Freshwater Bivalve Ecotoxoicology - Chapter 5 ppsx

AQUATIC TOXICITY TESTING WITH GLOCHIDIA, JUVENILE, AND ADULT LIFE STAGES OF FRESHWATER MUSSELS METHODS FORCONDUCTING ACUTEWATER-ONLYTOXICITYTESTS WITHGLOCHIDIA OFFRESHWATERMUSSELS Review

Trang 1

5 Laboratory Toxicity Testing with

Freshwater Mussels

Christopher G Ingersoll, Nicola J Kernaghan, Timothy

S Gross, Cristi D Bishop, Ning Wang, and Andy Roberts

INTRODUCTION

Numerous laboratory toxicity studies have been conducted with freshwater mussels in an attempt tounderstand the role of contaminants in the decline of field populations of mussels(Chapter 7).Inthese studies, early life stages of mussels of several species were highly sensitive to some metalsand ammonia in water exposures when compared to many of the most sensitive species of otherinvertebrates, fish, or amphibians that are commonly used to establish U.S Environmental Protec-tion Agency (USEPA) Water Quality Criteria (WQC) (Augspurger et al 2003; USGS 2005a,2005b) Importantly, results of these studies indicate WQC for individual chemicals establishedfor the protection of aquatic organisms may not be adequately protective of sensitive stages offreshwater mussels This chapter provides a summary of methods from over 75 laboratory toxicitystudies conducted with freshwater mussels and also provides an overview of a standardized methodfor conducting water-only acute and chronic laboratory toxicity tests with glochidia and juvenilefreshwater mussels (ASTM 2006a) Three life stages (glochidia, juveniles, and adults) have beenused to conduct laboratory toxicity tests with mussels Within this chapter, toxicity studies areseparated according to the medium of exposure (aqueous, sediment, and host fish) Each sectionbegins with a review of the methods used to conduct toxicity tests (e.g., obtaining organisms,duration of exposure, exposure chambers, and toxicity endpoints) Each section also discussesissues that have been identified regarding the routine application of the methods (e.g., to generatedata for the derivation of WQC) and discusses research needs The final section of this chapterreviews the use of the Asian clam (Corbicula fluminea) as a surrogate for assessing effects on nativeunionids Finally, a summary of future research needs for improving methods used to conduct acuteand chronic toxicity tests with freshwater mussels is provided

AQUATIC TOXICITY TESTING WITH GLOCHIDIA, JUVENILE, AND ADULT LIFE STAGES OF FRESHWATER MUSSELS

METHODS FORCONDUCTING ACUTEWATER-ONLYTOXICITYTESTS WITHGLOCHIDIA

OFFRESHWATERMUSSELS

Review of Methods

Conditions that have been used to conduct acute toxicity tests with glochidia of freshwater musselsare summarized inTable 5.1including the test conditions recommended in ASTM (2006a) Theprocedures outlined in Table 5.1 are consistent with acute toxicity testing methods for ﬁsh, macro-invertebrates, and amphibians (ASTM 2006c) and with acute toxicity testing methods for saltwater

4284x—CHAPTER 5—19/10/2006—19:50—CRCPAG—XML MODEL C – pp 95–134

95

Trang 2

TABLE 5.1

Summary of Test Conditions Used to Conduct Toxicity Tests with Glochidia of Freshwater Mussels

Conditions Johnson et al.(1990, 1993) Lasee (1991)

Huebner and Pynnonen (1992) a

Goudreau, Neves, and Sheehan (1993) Jacobson et al.(1997) Ruessler (1997) McCann (1993)Keller and

Klaine, Warren, and Summers (1997) USGS (Unpub-lished Data) Recommended Test Con-ditions in ASTM (2006a)

1 Species tested Utterbackia

imbecillis b Lampsilis

cardium c Anodonta

cygnea, Anodonta anatina

Villosa iris Multiple species d Multiple species e V iris U imbecillis Multiple species f NA g

2 Test type Static Static Static Renewal Static Static Static Static Static, renewal,

ﬂow-through Static, renewal, or ﬂow-through(depending on chemical

tested)

3 Test duration

(hours) 24 48 24, 48, 72, 144 24 24, 48 4, 24, 48 24 24, 48 6, 24, 48 6, 24 (up to 48 depending onviability of glochidia)

5 Light quality Ambient lab light NR h NR NR NR NR NR Ambient lab light Ambient lab light Ambient lab light

8 Test chamber 100-mL beaker 250-mL or

300-ml beaker 400-mL beaker Basket of meshnetting in 4-L

chamber

12-well plate 6-well plate 12-well plate 12-well plate 200-mL dish or

300-mL beaker

100-mL glass chamber (minimum)

Flush gills with syringe Cut gills andseparate

glochidia from marsupia

NR Flush gills with

syringe Flush gills withsyringe Flush gills withsyringe Flush gills with syringe

11 Age of test

organisms (hours)

12 Number of

organisms per test chamber

10 10 1000–3000 Several hundreds 50–75 50–100 40 50–100 About 1000 About 500 (1000 for repeated

sampling during a toxicity test)

13 Number of replicate

chambers per treatment

samples with about 100 glochidia

2, Counting 3 samples with about 100 glochidia

subsample with about

100 glochidia from each replicate

3, Counting a subsample with about 100 glochidia from each replicate

maintained above acceptable concentration

Trang 3

16 Dilution water Reconstituted

water, hardness 40–50 mg/L

as CaCO 3

Hardness

150 mg/L as CaCO 3

Tap water Dechlorinated

efﬂuent water Dechlorinatedtap water or

Clinch River water, VA

Reconstituted water, hardness 47–

76 mg/L as CaCO 3

Sinking Creek water, VA Hardness99–107 mg/L

as CaCO 3

Reconstituted water, hardness

170 mg/L as CaCO 3

Depends on experimental design

17 Water quality DO, pH,

hardness, alkalinity, conductivity

DO, pH, hardness, alkalinity, conductivity

pH, Ca, Cu, Zn DO, pH,

hardness, alkalinity, conductivity

DO, pH, ammonia, hardness, alkalinity, conductivity

18 Endpoint Survival (valve

closure with culture medium)

Survival (valve closure with NaCl)

Survival (valve closure with KCl)

Survival (valve closure with salt solution)

Survival (valve closure with saline solution)

19 Control survival

The Last Column Provides a Summary of Recommended Conditions That Can be Used to Conduct Toxicity Tests with Glochidia Based on ASTM (2006a)

quadrula, Quadrula pustulosa, Leptodea fragilis, Leptodea leptodon, Venustaconcha ellipsiformis, V iris.

Trang 4

bivalve mollusks (ASTM 2006c) Gravid female mussels are usually collected from the ﬁeld andheld in the laboratory before isolating glochidia to start a toxicity test (ASTM 2006a,Chapter 4).

Alternatively, Zimmerman and Neves (2002) suggested glochidia of some species (includingVillosa iris and Actinonaias pectorosa) could be extracted in the field from a female and transportedback to the laboratory in cool water where glochidia can remain viable for several days without areduction in ability to successfully attach to a host fish This procedure may be particularly usefulwhen glochidia of endangered species are extracted in the field and the female mussels are thenimmediately returned to their habitat Mature glochidia are typically flushed from the marsupium of

a female mussel using a syringe ﬁlled with water Glochidia have also been isolated by cutting asection of gill from the female mussel and then teasing out the glochidia in water (This technique isdestructive to the adult female and may not be appropriate for use in isolating glochidia forconducting toxicity tests.) No studies were identiﬁed where glochidia were isolated for toxicitytesting from conglutinates released into the water by female mussels

Before starting an exposure, the viability of glochidia is typically evaluated by a response

to the addition of a concentrated solution of NaCl or KCl Mature and healthy glochidia willsnap shut in response to the addition of a saline solution Immature glochidia isolated from themarsupium of a female will often still be enclosed in the egg membrane and will be fragileand tend to fracture (Chris Barnhart, Missouri State University, Springﬁeld, MO, personalcommunication) Tests are usually started if greater than 80 to greater than 90% viability ofthe glochidia is observed (Huebner and Pynnonen 1992; Jacobson et al 1997; Klaine, Warren,and Summers 1997; ASTM 2006a) If immature glochidia are isolated from a female mussel,these glochidia should not be used for testing Exposures are usually started the same day thatglochidia are isolated from a female by pooling glochidia from at least three females without

an extended acclimation period in the exposure water before the start of a toxicity test (ASTM2006a) The viability of glochidia isolated from each female should be evaluated before theyare pooled together Toxicity tests can be conducted with glochidia obtained from one female(e.g., when a limited number of endangered species are available for testing); however, theresults of tests conducted with a limited number of mussels should be interpreted with caution.Additional research is needed to determine the minimum number of females that should besampled to obtain glochidia to start a toxicity test This research might include an evaluation

of the variability in sensitivity of glochidia obtained from individual females using a variety

of toxicants

ASTM (2006a) provides a list of recommended test conditions for conducting toxicity tests withglochidia isolated from female mussels The list of recommended test conditions is based on thevarious methods outlined inTable 5.1and on the conditions used to conduct an inter-laboratorytoxicity test with glochidia (ASTM 2006a) ASTM (2006a) recommends that toxicity testswith glochidia should be conducted at 208C with a 16L:8D photoperiod at an illuminance ofabout 100–1000 lux (Table 5.1) The endpoint measured in toxicity tests with glochidia is survival(viability) as determined by the response of organisms to the addition of a solution of NaCl.Glochidia that close their valves with the addition of a salt solution are classiﬁed as alive (viable)

in a toxicity test For most species, the duration of a toxicity test conducted with glochidia should be

up to 24 hours with survival measured at 6 and 24 hours Control survival is typically greater than90% at the end of 24-hour toxicity tests conducted with glochidia Longer duration toxicity tests withglochidia (e.g., 48 hours) can be conducted as long as control survival greater than 90% is achieved.However, toxicity tests conducted for greater than 24 hours with glochidia may not be as ecologi-cally relevant given the short period of time between release of glochidia from a female mussel untilencystment on a host ﬁsh (ASTM 2006a; Chapter 4) Effect concentrations are typically calculatedbased on the percentage of viable glochidia in the control at a particular sampling time

ASTM (2006a) recommends the use of glass test chambers for conducting toxicity tests withglochidia Test chambers should be a minimum volume of 100 mL containing a minimum of 75 mL

of dilution water Static, renewal, or ﬂow-through conditions can be used depending on the

Freshwater Bivalve Ecotoxicology98

Trang 5

chemical being tested Glochidia are not fed during the toxicity test, and aeration of dilution water istypically not necessary Dilution water should be a source of water that has been demonstrated tosupport survival of glochidia for the duration of the toxicity test For site-speciﬁc evaluations, thecharacteristics of the dilution water should be as similar as possible to the site of interest Thenumber of replicates and concentrations tested depends in part on the signiﬁcance level selected andthe type of statistical analysis ASTM (2006a) recommends a minimum of three replicates should

be tested, each replicate containing about at least 500 glochidia (preferably 1,000 cate if survival is to be evaluated in subsamples of glochidia collected during the toxicity test).Survival can be determined throughout the toxicity test by subsampling each replicate (e.g., bysubsampling about 100 glochidia at 6 and 24 hours and then placing these organisms into one well

glochidia/repli-of a multi-well plate to determine survival with the addition glochidia/repli-of a salt solution)

Toxicity tests with glochidia have been conducted for up to 144 hours, but 24 and 48-hourexposures are most often used (Table 5.1) The relatively short duration of toxicity tests withglochidia is based on the relatively short duration between the release of glochidia into the watercolumn and encystment on the host and on the relatively short survival time of glochidia afterisolation from the female mussel(Table 5.2) If the life history of the glochidia for a particularspecies is not known (e.g., the host required for encystment or how long glochidia released from afemale mussel can remain in the water column before encysting on a host), it might be appropriate

to conduct toxicity tests with glochidia for longer than 24 hours as long as 90% control survival can

be achieved at the end of the test (ASTM 2006a)

Issues Regarding the Use of Methods

Glochidia and juvenile mussels of several genera have been found to be highly sensitive to somemetals and to ammonia in water exposures compared to many of the most sensitive genera of otherinvertebrates, ﬁsh, or amphibians that are commonly tested (Chapter 7, Cherry et al 2002;Augspurger et al 2003; USGS 2006a, 2006b) However, concerns have been expressed regardingthe use of toxicity data generated with glochidia in the derivation of WQC (Charles Stephan,USEPA, Duluth, MN; personal communication) These concerns mainly include: (1) the duration

of the toxicity tests, (2) the quality of the glochidia at the start of a test, and (3) the test acceptabilitycriteria The following section provides information that attempts to address these concerns Areas

of ongoing research or need for future research are also identiﬁed

Duration of the Toxicity Test

1 How long should acute tests with glochidia be conducted (i.e., based on the life history ofthe species)?

There are nearly 300 species of freshwater mussels in North America, and the length oftime that glochidia remain viable after release from the marsupium of a female into theenvironment depends on the life history of the species and the temperature of the water

exceed one week and may be dependent on temperature (Zimmerman and Neves 2002);however, some reports are anecdotal (Murphy 1942; Matterson 1948; Tedla andFernando 1969) Glochidia of some species released in conglutinates remain viable fordays or weeks after release into the environment (Chris Barnhart, personal communi-cation) Glochidia of several species, including Anodonta spp., remain viable while free

in the environment for 7–14 days (Howard and Anson 1922; Mackie 1984; Huebner andPynnonen 1992; Pynnonen 1995)

Table 5.2 provides a summary of laboratory studies that have evaluated survival times

of glochidia after removal from the marsupium of the female or survival time based onresults reported in toxicity tests conducted with glochidia For example, Zimmerman and

Trang 6

TABLE 5.2

Survival Time of Glochidia after Removal from Female Unionid Mussels

Duration of Viability

Actinonaias ligamentina 20 7 (O90); 8 (O75); 9 (O50) USGS (2004)

Alasmidonta heterodon 20 2 (O90); 2 (O75); 2 (O50) USGS (2004)

20 !1 (O90); 3 (O75) Bringolf et al (2005) Elliptio dilatata 20 !1 (O90); 1 (O75); !2 (O50) Bringolf et al (2005)

Lampsilis fasciola 20 6 (O90); 7 (O75); 8 (O50) Wang et al (2003)

20 1 (O90); 2 (O75); 3 (O50) Bringolf et al (2005)

20 2 (O90; 4 (O75); 5 (O50) Bringolf et al (2005) Lampsilis raﬁnesqueana 20 6 (O90); 6 (O75); 6 (O50) USGS (2004)

20 8 (O90); 9 (O75); 10 (O50) Wang et al (2003)

20 1 (O90); 3 (O75); 4 (O50) Bringolf et al (2005)

Leptodea fragilis 20 1 (O90); 3 (O75); 4 (O50) Wang et al (2003)

Leptodea leptodon 20 0.25 (O90); 1 (O75); 2 (O50) USGS (2004)

Potamilus alatus 20 6 (O90) 6 (O75); 6 (O50) Wang et al (2003)

Potamilus ohiensis 20 5 (O90); 6 (O75); 7 (O50) Wang et al (2003)

Quadrula quadrula 20 1 (O90); 1 (O75); 2 (O50) Wang et al (2003)

Quadrula pustulosa 20 !1 (O90); 1 (O75); 1 (O50) Wang et al (2003)

Utterbackia imbecillis 21 10 (O80); 14 (O50) Fisher and Dimock (2000)

1993) Venustaconcha

ellipsiformis

20 2 (O90); 3 (O75); 3 (O50) Wang et al (2003)

20 5 (O90); 5 (O75); 6 (O50) Wang et al (2003)

(continued)

Trang 7

Neves (2002) report that the viability of glochidia of V iris was greater than 75% for

8 days at 108C and 2 days at 258C, and viability of glochidia of A pectorosa was greaterthan 75% for 13 days at 108C and 5 days at 258C (Table 5.2) Similarly, glochidia ofUtterbackia imbecillis may survive up to 19 days but exhibit 50% mortality within13.5 days (Fisher and Dimock 2000) Survival of isolated glochidia from manyspecies listed in Table 5.2 is typically greater than 90% after two to three days;however, the viability of glochidia for a particular species should be determinedbefore the start of an exposure For example, glochidia of L teres and E capsaeformiswere viable for only four to six hours and glochidia of M nervosa and Q quadrula wereviable for one day after removal from the marsupium of the female (Table 5.2) There-fore, 24 hours is a reasonable time period to conduct toxicity tests with glochidia of manyspecies at 208C, although shorter or longer tests might be needed for a particular speciesdepending on glochidia survival time and the life history characteristics of the species(i.e., survival of glochidia in the control must be greater than 90% at the toxicity test)(ASTM 2006a)

The time between the release of glochidia from the marsupium of the female mussel toattachment of these glochidia on a host may only take a few seconds for some species,but hours are required for the gill tissue of a fish to migrate to form a cyst around theglochidia During that time, the glochidia may be exposed to water-borne toxicants.Anodontinae species releases glochidia directly into water, which remain viable fordays in order to effectively infest their host fish Therefore, a prolonged glochidial testwould have ecological relevance for these species Other species release glochidia inmucus strands that coat the bottom or remain suspended on vegetation, waiting for theirhosts to swim by, and still other species package glochidia in conglutinates that serve as alure to host fish Hence, glochidia of these species may also be in water for extendedperiods of time; however, it is not known how exposure to water-borne contaminantswould be influenced by the mucus or conglutinate surrounding the glochidia Toxicitytests conducted for 24 hours with glochidia may not be as ecologically relevant astoxicity tests conducted with juvenile mussels, but they may be useful for some purposessuch as deriving concentrations of a chemical that may be protective of the species Use

of glochidia to evaluate the relative sensitivity of a particular mussel species to chemicalswould be particularly useful when evaluating species where only a limited number ofadult mussels are available for methods development or for producing juvenile musselsfor toxicity testing Moreover, the host ﬁsh for some species of mussels or techniques fortransforming juvenile mussels in the laboratory may be unknown(Chapter 4)

TABLE 5.2 (Continued)

Duration of Viability

(1993)

a The value based on control survival in 24- or 48-hour toxicity tests.

b NR, not reported.

Trang 8

The relatively short duration of toxicity tests with glochidia is based on the relativelyshort duration between release of glochidia into the water column and encystment on thehost and on the relatively short survival time of glochidia after isolation from the femalemussel If the life history of a particular species is not known (e.g., the host required forencystment or how long glochidia released from a female mussel can remain in the watercolumn before encysting on a host), it might be appropriate to conduct toxicity tests withglochidia for longer than 24 hours as long as 90% control survival can be achieved at theend of the test.

2 How long can glochidia survive and still be able to attach to a host?

Glochidia of some species can still attach to a host for several days after release from afemale depending on temperature (Chris Barnhart, personal communication) Themaximum time at which greater than 50% of U imbecillis metamorphosed in a tissueculture medium was nine days after isolation from a female (Fisher and Dimock 2000).Zimmerman and Neves (2002) reported that glochidia can successfully attach to a host one

to two weeks after isolation from a female A future research project could be to conduct aseries of toxicity tests to determine if there is a change in sensitivity over time afterglochidia have been released into the environment Sensitivity of L siliquoidea glochidiaheld for 24 hours after isolation from a female was similar to newly-released glochidia inexposures to copper (Wang et al 2003) The sensitivity of glochidia held in an extra piece

of the marsupium in a refrigerator overnight was similar to the sensitivity of glochidiatested immediately after isolation from a female in toxicity tests conducted with zinc orcopper (Jerry Farris, Arkansas State University, State University, AK; personal communi-cation) Ultimately, it is more practical to base duration of exposure on survival of controlorganisms in the laboratory rather than on an estimate of the length of time glochidia cansurvive and still attach to a host (e.g.,Table 5.2)

3 Are there data that indicate that effect concentrations do not change very much during thelast half of a toxicity test (i.e., does the EC50 at 6, 24, 48, or 96 hours differ)?

There are limited studies with glochidia that have compared changes in toxicity over thistimeframe The toxicity of copper (Jacobson et al 1997; Wang et al 2003), ammonia(Wang et al 2003), and chlorine (Wang et al 2003) decreased over 48–96-hour exposures

In contrast, no change in the toxicity of several pesticides was observed in 24–48-hourexposures (Keller and Ruessler 1997; Bringolf et al 2005) If glochidia for a particularspecies are able to survive for more than 24 hours, then a 24-hour toxicity test should beconsidered Importantly, researchers are encouraged to design studies that generatetoxicity data throughout the exposure period (e.g., reporting 6, 24, and 48-hour responses)(ASTM 2006b) However, generating data for a six-hour exposure period is logisticallydifﬁcult in an eight-hour day

Quality of Glochidia at the Start of a Toxicity Test

1 How should the quality of glochidia be determined at the start of a toxicity test? Is theuse of a solution of NaCl (or KCl) to determine the percentage of glochidia exhibitingvalve closure an appropriate method to judge the acceptability of glochidia used to start

a toxicity test? Does the response of glochidia to a solution of NaCl (or KCl) relate tothe ability of glochidia to attach to a host? Is there an independent way of determining

if glochidia are alive or healthy at the start (or end) of a toxicity test?

Valve closure is an ecologically relevant endpoint that is critical for glochidia tosuccessfully transform on the host If glochidia do not snap shut, the glochidia should

be considered ecologically dead (Huebner and Pynnonen 1992; Goudreau, Neves, andSheehan 1993; McCann 1993; Jacobson et al 1997) The response of glochidia in

Trang 9

toxicity tests was similar when either KCl or fish plasma was used to make glochidiaclose at the end of an exposure (Huebner and Pynnonen 1992) Decreased response toKCl was considered an indication of reduced glochidia viability and thus reducedcapability to attach to the fish host (Pynnonen 1995) A significant correlationwas observed between the response of glochidia to KCl and the ability of glochidia

of U imbecillis to metamorphose to the juvenile life stage (Fisher and Dimock 2000).Zimmerman and Neves (2002) reported a correspondence between the response ofglochidia of V iris and A pectorosa to NaCl and the ability to infest a host fish.Jacobson et al (1997) reported glochidia of V iris that responded to the addition ofNaCl following an exposure to copper were able to attach to a host fish with noimpairment of subsequent metamorphosis to juvenile mussels Results of thesestudies indicate that addition of a solution of NaCl or KCl can be used to estimatethe condition of glochidia While either a solution of salt or fish plasma could be used

to determine the percentage of organisms closing, it is easier to work with NaClcompared to KCl or ﬁsh plasma

2 Should there be a holding time for glochidia after harvesting but before application of asaline solution to determine if glochidia that are initially closed might open?

Mature glochidia are not typically closed after being isolated from a female mussel.Glochidia that are closed after isolation from a female may reopen after being held inclean water a few hours (Goudreau, Neves, and Sheehan 1993; Dick Neves, TeresaNewton, USGS, LaCrosse, WI; personal communications)

3 Will immature, stressed, or unhealthy glochidia close when exposed to a salinesolution? Could glochidia be alive and successfully attach to a host but not closewhen exposed to a saline solution? Are broken glochidia frequently observed at thestart of a test? Would the presence of broken glochidia be indicative of stress duringharvesting?

Immature glochidia that are free of the egg membrane or mature and healthyglochidia will close when exposed to a salinity challenge However, immatureglochidia are generally enclosed in an egg membrane and are fragile and tend tofracture, thus should not be used for toxicity testing The best approach for avoidingthe use of immature glochidia in toxicity testing is to sample female mussels at a time

of the year when the organisms would be expected to be releasing mature glochidia(Jess Jones, US Geological Survey, Blacksburg, VA; personal communication).Stressed or unhealthy glochidia could either be opened or closed before the start of atest If stressed or unhealthy glochidia were to close when exposed to a salinity chal-lenge, then these individuals would be used in a toxicity test Measurement of theviability of glochidia in the control at the end of a toxicity test would help to identifystressed or unhealthy glochidia Results of reference-toxicant tests should also be used

to evaluate the health of the glochidia used to conduct the test (ASTM 2006a) Brokenglochidia have not been observed at the start of a test (Chris Barnhart, Jerry Farris, DickNeves, Teresa Newton, Ning Wang, USGS, Columbia, MO; personal communications).The presence of broken glochidia may indicate that the glochidia are immature andshould not be used for testing

Test Acceptability Criteria for Toxicity Tests with Glochidia

1 What criteria should be used to judge acceptability of a toxicity test conducted withglochidia?

ASTM (2006a) recommends that the age of glochidia should be less than 24 hoursold at the start of the toxicity test Viability of glochidia isolated at the beginning of a

Trang 10

toxicity test must be greater than or equal to 80% (preferably greater than or equal to90%) Average survival of glochidia in the control at the end of a test must be greaterthan or equal to 90% ASTM (2006a) also recommends that subsamples of each batch

of test organisms used in toxicity tests should be evaluated using a reference toxicant(e.g., NaCl or CuSO4) Data from these reference-toxicant tests can be used to assessgenetic strain or life-stage sensitivity of test organisms to select chemicals

2 Should glochidia be rinsed before use in a toxicity test? Would rinsing glochidia beforethe start of a test be stressful to the organisms?

Glochidia should be rinsed with culture or dilution water after removal frommarsupia to (1) eliminate tissues or excess mucus from the excised glochidia thathave a high potential for fungal growth and subsequently could affect the survival(toxicity tests) or transformation of glochidia (propagation) and (2) reduce thenumber of protozoans that may be present in the excised gill that could also affectglochidia survival or transformation (ASTM 2006a) Rinsed glochidia have beenobserved to successfully transform on ﬁsh or in artiﬁcial media and high controlsurvival in toxicity tests has been reported using glochidia that have been rinsed(Huebner and Pynnonen 1992; Johnson, Keller, and Zam 1993; Myers-Kinzie 1998;Milam et al 2005)

3 Should glochidia be acclimated to test conditions before the start of a toxicity test?Glochidia are not typically acclimated to the water-quality characteristics of thedilution water before the start of a toxicity test (Table 5.1) Most of these exposuresare started the same day that glochidia are isolated from marsupia of the females.Therefore, minimal time is available to acclimate glochidia to the dilution waterbefore the start of a test In order to maintain organisms in good condition andavoid unnecessary stress, ASTM (2006a) recommends that organisms should not besubjected to rapid changes in temperature or water quality before the start of a test.Glochidia can be acclimated in a mixture of 50% culture water and 50% test waterand gradually adjusted to the test temperature within about two hours before the start

of an exposure (ASTM 2006a) Investigators have held adult mussels under testconditions before isolation of glochidia (e.g., Huebner and Pynnonen 1992), whichwould result in acclimating glochidia to the selected exposure temperature in thetoxicity test However, brooding glochidia in the marsupium are in contact with thehemolymph of the female that is physically isolated from direct contact with water(Silverman, McNeil, and Dietz 1987) In addition, glochidia are typically releasedinstantaneously into the surrounding water from the marsupium of the female mussel.Therefore, holding the female mussels in the dilution water before isolating glochidiafor toxicity testing would probably have a minimal inﬂuence on the ability ofglochidia to acclimate to the conditions of the dilution water

METHODS FORCONDUCTING WATER-ONLYTOXICITY TESTS WITHJUVENILE

FRESHWATERMUSSELS

Review of Methods

ASTM (2006a) provides a list of recommended test conditions for conducting toxicity tests withjuvenile mussels The list of recommended test conditions is based on the various methods outlined in

(ASTM 2006a) ASTM (2006a) recommends that toxicity tests with juvenile mussels be conducted at208C with a 16L:8D photoperiod at an illuminance of about 100–1,000 lux (Table 5.3) Toxicity testsare typically started with newly-transformed juvenile mussels less than ﬁve days after the release

Trang 11

TABLE 5.3

Summary of Test Conditions Used to Conduct Toxicity Tests with Juvenile Freshwater Mussels

Conditions Johnson et al.(1990, 1993)

Jacobson (1990), Jacobson

et al (1993) Lasee (1991) Keller and Zam(1991)

Klaine, Warren, and Summers (1997) Scheller(1997) Myers-Kinzie(1998)

Dimock and Wright (1993)

1 Species

tested Utterbackia imbecillis b Villosa

nebulosa, Villosa iris, Anodonta grandis c

Lampsilis cardium d Mulitple

species e U imbecillis V iris Lampsilis

siliquoidea

U imbecillis, Pyganodon cataracta

L cardium Lampsilis ventricosa

U imbecillis Villosa

nebulosa

V iris Mulitple species f L siliquoidea, Epioblasma capsaeformis,

V iris g

NA h

through Renewal Renewal Artiﬁcial

stream Renewal Renewal, ﬂow through

Flow through Static, renewal or

ﬂow-through (depending on duration of exposure and chemical tested)

Petri dish

12-well plate Petri dish 120-mm diam tub with mesh bottom in 4-L chamber

132 by

90 by

130 mm chamber

Covered 250-mL izing dish

50-mm diam glass tub with mesh bottom

in 250-mL Chamber

Dish covered with mesh

30-mL beakers submer- ged in a 1-L glass beaker

50- or 300-mL beaker

300-mL beaker

Static or renewal: 50-mL beakers (minimum);

ﬂow-through: 300-mL beakers (minimum)

9 Test solution

volume (mL)

30 (minimum); ﬂow-through: 200 (minimum)

10 Procedure

for obtaining juveniles

Artiﬁcial media Fish host Fish host Fish hostor artiﬁcial

media

Fish host or artiﬁcial media

Fish host Artificial media Artificial media Fish host Fish host Artificialmedia Fish host Fish host Fish host Fish host Fish host

11 Age of test

organisms (days)

(minimum) Chronic:10 (minimum) (continued)

Trang 12

et al (1993) Lasee (1991)

Keller and Zam (1991)

Klaine, Warren, and Summers (1997) Scheller (1997) Myers-Kinzie (1998)

Dimock and Wright (1993)

Newton

et al.

(2003) Lasee (1991) Wade (1992) a Jacobson (1990)

Valenti

et al.

(2005) USGS (2004) USGS (2005a, 2005b, 2005c)

Recommended Test Conditions ASTM (2006a)

13 Number of

replicate chambers per treatment

(minimum) Chronic:3 (minimum)

cultured phyto plankton

Algae and silt

and sediment

None Instant algae mixture i

Acute:none Chronic Algae

oxygen is maintained above acceptable concentration

16 Dilution

Water Reconstituted water, hardness 40–50 mg/L as CaCO 3

Clinch River water, VA Hardness

150 mg/L as CaCO 3

Reconstituted water, hardness 47–76 mg/L as CaCO 3

Recon stituted water, hardness 99–107 mg/L as CaCO 3

Sinking Creek water, VA

Hardness 150 mg/L

as CaC0 3

Tennessee River Clinch River water, VA

100 mg/L as CaCO 3

170 mg/L as CaCO 3

Reconstituted water- hardness

170 mg/L as CaCO 3

DO, pH, hardness, alkalinity, conductivity

pH, hardness NR DO, pH,hardness,

alkalinity, conductivity

DO, pH, hardness, alkalinity, conductivity

ammonia, hardness, alkalinity, conductivity

DO, pH, ammonia, hardness, alkalinity, conductivity

18 Endpoints Survival

ment)

(move-Survival (gaped valves, foot activity or stained with neutral red)

Survival (foot or ciliary movement)

Survival (activity and heartbeat)

Survival (gaped valves with foot and ciliary activity)

Survival (heart beat and ciliary action)

Survival (foot or valve move ment)

Survival (foot, valve or ciliary activity, heartbeat)

Survival, growth, ratio of stressed to alive

Survival (foot or ciliary movement), growth (length and height)

Survival (Ciliary action)

Survival (extruded foot and gaping valves)

Survival, growth Survival (foot or shell movement) and growth (shell length)

Survival (fool or shell movement) and growth (shell length)

Survival (foot movement), growth (shell length)

19 Control

survival (%)

Chronic:O80 (should)

Trang 13

The last column provides a summary of recommended conditions that can be used to conduct toxicity tests with juvenile mussels as outlined in ASTM (2006a) In the last Column, Acute Tests are Tests Conducted for up to 96 hours and Chronic Tests are Tests Conducted for at least 21 days.

a See also Masnado, Geis, and Sonzogni (1995), McKinney and Wade (1996), Keller, Ruessler, and Kernaghan (1999).

b Formerly A imbedllis.

c See also McCann (1993) for two- to four-day exposures with Villosa iris, A pectomsa, M conradius.

d Formerly L ventricosa.

e A imbecillis, V lienosa, V villosa, U imbecillis, Lampsilis straminea daibomensis, L subangulata, Elliptic icterina See also Keller (1993), Keller and Ruessler (1997).

f V iris, E capsaeformis, L fasciola, L siliquoidea, L abrupta, L raﬁnesqueana, L leptodon.

g Bringolf et al (2005) has adapted this method to conduct 21-day toxicity tests with four-month old juvenile A ligamentina.

h NA, not applicable; NR, not reported.

i See USGS (2005a) for a description of the procedure used to prepare this instant algae mixture.

Trang 14

from the host; however, some toxicity tests have been started with two- to four-month-old juvenilemussels Acute toxicity tests with juvenile mussels are typically conducted for 96 hours with survivalmeasured at 48 and 96 hours Chronic toxicity tests started with two- to four-month-old juvenilemussels have been conducted for 21–28 days with measures of survival (based on movement of thefoot) and growth (based on shell length) Control survival is typically greater than 90% at the end of96-hour toxicity tests conducted with juvenile mussels and is typically greater than 80% at the end oftoxicity tests conducted for 10–28 days with juvenile mussels(Table 5.3;ASTM 2006a).

In acute static tests, glass test chambers should be a minimum volume of 50 mL containing aminimum of 30 mL of dilution water (ASTM 2006a) In chronic tests or in ﬂow-through tests, glasschambers should be a minimum volume of 300 mL containing a minimum volume of 200 mL ofdilution water Static, renewal, or ﬂow through conditions can be used depending on the chemicalbeing tested Juvenile mussels are not typically fed during acute toxicity tests Algae have been used

as a food source in toxicity tests conducted for 10–28 days (Table 5.3; ASTM 2006a)

The number of replicates and concentrations tested depends in part on the significance levelselected and the type of statistical analysis In 96-hour toxicity tests, ASTM (2006a) recommends aminimum of 20 organisms should be exposed to each concentration (e.g., four replicates eachcontaining a minimum of five juvenile mussels) It may be desirable to test only five juvenilemussels in each replicate when a limited number of test organisms are available or when testorganisms are relatively small (e.g., when juvenile mussels are small, it may be difficult toobserve more than about five test organisms simultaneously in a replicate test chamber under themicroscope) However, some investigators have tested 10–20 juvenile mussels in each replicate Inchronic toxicity tests, a minimum of three replicates should be tested, each replicate containing aminimum of 10 juvenile mussels

Toxicity tests with juvenile mussels are typically started with organisms that have been formed with a fish host (ASTM 2006a); however, artificial media has also been used to transformjuvenile mussels for use in toxicity testing (Johnson, Keller, and Zam 1993; Clem 1998; Hudson et al.2003) ASTM (2006a) recommends testing of juvenile mussels that have been transformed on a fishhost due to uncertainties regarding the sensitivity of juvenile mussels transformed using artificialmedia Numerous investigators have observed high mortality of juvenile mussels about four to sixweeks after transformation (e.g., Anne Keller, USEPA, Athens, GA; Don Cherry, Jerry Farris, TeresaNewton; personal communications) As a result of this problem, the duration of toxicity tests startedwith newly-transformed juvenile mussels is less than 14 days with survival or growth measured at theend of the exposures (Table 5.3) Food (mixtures of different species of algae) and sediment havebeen added to exposure chambers, but some investigators have found that newly-transformedjuvenile mussels will survive for at least 14 days without the addition of food (Table 5.3; ASTM2006a) For example, USGS (2004) determined the acute toxicity of copper in 48-hour tests withjuvenile L siliquoidea and Lampsillis rafinesqueana that had been held for 10 days under controlconditions (e.g., with the replacement of dilution water but without the addition of food) Similar48-hour EC50s were observed in tests conducted with juvenile mussels held for 10 days before testingcompared to tests started with newly-transformed juvenile mussels Results of these tests indicatethat the sensitivity of juvenile mussels did not change over the 10-day exposure without feeding.Hence, toxicity tests conducted for up to 10 days without feeding may provide reliable data forevaluating effects of chemicals on mussels in exposures longer than 4 days

trans-The high mortality of newly-transformed juvenile mussels in toxicity tests conducted forlonger than 14 days is likely related to a lack of understanding of the nutritional requirements

of mussels at this life stage Newly-transformed juvenile mussels depend on pedal feeding toobtain food (cilia on the foot are used to move food into the juvenile mussel) Juvenile musselsgradually begin to use a combination of pedal and filter feeding to obtain food until the musselseventually depend on filter feeding to obtain food by about six months in laboratory culturessupplied with a silt-clay sediment substrate However, in the field, juvenile mussels probablydepend on a combination of filter, deposit and pedal feeding in coarser substrates (Dick Neves,

Trang 15

personal communication) Research is ongoing to improve culturing methods for propagation,holding, and feeding of newly-transformed juvenile mussels(Chapter 4; Keller and Zam 1990;Gatenby, Neves, and Parker 1996, 1997; Henley, Zimmerman, and Neves 2001; ASTM 2006a).Once developed, these culturing methods should help to reﬁne methods for conducting chronicexposures with juvenile mussels.

Investigators have reported success in conducting toxicity tests for up to 28 days starting withtwo- to four-month-old juvenile mussels Valenti et al (2005) conducted 21-day exposures tomercury starting with two-month old juvenile V iris held in a small amount of sediment and fedalgae (Neochloris) USGS (2005a, 2005b, 2005c); Bringolf et al (2005) conducted toxicity testsstarting with two- to four-month-old juvenile A ligamentina, L siliquoidea, or V iris and observedcontrol survival greater than 88% in 21–28-day exposures to copper, lead, zinc, cadmium,ammonia, and several pesticides when a mixture of instant algae was used as a food source Theinstant algae mixture was prepared from commercial Instant Algae brand non-viable microalgaeconcentrates (Reed Mariculture, Campbell, CA including Nannochloropsis, Isochrysis, Pavlova,Tetraselmis, and Thalassiosira weissﬂogii)

Issues Regarding the Use of Methods

Concerns have been expressed regarding the use toxicity data generated with glochidia in thederivation of WQC (Charles Stephan, personal communication) Charles Stephan concluded thatacute methods for testing juvenile mussels (such as those outlined inTable 5.3)generally followstandard testing methods (e.g., ASTM 2006a, 2006b), and data generated from these types ofstudies should be useful in the derivation of WQC However, there were concerns identiﬁedregarding toxicity tests conducted with juvenile mussels including: (1) the life stage tested,(2) the determination of death at the end of a test, and (3) test acceptability criteria The followingsection provides information that attempts to address some of these concerns Areas of ongoingresearch or needs for future research are also identiﬁed

What Life Stage Should Be Used to Start Acute or Chronic Toxicity Tests

with Juvenile Mussels?

Toxicity tests have been started with newly-transformed juvenile mussels that have either beentransformed on a host or have been transformed with the use of an artiﬁcial medium (Table 5.3).Glochidia, newly-transformed juvenile mussels, and two- to four-month-old juvenile mussels havebeen successfully shipped via overnight carriers to other laboratories for use in toxicity testing(USGS 2004; Bringolf et al 2005; ASTM 2006a) Toxicity tests have been successfully conductedfor 10–14 days starting with newly-transformed juvenile mussels (Table 5.3), but exposures Startedwith newly-transformed juvenile mussels conducted for longer periods of time have resulted in highmortality in controls at about four to six weeks, probably due to nutritional limitations of the diet(e.g., Newton et al 2003) Valenti et al (2005), USGS (2005a, 2005b, 2005c) and Bringolf et al.(2005) conducted 21–28-day toxicity tests starting with two- to four-month-old juvenile mussels of

a variety of species and observed control survival greater than 88% when algae was used as afood source

How Should the Death of Juvenile Mussels Be Determined at the End

of a Toxicity Test?

Lack offoot or shell movement, lack of ciliary activity on the foot, lack of a heart beat, or a wide gapedvalve have been used to establish death in toxicity tests with juvenile mussels (Table 5.3) ASTM(2006a) recommends establishing death of juvenile mussels based on foot movement during a ﬁve-minute observation period under a microscope If it is suspected that juvenile mussels are avoidingexposure to a chemical in a toxicity test, it may be desirable to place the suspected live test organisms

Trang 16

into dilution water that does not contain any added test material for one to two days after the end of thetoxicity test to determine whether these test organisms are alive or dead (ASTM 2006a).

What Criteria Should Be Used to Judge Acceptability of a Toxicity Test Conducted

with Juvenile Mussels?

ASTM (2006a) recommends that average survival of juvenile mussels in the control at the end of a96-hour test must be greater than or equal to 90% An insufﬁcient number of tests have beenconducted with juvenile mussels for 10 or more days for ASTM (2006a) to provide speciﬁcguidance on control survival in longer-term tests However, a limited number of toxicitytests have reported control survival greater than 80% in tests conducted with juvenile musselsfor 10–28 days Therefore, ASTM (2006a) recommends that average survival of juvenilemussels in the control at the end of a test conducted for 10–28 days should be greater than orequal to 80% ASTM (2006a) also recommends that subsamples of each batch of test organismsused in toxicity tests should be evaluated using a reference toxicant (e.g., NaCl or CuSO4) Datafrom these reference-toxicant tests can be used to assess genetic strain or life-stage sensitivity oftest organisms to select chemicals

METHODS FORCONDUCTING WATER-ONLYTOXICITY TESTS WITHADULTFRESHWATERMUSSELSReview of Methods

Conditions that have been used to conduct toxicity tests with adult freshwater mussels are marized in Table 5.4 Specific standardized methods have not been developed for conductingtoxicity tests with adult mussels, but the procedures outlined in Table 5.4 are generally consistentwith guidance for conducting laboratory toxicity tests with early life stages of freshwater mussels(ASTM 2006a) Exposures have been conducted under static (Keller, Ruessler, and Kernaghan1999), renewal (Mane 1979; Holwerda and Herwig 1986), and flow-through (Naimo, Waller, andHolland-Bartels 1992a, 1992b; Imlay 1973; Kernaghan et al 2003) conditions A limited number ofspecies have been used to conduct toxicity studies with adults, and these mussels are typicallycollected from the field

sum-Adults have been held under laboratory conditions from one day to several monthsbefore the start of toxicity testing Toxicity tests are conducted under a wide variety ofconditions, with exposure chambers ranging from 10 to 1,500 L Due to the relatively lowabundance of adult mussels, the number of replicates per test concentration and the number

of mussels tested is generally low Replication ranges from one to six chambers, eachcontaining between 9 and 125 organisms The tests have been conducted from 48 hours

to 8 months, and a variety of endpoints have been used to evaluate toxicity Survival, asmeasured by cessation of siphoning activity and inability to react to stimulation, is acommon endpoint assessed in most adult toxicity studies In addition, sublethal endpoints,such as respiration rate, condition indices, glycogen content, and other biochemical par-ameters, are frequently measured in toxicity tests conducted with adults Water qualityanalysis, including temperature, dissolved oxygen, pH, conductivity, hardness, and alkalinity,are routinely measured during adult toxicity tests In addition, bioaccumulation has also beendetermined (e.g., Holwerda and Herwig 1986; Naimo, Waller, and Holland-Bartels 1992a,1992b) Control survival for adult toxicity studies is typically greater than 80 to greater than90% at the end of the exposures

Issues Regarding the Use of the Methods

Issues regarding the use of adult mussels in toxicity tests are similar to those for toxicity tests withglochidia and juvenile mussels Some issues that have been raised in relation to glochidia or

Trang 17

TABLE 5.4

Summary of Test Conditions for Conducting Toxicity Tests with Adult Freshwater Mussels

Conditions Ruessler (1997)Keller and and Pawar (1979)Mane, Kachole,

Naimo, Atchison, and Holland-Bar- tels (1992b) Imlay (1973)

Raj and Hameed (1991), Jacobson (1990) Herwig (1986)Holwerda and Farris et al (1991) Kernaghan et al.(2003)

Nicola ghan, University

Kerna-of Florida, FL (Unpublished Data)

Chris Ingersoll, USGS, Columbia,

MO (Unpublished Data)

1 Species tested Villosaosa lienosa,

Elliptic icterina, Utterbackia imbecillis a

Indonaia caeruleus Lampsilis

ventricosa Amblycoryphacarinata,

Lampsilis radiata silquoidea, Fusconaia ﬂava, Amblema plicata

Villosa iris, Actinonaias pectorosa, Pyganodon grandis, Lampsilis fasciola, Medionidus conradius

Anodonta anatina Elliptio dilatata,

M.conradicus, Pleurobema oviforme, Villosa iris

E buckleyi E buckleyi Amblema plicata

2 Test type Static Static-renewal Flow-through

diluter Flow-through Static-renewal Static-renewal Static-renewal Flow-through Static-renewal Flow-through

3 Test duration 72–96 hours 48 hours 14, 28 days 36 days-8 months 96 hours, 10, 20,

30 days 7 months 30 days 56 days 7, 14, 21, 30,60 days 4, 56 days

7 Photoperiod 12L:12D NR NR Natural conditions NR NR 10L:14D Natural conditions 16L:8D 16L:8D

8 Test chamber 23-L aquaria Plexiglass aquaria 57-L glass aquaria 20-L stainless steel

chamber Plastic containers NR 75-L ﬁberglassoval stream 1500-L plasticcontainers 46-L glass aquaria 40-L glass aquaria

9 Test solution

10 Number of

organisms per test chamber

11 Number of

replicate chambers per treatment

Trang 18

TABLE 5.4 (Continued)

Conditions Ruessler (1997)Keller and and Pawar (1979)Mane, Kachole,

Naimo, Atchison, and Holland-Bar- tels (1992b) Imlay (1973)

Raj and Hameed (1991), Jacobson (1990) Herwig (1986)Holwerda and Farris et al (1991) Kernaghan et al.(2003)

Nicola ghan, University

Kerna-of Florida, FL (Unpublished Data)

Chris Ingersoll, USGS, Columbia,

MO (Unpublished Data)

14 Dilution water Reconstituted

water with a hardness of

76 mg/L as CaCO 3

River water Reconstituted

water with a hardness of

165 mg/L as CaCO 3

Dechlorinated city water and lake water

Filtered river water Tap water River water Well water Well water

(hardness

260 mg/L as CaCO 3 ) and reconstituted water (hardness

80 mg/L as CaCO 3 )

Mixture of well water (hardness

260 mg/L as CaCO 3 ) and pond water to a hardness of about 190 mg/L

as CaCO 3 )

15 Water quality pH, hardness,

conductivity Temperature Temperature, DO,pH, alkalinity,

hardness, conductivity

Temperature, DO,

pH, hardness, alkalinity

Temperature,DO,

pH, salinity DO, pH, Ca, Mg,Na, Fe,

HCO 3 ,Cl, SO 4

Temperature, DO, pH, conductivity, hardness, alkalinity

Temperature, DO, pH, conductivity

Temperature, DO,

pH, ammonia, conductivity, hardness, alkalinity,

16 Endpoint Survival (cessation

of siphoning activity and inability to react

to stimulation)

Survival and effects on neurosecretory cells, digestive gland and intestine

Respiration rate, food clearance rate, ammonia excretion rate, assimilation efﬁciency, tissue condition index, oxygen

to nitrogen ratio

Survival Survival,

respiration rate and body weight

Carbohydrate and lipid content, lactate, succinate, acetate, and propionate

Survival and cellulolytic activity

Survival, body condition index, glycogen concentration, sex steroid conentration

Survival, body condition index, soft tissue index, glycogen concentration, sex steroid conentration

Survival, glycogen concentration, behavior

Trang 19

juvenile toxicity tests are also applicable to adult toxicity tests These concerns mainly include: (1)the length of time that adults can be held in a laboratory, (2) the conditions for maintaining adults inthe laboratory, (3) the evaluation of the health of adults, and (4) the similarities in sensitivity tocontaminants between different populations of mussels The following section attempts to addresssome of these concerns Areas of ongoing research or needs for future research are also identiﬁed.How Long Can Adults Be Held in the Laboratory?

Dunn and Layzer (1997) reported the results of several long-term holding experiments usingﬁshery ponds and raceways Survival of adults varied by species and according to holdingconditions In the raceway experiments, survival ranged from 43 to 100% after one year.Survival of mussels held in ponds appeared more variable, ranging from 0 to 100% Otherresearchers report that adults can be successfully maintained in the laboratory for a period ofseveral months (e.g., Chris Barnhart, Jerry Farris, Dick Neves, Teresa Newton; personalcommunications)

What Conditions Should Be Used to Maintain Adults in the Laboratory?

Holding conditions for adults vary by species and season Temperatures tested ranged between 10and 258C, and mussels have been maintained in systems supplied by pond, river, or well water.Conditions that most closely mimic those in the environment from which the mussels werecollected are recommended Some research facilities have relied upon natural sources of food inthe pond or river water to maintain an adequate diet for the captive mussels Researchers at VirginiaTech have successfully developed a cultured algal diet to feed the mussels (Gatenby 2000).How Should the Health of Adults Held in the Laboratory Be Evaluated?

Adults to be used in toxicity studies are only occasionally screened for background contaminationlevels (Nicola Kernaghan, University of Florida, Gainesville, FL; personal communication) Thehealth of adults can be evaluated by making observations of activity, behavior, and orientation ofmussels in a substrate (Jerry Farris and Teresa Newton, personal communications) Further healthassessments may be achieved with the use of biochemical indicators, such as glycogen(Chapter 10)

Several studies have used glycogen as a measure of the energetic status of mussels and as an indicator

of their physiological condition after exposure to contaminants (Hemelraad and Herwig 1990;Holopainen and Lamberg 1997; Kernaghan et al 2003)

Are There Similarities in Sensitivity to Contaminants between Populations of Mussels

of the Same Species?

Some species of mussels appear to be more sensitive to certain contaminants than others, andseveral studies have been conducted to compare species sensitivities (Imlay 1973; Keller andRuessler 1997) SeeChapter 7 for comparison of toxicity endpoints between species However,

no studies have been conducted to date comparing the sensitivity of different populations of thesame species of mussel

METHODS FOR CONDUCTING SEDIMENT TOXICITY TESTS

WITH FRESHWATER MUSSELS

REVIEW OFMETHODS

Conditions used to conduct sediment toxicity tests with freshwater mussels are summarized in

mussels in the ﬁeld Only a few sediment toxicity tests with mussels have been conducted, and

Trang 20

TABLE 5.5

Summary of Test Conditions for Conducting Sediment Toxicity Tests with Freshwater Mussels

USGS (2005c), Nile Kemble, USGS, Coumbia, MO (Unpub- lished Data)

costata, Villosa villosa

Utterbackia imbecillis (formerly Anodonta imbecillis)

raﬁnesqueana

7.5 cm in height, closed on one end, with 100 um Nitex screen)

in 5-L glass aquaria

Glass cylinder (5 cm in diameter, closed on one end, with 100 um Nitex screen) in 250-mL crystallizing dishes

Polycarbonate tube (4.5 cm in diameter, 11 cm in height, closed one end with 153 um mesh Nitex screen) in 12 ! 8 !

13 cm chamber

300-mL beaker

of sediment, and 1.5 cm of sand 260 mL of overlying water and15 mL of sieved sediment, two

volume additions/day of overlying water

chambers per treatment

Định dạng
Số trang	40
Dung lượng	675,85 KB