Contaminant Sensitivity of Freshwater Mussels

Transcription

1 Environmentl Toxicology nd Chemistry, Vol. 26, No. 10, pp , SETAC Printed in the USA /07 $ Contminnt Sensitivity of Freshwter Mussels CHRONIC TOXICITY OF COPPER AND AMMONIA TO JUVENILE FRESHWATER MUSSELS (UNIONIDAE) NING WANG,* CHRISTOPHER G. INGERSOLL, I. EUGENE GREER, DOUGLAS K. HARDESTY, CHRISTOPHER D. IVEY, JAMES L. KUNZ, WILLIAM G. BRUMBAUGH, F. JAMES DWYER, ANDY D. ROBERTS, TOM AUGSPURGER, CYNTHIA M. KANE, RICHARD J. NEVES,# nd M. CHRIS BARNHART Columbi Environmentl Reserch Center, U.S. Geologicl Survey, Columbi, Missouri U.S. Fish nd Wildlife Service, Columbi, Missouri U.S. Fish nd Wildlife Service, Rleigh, North Crolin U.S. Fish nd Wildlife Service, Gloucester, Virgini #Virgini Coopertive Fish nd Wildlife Reserch Unit, U.S. Geologicl Survey, Virgini Polytechnic Institute nd Stte University, Blcksburg, Virgini Deprtment of Biology, Missouri Stte University, Springfield, Missouri 65897, USA (Received 17 October 2006; Accepted 9 April 2007) Abstrct The objectives of the present study were to develop methods for conducting chronic toxicity tests with juvenile mussels under flow-through conditions nd to determine the chronic toxicity of copper nd mmoni to juvenile mussels using these methods. In two feeding tests, two-month-old ftmucket (Lmpsilis siliquoide) nd rinbow mussel (Villos iris) were fed vrious live lge or nonvible lgl mixture for 28 d. The lgl mixture ws the best food resulting in high survivl ( 90%) nd growth. Multiple copper nd mmoni toxicity tests were conducted for 28 d strting with two-month-old mussels. Six toxicity tests using the lgl mixture were successfully completed with control survivl of 88 to 100%. Among copper tests with rinbow mussel, ftmucket, nd oyster mussel (Epioblsm cpseformis), chronic vlue ([ChV], geometric men of the no-observed-effect concentrtion nd the lowest-observed-effect concentrtion) rnged from 8.5 to 9.8 g Cu/L for survivl nd from 4.6 to 8.5 g Cu/L for growth. Among mmoni tests with rinbow mussel, ftmucket, nd wvy-ryed lmpmussel (L. fsciol), the ChV rnged from 0.37 to 1.2 mg totl mmoni N/L for survivl nd from 0.37 to 0.67 mg N/L for growth. These ChVs were below the U.S. Environmentl Protection Agency 1996 chronic wter qulity criterion (WQC) for copper (15 g/l; hrdness 170 mg/l) nd 1999 WQC for totl mmoni (1.26 mg N/L; ph 8.2 nd 20 C). Results indicte tht toxicity tests with two-month-old mussels cn be conducted for 28 d with 80% control survivl; growth ws frequently more sensitive endpoint compred to survivl; nd the 1996 chronic WQC for copper nd the 1999 chronic WQC for totl mmoni might not be dequtely protective of the mussel species tested. However, recently revised 2007 chronic WQC for copper bsed on the biotic lignd model my be more protective in the wter tested. Keywords Freshwter mussels Juvenile mussels Chronic toxicity Copper Ammoni INTRODUCTION The erly life stges of freshwter mussels re cutely more sensitive to some chemicls when compred to commonly tested qutic orgnisms [1 4]. However, only limited dt re vilble to evlute the chronic toxicity of chemicls to mussels nd to compre the cute nd chronic effect of these chemicls on mussels [5 7]. A primry limittion for conducting long-term toxicity tests in the lbortory with juvenile mussels is the high mortlity of newly trnsformed juvenile mussels frequently observed few weeks fter relesed from fish host [7]. As result of this problem, Vlenti et l. [5,6] used older, more developed juvenile mussels nd reported high control survivl in 21-d wter-only toxicity tests. Little is known bout the quntity or qulity of food source tht provide conditions for sustining popultions in the wild or in the lbortory [8,9]. Lbortory-cultured green lge hve been used to propgte juvenile mussels in the lbortory [10 12]. Commercilly vilble nonvible lge (Instnt Alge products Nnnochloropsis, nd Shellfish Diet, Reed Mriculture, Cmpbell, CA, USA) lso hve been used for the * To whom correspondence my be ddressed (nwng@usgs.gov). lbortory culture of juvenile nd dult mussels [7,13]. The nonvible lgl products re pure mrine microlge tht re concentrted for esy storge. The single lg Nnnochloropsis is used extensively in the quculture industry for growing smll zooplnkton such s rotifers. Shellfish Diet is mixture of four mrine microlge (Isochrysis, Pvlov, Tetrselmis, nd Thlssiosir) tht hve been used successfully for culturing vriety of mrine shellfish including oysters, clms, mussels, nd scllops. In our preliminry feeding test, combintion of the single lg nd Shellfish diet ws found to support 80% juvenile mussel survivl for 50 d. Additionl studies were needed to evlute the effects of foods nd feeding rtes on the survivl nd growth of juvenile mussels over 28-d period to optimize conditions for conducting long-term toxicity tests. A series of studies were undertken to refine methods for conducting cute nd chronic toxicity tests with erly life stges of mussels [3,7,13 15]. Bsed on these studies nd previous literture, Americn Society for Testing nd Mterils (ASTM) recently published n interntionl Stndrd Guide For Conducting Lbortory Toxicity Tests With Freshwter Mussels [16]. As one of series of ppers developed to ssess con- 2048

2 Chronic toxicity of copper nd mmoni to freshwter mussels Environ. Toxicol. Chem. 26, Tble 1. Food types nd feeding levels in feeding test one strting with two-month-old ftmucket (Lmpsilis siliquoide) Food type Sources Feeding level (low, medium, high) Live lg (Neochloris oleobundns) MSU, Springfield, Missouri, USA 20,000; 40,000; 60,000 cells/ml Live lg (N. oleobundns) sediment b MSU As bove but with sediment Live lg (Selenstrum cpricornutum) ABS c, Fort Collins, Colordo, USA 20,000; 40,000; 60,000 cells/ml Live lg (Nnnochloropsis ocult) Virgini Tech d, Blcksburg, Virgini, USA 30,000; 60,000; 90,000 cells/ml Nnno: Nonvible lg (Nnnochloropsis) Reed Mriculture, Cmpbell, Cliforni, USA 1, 2, 3 ml Algl mixture: Nnno Shellfish Diet Reed Mriculture 1, 2, 3 ml MSU Missouri Stte University. b Fine sediment ws dded s thin lyer in the bottom of the test chmbers t the beginning of the test. c ABS Aqutic Biosystems. d Polytechnic Institute nd Stte University. tminnt sensitivity of erly life stges of freshwter mussels, the present pper summrizes the results of 28-d feeding studies nd toxicity tests with two-month-old juvenile mussels during the process of developing the ASTM stndrd. Specificlly, the objectives of the present study were to evlute survivl nd growth of juvenile mussels fed vrious foods t different feeding levels over 28-d period in intermittent flowthrough systems, routinely used for cute nd chronic toxicity tests with fish nd qutic invertebrtes, nd to determine chronic toxicity of copper nd totl mmoni to juvenile mussels in 28-d tests following stndrd methods [16]. The two toxicnts were selected becuse of their wide occurrence in contminted qutic environments, becuse cute toxicity dt hve indicted the erly life stges of mussels were sensitive to these toxicnts [1 3], nd becuse no (totl mmoni) or limited (copper) chronic toxicity dt were vilble for freshwter mussels. MATERIALS AND METHODS Culture of juvenile mussels Grvid femles of five mussel species were collected from strems nd rivers in Virgini (rinbow mussel, Villos iris; wvy-ryed lmpmussel, Lmpsilis fsciol), Tennessee (oyster mussel, Epioblsm cpseformis), nd Missouri, USA (ftmucket, L. siliquoide; pink mucket, L. brupt) between Mrch nd June of 2004 nd Adult mussels were collected from reltively uncontminted loctions where these species were bundnt nd pprently helthy, nd reproduction nd recent recruitment were evident. Glochidi isolted from t lest three femle mussels were pooled for the production of juvenile mussels from host fish in lbortories t Virgini Polytechnic Institute nd Stte University (Virgini Tech, Blcksburg, VA, USA) or Missouri Stte University (Springfield, MO, USA; see Wng et l. [13] for description of the methods used to trnsform juvenile mussels). Trnsformed juveniles were rered with lbortory-cultured lge (Neochloris oleobundns) for two months either in recirculting quculture system contining fine sediment [11] or in compct recirculting system [12] before testing. Feeding tests Feeding test one. The first feeding test ws conducted for 28 d strting with two-month-old (two months fter trnsformtion) ftmucket nd vrious lgl sources. The juvenile mussels were shipped overnight to the Columbi Environmentl Reserch Center (CERC, U.S. Geologicl Survey, Columbi, MO) for testing. Once juvenile mussels were received, the temperture of the wter ws djusted to the test temperture by plcing the continers contining mussels into wter bth t 20 C. Approximtely 50% of the wter in the continers then ws replced grdully with test wter (ASTM reconstituted hrd wter [17]) t lest three times over 2- or 3-d cclimtion period, during which juveniles were fed live lge (N. oleobundns) twice dily. At the beginning of the feeding test, 10 juveniles with foot movement were imprtilly trnsferred into ech of thirty 300-ml glss bekers using 1-ml syringe with 2.5-cm long, 16-guge needle connected to 60-cm-long Tygon tubing (1.0-mm inner dimeter, Sint-Gobin Performnce Plstics, Akron, OH, USA) with glss cpillry tube (1.17-mm inner dimeter) t the end [16]. The test ws conducted in wter-renewl system [18], which delivered 60 ml of dditionl wter into ech beker every 4 h. Ech beker hd 2.5-cm hole in the side covered with 50 mesh (279 micron opening) stinless-steel screen nd contined 200 ml of wter. At ech cycle of wter ddition, excess wter in the bekers flowed out through the screen, with no wter exchnge mong replicte bekers (in contrst to previous studies conducted by Vlenti et l. [5,6], where there ws wter exchnge between replicte exposure chmbers). Bekers were plced in temperture-controlled wter bths t 20 1 C. Juvenile mussels were fed three species of lbortory-cultured live lge or two nonvible lge concentrtes t three feeding levels (Tble 1): Low level, which pproximted the feeding level used for the mussel culture in continuous feeding nd clculted from informtion (e.g., the density of lge) on lge concentrte continers provided by the mnufcturers; medium level (two times the low level); nd high level (three times the low level). Ech tretment hd two replictes. Juvenile mussels were fed twice dily in the morning nd fternoon immeditely fter n ddition of wter. In ddition, juveniles in one tretment were not fed lge but received only thin lyer of fine sediment t the strt of the test (Tble 1; see Jones et l. [11] for the description of the sediment chrcteristics), nd juveniles in nother control tretment received neither food nor sediment during the test. Fresh live lge were provided by Virgini Tech nd Missouri Stte University nd were delivered twice to our lbortory t the beginning nd middle of the 28-d feeding test. Ech btch of lge ws used for two weeks nd kept in refrigertor t pproximtely 3 C. The single lg food (Nnno; Tble 1) ws prepred by dding 1 ml of Nnnochloropsis concentrte into 1.8 L of well wter, nd the combintion of Nnno nd Shellfish Diet (lgl mixture; Tble 1) ws prepred by dding 1 ml of Nnnochloropsis concentrte nd 2 ml of Shellfish Diet concentrte into 1.8 L of wter. Ech btch of

3 2050 Environ. Toxicol. Chem. 26, 2007 N. Wng et l. Tble 2. Summry of test conditions for conducting chronic toxicity tests with juvenile mussels in ccordnce with methods outlined in Americn Society for Testing nd Mterils (ASTM) [16] Test chemicls Copper sulfte or mmonium chloride Test type Flow-through Test durtion 28 d Temperture 20 1 C Light qulity Ambient lbortory light Light intensity 200 lux Photoperiod 16:8 h light:drk Test chmber 300-ml glss beker Test solution volume 200 ml Renewl of solution Additionl 120 ml of solution to ech beker once every 4 h Age of test orgnism 2 months Number of orgnisms per test chmber 10 Number of replicte chmbers per concentrtion 4 (except the test with wvy-ryed lmpmussel, n 3) Feeding Twice dily with live lge or with nonvible lgl mixture Chmber clening None Aertion None Dilution wter Reconstituted ASTM hrd wter (hrdness mg/l s CCO 3, lklinity mg/l s CCO 3 [17]) Dilution fctor 0.5 Test concentrtion Copper: 0, 3.125, 6.25, 12.5, 25, nd 50 g/l Totl mmoni: 0, 0.5, 1, 2, 4, nd 8 mg N/L or 0, 0.125, 0.25, 0.5, 1.0, nd 2.0 mg N/L Chemicl residues Copper weekly nd totl mmoni once every 3 to 5 d Wter qulity Dissolved oxygen, ph, conductivity, hrdness, nd lklinity t the control, medium-, nd high-exposure concentrtions weekly Endpoint Survivl (foot movement) nd growth (shell length) Test cceptbility criterion 80% control survivl on dy 28 lgl foods ws used for one week nd kept in refrigertor t pproximtely 3 C. Wter qulity ws determined weekly on composite smples of ech tretment using stndrd methods [19]. Wter hrdness rnged from 160 to180 mg/l s CCO 3, lklinity rnged from 110 to120 mg/l s CCO 3, ph rnged from 8.4 to 8.6, dissolved oxygen ws 8.0 mg/l, nd totl mmoni ws 0.07 mg N/L cross ll tretments. Ambient lbortory light ws pproximtely 200 lux, with 16:8 h light:drk photoperiod. Survivl (foot movement within 5 min [16]) ws determined t the end of ech test using dissecting microscope. Surviving mussels in the two replictes of ech tretment were pooled nd preserved in 70% ethnol for subsequent growth mesurements. The mximum shell length of ech mussel ws mesured to the nerest mm using digitizing system with video micrometer softwre (Imge Cliper, Resolution Technology, Dublin, OH, USA). Becuse of the limited repliction in this feeding test, the differences in survivl nd growth were not sttisticlly compred mong tretments. Feeding test two. The second feeding test ws conducted for 28 d with two-month-old rinbow mussels nd the best food found in feeding test one. The test ws conducted in n intermittent flow-through diluter system [20]. The system provided pproximtely 120 ml of wter to ech glss beker every 4 h. An in-line flow splitter ws ttched to ech delivery line to prtition the wter flow evenly to ech of replicte bekers. Test chmbers, cclimtion of test orgnisms, wter temperture, light qulity, photoperiod, nd test wter were s described bove for feeding test one. Ten juveniles were trnsferred imprtilly into ech of four replicte bekers per feeding level. A smple with pproximtely 10 juveniles lso ws imprtilly collected t the beginning of the feeding test, nd preserved in 70% ethnol to record initil shell length. Juveniles were fed the lgl mixture s described in feeding test one t four feeding levels: Mnully dding 2 or 4 ml of the food into beker twice dily, or utomtic delivery of 1 or 2 ml of the food using Hmilton syringe pump (Hmilton Compny, Reno, NV, USA) six times dily, with ech wter ddition. In ddition, juvenile mussels in two bekers were not fed during the test. Wter qulity ws determined weekly on composite smples of ech tretment. The hrdness rnged from 160 to180 mg/l s CCO 3, lklinity rnged from 110 to120 mg/l s CCO 3, nd ph rnged from 8.2 to 8.4. Dissolved oxygen ws 7.9 mg/l nd totl mmoni ws 0.05 mg N/L. Survivl ws determined t the end of the feeding test. Surviving mussels in ech replicte beker were preserved in 70% ethnol for subsequent growth mesurement s described bove. Differences in men survivl nd growth mong feeding tretments were nlyzed using nlysis of vrince nd Tukey s test [21]. The level of sttisticl significnce ws set t p Toxicity test Test conditions used to conduct the chronic toxicity tests with juvenile mussels were summrized in Tble 2. Copper sulfte (CuSO 4, 99.9% purity; JT Bker, Phillipsburg, NJ, USA) nd mmonium chloride (NH 4 Cl, 99.5% purity; Fisher Scientific, Houston, TX, USA) were used s toxicnts. Tests were conducted in flow-through diluter system s described in feeding test two, which delivered five exposure concentrtions with dilution fctor of 0.5 plus control, nd provided pproximtely 120 ml of test solution to ech replicte beker per concentrtion every 4 h. Bekers were plced in temperture-controlled wter bths. Toxicnt stock solution ws delivered with ech cycle of the diluter by the Hmilton syringe pump. Test chmbers, cclimtion of test orgnisms, light qulity, photoperiod, nd dilution wter were s described bove for the feeding tests. At the beginning of toxicity test, 10 juveniles exhibiting foot movement were imprtilly trnsferred into ech beker. Approximtely 20 juveniles lso were imprtilly smpled nd

4 Chronic toxicity of copper nd mmoni to freshwter mussels Environ. Toxicol. Chem. 26, Tble 3. Men shell length of juvenile mussels t the beginning of the toxicity test (n 10 to 20) nd men wter qulity chrcteristics over 28-d period of ech test (n 5 to 7 smpling times). Stndrd devition is in prentheses Test Species Men length (mm) ph Hrdness (mg/l s CCO 3 ) Alklinity (mg/l s CCO 3 ) Conductivity ( S/cm) Cu1 Ftmucket 0.76 (0.08) 8.3 (0.1) 171 (3.0) 122 (6.4) 577 (12) Cu2 Pink mucket 0.59 (0.08) Cu3 Rinbow mussel 0.90 (0.08) 8.3 (0.1) 171 (8.4) 119 (1.5) 589 (8.3) Cu4 Ftmucket 0.63 (0.12) 8.7 (0.2) 165 (4.1) 124 (1.8) 563 (21) Cu5 Oyster mussel 0.84 (0.27) 8.2 (0.1) 162 (2.5) 88 (11) 589 (11) TN1 Ftmucket 0.76 (0.08) 8.4 (0.2) 168 (5.2) 118 (5.6) 586 (7.2) TN2 Pink mucket 0.59 (0.08) TN3 Rinbow mussel 0.90 (0.08) 8.2 (0.1) 175 (4.2) 121 (3.3) 610 (9.5) TN4 Ftmucket 0.62 (0.13) 8.2 (0.1) 161 (4.4) 92 (16) 556 (45) TN5 Wvy-ryed lmpmussel 0.66 (0.07) 8.2 (0.1) 163 (3.6) 92 (12) 579 (24) Wter qulity dt were the sme in copper test one (Cu1) nd two (Cu2) or in totl mmoni test one (TN1) nd two (TN2), which were conducted concurrently in one diluter system with the sme source of dilution wter. preserved in 70% ethnol to record initil shell lengths. In the first four tests with ftmucket (copper test one [Cu1] nd totl mmoni nitrogen test one [TN1]; Tble 3) nd pink mucket (Cu2 nd TN2), juveniles were fed live lge (N. oleobundns), previously used for the culture of these juveniles nd provided by Missouri Stte University. A smple of fresh lge ws kept in refrigertor t 3 C nd used for pproximtely two weeks. The fresh lge were dded into bekers twice dily, in erly morning nd in lte fternoon, t feeding rte of pproximtely 40,000 cells/ml, which ws double the mount of food used to culture these juveniles in continuous feeding systems [12]. Becuse of the high control survivl nd growth of juveniles fed the lgl mixture in feeding tests, the lgl mixture ws used in six subsequent toxicity tests (Cu3, Cu4, Cu5 nd TN3, TN4, nd TN5; Tble 3), where juveniles were fed 2 ml of the lgl mixture twice dily. Wter qulity ws determined weekly on composite wter smples collected from the control, medium-, nd high-exposure concentrtions. Additionlly, totl mmoni ws mesured periodiclly during copper tests nd never exceeded 0.05 mg N/L. Mesured dissolved oxygen ws bove 7.0 mg/l during ll tests. Men vlues of hrdness nd lklinity over the 28-d period of ech test (Tble 3) were generlly within the rnge recommended for ASTM hrd wter (hrdness mg/l s CCO 3, lklinity mg/l s CCO 3 [17]). Men ph vlues rnged from 8.2 to 8.7 for copper tests nd from 8.2 to 8.4 for mmoni tests (Tble 3), which were bove the listed rnge of 7.8 to 8.0 in ASTM [17]. To mintin more stble ph vlue in three tests (Cu5, TN4, nd TN5; Tble 3), the dilution wter ws initilly djusted between 8.0 nd 8.2 in 700-L polypropylene tnk before the wter ws delivered to the flow-through diluter. The ph ws djusted by injecting dilute hydrochloric cid (HCl) into the tnk using ph pump control system with proportionl output (Brnnt HD PH-P1, Brrington, IL, USA). However, the ddition of HCl to the dilution wter lowered the lklinity in these three tests (Tble 3). During the 28-d exposure period, survivl ws determined on test dys 4, 10, 21, nd 28. In order to observe the juveniles under the microscope during test, it ws necessry to remove some of the test wter from the beker. Gently swirling the beker creted slight vortex in the wter nd concentrted the juveniles in smll re, mking it esier to see ll of the orgnisms simultneously in the field of view under the microscope. Juvenile mussels exhibiting foot movement within 5-min observtion period were clssified s live. The cceptbility criterion ws 90% control survivl in 4-d exposures nd 80% control survivl in 10-, 21-, nd 28-d exposures [16]. Surviving juveniles t the end of 28-d tests were preserved in 70% ethnol for growth mesurement s described bove. For dissolved copper nlysis, wter ws collected weekly from composite smples of ech of the six exposure concentrtions. Ech wter smple ws cidified to 1% (v/v) ultrpure nitric cid. Copper concentrtions were determined by inductively coupled plsm-mss spectrometry (PE/SCIEX ELAN 6000, PerkinElmer, Norwlk, CT, USA). Smples were utomticlly delivered to the inductively coupled plsm-mss spectrometry by mens of softwre-controlled CETAC ASX- 500/ADX-100 utosmpler/utodiluter system (CETAC Technologies, Omh, NE, USA; see Wng et l. [13] for dditionl detil). Anlyticl precision for quntittive inductively coupled plsm-mss spectrometry ws determined by nlyzing smples in duplicte during the instrumentl run nd determining the reltive percent differences, which were 2.7% for ll nlysis duplictes. Recoveries of copper spiked into wter smples nd nlyzed by quntittive inductively coupled plsm-mss spectrometry rnged from 98 to 103%. Instrumentl detection limit ws g/l, nd the method detection limit ws 0.2 g/l. During mmoni tests, totl mmoni nitrogen of ech exposure concentrtion ws mesured every 3 to 5 d using n Orion Ammoni Electrode nd Orion EA940 meter (Thermo Electron, Beverly, MA, USA). The meter for totl mmoni nlyses ws clibrted ech time before mesuring smples with 0.1 nd 1 mg/l or 1 nd 10 mg/l independent clibrtion verifiction stndrds, depending on the rnge of totl mmoni concentrtions to be mesured. The percent recovery of the stndrds rnged from 90 to 100%. For mmoni nitrogen concentrtions in wter smples, minimum reporting limit of 0.1 mg N/L ws selected bsed on the method detection limit of 0.02 mg N/L nd method quntittion limits of 0.06 mg N/L. Men mesured copper nd totl mmoni concentrtions (Tble 4) were similr to nominl concentrtions (Tble 2) throughout the 28-d tests. The men mesured concentrtions for ech test were used to clculte the cute nd chronic effect concentrtions. The medin effective concentrtions (EC50s) for survivl on test dys 4, 10, 21, nd 28 were clculted with Toxstt softwre [21], using Probit model when pproprite, nd either Spermn-Krber or trimmed Spermn-

5 2052 Environ. Toxicol. Chem. 26, 2007 N. Wng et l. Tble 4. Men mesured concentrtions of copper (Cu, g/l) nd totl mmoni (TN, mg N/L) over 28-d exposure periods in toxicity tests with juvenile mussels. Stndrd devition is in prentheses Men mesured concentrtion Test Species n Control Low Med-low Medium Med-high High Cu1/2 Ftmucket/pink mucket (0.2) 3.4 (0.3) 5.8 (0.5) 13 (2.6) 25 (4.5) 56 (11) Cu3 Rinbow mussel (0.2) 4.1 (0.9) 6.4 (2.2) 15 (7.3) 21 (2.1) 51 (3.0) Cu4 Ftmucket (0.4) 3.1 (0.9) 6.6 (2.8) 11 (2.1) 21 (1.6) 45 (2.6) Cu5 Oyster mussel (0.2) 3.1 (0.7) 6.7 (1.1) 13 (2.0) 23 (4.6) 50 (6.1) TN1/2 Ftmucket/pink mucket (0.09) 0.79 (0.14) 1.6 (0.34) 3.3 (0.45) 7.4 (0.82) TN3 Rinbow mussel (0.07) 0.81 (0.10) 1.7 (0.22) 3.5 (0.29) 7.6 (0.44) TN4 Ftmucket (0.02) 0.28 (0.10) 0.49 (0.03) 1.0 (0.07) 2.0 (0.16) TN5 Wvy-ryed lmpmussel (0.02) 0.34 (0.16) 0.44 (0.11) 1.0 (0.13) 2.0 (0.12) Copper test one (Cu1) nd two (Cu2) or totl mmoni test one (TN1) nd two (TN2) were conducted concurrently in diluter system with the sme dilution wter. Krber method otherwise [22]. The no-observed-effect concentrtion nd the lowest-observed-effect concentrtion for survivl nd growth t the end of 28-d toxicity tests were determined by nlysis of vrince, with men comprison mde by one-tiled Dunnett s test or by Steel s mny-one rnk test [21,23]. To focus on effects on growth tht occurred t concentrtions less thn those ffecting survivl, exposure concentrtions bove the lowest-observed-effect concentrtion for survivl were excluded from sttisticl nlysis for growth [23]. The level of sttisticl significnce ws set t p Chronic vlue (ChV) ws clculted s the geometric men of the no-observed-effect concentrtion nd lowest-observed-effect concentrtion. A liner interpoltion method [23] lso ws used to estimte 10 nd 25% inhibition concentrtions for survivl nd growth t the end of 28-d toxicity tests [21]. When clculting 10% inhibition concentrtion nd 25% inhibition concentrtion, survivl nd growth dt from ll concentrtions were included in the nlysis [23]. RESULTS AND DISCUSSION Feeding test one with live nd instnt nonvible lge Men 28-d survivl of juvenile ftmucket fed vrious foods t the low feeding levels rnged from 70 to 90% (Tble 5), wheres the men survivl in no-feeding tretments ws 35% (wter-only) nd 25% (with sediment). Higher feeding levels generlly did not increse the survivl or growth of juvenile mussels (Tble 5). The best survivl ( 90%) nd growth were observed in feeding tretments with the lgl mixture (Tble 5). Lower survivl nd growth of juvenile mussels in tretments with live lge might hve resulted from underfeeding. Although the feeding levels were one to three times those used for the culture of juvenile mussels in continuous feeding, underfeeding my hve occurred becuse the lge were dded twice dily nd some of the lge would hve been flushed from the bekers with ech cycle of wter ddition into the beker every 4 h. In ddition, using lge held for up to two weeks might hve influenced the qulity of the lge. Becuse the live lge hve been used to successfully culture juvenile mussels [11,12], it is likely tht live lge cn be used s food source in toxicity tests. However, more studies re needed to evlute the quntity nd qulity of live lge for use in flow-through toxicity tests. Feeding test two with the lgl mixture Results of the first feeding test indicte tht the lgl mixture ws good food source for the juvenile mussels held under flow-through conditions. Further study on the feeding levels of the lgl mixture ws conducted in the second feeding test. Men survivl of juvenile rinbow mussels t ech of the feeding levels rnged from 85 to 100%, nd men shell length incresed 47 to 71% over the 28-d test (Tble 6). In contrst, men survivl in no-feeding tretment ws 50% nd men shell length incresed only 16% (Tble 6). Among the four mnul nd utomtic feeding tretments, men survivl nd growth were not significntly different. The results indicte tht the lgl mixture cn be used to mintin high control survivl nd growth of juvenile mussels in flow-through conditions nd tht lower mnul feeding level (i.e., dding 2 ml of the food mixture twice dily) cn be used in subsequent chronic toxicity tests. Advntges of using the lgl mixture include low cost, long storge time (three months in refrigertor), nd esy preprtion compred to mintining lbortory cultures of live lge. Tble 5. Men 28-d survivl nd growth of juvenile ftmucket (Lmpsilis siliquoide) fed vrious foods t low, medium (2 times the low level), nd high (3 times the low level) feeding levels in feeding test one. Stndrd devition is in prentheses Tretment Men survivl (%; n 2) Low Medium High Men shell length (mm) Low Medium High Live lg (Neochloris oleobundns) (0.09) 0.82 (0.09) 0.83 (0.10) Live lg (N. oleobundns) sediment (0.14) 0.74 (0.13) 0.75 (0.15) Live lg (Selenstrum cpricornutum) (0.17) 0.87 (0.10) NR b Live lg (Nnnochloropsis ocult) (0.08) NR 0.81 (0.11) Nnno (0.11) 0.79 (0.18) 0.75 (0.15) Algl mixture (0.07) 0.83 (0.09) 0.84 (0.19) Surviving juveniles of two replictes per food were pooled for growth mesurement (n 10 to 20). b Growth dt were not reported when men survivl ws below 50% in feeding tretment.

6 Chronic toxicity of copper nd mmoni to freshwter mussels Environ. Toxicol. Chem. 26, Tble 6. Men 28-d survivl nd growth of juvenile rinbow mussels (Villos iris) fed the lgl mixture t vrious feeding levels in feeding test two. Stndrd devition is in prentheses Growth Tretment n Survivl (%) Length (mm) % Increse b Mnul feeding: 2 2 ml (0) X 1.54 (0.07) X 71 Mnul feeding: 2 4 ml 4 85 (24) X 1.50 (0.06) X 67 Auto feeding: 6 1 ml 4 95 (10) X 1.37 (0.16) X 52 Auto feeding: 6 2 ml 4 98 (5.0) X 1.32 (0.13) X 47 No food 2 50 (42) 1.04 (0.09) 16 Mens with sme letter in column re not significntly different (the dt from no-food tretment were not included in this nlysis). b Men shell length of juveniles t the beginning of the feeding test ws mm (n 10). Toxicity tests Men survivl of juvenile mussels in the first two copper tests (Cu1 nd Cu2) nd two mmoni tests (TN1 nd TN2) ws 90% on test dys 4, 10, nd 21, but declined to 30% by the end of the 28-d test. The four toxicity tests were conducted before the feeding tests, nd live lge were used s food source. It is likely tht mussels were underfed in the first four toxicity tests, s discussed bove in feeding test one. Low control survivl lso ws observed fter 21 d in long-term toxicity test conducted with four-month-old ftmucket fed live lge (Robert Bringolf, North Crolin Stte University, Rleigh, NC, USA, unpublished dt). In contrst, ll of the six subsequent toxicity tests using the lgl mixture were successfully completed over 28-d exposures, with control survivl rnging from 88 to 100% (Tbles 7 nd 8). In ddition, compred to the initil length (Tble 3), the men length of juveniles in the controls t the end of tests (Tbles 7 nd 8) incresed substntilly, rnging from 32 to 83% increse. An exception ws the wvy-ryed lmpmussel, with only 9.1% increse. The ChVs for growth in three of the six tests were lower thn the ChVs for survivl (Tbles 7 nd 8), indicting tht the growth endpoint ws more sensitive thn survivl nd cn be useful sublethl endpoint for chronic toxicity tests with juvenile mussels. The copper ChV for growth rnged from 4.6 to 8.5 g/l for rinbow mussel, ftmucket, nd oyster mussel (Tble 7), lthough the totl mmoni ChV for growth rnged from 0.37 to 0.67 mg N/L for rinbow mussel, ftmucket, nd wvy-ryed lmpmussel (Tble 8). The 10% inhibition concentrtion for growth in ech copper or mmoni test ws generlly similr to the ChV for growth, nd ws 16 to 48% lower thn the 25% inhibition concentrtion (Tbles 7 nd 8). All of these chronic effect concentrtions (ChVs, 10% inhibition concentrtions, nd 25% inhibition concentrtions) were below the U.S. Environmentl Protection Agency s (U.S. EPA) 1996 hrdness-dependent chronic wter qulity criterion (WQC) for copper (15 g/l t hrdness of 170 mg/l [24]) or the 1999 temperture- nd ph-dependent chronic WQC for mmoni (1.26 totl mmoni N/L t ph 8.2 nd 20 C [25]). Results of these toxicity tests indicte tht the ntionl WQC might not dequtely protect the erly life stges of freshwter mussels tested in the present study from chronic exposure to copper or totl mmoni. The U.S. EPA published the 2007 revised WQC for copper [26], which is bsed on the biotic lignd model nd dependent on number of wter qulity prmeters (e.g., dissolved orgnic crbon, ph, temperture, mjor ctions nd nions). The biotic lignd model bsed chronic copper criterion is 3.9 g/l for the reconstituted ASTM hrd wter used in the present study (20 C, ph 8.3, nd the estimted wter qulity prmeters bsed on the specified formuls [26]), is pproximtely four times lower thn hrdness-bsed copper criterion [24], nd my be more protective of the mussel species tested. However, dditionl studies re needed to determine how dissolved orgnic crbon, other wter qulity prmeters, nd dietry exposure to copper influence the chronic toxicity of copper to mussels. The toxicity dt used in the derivtion of the WQC hve not routinely included dt generted for freshwter mussels. Recent studies hve demonstrted tht use of cute toxicity dt for erly life stges of mussels would lower the cute WQC for copper [2] nd totl mmoni [1]. The chronic toxicity dt in the present study should be considered to reevlute the chronic WQC for copper nd totl mmoni. Mussels my not be more sensitive to other chemicls. Chronic Tble 7. Men survivl nd growth (shell length, n 4) of juveniles of three mussel species t the end of the 28-d copper toxicity tests. Chronic vlue (ChV) nd 10% nd 25% inhibition concentrtions (IC10, IC25 with 95% confidence intervl [CI]) re presented for ech endpoint. Stndrd devition is in prentheses. An sterisk (*) indictes significnt reduction reltive to control. Concentrtions bove the concentrtion cusing significnt reduction in survivl were excluded from the hypothesis test for growth effect Rinbow mussel Ftmucket Oyster mussel Men concn. Survivl (%) Length (mm) Men concn. Survivl (%) Length (mm) Men concn. Survivl (%) Length (mm) (10) 1.31 (0.11) (5) 1.15 (0.11) (0) 1.11 (0.09) (7.5) 1.34 (0.11) (14) 1.30 (0.19) (14) 1.05 (0.08) (13) 1.13 (0.05)* (12) 1.09 (0.12) (14) 0.97 (0.05)* (29)* (17)* (7.2)* (19)* 21 5 (10)* 23 0* 51 0* 45 0* 50 0* ChV ChV ChV IC10 (CI) 4.9 ( ) 5.7 ( ) IC10 (CI) (4.8 12) IC10 (CI) ( ) IC25 (CI) 6.3 (5.5 14) 7.5 ( ) IC25 (CI) 8.0 (5.5 11) 12 (10 15) IC25 (CI) 7.6 ( ) 7.6 ( )

7 2054 Environ. Toxicol. Chem. 26, 2007 N. Wng et l. Tble 8. Men survivl nd growth (shell length) of juveniles of three mussel species t the end of 28-d mmoni toxicity tests (n 4, except for test with wvy-ryed lmpmussel, n 3). Chronic vlue (ChV) nd 10% nd 25% inhibition concentrtions (IC10, IC25 with 95% confidence intervl [CI]) re presented for ech endpoint. Stndrd devition is in prenthesis. An sterisk (*) indictes significnt reduction reltive to control. Concentrtions bove the concentrtion cusing significnt reduction in survivl were excluded from the hypothesis test for growth effect Wvy-ryed lmpmussel Ftmucket Rinbow mussel Men concn. Survivl (%) Length (mm) Men concn. Survivl (%) Length (mm) Men concn. Survivl (%) Length (mm) (0) 1.52 (0.11) (5.0) 0.95 (0.18) (0) 0.72 (0.03) (5.0) 1.32 (0.05)* (15) 0.84 (0.07) (15) 0.77 (0.04) (5.0) 1.10 (0.08)* (10) 0.95 (0.11) (5.8) 0.74 (0.04) (24)* (21)* (15) 0.72 (0.09) * (39)* (36)* * (25)* * ChV ChV ChV IC10 (CI) 0.89 ( ) 0.4 IC10 (CI) ( ) IC10 (CI) ( ) IC25 (CI) 1.0 ( ) 0.73 ( ) IC25 (CI) 0.32 ( ) 0.44 ( ) IC25 (CI) 0.39 ( ) 0.57 ( ) Fig. 1. Medin effective concentrtions (EC50s) for copper nd totl mmoni over 4-, 10-, 21-, or 28-d exposures in tests strting with two-month-old juvenile mussels. Blck circles represent chronic vlues for survivl nd growth of juvenile mussels on test dy 28. Dshed line represents the U.S. Environmentl Protection Agency s 1996 hrdness-dependent chronic wter qulity criterion (WQC) for copper (hrdness 170 mg/l [24]) or 1999 temperture- nd ph-dependent chronic WQC for mmoni (ph 8.2, 20 C [25]). The symbol ( ) bove br indictes tht the EC50 vlue ws greter thn the highest test concentrtion. tests conducted by others for chlorine [6] nd chlorpyrifos (insecticide) [15] indicte the mussels re much less sensitive to these compounds nd likely would be protected by current chronic WQC for chlorine nd chlorpyrifos. The EC50s for copper or totl mmoni generlly decresed over the exposure periods of 4, 10, 21, nd 28 d (Fig. 1). The 28-d ChVs for survivl nd growth typiclly were lower thn 28-d EC50s for survivl nd were lower thn the 1996 chronic WQC for copper or the 1999 chronic WQC for mmoni (Fig. 1). Toxicity tests evluting juvenile mussel survivl nd sublethl responses hve been conducted for 10 to 21 d [3,5,6, 15,27,28]. The 10-d EC50s in our previous sttic-renewl toxicity tests (without feeding) with two-month-old ftmucket nd rinbow mussels ( g Cu/L, mg totl mmoni N/L [3]) were similr to those in the present flow-through tests (Fig. 1), but were two to three times higher thn the 28-d ChVs (Tbles 7 nd 8; Fig. 1). These results indicte tht prolonged test period nd sublethl endpoint re needed to estimte chronic toxicity of toxicnt to juvenile mussels. Becuse freshwter mussels re long-lived orgnisms with some species often living over 30 yers, concern hs been expressed tht 28-d chronic test with erly life stges of mussels my not

8 Chronic toxicity of copper nd mmoni to freshwter mussels Environ. Toxicol. Chem. 26, Tble 9. Acute-chronic rtio (ACR) for copper or totl mmoni clculted from 4-d men medin effective concentrtions (EC50s) in stticrenewl or flow-through tests with newly trnsformed or two-month-old juvenile mussels, nd 28-d chronic vlues (ChVs) for survivl nd growth in flow-through tests with two-month-old juvenile mussels Chemicl Species EC50 Young/sttic Older/sttic Older/flow b Men ChV Older/ flow ACR Copper ( g/l) Rinbow mussel c Ftmucket 21 c 46 c Oyster mussel 12 c NT d Ammoni (mg N/L) Rinbow mussel c 8.0 e Ftmucket Wvy-ryed lmpmussel 7.4 NT 2.0 e Vlue from the previous sttic-renewl tests with newly trnsformed (young) or two-month-old (older) juveniles [3]. b Vlue from flow-through tests with two-month-old juveniles (Fig. 1, this study). c Men vlue of two to five tests [3]. d NT Not tested. e Vlue greter thn the highest exposure concentrtion ws not included for clculting men EC50. represent dequtely the concentrtions of chemicls elicit hrmful effects on the long-lived species [2]. McKim [29] evluted reltionships between full life-cycle toxicity tests nd erly life stge toxicity tests. Bsed on 72 life-cycle tests with four freshwter species nd 37 orgnic nd inorgnic chemicls plus one sltwter species nd 11 orgnic chemicls, McKim [29] concluded tht, in 83% of these tests, the erly life stge portion of the life cycle gve the sme mximum cceptble toxicnt concentrtion ( rnge of no-observed-effect concentrtion nd lowest-observed-effect concentrtion) s the full life cycle, lthough the remining 17% of the erly life stge tests showed more or less sensitivity thn the life cycle test by fctor of two. The vrition of fctor of two is lmost meningless becuse the mximum cceptble toxicnt concentrtions for specific toxicnts, species, nd wter combintions esily cn vry by fctor of two [29]. However, given tht the evlution by McKim [29] did not include mussel toxicity dt, there is uncertinty s to how well 28-d toxicity test with juvenile mussels cn be used to predict effect of toxicnt in long-term exposures with other life stges of mussels, where effects on survivl, growth, or reproduction might occur. Becuse totl mmoni concentrtions could decline up to 40% during 96-h sttic-renewl tests with juvenile mussels, the EC50s clculted bsed on nominl mmoni concentrtions might underestimte mmoni toxicity [3]. However, similr 96-h EC50s were observed in mmoni tests conducted concurrently with two-month-old juvenile mussels under sttic-renewl (fluctuting totl mmoni concentrtion; Tble 4 in Wng et l. [3]) nd flow-through (reltively constnt mmoni concentrtions; Fig. 1) conditions: The EC50 for ftmucket ws 4.9 mg N/L in the sttic-renewl test nd 4.6 mg N/L in the flow-through test; the EC50 for pink mucket ws 2.3 mg N/L in the sttic-renewl test nd 2.8 mg N/L in the flow-through test; nd the EC50 for rinbow mussel ws 11 mg N/L in the sttic-renewl test nd 8.0 mg N/L in the flow-through test. Results of these studies indicte tht the mmoni toxicity dt generted from sttic-renewl tests with somewht vrible mmoni concentrtions were comprble to those from flow-through tests with more consistent totl mmoni concentrtions. Conducting chronic toxicity tests is more expensive nd time-consuming compred to cute toxicity tests. In ddition, specific methods my not be vilble for conducting chronic toxicity tests with some mussel species. Therefore, the cute chronic rtio (ACR) my be useful to estimte chronic effect from cute toxicity dt. We clculted ACR for copper or totl mmoni bsed on cute nd chronic toxicity dt for four mussel species tested in our previous [3] nd present studies (i.e., 4-d EC50 for survivl divided by the 28-d ChV for survivl nd growth). The ACR for copper rnged from 4.0 to 4.7 for rinbow mussel, ftmucket, nd oyster mussel, wheres the ACR for totl mmoni rnged from 11 to 18 for rinbow mussel, ftmucket, nd wvy-ryed lmpmussel (Tble 9). These vlues were reltively consistent mong species, nd were within the rnge of ACR for copper or totl mmoni reported in the U.S. EPA WQC [24,25]. CONCLUSION In summry, stndrdized chronic toxicity tests with twomonth-old juvenile mussels were conducted successfully in flow-through diluter systems, nd ll chronic effect concentrtions of copper nd totl mmoni for survivl nd growth were below the U.S. EPA 1999 ph- nd temperture-dependnt chronic WQC for totl mmoni nd the 1996 hrdness-dependent chronic WQC for copper. Results indicte tht the erly life-stges of freshwter mussels re chroniclly sensitive to copper or mmoni, nd the U.S. EPA 1996 chronic WQC for copper nd 1999 chronic WQC for mmoni my not be dequtely protective of the mussel species tested. For the wter tested in the present study, the 2007 biotic lignd model bsed chronic WQC for copper is lower thn the 1996 hrdness-bsed WQC nd my be more protective of chronic toxicity to mussels. Additionl studies lso re needed to test the ssumption tht 28-d exposures with juvenile mussels cn be used to predict the effects of toxicnts on other life stges of mussels in long-term tests. Acknowledgement We thnk J. Arms, E. Brunson, B. Kiser, R. Mir, T. My, C. Mebne, nd D. Whites for technicl ssistnce. Funding for this reserch ws provided in prt by U.S. Fish nd Wildlife Service (USFWS, Environmentl Contminnts Progrm, Project N037) nd the U.S. EPA (Project DW ). We lso thnk J. Firchild, C. Schmitt, nd three nonymous reviewers for helpful comments on n erlier version of the mnuscript. This pper hs been reviewed in ccordnce with USGS nd USFWS peer review policy. Publiction does not signify tht the contents reflect the views of the USFWS. References to trde nmes or mnufcturers do not imply government endorsements of commercil products. REFERENCES 1. Augspurger T, Keller AE, Blck MC, Cope G, Dwyer FJ Derivtion of wter qulity guidnce for protection of freshwter

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