UIA) conce ntra tions and unequal survival respon se s among select replicates and treatments (see Figure s 5 , 6 and 7) , particularly in the lowe r test concentrations, cast s doubt on the reliability of associating effects with a particular "mean" TAN

concentration for this t est

. Temporal variability in TAN concentrations was the primary reason

GLEC calculated and reported cumulative daily average TAN concentration

, which more closely reflects the average concentration experienced by snails over the course of the entire 28

- da y exposure period ( data provided in Table 4). Despite the highly variable survival response s within the nominal 1.9 mg TAN/L treatment (0.81 mg TAN/L measured), Figure 9 a shows a relatively strong concentration

- response relationship after 28 days

. Howev er, because of uncertainty associated with both the rapid mortality in two replicates as well as the overall high

level inter

- replicate variability within this treatment, it was decided that a more conservative approach would be to exclude this treatment c ompletely from the analysis.

Nevertheless, g iven the above uncertainty , and given tha t the sample size was low for the test (N = 6 per replicate, 24 per treatment versus the ASTM recommended N=10 per replicate, 40 per

treatment )

, a 28

- day ammonia survival effects concentration is proposed in place of the

calculated EC20 values reported in Table 6 of this report. The value of 1.86 mg TAN/L ( 28

- day

measured cumulative average concentration of the nominal 3.8 mg TAN/L exposure treatment)

was selected based o n the strong negative effect of ammonia on snail survival (67% reduction or more) over time at this and all higher test concentrations. The 28

- day ammonia survival effects concentration, when normalized to 25 o C and pH 8 assuming an average exposure temper ature of

24.7 o C an d pH 8.15, is 2.28 mg TAN/L. This effects concentration should be considered a

maximum “unacceptable” 28

- day survival effect concentration for the species above which substantial mortality is expected

. Owing to the uncertainty in this v alue, however, it is not

recommended for FCV calculation as originally intended, but should instead be considered as13

contributing to the weight of evidence indicating the sensitivity of g ill

- bearing (non

- Pulmonate)

snails to prolonged ammonia exposure in g eneral

.

Finally, d ue to the low number of snails originally collected to support testing

, GLEC was not

able to repeat the test

. However, GLEC advises that this test should be repeated in order to better

define the 28

- day survival EC20 for the species.

SUMMARY AND CONCLUSIONS

A 28

- day ammonia toxicity test was completed with the non

- pulmonate snail species Pleurocera

canaliculata . Mature, mixed age snails were collected from the Ohio River and acclimated to test conditions for one month prior to testing . The 28

- day survival EC20 for this snail species

was determi ned to be as low as 0.5 5 mg TAN/L at pH 8 and 25 o C ; how ever, due to uncertainty

in the point estimate , a 28

- day ammonia survival effect concentration of <2.28 mg TAN/L representing a maximum “un acceptable” survival effect concentration is recommended instead.

A corresponding ACR for the test is calculated as the quotient of the normalized 96

- h r EC50

estimated from the acute range

- finding test (13.56 mg TAN/L) and the normalized 28

- day

ammonia survival effects concentration of <2.28 mg TAN/L, resulting in an ACR of >5.9

.

Based on these results, GLEC concludes that for P. canaliculata , the low 28

- d ammonia survival effects concentration of <2.28 mg TAN/L confirms that:

1) The sensitivity of mature , mixed age P. canaliculata to ammonia toxicity via a 28

- day test approaches th ose of their more specialized and/or Federally

- Listed counterparts (e.g., Idaho springsnail, Bliss Rapids and pebblesnails) ; and, 2) May also approach that of the highly ammonia

- se nsitive freshwater unionid mussel species: wavy

- rayed lamp mussel, Lampsilis fasciola ; fatmucket, Lampsilis siliquoidea ; and rainbow mussel, Villosa iris

– see Wang et al. (2007a, b) and Wang et al. (2008).

Additional 28

- day toxicity tests are recommended for this and other non

- pulmonate snails to further substantiate the preliminary findings obtained in this and other similar studies with freshwater gill

- bearing snails.14

REFERENCES

APHA. 1995. Standard Methods for the Examination of Water and Wastewater. 20th Edition.
American Society for Testing and Materials. 1995. Standard Test Methods for Measuring the
Toxicity of Sediment Associated Contaminants with Freshwater Invertebrates. E1706

- 95b. In
Annual Book of Standards. Vol. 11.05. Philadelphia, PA.

ASTM

. 2010a . Standard Guide for Conducting Early Life

- Stage Toxicity Tests with Fish.

E1241

- 05 . In: Annual Book of Sta ndards. Vol. 11.06. West Conshohocken , PA.

ASTM. 20 10b . Standard Guide for Conducting Laboratory Toxicity Tests with Freshwater

Mussels . E245 5

- 06. In Annual Book of ASTM Standard s. Vol. 1 1.06. West Conshohocken , PA.

Besser JM, Hardesty DL, Greer, IE, Ingersoll CG. 2009. Sensitivity of Freshwater Snails to
Aquatic Contaminants: Chronic Toxicity of Copper, Ammonia and Pentachlorophenol to
Endang ered Snail Species and Surrogates . Report submitted to USEPA Duluth, MN by USGS,
Columbia, MO (CERC

- 8335

- FY07

- 20

- 10).

Newman M C. 1995. Advances in Trace Substance Research. Quantitative Methods in Aquatic
Ecotoxicology. CRC Press, Inc., Boca Raton, FL. pp. 139

- 140.

United States Environmental Protection Agency. 1999. 1999 Update of ambient water quality

criteria for ammonia. EPA

- 822

- R

- 99

- 014. National Technical Information Service, Springfield,

VA.

Wang N, Ingersoll CG, Hardesty DK, Ivey CD, Kunz JL, May TW , Dwyer FJ, Roberts AD,
Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007a. Acute Toxicity of Copper, Ammonia,

and Chlorine to Glochidia and Juveniles of Freshwater Mussels (Unionidae). Environ

, Toxicol

. Chem

. 26:2036

- 2047.

Wang N, Ingersoll CG, Greer IE, Hardesty DK, Ivey CD, Kunz JL, Brumbaugh WG, Dwyer FJ,
Roberts AD, Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007b . Chronic T ox icity of
Copper and Ammonia to Juvenile Freshwater M ussels (Unionidae). Environ

. Toxicol

. Chem

. 26:2048

- 2056.

Wang N, Er ickson RJ, Ingersoll CG, Ivey CD, Brunson EL, Augspurger T, Barnhart MC. 2008.
Influence of pH on the Acute Toxicity of Ammonia to J uvenil e F reshwater M ussels (Fatmucket,
Lampsilis siliquoidea ). Environ

. Toxicol

. Chem

. 27:1141

- 1146.15

Table 1 . Water quali ty summary statistics for 96

- hr ammonia toxicity range

- finding test
with mature, mixed

- age P. canaliculata

.

Statistic Concentration

(mg TAN/L)
pH Temp (°C) D.O. (mg/L) Nominal Measured
Average Control 0.06 8.19 25.1 7.6

Std. Deviation n.a. a 0.06 0. 94 0.51

Minimum 0.06 8.11 23.7 6.8

Maximum 0.06 8.26 26.6 8.1

Average 3.0 2.62 8.25 24.7 7.5

Std. Deviation 1.64 0.08 1.0 0.46

Minimum 0.57 8.19 23.0 6.8

Maximum 4.10 8.47 26.3 8.1

Average 6.0 3.57 8.23 24 .7 7.3

Std. Deviation 1.41 0.05 1 .0 0.74

Minimum 2.05 8.15 23.6 6.4

Maximum 5.20 8.29 26.7 8.0

Average 12.0 7.64 8.19 24.3 7.3

Std. Deviation 2.04 0.07 0.87 0.34

Minimum 5.83 8.12 22.8 6.8

Maximum 10.2 8.30 25.3 7.7

Average 25.0 17.0 8.15 24.6 7.6

Std. Deviation 0.92 0.0 4 1.21 0.29

Minimum 15.9 8.10 23.0 7.1

Maximum 17.7 8.21 26.7 7.9

Average 50.0 32.8 8.07 24.7 7.5

Std. Deviation 3.52 0.06 0.96 0.66

Minimum 28.4 7.98 23.6 6.3

Maximum 36.1 8.17 26.4 8.0

a All measurements were below detection limits. Concen trations set to 0.06 mg/L based on

re

- analysis of archived samples using revised standard curve equation (see text in Results

and Discussion under 28

- Day Toxicity Test

– TAN Measurement and Concentrations ).16

Table 2

. Results of the 96

- h r range

- finding tes t with mature , mixed

- age P. canaliculata (N=6 snails per treatment).

Nominal Concentration (mg TAN/L) Measured Concentration (mg TAN/L) P. canaliculata

Survival (Number Alive out of 6)

Control 0.06 6

3 .0 2.6 2 6

6.0 3.57 5

12 7.64 4

25 17.0 1

50 32.8 0
96 h EC50 (95% CI) 9.057 (5.996

– 15.042) a

a Calcul a ted using EPA’s Probit Analysis Program (Version 1.5)

.17

Table 3 . Overall water quality summa ry statistics for 28

- day ammonia toxicity test with mature, mixed

- age P. canaliculata

. Values represent su mmary statistics derived using all measured values within a treatment (nominal) for a given parameter

.

Statistic pH Temp

(°C) D.O. (mg/L) Specific

Conductance (μS/cm)

Control Average 8.22 24.8 7.7 307

Minimum 7.86 21.7 5.6 274

Maximum 8.73 26.1 9 .0 324 0.90 mg TAN/L Average 8.17 24.7 7.3 322

Minimum 7.43 21.2 2.1 311

Maximum 8.7 26.1 8.9 340 1.9 mg TAN/L Average 8.14 24.6 7.1 330

Minimum 7.52 21.3 3.3 311

Maximum 8.64 26.2 10.1 354 3.8 mg TAN/L Average 8.13 24.7 7.1 342

Minimum 7.27 21. 8 1.9 317

Maximum 8.63 26.3 8.6 363 7.5 mg TAN/L Average 8.18 24.7 7.6 361

Minimum 7.8 21.6 6.0 337

Maximum 8.67 26.3 9.4 387 15 mg TAN /L Average 8.07 24.5 7.2 415

Minimum 7.43 21.7 3.3 399

Maximum 8.33 26.1 8.9 42818

Table 4

. Daily average measu red t otal ammonia concentrations (mg TAN/L ) by nominal treatment level.

Number in parentheses following the coefficient of variation (CV) is the number of available TAN

measurements at t hat concentration on that day, i.e., N = 1 to 4 based on number of re plicates sampled per treatment. A value of (0) indicates that the TAN concentration for that day was estimated by interpolating the average concentrations from the closest days for which

measurements were taken (see text).

Test
Day Daily Average Measured T AN Concentration (mg TAN/L)

Control Nominal 0.90 mg

TAN/L Nominal 1.9 mg

TAN/L Nominal 3.8 mg

TAN/L Nominal 7.5 mg

TAN/L Nominal 15 mg

TAN/L

TAN CV TAN CV TAN CV TAN CV TAN CV TAN CV

1 0.06 0.00(4) 1.36 0.10(4) 2.57 0.11(4) 3.94 0.04(4) 7.15 0.02(4) 1 0.93 0.01(4)

2 0.06 0.00(4) 0.39 0.29(4) 0.88 0.28(4) 2.33 0.20(4) 4.06 0.04(4) 10.55 0.05(4)

3 0.06 0.00(2) 0.06 0.00(2) 0.74 0.03(2) 1.74 0.16(3) 3.69 0.03(2) 8.85 0.04(2)

4 0.06 na(0) 0.12 na(0) 0.62 na(0) 1.52 na(0) 3.58 na(0) 8.99 na(0)

5 0.06 0.0 0(4) 0.19 0.16(4) 0.50 0.02(3) 1.31 0.12(4) 3.47 0.05(4) 9.14 0.03(4)

6 0.06 na(0) 0.16 na(0) 0.53 na(0) 1.18 na(0) 3.49 na(0) 8.88 na(0)

7 0.06 0.00(2) 0.13 0.21(2) 0.56 0.03(2) 1.04 0.43(2) 3.50 0.03(2) 8.62 0.04(2)

8 0.06 na(0) 0.18 na(0) 0.68 na(0) 1.28 na(0) 4.18 na(0) 9.22 na(0)

9 0.06 0.00(2) 0.22 0.77(2) 0.79 0.08(2) 1.52 0.74(2) 4.86 0.03(2) 9.82 0.10(2)

10 0.06 na(0) 0.17 na(0) 0.55 na(0) 1.11 na(0) 3.75 na(0) 9.94 na(0)

11 0.06 na(0) 0.11 na(0) 0.30 na(0) 0.70 na(0) 2.64 na(0) 10.06 na(0)

12 0.06 0.00(2) 0.06 0.00(2) 0.06 0.00(2) 0.29 0.08(2) 1.53 0.06(2) 10.18 0.12(2)

13 0.06 na(0) 0.09 na(0) 0.39 na(0) 0.81 na(0) 3.44 na(0) 12.48 na(0)

14 0.06 0.00(2) 0.13 0.09(2) 0.71 na(1) 1.34 0.18(2) 5.36 0.02(2) 14.79 na(1)

15 0.06 na(0) 0.18 na(0 ) 0.76 na(0) 1.80 na(0) 5.54 na(0) 16.82 na(0)

16 0.06 0.00(4) 0.23 0.79(4) 0.81 0.55(3) 2.27 0.22(4) 5.72 0.02(4) 18.84 na(1)

17 0.06 na(0) 0.38 na(0) 1.10 na(0) 2.61 na(0) 6.00 na(0) 17.82 na(0)

18 0.06 na(0) 0.54 na(0) 1.39 na(0) 2.95 na(0) 6.27 na(0 ) 16.80 na(0)

19 0.06 0.00(4) 0.69 0.39(4) 1.69 0.40(4) 3.29 0.19(4) 6.54 0.07(4) 15.77 0.07(4)

20 0.06 na(0) 0.30 0.92(4) 1.07 0.13(3) 1.69 na(0) 4.78 na(0) 14.80 na(0)

21 0.06 0.00(4) 0.30 0.55(4) 0.93 0.21(3) 2.17 0.07(3) 4.78 0.03(3) 13.83 na(0)

22 0.06 0.00(4) 0.52 0.61(4) 0.69 0.56(3) 2.14 0.09(3) 4.56 0.02(3) 12.86 na(0)

23 0.06 0.00(4) 0.49 0.05(4) 0.97 0.49(3) 2.17 0.37(3) 5.16 0.04(3) 11.89 na(1)

24 0.06 0.00(4) 0.50 0.04(4) 0.79 0.57(3) 2.35 0.03(3) 4.44 0.05(2) 10.01 na(1)

25 0.06 0.00(4) 0.55 0.09(4) 0.66 0.95(3) 2.32 0.00(3) 4.49 0.04(2) 11.35 na(1)

26 0.06 0.00(4) 0.48 0.13(4) 0.49 1.21(3) 1.99 0.01(3) 4.17 0.06(2) 10.06 na(1)

27 0.06 0.00(4) 0.48 0.10(4) 0.76 0.34(3) 2.18 0.02(3) 4.51 0.04(2) 12.17 na(1)

28 0.06 0.00(4) 0.56 0.23(4) 0.76 0.54(3) 2.04 0.07(4) 4.26 0.07(4) 10.04 0.01(4)

Treatment Mean Concentration (mg TAN/L) a

x  ̄ 0.06 0.34 0.81 1.86 4.50 11.98

Max 0.06 1.36 2.57 3.94 7.15 18.84

Min 0.06 0.06 0.06 0.29 1.53 8. 62

a Treatment mean concen trations represent the 28

- day average ( x  ̄ ) concentration. Maximum and minimum values represent the highe st and lowest daily average concentration for a particular treatment level. The corresponding raw data of measured TAN for all replicates w ithin treatments is provided in Appendix 6.19

Table 5. Daily average calculated unionized ammonia concentration (mg UIA /L) by nomina l treatment level. Number in parentheses following the coefficient of variation (CV ) is the number of available

measurements at that concentration on that day , i.e., N = 1 to 4 based on number of replicates sampled per treatment . A value of (0) indicates that the UIA concentration for that day was estimated by interpolating the average concentrations from the closest days for w hich measurements were taken (see text).

Test Day Daily Average UIA Concentration (mg NH 3 /L)

Control Nominal 0.90 mg

TAN/L Nominal 1.9 mg

TAN/L Nominal 3.8 mg

TAN/L Nominal 7.5 mg

TAN/L Nominal 15 mg

TAN/L

TAN CV TAN CV TAN CV TAN CV TAN CV TAN CV

1 0 .0029 na(1) 0.0683 na(1) 0.1564 na(1) 0.2026 0.06(2) 0.4363 na(1) 0.5536 na(1)

2 0.0064 0.21(4) 0.0268 0.26(4) 0.0539 0.19(4) 0.1207 0.37(4) 0.3380 0.10(4) 0.7566 0.10(4)

3 0.0046 0.09(2) 0.0047 0.01(2) 0.0477 0.70(2) 0.1288 0.50(3) 0.2522 0.06(2) 0.5220 0.07(2)

4 0.0049 na(0) 0.0112 na(0) 0.0441 na(0) 0.1133 na(0) 0.2692 na(0) 0.5739 na(0)

5 0.0053 0.071(4) 0.0176 0.168(4) 0.0405 0.16(3) 0.0978 0.41(4) 0.2861 0.11(4) 0.6258 0.09(4)

6 0.0050 na(0) 0.0131 na(0) 0.0427 na(0) 0.1069 na(0) 0.2813 na(0) 0.5 861 na(0)

7 0.0048 0.06(2) 0.0086 0.20(2) 0.0449 na(1) 0.1159 na(1) 0.2764 0.09(2) 0.5464 0.09(2)

8 0.0040 na(0) 0.0089 na(0) 0.0391 na(0) 0.0961 na(0) 0.2357 na(0) 0.4081 na(0)

9 0.0031 0.04(2) 0.0092 1.16(2) 0.0332 0.25(2) 0.0763 1.26(2) 0.1949 0.07(2 ) 0.2697 0.64(2)

10 0.0033 na(0) 0.0077 na(0) 0.0230 na(0) 0.0566 na(0) 0.1715 na(0) 0.4100 na(0)

11 0.0034 na(0) 0.0061 na(0) 0.0128 na(0) 0.0369 na(0) 0.1481 na(0) 0.5502 na(0)

12 0.0036 0.32(2) 0.0045 0.55(2) 0.0025 0.59(2) 0.0172 0.32(2) 0.1248 0.14 (2) 0.6904 0.04(2)

13 0.0060 na(0) 0.0103 na(0) 0.0202 na(0) 0.0597 na(0) 0.4211 na(0) 1.1456 na(0)

14 0.0084 0.05(2) 0.0161 0.07(2) 0.0378 na(0) 0.1021 0.14(2) 0.7174 0.24(2) 1.6007 na(1)

15 0.0077 na(0) 0.0178 na(0) 0.0554 na(0) 0.1413 na(0) 0.7037 na (0) 1.5463 na(0)

16 0.0070 0.05(4) 0.0196 1.03(4) 0.0731 0.78(3) 0.1805 0.59(4) 0.6899 0.36(4) 1.4920 na(1)

17 0.0065 na(0) 0.0329 na(0) 0.0874 na(0) 0.2221 na(0) 0.7079 na(0)

18 0.0061 na(0) 0.0462 na(0) 0.1017 na(0) 0.2638 na(0) 0.7259 na(0)

19 0 .0057 0.14(4) 0.0595 0.50(4) 0.1160 0.46(3) 0.3054 0.19(4) 0.7439 0.52(4)

20 0.0053 na(1) 0.0305 0.96(4) 0.1185 0.20(3) 0.1340 na(1) 0.6541 na(1)

21 0.0061 0.19(4) 0.0308 0.63(4) 0.0926 0.27(3) 0.2432 0.11(3) 0.4529 0.16(3)

22 0.0071 0.10(4) 0.053 4 0.65(4) 0.0660 0.64(3) 0.1944 0.29(3) 0.4511 0.20(3)

23 0.0162 0.06(4) 0.1080 0.35(4) 0.2208 0.48(3) 0.4590 0.59(3) 1.0445 0.29(3)

24 0.0061 0.06(4) 0.0473 0.06(4) 0.0676 0.76(3) 0.2250 0.19(3) 0.4247 0.08(2)

25 0.0075 0.04(4) 0.0655 0.10(4) 0.0 734 1.08(3) 0.2417 0.20(3) 0.3641 0.15(2)

26 0.0066 0.10(4) 0.0510 0.11(4) 0.0502 1.25(3) 0.2269 0.03(3) 0.2259 0.27(2)

27 0.0080 0.19(4) 0.0510 0.12(4) 0.0924 0.39(3) 0.2779 0.06(3) 0.4081 0.33(2)

28 0.0070 0.26(4) 0.0568 0.33(4) 0.0621 0.77(4) 0 .2547 0.05(4) 0.2289 0.70(4)

Treatment Mean Concentration (mg UIA/L) a

x  ̄ 0.0060 0.03156 0.06701 0.1679 0.4278 0.7673

Max 0.0162 0.1080 0.2208 0.4590 1.0445 1.6007

Min 0.0029 0.0045 0.0025 0.0172 0.1248 0.2697

a Treatmen t mean concentrations represent the 28

- day average ( x  ̄ ) concentration. Maximum and minimum values represent the hi ghest and lowest daily average concentration for a particular treatment level. The corres ponding raw data used to calculate UIA c oncentrations for all replicates within tr eatments is provided in Appendices 5 (pH and temperature) and 6 (TAN concentrations), where UIA is calculated using the equations provided in the 1999 ambient water quality criteria document for ammonia

- freshwater (EPA 1999).20

Table 6 . Concentration

- response results for P. canaliculata 28 day survival in response to

TAN , with and without replicates from nominal 1.9 mg TAN/L treatment

. Toxicity predictions obtained from a 3

- parameter logistic model using the EPA

T RAP (v. 1.21) analysis program.

28 Day Dose

- Response Results (all replicates every treatment)

Model Fit

Parameter Estimate Standard Error 95% LCL 95% UCL Log(EC 50 )

- 0.06

- 0.06 0.11

- 0.30 Slope 1.19 1.19 0.53 0.10

Y

- Intercept 0.91 0.91 0.11 0.68

Effec t Concentrations (EC)

EC EC

x 95% LCL 95% UCL

50 0.8750 0.5061 1.513

20 0.4475 0.1657 1.208

10 0.3023 0.0808 1.131

5 0.2106 0.0411 1.078 Normalized EC20 (pH=8, 25°C)

EC EC

x 95% LCL 95% UCL

20 0.5508 0.2039 1.487

28 Day Dose

- Response Results (minus replicates from nominal 1.9 mg TAN/L treatment)

Model Fit

Parameter Estimate Standard Error 95% LCL 95% UCL

Log(EC 50 ) 0.09 42 0.12995

- 0.17996 0.36838

Slope 1.3377 0.69655

- 0.13189 2.8073

Y

- Intercept 0.89187 0.07 06 0.74297 1.0408

Effect Concen trations (EC)

EC EC

x 95% LCL 95% UCL

50 1.2423 0.6608 2.336

20 0.6841 0.2017 2.320

10 0.4826 0.0984 2.367

5 0.3499 0.0505 2.423 Normalized EC20 (pH=8, 25°C)

EC EC

x 95% LCL 95% UCL

20 0.8584 0.2531 2.91121

Figure 1

. TRAP logistic equation re sults of 9 6

- h r range

- finding test at pH 8.19 and 24.7°C. Ninety

- six hour EC50 normalized to p H 8.0 and 25°C is 13.56 (9.91

- 18.53) mg TAN/L. TAN concentration represents average concentration calculated from the measurement of samples taken over the 96

- hr test period . Statistics below figure represent output from TRAP where “initial” represents the input valu e required as a first guess to generate a model and where “final” represents actual statistic given by the fitted model equation

.22

Figure 2

. Graphical display of the variability in measured water temperatures over the 28

- day test period amongst replicate chambers within the various test treatments ( based on nominal TAN concentrations ).23

Figure 3

. Graphical display of the variability in meas ured pH of test water over the 28

- day test period amongst replicate chambers within the various test treatments ( based

on nominal TAN concentrations)24

Figure 4

. Graphical display of the variability in measured dissolved oxygen concentrations

over the 2 8

- day test period amongst replicate chambers within the various test

treatments ( based on nominal TAN concentrations)

.25

Figure 5 . Graphical display of the variability in measured TAN concentrations over the 28

- day test period amongst replicate chambers with in the various test treatments ( based on nominal TAN concentrations). Line represents the daily average TAN from all replicates within a treatment when measured

, and int erpolation between

the daily average concentration s when measurements were not mad e.26

Figure 6. Graphical display of the variability in calculated unionized ammonia (UIA)

concentrations over the 28

- day test period amongst replicate chambers within the various test treatments ( based on nominal TAN concentrations). Line represents

the d aily average UIA from all replicates within a treatment when measured

, and interpolation between the daily average concentrations when measurements were

not made.27

Figure 7 . Twenty eight day survival of P. canaliculata across an ammonia concentration gr adient with replicate values averaged. P. canaliculata in the control treatment were maintained for an additional 14 days after test completion to

further assess controls

.28

Figure 8. Twenty

- eight day survival of P. canaliculata across an ammonia concen tration gradient by replicate chamber. Note the anomalous early mortality in replicates 3

and 4 of the nominal 1.9 mg TAN/L treatment

.29

Figure 9

. Graphical display of concentration

- response results for P. canaliculata after 28 days in response to TAN

, (a) all replicates from all treatments included

, and (b) minus the four replicates from the second lowest (nom inal 1.9 mg TAN/L)

treatment . TRAP logistic equation results are as those presented in Table 5.30

APPENDIX 1

. Justif

ication for selection of test species and species distribution.

GLEC’s test species selection process utilized a “tiered” approach to identify possible candidates with the widest distribution range in the continental United States and with habitat that wo uld most likely receive discharge from municipal wastewater treatment plants, i.e. , both small and large rivers. Step one in the screening process was to identify families of snail species that were

non

- pulmonates (gill

- bearing species; formerly referred to as prosobranchs). A USEPA document: USEPA. 1982. Freshwater snails (mollusca: gatropoda) of North America. EPA

600/3

- 82

- 026, was used as the primary tool for identifying ten families of non

- pulmonates in North America representing 49 genera and 349 spe cies. This identification guide was used to eliminate those families which were amphibious breathers (Ampullariidae), were not freshwater species (Neritinidae), had highly

- specialized habitats requirements (Micromelaniidae, cave

dwelling) or were not wide ly distributed (Pomatiopsidae and Thiaridae). From the initial list of ten families, the EPA document was utilized a third time to identify the genera within those remaining families that had large species distributions. As a result of this third tier sc reen
, 20 genera were identified as worthy to pursue given the requirements of the project.

The 20 genera identified were entered into the NatureServe Explorer website (http://www.natureserve.org/explorer/servlet/NatureServe?init=Species). NatureServe Exp lorer is a source for information of over 70,000 plants and animals of the United States and Canada. When the genera were entered, only the top three species within each genus in terms of the greatest geographical distribution (most states and/or area) we re noted to create a list of possible test species. This list did not include any invasive species or species that were listed as critically endangered or threatened. From this fourth screen 10

- 15 species were identified that could be

used for ammonia to xicity testing.

GLEC staff then contacted experts who could help in the process of choosing from the list the best test species with known distributions in large rivers that were not highly specialized, and were readily available for collection and subseq uent testing. Dr. Paul Johnson, from the Alabama Aquatic Biodiversity Center was fundamental in identifying species from the list that were relatively ubiquitous and were expected in large rivers. He also provided assistance with

his extensive snail cult uring experience to help eliminate those species that would prove too difficult to handle for testing. Dr. Thomas Watters from the Ohio State University’s Division of Mollusks in the Department of Evolution, Ecology and Organismal Biology provided assista nce with identifying species that were present in Ohio and available for collection. Dr. Watters also provided GLEC staff with contact information for additional experts who knew of specific locations of candidate species. In addition

, he allowed us to o bserve and identify juvenile specimens in his culture tank of Pleurocera canaliculata , providing ease of identification in the

field. Based on Dr. Watter’s and Johnson’s recommendations

, three species of snails were identified for testing. Patricia Morri son of the U.S. Fish and Wildlife Service, Ohio River Islands National Wildlife Refuge provided assistance in identify locations along the Ohio River where two of the three species ( Birgella s ubglobosus and P. canaliculata and) could be collected

for testi ng. The third species Campeloma decisum is also known to inhabit large rivers and can be found in large numbers in the Cuyaho ga River, according to Dr. Martin Huehner, EnviroScience, Inc. Another benefit of the three test species selected is that they re present three of the most widely

- distributed families of non

- pulmonate snail species in the U.S. B.31

subglobosus, P. canaliculata , and C. decisum are from the families Hydrobiidae, Pleuroceridae, and Viviparidae, respectively.

Maps showing the distributio n of the selected snail test species (depicted as a pattern dots) overlaid on a base layer that is a gradient scale of the number of snail families (non

- pulmonates) present in each state are provided for reference (attached separately below in this Appendi x for convenience). All data are from NatureServe Explorer.32

Map 1 . Distribution of globe siltsnail ( B. subglobosus ) within the conterminous U.S.33

Map 2 . Distribution of silty hornsnail ( P. canaliculata ) within the conterminous U.S.34

Map 3 . Distrib ution of pointed campeloma ( C. decisum ) within the conterminous U.S.35

APPENDIX 2

. Summary of acquisition, acclimation, maintenance, and health of test snail

species.

Birgella subglobo sus (globe siltsnail)

General Species In formation: The genus Birgella currently contains a single widespread species (Freshwater Mollusk Conservation Society, 2004) that has a wide distribution across the United States (see Map 1, Appendix 1). It has been recorded from Alabama, Arkansas, Iowa, K entucky, Michigan, Missouri, and New York, and is found in large streams, rivers and lakes.

The globe siltsnail is part of the larger Family Hydrobiidae which is composed of over 500 genera and several thousand species (largely freshwater) worldwide. The fauna of the United States and Canada from this large taxonomic group is among the most diverse of the world, with 49 genera and 500 plus species (350 currently named). The most widespread genera are Amnicola, Birgella, Cincinnatia, Probythinella, Pyrgulo psis, Somatogyrus and Tryonia . The largest genus is Pyrgulopsis with over 140 known species, the vast majority of which are described as being restricted to springs and small streams of the arid western areas of the country (Freshwater Mollusk Conservation Society, 2004).

The shell morphology varies widely from flattened planispiral shaped to conic to elongated. The size varies from about a 1 mm to almost 10 mm in height. Anatomically the group is very diverse with most generic and species designations pri marily determined by anatomical characters, particularly penial morphology. The group feeds on detritus and bacterial/algal films and can inhabit slow to fast moving water. All the North American taxa have separate sexes and lay single egg capsules, howeve r the introduced species, Potamopyrgus antipodarum is parthenogenetic, which partially accounts for its success as an invasive species (Freshwater Mollusk Conservation Society, 2004). Acquisition: Approximately 300 mature snails were collected August 11, 2010 along the Ohio River at Belleville, Wood County, West Virginia (Latitude: 39 o 08’21.03”; Longitude: 81 o 44’25.63”, elevation 595 feet). Snails were collected by hand using mask and snorkel. Snails were bagged (approximately 50 per bag) in site water s uper

- saturated with pure oxygen, and subsequently shipped (styrofoam insulated boxes cooled with ice) overnight to the laboratory in Traverse City, Michigan. Temperature of Ohio River water upon collection was approximately 24 o C. Water quality conditions of the bagged site water (with snails) measured upon receipt at the laboratory

were: Specific conductivity

– 501 μ mhos/cm Dissolved oxygen

– 20 mg/L pH

– 7.25 Water temperature

– 23.4 o C Alkalinity

– 74 mg CaCO 3 /L

Hardness

– 168 mg CaCO 3 /L Holding and Acc limation Process: Upon receipt at the laboratory, snails were immediately transferred to a dedicated on

- site36

holding/test facility and released into a 20 gallon tank filled with the Ohio River water (gently aerated) in which they were shipped. The snails were initially allowed to feed ad libitum only on the periphyton colonizing the sides of the holding tank, after which (beginning August 17, 2010) they were fed nutrients and PVC tiles covered with periphyton from in

- house fathead minnow

culture tanks.

One day after arrival at the laboratory (on August 13, 2010), approximately 25 percent of the Ohio River water was replaced with Lake Michigan acclimation/test dilution water. Snails were maintained for two days under these conditions, and on August 16, 2 010, another 25 percent of holding water was replaced with acclimation/test dilution water (Lake Michigan water) resulting in the following conditions: Specific conductivity

– 468 μ mhos/cm Dissolved oxygen

– 7.7 mg/L pH

– 7.80 Water temperature

– 24.1 o C

Al kalinity

– approximately 124 mg CaCO 3 /L

Hardness

– 168 mg CaCO 3 /L

Five days after receipt at the laboratory (on August 17, 2010), and periodically thereafter, the snails received supplemental nutrients via the addition of a natural organic matter slurry mix from an existing in

- house fathead minnow culture fed frozen brine shrimp . Ammonia measured in holding water was not detectable (<0.06 mg TAN/L) throughout acclimation and holding.

One week after receipt (August 18, 2010), the snail holding tank began receiving flowing Lake Michigan test dilution water at 160 mls/min.

Maintenance and Health: Snails were allowed to graze on algae

- laden tiles collected from the in

- house fathead minnow

culture tanks ad libitum , and periodically received supplemental nutr ients (natural organic matter slurry mix) as described above. Water quality during holding consisted of the following ranges:

DO

– 7.4 to 7.8 mg/L pH

– 8.13

- 8.42 Water temperature

– 23.2 to25.1 o C

Approximately 2 weeks after receipt (August 26, 2010), 13 0 dead snails were removed from the tank leaving only 167 survivors. Excessive mortality continued through August 30, 2010 when plans for testing this particular snail species were aborted after further consultation with EPA.

Other Information:

None37

Pleurocera canaliculata (silty hornsnail)

General Species Information: The silty hornsnail (Family Pleuroceridae) is also relatively widely distributed across the United States (see Map 2, Appendix 1). It has been recorded in the Mississippi River and Gre at Lakes drainages (including into Ontario and British Columbia, Canada), in the Hudson River basin, and in the Gulf of Mexico drainages. It prefers slow current areas of streams and rivers near the bank where it often leaves trails in the substrate (Fresh water Mollusk Conservation Society, 2004).

The shells of this species are narrowly to elongately conic or cylindrical. Aperture is rectangular, with a short anterior canal, 20

- 40 mm in height. Shell sculpture varies from smooth to with spiral ribs, lirate s or malleations and occasionally short blunt spines.

Pleurocerids in general are all dioecious (separate sexes), the males lack penises, and the females

possess an egg

- laying pore or groove on the right side of the foot. Little is currently known about

t he ecology of this family of snails. Snails of the genus Pleurocera prefer the silty, low

- flow areas along the bank of streams, while Elimia and Leptoxis are commonly found in fast

- moving

waters in the mid

- channel, attached to or underneath rocks. Pleuroce rids are primarily algal grazers. They often occur in large numbers, frequently far outnumbering species of other gastropod families in freshwater streams. Elimia can be particularly abundant in some locales, often numbering hundreds per square meter. Pleu rocerid adults have thick shells, making them less susceptible to predation than thinner

- shelled gastropods like physids (Freshwater Mollusk Conservation Society, 2004). Acquisition: Approximately 280 snails (238 large and 46 smaller snails) were collecte d August 11, 2010 along the Ohio River at Belleville, Wood County, West Virginia (Latitude: 39 o 08’21.03”; Longitude: 81 o 44’25.63”, elevation 595 feet). Snails were collected by hand using mask and snorkel. Snails were bagged (approximately 50 per bag) in site water super

- saturated with pure oxygen, and subsequently shipped (styrofoam insulated boxes cooled with ice) overnight to the laboratory in Traverse City, Michigan. Temperature of Ohio River water upon collection was approximately 24 o C. Water quali ty conditions of the bagged site water (with snails) measured upon receipt at the laboratory were: Specific conductivity

– 504 μ mhos/cm Dissolved oxygen

– 13.4 mg/L pH

– 7.09 Water temperature

– 24.3 o C Alkalinity

– 74 mg CaCO 3 /L

Hardness

– 176 mg CaCO 3 /L Holding and Acclimation Process: Upon receipt at the laboratory (August 12, 2010), all snails were immediately transferred to a

dedicated on

- site holding/test facility and released into a 20 gallon tank (large snails) or 500 ml beaker (small snails) filled with the Ohio River water (gently aerated) in which they were shipped. As above, all snails were initially allowed to feed ad libitum only on the periphyton colonizing the sides of the holding tank or beaker, after which (beginning August 17, 2010) they were fed PVC tiles covered with periphyton from in

- house fathead minnow culture tanks. Note:38

as many as 40 large snails were placed (upon arrival) in a separate large beaker and fed an algae and commercial fish flake food slurry as a small feeding trial.

One day after receipt at the laboratory (August 13, 2010), approximately 25 percent of the Ohio River water was replaced with Lake Michigan acclimation/test dilution water. Snails were maintained two days under these conditions, and on August 16, 2010, another 25 percent of holding water was replaced with acclimation/test dilution water resulting in the following

conditions: Specific conductivity

– 471 μ mhos/cm Dissolved oxygen

– 6.2 mg/L pH

– 7.29 Water temperature

– 24.2 o C Alkalinity

– approximately 12 4 mg CaCO 3 /L

Hardness

– 156 mg CaCO 3 /L

Five days after receipt at the laboratory (August 17, 2010) and periodically thereafter the snails received supplemental nutrients via the addition of a natural organic matter slurry mix from an existing in

- house fat head minnow culture and PVC tiles coated with periphyton. Ammonia measured in holding water was not detectable (<0.06 mg TAN/L) throughout acclimation and holding.

One week after receipt (August 18 th , 2010), the snail holding tank began receiving flowing Lake Michigan test dilution water at 160 mls/min (after 25% of water was replaced with L MI water 3 times).

Maintenance and Health: Snails were allowed to graze on algae

- laden tiles collected from the in

- house fathead minnow

culture tanks ad libitum , and periodically received supplemental nutrients (natural organic matter slurry mix) as described above. Water quality during holding consisted of the following ranges:

DO

– 7.4 to 8.3 mg/L pH

– 8.13

- 8.37 Water temperature

– 23.3 to 26.2 o C

By August 26, 20 10 (approximately 2 weeks after receipt), t here were a total of 236 large and 45 smaller (presumed juvenile) snails remaining in the holding tank (<0.1 mortality). By September 14, 2010 (p rior to initiation of the range

- finding test with this species), th ere were 205 large and 45 small snails remaining. Survival of large sna ils immediately prior to 28

- day toxicity testing (or after approximately 6 weeks holding) was 80 percent (98 percent for small snails).

Other Information:

A 96

- h r range

- finding test w as initiated on September 14, 2010 with large and small P.

canaliculata . The range

- finding test included a sample size of three snails tested in duplicate chambers (N= 6 snails total of each size) at each of five concentrations with a control. The

average measured ammonia concentrations (N=3) for the test were 2.6, 3.6, 7.6, 17.0, and 32.8 mg TAN/L, respectively. The resulting 96

- hour EC50 was 9.1 mg TAN/L with 95 percent confidence intervals ranging from 6.0 to 15.0 mg TAN/L for the large P. canaliculata (see Table 2 of this report), and 7.9 mg TAN/L with 95 percent confidence intervals ranging from 4.8 to 13.4 mg TAN/L for the small snails. The average test pH was 8.18. The average test temperature39

was 24.8 o C.

From these preliminary results, it appears that smaller, possibly juvenile, P. canaliculata are at least as acutely sensitive to ammonia as larger, presumably more mature snails.40

Campeloma decisum (pointed campeloma)

General Species Information: Combined with all its probable synonyms, Campelo ma decisum is widespread throughout eastern North America, ranging from well into Canada and down to Florida (see Map 3, Appendix 1). This snail species prefers the sandy bottoms of rivers where the current is sufficient to oxygenate the water column (Fres hwater Gastropods of North America

at: http://spinner.cofc.edu/~fwgna/species/viviparidae/c_decisum.html?referrer=webcluster&

, accessed January 2011).

The burrowing habit and peculiar radular morphology displayed by these snails imply an ability to filter feed, as has been documented for other snails in the Family Viviparidae, but the snails are assumed to have retained the ability to graze or harvest deposi ts on soft sediments as well. This species has also been observed feeding directly on carrion (Dr. Martin Huehner, EnviroScience, Inc., personal communication).

Campeloma are ovoviviparous brooders where parthenogenesis has evolved. Some populations

of C ampeloma appear comprised entirely of parthenogenic females, others appear to reproduce entirely by outcrossing, and some populations display a mixture of the two modes. Only one year is required for maturation in some populations, two in others, and both semelparous and iteroparous reproduction have been reported (see references associated with weblink provided above). Acquisition: Approximately 400 snails were collected from the Upper Cuyahoga River via subcontract with

Dr. Martin Huehner of EnviroScien ce, Inc., on September 28, 2010. The undisclosed collection site has very clean water that arises from a large watershed managed by the City of Akron for drinking water. Snails were collected via scuba diving by sieving sediments. Captured animals

were mai ntained overnight in clean plastic buckets filled with continually aerated ambient river water before delivery to Great Lakes Environmental Center in Columbus, Ohio for overnight shipping to their laboratory in Traverse City, Michigan, as described above. Water quality conditions of the bagged site water (with snails) measured upon receipt at the laboratory were: Dissolved oxygen

– 11 mg/L Water temperature

– 16.4 to 17.8 o C Holding and Acclimation Process: On September 30, 2010, snails were transferred to the dedicated holding/test facility and released (N = 201 and 196 snails, respectively) into two 20 gallon tanks filled with the Upper Cuyahoga River water (gently aerated) in which they were shipped. The bottoms of the tanks were covered with approximate ly 1 inch of inert sand for burrowing and cover. Snails were fed a natural organic matter slurry mix from in

- house fish culture tanks as described above.

On October 1, 2010, approximately 25 percent of the upper Cuyahoga River water was replaced

with Lak e Michigan acclimation/test dilution water. Holding tank water temperature was 23 o C. The same regimen was implemented over the course of next two to four days until approximately one week after receipt, the snail holding tanks began receiving flowing Lak e Michigan test dilution water at approxi m ately 160 mls/min.41

Maintenance and Health: Snails were fed the natural organic matter slurry mix two to three times per week for the first two weeks. Survival was 96 percent until October 15, 2010, declining pro gressively thereafter to 69 percent by October 28 th . A one

- inch layer of natural Boardman River organic sediment was added to the inert sandy layer at this time to improve foraging and cover.

At this time, based upon consultation with an outside expert, diet was switched to a commercial spirulina

- based flake fish food, and, with EPA approval, GLEC initiated a 1°C per day temperature reduction in holding tanks from 25°C to 20°C

– the new target water temperature for the pending 28

- day toxicity test with th e species.

Under the new holding and feeding conditions, mortality was reduced to just a 20 percent loss of

animals from November 8 th until December 2 nd , 2010, when a 96

- h r range

- finding test was initiated (see below).

Plans for testing this particular snail species were subsequently aborted after further consultation with EPA based partially on only 67 percent survival of Campeloma by December 7, 2010.

Other Information:

A 96

- h r range

- finding test was initiated on December 2, 2010 with the snail speci es. From these preliminary results, it appears that this snail species ( C. decisum ) is at least as acutely sensitive to

ammonia as P. canaliculata

.42

APPENDIX 3

. C haracterization of dilution water from East Arm Grand Traverse B ay,

Lake Michigan a

.

Parameter Level of Detection (mg/L) 2/12/2010 Result (mg/L) 2/16/2010

Result (mg/L)

Total Aluminum 0.05 ND 0.05

Dissolved Aluminum 0.05 ND ND

Total Antimony 0.002 ND ND

Total Arsenic 0.002 0.003 ND

Total Barium 0.05 ND ND

Total Be ryllium 0.001 ND ND

Total Cadmium 0.001 ND ND

Total Calcium 0.1 42.6 32.8

Total Chromium 0.005 ND ND

Total Cobalt 0.02 ND ND

Total Copper 0.004 ND ND

Total Iron 0.05 ND ND

Total Lead 0.002 ND ND

Total Magnesium 5.0 13.7 12.3

Total Manganese 0.05 N D ND

Total Mercury 0.0005 ND ND

Total Nickel 0.05 ND ND

Total Potassium 0.1 1.42 1.33

Total Selenium 0.005 0.006 ND

Total Silver 0.001 ND ND

Total Sodium 0.5 8.83 7.75

Total Thallium 0.002 ND ND

Total Vanadium 0.004 ND ND

Total Zinc 0.05 ND ND

a Analysis of City Tapwater (from adjacent East Arm Grand Traverse Bay

- 02/11 ) indicated no detects for all carbamates, chlorinated acid herbicides and pesticides, and very low levels of only a few volatile organic compounds. Chloride and
sulfate concentrat ions were 7 and 20 mg/L, respectively, and nitrate and nitrite (as N) were below reporting limits of 0.4 and 0.05 mg/L, respectively.
ND = Below level of detection.43

APPENDIX 4

. Cross validation of archived samples using new st andard curve

.44

APPENDIX 5 A

. Water quality measurements Treatment A (control)

.45

APPENDIX 5 B. Water quality measurements Treatment B (nominal 0.9 mg TAN/L)

.46

APPENDIX 5 C. Water quality measurements Treatment c (nominal 1.9 mg TAN/L)

.47

APPENDIX 5 D. Water quality measurements Treatment D (nominal 3.8 mg TAN/L)

.48

APPENDIX 5 E. Water quality measurem ents Treatment E (nominal 7.5 mg TAN/L)

.49

APPENDIX 5 F. Water quality measurements Treatment F (nominal 15 mg TAN/L)

.50

APPENDIX 6

. TAN measurements

.51

APPENDIX 7

. Tr imatrix duplicate TAN analysis using Phenate Method.52

APPENDIX 8 A. P. canaliculata survival

– control treatment through treatment B (nominal 1.9 mg TAN/L).53

APPENDIX 8 B. P. canaliculata survival

– treatments C through E (nominal 3.8 through 15 mg TAN/L)

.54

APPENDIX 9

. P. canaliculata growth measurements.