Date: February 23, 2011

To: Lisa Foersom Huff, Toxicologist, U.S. Environmental Protection Agency, Office of Water

From: John Besser, Research Fish Biologist, USGS Columbia Environmental Research Center

Subject: Ammonia toxicity to pebblesnails (Fluminicola sp.)

Hi Lisa,

Per your request, I am attaching a preliminary data summary for 28- day water -only toxicity tests conducted in 2010 to evaluate the toxicity of ammonia to Fluminicola sp., a species of pebblesnail (Gastropoda: Hydrobiidae) that were collected from the Snake River in Idaho and cultured in our laboratory. We reported results of previous ammonia toxicity tests with this species in a recent report to USEPA [Besser, J.M., Hardesty, D.L., Greer, I.E., and Ingersoll, C.G., 2009, Sensi tivity of freshwater snails to aquatic contaminants: chronic toxicity of copper, ammonia and pentachlorophenol to endangered snail species and surrogates. Final Administrative Report CERC -8335- FY07 -20-10 submitted to U.S. Environmental Protection Agency., U.S. Geological Survey, Columbia MO]. Survival of pebblesnails was highly variable in these previous tests, which were conducted with a limited number of mixed- age individuals (juveniles and adults)

. The attached PDF file includes: (1) the work plan for the 2010 pebblesnail tests, including a table

summar izing test conditions (Table 1) ; (2) a table summarizing measurements of ammonia, pH, temperature, and other water quality parameters during the tests (Table 2); and (3) a table summarizing toxicity data and statistical analyses (Table 3).

Methods for the pebble snail te sts with ammonia conducted in 2010 (Table 1) were similar to those reported for tests with another laboratory-cultured Hydrobiid snail (formerly ‘Idaho springsnail’, Pyrgulopsis idahoensis; now Jackson Lake springsnail, P. robusta) by Besser et al. (2009). The 2010 t est was conducted with two size classes of juvenile pebblesnails: ‘small’ snails (reps 1 -4; 10 per rep) had mean shell length of 1.34 mm at the start of the study; and ‘large’ snails (reps 5-8; 10 per rep ) had mean starting shell length of 1.81 mm. Both size groups were exposed in the same flow -through exposure system with nominal total ammonia concentrations up to 8.0 mg N/L at 20 degrees C in ASTM hard reconstitu ted water . An automated pH controller adjusted the pH of incoming test water to 8.25. Ammonia concentrations, pH, and temperature were maintained close to target values during the test (Table 2).Endpoints of survival and growth (shell length) were determined after 28 days (Table 3)

. Survival of snails in the small size group was relatively low in the control group (75%) and in the lower ammonia treatments (60 -68%), but survival in the large s ize group was high (93-100%) in both controls and low ammon ia treatments . Toxicity data were converted to ranks and analyzed by one- way analysis of variance , with Dunnett’s test used for comparisons to controls. For both size groups, snail s urvival differed significantly among treatments and was significantly lower than controls in the two highest ammonia treatments (4.0 and 8.0 mg N/L nominal total ammonia). Snail shell length differed significantly among treatments for the small size group but not the large size group. No treatment means for shell length di ffered significantly from controls in either size group.

Attempts to model concentration

- response curves for survival using TRAP software were generally not informative , due to the lack of treatments with partial mortalities. However, the geometric mea n of the survival NOEC of 2.06 mg N/L total ammonia (0.105 mg N /L unionized ammonia) and L OEC of 3.67 mg N/L total ammonia (0.220 mg N/L unionized ammonia) can be used to estimate a survival chronic value of 2.75 mg N/L total ammonia (0.152 mg/N/L unionize d ammonia) . Th is chronic value is consistent with the total ammonia LC50 of 2.38 ug N/L for this species reported by Besser et al. (2009) . In contrast , the LC20 of 1.02 mg N/L estimated from the previous study was clearly in the no

- effect range of the 2010 studies.Toxicology Branch Study Outline USGS Columbia Environmental Research Center (CERC)

Study code: 10-20-10
Title: Chronic toxicity of ammonia to pebblesnails ( Fluminicola sp.)

CERC project number: 8335C2F Basis+ project number: 3 USGS program element : 31070 CERC proposal: 05-TOX-04 Lead Investigator: John Besser
Lead Technician: Rebecca Consbrock
Date: April 16, 2010 Reviewed by: Chris Ingersoll, Ning Wang, Dave Mount Background:
Previous studies (Besser et al. 2009) suggested that an undescribed species of pebblesnail, Fluminicola sp. (PS) from the Snake River of Idaho may be highly sensitive to toxicity of ammonia in 28-d water toxicity tests. This species was originally intended to serve as a surrogate for other Idaho snail species listed under the U.S. Endangered Species Act, including the Idaho springsnail (formerly Pyrgulopsis idahoensis ; now merged with the non-listed Jackson Lake springsnail, P. robusta ) and the Bliss Rapids snail, Taylorconcha serpenticola (USFWS 1995). Besser et al. (2009) reported an LC50 for total ammonia of 2.4 mg N.L for a mixed-aged group of PS, although this result had a wide 95% confidence interval (0.8-8.0 mg N/L) due to high variability in survival between replicates. This result has drawn broad interest because it could (if verified) influence the proposed revision of the national water quality criterion for ammonia (USEPA 2009). This objective of the current study is to conduct 28-d ammonia tests using laboratory-cultured PS with greater degree of replication and, if possible, with multiple age/size groups. For additional detail, see CERC Study 07-20-10. Additional background for this study is provided in the document ‘Scope of Work or Development of Methods for
1Propagation of ESA-listed and Surrogate Species of Freshwater Snails for Use in Toxicity Testing, an interagency agreement between USGS/CERC and the U.S. Environmental Protection Agency (dated May 12, 2008).

Methods:
Toxicity tests with PS will closely follow methods used for previous chronic ammonia toxicity studies with reported by (Besser et al. 2009). The first of these tests will be conducted with mixed-age juveniles (about 6 months old). If PS cultures continue to be productive, a second test will be conducted with uniform-aged neonates (4-8 weeks post-hatch). Tests will consist of five ammonia concentrations, plus a control, with four or more replicate test chambers for each exposure concentration and ten test organisms per replicate. The first four replicates will be stocked with smaller animals from the mixed-age test cohort, making an attempt to obtain uniform-sized animals across treatments and replicates. The remaining four replicates will be stocked with the largest animals available, in order to maximize size differences between reps 1­ 4 and 5-8. Archive samples will be collected from both size groups and preserved in formalin for length determinations. This experimental design will allow estimation of ammonia effect concentrations on each size group by analysis of concentration-response curves and by determination of statistical differences among treatments by analysis of variance.
Test conditions are summarized in Table 1. Tests will be conducted in proportional diluters with hard reconstituted water (160 to 180 mg/L hardness as CaCO 3 ; ASTM 2009) at 20°C. Tests will be conducted at a target pH of 8.2 by using pH-adjusted ammonia stock solutions (mixtures of ammonium chloride and ammonium hydroxide) and by adjusting pH of incoming dilution water using automated pH controllers (Wang et al. 1997). Snails will be fed a suspension of flake fish food (Tetra-Fin; 6 mg/m L). Total ammonia concentrations and pH will be monitored in all treatments at least weekly. Tests will last 28 days, with endpoints of survival (movement and/or opercular response) and growth (shell length and dry weight).

References American Society for Testing and Materials. 2009. Standard guide for conducting acute toxicity
2tests on test materials with fishes, macroinvertebrates, and amphibians. Standard E729-96 (2007). In Annual Book of ASTM Standards, Vol 11.06 West Conshohocken,, PA. Besser, J. M., et al. (2009). Sensitivity of freshwater snails to aquatic contaminants: chronic toxicity of copper, ammonia and pentachlorophenol to endangered snail species and surrogates. Final Administrative Report (CERC-8335-FY07-20-10) submitted to U.S. Environmental Protection Agency., U.S. Geological Survey, Columbia MO.
USEPA (2009). Draft 2009 Update Aquatic Life Ambient Water Quality Criteria for Ammonia – Freshwater, EPA-822-D-09-001. Washington DC, U.S. Environmental Protection Agency, Office of Water.
USFWS 1995. Snake River Aquatic Species Recovery Plan, U.S. Fish and Wildlife Service, Boise, Idaho.
Wang N., et al. (2007). "Chronic toxicity of coppe r and ammonia to juvenile freshwater mussels (Unionidae)." Environ Toxicol Chem 26:2048-2056.
Wang N, et al. 2008. Influence of pH on the acute toxicity of ammonia to juvenile freshwater mussels (Fatmucket, Lampsilis siliquoidea ). Environ Toxicol Chem 27:1141-1146.

3T able 1. Summary of test conditions for conducting a chronic water-only 28-d ammonia toxicity test with Pebblesnail ( Fluminicola sp.; Besser et al. 2009).

Parameter Conditions 1. Test organisms: Pebblesnail ( Fluminicola sp.) 2. Test type: Flow-through chronic water-only toxicity test

3. Test Duration: 28 d

4. Toxicant: Ammonia (mixture of ammonium chloride & ammonium hydroxide, adjusted to pH 8.2; Wang et al. 2008)
5. Dilution series Control and five ammonia concentrations in 50% dilution series: Maximum total ammonia concentration = 8 mg N/L 6. Temperature: 20  1  C 7. Lighting: Ambient laboratory light (about 200 lux, 16 hr light:8 hr dark)

8. Aeration: None

9. Feeding Flake fish food suspension (6 g/L), with the initial feeding rates: 0.5 mL/beaker/d for juveniles (feed neonates M-W-F only )

10. Test water: Hard reconstituted water (hardness 160-180 mg/L; ASTM 2009); pH of test water adjusted to pH of 8.2 (Wang et al. 2007, 2008) 11. Water replacement: 125 ml per chamber every 4 hr (3.8 volume-additions/day)

12. Test chamber: 300 mL screened beaker (200 mL water volume)
13. Age of organism: Test 1: juveniles (about 6 months old) Test 2: neonates (about 4-8 weeks old). 14. Organisms / chamber: 10

15. Chambers / treatment 4-8

16. Water quality: pH of incoming test water (daily); pH, temperature, dissolved oxygen, and pH in test beakers (at least weekly); alkalinity and hardness (biweekly). 17. Toxicant analyses Total ammonia by ion-selective electrode (at least weekly). 18. Endpoints: Survival, growth (shell length and dry weight), and biomass

4TABLE 2. AMMONIA CONCENTRATIONS AND WATER QUALITY PARAMETERS(USGS-CERC Study 10-20-10)Treatment (Total ammonia)Control0.5mg/L1.0mg/LVariableN Mean Min Max Mean Min Max Mean Min MaxTemperature (deg. C) 5 20.1 19.3 20.5 20.2 19.3 20.5 20.1 19.3 20.4Dissolved oxygen (mg/L) 5 8.0 7.1 8.7 8.0 7.5 8.7 7.9 7.4 8.7Conductivity (uS/cm) 5 608 599 619 608 601 617 615 605 622Alkalinity (mg/L)3 111 106 114 112 106 116 113 106 118Hardness (mg/L)3 173 168 180 178 170 190 176 172 180pH5-8 8.26 8.12 8.48 8.21 8.16 8.37 8.22 8.14 8.33Total ammonia (mg N/L) 10 0.12 0.09 0.19 0.57 0.43 0.73 0.97 0.75 1.28Unionized ammonia (mg N/L) 4-6 0.007 0.002 0.011 0.036 0.030 0.041 0.059 0.047 0.068Treatment (Total ammonia)2.0mg/L4.0mg/L8.0mg/LVariableN Mean Min Max Mean Min Max Mean Min MaxTemperature (deg. C) 5 20.1 19.3 20.4 20.1 19.4 20.5 20.2 19.5 20.5Dissolved oxygen (mg/L) 5 7.8 7.0 8.6 7.9 7.4 8.7 8.0 7.4 8.6Conductivity (uS/cm) 5 622 614 634 634 627 645 681 673 697Alkalinity (mg/L)3 113 108 116 115 110 120 115 108 120Hardness (mg/L)3 176 172 182 174 170 180 175 170 182pH5-8 8.18 8.08 8.32 8.20 8.15 8.33 8.25 8.13 8.41Total ammonia (mg N/L) 10 2.06 1.66 2.63 3.67 2.95 4.80 8.80 7.33 10.81Unionized ammonia (mg N/L) 4-6 0.105 0.055 0.138 0.220 0.175 0.247 0.486 0.183 0.671TABLE 3. TOXICITY MEANS AND RESULTS OF RANK ANOVA (USGS-CERC Study 10-20-10)

Small snails (Reps 1-4; mean starting shell length 1.3494)

Total Ammonia (mg N/L) Survival (of 10); p=0.005 Length (mm); p=0.003

Nominal Measured N Mean Std. Error N Mean Std. Error

Control 0.12 4 7.50 1.04 4 1.5618 0.0276 0.5 0.57 4 6.75 2.25 3 1.7729 0.0119 1.0 0.97 4 6.25 0.85 4 1.7137 0.0351 2.0 2.06 4 6.00 2.12 3 1.6118 0.0530 4.0 3.67 4 0.00 0.00 * 0

-- -- 8.0 8.80 4 0.00 0.00 * 0

-- --

Large snails (Reps 5-8; mean starting shell length 1.8075)

Total Ammonia (mg N/L) Survival (of 10); p<0.0001 Length (mm); p=0.18

Nominal Measured N Mean Std. Error N Mean Std. Error

Control 0.12 4 9.25 0.48 4 2.3572 0.0553 0.5 0.57 4 10.00 0.00 4 2.2923 0.0835 1.0 0.97 4 9.75 0.25 4 2.4014 0.0777 2.0 2.06 4 10.00 0.00 4 2.2679 0.0486 4.0 3.67 4 1.00 0.71 * 2 2.0588 0.0468 8.0 8.80 4 0.00 0.00 * 0

-- --

* Asterisk indicates mean significantly less than control (Duinnett's test).