Tributyltin Effects on Juvenile Mussel Growth

Juvenile mussels (Mytilus edulis <20 mm) were exposed to three concentrations of tributyltin (TBT) in two site-specific, flow-through bioassays with unfiltered seawater. Mean TBT concentrations were 70, 80 and 200 ng/l in Test I (196 days) and 40, 50 and 160 ng/l in Test II (56 days). Treatments did not significantly affect juvenile mussel growth during the first 56 days of exposure in either test. After 63 days, all treatments significantly reduced growth in Test I. No significant mortalities occurred at any TBT concentration in either test. Increases in weights and lengths of Tank Control animals in Test II were much greater than during the first 56 days of Test I. Further, weight increases in the Pier Control were almost four times greater than in the Tank Controls during Test II. These data suggest that test animals were probably under significant stress induced by the Sioassay test system. The data also suggest that the effects of TBT on juvenile mussel growth may have been overestimated in this and other studies.

laboratory studies with associated growthinhibiting stresses. Third, reported reductions in Juvenile mussels (myti e .20 mm) were mussel growth rates may be attributable to stress exposed to three concentrations of tributyltin from urmeasured or unknown factors (10). (TBE) in two site-specific, flow-through bioassays with unfiltered seawater. Mean TBT concent-ations ExTrapolation to the environment is difficult were 70, 80 and 200 ng/l in Test I (196 days) and (8, 10, 11, 12) due to differences in conditions 40, 50 and 160 ng/l in Test II (56 days). Treat-between the above laboratory studies with TBT (4, ments did not significantly affect juvenile mussel 5) and the field. The above field study (7) pro-X growth during i days ofexoure in vided environmentally realistic test conditions, , ' After 63 days, all treauents siga-" yet still failed to confirm a cause-and-effect ficantly reduced growth in Test I. No significant relationship between TT and mussel growth rateC mortalities occurred at any TBT concentration in because of unmeasured variables, uncontrolled _\ther test. --increases in weight n and Eand an inappropriate control site. Ld ted Tank ontrol anmals in Test II were muh greater sample sizes and short-term exposures in previous than during the first 56 days of Test 1. Fur-her, laboratory and field stud.ies only permit estLrating _ weight increases in the Pier Control were almost order-of-magnitude effects (10) on growth of C four times greater than in the Tank Controls during mussels exposed to high TBT concentrations. These Test II. These data suggest that test animals were studies have not adequately assessed the subtle probably under significant stress induced by the effects of realistic exposure states for low TBT bioassay test system.
The data also suggest that concentrations of interest (13) near those C the effects of TBT on juvenile mussel growth may predicted to be safe (50 ng/i) in the es-"arine I have been overestimated in this and other studies. envirO-nt (14).

Two site-specific, flcw-through bioassays with -INMDC!ICN
TBT were conducted in San Diego Bay using a Portable Environmental Test System (PETS) to Growth represents the integrated response of evaluate the long-term effects of low TBT conceninternal biological process. It is generally be-trations on juvenile mssel growth. This approach lieved that in any envirorment the added stress of combined the advantages of controlled laboratory toxicants reduces animal growth rates and that dosing with realistic field test conditions in an juveniles are more sensitive than adults. signifi-attempt to provide more meaningful results than r cant reductions in growth rate could adversely previous studies. This report addresses results of affect the population (1).
A good measure of those site-specific bioassays and their r stress in juvenile mussels is shell growth since it significance in relation to previous laboratory and is a significant part of total somatic production field studies. .. (2) and there is no interference by gametogenesis (3 ) .

MT O S A D N, % 1 T .% Z S
A number of investigators lave studied the PETS was evaluated over a 7-month period in effects of tributyltin (TBT) on mussel (Mtilus San Diego Bay using TBT leachates. A more detailed edulis) growth. Several laboratory studies (4, 5, description of the test site and the physical/ 6) and a single field study (7) have reported chemical parameters monitored are presented elsereduced mussel growth at TST concentrations of 230 where (15). As part of that evaluation, two overng/l and greater in tests ranging from 7 days to 5 lapping tests were conducted with juvenile mussels months. The interpretation and envirormental sign-(M. edulis). Test I lasted 196 days (June to ificance of these data are unclear (8).
Test II lasted 56 days (Octch-e to these high concantrations are not characteristic of December 1986). Test II .as conduce concur--ntY most harbor environments and are restricted to with the last 56 days of Test I. During that ti.re enclosed basins with poor tidal e.'rhange and large mussels from each test were in the same tanks ar.d a ibers of organotin-painted vessels (9).
Second, subjected to identical experimental conditions. mussel growth in the field may be different than in Temperatures in PETS tanks ranged from 15.0 -88 3 o192 8 3 0.
25.9 0 C R -22.3 0 C) in Test I and 15.0 -21.7 0 C (re weight and leth data among replicate TT moncen--18.6 C)-in Test 1I. Bay temperatures at the trations at each sampling interval to test the seawater intake ranged from 14.0 -24.9 0 C (R = null hypothesis: TBT exposure has no effect on 21.9 0 C) during Test I and 14.0 -20.8 0 C (Rjuvenile mussel growth. If the null hypothesis was 17.8 0 C) during Test II. In a single 24-hour study rejected, Duncan's new wultiple-range test was used in December, temperature ranged from 13.5 -16. 9° to determine which TBT concentrations significantly in PET tanks and 15 -16 CO at the seawater intake. affected growth. In addition, a series of linear regression analyses were performed on log-11e eqpei wtal design cnsisted of a control transformed data to compare the slopes of estimated and three TBE test concentrations with three repligrowth rates. If slopes differed by more than two cates each and approximataly 50 animals per treatstandard deviations (P < 0.05), growth rates were ment. Mean TBT concentrations (±s.d.) were 70 considered significantly different. (±40), 80 (±40) and 200 (±70) ng/1 in Test I and 40 (±15), 50 (:19) and 160 (±66) nq/1 in Test II. TheSe concentrations represented nominal 10, 25 FESULTS and 100% leachate solutions, respectively, but measured concentrations were markedly different Growth rate estimates from changes in mussel than expected (15).
Mean TBT concentration in weights and lengths over time are given in Figure 1 control seawater was approximately 10 ng/l. TBT and Table 1. Figure 2 gives the slopes of regresconcentrations were measred by hydride derivatiza-sions performed on lo-transformed 196-day weight tion and atomic absorption detection (5) and and length data from Test I. In Test I significant reported as tributyltin chloride. All plastic reductions in growth were found between 63-196 days holding trays were leached for 2 weeks in the at all three TBT concentrations. Growth was inlaboratory with filtered flow-through seawater.
creasingly suppressed with increasing TBT concen-M9Lssels were initially selected by length (-10-15 tration. At the highest concentration mussel m), takin great care to randomly distribute them growth rate was approximately half the Tank Control within the replicate plastic holding trays. Therv rate. After 196 days, mussel weights and lengths were no significant differences in weights or in the Tank Controls and the 200 ng/l TBT treatlengths among replicates at the start of either ments increased by 450% and 250%, and 65% and 37%, 0 test. All test animals were acclimated for 2 eks respectively. MLssel weights in the to lowest TE 0 in PEI'S control tanks before the experiment began.
treatments increased by 355% and lengths by 53%. C Both the multiple-range test on weekly length and , weight measurements and the linear regression Test I began with 192 juvenile mussels, 16 analyses on the 196-day growth rates gave the folanimals per test tank (48 per treatment). Some lowing results: Contr-ol ? (70 -80) , 200 ng/l TBT > animals died and some escaped; the 163 survivors treatments. That is, control growth was signifibad initial lengths of 10.7 -17.0 ma ( -14.4 mm) cantly different than treatment growth at all TBT G, and weights of 124 -563 mg (R -313 mg).

Mussels concentrations.
There was no significant C were collected from plexiglas panels that were difference in growth between the two lowest cncen-< suspended at the test site in January. This was trations. serial ANOVAs showed there was also a rr done to ensure that all test animals were from the significant difference among treatment replicates. same spawning season and were approximately the same age.
In Test I there were no significant differences in lengths, weights or growth rates when TBT Test 11 began with 234 mussels, 18 animals per treatments were co.ared to Tank Controls. After tank (54 per treatment) and 18 for the Pier 56 days, weights of Tank Controls and the 160 nq/l Control. Oe mussel escaped; the 233 survivors had TBT treatments increased by 99% and 71%, ir initial lengths of 10.1 -15.0 mm ( -12.6 mm) and respectively. Lengths increased by 30% and 18%, weights of 142 -553 mg (x -287 mg).
The purpose respectively. At the highest TBT concentration Ir of the Pier Control was to determine if the test growth rate was approximately 70% of the control. 2 system affected growth. Without sufficient numbers There were significant differences in weights, of mussels from the same site, Test II animals were lengths and growth rates between the Pier Control collected from the rubber tire fenders on the and Tank Controls. Pier Control mussels increased Coronado Say Bridge approximately 1 km from the in weight by 378% and length by 73% after 56 days. test site.
In the Pier Controls and Tank Controls respectively, growth rates by weight were 140 and Whole-animal wet weights and lengths were 40 mg/wk, and by length were 1.13 and 0.48 mU/wk. measured weekly using vernier cAiipers and an After 56 days, weights for Tank Controls in Test I electronic balance. Byssal. threads were carefully and Test II iresed by 70% and 99%, respectively. broken prior to removing mussels from the trays --for measurements. Presence/absence of byssal threads was recorded weekly as another measure of In Test I, byssal thread production decreased envirormental stress (16,17,18).
to a minim= by day 49, when half of the mussels exposed to the highest TBT concentration produced Q Statistical analyses were conducted only on no byssal threads. Byssal th-read production re-o survivor data. For each treatment cuulative mained suppressed until day 140. From then on percent increases in lengths and weights were calthere were no obse.zable diferenses in byssal culated to normalize size effects and to estimate thread production at any concentration. No dif-relative growth rates for graphical presentation. ferences in byssal thread production were obser-ed Serial M2VAs (P < 0.05) were performed on pooled in Test 11.
'Q4  DTsazs::cN rates between the Pler Control ard =1-e Tank Ctnrol suggest that test or.ditions were not as enrr Cncaared tz =ntrols, sicnif icant reductions mentally realistic as expected due to systemin uvenile mussel growth rate were measured in ireuced ztxress. oi::rece _n rc--wth between Test PETS after 196 lays at TBT concentrations much I and 1_ during the first 56'days of exposure lcwer than prerrim-_s>_ reported in other laL-cratorI indicate that test conlditions were mar:edly or field studies (4, 5, 6, 7).  cf: the differences in results can be attributed to t!here is increased respiraticn and reduced filtrasystem-induced stess and differences in tesz contion in mussels. Above 250C there are adverse ditions. Salazar et al. (15)  r sys-cem and suggested that tahk conditions were more part of Test I suggests that juvenile mussels were favorable fcr mussel grcuth in Test II than Test I. under temperature stress. When temperature ie-r This was due to lower mean BT concentrations, creased in the latter part of Test I, growth ratas more optimum temperatures and reduced biomass increased in all t.-rat:ents. San Diego Bay si.r~er during the last 56 days when the tests overlapped.
te_..eratures may reduce grswrth because they ap-r Grur.ah rates and byssal thr ead production of Test I proach levels that adversely affect mussel mussels increased dramatically durinq this period.
physiology. Adverse temperature effects were ac-Baynre ard Thoascrn (19) have described scme of the gra-vatad by PET tanks which raised the temper:tre ;hysiological -onsequencas of maintaining M. edulis hic.er than ambient. Measured daily fluctuation ".as .n t-he laboratory as weil as the specific effects aLmost four times higher in t tanks than in t-he of temperature and nutritive stress on reducino bay. both growth rate and reproduction effectiveness (20, 21).
Hcniever, growth rates of Tark Cntrol anmals were higher in Test II than the fie 56 days of Test I.  have shown that acc-11ation of TT by mussels in te laboratory is different frm that in the field.

TBT CCNCENTRATION (ng/I)
If acc-mulated TNT affects mussel cgowrh rate, these results sugest that grow-ch rates of m-ssels expsed = TBT in the laoratory ',uld be different WEIGHT SLOPE (= 95% CL) n in th field. for j.e l-erfn-a mussel and in T. tes these decreases arm probably an arx-fact of statissnticainalyses. They also reported a 66-day :C-10 of apprsximatelv 125 nq/l TBT fcr adult mussels.
-There were no sicnificant mortalities attri-u-able to Ter eoposure in either Test : or :1 of the PTS studies at concentratlons up to 200 ng/.. TBT C effect-on survi-mal and grrowt. may have been over-The major ccnrtributLing factors to nutritive estLma-_ed in the Valk:.rs et a . (5) and other stres in ?EIS mussels wera probably reduced .nyto-laburatzr" tests due to.utit-e stress. plankton levels and reduced suspended sediment =pred to amient bay water. 1ee-y intanca Str--o=n-n and Bongard (6) used juveniles much revealed large amctnts of sediment trapped in the smaller than in PET and retc-:ed significant plumbing that never reached test tanks. As reuctic-. in mussel shell growth after only 7 days N .diced by a=miatad sediment in the bc-of expcsu-e to 400 q/l TT. While t!-e laser measurethe PETS tanks however, there was much more wn technie is interesting, repor-irg effects at suspended sediment in PETS than in laborator-y such hi h levels in a test of such short duration Sbiies.

MI"
derives additional raztrition from suspended particulates.
'hese authors predicted gro'd-h rate increases of 30 -70% with the addition of only 5 m;/l suspended sediment. Waldcck and Thain (25) pxvvided additional-data showing a 72% anhancamert in oyster grwt !. with 75 mg/l suspended sediment. Mo~m of the previous laboratory growth studies with mussels exposed to TBT included suspended sediment. This may have resulted in nutritive stress. Nussels in any envirarment with suspended sediment and a nac.-ral diet may be , ler less tctal stress, grow fa_-ar axd pcentilly more resistant to MrT.
S---q I developed for field use (in-situ). Thretically, mussel grwth rates could be used to care dosed Field measurements provide a realistic test versus undosed animals with a control group close platform for long-term staiies, but generally lack enough to be a true control .n all other srnrironthe control necessary. for experimentation and mental parameters yet far enough away to be establishing cause-and-effect rela ioships parti-unaffected by TBr. cularly with TBT (12).
In a San Diego Bay field test Staphenm et al. (7) exposed mussels at four sites along a known TST concentration gradient of -2 km. Significant differences in growth were This research was sponsored by the Naval abserved after 150 days exposure to 230 n/l TnT. Facilities Engineering Ccunad, Office of Ciief of The control site, hLever, was inappropriate in Naval Pesearch and the David Taylor Naval Pesearch that it differed in many parameters other than TnT and Developmnt Canter. We wish to thank P. Stang, concentration. In addition, there were many other A. Valkirs and M. Stallard for chemical analyses variables along that TBT gradient which may have and G. Pickwell, K. Ri-hter and P. Seligman for affected mussel growth. Since mussel growth can editorial assistance. A special thanks is due to exhibit ext-eme local variation (22,28,29), the B. Davidson for maintaining the test system, utility of using mussel growth as an index of conducting statistical analyses and editorial -ussel stress at different sites in the field withsp . out appropriate experimental control must be challerged.
White (30) has cautioned against the arbitrary use of mussel monitoring systems without d inh el to be tastd   Koen. 1986. Gmte pr-duc--ion, deection of significant TNT effects at crnr---a-somatic growth and multiple-locus enzyme tins much loer than previously reported. It must heterozygosity in Mv-'u edulis. Mar. Biol. be emphasized that system-induced stresses also 90:209-214. reduce juvenile mussel gr=.th. It was Lpossible to quantify the relative effects of each.
The 70 (4) 7-n, J. E. and X. J. Waldcck. -9_5. The na/l TET that reduced grcwth rates in this study growth of bivalve spat exposed to organotin would not have an effect on mussel growth rates lehatas fb antivouaiq .eaits. Intargaticnal under most environmental conditions . Only under C i for te Eporation of the Sea (pr'rin.). very stressful environmental conditions similar to those in MIS experimental tanks would this n- The authors feel that results from the three Ichikawa and M. Martin.

1986.
Growth laboratory studies, the field study and this siteabnormalities in mussels and oysters from areas specific bioassay were as much A function of un-with high levels of tributyltin in San Diego Bay. dosing system with TST-ccated panels s'-.,ilar to that -sed in the laboratory and Ln .!TS should be