MUSSELS AS BIOINDICATORS: EFFECTS 15 OF TBT ON SURVIVAL

MUSSELS AS BIOINDICATORS: EFFECTS 15OF TBT ON SURVIVAL,BIOACCUMULATION, AND GROWTHUNDER NATURAL CONDITIONS

Michael H. Salazar1 and Sandra M. Salazar2

1Applied Biomonitoring, 11648 72nd Place NE, Kirkland, Washington 98034,USA2 EVS Consultants, 200 West Mercer Street, Seattle, Washington 98119, USA

Abstract 30515.1. Introduction 30615.2. Methods 30715.3. Mussels as bioindicators 310

15.3.1. General trends 31015.3.2. Survival 31115.3.3. Bioaccumulation 31215.3.4. Growth 314

15.4. Laboratory vs field 31715.5. Influencing factors 319

15.5.1. Handling effects 31915.5.2. Juveniles vs adults 32115.5.3. Natural factors 322

15.6. Mussel bioindicator model 32315.7. Conclusions 325Acknowledgments 326Epilogue 326References 327

ABSTRACT

During nine field-transplant experiments(1987-1990), juvenile mussels were exposedto mean tributyltin (TBT) concentrationsfrom 2 to 530 ng L-1 for 12 weeks undernatural conditions in San Diego Bay.

Mussels were used as biological indicatorsand monitored for survival, bioaccumu-lation, and growth. Mussel growth was theprimary biological response used to quan-tify TBT effects. Chemical analyses wereused to estimate TBT contamination in waterand tissues.

Organotin. Edited by M.A. Champ and P.F. Seligman. Published in 1996 by Chapman & Hall, London. ISBN 0 412 58240 6

306 Mussels as bioindicators: effects of TBT on survival, bioaccumulation and growth

Integrating intensive measurements ofchemical fate and biological effects increasedthe environmental significance of the data.Multiple growth measurements on in-dividuals increased the statistical power.Size effects were minimized by restrictingtest animals to 10-12 mm in length, andmethods were developed to minimizehandling effects. This monitoring approachalso permitted documenting temporal andspatial variability in TBT and its effects thathave not been previously reported. Survival,bioac-cumulation, and growth weregenerally higher than predicted fromlaboratory studies. Survival was not directlyaffected by seawater or tissue TBT con-centrations. Growth was significantly relatedto both seawater and tissue TBT, with thebioconcentration factor inversely pro-portional to seawater TBT concentration.Threshold concentrations always causingsignificant reductions in juvenile musselgrowth are estimated at 100 ng L-1 TBT forseawater and 1.5 Fg g-1 TBT for tissue, butgrowth could be affected by much lowerconcentrations of TBT under the mostadverse conditions. Temperatures above20EC were also found to reduce juvenilemussel growth rates.

15.1. INTRODUCTION

Biological indicators provide integrated in-formation about environmental contami-nation and effects that cannot be definedwith chemical analysis of water samples.Nevertheless, chemical analyses quantifycontamination in a way that is essential inexplaining biological effects. Measuringsurvival, bioaccumulation, and growthquantifies both contamination and effects. Itis important to make the distinction betweenthe use of biological indicators as detectors ofenviron-mental contamination by monitoringtissue accumulation with chemical analysesversus their use as indicators of environ-mental effects by measuring other biologicalresponses (Waldock et al., Chapter 11).

Mussels have been used as biologicalindicators in many field monitoring pro-grams because of their cosmopolitan dis-tribution and ability to concentrate manydifferent contaminants. Their demonstratedutility in transplant experiments andmonitoring is another significant advantage.Mussels have been used most extensively tomonitor contamination by measuring tissueaccumulation with chemical analyses(Phillips, 1980). They have also been used tomonitor the biological effects of con-tamination by measuring biologicalresponses related to growth, physiology, andreproduction (Bayne et al., 1985). It should berecognized that bioaccumulation can beregarded as both a chemical and a biologicalprocess; however, biological effects are notnecessarily related to tissue accumulation.We suggest that caged mussels should beregarded as a bioindicator system. Thisindicator system consists of the entire suite ofbiological responses.

Bioaccumulation is the process throughwhich organisms integrate exposure to en-vironmental concentrations of bioavailablecontaminants. The results of these integratedchemical and biological processes can bequantified with chemical analyses. Labora-tory and field studies have shown thattributyltin (TBT) is highly toxic to molluscsand that filter-feeding bivalves readilyaccumulate TBT (Hall and Bushong, Chapter9; Laughlin et al., Chapter 10; Waldock et al.,Chapter 11; Gibbs and Bryan, Chapter 13;Henderson and Salazar, Chapter 14;Laughlin, Chapter 16). Natural musselpopulations have been used as indicators ofTBT contamination by measuring tissueconcentrations of TBT (Wade et al., 1988;Short and Sharp, 1989; Uhler et al., 1989;Roberts et al., Chapter 17). Chemicalmeasurements of TBT concentrations inseawater and tissues of mussels from naturalpopulations (Grovhoug et al., Chapter 25) ortransplants (Zuolian and Jensen, 1989) aremore informative than measuring seawateror tissue levels alone. The combination of

Methods 307

seawater and tissue measurements of TBTprovides a quantitative relationship betweenthe chemistry of the environment and thechemistry of the organism. However, therelative influence of environmental andbiological factors on this relationship ishighly variable and difficult to predict (Cainand Luoma, 1990).

Survival and growth are biological re-sponses that also integrate exposure to en-vironmental concentrations of bioavailablecontaminants. These responses are moredirectly related to animal health and do notdepend on chemical analysis. Survival is theleast sensitive to environmental effectsbecause it is an all-or-nothing response.Therefore, survival data are not alwaysinformative. Growth is more sensitivebecause there is a graded response toenvironmental conditions that can bequantified through repetitive, non-destructive measurements. Reduced growthrepresents adverse environmental effects andpossible effects on the population. Bothnatural and pollution-related stresses havebeen shown to reduce mussel growth rates(Bayne et al., 1985). Reduced mussel growthhas been associated with TBT in laboratoryand field studies (Thain and Waldock, 1985;Stephenson et al., 1986; Salazar and Salazar,1987; Stromgren and Bongard, 1987; Valkirset al., 1987; Salazar and Salazar, 1988).Juvenile mussel growth was the mostsensitive indicator of TBT measured in SanDiego Bay microcosm experiments (Salazarand Salazar, 1987; Salazar et al., 1987;Henderson and Salazar, Chapter 14).Juvenile mussels have advantages overadults as bioindicators: (1) they grow fasterand provide a greater range of response; (2)the growth process is not affected bygametogenesis (Rodhouse et al., 1986); (3)bioaccumulation in short-term tests with fast-growing juveniles more accurately reflectsrecent environmental changes (Fischer, 1983,1988); and (4) they may be more sensitive toTBT (Hall and Bushong, Chapter 9).

Survival, bioaccumulation, and growth incaged mussels can be routinely measured in

field-transplant experiments. Field-transplant experiments provide an op-portunity to corroborate and validate musselperformance under laboratory conditions. Itis important to measure performance undernatural conditions because that is where thebiological effects, if any, will occur. The mainadvantage in using trans-planted animalsover monitoring naturally settled popu-lations is the experimental control. Ex-perimental control is achieved by usingmussels of similar genetic and environmentalstocks at all test sites, pre-selecting testanimal size or age group, and monitoringindividual animals during the test. Animalscan also be transplanted to areas where theymight not normally be found. Serialtransplants and monitoring facilitate theexamination of both short- and long-termtrends in contaminant distribution andrelated effects.

The work reported here had three primarygoals: (1) determine the effects of TBT onsurvival, bioaccumulation, and growth injuvenile mussels under natural conditions;(2) identify long-term trends in the distri-bution of TBT and its effects; and (3) refinethe use of the juvenile mussel bioindicatorfor environmental assessment.

15.2. METHODS

Nine field-transplant tests were conducted inSan Diego Bay between 1987 and 1990(Salazar and Salazar, 1991c). Juvenilemussels (Mytilus sp.) were transplanted fromthe collection site to test sites and monitoredduring 12-week exposure periods. Test datesare shown in Fig. 15.1. Serial musselmeasurements were made in the field, andwater samples collected weekly during tests1-4 and on alternate weeks (biweekly) duringtests 5-9. Growth measurements includedwhole animal wet weights and lengths.Caged mussels were removed from the waterfor .20 min during these measurements, andbyssal threads were carefully cut with scis-sors. Water was measured for chlorophyll-a

Methods 309

and TBT concentrations (Venrick andHayward, 1984; Stallard et al., 1988). Meanchlorophyll-a and seawater TBT con-centrations for each test were determined fromweekly or biweekly measurements. Tem-perature was measured at half-hour intervalswith in-situ monitors. Whole animal wetweights, lengths, shell weights, and tissue wetweights were measured at the end of eachstudy. Tissues for a given site were pooled forTBT analysis on a wet-weight basis (Stallard etal., 1988). Average weekly growth rates basedon weight (mg week-1) and length (mm week-1)were calculated from regression analyses foranimals transplanted at each site. Only weightgrowth rates will be discussed because there isa greater range in response than with lengthgrowth rates (25 X compared to 10 X), andweight measurements are more accurate.

Naturally settled mussels were collectedfrom pilings where mean TBT concentrationsin seawater and mussel tissues were low, .5 ngL-1 TBT and .0.15 Fg g-1 TBT wet wt, respec-tively. The collection site, a Navy site, islocated near the mouth of San Diego Bay andcharacterized by high current speeds, near-shore ocean temperatures, few vessels, and lowlevels of contaminants in mussel tissues.Mussel growth rates in this area are among thehighest in San Diego Bay. The collection sitewas used as a test site for comparative pur-poses. Animals were sorted by length forconvenience. Test mussels were 10-12 mm inlength (0 .11.0 mm) and initial mean weightsranged from 100 to 250 mg (0 .175 mg).Eighteen mussels were caged and transplantedto each site. There were no statisticallysignificant differences in weights or lengthsamong these mussel groups at the start of anytest. Animals were continuously submergedeither 1 m below the surface or 1 m above thebottom. The 18 monitoring sites included 11Navy sites with low seawater TBTconcentrations and seven marina sites withhigh seawater TBT concentrations (Grovhouget al., 1986). The most significant refinements inmethods include the use of (1) field trans-plants; (2) serial growth measurements on

individuals; (3) minimizing the size rangeand maximum size of test animals; and (4)synoptic measurements of bioaccumulationand growth.

Bioaccumulation in transplanted andnatural mussels was compared to determineif the transplanted bioindicator was respon-ding like natural populations of undisturbedmussels. Bioaccumulation in juveniles andadults was compared to determine if therewere size or age differences. During tests 4,6, and 9, juvenile (0 length <35 mm) andadult mussels (0 length >50 mm) werecollected from natural populations at onemarina and one Navy transplant site. Thesesites were characterized by the highest andlowest concentrations of TBT in seawater,respectively. This Navy site was also thecollection site. Tissues from these mussels,along with tissues from juvenile transplants(tests 4, 6, and 9) and adult transplants (test9), were analyzed for TBT. In a different ex-periment, handling and measurement effectson growth of juvenile mussels were assessedat five sites, including the most contaminatedmarina site. Growth rates of animalsmeasured weekly were compared withgrowth rates from those animals measuredonly at the beginning and end of the 12-weekexposure (untouched). Some comparisonswere also made with animals measured onalternate weeks (biweekly).

The data for survival, seawater TBTconcentration, tissue TBT concentration, andgrowth were pooled by test for both marinaand Navy sites to calculate regional meansfor comparison with other San Diego Baymonitoring studies (Grovhoug et al., Chapter25). Regional means for seawater TBT andgrowth rates were compared with a one-wayanalysis of variance on a per-test basis aswell as for data pooled across tests. Regionalmeans for tissue TBT and individual site datawere pooled across tests and compared usinga one-way analysis of variance and multiplelinear regression analyses. Although theseregional divisions are somewhat arbitrary,they can be used to illustrate the effects of


low and high exposure to TBT in seawater andconvey important general information aboutthe physical-chemical characteristics of thesites in each region. Details regarding temporaland spatial variability, site locations, and site-specific responses are presented elsewhere(Salazar and Salazar, 1991a,b,c).

Graphical methods are used to display thegeneral relationships among environmentallevels of TBT and mussel survival, bio-accumulation, and growth. Each data pointrepresents a 12-week exposure and theresponse of approximately 18 animals. Thesignificance of each relationship was deter-mined by linear regression analyses. The signi-ficance of the regression (p); the correlationcoefficient, which provides a measure ofassociation intensity between the two variables(r); and the total variation in the dependentparameter that is explained by the fittedregression (r2) were used to compare re-gressions. Stronger relationships are indicatedby higher p, r, and r2 values. Lines and slopescan also be compared on a relative basis. The r2

statistic estimates the predictive strength ofeach relationship and possible environmentalsignificance. Statistical significance was deter-mined at the 95% confidence level.

15.3. MUSSELS AS BIOINDICATORS

In recognition of the limitations of laboratorybioassays and chemical monitoring, there hasbeen a shift in emphasis toward biologicalmonitoring and field bioassays (Chapman,1983; Chapman and Long, 1983; Phillips andSegar, 1986; Parrish et al., 1988). Specificproblems with the interpretation and environ-mental significance of TBT studies have beendiscussed previously (Stebbing, 1985; Salazar,1986, 1989; Salazar and Champ, 1988). Usingmussels as biological indicators of contami-nation and effects is a potentially powerfultool, but there are many pitfalls regardinginterpretation and environmental significance(Phillips, 1980; White, 1984). Many of these

pitfalls were avoided in this mussel bio-indicator study by integrating chemical andbiological measurements and by demon-strating the differences between usingmussels as indicators of contaminationversus their use as indicators of effects. Moststatus and trends programs in the UnitedStates have used biological indicators asdetectors of environmental contamination ofTBT by monitoring tissue accumulation withchemical analyses (Wade et al., 1988; Shortand Sharp, 1989; Uhler et al., 1989). Thepredictive value of these studies is extremelylimited. Measuring seawater TBT in additionto accumulation in natural populations(Grovhoug et al., Chapter 25) or fieldtransplants (Zuolian and Jensen, 1989)establishes a quantitative relationship bet-ween environment and organism that ismuch more informative. Such studies areusing mussels as detectors of environmentalcontamination. By measuring survival andgrowth in addition to bioaccumulation, weused mussels as indicators of environmentalcontamination and effects (Waldock et al.,Chapter 11). The approach was furtherintegrated by including chemical measure-ments of seawater. This type of integration isvery important.

15.3.1. GENERAL TRENDS

Mean concentrations of TBT in seawater atthe 18 sites ranged from 2 to 530 ng L-1 andtissue TBT concentrations from 0.1 to 3.2 Fgg-1 wet wt. Mean 12-week temperatures ran-ged from 14.3 to 25.7EC and chlorophyll-afrom 0.79 to 6.07 Fg L-1 Mean temperaturesfor winter tests ranged from 14.3 to 14.8ECwhile mean temperatures for summer testsranged from 20.1 to 25.7EC. Chlorophyll-awas lowest in the winter. End-of-test survivalranged from 50 to 100%. Mean growth ratesof caged mussels ranged from 17 to 505 mgweek-1 (0.2 to 2.5 mm week-1). The lowestgrowth rates were measured at the mostcontaminated marina site. Mussels increased

Mussels as bioindicators 311

from .190 to 420 mg (11 to 14 mm in length).This was an increase of only 230 mg in weight(3 mm in length) after 12 weeks. The highestgrowth rates were measured at the Navy sitenearest the mouth of the bay. Musselsincreased from .160 to 6200 mg (11 to 40 mmin length). These mussel growth rates areamong the highest reported (Kiorboe et al.,1981). The minimum concentration of TBT inseawater predicted to always reduce juvenilemussel growth was estimated at 100 ng L-1

TBT. The minimum concentration of TBT intissues predicted to always reduce juvenilemussel growth was estimated at 1.5 Fg g-1 (wetwt). These concentrations were estimated fromstatistical analyses of mussel responses underrepresentative conditions in San Diego Bay.Because biological responses are so sitespecific, site selection influenced the signi-ficance of each relationship and comparisonsbetween regions. Therefore, the most meaning-ful comparisons are those between sites. Bothwater and tissue TBT con-centrations aresimilar to those predicted to reduce oystergrowth (Waldock et al., Chapter 11). Acomparable concentration of tissue TBT wasreported to adversely affect adult musselphysiology (Page and Widdows, 1990).

Regional differences and temporal changesin survival, seawater TBT, tissue TBT, andgrowth for the marina and Navy sites areshown in Fig. 15.1. Seawater and tissue TBTconcentrations generally decreased whilemussel growth rates increased over time. Al-though the lowest growth rates wereassociated with the highest concentrations ofseawater TBT, extremely low growth rateswere also associated with winter seawatertemperatures <15EC. Seawater and tissue TBTconcentrations were significantly higher andgrowth rates significantly lower at marina sitesthan Navy sites. There were no differences insurvival. There was a significant decrease inseawater and tissue TBT concentrations atmarina sites and Navy sites. This wasassociated with a general increase in growth.These changes were not consistent at all sites,however, and argue against pooling (Salazar

and Salazar, 1991b). There was also asignificant decrease in growth rates duringtest 8 that demonstrates the significance ofnatural factors in influencing mussel growthrates. The mean concentration of seawaterTBT at marina sites Navy sites as well as thedecreases in sea-water and tissue TBTconcentrations were similar to those des-cribed for San Diego Bay by Grovhoug et al.(Chapter 25) except that we did not findsignificant decreases in tissue TBT con-centrations at all Navy sites. Neither tissueTBT concentrations, survival, nor growthrates consistently followed sea-water TBTconcentrations. However, the ratio of tissueto seawater TBT, growth rates, and survivalgenerally increased at both marina and Navysites after test 4.

15.3.2. SURVIVAL

Although survival is not a sensitive indi-cator of environmental effects, important in-formation was gained for some sites whereexceptionally high or low survival wasdifferent than expected based on othermeasurements and indicators of waterquality. Survival is not significantly cor-related with seawater or tissue TBTconcentration. The four lowest survivalmeasurements were between 50 and 65%.They were associated with mean con-centrations of TBT in sea-water between 34and 130 ng L-1 at one marina and one Navysite. Conversely, the five highest mean con-centrations of TBT in seawater were be-tween 169 and 530 ng L-1. They were mea-sured at a different marina site and wereassociated with mussel survival between 94and 100%. There was no difference inmeasured survival between marina andNavy sites even though seawater and tissueTBT concentrations were significantly higherat marina sites (Fig. 15.1). Survival washigher in tests 5-9 (96.8%) than in tests 1-4(90.2%), but the difference is not statisticallysignificant. One hundred percent survival


was measured in 42% of the transplant in tests1-4 and in 70% of the transplant in tests 5-9.The increase in survival after test 4 wasassociated with the decline in TBT con-centrations in marina seawater followingrestrictions imposed in January 1988 (State ofCalifornia, 1988) and reduced handling aftertest 4 when weekly growth measurementswere changed to alternate weeks. Sincehandling was reduced at the same timeseawater TBT concentrations decreased, therelative effects of each on survival are unclear.

15.3.3 BIOACCUMULATION

There is a significant positive linear re-lationship between TBT accumulation injuvenile mussel tissues and seawater TBT con-

centration (Fig. 15.2). Based on regression an-alysis of all data, only 38% of the variance intissue TBT concentration can be explained byseawater TBT concentration. The regressionequations calculated using all data and onlydata associated with seawater values >105ng L-1 are very similar. Six of the seven datapoints for seawater TBT concentrations >105ng L-1 are from the most contaminatedmarina and strongly influence the regressionfor all data. The data appear to fall into twoseparate groups. The slope of the regressionfor tissue TBT values associated with sea-water TBT concentrations <105 ng L-1 is al-most five times higher than the slope of theregression for tissue TBT values associatedwith seawater TBT concentrations >105 ngL-1. The ratio of tissue TBT to seawater TBT


concentration estimates a bioconcentrationfactor (BCF). BCF values ranged from 4,700 to105,600 with the majority between 20,000 and40,000 (Fig. 15.3). An inverse exponentialfunction best describes the relationshipbetween BCF and seawater TBT concentration.The higher ratios of tissue TBT to seawaterTBT at lower seawater TBT concentrationsshown in Fig. 15.3 were suggested by theregressions in Fig. 15.2 and the shift in ratiosfrom the trends in Fig. 15.1. Lowerconcentrations of TBT in seawater wereassociated with higher BCF values. Atseawater TBT concentrations <105 ng L-1, BCFvalues range from 5,000 to .100,000. Aboveseawater TBT concentrations of 105 ng L-1, BCFvalues were <9,000. This inverse exponentialrelationship between TBT concentration inseawater and in mussel tissue was found inlaboratory studies (Laughlin et al., 1986;Laughlin and French, 1988), microcosm studies(Salazar et al., 1987), other field-transplantstudies (Waldock et al., Chapter 11; Zoulianand Jensen, 1989), and natural San Diego Baypopulations (Grovhoug et al., Chapter 25).

Using tissue accumulation in field bio-indicators to quantify environmental levelsof contamination is a potentially powerfultool, but the limitations of this approachmust be recognized (Phillips, 1980). Since therelationship between seawater and tissueTBT concentrations (BCF) is not constant,accurate predictions of one using the otherare not possible. Even accumulating elevatedlevels of contaminants does not a prioriindicate environmental effects on thebioindicator or other species (Peddicord,1984). Initial reports on mussels asbioaccumulators emphasized the utility ofidentifying order-of-magnitude differencesin seawater contamination and minimizedpotential interference from extraneous en-vironmental factors (Goldberg et al., 1978,1983; Farrington et al., 1983). Other reportshave outlined potential problems in usingtissue concentrations of contaminants for en-vironmental prediction (Phillips, 1980;Luoma, 1983; White, 1984; Phillips andSegar, 1986; Cain and Luoma, 1990). Bio-accumulation is an important link between


organism and its environment, but therelationship between concentrations of TBT inseawater and mussel tissue is very complex.Bioavailability of TBT may be affected by anumber of natural factors, and the relationshipbetween bio-availability and chemical analysisof TBT is unclear (Laughlin et al., 1986; Salazar,1986, Laughlin and French, 1988).

15.3.4. GROWTH

Juvenile mussel growth is significantly relatedto both seawater and tissue TBT concen-trations. The statistical relationship is strongerfor seawater TBT concentration. The relation-ship between juvenile mussel growth rate andseawater TBT concentration is negative andexponential (Fig. 15.4). Based on regressionanalysis, .52% of the growth variance can beexplained by seawater TBT concentration.There is a high degree of variability at the low-

est concentrations of seawater TBT. The sevendata points for seawater TBT concentration<100 ng L-1 strongly influence the significanceof the regression for all data. They alsodemonstrate the influence of the mostcontaminated marina where six of the sevenhighest measurements were made. Theequations for all data and seawater TBT > 100ng L-1 are quite similar. A power fit bestdescribes the relationship for seawater TBTdata >100 ng L-1 (p = 0.0001, r = -0.97, r2 =0.94). There is also a significant negativeexponential relation-ship when the seawaterTBT data <100 ng L-1 are analyzed separately(p<0.0025, r = -0.36, r2 = 0.13), but only 13%of the growth variance can be explained byseawater TBT concentration. Some statisticallysignificant relationships (p = 0.0357) were alsofound with seawater TBT data <70 ng L-1, butthe r2 value was <0.09 and suggests little en-vironmental significance.


Although it has been suggested that bi-valve growth is directly affected by ac-cumulated TBT (Waldock and Thain, 1983),it is not clear from the weak relationshipsfound in these field-transplant studies ifaccumulated TBT is regulating growth rateor growth rate is regulating the accumu-lation of TBT. There is a significantnegative exponential relationship betweenjuvenile mussel growth and tissue TBTconcentration (Fig. 15.5), but variability ingrowth is high at all tissue TBT concen-trations. Based on regression statistics, TBTin seawater has a more direct effect onmussel growth than TBT ac-cumulated inmussel tissues. Only 16% of the variance ingrowth can be explained by tissue TBTconcentration. There are no significantregressions when the analyses are limitedto data <1.5 Fg g-1 TBT in tissue (wet wt).Many high growth rates are associatedwith elevated concentrations of TBT in

tissue even at low concentrations of TBT inseawater.

Growth has a significant effect on thebioaccumulation process as shown by thesignificant linear relationship betweengrowth rate and BCF. The highest growthrates are associated with the highest BCFvalues (Fig. 15.6). The highest growthrates are also associated with the lowestconcentrations of TBT in seawater andreduced stress from decreased handling.These data suggest that growth rate affectsbioaccumulation. At similar seawater TBTconcentrations, faster growing musselsmay accumulate more TBT. If mussels donot grow, they will not accumulate muchTBT. Growth rates were very low in tests 2and 8 due to winter conditions, but ac-cumulation was similar even though TBTconcentrations were significantly lower intest 8.


Using biological responses such as growthin field bioindicators to quantifyenvironmental effects is also a potentiallypowerful tool, but, as with bioaccumu-lation, the limitations must be ack-nowledged (White and Champ, 1983; Cairnsand Buikema, 1984; Malins et al., 1984;Moller, 1987; Cairns, 1988). Based onstatistical analyses, the data show that TBTaccumulated in mussel tissue has less of aneffect on mussel growth than TBT inseawater. These relationships demonstratethat other factors have a significant effect ongrowth rate. Nevertheless, growth rates canprovide other information and perhaps beused to calibrate bioaccumulation (Fischer,1983, 1988). Variable growth rates couldexplain some of the apparent anomalies intissue accumulation shown here. Forexample, mussels severely stressed by TBTor other factors will not grow and will notaccumulate much TBT. Without supportingmeasurements of growth and seawater TBTconcentrations, only analyzing tissues could

be very misleading. Survival was the leastsensitive indicator measured here, but ithelped explain the results. Clearly, naturalfactors can affect survival, bioaccumu-lation,and growth in juvenile mussels.

15.4. LABORATORY VS FIELD

There is a tremendous gap betweencorrelations and causality when usingbiological indicators in the field. Biologicalresponses under natural conditions are oftenvery different from biological responsesmeasured under controlled laboratoryconditions, and they are affected by manydifferent factors (White and Champ, 1983;Mallet et al., 1987; Salazar, 1989). Forexample, conditions in laboratory and fieldtests appear to be as responsible forobserved biological responses as the toxicantbeing tested. Temperature and nutritivestress have been shown to cause adversephysiological changes in mussels under

Laboratory vs field 317

laboratory conditions (Bayne andThompson, 1970; Bayne, 1973). Musselsurvival, bio-accumulation, and growthwere generally much higher in these fieldstudies than in comparable laboratorystudies. Laboratory-induced stress may haveenhanced the effects of TBT and account forthe reduced performance of laboratoryanimals.

Survival of juvenile mussels in the field-transplant experiments was higher and didnot demonstrate the dose dependencyshown for adult mussels in laboratorytoxicity tests (Thain, 1983; Valkirs et al.,1987; Salazar and Salazar, 1989). Thesedifferences are best demonstrated bycomparing results from a laboratoryexperiment with conditions most similar tofield exposures (Fig. 15.7). In this laboratoryexperiment adult mussels were exposed toTBT for 66 d in a flow-through system(Valkirs et al., 1987). Predicted decreases insurvival with increasing seawater TBTconcentrations found in the laboratory were

not observed in the field-transplantedmussels. This apparent dose dependency inthe laboratory may be due to a limitednumber of test concentrations and the use ofextremely high seawater TBT concentrations(>500 ng L-1) that strongly influencestat is t ica l analyses and yet areenvironmental ly unreal ist ic . Dosedependency may not be manifested in fieldstudies due to natural factors that modifymussel performance. At similar TBTexposure concentrations, survival washigher in field-exposed animals, eventhough the exposure period was longer andthe test animals were also exposed to highconcentrations of other contaminants.

It is extremely difficult to compare tissueTBT concentrations in mussels from thesestudies with tissue concentrations in musselsfrom laboratory studies. Analytical methodsand units for reporting results are highlyvariable (Salazar, 1986; Page and Widdows,1990). Another potential problem is thattotal contaminant content per individualmay be more biologically significant than


measured tissue concentrations (Fischer,1983, 1988; Cain and Luoma, 1990). Oneapparently common unit for comparison isBCF. This convention is commonly used inthe TBT literature. The majority of BCFvalues calculated for mussels in this andother field-transplant studies, microcosmexperiments, and natural populations are.30,000 (Salazar et al., 1987; Zuolian andJensen, 1989; Grovhoug et al., Chapter 25).Only one laboratory experiment hasprovided comparable BCF values, and it wasconducted under flow-through conditionswith apparently healthy animals (Waldocket al., Chapter 11). In most other laboratoryexperiments, BCF values were much lowerwith a reported maximum of .7000(Laughlin et al., 1986; Laughlin and French,1988). The combined stresses of static-renewal conditions, overcrowding, and poornutrition probably affected animal healthand limited the ability to accumulate TBT.These obser-vations support measuringgrowth to quantify animal health andcalibrate bio-accumulation.

Comparing BCF values from thelaboratory and field can be misleadingbecause they do not represent the sameconditions. Since mussels may be exposed toTBT through various routes, such asseawater and food, field exposures includeTBT from all sources. Technically, the BCFonly accounts for the TBT concentration inseawater, which is generally the primarysource in laboratory studies. The bio-accumulation factor (BAF) includes TBTfrom seawater and food. Higher BCF valuesfrom these field-transplant studies could beattributable to measuring only the TBTdissolved in seawater. These values coulddecrease substantially after ac-counting forTBT associated with the food, if food is amajor source of accumulated TBT.Quantifying the food and water componentsof the BAF is important for understandingthe bioaccumulation pro-cess and explainingeffects attributable to TBT.

Laboratory-held mussels generally growmore slowly than mussels main-tained

under natural field conditions (Kiorboe et al.,1981). Temperature and nutritive stressesoften associated with la-boratoryexperiments may cause physio-logicalchanges (Bayne and Thompson, 1970; Bayne,1973) that preclude optimum growth ratesand influence the relative effects oftoxicants. In Fig. 15.8 juvenile musselgrowth in our field-transplant studies iscompared with juvenile mussel growthunder laboratory (Thain, 1986) and flow-through microcosm conditions (Salazar andSalazar, 1987; Salazar et al., 1987). The rateof juvenile mussel growth in field-transplantexperiments is much higher than inlaboratory or microcosm animals. Growthrates under laboratory and microcosmconditions were very similar and suggest alinear relationship between seawater TBTconcentration and mussel growth. The fielddata show an exponential relationship.Growth rates of transplanted musselsapproach those of juvenile mussels underlaboratory and microcosm conditions onlywhen exposed to seawater TBTconcentrations >100 ng L-1. At seawater TBTconcentrations <100 ng L-1 growth rates fortransplanted mussels are as much as anorder of magnitude greater. Microcosmstudies suggest significant re-ductions injuvenile mussel growth at sea-water TBTconcentrations between 70 and 80 ng L-1

(Salazar and Salazar, 1987; Salazar et al.,1987). However, these mussels were stressedby overcrowding, high temperatures, andinadequate food associated with the testsystem and experimental procedures. Thesestressful conditions in laboratory andmicrocosm tanks may overestimate theeffects of, TBT in most San Diego Bayenvironments. They may accuratelyrepresent the most stressful San Diego Bayenvironments.

Laboratory vs field 319

15.5. INFLUENCING FACTORS

15.5.1. HANDLING EFFECTS

Experimental procedures in the field canalso induce stress that affects test results.Transplanting mussels to different en-vironments and removing them from thewater for growth measurements bothmodif ied performance. This wasdemonstrated by comparing growth rates ofmussels measured weekly to thosemeasured only at the end of the test( u n t o u c h e d ) a n d b y c o m p a r i n gbioaccumulation in transplanted and na-tural populations of mussels. Untouchedmussels had significantly higher growthrates than animals measured weekly (Fig.15.9). The results from five different sitesconfirm that frequency of measurementaffects mussel growth rates. The largest

differences were measured at the threehighest concentrations of TBT in seawaterwhere growth rates were approximatelydouble those measured weekly. This sug-gests that stress attributable to handlingenhanced the effects of TBT on juvenilemussel growth rates. Rates from biweeklymeasurements are similar to end-of-testmeasured rates and suggest they may be areasonable indicator of growth in the naturalpopulation.

Measurements were changed fromweekly to biweekly after test 4 to reduce thehandling stress affecting growth rates.Increases in survival, growth, and a shift inthe relationship between concentrations ofTBT in seawater and in tissue after test 4suggest that handling affected survival,bioaccumulation, and growth (Fig. 15.1).


Even though concentrations of TBT inseawater were relatively constant, thelargest increase in mean growth rateoccurred between tests 4 and 5 when mea-surements were changed from weekly tobiweekly. The dramatic increases ingrowth and survival after test 4 suggestthat switching to biweekly measurementshad as much or more of an effect ongrowth as the concentration of TBT inseawater.

Collectively, these results suggest thattransplanting and frequency of measure-ment both affect bioaccumulation. Theeffects of handling on bioaccumulation arealso shown in the comparison betweentransplanted mussels and the naturalpopulation (Fig. 15.10). At the marina site

with the highest concentrations of seawaterTBT, transplanted juveniles accumulatedmuch more TBT than natural juveniles intwo of the three comparisons. Differencesin growth rates associated with lowerconcentrations of seawater TBT and re-duced handling could explain higheraccumulation than expected at lower sea-water TBT concentrations. Although onlyone comparison was made, transplantedadults accumulated more TBT than naturaladults. Juvenile transplants measured forgrowth on a biweekly basis did not ac-cumulate significantly different amounts ofTBT than transplants measured only at theend of the test. Although only one com-parison was made, accumulation of TBT injuvenile mussel transplants measured bi-

Influencing factors 321

weekly was slightly higher than in trans-plants measured only at the end of the test.At the Navy site concentrations of tissueTBT were significantly lower than at themarina site and generally too low formeaningful comparisons.

15.5.2. JUVENILES VS ADULTS

It is often assumed that juveniles of mostspecies are more sensitive to contaminantsthan adults. Hall and Bushong (Chapter 9)suggest such a difference in sensitivity toTBT based on laboratory studies. In thefield, rapid growth and low mortality havebeen associated with smaller animals whileslow growth and high mortality have beenassociated with larger individuals(Freeman and Dickie, 1979). Juvenilemussels in our field studies exhibitedhigher survival when exposed to TBT than

adults in a laboratory study (Valkirs et al.,1987). These apparent differences betweenjuveniles and adults could be attributableto the differences in laboratory and fieldexperiments. However, test animals in thisparticular laboratory study were undersevere nutritive stress, and mortalitieswere highest among the largest mussels.Their findings and our results are con-sistent with relationships established fromphysiological measure-ments in the labora-tory and field observations. Laboratory ex-periments demonstrate increasing energylosses from respiration as a function ofmussel size (Bayne et al., 1985). Theseenergy losses are enhanced by increasingtemperature and decreasing food ration.Additional energy losses occur with game-togenesis. Therefore, under natural con-ditions the highest mortalities and lowestgrowth rates would be expected in the


largest mussels during gametogenesiswhen temperatures are highest andchlorophyll-a lowest (Freeman and Dickie,1979; Incze et al., 1980; Bayne et al., 1985;Mallet et al.,. 1990). The introduction ofTBT or any other contaminant could signi-ficantly increase the effects of these stress-ful conditions.

It is well established that juvenilemussels grow faster than adults (Rodhouseet al., 1986), but reported effects of size andage on bioaccumulation are conflicting(Phillips, 1980). Fischer (1983, 1988) sug-gests that bioaccumulation in juvenilemussels represents a short-term inte-gration while bioaccumulation in adults re-presents a longterm integration. Manyauthors have reported that smaller musselsaccumulate more heavy metals than largermussels (Lobel and Wright, 1982; Ritz et al.,1982; Calabrese et al., 1984; Amiard et al.,1986). Juvenile oysters have been shown toaccumulate more TBT than adult oysters(Ebdon et al., 1989). In our study juvenilemussels generally accumulated more TBTthan adults (Fig. 15.10). Our field-transplanted juveniles also accumulatedmore TBT than adult mussels in anotherSan Diego Bay monitoring study of naturalpopulations (Grovhoug et al., Chapter 25).The differences in accumulated TBT maybe due to the combined effects of size,handling, and natural factors.

15.5.3. NATURAL FACTORS

Even in the absence of contaminants, na-tural factors affect survival, bioaccumu-lation, and growth. They could also alterthe effects of TBT. Important natural fac-tors include temperature, food, currentspeed, salinity, suspended sediment, andtidal position (Seed, 1976; Newell, 1979;Kiorboe et al., 1981). In our field-transplantstudies there is no statistically significantrelationship between chlorophyll-a (food)and growth rate, but chlorophyll-a mea-surements may not provide the best es-

timate of available food. Particulate or-ganic carbon has been related to growth insouthern California coastal waters (Pageand Hubbard, 1987). Our weekly and bi-weekly chlorophyll-a measurements mayhave been too infrequent to detect a stat-istically significant effect. On the otherhand, chlorophyll-a levels in San DiegoBay may not be a limiting factor. Due to apaucity of freshwater inputs, salinity is nothighly variable in most areas of San DiegoBay and is probably not a significant factorin mussel growth rates. Salinity could bean important factor in other more typicalestuaries. Current speed appeared to bevery important, but measurements weretoo infrequent to quantify a significant re-lationship. Suspended sediment was notmeasured but has been very important inother TBT studies (Waldock and Thain,1983).

Temperature had more of an effect ongrowth than any natural factor quantifiedin our studies. There is a significant linearrelationship between temperature andgrowth rate (p<0.0005), but only 11% of thevariance in growth can be explained bytemperature. The interaction between tem-perature and seawater TBT on growth ofjuvenile mussels is shown in Fig. 15. 11.The three-dimensional surface plot pre-dicts optimum growth near 20EC and atthe lowest concentrations of TBT in sea-water. Optimum growth near 20EC hasbeen reported in several other laboratoryand field studies (Incze et al., 1980;Almada-Villela et al., 1982; Bayne et al.,1985). The lowest growth rates are pre-dicted at the highest TBT concentrations(>100 ng L-1) and temperature extremes(>22EC, <16EC). Therefore, the highest andlowest temperatures may impose naturallimits on mussel growth rates and survivalin San Diego Bay (Wells and Gray, 1960). Itis possible that high summer temperaturesalone adversely affect survival, bioaccumu-lation, and growth, and limit natural pop-ulations in the southern portion of SanDiego Bay.

Mussel bioindicator model 323

Comparing the correlation coefficientsof growth rate versus temperature and con-centrations of TBT in seawater shows howthe relationships change (Fig. 15.12). Thisalso establishes a statistical correlation forthe graph in Fig. 15.11. There is a signi-ficant linear relationship between growthand temperature in the range of 14-20EC(p<0.0001, r = 0.50, r2 = 0.25). The relation-ship improves when the seawater TBT data<100 ng L-1 are analyzed separately(p<0.0001, r = 0.57, r2 = 0.32). The combina-tion of TBT masking temperature effectsand too few exposures >20EC precludeddetecting a statistically significant relation-ship at higher temperature ranges. Consi-dering all the data, growth rates are cor-related with both seawater TBT concentra-tion ® = -0.72) and temperature (r = +0.33);although TBT concentration is more im-portant. Using only seawater TBT data<100 ng L-1, the importance of seawaterTBT concentration is reduced by half.

Figure 15.12 also shows the dramatic shiftfrom a positive to a negative correlation attemperatures above 20EC that is consistentwith the literature. TBT appears to modifythe effects of temperature and temperatureappears to modify the effects of TBT onmussel performance.

15.6. MUSSEL BIOINDICATOR MODEL

White (1984) has cautioned against the ar-bitrary use of mussel monitoring systemswith out developing a model to be tested.The mussel bioindicator model in Fig.15.13 emphasizes the importance ofnatural factors in modifying the environ-mental effects of TBT in San Diego Bay anddepicts the inherent cycles of natural fac-tors, TBT inputs, and mussel biology. It issuggested that natural factors act directlyon TBT by altering bioavailability anddirectly on mussels by altering bio-chemistry and physiology. Other con-taminants are also involved. For example,the marina with the highest seawater TBTconcentrations also had the copper con-centrations (.10 Fg L-1) (Krett Lane, 1980;Johnston, 1989). These copper con-centrations were approximately 150 timeshigher than seawater TBT concentrationsand about three times higher than the USEnvironmental Protection Agency (USEPA) water quality criterion (US EPA,1985). Similar copper concentrations re-duced mussel growth rates in laboratorystudies (Stromgren, 1982; Manley et al.,1984). Even though they are much lesstoxic than TBT, accumulated petroleumhydrocarbons were shown to have more ofan adverse effect on mussel physiologythan accumulated TBT (Widdows et al.,1990).


In addition to being modified by naturalfactors and TBT, mussel biology is also af-fected by internal biochemical and physio-logical cycles. These factors act in concert tomodify mussel growth, bioaccumulation,and survival. The key to calibrating the mus-sel bioindicator is separating the effects ofnatural and biological factors from theeffects of TBT. The clear arrows in the model(Fig. 15.13) represent the direct effects ofrelatively uncontaminated environments onmussel growth and survival. The dark ar-rows show direct effects from TBT con-tamination on growth, bioaccumulation, andsurvival.

This mussel bioindicator model has beendeveloped based on field studies in SanDiego Bay and existing knowledge of mus-sels and TBT. It demonstrates the difficultiesin quantifying bioindicator responses to TBT

by illustrating the complex and dynamic in-teractions among various factors that affectmussel growth, bioaccumulation, and sur-vival. All these factors must be measured totest and verify the model and calibrate thebioindicator. The model presented herecould be used for other contaminants andother bioindicators; however, given theunique and variable mussel responses toTBT exposures in San Diego Bay, extremecaution should be used in extrapolatingspecific results to other environments,contaminants, or bioindicators.

Conclusions 325

15.7. CONCLUSIONS

This TBT study in San Diego Bay demon-strates the utility of juvenile mussels as bio-logical indicators. The most significant re-finement in approach was the integration ofchemical and biological measurements.Chemical measurements of TBT in seawaterand mussel tissues were combined with bio-logical response measurements of survivaland growth of transplanted mussels. Mus-sels were used as indicators of contamina-tion and effects. By using this approach andtransplanting mussels under natural testconditions, we were able to quantify thedistribution and effects of TBT, identifyshort- and long-term trends, and refine the

use of mussels as biological indicators. Thefrequency of water sampling combined withmultiple measurements on the same indi-vidual mussels of a very restricted sizerange facilitated defining statistically signi-ficant relationships. The lack of replicationfor tissue residue may have precludeddefining a better relationship between tissueTBT concentration and juvenile musselgrowth rates. It should be emphasized that astatistically significant relationship does notprove an environmentally significant rela-tionship. Conversely, not finding a statis-tically significant relationship could be at-tributed to the influence of various factorsidentified here. Zar (1974) states:

Although in many cases there is amathematical dependence of Y on X,it cannot automatically be assumedthat there is a biological cause-and-effect relationship. Causal relation-ships are concluded only with someinsight into the natural phenomenonbeing investigated and may not beconcluded by statistical testing alone.It must also be remembered that aregression function is mathematicallynothing more than a line forced to fitbetween a set of data points, and maynot at all describe a naturalphenomenon. Although an empiri-cally derived regression functionoften provides a satisfactory andsatisfying description of a naturalsystem, sometimes it does not.

Identifying some of the natural andpollution-related factors affecting musselperformance when exposed to TBT undernatural conditions is only the first step incalibrating the field bioindicator and veri-fying the model. Crucial questions regardingenvironmental fate and effects of TBTremain unanswered. Factors identified inthis study as affecting mussel performanceneed further investigation. Other contami-nants and natural factors require additionalstudy. Differences in results between the


laboratory and the field require meaningfulexplanations. All test conditions modifyingresults must be identified and their relativeinfluence quantified.

ACKNOWLEDGMENTS

We thank Team Hydride (S. Frank, J.Guerrero, M. Kram, M. Stallard, P. Stang, A.Valkirs, and J. Vaughn) for measuring TBTin >700 water samples. We also thank S.Cola and L. Kear for measuring TBT in >100tissue samples, B. Davidson for assistanceand the Office of Chief of Naval Researchthrough Naval Sea Systems Command.

EPILOGUE

While it is beyond the scope of this chapterto provide all the significant findings andparadigms established since this study wasconducted relative to TBT and mussels, wewould like to provide an updated perspec-tive. This new perspective will emphasizenew results and new ideas concerning thethree primary goals of our original work: (1)effects of TBT on juvenile mussels, (2) trendsin the distribution of TBT and its effects, and(3) refinements in the mussel bioindicatorsystem.

Most importantly, threshold concen-trations predicted in this chapter to alwaysreduce growth in juvenile mussels have notchanged. Threshold concentrations for noeffects remain speculative based on ourdata, but are probably lower than originallypredicted. Although the data provided inthis chapter regarding the size effects on bio-accumulation of TBT were only preliminary,additional San Diego Bay studies conductedin 1990-1991 and 1993, and a Puget Soundstudy, have confirmed that smaller musselsconsistently accumulate higher concentra-tions of TBT in their tissues and consistentlyhave higher rates of survival (Salazar andSalazar, 1995; Salazar et al., 1995). The inter-action between size effects, concentrationeffects, and natural factors in determiningthe time required for TBT to reach equili-

brium in mussel tissues remains unclear.Although the 1990-1991 study showed thatsmall, fast growing mussels could reachequilibrium in less than 3 weeks at low TBTconcentrations, we still recommend an ex-posure period of 60-90 d based on the timeto reach equilibrium for larger mussels athigh TBT concentrations (Salazar andSalazar, 1995). The trends in distribution ofTBT and its effects have not changed. The1993 San Diego Bay study shows that musselgrowth rates were higher and tissue TBTconcentrations lower than in 1990. Thissuggests that mussel growth rates haveincreased as a result of the decreases in TBT.

The mussel monitoring system describedin this chapter is operational. A successfultest could be conducted using the protocolsoutlined in Salazar and Salazar (1995). Wecontinue to add improvements in logistics,handling, and interpretation from insightgained in subsequent tests. From 1990 to1995, we have conducted transplants usingsix different bivalve species and over 15,000individuals. The major conceptual refine-ments in experimental design that we ori-ginally introduced in this chapter remainunchanged. Since a 2 mm size range is notalways achievable, 10 mm is recommendedas a target size range. Absolute size is lessimportant than minimizing the range. Otherlogistic refinements include using dispos-able mesh bags with individual compart-ments instead of rigid plastic cages, andinterfacing digital calipers and balanceswith laptop computers to facilitate dataentry in the field.

Although our standard protocol includesmeasuring whole-animal wet-weights andlengths, shell weights, and tissue weights,we only presented the whole-animal wet-weight data in this chapter. Several sub-equent studies have shown that under cer-ain conditions, estimates of growth based ontissue weights are more informative thanwhole-animal growth. We are in the processof reanalyzing the tissue weight data fromthese nine transplant experiments to see ifthese data can provide any further clari-

References 327

ication regarding TBT effects on mussels.Our most recent statistical analysis of copperand zinc tissue data collected during tests 5,6, 7, 9 have shown significant relationshipswith juvenile mussel growth rates (Salazarand Chadwick, 1991; Salazar and Salazar,1995). The data further indicate that, inaddition to TBT, smaller mussels accumu-ated higher concentrations of several metalsthan larger mussels (Salazar and Salazar,1995; Salazar et al., 1995). Some of the re-ationships were more informative by analy-ing the data on a per animal basis (content)rather than on a concentration basis.

In the final analysis, bivalves in cagescould be viewed as an exposure system tomake any clinical measurements. Thisversatility is another advantage of usingbivalve transplants. Further, the discri-minating power of bivalve monitoring iswell beyond the order-of-magnitude dif-ferences initially suggested by Goldberg etal. (1978) as a limit for detecting significantdifferences in concentrations of tissue con-taminants. A number of studies, includingour own, have often found statisticallysignificant differences in both bioaccumu-lation and growth that we believe areenvironmentally significant. Some of thesesite-specific data differ by a factor of 2 orless. In-situ, studies with caged bivalvesfacilitate measurements that make this fieldbioassay similar to laboratory bioassays interms of experimental control and predictivepower, and retain the environmental realismof traditional field monitoring. Approachesthat combine the integrating ability of filterfeeding bivalves, the versatility of in-situcaging, and the relationships between bio-accumulation and bioeffects as demon-strated here, have a number of applicationsfor environmental monitoring, risk assess-ment, and establishing regulatory criteria.

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MUSSELS AS BIOINDICATORS: EFFECTS 15 OF TBT ON SURVIVAL

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