Southeastern Naturalist 239 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 22001188 SOUTHEASTERN NATURALIST 1V7o(2l.) :1273,9 N–2o5. 62 Acute Toxicity of Chloride, Potassium, Nickel, and Zinc to Federally Threatened and Petitioned Mollusk Species Kesley J. Gibson1,3,*, Jonathan M. Miller1, Paul D. Johnson2, and Paul M. Stewart1 Abstract - Loss of freshwater mollusk populations nationally has prompted the use of these species in establishing USEPA water quality criteria (WQC). The objectives of this study were to determine the sensitivity (EC50) of 5 at-risk mollusk species endemic to the Mobile River Basin to chloride, potassium, nickel, and zinc. Villosa nebulosa (Alabama Rainbow) was the only species evaluated in the study with an EC50 value included under current WQC for chloride and nickel. All species in the current study were more sensitive to potassium than other mollusk species previously tested, although there is currently no established WQC for that contaminant. For zinc, all species but Leptoxis ampla (Round Rocksnail) had EC50 values included under existing criteria. Results suggest current WQC may be insufficient for basins containing localized endemic species in a relatively small geographical space, such as the Mobile River Basin. We urge broader testing of highly regionalized aquatic species to aid in establishing national WQC. Introduction About 72% of freshwater mussels and 74% of freshwater gastropods are considered species of conservation concern in North America, and many species continue to decline due to anthropogenic factors (Johnson et al. 2013, Williams et al. 2008). Freshwater mollusks are among the most threatened and sensitive aquatic organisms to environmental change, and gastropods, in particular, comprise a substantial percentage of freshwater biodiversity. Therefore, it is necessary to include freshwater mollusks as test organisms in toxicity testing (Farris and van Hassel 2007). Juvenile mussels were rarely considered in setting USEPA water quality criteria (WQC), but have recently been mentioned in toxicity reports as a result of their increased availability due to improvements in propagation and rearing methods (Augspurger 2013, Augspurger et al. 2009). Caenogastropods (gill-breathing) were rarely included in toxicity tests (e.g., Archambault et al. 2014, Johnson et al. 2013, Keller et al. 2007), but freshwater pulmonate gastropods (mantle-breathing) were used in establishing WQC (Besser et al. 2009). Traditionally, the mollusks evaluated in toxicity tests were commonly encountered species with wide distributions and not regionalized endemics, particularly among freshwater gastropods. However, many of these species are highly stenotypic and have been largely ignored in toxicity testing (Johnson et al. 2013, Ó Foighil et al. 2011). Molluscan diversity is regionally variable; many localized endemics are restricted to discrete river basins, 1Troy University, Department of Biological and Environmental Sciences, Troy, AL 36082. 2Alabama Aquatic Biodiversity Center, Marion, AL 36756. 3Current address - Harte Research Institute for Gulf of Mexico Studies, Texas A&M University-Corpus Christi, Corpus Christi, TX 78412. *Corresponding author - Kesley.Gibson@tamucc.edu. Manuscript Editor: Lance Williams Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 240 especially in the southeastern US, which has the greatest mollusk diversity globally (Johnson et al. 2013, Williams et al. 2008). The Mobile River Basin has one of the highest numbers of ecologically stenotypic aquatic fauna of any basin in the US, including 34 mussel and 105 gastropod species not found elsewhere (Neves et al. 1997, Ó Foighil et al. 2011, Williams et al. 2008). The loss of freshwater mollusk populations nationally, especially highly stenotypic species, has prompted the relatively recent use of these species in establishing (e.g., potassium) or updating outdated (e.g., chloride, nickel, and zinc) USEPA WQC. Chloride is a byproduct of oil and gas production, an ingredient in many products including some pesticides, and a common component of effluents released from wastewater treatment plants, urban run-off, and mining operations (Kelly et al. 2008, Soucek 2007). For example, industrial plants, such as the Olin Chlor-Alkali Products plant in McIntosh, AL (a superfund site), known for manufacturing chlorine and caustic soda (e.g., lye), used sodium chloride as an important intermediate chemical in various stages of the manufacturing processes (Quirindongo et al. 2006). Groundwater surrounding the plant was polluted by several contaminants, including chloride, that exceeded the maximum contaminant level (Alabama Department of Environmental Management 2003). Evaluation of this site focused solely on exposure risks for humans and were deemed reasonable; however, exposure risks of these pollutants to aquatic fauna were not addressed. In the northern US, salt used to prevent hazardous ice from accumulating on roads is carried into streams and detected at levels harmful to aquatic organisms up to 172 m from highways, indicating impacts are not localized adjacent to the road (Karraker et al. 2008, Kaushal et al. 2005). Current USEPA WQC for acute chloride exposure (published over 25 years ago) is 860,000 μg/L (USEPA 1988). No studies of mussels or caenogastropods were included in determining this limit (Augspurger 2013); however, 2 species of pulmonate gastropods were evaluated (Birge et al. 1985, Patrick et al. 1968). Like chloride, potassium is also commonly found in urban run-off, as well as agricultural fertilizer, livestock waste, and sewage leaks, where it enters aquatic systems (Romano and Zeng 2007). Over 90% of potassium produced in the US in 2005 was utilized by the fertilizer industry (Ober 2006). Potassium has been perceived to be of lower toxicity than other contaminants (e.g., nitrate or ammonia) (Romano and Zeng 2007). However, potassium was reported to be the most toxic cation when compared to calcium or sodium (Trama 1954); potassium is about 10 times as toxic as sodium (McKee and Wolf 1963). Imlay (1973) suggested that naturally high potassium concentrations decreased the diversity of mussel populations in the Missouri River Basin. Any river or stream with a potassium concentration of ≥7 mg/L lacked mussels, while mussels could be found in rivers with concentrations less than 4 mg/L (Imlay 1973). There is currently no USEPA WQC for potassium. Mollusks are sensitive to heavy metals, such as nickel and zinc, and are among the first aquatic invertebrates to be lost from streams following heavy metal contamination (Nebeker et al. 1986, Shuhaimi-Othman et al. 2012). In response to increased levels, mollusks can sequester heavy metals in their tissues (e.g., foot or mantle) and shells, but these levels can accumulate to toxic concentrations Southeastern Naturalist 241 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 (Oehlmann and Schulte-Oehlmann 2003, Richardson et al. 2001, Salánki et al. 2003). Nickel concentrations in many aquatic systems have nearly doubled every decade since 1930, and increased concentrations can interfere with detoxification processes in the liver, often by decreasing filtration rates (Sreedevi et al. 1992, Stuijfzand et al. 1995). Zinc, an essential metal used in many biological functions (i.e., DNA synthesis, gene expression, etc.), can have harmful effects (i.e., decreased filtration rates, gill damage, etc.) on mollusks in excess concentrations (Kraak et al. 1994, Reed-Judkins et al. 1997). Minear et al. (1981) reported that mean zinc concentration in wastewater treatment plant effluents in the US was 0.7 mg/L (min– max = 0.0001–28.7 mg/L), while the zinc concentration of stormwater runoff was 0.01–2.4 mg/L (Cole et al. 1984), both of which exceed levels harmful to aquatic life (Agency for Toxic Substances and Disease Registry 2005). Currently, the EPA has an established WQC for nickel of 470 μg/L and 120 μg/L for zinc, but criteria have not been updated since 1995 (USEPA 1995). No studies to develop USEPA WQC for these metals considered freshwater mussels and few included caenogastropods (Augspurger 2013, Besser et al. 2009). Our objectives for the current study were to (1) determine the sensitivity (EC50) of 5 Mobile River Basin mollusks to chloride, potassium, nickel, and zinc; (2) compare our results to current USEPA WQC for chloride, nickel, and zinc and to other previously published studies; and (3) provide data that may be useful in establishing future potassium WQC. Methods For this study, we employed 3 lotic freshwater mussel species (Hamiota perovalis (Cole) [Orangenacre Mucket], Villosa nebulosa (Conrad) [Alabama Rainbow], and Villosa umbrans (Lea) [Coosa Creekshell]) and 2 lotic caenogastropod species (Leptoxis ampla (Anthony) [Round Rocksnail] and Somatogyrus sp. [pebblesnail]) endemic to the Mobile River Basin in toxicity testing. Orangenacre Mucket and Round Rocksnail are federally listed as threatened under the Endangered Species Act (ESA; USFWS 1993, 1998). Alabama Rainbow and Coosa Creekshell have been formally petitioned for federal protection (Center for Biological Diversity 2010). Preliminary COI and 16S genetic analyses suggest Somatogyrus sp. cf. coosaensis (Coosa Pebblesnail) is likely an undescribed species and its status is currently under review (E.E. Strong, Smithsonian Institution, Department of Invertebrate Zoology, Washington, DC, and P.D. Johnson, unpubl. data). The Alabama Aquatic Biodiversity Center (AABC), Marion, AL, propagated and supplied mussels using host-fish infections and standard culturing methods (Barnhart 2006). The AABC also propagated Round Rocksnail, and we collected pebblesnails from the Cahaba River (32º57'35''N, 87º08'26''W). Juvenile mussels were 30–60 d post-transformation, and gastropods were 5–8 months post-hatch. Pebblesnails are annuals; juvenile gastropods hatch in April–May, and most adults die soon after the reproductive season has concluded (Johnson et al. 2013). We kept the organisms in a holding aquarium with reconstituted soft-dilution water prepared following ASTM (2007) guidelines and completed testing within Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 242 14 d of arrival (most within 7 d of arrival). The study organisms were not fed during the 96-h acute-toxicity tests (ASTM 2013, USGS 2004) and were acclimated a minimum of 24 h before trials by placing them in dilution water and adjusting the temperature no more than 3 °C/h until 25 °C was reached (ASTM 2013, Wang et al. 2007). The temperature difference between the culture water and dilution water was minimal (no more than 5 °C difference); thus, we expected the acclimation time to be sufficient. Experimental conditions We performed static-renewal acute-toxicity tests following the ASTM Standard Guide for Conducting Laboratory Toxicity Tests with Freshwater Mussels (E2455-06) (ASTM 2013). We made reconstituted soft-dilution water following ASTM (ASTM 2007) protocols, which include additions of sodium bicarbonate (NaHCO3), calcium sulfate (CaSO4), magnesium sulfate (MgSO4), and potassium chloride (KCl). We measured physiochemical variables for each batch of dilution water after aeration and prior to toxicity testing. Mean physiochemical variables of dilution water were as follows: pH = 7.3 (min–max = 7.1–7.4), hardness = 41 (min– max = 40–42) mg CaCO3/L, alkalinity = 33.5 (min–max = 33–34) mg CaCO3/L, and conductivity = 168 (min–max = 130–189) μΩ/cm. We used soft water in testing to mimic historic hardness values (1974–1987: 74 mg CaCO3/L [min–max = 6–194 mg CaCO3/L]; Pitt and Dee 2000) in the Cahaba River Basin, where both of the federally threatened species tested in the current study are endemic. We mixed toxicant solutions of NaCl (ACS grade, Lot #A2246-35, Carolina Biological Supply Company, Burlington, NC), KNO3 (ACS grade, Lot #AD-13298-40; Carolina Biological Supply Company), NiCl2 (Reagent grade, Lot #AD-13324-13; Carolina Biological Supply Company), and ZnSO4 (Reagent grade, Lot #AD-14021-15; Carolina Biological Supply Company) 1–2 hours prior to starting the trails (Wang et al. 2007). We tested 3 replicates of 10 individuals each in 300 mL of dilution water (controls) or toxicant solution in 600-mL Pyrex® beakers and refreshed the dilution water or toxicant solution after 48 h. For endpoint determination at the end of 96-h exposure, we placed mussels with closed valves under a microscope, looked for a heartbeat or foot movement, and, if we observed neither, considered them dead. We observed gastropods for movement for 5-min (Archambault et al. 2014, ASTM 2013) or conducted a “tickle” test, performed by touching the organisms with a soft pick to provoke a reaction. We used an eyelash stick to prevent any excess pressure being placed on the foot and causing a false reaction. We re-checked non-decaying individuals for survival after being placed in fresh dilution water for 30 min. Data and toxicant concentration analysis We determined the 96-h EC50 values for the 4 toxicants using ToxStat® 3.5 from West, Inc. (https://www.msu.edu/course/zol/868/) and the trimmed Spearman– Karber method (Hamilton et al. 1977). We used cessation of ventilation or general movement as the endpoint; thus, we calculated EC50 values instead of LC50 values. Accuracy of toxicant concentration was calculated using the exposureaccuracy formula: exposure accuracy = (Pm) / (Pt) * 100, where Pm is the measured Southeastern Naturalist 243 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 toxicant concentration (i.e., chloride, potassium, nickel, or zinc) and Pt is the target concentration (Archambault et al. 2014). We did not calculate accuracy for each concentration; rather we calculated accuracy for the highest toxicant concentration that could be measured using a Hach DR 2800 Spectrophotometer (DOC022.53.00725; Hach, Loveland, CO) following the chloride method 8113, potassium method 8049, nickel method 8150, or zinc method 8009. Therefore, EC50 values reported here are nominal concentrations. For tests with mussels, mean exposure accuracy was 98% (min–max = 88–107%) for chloride, 95% (min–max = 92–97%) for potassium, 114% (min–max = 108–120%) for nickel, and 89% (min–max = 84–94%) for zinc (Table 1). For tests with gastropods, mean exposure accuracy was 120% (min–max = 80–159%) for chloride, 127% (min–max = 120– 133%) for potassium, 95% (min–max =88–101%) for nickel, and 104% (min–max = 88–120%) for zinc. Median effective concentration comparisons We compared our calculated median effective concentrations (EC50) to current USEPA WQC for chloride, nickel, and zinc and other previously published studies used in determining USEPA WQC or that met the criteria for acceptable tests by ASTM 2013. We included only toxicity studies found in the peer-reviewed literature that met ASTM (2013) standards and were between 48-h and 96-h exposures to mollusks in comparison to our trails. These criteria greatly decreased the number Table 1. Exposure-accuracy calculations for chloride, potassium, nickel, and zinc for toxicants tested. Measured Target Exposure Toxicant Species concentration (μ/L) concentration (μ/L) accuracy (%) Chloride Alabama Rainbow 2,569,000 2,400,00 107 Orangenacre Mucket 440,000 500,000 88 Round Rocksnail 159,000 100,000 159 Pebblesnail 2,400,000 3,000,000 80 Potassium Alabama Rainbow 77,271 80,000 97 Orangenacre Mucket 924 1000 92 Round Rocksnail 1200 1000 120 Pebblesnail 4000 3000 133 Nickel Alabama Rainbow 1300 1200 108 Orangenacre Mucket 120 100 120 Round Rocksnail 88 100 88 Pebblesnail 162 160 101 Zinc Alabama Rainbow 5400 6400 84 Orangenacre Mucket 1130 1200 94 Round Rocksnail 140 160 88 Pebblesnail 120 100 120 Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 244 of studies available for comparison using glochidia because most test durations were only 24 h for glochidia. Macroinvertebrates and fish have been found to be more tolerant overall, so we excluded these taxa from our comparison. For a more exhaustive list of toxicity studies, see Gibson (2015). Results Mollusk acute toxicity Mussel survival in the control treatment was >95% for all tests conducted, suggesting acceptability for these tests per the ASTM guidelines (2013). Gastropod survival in the control treatment was 100% for all exposures; gastropods were active and observed scaling beaker walls in all controls. We observed deceased mussels gaping slightly with no heartbeat or foot movement, while living mussels were tightly closed with a heartbeat or foot movement observed. Gastropods that died during the exposure period were generally found clustered at the bottom of the beakers. Individuals observed in the bottom of the beakers often had no soft tissue remaining, but if tissues were present, they appeared “bloated” and protruding from the shell. Chloride. For Alabama Rainbow, the EC50 value was 1,538,452 μg/L (95% CI: 1,462,493–1,618,357 μg/L), but Orangenacre Mucket was more sensitive and had an EC50 value of 452,491 μg/L (95% CI: 379,524–539,487 μg/L) (Table 2). We observed Orangenacre Mucket widely gaping in the lowest concentrations of chloride and they showed no reaction when touched with an eyelash pick, indicating high levels of stress; however, we still observed a heartbeat in some speciemens. The EC50 value (including the 95%-confidence intervals) for Orangenacre Mucket was below the current USEPA WQC of 860,000 μg/L (USEPA 1988), while the EC50 value for Alabama Rainbow was above the WQC. Round Rocksnail had an EC50 value of 3414 μg/L (95% CI = 387–30,117 μg/L) and the pebblesnails had an EC50 value of 190,595 μg/L (95% CI = 136,515– 266,098 μg/L) for chloride. The wide confidence interval for the EC50 value for Round Rocksnail may be due to 50% death occurring in the lowest concentration (100 μg/L) and no complete kills in the highest concentration (500,000 μg/L). The trial could not be repeated because additional cultured test-organisms were unavailable. Both gastropod species had EC50 values below the current WQC. Potassium. Alabama Rainbow had an EC50 value of 15,966 μg/L (95% CI = 12,450–20,476 μg/L), while Orangenacre Mucket had an EC50 value of 11,938 μg/L (95% CI = 10,089–14,134 μg/L; Table 3). Mortality was never >50% in the Round Rocksnail trial; thus, we could not calculate an EC50 value for this study. At 100 μg/L, we classified 50% of the test organisms as dead at the end of the trial but only a third of the test organisms died at the highest concentration (1000 μg/L), so we estimated that the EC50 value for Round Rocksnail was above 1000 μg/L. We observed partial kills (≤33%) at all 5 concentrations. Unfortunately, we had insufficient numbers of cultured organisms available to conduct retests. The pebblesnails had an EC50 value of 7285 μg/L (95% CI = 5739–9245 μg/L) (Table 3), which is lower than either mussel species tested in the current study. We observed movement without “tickling” at most concentrations. Southeastern Naturalist 245 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 Nickel. The EC50 value for Alabama Rainbow was 510 μg/L (95% CI = 326–798 μg/L), which was higher than the EC50 value for Orangenacre Mucket (313 μg/L; 95% CI = 216–453 μg/L). The EC50 value for the pebblesnails was 301 μg/L (95% CI = 256–358 μg/L), similar to Orangenacre Mucket. Round Rocksnail had an EC50 value of 33 μg/L (95% CI = 12–88 μg/L), the lowest EC50 value of the organisms used in this study (Table 4). All EC50 values, including confidence intervals, were below the current WQC of 470 μg/L for nickel except for Alabama Rainbow, which had confidence intervals that encompassed the current WQC. Zinc. Coosa Creekshell had an EC50 value of 1302 μg/L (95% CI = 1068–1588 μg/L) for zinc—the highest observed, followed by Alabama Rainbow, with an EC50 Table 2. Median effective concentrations (EC50) for 96-h acute-toxicity tests of chloride on mollusk species. Studies included in comparisons were previously used in determining USEPA WQC or met the criteria for acceptable tests by ASTM 2013. To date, chlorine has been more commonly used in acute-toxicity testing on freshwater mollusks than chloride, but chloride is a commonly used anion in toxicity testing. We did not use chlorine for comparison in the current study because this is a compound that can stand alone and has different effects on organisms than chloride, which must be bonded to a cation (Das and Blanc 1993). Additionally, the USEPA differentiates between chlorine and chloride, and they have separate WQC (see http://www.epa.gov/wqc/national-recommendedwater- quality-criteria-aquatic-life-criteria-table). Age: A = adults, J = juveniles, NR = not reported. Hardness 96-h (mg/L EC50 Mollusk species Common name Age CaCO3) (μg/L) References Bivalves Lampsilis fasciola Wavyrayed J 170–192 3,980,000 Bringolf et al. 2007 Rafinesque Lampmussel Lampsilis siliquoidea Fatmucket J 170–192 4,560,000 Bringolf et al. 2007 (Barnes) Musculium transversum Long Fingernail Clam A 100 168,000 Anderson 1977 Say Musculium transversum Long Fingernail Clam A 263 254,000 Anderson 1977 Musculium transversum Long Fingernail Clam J 263 472,000 Anderson 1977 Musculium transversum Long Fingernail Clam J 243 907,000 Anderson 1977 Musculium transversum Long Fingernail Clam J 234 1,655,000 Anderson 1977 Musculium transversum Long Fingernail Clam J 48 1,930,000 USEPA 2010 Elliptio lanceolata Yellow Lance J 160–180 2,100,000 Augspurger et al. (Lea) 2014 Villosa constricta Notched Rainbow J 160–180 3,900,000‒ Augspurger et al. (Conrad) 4,100,000 2014 Villosa delumbis Eastern Creekshell J 170–192 5,230,000 Bringolf et al. 2007 Conrad Villosa iris Rainbow J 163 1,660,000 Pandolfo et al. 2012 Hamiota perovalis Orangenacre Mucket J 43 452,491 Current study Villosa nebulosa Alabama Rainbow J 43 1,538,452 Current study Gastropods Physa gyrina Say Tadpole Physa NR 100 2,540,000 Birge et al. 1985 Physa heterostropha Pewter Physa NR NR 451,000 Patrick et al. 1968 Leptoxis ampla Round Rocksnail J 43 3414 Current study Somatogyrus sp. Pebblesnail A 43 190,595 Current study Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 246 value of 436 μg/L (95% CI = 250–759 μg/L). The pebblesnails had an EC50 value of 329 μg/L (95% CI = 276–394 μg/L), and Round Rocksnail had the lowest EC50 value, at 67 μg/L (95% CI = 39–116 μg/L) (Table 5). Round Rocksnail was the only species tested that exhibited values below the current WQC for zinc (120 μg/L). Table 4. Median effective concentrations (EC50) for 96-h and 48-h acute-toxicity tests of nickel on mollusk species. Studies used for comparison were previously used in determining USEPA WQC or met the criteria for acceptable tests by ASTM 2013. A = 48-h LC50 value, * = pulmonate gastropod. Hardness 96-h (mg/L EC50 Mollusk species Common name Age CaCO3) (μg/L) References Bivalves Utterbackia imbecillis Paper Pondshell Juveniles 60 190 Keller and Zam 1991 Utterbackia imbecillis Paper Pondshell Juveniles 80 252 Keller and Zam 1991 Utterbackia imbecillis Paper Pondshell Juveniles 60 240A Keller and Zam 1991 Utterbackia imbecillis Paper Pondshell Juveniles 80 471A Keller and Zam 1991 Hamiota perovalis Orangenacre Mucket Juveniles 43 313 Current study Villosa nebulosa Alabama Rainbow Juveniles 43 510 Current study Gastropods Amnicola sp. Duskysnails Embryo 50 11,400 Rehwoldt et al. 1973 Amnicola sp. Duskysnails Adult 50 14,300 Rehwoldt et al. 1973 Lymnaea stagnalis (L.) Swamp Lymnaea Juveniles 100 ~900* Nebeker et al. 1986 Physa gyrina Tadpole Physa NR 26 239 Nebeker et al. 1986 Leptoxis ampla Round Rocksnail Juveniles 43 33 Current study Somatogyrus sp. Pebblesnail Adults 43 301 Current study Table 3. Median effective concentrations (EC50) for 96-h and 48-h acute toxicity tests of potassium on mollusk species. Studies used for comparison met the criteria for acceptable tests by ASTM 2013. NR = not reported, A = 48-h LC50 value, and ** = estimated value. Hardness 96-h (mg/L EC50 Mollusk species Common name Age CaCO3) (μg/L) References Bivalves Dreissena polymorpha Zebra Mussel Adults NR 150,000A Fisher et al. 1991 (Pallas) Dreissena polymorpha Zebra Mussel Adults 40 129,000– Waller et al. 1993 163,000A Obliquaria reflexa Threehorn Wartyback Adults 40 >2000A Waller et al. 1993 (Rafinesque) Villosa vibex Southern Rainbow Juveniles NR 24,000 Keller et al. 2007 Hamiota perovalis Orangenacre Mucket Juveniles 43 11,938 Current study Villosa nebulosa Alabama Rainbow Juveniles 43 15,966 Current study Gastropods Physa heterostropha Pewter Physa NR NR 940,000 OECD 2001 Leptoxis ampla Round Rocksnail Juveniles 43 >1000** Current study Somatogyrus sp. Pebblesnail Adults 43 7285 Current study Southeastern Naturalist 247 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 Discussion Chloride. Three of the 4 Mobile River Basin species tested in the current study displayed a lower EC50 value to chloride than previously tested freshwater mollusks (Table 2), suggesting these species are highly sensitive to chloride (Table 2). Of these species tested, only Alabama Rainbow had values above the current WQC of 860,000 μg/L. The pebblesnails had an EC50 value about 4.5 times lower than the current criteria, and Round Rocksnail was the most sensitive gastropod species, with an EC50 value about 250 times lower than the current criteria and lower than average background levels of chloride measured in all watersheds in Alabama except the Yellow River watershed (Table 6). Only 2 gastropods have been previously used in setting WQC for chloride and both were pulmonates (Physa spp.) with Table 5. Median effective concentrations (EC50) for 96-h and 48-h acute-toxicity tests of zinc on mollusk species. Studies used for comparison were previously used in determining USEPA WQC or met the criteria for acceptable tests by ASTM 2013. NR = not reported, A = 48-h LC50 value. Hardness 96-h (mg/L EC50 Mollusk species Common name Age CaCO3) (μg/L) References Bivalves Corbicula fluminea Asiatic Clam NR 64 6040 Cherry et al. 1980 (O.F. Müller) Actinonaias pectorosa Pheasantshell Juveniles 40 360–370A McCann 1993 Conrad Actinonaias pectorosa Pheasantshell Juveniles 160 1060–1186A McCann 1993 Actinonaias pectorosa Pheasantshell Glochidia 170 309A Cherry et al. 1991 Medionidus conradicus Cumberland Glochidia 170 570A Cherry et al. 1991 I. Lea Moccasinshell Ptychobranchus fasciolaris Kidneyshell Glochidia 170 212A Cherry et al. 1991 Rafinesque Utterbackia imbecillis Paper Pondshell Juveniles 60 268 Keller and Zam 1991 Utterbackia imbecillis Paper Pondshell Juveniles 80 438 Keller and Zam 1991 Utterbackia imbecillis Paper Pondshell Juveniles 60 355A KellerandZam1991 Utterbackia imbecillis Paper Pondshell Juveniles 80 588A KellerandZam1991 Villosa iris Rainbow Juveniles 50 339A McCann 1993 Villosa iris Rainbow Juveniles 160 1122A McCann 1993 Villosa nebulosa Alabama Rainbow Glochidia 170 656A Cherry et al. 1991 Villosa umbrans Coosa Creekshell Juveniles 43 1302 Current study Villosa nebulosa Alabama Rainbow Juveniles 43 436 Current study Gastropods Amnicola sp. Duskysnails Embryo 50 20,200 Rehwoldt et al. 1973 Amnicola sp. Duskysnails Adult 50 14,000 Rehwoldt et al. 1973 Helisoma campanulatum Ramshorn Snail Adult 20 870–1270 Wurtz 1962 Helisoma campanulatum Ramshorn Snail Adult 100 1270–3030 Wurtz 1962 Physa gyrina Tadpole Physa Adult 36 1274 Nebeker et al. 1986 Physa heterostropha Pewter Physa Juveniles 20 303–434 Wurtz 1962 Physa heterostropha Pewter Physa Juveniles 100 434–1390 Wurtz 1962 Leptoxis ampla Round Rocksnail Juveniles 43 67 Current study Somatogyrus sp. Pebblesnail Adults 43 329 Current study Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 248 Table 6. Average background levels for water physiochemical variables and toxicant levels in Alabama measured by the US Geological Survey (https:// www.waterqualitydata.us/portal/) from 2009–2014. Data is not available for all watersheds or toxicants. We omitted watersheds that are classified as solely brackish. However, those watersheds included for comparison may have some sampling stations that are brackish depending on water level. NR = not reported, A = single value was reported so no range can be calculated, and B = no mussels are found in this watershed due to the acidic conditions. Conductivity Hardness Alkalinity Chloride Nickel Zinc Watershed DO (mg/L) pH (umho/cm) (mg/L) (mg/L) (μg/L) (μg/L) (μg/L) Alabama River 7.15 7.23 140 56 53 6572 26A (0.12–13.10) (5.21–8.79) (31–458) (9–137) (5–143) (2320–41,200) Apalachicola–Chipola–Flint 5.46 6.76 109 26 35 4750 (3.65–6.44) (6.26–6.93) (88–135) (12–87) (20–60) (2770–6220) Black Warrior River 8.55 7.49 323 320 77 19,758 (3.53–14.88) (5.10–8.90) (1–646) (3–1,400) (13–223) (1459–47,937) Blackwater RiverB 7.87 4.94 19A 12 (7.85–7.93) (4.92–4.99) (4–37) Cahaba River 8.93 7.83 244 134 115 9583 (6.28–16.46) (6.83–8.80) (81–417) (75–199) (69–176) (3180–24,478) Choctawhatchee River 8.93 7.08 82A 23 21 5805 (6.94–12.76) (6.11–7.58) (16–32) (14–32) (5210–6400) Coosa River 5.7 7.49 193 62 66 5326 (0.04–15.47) (6.50–9.47) (0–653) (6–160) (6–157) (796–30,899) Escambia River 6.9 7.14 87 32 38 17,907 (0.2–10.77) (6.38–8.06) (40–158) (23–48) (31–47) (3750–46,000) Mobile River 5.87 6.74 4155 330 41 914,231 3A (0.06–12.30) (6.05–8.66) (6–45,850) (4–3400) (1–456) (1000–11,590,000) Pascagoula River 8.08 5.27 127 15 3 6,314 (3.9–12.60) (3.20–8.30) (3–8900) (2–160) (1–12) (1000–137,000) Perdido River 7.97 7.13 173A (6.02–10.78) (6.83–7.69) Tallapoosa River 6.29 6.6 49 18 26 4301 (0.14–10.80) (5.79–7.79) (0–258) (8–68) (7–72) (1510–24,500) Tennessee River 6.43 7.28 163 101 83 8847 790 1303 (1.91–13.84) (6.17–8.31) (2–383) 8–178) (3–157) (725–35,680) (740–840) (0–2600) Tombigbee River 7.81 7.17 113 39 42 6125 (3.00–14.82) (6.42–8.86) (0–402) (4–128) (5–126) (1620–38,400) Yellow River 5.45 6.8 68 23 32 3499 (0.18–8.37) (6.03–7.76) (27–169) (4–80) (12–43) (2640–4100) Southeastern Naturalist 249 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 higher LC50 or EC50 values than for the caenogastropods evaluated in the current study (Table 2), suggesting that previously tested pulmonate gastropods display lower sensitivity and likely tolerate higher chloride concentrations. Recent studies (e.g., Elphick et al. 2011, Gillis 2011, Soucek et al. 2011) have reported that chloride toxicity decreased with increasing hardness; thus, soft-water ecosystems, such as portions of the Mobile River Basin in Piedmont physiography (e.g., Tallapoosa River Basin), may be at a greater risk for chloride toxicity. Most species previously used in establishing USEPA WQC reported LC50 or EC50 values corresponding to higher water hardness values than in the current study (Table 2). This finding suggested that sensitive mollusk species endemic to soft-water environments may be at a greater risk of decline when exposed to chloride. Potassium. Potassium has been suggested as a possible molluscicide for the invasive, non-native species Dreissena polymorpha Pallas (Zebra Mussel) (Wildridge et al. 1998), but our study’s data suggest that native mussels may be more sensitive than Zebra Mussel; thus, potassium should not be used for a molluscicide. Waller et al. (1993) reported 48-h LC50 values for Zebra Mussel varying from 129,000 μg/L to 163,000 μg/L, which was higher than the values for our study organisms. Keller et al. (2007) reported a 96-h LC50 value of 24,000 μg/L for juvenile Villosa vibex (Conrad) (Southern Rainbow)—a species that is distributed across multiple river basins, including the Mobile River Basin—which was also higher than the values we report here. These findings suggest that stenotypic Mobile River Basin species, Alabama Rainbow and Orangenacre Mucket, were more sensitive to potassium than any mussels previously evaluated, including the invasive Zebra Mussel. The caenogastropods used in the current study were more sensitive to potassium than Physa heterostropha Say (Tadpole Physa), a pulmonate gastropod (OECD 2001). We did not compare our results for Round Rocksnail to other published EC50 values because we could determine no clear EC50 value for potassium for this species. However, Round Rocksnail is expected to be much more sensitive than most other species tested. This assumption was supported by the other trials in our laboratory including those reported herein (chloride, nickel, and zinc) and in Gibson et al. (2016) (sodium dodecyl sulfate). Nickel. The current USEPA WQC for nickel is 470 μg/L (USEPA 1995), a higher concentration than 3 of the 4 EC50 values recorded for Mobile River Basin endemics in the current study (Table 4). Alabama Rainbow had an EC50 value similar to the current WQC value; the confidence interval reported overlapped the WQC, suggesting effects from nickel sensitivity for this species may be near the range of the current criteria. However, Orangenacre Mucket, Round Rocksnail, and the pebblesnails had EC50 values below the WQC for nickel, suggesting these species would not be protected under the current criteria. Excluding Round Rocksnail, which had an EC50 value 14 times lower than the current WQC for nickel, EC50 values reported in the current study were comparable to most previously reported values for freshwater mollusks (Table 4), such as juvenile Utterbackia imbecillis Say (Paper Pondshell; Keller and Zam 1991). However, Amnicola sp., a caenogastropod, had reported 96-h LC50 values of 11,400 μg/L and 14,300 μg/L for embryos Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 250 and adults, respectively (Rehwoldt et al. 1973), which is much higher than the 96-h EC50 values reported in the current study and suggests that Amnicola sp. is more tolerant to nickel than the mollusks used in the current study. Background levels for nickel were only reported for 2 watersheds in Alabama—the Alabama River and Tennessee River (Table 6). Background levels for the Tennessee River watershed were higher than any EC50 value found in the current study; however, average hardness was more than double the hardness of test water. Only Round Rocksnail had a similar EC50 value to average background nickel concentrations in the Alabama River watershed; all other reported EC50 values in the current study were significantly higher. Zinc. The current USEPA WQC for zinc is 120 μg/L (USEPA 1995) and appears to be a sufficiently protective standard for 3 of the 4 mollusk species evaluated in our study. Round Rocksnail was the only species we tested that had an EC50 value below the current WQC; it was sensitive at a concentration of 67 μg/L—nearly half the current criterion. Alabama Rainbow, Coosa Creekshell, and the pebblesnails all had EC50 values more than twice the WQC, suggesting that these species would be protected under the current criteria. Unfortunately, we were unable to determine the EC50 value for zinc for the Orangenacre Mucket because we had an insufficient number of juveniles available for the trial. Cherry et al. (1991) reported a 48-h LC50 value of 656 μg/L for zinc using hard water (hardness of 170 mg CaCO3/L) for Alabama Rainbow glochidia. However, Alabama Rainbow does not occur in the Tennessee River drainage; thus, we suspect that this determination is incorrect and that the report likely concerned Villosa iris (I. Lea) (Rainbow Mussel; P.D. Johnson, pers. observ.). The observed LC50 of 656 μg/L for putative Rainbow Mussel was slightly higher than the 96-h EC50 value determined for juvenile Alabama Rainbow in the current study using soft water (hardness 43 mg CaCO3/L; Table 5). McCann (1993) reported a 48-h LC50 value of 339 μg/L using soft water (hardness of 50 mg CaCO3/L) for juvenile Rainbow Mussel, but when water hardness was increased to 160 mg CaCO3/L, the LC50 value for this species increased to 1122 μg/L. This result suggests that Rainbow Mussel and Alabama Rainbow are similar in zinc sensitivity, and these findings support the contention that toxicity increases in soft water (ASTM 2013). Regarding gastropods, Round Rocksnail had an EC50 value in the current study much lower than any previously reported EC50 value for freshwater gastropods or mussels. There may be other organisms with this level of sensitivity to zinc that have yet to be tested, particularly among other caenogastropods. The pebblesnails had an EC50 value similar to the EC50 value for Alabama Rainbow reported in the current study and LC50 values for Paper Pondshell reported by Keller and Zam (1991). When compared to other gastropods, the pebblesnails had a similar EC50 value to Tadpole Physa in soft water (Wurtz 1962), suggesting similar sensitivities to zinc. However, Wurtz (1962) reported higher LC50 values for the pulmonate Helisoma campanulatum Say (Bellmouth Rams-horn) than either gastropod species reported in our current study (Table 5). Like the situation with nickel, background levels of zinc have not been reported for all watersheds (Table 6). Background levels of zinc have been reported for 2 watersheds: the Mobile River watershed had Southeastern Naturalist 251 K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 a background level of 3 μg/L, but the Tennessee River watershed had an average background level of 1303 μg/L, which was similar to the EC50 value for Alabama Rainbow, but was higher than the other EC50 values in the current study. Average hardness was higher in both watersheds than in the test water. Toxicity results from our study indicate that pulmonate gastropods are more tolerant than caenogastropods to polluted waters or toxicant solutions that may be more bioavailable to cross the ctenidium (a comb-like gill). The lack of a defined gill could limit exposure of pulmonate gastropods to particular toxicants. Future testing should consider using caenogastropods, or contrast pulmonates and caenogastropods to determine comparative sensitivity to toxicants. Freshwater mollusks are particularly sensitive to environmental change, which has made them the most threatened fauna in North America (Johnson et al. 2013, Williams et al. 2008). In addition to water-quality sensitivity, many species are narrow-range endemics, which makes their use as test organisms in toxicity testing more important. To our knowledge, our investigation is the only toxicity study to include both federally threatened and highly endemic mollusk species, which highlights the need to examine more narrowly stenotypic mollusk species in WQC development. Stenotypic species, such as the Mobile River Basin endemics used herein, might display increased sensitivity because their historic distributions were limited to restricted areas within a relatively small regional drainage. Each species tested in this study occupied only a portion of the Mobile River Basin (e.g., Cahaba River Basin gastropods) and no species tested can be found throughout the entire basin. This speciation in localized water-quality systems would more likely result in increased sensitivity to some toxicants not historically associated with their limited distribution. Gillis (2011) suggested that mussels from different watersheds may demonstrate different sensitivities to toxicants. This hypothesis has been demonstrated in multiple faunal groups including fish and amphipods (e.g., Leuven et al. 2011, Prenter et al. 2004, Rieman et al. 2003). Our results from this initial study support the possibility that mollusk species that occupy highly regionalized watersheds, such as the Mobile River Basin, may display increased sensitivity to certain toxicants in comparison to other broadly distributed species with historic ranges that spanned multiple major watersheds. This characteristic is extremely important because most WQC are based on toxicity studies using widely distributed species that may be more tolerant than the rare ones we studied. Development of modern propagation and rearing techniques now make research using threatened and highly endemic species possible. We encourage other investigators to include narrowly endemic species in future toxicity assessments. Acknowledgments We thank Dr. Chris Barnhart (Missouri State University), Dr. Ning Wang (USGS), and Jennifer Archambault (North Carolina State University) for their advice on this project. Our manuscript benefited greatly from the input of 2 anonymous reviewers. This research was conducted under FWS permit #TE130300-4. Funding was provided by the ALFA Fellowship at Troy University. Southeastern Naturalist K. Gibson, J.M. Miller, P.D. Johnson, and P.M. Stewart 2018 Vol. 17, No. 2 252 Literature Cited Agency for Toxic Substances and Disease Registry (ATSDR). 2005. Toxicological profile for Zinc. US Department of Health and Human Services, Public Health Service, Atlanta, GA. Available online at https://www.atsdr.cdc.gov/toxprofiles/tp60.pdf. Accessed 1 November 2017. Alabama Department of Environmental Management (ADEM). 2003. 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