Stanford Schwimer: Page 2 INTRODUCTTON The determination of metal concentrations in marine organ- isms is becoming increasingly nécessary for ecological study due to interactions of essential trace metals with toxic heavy metals. The significance of trace metals in the marine biosphere was documented many years ago (CORNEC, 1919; CLAPKE ARD WHEELER, 1922), and research in this field has flourished with the improv- ment of analytical methods. Reviews by VINCGRALOV (1953) and COLDBERG (1967) demonstrate increasing importance of heavy metals in the marine environment. The accelerated research in heavy metal analysis has led to greater knowledge of the elemental levels in several marine organisms (PROOKS AND RUNSBY, 1965; CULKIN AND RILEY, 1958; — BERTINE AND GOLDBERG, 1971; GRAHAN, 1972). However, the marine environment being extremely complex, diverse, and in constant flux, comparatively few organisms have been analyzed for their elemental content. The objectives of this study were: 1) to determine the levels of Ag, Al, Ba, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr, and Zn in the herbivorous gastropod Olivella biplicata (SOWERBY, 1925), the predatory gastropod Polinices lewisii (GOULD, 1847), and the predatory starfish Pisaster brevispinus (STIMPSON, 1857) (these latter two species prey upon Olivella (see EDMARDS, 1969)); 2) to clarify the relationship of metals between the two trophic levels; 3) to define the range and variability of metal con- centrations within the same species with respect to geographic location, and 4) to determine the difference in elemental con¬ centrations between certain tissues. Stanford Schwimer: Page 3 METHOD. — Olivella, Polinices, and Pisaster were collected subtidally on sandy beaches (Figure 1) and placed in plastic bags. In the laboratory, the animals were placed in filtered sea-water aquaria and allowed to purge themselves of sediments for a minimum of twenty-four hours. The gastropods were boiled in distilled water for three to five minutes. This procedure allowed the soft parts to be easily separated from the shells with a plastic fork or stainless steel forceps. Both the shell and soft tissue were oven dried at 65 degrees Centigrade for a minimum of forty-eight hours. he starfish were dissected with stainless steel scissors immediately after purging in sea-water. Part of one ray (not including hepatic caecum or gonads), hepatic caecum, gonadal tissue, and a combination of the pyloric stomach, cardiac stomach and rectal caeca were dissected, placed in tared beakers, and oven dried. When dry, the soft parts were ground to a fine powder with morter and pestle. The Pisaster ray homogenate contained particles of about 0.5 centimeters across or smaller. The gastropod shells were not ground. The Polinices shells were separated into four parts: operculum, anterior, posterior, and spire. The Olivella shells were digested whole. Aliquots were weighed into tared beakers and digested with 70% or 90% nitric acid. Shells were digested with concentrated hydrochloric acid. The samples were left at room temperature Stanford Schwimer: Page 4 for one hour, refluxed for one hour, and evaporated to 5 ml. Two to three milliliters of 30% hydrogen peroxide added drop¬ wise oxidized any remaining organic matter. One milliliter of hydrochloric acid was added and'the samples were adjusted to a final dilution (1%) with distilled water. The samples were analyzed by atomic absorption. Reagent blanks were run with each group of samples. Readings of less than 2% absorption were discarded due to probable background effects. Means and 95% confidence intervals were determined using the expression t VX/N, where Vx/N is the standard error of the mean for NX30, and t is the Student's t value for N-1 degrees of freedom (ELLIOTT, 1971). LESULPS AND DISCUSSION Means and 95% confidence intervals for Olivella, Polinices, and Pisaster soft and hard parts are listed in Table 1. Con- centrations of aluminum, cadmium, copper, iron, lead, manganese, nickel, silver, and zinc for Olivella and Polinices soft parts, and selected Pisaster tissues from different localities are presented in Figure 2 and Figure 3, respectively. The results are discussed individually for each element with respect to geographical location, trophic level, and physiological parts. Ba, Ca, k, Mg, Na, and Sr values are not discussed but are listed in Table 1. All elemental values are expressed as Ag/g (ppm) and ppm is used throughout the text. Stanford Schwimer: Page 5 Aluminum: Polinices had highest levels at Fisherman's Wharf (50.6 + 7.5 ppm) and Olivella had highest values at Elkhorn Slough (Os.5 + 12.3 ppm) and lowest at Monterey Outfall (59.6 + 19.2 ppm). These values are similar to the 50 ppm level described by VINCGRADOV (1953). The high values of Olivella at Elkhorn Slough are probably due to the greater amount of clay particles in suspension in this area. Cadmium: Y (1956) found cadmium levels in Mollusca EULLIN AND RILE of 3 ppm, and levels in Echinodermata of 1 ppm. Results indi¬ cate that these levels are consistent with Olivella (2.3 + 1.9 ppm -- 4.2 + 5.1 ppm) and Polinices (0.3 + 0.5 ppm -- 1.6 f 1.3 ppm). GRAHAM (1972) found cadmium levels in the gastropod Tegula funebralis (ADANS, 1854) at Fisherman's Wharf of 2.2 ppm. Pisaster hepatic caecum was found to have high Cd levels (46.3 + 20.6 ppm) at the Monterey Outfall. Cadmium is a known contaminant (NILSSON, 1970). Besides being a general cytotoxic agent, cadmium competes with zinc and copper for the same binding sites. Zinc and cadmium occur synergistically in nature; these two elements are likely iso- morphic in metallothionein (PASSOW, 1969). Copper: Levels of 4 ppm to 50 ppm (VINCGRADCV, 1953) are much higher than copper levels in sea-water, 3 x 10-3 g/1 (GOLDBERG, Stanford Schwimer: Page 6 1963), yet much lower than copper levels observed in experimental animals. Fisherman's Wharf had the highest values of copper: Olivella - 177.3 + 16.5 ppm; Polinices - 115.4 + 11.3 ppm. GRAILIM (1972) and FITZ (1971) found copper concentrations at Fisherman's Wharf in Tegula funebralis and Emerita analoga (STIMPSON, 1857) to be 175 + 9.C ppm and 71 - 90 ppm, respectively. Pisaster had higher copper levels at Fisherman's Wharf than at the Monterey Sewage Outfall. It is not known why these levels are highest at the Wharf. Perhaps high copper values are re¬ lated to pollution from boats. Copper, a highly electronegative metal, is classified as a metallo-enzyme (BOWEN, 1966). This high electronegative quality may act to destroy or deactivate enzymes. ADELSTEIN AND VALLEE (1962) and UNDERWCOD (1971) cited that haemocyanin, an important copper containing respiratory pigment in various gastropods and other marine species, was a constituent in blood plasma. On this basis one would expect to find this element concentrated in gastropod soft parts. Iron: Olivella had the lowest iron levels (358 + 47 ppm) at the Monterey Sewage Outfall, while Polinices levels remained equal at both localities. These values, although lowest, are still above the 200 ppm level for Mollusca and 300 ppm level for Echinodermata specified by VINOGRADOV (1953). CULLIN AND RILEI 1758) and Lit¬ (1958) show that Littorina littorea (LINNAEUS, 17 (171 torina littoralis (LINNAEUS, 1758), gastropods from the Irish Sea, to have values within VINCGRADOV'S limits, 171 ppm and Stanford Schwimer: Page 7 229 ppm, respectively. Gastropod shells were found to be highest in iron at the Fisherman's Wharf site. Olivella shells had mean values of 74.6 C.2 ppm, while anterior parts of the Polinices shell had a value of 81.1 + 22.4 ppm. Pisaster data suggested that the soft parts were concentrating more iron at the Outfall, while the ray was higher at Fisherman's Wharf. Iron is found in high concentrations in clay, and so the high values may be natural levels. The high values at Fisherman's Wharf could, however, be attributed to the iron structures located there. Iron, like many other elements, is essential for life. As a metal-activated enzyme, iron is able to activate many oxi¬ dases involving molecular oxygen. However, iron is more common- ly considered a metallo-enzyme, because it is more firmly bound to a protein in constant stoichiometric ratios rather than loosely held. One such metalloprotein, echinochrome, is found in Echinoderm blood (CANNAN, 1927). Lead: Levels of lead were found to be greatest for Olivella at Fisherman's Wharf (8.2 + 3.6 ppm) and Polinices (5.0 + 2.4 ppm) soft parts. GRAHAM (1972) found the gastropod Thais emarginata (DESHAYES, 1839) to have levels of 9.8 + 4.0 ppm in lead at Fisherman's Wharf. Olivella shells were lowest at the Wharf, with highest values at Elkhorn Slough. Polinices shells also had highest concentrations at Elkhorn Slough. Pisaster tended to concentrate lead in the rays, Fisherman's Wharf being higher than Monterey Outfall (30.9 + 4.3 ppm and 15.9 + 4.2 ppm, Stanford Schwimer: Page 8 respectively). These high values for the shells and Pisaster rays may be due to light scatter. BOWEN (1966) classified lead as a very high potential pollutant. Lead occurs chiefly as a contaminant, and can pro¬ duce toxic effects by combining with cellular membranes to altei permeability (PASSOW, 1969). Cadmium and copper may also pro- duce toxic effects by acting on membrane permeability (PASSON, 1969). Lead contamination is very high (0.07 - 0.35Ag Pb/ng sea-water) in the marine environment (PATTEPSON, 1971). Soil contains 10 ppm (BOWEN, 1966), and stated levels for Mollusca and Echinodermata are 0.7 ppm and 187 ppm, respectively (VILO- GRADOV, 1953). The highest input of lead into the biosphere is mostly due to automobile exhaust emission. kanganese: Manganese has been reported at 60 ppm in Echinodermata and 10 ppm in Mollusca (VINCGRADOV, 1953). Polinices was found to have the highest concentration of Mn at Elkhorn Slough (27.5 + 5.5 ppm), while levels in Olivella soft parts remained consistant, as did levels in rays of Pisaster. Manganese levels in Pisaster soft parts were very low (non-detectable —- 3.0 + 1.4 ppm). Manganese is another element that is essential for life. It is considered a metal-activated enzyme (VALLEE, 1955), and was found to activate certain phosphate transferases and decarboxyl- ases (BOWEN, 1966). Organically bound Mn is found in Mollusca (VINCUMADEV, 1953), one of these proteins being pinnaglobin (ECERI, 1963). Stanford Schwimer: Page 9 Nickel: Olivella bodies had highest concentrations at Fisherman's Wharf (1.8 + 1.7 ppm), and Polinices was highest (1.9 + 1.5 ppm) at Elkhorn Slough. These values compare with 4 ppm for Mollusca (VINOGRADOV, 1953). Olivella shells were much higher than the soft parts (16.9 + 2.9 ppm), and the same is true for Polinices shells (16.2 + 8.4 ppm). Pisaster rays were found to have higher concentrations than other tissues, however, these high values may be due to light scattering caused by the large amounts of calcium in the samples. Nickel affects several enzymes in vitro- activation of arginase, carboxylase, trypsin, citritase, and inhibition of acid phospotase- but is not necessary for proper functioning. Silver: VINOGRADOV (1953) stated Mollusca levels as O ppm, and Echin- odermata levels as 3? ppm. Since sea-water contains only 3x 10-44g/1 (GOLLBERG, 1963), these relative low levels are quite high enrichment factors. Olivella bodies were found to be much higher at Monterey Outfall than any other area (10.7 + 4.8 ppm). The rays of Pisaster contained equal concentrations and were higher than any other tissue. This high value in the rays may be due to scatter. VEITH (1971) reported effluent levels of many metals from the Monterey Sewage Cutfall, silver having a concentration in the effluent of 25 ppm. This high input directly into the biosphere could possibly explain the high Olivella values. ffinity for Silver, an electronegative metal, has a strong imino, amino, and sulphydryl groups (ECWEN, 1966). These groups Stanford Schwimer: Page 10 are most likely reactive sites on many enzymes, and hence silver has the capacity to deactivate the enzyme. It follows that silver is therefore a poison, by virtue of its reactivity with proteins, especially enzymes. Silver competes with copper, but probably does not inhibit the copper enzyme (CHRISTIAN ARD FELDMIN, 1970). Zinc: Polinices soft parts had highest concentrations at Elkhorn Slough (288 + 18 ppm) and Olivella bodies were highest at Fisherman's Wharf (127 + 14 ppm). GRAHAM (1972) found zinc levels in Tegula funebralis to be 198 + 7 ppm. These values all correspond with those in VINCGRADOV (1953) of 200 ppm for Mollusca and 25 ppm for Echinodermata. Pisaster displays an interesting pattern as to zinc concentration. The Wharf site was higher in all tissues except for the gonads. At the Outfall, the gonads were excessively high, with values averaging 511.9 + 28.1 ppm. Echinoderm gonads are known to contain high zinc levels (personal correspondence, MARTIN). Those Pisaster collected at the Outfall had less developed gonads than those from Fisher- man's Wharf. This may be the reason why specimens from these two localities differed in Zn concentrations. Another possibility could be the high zinc effluent from the Monterey Outfall of 820 ppm (YEITH, 1971). Zinc, a metallo-enzyme, is essential to most life forms (EOWEN, 1966). Zinc can displace copper from a protein, and hence act as an antagonist, or can compete in some proteins with Stanford Schwimer: Page 11 cadmium for binding sites. Trophic Magnification: Much attention has been given to the problems of biological magnification of DDT, DDE, PCB's (polychlorinated biphenyls), and other halogenated hydrocarbon concentrations (WOCDWELL, 1967; JOHNSON, et al, 1971; NIMTO, et al, 1971). Upon analy- sis of Table 1, it seems evident that heavy metals do not con- centrate through the trophic levels of the organisms studied. SUMMARY Although concentrations of heavy and trace metals are not magnified through the studied trophic levels, this possiblity may still exist. This is suggested from the high elemental levels observed at Fisherman's Wharf (Cu and Pb), Monterey Sewage Outfall (Ag, Cd, and Zn), and Elkhorn Slough (Fe). These high values, possible consequences of pollution, should not go unchecked. Much study is still needed as to toxicity, base levels, and the possible biological magnification of heavy and trace metals. Schwimer: Page 12 Stanfor ACOMLEDCEN I sincerely thank Dr. John H. Martin for his guidance and instruction of atomic absorption spectroscopy and related analytical methodology, Dr. Donald P. Abbott for expert ad- vice and suggestions, Dave Phillips for inspiration and understanding in the beginning ..., and all of the scientists, including George Mpitsos, of Hopkins Marine Station. Finally, I wish to thank Marsh Youngbluth for his con- stant assistance in statistical analysis, general research, the writing of this paper, and especially for his unrelentless prowess on the volleyball court. Stanford Schwimer: Page 13 LITERATURE CITED Adelstein, S. J. & Bert L. Vallee 1962. Mineral Metabolism. Volume 2E: 37. (C. L. Comar & F. Bronner, eds.). Academic Press, New York. Berting K.K. & Edward D. Goldberg. 1971. Trace Elements in Clams, Mussels and Shrinp. In¬ stut. Royal des Sciences Naturelles de Belgique, Brussels, Belgium. Boeri, Enzo 1963. Non Porphyrin-Metalloproteins. In Comprehensive Biochemistry 8: 38. (M. Florkin & E. H. Stotz, eds.). Elsevier, Amsterdam. Bowen, H.J.M. 1966. Trace elements in Biochemistry. Academic Press. London & New York. 241 pp. Erooks, Robert R. & Martin G. Rumsby 1965. The biogeochemistry of trace element uptake by some New Zealand bivalves. Limnol. Oceanogr. 10:521-527. Cannan, Robert Keith 1927. Echinochrome. Biochem. Journ. 21: 184-189. Christian, Gary D. & Fredric J. Feldman 1970. Atomic Absorption Spectroscopy; Applications in Agriculture, Biology, and Medicine. John Wiley and Sons, New York. 490 pp. Clark, F.W. & W.C. Wheeler. 1922. Inorganic constituents of marine invertebrates. U.S. Geol. Surv. Profess. Papers, 124:1-62. wimer: Page 14 sch Stanford Cornec, Ernest 1919. Spectrographic studies of the ash of marine plants. Compt. Rend., 168: 513-514. Culkin, F & J.P. Riley. 1958. The occurrence of Ballium in Marine Organisms. Journ. Mar. Biol. Ass. U. K., 37: 607-615. Edwards, D. Craig. 1969. Predators on Olivella biplicata, including a species specific predator avoidance response. The Veliger, 11: 326-333. Elliott, J.M. 1971. Some methods for the statistical analysis of samples of Benthic Invertebrates. Freshwater Biological Association Scientific Publication No. 25: 83-86. Fitz, J.D. 1971. Trace Metal concentrations in the sand crab Emerita analoga (Stimpson), its eggs, and the sand it in- habits from the Monterey Bay area. (Unpublished M.S. on file at Hopkins Marine Station Library). Goldberg, Edward D. 1963. "The Sea", Vol 2: 3. (M.N. Hill, ed.), Interscience, London. Goldberg, Edward D. 1967. Review of trace element concentrations in marine organisms. Puerto Rico Nucl. Center, 35 pp. Johnson, B. Thomas, Saunders, C. Richard, Sanders, Herman C., and R.S. Campbell. 1971. Biological magnification and Stanford Schwimer: Page 15 degradation of DDT and aldrin by freshwater invertebrates. J. Fish. Res. Bd. Canada, 28: 705-709. Mullin, J.B. & J.P. Riley 1956. The occurrence of cadmium in seawater and in marine organisms and sediments. Journ. Mar. Res., 15:103. Nilsson, Robert 1970. Aspects on the toxicity of cadmium and its compounds; A review. Ecological Research Committee. Eulletin No. 7. Nimmo, D.R., Wilson, P.D., Blackman, R.R. and A.J. Wilson, Jr. 1971. Polychlorinated biphenyl absorbed from sediments by fiddler crabs and pink shrimp. Nature, 231:50-52. Passow, Hermann. 1909. In Effects of metals on cells, subcellular elements and macromolecules. (Maniloff, Jack, Coleman, James R., and Morton W. Miller eds.). Rochester Conference on Toxi¬ city, 2nd, 291-344 pp. Patterson, Clair 1971. Lead. In Impingment of man on the oceans. Donald W. Hood, ed. New York, Wiley-Interscience. 738 pp. Shuster, Carl N. & Benjamin H. Pringle 1968. Effects of trace metals on estuarine mollusks. Proc. Ist Mid-Atl. Indust. Waste Conf., Univ. Delaware CE-5: 285-304. Underwood, Eric J. 1971. Trace Elements in humans and animal nutrition. 3rd Ed. Academic Press, New York. 543 pp. age 16 Star Vallee, Bert L. 1955. Zinc and metalloenzymes. Advanc. Protein Chem., 10:317 Veith, Robert G. 1971. Trace metals in Monterey Peninsula sewage (unpub¬ lished M.S. on file at Hopkins Marine Station Library). Vinogradov, A.P. 1953. The elementary composition of marine organisms. Sears. Found. Marine Res. Mem. 2, New Haven. 647 pp. Woodwell, George M. 1967. Toxic substances and ecological cycles. Scientific American, 216:24-31. Stanford Schwimer: Page 1 IGURF ETANATIONS Figure 1: Sampling Sites round Monterey Bay, California. Area A: Fisherman's Wharf, terey; Area B: Monterey Sewage Cutfall, Monterey; Area C: khorn Slough, Moss Landing. Figure 2: Comparison of Olivella biplicata (clear) and Folinices lewisii (stipple) soft parts: Means, Medians, Range, and 95% Confidence Intervals (see Figure 3 for symbol explanation). Figure 3: Comparison of Pisaster brevispinus tissues: Pay (clear), Gonad (positive diagonal slope), Stomach (negative diagonal slope), and Hepatic Caecum (stipple). Means, Medians, Range, and 95% Confidence Intervals (see legend for symbol explanation). o OO O o + + + 1 +1 +4 aitatata- . . 0 oo o ) . DO t o o8 +1 + + + + +1 + +++1 +1 + + + + + tt +1 + OT oo . o O QO - + OO 1 c o0 O O O o o ataaa- * . 10 — + + +1 + + k oc * o oo oO o - oo + + ++ o o o .. C aoo ooo aoo ooo oo mo s y y oo on og og o o o o o o o 35 5 5 Od Od + + 55 — 0 U. c o 0 0 1 — + —— 0 +1 — — — 33 — — — — — — O0 O ( OO O O Od Od o ooo . 0 u 5 oou oou 0 c .. .. 60 50 0 .. .. .. .. .. .. 0 U 1 U U 5. 0 U O O O O O O 2 Od 5 5 — — — — — c oc 0a O0 OC oc E — — — 5 — — — — — P + + C 0 0. O. U 5 U . -— -— — —— c o aaa- . a — — — — — — — — 5 U OOo 0 0 — — — — — — - — oo — —— 90 00 ooo . ooc a O aoo a o 8 + S Od .. .. . oo . o - o0 . 5 — — — — — 6 . oOo o o O d oc + ot oo oP od O O 8 C o 0 + + + +1+1+ +++ t +1 + + + ++ 0 +1 O O C o O o O e ooc O o o ooc Oo o O O .. O 1 — . 1 a +I+1+ +1 +1 + + + — ood oc 58 0. o oooo + +1+ | tir kir OC O O 1 O oc o oo 8 ao u o o 0 ap e . o . O V O o 1 ti ti tit +1 + +1 +1 + 7 +1 + ++1 - o . . c o aoo + ONN C ao O oc . . wooooo oo . . o o a — — — — +I +1 + + +1 +1 +1 tt +1— +1 +1 + +1 + o + + ..oc . . N v .O od o oo 0 aa ooo oo o oc . 0 0 o 1 1 44 +1 + +1 7 74 ++ • - O . .. .. 1 C o o o o oo HO O o wo op e 0 OC o ++ + + 1 oo.o.m * — oo d C a o 0 . C . oo tt it tit 17 4 +++1 + O ooo S o .. oc ed o o o o o o od 1 +1 + +1 + +1+ +1 iooo . O 8 8 — 5 ooo o 4 + +1+1 +1 + + + +1 +1 +1 +1 1 O u. o o o o o o oo o — 5S' 50 34' 90 FIGURE 1 122°00' 121055 12150' AREAC MONTEREY BAY AREA / LAREA A 122 00 12150 ss Stanford Schwimer: Page 21 55 310 5o 95 36 eu 0 F ++ 00 — 1 I 4 8 5. 2 10 I taakaa- 2 a 10.0 - 1 - l. 0 — c 8A A gore 2250.3 Ac 8 MN I c Stanford Schwimer: Page 22 ZN 1 17 c A A 1 8 100 10 — —:— B 2292. 19443 ed P KB 4 — 2 — . + 4 A u 1139 22 1 Stan Schwimer: Page 23 HIEH — 15% MEAN — t.f. MEDIAN° 0 — NN T - 100 1.0 - 0. 1 K A