Marine Environmental Research 32 (1991) 51-59

A Field Bioassay Approach to Determining Tributyltin Toxicity to Oysters in California

M a r k S tephenson

California Department of Fish and Game, Moss Landing Marine Laboratories, Moss Landing, California, 95039, USA

A BSTRA CT

Field experiments were conducted with oysters, Crassostrea gigas to determine the toxicity of tributyltin ( TBT). Oysters were transplanted to over one hundred stations distributed in 25 marinas usually in a transect of .four or five stations along a gradient next to vessels extending to station locations distant from vessels. In almost every bay in California there are many vessels, the TBT levels in water are > 50ng/liter (parts per trillion), and the oysters exhibit a chambering response similar to the chambering response that was indicative of the destruction of the oyster industry in France. Although oysters were transplanted to stations that had extreme environmental conditions (near sewer and petroleum refinery discharges) no evidence of chambering was observed indicating that the chambering response does not occur with every type of environmental stress. In addition, oysters transplanted to a marina that had been abandonedJbr 18 months did not show a chambering response, whereas those transplanted in marinas that had vessels always showed a chambering response. The use of field techniques in determining toxicity responses worked in this case but the response could have been more clearly defined if more stations were added in each marina and the dose response determined on a marina by marina basis. More TBT measurements in water would have also helped to refine the dose-response relationship. A first estimate o f a toxicity value can be determined from these data to be less than 40 ng/liter.

I N TRO D U C TION

TBT has been implicated in the destruction of marine resources in Europe and the US. In France and the UK, commercial oyster growing habitat has

51 Marine Environ. Res. 0141-1136/91/$03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain

52 Mark Stephenson

been degraded and attributed to TBT (Alzieu et al., 1980, 1986; Waldock et al., 1987; Thain et al., 1987a,b). In Coos Bay, Oregon, (Wolniakowski et al., 1987) and Morro Bay, California (Stephenson, unpublished data) oyster growing areas have been abandoned or the oysters show a shell thickening deformation characteristic of TBT toxicity. In light of the great potential for environmental degradation, it is important that realistic TBT water quality criteria be established to protect marine life.

This paper assesses the impact of TBT on oysters in California and presents a case for using a field bioassay approach for setting water quality criteria and monitoring TBT. Water quality criteria are traditionally derived from laboratory data, but this approach has been criticized because these studies do not reflect what happens in nature (White & Champ, 1983; Salazar, 1986). Field bioassays have the advantage of being environmentally relevant but lack the controls inherent in laboratory toxicity testing. For example, in the present study environmental variables such as temperature, food supply, salinity, and the concentration of many pollutants could not be controlled. A hypothesis was tested that the toxicity response from TBT would be much stronger than that from other variables. If this were true then the toxicity response next to vessels would be strong and consistent in all the marinas studied. Water quality criteria could then be generated. To test this hypothesis, oyster toxicity and TBT concentrations in water were monitored at a large number of sites many of which represented extreme environmental conditions. Oysters were placed in 26 bays, estuaries, and near-shore locations including highly influenced areas (near sewage, petroleum refinery and harbor discharges), abandoned harbors, and off-shore areas.

METHODS

Three Crassos t r ea g igas oyster transplantation experiments were conducted. The first experiment was started in October 1986. Small oysters, 3.5 + 1 mm (mean + standard deviation) in length, were transplanted to bays in 2 mm mesh bags and retrieved 2 to 3 months later.

In the second experiment, larger oysters, 18"0 + 2"5 mm in length (weight 0.13 g), were used for Humboldt Bay and Crescent City stations. In the third experiment larger oysters, 25 + 3.1 mm in length (weight 0.64 g), were used for all stations between San Diego and San Francisco Bay. In the second and third experiment oysters were transplanted in 2 mm mesh bags in August 1987 for a duration of 4-5 months. These three oyster experiments are described in more detail in Smith et al. (1987) and Stephenson et al. (1988).

In the laboratory the oysters were dissected, the tissue weighed, and the soft tissue homogenized. The shells were sectioned longitudinally and the

TBT toxicity to oysters in California 53

length and the degree of chambering determined. An L/D (length/depth measurement of upper shell) ratio of >_ 12 indicates a commercially viable oyster (Thain et al., 1987a,b).

The tissue was analyzed for TBT using a technique developed by Stephenson and Smith 0988) that has been intercalibrated with other techniques (Stephenson et al., 1987). The method uses a sodium hydroxide wash to exclude di- and mono-alkyl tins but probably includes some other tri- or tetra-alkyl tin compounds as well as TBT. The intercalibration showed the results to be in fair agreement with other techniques. All concentrations are expressed on a dry weight basis.

The water was analyzed by hydride derivatization according to the protocols described by Valkirs et al. (1976). The values reported are single measurements made at the time of oyster collection.

RESULTS

Oysters transplanted near refineries, sewer outfalls, and control areas (distant from obvious pollution sources) do not seem to exhibit a toxic response in their L / D (using an L / D of > 12 as a normal oyster) similar to that found in marinas (Tables 1 and 2). Interestingly, normal oysters were found at the abandoned marina at Palo Alto (no vessels were at this site for the preceeding 18 months).

In these studies, every one of these oysters that were placed in 25 different marinas developed abnormal chambering responses. Likewise, TBT levels of > 50 ng/liter were found in all 25 sites.

TABLE 1 First Oyster Chambering Experiment

Location of oyster Chambered No growth Normal (%) (%) (%)

Marina abandoned for 12 months (Palo Alto) 0 0 100

Refinery 0 0 Castro Cove 0 0 100

Sewage discharge Alviso Slough 0 0 100

Control Area Pt Pinole 0 0 100

Marinas Oakland Inner Harbor 100 0 0 Lauritzen Canal 90 10 0 Richmond Inner 70 30 0

TABLE 2 Second and Third Transplant Experiments

Station name Station no. Wet weight TBT water L/D TBT oyster (g) (ng/liter) (ng/g dry)

Crescent City inner boat 2. i 0.17 38 11.53 NA a Humboldt Shell Oil 101-3 1"32 25 12,5 464 Humboldt Woodley I C.G. 101.4 2.25 9 10,88 98 Humboldt Woodley I 102.5 1.46 33 10 170 Humboldt Small Boat Basin 103'2 0-71 34 1603 47 San Francisco Bay Unical 301-4 1.32 33 14.18 43 San Francisco Bay Santa Fe 303-2 0.54 49 4-79 209 San Francisco Lauritzen 303.3 0.41 49 6 140 San Francisco Santa Fe end 303-4 0"35 107 7.33 1 200 San Francisco Richmond inn. 303"6 0.42 38 4.73 278 San Francisco Alameda 307'2 0.29 70 6-32 160 San Francisco Oakland M. 307,3 0.94 43 5.49 2 503 San Francisco Oakland Emb. 307'6 0-96 NA 4.59 363 San Francisco Dumbarton 321 2.23 NA 12-79 52 San Francisco Palo Alto 323.3 1.11 NA 13-88 120 Santa Cruz Harbor Mouth 400"1 0"17 93 8-43 NA Santa Cruz Harbor T-East 400-4 0.35 107 6.51 NA Santa Cruz Harbor V 400-5 0.28 202 7.64 755 Santa Cruz Harbor J 400'6 0-23 NA 8'49 857 Moss Landing North Harbor 401"3 1"38 42 5"36 1 547 Moss Landing Parsons 402,2 2.91 10 11.82 102 Moss Landing Skippers 402.5 4-83 27 9.58 606 Moss Landing H W l Bridge 403 3.95 23 4.26 152 Moss Landing S. Harbor 403.5 0.42 43 4-97 886 Monterey Comm. Wharf 421.4 0.74 NA 5.24 1 200 Monterey Pier D 421.7 0.14 NA 10-14 NA Monterey Pier B 421.8 0.19 NA 6.38 1 378 Carmel Control 423.2 0.97 NA 18'11 80 Carmel Sewer 423.6 2 NA 18.16 NA Morro Bay Duch Rest. 429.2 0.65 26 6-64 80 Morro Bay State Park 429-3 0.2 77 4-97 NA Santa Barbara Harbor pat. 470 0'21 58 4.31 928 Santa Barbara Dry Dock 471 0.22 101 4,26 1 537 Santa Barbara 3C Dock 472 0.33 118 3-83 1 270 Santa Barbara 4 Dock 473 0.25 138 3"9 550 Carpenteria Dock 475 4-49 9 11"98 34 Ventura Boat Works 485 0.34 186 4.8 884 Ventura Sports fishing 486 0.45 106 4-39 2 056 Ventura Channel I 488 0.33 260 3.94 3 262 Port Hueneme Wharf B 506"1 0.45 60 4-12 797 Port Hueneme Entrance 506.3 0-99 48 4.24 1 452 Port Hueneme Wharf A 507.5 0.68 77 3.67 451 Port Hueneme Boat Launch 507.7 1.15 81 4.37 2 104 Marina del Rey Patrol 554 0.45 45 3"6 965 Marina del Rey G 555 0.27 134 8-81 NA Marina del Rey D 555-2 0.21 270 3-9 NA Marina del Rey Entrance 555.3 2.52 20 5-8 144 Marina del Rey E 556 0-46 141 4.34 648 Kings Harbor E 559"1 0.16 108 4-64 NA Kings Harbor H 559.2 0.21 139 5.79 NA

TABLE 2--contd.

Station name Station no. Wet weight TBT water LID TBT oyster (g) (ng/liter) (ng/g dry)

LA-Long Beach Nat. Steel LA-Long Beach West Basin LA-Long Beach Todd LA-Long Beach Pacific Ave. LA-Long Beach Berth 49 LA-Long Beach Berth 151 LA-Long Beach Slip 240 LA-Long Beach W. Channel LA-Long Beach Fish Harbor LA-Long Beach Watchhorn LA-Long Beach Tide Gauge LA-Long Beach Consolidated Anaheim Navy Bridge Anaheim Navy Marsh Anaheim Fuel Anaheim Edinger Anaheim Warner Ave. Anaheim Harbor Ln. Newport Bay Entrance Newport Bay Turning Basin Newport Bay Hwy. 1 Bridge Newport Bay Rhine Channel Newport Bay Upper Rhine Newport Bay Santiago Cr. San Diego Sweetwater Ramp San Diego Sweetwater 2 San Diego 7th st 32 San Diego 7th st San Diego Glorieta Bay B San Diego Glorieta Bay C San Diego Glorieta Bay Patrol San Diego E. Basin G San Diego E. Basin L San Diego E. Basin Storm San Diego W. Basin Sheraton San Diego W. Harbor I mid San Diego W. Harbor I bridge San Diego Naval T. Center mid San Diego Naval T. Center end San Diego Rubin Rest. San Diego T. Hamms San Diego Comm. Basin 5mph San Diego N. Harbor Dr. San Diego Kettenburg San Diego Comm. Basin B San Diego S.I. Fuel Docks San Diego S.I. Bait Tanks San Diego Degaussing San Diego Zuniga

601 0"14 71 5-49 NA 602 0.26 NA 4'67 NA 602-5 0-19 NA 7"71 NA 602-7 0.32 NA 6'82 3 216 602.8 0.34 45 6'29 NA 603 0.21 61 6"06 420 603.6 0'39 41 6"44 NA 603"8 0-22 NA 9-22 NA 606-2 0.1 113 8-27 NA 606-3 0-15 232 7"55 NA 609 1"01 16 7"46 90 616 0.31 14 7.72 NA 707 0.94 52 4-9 80 708 0.97 34 7.96 286 710.2 0"39 19 4"75 422 713 0.4 48 4'95 334 715 1-42 40 3.51 2776 717 0-38 54 3"48 NA 721 0"53 62 5'43 206 723.4 NA 123 5 ! 011 724 0"53 89 4"09 NA 725 0.24 NA 4-96 NA 726 0.27 214 5"13 NA 726-4 1-34 NA 7.67 220 881.6 4-79 22 6-71 254 881.8 1"56 18 9"13 292 882"5 2 32 6-6 269 883"6 0"8 19 11"18 213 884-1 0"31 86 4.56 870 884'2 0.49 29 3-94 395 884'3 1.03 37 5'28 527 893.3 0-3 130 4.76 379 893"4 0.49 63 5"05 323 894 0"68 10 5"3 966 894.4 0'36 152 5"43 478 894.6 0"14 89 6'5 NA 894.7 0.3 44 3"84 4 015 894.8 1.3 58 4.91 2816 894.9 0.62 49 4"79 236 895 0"54 20 4.31 752 895.3 0'45 22 4-04 1 288 897 0-65 60 5-62 402 897.5 0-16 200 6"41 317 898 0-25 280 6-59 1 305 898-3 0-41 73 3"85 355 899-4 0-37 197 4.64 880 900 0"91 49 4.55 1 054 901 2.99 19 6.79 559 902 7.02 NA 11"84 281

" NA = not analyzed.

56 Mark Stephenson

TABLE 3 Second and Third Oyster Transplant Experiment (Summary of Table 2)

TBT Oyster Oyster Oyster water tissue LID TBT

(ng/liter) weight (g) (ng/g dry wt)

Control sites in bays Humboldt Bay Woodley I 33 2.3 12 34 San Francisco Bay

Dumbarton Br. NA a 2.2 13 52 Moss Landing-Parsons SI. 10 2.9 12 100 Carpenteria Marsh 9 4.5 12 34 San Diego-Zuniga Jetty < 5 7.0 12 280

Off-shore Carmel Bay NA 1.0 18 395

Near refinery discharge Unical 33 1.3 14 43

Near sewage discharge Palo Alto NA 1-1 14 248

Naval facility 7th Street Base 19 0-8 12 210

Marinas 50-400 0"15-0.27 3.9-7.6 760-10000

NA = not analyzed.

The relationship between oyster final tissue weight, L/D, and TBT concentration in the water shows that there is a critical level of 30-40 ng/liter past which there are no normal oysters (LID > 12 and weight > 1.5 g) (Fig. 1). This critical level corresponds to approximately 500 ng/g (dry weight) in the oysters tissues (Fig. 1). A no-effect level, where all the normal oysters are found, is difficult to estimate from these few data but appears to be less than 10 ng/liter.

DISCUSSION AND CONCLUSION

Most of the bays in California have many vessels (as many as 12000 in some bays) and the TBT levels are quite high (above 50 ng/liter in the harbors with vessels as of January 1988). Results presented in this paper indicate that there are no normal oysters found where the waters exceed 30 to 40 ng/liter. Oysters appear to be good indicators of TBT contamination. At every marina that had vessels, oysters had growth anomalies and conversely, they did not exhibit the same anomalies at any non-marina setting including sites at sewer and petroleum refinery discharges. This relationship held even though oysters were placed in 25 different embayments subject to a wide

TBT toxicity to oysters in California 57

5 .

4

3 -

2

B

m

B=l

B m

B

~ E I ~ B BI

• do ~o 300 TBT (rig/liter IN WATER)

20

t NORMAL OYSTERS 012 L/D)

m

Im lIB

I! i~1

o do 2o~ 300 TBT (rig/literlN WATER)

8

: '" 0 • n I . ,B ,

0 1000 2000 3000 4000 OYSTER TBT (ng/g d~)

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NORMAL OYSTER5 (>12 L/D)

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Fig. 1. Relationships between TBT and oyster toxicity measurements.

variety of stresses from other pollutants. Salazar and Champ (1988) have recently criticized the use of Ci~assostrea gigas as an indicator of TBT toxicity citing non TBT factors causing shell thickening. We find no evidence of this. They also criticize the use ofC. gigas in areas where it is not native. C. gigas is the most successful mariculture bivalve in the world having been used in almost every temperate marine environment, which speaks of its hardiness. It appears to survive and thrive in temperate climates. Even though it will not reproduce successfully it does not follow that the adults living in temperate areas are stressed and are prone to chambering. C. gigas appears to work as an indicator species along with some marine snails to act as an early warning system to indicate TBT toxicity.

Although one can obtain an estimate of the levels of TBT necessary to cause shell deformities in oysters from this data, the data show more variability than the typical laboratory experiment. This is inherent in the field approach and the experimental design that was chosen. Data from 25 embayments were lumped to include much between embayment variability. This variability could be reduced by making the same plots at each embayment, however, more data points would be needed at each embayment. The other major source of variability was the limited number of TBT measurements in seawater. At some locations, especially near the mouth of embayments it is difficult to characterize a station with only one

58 Mark Stephenson

sample. In fact, order of magnitude differences are inherent due to tidal fluctuations at some stations where a mixture of bay and off-shore waters occur (Clavell et al., 1986). This variability could be reduced by either taking more samples at each station or eliminating some stations where high levels of variability would be expected.

Preliminary criteria to protect oysters from TBT could be developed from these data. At 40 ng/liter the effect of TBT on oysters is absolute in that no oysters developed normally and were commercially viable. At 10ng/liter there are still some oysters that have abnormal development. Alzieu et al. (1989) report that their unpublished data indicate that concentrations of 2 ng/liter can cause deformities. This is not inconsistent with the results of this study. In this study there were no samples that contained 2-10 rig/liter so an evaluation at this level was not possible. There needs to be more data collected in marinas that have water levels of less than 20 ng/liter to refine this criterion. TBT was restricted to large vessels ( > 2 5 m in length) in California in January 1988. In a few years many of the harbors should have TBT values < 50 ng/liter and should be resampled in order to refine this criterion.

A C K N O W L E D G E M E N T

This study was sponsored by the California State Water Resources Control Board.

REFERENCES

Alzieu, C., Thibaud, Y., Heral, M. & Boutier, B. (1980). Evaluation des resques dus a I'emploi de peintures anti-salissures dans ies zones conchylicoles. Rev. Tray. lnst. Peches Maritimes, 44, 301-49.

Alzieu, C., Sanjuan, J., Deltreil, J. P. & Borel, M. (1986). Tin contamination in Aracachon Bay: Effects on oyster shell anomalies. Mar. Poll. Bull., 17(11), 494-7.

Clavell, C., Seligman, P. F. & Stang, P. F. (1986). Automated analysis of organotin compounds: a method of monitoring butyltins in the marine environment. In Proc. Organotin Symposium of the Ocean's '86 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1152-4.

Salazar, M. H. (1986). Environmental significance and interpretation of organotin bioassays. In Proc. Organotin Symposium of the Ocean's '86 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1240-5.

Salazar, M. H. & Champ, M. A. (1988). Tributyltin and water quality: A question of environmental significance. In Proc. Oceans 1988 Conference, Organotin Symposium (Vol. 4), Marine Technical Society, Washington, DC, USA, pp. 1497-1506.

TBT toxicity to oysters in California 59

Smith, D. R., Stephenson, M., Goetzl, J., Ichikawa, G. & Martin, M. (1987). The use of transplanted juvenile oysters to monitor the toxic effects of tributyitin in California Waters. In Proc. Organotin Symposium of the Ocean's '87 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1511-16.

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Stephenson, M., Smith, D. R., Goetzl, J., Ichikawa, G. & Martin, M. (1986). Growth abnormalities in mussels and oysters from areas with high levels of tributyltin in San Diego Bay. In Proc. Organotin Symposium of the Ocean's '86 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1246-51.

Stephenson, M. D., Smith, D. R., Hall, L. W. Jr., Johnson, W. E., Michel, P., Short, J., Waldock, M., Huggett, R. J., Seligman, P. and Kola, S. (1987). An international intercomparison of butyltin determinations in mussel tissue and sediments. In Proc. Organotin Symposium of the Ocean's '87 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1334-8.

Stephenson, M. D., Aronson, L., Goetzl, J., Ichikawa, G., Paulson, K., Moore, D. & Martin, M. (1988). Report on TBT in California Harbors. A report submitted to the California State Water Quality Control Board, August, 1988.

Thain, J., Waidock, M. J. & Waite, M. E. (1987a). Toxicity and degradation studies of tributyltin (TBT) and dibutyltin (DBT) in the aquatic environment. In Proc. Organotin Symposium of the Ocean's '87 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1398-1404.

Thain, J., Waldock, M. J. & Waite, M. E. (1987b). Changes in concentration of organotins in UK rivers and estuaries following legislation in 1986. In Proc. Organotin Symposium qf the Ocean's '87 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1352-6.

Valkirs, A. O., Seligman, P. F., Stang, P. M., Homer, V., Lieberman, S. H., Vafa, G., & Dooley, C. A. (1976). Measurement of butyltin compounds in San Diego Bay. Mar. Poll. Bull., 17, 319-24.

Waldock, M. J., Thain, J. & Waite, M. E. (1987). The distribution and potential toxic effects of TBT in UK estuaries during 1986. Appl. Organometallic Chem., I, 287-301.

White, H. H. & Champ, M. A. (1983). The great bioassay hoax and alternatives. In Hazardous and industrial solid waste testing: Second Symposium, ASTM STP 805, R. A. Conway & W. P. Gullege. American Society for Testing Materials, pp. 299-312.

Wolniakowski, K. U., Stephenson, M. D. & Ichikawa, G. I. (1987). Tributyltin concentrations and oyster deformations in Coos Bay, Oregon. In Proc. Organotin Symposium of the Ocean's '87 Symposium, Marine Technical Society, Washington, DC, USA, pp. 1438-42.