A stress syndrome in the hard clam, Mercenaria mercenaria

JOURNAL

OF INVERT,?BRATE

A Stress

Syndrome

PATHOLOGY

in

20,

the H.

Graduate

School

242-251

(1%“)

Hard

Clam,

PERRY

JEFFRIES

of Oceanography, Kingston, Rhode Received

Mercenuriu

Uniuersity Island 02881

January

of Rhode

mercenaria

Island,

21,1972

The hard clam, Mezenuriu mercerka, shows a general response to environmental variation and infection. The molar ratio of free t.aurine to glycinc in gill and mantle tissues climbs above 3, while a-amino acids and carbohydrates decrease. Subtle adjustments in the total pattern of free amino acids and fatty acids also occur. but these can be readily seen by changes in biochemical diversity and equitability. In an estuary long polluted with hydrocarbons. the calm population has a patch> distribution and a short lifespan. These variations are attributed to a culmination of abnormal complications superimposed on natural responses to a seasonally changing environment. The process apparently starts after a tarlike irritant collects in cpitheCal tissue and eventually plugs the renal sac. This leads, indirectly, to invasion of shells by PolydoTa sp., a polychaetc which is rarely found in hard clams. .4 syndrome with many facets soon becomes clear, but the situation can be identified and its results predicted by simply observing the responses of taurinc and glycine in stressed and normal populations. When the molar ratio of taurine to glycine is less than 3, the population is normal; if it lies between 3 and 5, a chronir stress is indicated; and zt values greater than 5 the siiuation is acute

Marine invertebrates react to their surroundings in a number of ways. It is often difficult if not, impossible to separate a natural response in a seasonally changing environment from the effects of pollution and other artificial complications. To make this important distinction, WC need a set of readily measured norms that collectively show the status of homeostatic regulation within a species and its community. Each departure would then rcprcwnt a symptom of internal stress but, not necessarily disclose the causatiw factor. If these symptom* are taken togctlicr as a syndrome, the identification of external factors might well be accurate. In this context, hard clam, ibfewena& viewenavia, populations from polluted and clean habitats of Narragansett Bay, Rhode Islan~l, are compared. A st,ress syndrome in the polluted stock is derived from a series of observations on morphological changes, histological variations, infection, carbohydrate levels 242

and the patt.erns of free amino acids and fatty acids. Mort,ality is high in the polluted population. Observations over the last 20 years by the Rhode Island Department of Natural Resource haw shown that the stock deof larvae from pends on recruitment nearby, ul~polluted waters; oUlerwise the polluted clams woulcl soon disappear (G. Gray, pcwonal communication). Abundances are patchy in the pollutecl population, leading Saila et al. (1967) to conclude, after statistical analysis, that much of the yariatiori is due to factors other than 4imcnt and its associated properties. The causative factor now appears to be an irritant that may be dcriwd from hydrocarl~ons discharged into the area. A1n understanding of the ensuing responw~ should 1~1generally useful in helping uq to predict the f&e of invertebrates sub.jected to combination5 of cnvironmcntal st ressorS.

STRESSED ~MATERIALS

AXD

METHODS

Clams were collected with a commercial (rocking chair’) dredge in the West Passage of Narragansett Bay, 10.5 km from its mouth, and at Sabin Point in the Providence River, a tributary of Narragansett Bay. At each station, 60 kg of clams were dredged, and the individuals ranging from 6 to 11 cm in length were saved. L\bout 100 clams were opened and rinsed; gonadal examinations indicated that approximately :$ of the specimens were femalts. The gills and mantle were washed carefully, homogenizecl, freeze dried, ground to a fine powder, and stored for analysis at -2O’C in desiccators. Clams were also maintained in wooclen tanks supplied with clean, running seawater (30-32 y&l. The tanks have no natural sediment for the clams to burrow into, and detrit,us accumulated at a phenomenal rate (132-162 g nl-Z (1-l; Blake and tJeffries, 1971’). They were washed monthly, but cv~n so shell cliscoloration and anaerobic conditions occurred in the sediment. This environment represents, therefore, another stst of stressed conditions. Ilipids were extractecl by the procedure of Bligh and Dyer (1959). Methyl esters of the fatty acids were prepared by treating th(, lipids first with 0.5 x sodium methoxidc in methanol! then with 14% boron trifluoride in methanol-an adaptation of met’hods de~cribcd by Luddy et al. (1960, 1968). Gas chromatographic conditions were : dualflame ionization detectors; stainless steel columns~ 1.86 x 3.2 mm, packed with 17% di(thylene glycol succinate on Chromosorb My (HP), 100/120 mesh ; He flow 30 m/ nun; in,iector and cletcctor temperatures 2.50°C; ancl column temperature programmed from 165’ to 19O’C at 4’/min :tfter a 2-min init’ial period. Peaks were i(hntified from linear relationships between log retention time and carbon-number, and tll(y WYC intcgratecl electronically. Quantit:Ltivc rflsults with NH Fatty Acid Stand-

CLAMS

243

ards Bj D, and F, and Hormel Institute mixture GLC 1 agreed with stated composition with a relative error less than *5% for the ma,ior components (>lO%). A sample chromatogram was discarded if, in replicate analysis, any component varied by more than &5% of its mean value. Free amino acids were extracted by homogenizing 0.5 g of dry tissue with 15 ml water, centrifuging, washing the residue with 10 ml water, combining supernatants, and aclding 15 ml of 95% ethanol to precipitate proteins. After standing for 1 hr, t#he extract was centrifuged and the supernatant was concentrated by rotary evaporation. Analysis by ion-exchange chromatography was made with a Technicon amino-acid analyzer, Model NC-l, operated according to the manufacturer’s instructions for Type B (high resolution) Chromobeads. Uncertainties averaged less than *6:4~ (Reid, 1966) for all components, significantly better than previously reported for acidic and basic components (Jeffries, 1969), probably because an aqueous extract was analyzed, rather than the supernatant from the Bligh am1 Dyer extract. Carbohydrates were extracted into hot trichloroacetic acid solution (Kemp and Kits van Hei,iningen, 1954) and estimated calorimetrically in phenol, with anhydrous dcxtrost> as a standard (Dubois et al., 1956). RESULTS

EnvironwLental

Salinity at’ the bottom usually ranges between 30 and 32 & in the Bay and betKeen 28 and 31 % in the River (Hicks, 1959) ; in both areas temperature ranges from freezing to approximately 22’C, sediment consists of sa,nd for the most part (MeMaster, 1960) and depth is 2-5 m. Detailed studies of the phytoplankton in the Providence River have not been made, but, to the best of our knowleclge there is no reason to suspect that food availability in

244

JEFFRIES

TABLE

Amino

acid

Taurine Glycine Alanine Glutamic Aspartic Leucine Threonine Valine Isoleucine Seritke Phenylalanine Arginine Tyrnsine Cysteic Histidine Lysine Methi<>nille Proline Ornit hine Total n Micromoles

per

gram

1

Normal

Stressed

2O!i.4 81.1 76.3 54.9 27.0 23.3 21.3 16.2 14.2 12.6 12.4 12.2 11.6 7.3 6.5 4.8 3.9 + + 591.0

247.-i

dry

37.6 52.2 43.4 28.8 16.1 17.i 12.0 9.6 10.2 8.0 13.4 7.9 8.4 4.0 5.6 2.0 + + 524.4

tion, but the total amino acid concentration is less than in normal clams owing to lower amounts of most a-amino acids-glycine. alanine, and leucine in particular. With taurine not included, total concentration in stressed clams is only 276.9 pmolcs/g compared to 385.6 pmoles/g iu normal clams. Differences between the two stocks are persistent. They may become even more pronounced from April to November, because the general seasonal pattern is more taurine and less a-amino acid (Table 2’~. Consequently the two stocks can be separated at any time by the molar ratio of taurine to glycinc, which ranges from 2.5 to 2.9 in normal clams and from 5.0 to 6.6 in Stressed clams. Fatty -4cids During spring both stocks have very similar fatty acid patterns (Table 3). The only consistent trends are comparatively low levels of 20: 5 uncler stress and a tendency

we,ight.

the two sampling areas would bc sufficiently different to account for the variations observed here (Pratt 1953, 1959; Pratt and Campbell, 1956 1. Pollution is thus the ma,jor environmental difference between the two stations. The Providence River receives all manner of industrial and domestic wastes, not the least of which is oil from tankers and storage facilities (Farrington, 1971). The Bay station, on the other hand, is in an open, wellflushed region of Narragansett Bay, not, far from the ocean. For convenience the River is termed “abnormally stressed” ancl the Bay habitat is “normal.”

Distributions of free amino acids in clams collected during spring are shown in Table 1. In stressed clams, the most noticcable difference is a high taurine concentra-

Normal Taurine Nov. July Nov.

Glycine Nov.

July Nov.

Alanine Nov. July Nov.

Stressed

-c

212.6 245.2 264.1

(216.i) (221.3)

277.2 31.5.4

(290.2) (2Q2.9)

84.2 73.6 54.3

(95.3)

(i6.4)

57.8 54.2

49.0

c57.21

48.5

(3i.3)

34.8

(44.6)

31.0

(34.3)

-‘ C-52.6) (.i2,7)

.~ C’

5.5.4

a Micromoles per gram dry \I-eight. ,SIn parentheses, corresponding natural (field) populations are given. c Data not available for Nnvember

valltes 1968.

for

STRESSED

for slightly higher ratios of palmitoleic acid ( 16: 1) to palmitic acid (16:O) in stressed clams (0.4-0.6) than is normal (0.2-0.6). Compared with other marine invertebrates, the hard clam has a remarkably stable fatty acid composition. At low temperaturcs, polyunsaturates increase in various bpecies but here this does not appear to be tlw caw.

One peculiarity is 20:4, a minor componclnt usually comprising less than 0.6% of the total. Late in the fall, the amount may go up to 13.9%. Apparently there is a connect’ion with 20: 5; because when 20: 4 increases, 20:5 falls off an equal amount, the total remaining fairly constallt, at about 17-1974. Whenever this reversal occurs, 15: 1 increases several times, reaching concentrations above 6%.

weight acids.

ratio

Labomtorg

of palmitoleic

t’o palmitic

Conditions

In normal clams held for a year in running seawater, taurine increases from 212.6 to 264.1 pmoles/g, but this is still far less than the 315.4 pmoles/g found in stressed transplants held in identical aquaria (Table 2). Conversely, glycine in the normal transplants decreases from 84.2 to 54.3 pmoles/g, which is probably near the lower level reached by this amino acid in M. rnercen&I. .Uanine also decreases, approaching a TABLE

3

F.ITTY ACID COMPOSITIONS OF Uercenariu mewmnwiu FROM NORMAL MJD STRESSED H.\HIT~\TS IN ~~~~~~~~~~~~~~~~~ B.iy, APKK Fatty

Figure 1 shows the ranges of metabolites having substantially different concentrations in the two stocks. As indicated in the left and right portions of the diagram, a symptom of stress may be either a lower or higher than normal range. Glycine and carbohydrates are clearly lower in stressed than in normal clams. Alanine is also low, and the difference with normal at any one time may be great, but there is an equally large seasonal variation, and so the ranges ov(&p. For the components having higher than normal concentrations under stress, only taurinc is exclusive: there is no overlap into the, normal. The other components in Fig. 1 merely show tcnclencies toward high or low concentrations; but as persistent trends they are, nevertheless, the more prevalent and noticeable symptoms collectively describing a synclrome. Thus in stressed clams, total free amino acids and palmitic acid tend to be fomid in low concentrations; high values are found for palmitoleic acid and the

245

CLAMS

acid*

14:u 1.5 : 0 l>:l 16:O 16:l 16:2 17:o 17:l 18:O 1S:l 18:2 19:l 2o:oc 20: 1,’ 20:2 20:4 20:*5 T‘:()e 22:4/ 22:s 22:6 Other Cx~-~z~

1969

Normal

Stressed

1 .o 1.3 1.2 12.2 7.0 0.5 4.2 0.5 6.5 5.9 0.3 0.2 0.6 9.4 1.4 0..5 19..5 3.6 1.1 3.4 16.3 3.3

a Percent of total fatty b Shorthand designation number double bonds. c Includes 18:3. ,i Includes 18:4. * Includes 22: l(?). 1 Includes 24: 1. LI 21:4, 21:-S, 23:1(?).

1 .o 1.3 3 0 12.8 6.0 1.0 4.6 2.0 6.2 6.1 0.i

0 1 0.5 8.4 1.1 0 *5 16.i 4.6 1.3 2.5 15.4 4.1 acid for

methyl number

esters. C atoms;

246

JEFFRIES

STRESS LOW GLYCINE

Concentration

SYMPTOM HIGH

Concentration

TAURINE

elm

Elcl zoo

ALANINE

TOTAL AMINO

TAURlNE/ GLYCINE RATIO

OYiiiiT

FREE ACIDS O-iii2 400

16: 1 600

minimum of approximately 31 pmoles/g after a year in the laboratory. From this it is concluded that glycine and alanine reach minima in the laboratory, regardless of the clam’s environmental history. Taurine, however, is exceptional: first because it increases under stress, and second because there is an additive elect involving the responses to abnormalities originating prior to transplanting. This latter abnormality appears to be associated with a mud-blister infection due to a polychaete (PoZ,udo~~ sp.) that is found only in the stressed clams. Biochemical Dominance-Diversity Awapara (1962) states that free amino acid patterns are “fingerprints” representing a picture of an organism’s metabolic activities-a balance between formation and utilization. Fingerprints are difficult to describe, however, especially when the whole picture is considered. StaGstical procedures have their place (Jeffries, 1969 I, but we can also apply the diversity concepts used by ecologists to compare species distributions and community structure.

Ll

cl

OYil&? 0

FIG. 1. Symptoms of stress in hard clams are shown by key lower than normal (left) or higher (right). Scales are micromoles free amino acids, percent of dry weight for carbohydrates, and methyl esters. April-November 1969.

300

IO

metabolites ranging either per gram dry weight for pervent of total fatty acid

Most formulations of diversity take into account how many species are present and their individual abundances. The more species there are and the more even their abundances become, the higher the diversity. A fundamental problem exists, however, because the number of species found in a sample increases with sample size, and as sample size increases common species become all the more common (Hairston, 1969). Although this is not nearly as serious with the Shannon-Wiener and rarefaction methods as it is for the logarithmic series (Sanders, 19681, there arc obvious difficulties when one compares the results of several investigators. Biochemical diversity is not so limited: the major chemical species, such as amino acids and fatty acids, are ubiquitous monomers and they are measured with rcspect to independent standards. With a fixed number of biochemical “speties,” all we need to be concerned with is the proportion of cacll-the evenness component of ecological species diversity. Dominance, the opposite of evenness, is easier to envision than diversity and it is readily cal-

STRESSED

culated (*Jeffries, 1970). The index of biochemical dominance, D,,,n, for n-many constituents indicates the departure from a distribution in which each component has the same relative concentration. The reciprocal of dominance x 100 is the index of biochemical diversity, D,.n, which gives values directly related to diversity in the usual sense, c.g., H (S’), the Shannon-Wiener index. A4sshown in Table 4, biochemical dominance of free amino acids is greater in stressed than in normal clams, and it increases seasonally, with maxima found in laboratory transplants. These patterns simply reflect an increase in taurine and a decrease in a-amino acids, both seasonally and abnormally induced. Conversely, dominance among the fatty acids is greater in normal than in stressed clams; spring and fall values for each stock arc about equal; and there is a considerable drop in the t’ransplants. Equitability (c’), or evenness of the distribution in relation to a random model TABLE 4 BIWHEMICAL

AMIDE

D~MIN.~NCE

IN

ok

Normal -Amino acids April November Transplants Fat,ty acids April November Transplants

AMINO

HM~D CL.~MS FROM -*ND NORM.IL H.U~IT.ITS

29.1 34.4 36.3

(23.8) (28.3) (28.0) 27..Y 27.3 23.5

AND

F.ITT~

STRESSED

Stressed

31.5 35.7 37.6

(21.7) (26.3) (28.2) 25.0 24.2 23.5

U Biochemical dominance of free amino acids (DE,,?) is greater in clams from stressed habitats; increases are apparent seasonally and after transplantation to laboratory tanks; values in parentheses are the same data recalculated without taurine (DI,,Is). Among the fatty acids, DmIV is greater in normal than stressed clams and the transplants are uniformly low; in these calculations, 16: 2 and 17: 1 have been combined and 19: 1 eliminated (cf. Table 3).

247

CLAMS

TABLE 5 EQUITABILITY

ACIDS

OF FREE

(6) RELATIONSHIPS

.\ND

FATTY

ACIDS

STRESSED

IN

0.75 0.54 0.44

AMINO

AND

CLAMP

Normal Amino acids April November Transplants Fatty acids April November Transplants

NORMAL

(0.97) (0.80) (0.82) 0.86 0.87 0.99

Stressed

0.60 0.45 0.38

(1.07) (0.89) (0.81) 0.94 0.97 0.99

a The relationships of free amino acids depend on whether taurine is included in the calculation or eliminated (values in parentheses). With taurine included, normal is greater than stressed, but if taurine is eliminated, 6 then taking into account only the a-amino acids, stressed is greater than normal. Equitability among the free amino acids decreases seasonally and when clams are brought into the laboratory (transplants) ; the opposite trend is indicated for fatty acids.

(Lloyd and Ghelardi, 1964), also reveals the extent to which taurine dominates the free amino acids (Table 5). With taurine included, c is 0.60 in stressed clams and 0.75 in normal specimens (April), but with taurine eliminated from the calculations, the corresponding values are 1.07 and 0.97. A value near 1 means that concentrations within the a-amino acid pool are randomly distributed, a situation analogous to that predicted by MacArthur’s model for random, nonoverlapping niches among the species of a community. In autumn, however, l drops well below unity, reaching 0.80 in normal clams ancl 0.89 under stress. Fatty acids distributions behave oppositely : stressed is greater t’han normal, and the t’rend is toward higher values seasonally and in the laboratory. Under these conditions, the approximation to a random distribution is remarkable. DISCUSSION

Environmental conditions in the Providence River (the stressed habitat) have ob-

248

JEFFRIES

viously deteriorated over the past 50 years or so. Farrington (1971) reports high concentrations of hydrocarbons in river sediment that he traces to petroleum and sewage. Upon being released into the river, normal alkanes undergo a series of quick reactions, leaving behind cyclic alkanes and aromatic compounds that degrade slowly and become incorporated into sediments and molluscs. This complex mixture appears in a gas chromatogram as a broad hump, or envelope, beneath the n-alkane peaks (Blumer et al., 1970). A black, tarlike discoloration is found in many clams from the Providence River. Much of it is resistent to lipid solvents, and it lasts for at least a year aft’er the clams have been transplanted to a clean, natural habitat or the laboratory tanks used here. We suspect that the discoloration is a polymer derived from petroleum products and hydrocarbons in other pollutants. Amoebocytes are able to remove the pollutant, perhaps directly from solution (pinocytosis) or by phagocytosis if it is in particulatc form. Masses of’black amoebocytes arc seen in histological sections of mantle and kidney tissues from stressed clams (Fig. 2) ; in normal clams these cells are far less numerous, especially in the kidney, and they arc not blackened. This black material collects in t’he renal sac, in quantities that would appear to plug the tubules and interfere with normal kidney function (Fig. 2). There is also a high incidence (510%) of a mucl-blister infestation associated with the discoloration, which S. Feng (personal FIG. 2, Mercemrh waercenariu from the stressed environment. In the mantle (X250) and kidnq (X98) (upper and middle photographs), note the masses of blackened amocbocytw standing out clearly in histological sections stained with hematoxylin cosin. In the lower photograph (kidney, ~4). ~a macroscopic, transverse swtion through the visceral mass, kidney. and pericardial cavity; this black material has occluded the renal sac and tubule, Magnifications approximate ; photographs by Mr. Paul Yevich.

STRESSED

communication) indicates is PoZyClom sp., a common parasite in the American oyster, C’rassostreu z~~T@GLc, but rare in n~olluscs that burrow into sediments. This may indicate t,hat clams in the river emerge from sediments-in response to irritants, low oxygen, and other conditions associated with pollution-exposing themselves to attack by Polydora larvae, which are abundant in Narragansett Bay during summer (Jeff ries, 19641. In addition, discolored clams often have a calcareous ridge extending longitudinally on the inner surface of the shell. Ridges of this nature are formed by the mantle when it retracts in response to an irritant. Thus we xc an interrelation of natural and abnormal factors in the Providence River, culminating in conditions that the clam camlot endure. The shells gape open in about 1% of the individuals, exposing their dead and clecomposing soft parts. Mortality i5 most noticeable among larger specimens approximately 11 cm long. Empty shells larger than this are rare, and, because shellfishing has been prohibited in the river for years, small size would indicate an early (l&h, compared to Narragansett Bay, xvliere living clams 12 cm and longer are oft(ln found. Young clams are abundant in the river, hut it, is obvious that when 1% of the adults dies cvcry day or so during the warm months, the population might disappear in a short time. A year after the investigation, only a few clams could be found in a once i)roduptive portion of the sampling area. 1Iass mortality is a common phenomenon in the palcontological recorcl (BrongcrsmaSanders, 19571, which can often be attributetl to thp effects of a persistent environmental change. The question is: how can we separate responses to normal environmental variation from the effects of cataclysmic natural proccsscs and incipient abnormalities such as pollution?

CLAMS

249

reactions are superimposed on natural seasonal variation, so whatever the natural burden on homeostasis may be, environmental abnormalities serve to make it even worse. Because the effects of natural and artificial stressors differ only in magnitude, we must recognize a generalized stress syndrome. When a certain severity is reached, the population dies. In its simplest form, this syndrome can be identified when the molar ratio of taurine to glycine is greater than 3. A similar pattern in the American oyster infect’ed wit,h Minchiniu ,nelsoni and Bucephaks sp. has been described by Feng et al. (1970). From their Table 1, ratios of taurine to glycine in the hemolymph can be calculated: the ratio in normal oysters ranges from 0.5 to 2.0 with a mean of 1.2; in infectecl oysters the range is 1.3 to 9.1; the mean is 4.0; ancl the concentration of total free amino acids is low. Feng et al. propose a compensatory mechanism in which kcto acids are converted to amino acids that have been depleted by parasites; meanwhile an elevated taurine level helps the host to maintain osmotic balance with the external environment. Polydora, however, is not a true parasite in the sense that a nutritional benefit is derived from the host’s metabolism. But the average taurine/glycine ratio in stressecl clams is even higher than in the infected oysters studied by Feng et al., and normal clams held in the laboratory for one year have a 4.9 average value. It follows that this ratio indicates a general reaction by the clam to disease, trauma, laborat’ory conditions, pollutants, starvation and seasonal variations in the environment. Moreover the magnit,ude points to causative factors: values between 3 and 5 result from physical-chemical abnormalities in the environment; anything greater than 5 indicates clisease. The extreme may also disclose a severely weakened condition that

250

JEFFRIES

Variable

Normal

Composition Taurine (pmoles/g) Glycine (pmoles/g) Taurine/glycine molar

<230,

Dominance-di0ersii.y D,,, amino acids Taurine included (Dmu) No taurme (I&,,iG) C-Amino acids Taurine included No taurine D ,,,i9 fatty acids c-Fat,ty acids Gross ahatowy Polydora sp. Color of tissues Inner surface of shell Renal sac

28-30g0,

temperature

increase

>240,

>70 2.3-2.9

rat,io

Alanine (Fmoles/g) Total free amino acids (pmoles) Total carbohydrates (yC) 16:0%, 16: l/16:0 wt ratio 20 : 4cz

0 Salinity

Seasonal

Symptomatic

*Seasonal ,Seasonal > 15 Generally 6.2-0.6 Cienerally

decrease decrease

,Seasonal 29-35 23-28

decrease

80-40 590-490

> 13 < 1

300 in lab-

ca. 4.9 in laboratory; 5.0-6.6 if infected Seasonal decrease 60-30 Seasonal decrease 530-486 <15 Cienerally < 13 0.50.6 May exceed 9, associated with a drop of 20:5 from about 26 to 6 Seasonal 3-36; 21-26;

2.i%29; 0.991.0

Absent White-yelloN &Smooth Open

decrease max 38 (laboratory) max 2X (laboratory)

23 ,.j in laboratory ; 1.0 in laboratory

Infected (iray-black Calcareous ridges May be occluded like deposit

April-November,

made it possible for Pol,ydou~ to become prevalent in the first place. Other symlltoms include group responses of entire classes of metabolites, and it is proposed that dominance-diversity relationships arc an easy and sensitive way of examining these complex patterns. For practical purposes, the taurine/glycinc ratio may be sufficient to recognize abnormalities in the hard clam. In view of our incomplete understanding of the many ways that marine organisms react to their surroundings, it does seem useful to keep in mind the various symptoms listed in Table 6. The manner in which these symptoms are interrelated, their mechanisms and regional

exceed

<60 >3,0;

o..j-0.8 0.8-1.0 27-31 0.8-1.6

14-22’C;

may oratory

1969;

Narragansett

with

a tar-

Bay.

variations offer many opportunities ther investigation.

for fur-

hKSO\VLEDGMEiYT

This investigation was supported by Environmental Prot,ection Agency Grant, 18050 DTX and by an institutional grant to the University of Rhode Island by the Office of Sea Grant, Programs. I am indebted to Dr. Sung Y. Feng for advice on molluscan pathology, to Dr. Donald K. I’helps and Mr. George Gray for information on the ecology of the hard clam in Rhode Island wat,ers. 10 Dr. John W. Farrington for discussions on I he plwmistry of hydrocarbons in the Providenw Riwr and to Mr. Paul Yevich for his unpublished obwrvations on changes in the clam’s normal histology taking nlace in this river. Miss Gale ?Cigrelli assisted in &e laboratory,

251

STFtESSED CLAMS

J. 1962, Free brates: a comparative t ion and metabolism. (J. T. Holden. ed.). si(trdam.

AWAP,~RA,

H.

JEFFRIE~,

REFERENCES amino acids in invertestudy of their distribu1rb “Amino Acid Pools” pp. 158-175. Elsevier, -4m-

N. J., .WII JWFRIES, H. P. 1971. The st,ructure of an experimental infaunal community. .I. Exp. Mar. 3iol. Ecol., 6, l-14. I%I,IG~~. E. G., ;)w DYER, W. J. 1959. A4 rapid method of total lipid extraction and purification. CW. J. Biocl~em. Physid.. 37, 911-917. ~~I,UWX, &I., So~z.4~ G,. .\sn S.4ss. J. 1970. Hydro(*arbon pollution of rdiblr shellfish by an oil spill. ilffl~,. BGI~.. 5, 195-202. I~I~~)~I~I~:R~s~~-~~~D~Rs, M. 1957, Ma3s mortality in th(l sea. In “Trpa&c on Marine Erology and P:~lcow~logy.” Vol. 1. Ecology, (J. W. HedgeIwth, pd.), Geol. Sot. AwLer. Mem.. 67, pp. Q41-1010. DULWIS, M., GILLES, K. A,, H.NILTOY, J. Ii., RIG BIGW, I?. A,, .
tionships plankton.

P. 1970. Dominancediversity of free amino acids in coastal Camp. Riochem. PhysioZ.. 37,

relazoo215-

223. KWIP,

A.. AND KITS VAN HEIJNINGEN, A. J. N. 1954A calorimetric micro-method for the determination of glycogen in tissues, Biochem. J., 56,

646-648. R. A., AND RIEMENSCHNEIDE. conversion of lipid compofatty acid methyl esters. J. Sot., 37,447451. J~urx)y. I?. E., HERB, S. F., AND MAGIDAM, P. 1968. A rapid and quantitative procedure for the preparation of methyl esters of butter-oil and other fats. J. Amer. Oil Chem. Sot.. 45, 549-

I~UDDY,

I?. E.,

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