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Short-term low-salinity response in lead-exposed lobsters, Homarus americanus (Milne Edwards)
J. Exp. Mar. Biol. Ecol., 1983, Vol. 69, pp. 283-295
283
Elsevier
SHORT-TERM LOW-SALINITY LOBSTERS, HOMARUS
RESPONSE IN LEAD-EXPOSED
AMERZCANUS
E. GOULD and R.A.
(Milne Edwards)
GREIG
National Marine Fisheries Service, Northeast Fisheries Center, Mivord Laboratory, Milford. CT 06460, U.S.A. Abstract: The only effect attributable solely to lead exposure (50 ng .I-‘, 30 days) in lobsters, Homarus americanus (Milne Edwards), was decreased pentose shunt activity (G6PDH) in the antenna1 gland, which
contained the greatest concentration of lead taken up by the tissues analyzed. Similarly-exposed lobsters were subsequently held for 2 days in clean, aerated sea water at either ambient or low salinity (27 or 17x., respectively), after which time the lead concentration in the antennal gland had dropped by one-third and G6PDH had returned to control values. Low-salinity effects far outnumbered lead-induced effects and were found primarily in the tail muscle, corroborating findings in an earlier study with cadmium. In that earlier study, cadmium produced more metabolic disturbance than did the lead exposure reported here. Lead exposure at low salinity abolished several normal hyposaline reactions seen in control animals.
In a continuing study of biological effects in marine animals after long-term exposure to low concentrations of heavy metals, this laboratory has begun to examine subsequent challenge by extremes of a normal environmental variable for the purpose of learning how such sublethal exposures may affect an animal’s capacity to adapt to its surroundings. Salinity was the variable chosen for the first exposure series. The test animal was the American lobster, Homarus americanus (Milne Edwards), a good subject for metabolic study at low salinity because considerable hyperosmoregulation has been observed at the lower end of its salinity-tolerance range (Dall, 1970). The stronger the adaptive response, the more likely is any detection of stress-induced change in that response. In an initial study, lobsters were exposed for 1 month to low concentrations of cadmium and subsequently held for 1 wk in clean sea water of either ambient or low salinity (Gould, 1980). Low-salinity effects were more prominent than the cadmium effects, probably because the 7-day holding (clearing) period at the different salinities may have diminished or eliminated some of the metal effects. We report here the results of an analogous study with lead, another element of environmental concern, and compare the effects of the two metals. Like cadmium or mercury, lead is bioaccumulative, toxic, and has no known physiological role as a trace metal (Wood, 1974; Waldichuk, 1980). In the marine environment it is found primarily in sediments (Campbell & Loring, 1980) although the continuous input to oceanic 0022-0981/83/$03.00 0 1983 Elsevier Science Publishers B.V
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waters by rivers and by atmospheric fallout ensures the presence of lead in the water column; as high as 140 pg. 1-i has been reported for waters of the New York Bight Apex (Klein &al., 1974). Edible species of moliuscs and crustacean shellfish in contaminated estuaries of the U.K. have been found to have high concentrations of lead in their tissues, averaging 3.5 to 5.2 mg* kg-’ (Popham et al., 1980). Marine animals, in particular the invertebrates, have a variety of mechanisms for metabolizing or sequestering toxic metals (Fowler et al., 1981), which may differ from tissue to tissue. Moreover, different tissues in the same animal react differently to osmotic variation, and whole-animal respiration rates are the resultant of several different organs (Munday & Poat, 1971). These rates individually may be greater or lesser than the sum of all; in hyposmotic gill mitochondria from marine crabs, for example, King (1966) found that activity of citric acid cycle enzymes increased > 200-fold, whereas overall respiration increased only 35 Ye. It is at the subceilular level that one finds the earliest, most sensitive response to stress, and where the first adaptive mechanisms appear. Bayne et al. (1979) have pointed out that “Cytological and biochemical responses are likely to prove more sensitive to environmental changes than some physiological [or ‘whole-animal’ response] indices.” In the study reported here, the holding period at different salinities was shortened to 2 days to minimize clearing. The lead exposure concentration was set at 50 pg. 1~ ’ rather than the 10 pg. I- ’ used for cadmium, because we have found that lead produces fewer metabolic effects than does cadmium in adult marine animals such as the winter flounder, Pseudopleuronectes americanus (Gould, 1977). In crustaceans, the primary repository organs for metals are the hepatopancreas and the antenna1 gland, which functions largely as a kidney (e.g. Prosser, 1973); these tissues and gills were analyzed for the presence of lead. Heart and tail muscle from lobsters held at ambient salinity were also analyzed for lead content, although the muscle samples were not expected to accumulate significant amounts of the metal. To examine metabolic reaction in the lobster to the imposed stresses of lead exposure and low salinity, and for comparison with data from the cadmium study, we looked at the antennal gland, heart, tail muscle, and male gonad. Measurement was made of enzyme activities that changed significantly after cadmium exposure and low salinity in that study (Gould, 1980). These were enzymes involved in the mobilization or expenditure of energy (pyruvate kinase, PK, EC 2.7.1.40; isocitrate dehydrogenase, IDH, EC I. 1.1.42), in biosynthetic (glucose-6-phosphate dehydrogenase, G6PDH, EC 1.1.1.49) and redox (malate and lactate dehydrogenases, MDH and LDH, EC 1.1.1.37 and 1.1.1.27) activities, and in amino-acid metabolism (aspartate aminotransferase, AAT, EC 2.6.1. I).
LEAD-EXPOSED
LOBSTERS
METHODS
ANIMAL
AND
AT LOW SALINITY
285
MATERIALS
EXPOSURE
Lobsters were collected by trap from the waters of Long Island Sound near Milford, Connecticut (U.S.A.), and acclimated in the laboratory in flowing, filtered sea water for 2 wk before experimental exposure in the summer of 1979. They were fed chopped clams (Spisula solidissima (Say)) daily during acclimation and throughout the exposure period. In duplicate experiments, the lobsters were exposed to 50 pg * l- ’ lead as Pb(NO,)z in a flow-through diluter system (Mount & Brungs, 1967) providing the intermittent delivery of either metal-containing or control water at a flow rate of 1.5 1. min- ’ to each tank throughout the test exposure. Sixteen lobsters served as controls (four per 285-l fiberglass tank) and the same number for lead exposure in each of the duplicate test periods. Salinity and temperature ranges during the two 30-day exposures were 25.9-27.3x, and 21-25 “C, respectively. At the end of each 30-day exposure, half of the controls and half of the lead-exposed lobsters were removed and their tissues dissected, packaged, and frozen-stored at - 80 ‘C to await testing. The remaining lobsters for each exposure were placed in two large fiberglass tanks, one for controls and the other for metal-exposed animals, containing aerated sea water at 21 “C of either ambient (26.9x,) or low (17%,) salinity. At the end of each 2-day holding period, the lobsters were removed and handled as above. TISSUE
PREPARATION
AND
ENZYME
ASSAYS
Tissues were homogenized and centrifuged and assays were performed with reaction media providing maximal activity, all as specifically described in Gould (1980). The supernatants represented tissue dilutions of 10 x for antennal gland (AG) and male gonad (MG), 5 x for tail muscle (M), and 20 x for heart (H). Amounts of these tissue preparations or further dilutions thereof for each 3-ml reaction mixture were: MDH 0.05 ml 100 x (AG and H) and 0.1 ml 100 x (MG), and 0.1 ml 50 x (M); LDH - 0.1 ml 10 x (AG and MG), 0.1 ml 40 x (H), and 0.1 ml 50 x (M); PK - 0.05 ml 5 x (M) and 0.1 ml 20 x (H); AAT - 0.1 ml 20 x (H); and G6PDH - 0.1 ml 10 x (AC). CHEMICAL
ANALYSIS
Tissue analyses for lead employed wet (acid) digestion followed by atomic absorption using a graphite furnace (Greig etal., 1982). This procedure was employed in a collaborative study on fish tissue (involving about 20 laboratories in various countries) conducted by the International Council for the Exploration of the Sea (Copenhagen, Denmark), in which our results compared favorably with the majority of the participating laboratories. In the lead-exposure study reported here, analytical precision could not be determined on the small, individual samples; in other, pooled samples for which
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replicate analyses could be made, however, relative standard deviation was 15-20 “/, Reagent blanks analyzed with these lobster tissue samples were no more than one-tenth of the lowest observed concentration of lead found in the tissue samples.
RESULTS
In lobster tail muscle, no effects attributable solely to lead exposure were seen. The 2-day low-salinity stress alone, however, produced a significantly increased energy expenditure in this tissue. Fig. la, b, and c, respectively, illustrate the elevated redox activity (MDH, P >O.OOl)and glycolytic rates (PK, P > 0.05, IDH, P > 0.01) in unexposed lobsters held at low salinity. In the lead-exposed lobsters, on the other hand, these low-salinity effects were abolished for MDH and PK. LDH, which was not affected by either lead or low salinity alone, was synergistically depressed (P> 0.001) in the lead-exposed lobsters at low salinity (Fig. Id). In heart muscle, also, no lead effects were observed at ambient salinity. At low salinity, AAT increased in the controls (P> 0.01) but not in the lead-exposed animals (Fig. 2a). IDH activity was unchanged by either low salinity or lead exposure alone, but the two stresses acted synergistically to elevate IDH (P> 0.02, Fig. 2b). Although the data for MDH looked like synergistic induction (3999 vs. 4862), the range of variation was too great to indicate statistical significance. Neither LDH nor PK changed under any of the test conditions. The antenna1 gland provided the only clear-cut effect due to lead exposure alone, a depression of G6PDH (P> 0.025) in the lead-exposed lobsters at ambient salinity (Fig. 3b). Low-salinity effects were limited to a synergistic depression of MDH (P> 0.05) in the lead-exposed animals (Fig. 3a). A greater sample number (antenna1 glands were pooled) might have shown both MDH and G6PDH to be elevated in the low-salinity controls; in both cases, however, the apparent increase was not statistically significant. Neither low salinity nor lead exposure alone affected LDH. No effects due to lead exposure alone were observed in the male gonad. LDH dropped significantly (P> 0.05) in controls at low salinity, but increased (P> 0.02) in lead-exposed lobsters at low salinity (Fig. 4b). A synergistic effect in the lead-exposed lobsters at low salinity was seen in the elevated MDH (P> 0.01).
LEAD-EXPOSED
LOBSTERS AT LOW SALINITY
A8 CONTROLS
Pb-EXPOSED
287
La
CONTROLS
a 26 -
looO-
7
~
P
a
li A8
600 -
La
‘b-EXPOW
Fig. 1. Effects of sublethal lead exposure (50 pg. I-‘, 30 days) and of low salinity, indi~dual~y and combined, on enzyme activities in lobster tail muscle: enzyme activity units are ymol substrate turnover. min- ’ . mg- ’ biuret protein; in Fig. a the bar graphs are identified as either 3O-day exposure alone or + 2-day holding period at either ambient (27x,, AS) or low (17x,, LS) salinity; bar height represents arithmetic mean, and the ticked vertical lines, standard error; numbers atop the SE line are sample number; level of confidence, derived from Student’s t-test, is inserted in each arrow connecting a pair of bars representing compared data sets; these conventions are also used in Fig. 2-4.
IDH 1.1.1.42 250-
AAT 15
2.8.1.1 800-
7
11
i
f
1506
500-
[’ C
50-
k
-I
AS
CONTROLS
ofsublethal lead
Fig. 2. Effects
] LSf
and low salinity, indiwdually in lobster heart
I K.37 1200
1000-
on
enzyme activities
0.05 t 4\
t
t
and combined,
%PDH 1.1.1.4s
P
@
Pb-EXPOSED
CONTROLS
Pb-EXPOSED
exposure
LS -
AS -
01
T
/-I
I i
4 -i 5
I+
800
-
CONTROLS
Fig. 3. Effects of sublethal
‘b-EXPOSED
lead exposure
CONTROLS
and low salinity. individually in lobster antenna1 gland.
and combined,
Pb-EXF
on enzyme activities
LEAD-EXPOSED
289
LOBSTERS AT LOW SALINITY
NS 2SOr
(880-1271)
5
1.1.1.27
I
_
NS
\ \
3_w35-291) I-
(157-256)+
T
r i
750
-
500
-
250
-
/
5
1! !(16,QQ)
st ILS LO! 5 AS o-
CONTR
1
IO1
Pb-EXPOSE
,
1
rn0L.s
AS 0
Pb-EXPOSED
Fig. 4. Effects of sublethal lead exposure and low salinity, individually and combined, on enzyme activities in lobster male gonad.
DISCUSSION
A summary of the effects of lead exposure and low salinity on enzyme activity in lobster tissues is presented in Table I. For ease in comparing the effects of lead and of cadmium in the presence and absence of low salinity, Table II shows an analogous summary of the earlier study with cadmium-exposed lobsters (Gould, 1980). Lowsalinity effects outnumbered lead-induced effects and were found primarily in the tail muscle, as was true for the cadmium-exposed lobsters. In contrast to the numerous cadmium effects reported in the earlier study (Table II), however, the only metal effect produced by lead exposure alone was the lowered G6PDH activity in antennal gland, where the greatest bulk of lead taken up by the various tissues was located.
290
E. GOULD
AND R.A. GREIG TABLF
Classification
of effects
of lead exposure combination.
I
(50 fig. I- ‘, 30 days) and low salinity on enzyme activities in lobster tissues.
( 17?;,, 2 days), alone and m
3. Effect of Pb + low salinity (LS) combined
I. Low-salinity
effect alone (in controls)
Response type:
2. Pb effect alone (at ambient salinity, 27%,)
Elevated MDH IDH
.4ntagonism Synergism
LS > Pb
Pb > LS
.4bolition of depressed activity
activity IDH MDH
(M) (M)
(H) (MG)
G6PDH
(AG)
LDH
(MG)
PK (M) AAT (H) Response type:
Depressed LDH
TISSUE
(MG)
CONCENTRATIONS
.Abolition of cievated activity
activity
G6PDH
(AG)
LDH MDH
(M) (AC)
MDH
(M)
PK (M) .4AT (H)
OF METAL
The relative distributions of lead and cadmium in lobster antenna1 gland and hepatopancreas apparently differ. Ray et al. (1981) reported that the hepatopancreas ( < 5 “/, of the total wet weight of the lobster) contained 93-99 y0 of the total cadmium in lobsters taken from cadmium-polluted waters near a lead smelter, which produces cadmium-laden dust (Sergeant & Westlake, 1980). Ray’s group (198 1) found the antennal or green gland to rank second or third in total cadmium content, although it is only 0.01 y0 of the animal’s total wet weight. By contrast, in the lead-exposed lobsters reported here, the antennal gland contained the highest concentration of lead of those tissues analyzed (Table III). Moreover, of the seven hepatopancreas specimens from lead-exposed lobsters analyzed for metal uptake, five had no detectable lead. Concentration of cadmium in the hepatopancreas suggests an active sequestering mechanism for this metal in the lobster: intracellular calcium-phosphorus concretions in the crustacean hepatopancreas, as well as in bivalve kidneys, have been shown to contain high concentrations of metals (Fowler et al., 1981). In bivalve kidneys, especially, these concretions have the major role in the accumulation, detoxification, and excretion of cadmium (Carmichael & Fowler, 1981) and other metals (George et al., 1980). For marine invertebrates in general, lysosomes and metallothionein-like proteins are also important in compartmentalizing or sequestering heavy metals (Fowler et al., 1981). The prominent concentration of lead in lobster antenna1 gland, on the other hand,
TABLE II
Response type:
Response type:
LDH GPI PK LDH AAT LDH
MDH IDH MDH MDH
(M) (M) (M) (H) (H) (MG)
(M) (M) (AG) (MG)
1. Low-salinity effect alone (in controls)
(M) (H) (H) (H) (AG)
Depressed activity
MDH MDH LDH GPI MDH
Elevated activity
2. Cd effect alone (at ambient salinity, 27%,J
MDH (M) IDH (M)
Additive effect
MDH (AG) LDH (AG)
GPI (MG)
Synergism
Cd>LS
GPI (H) LDH (H)
Abolition of elevated activity
GPI (M) LDH (MG)
Abolition of depressed activity
LS > Cd
Antagonism
3. Effect of Cd + low salinity (LS) combined
Classification of effects of cadmium exposure (10 fig. I-‘, 30 days) and low salinity (17x,,, 7 days), alone and in combination, on enzyme activities in lobster tissues (from Gould, 1980).
M
N.D.h 2.9 + 1.0
(3) (5)
---.--..-
N.D. 8.8 f 6.1
(4) < I.0 i 0.3 2.5 & 0.9 (5)
(4)” (5)
..-- _...
Gills
” ND., not detectable.
” Unrts: (sample _VJ arnhmcttc mean 2 sk
+ 7 days low satinity: Controls Exposed
+ 2 days ambient salinity: Controls Exposed
Exposed
30 days: Controls
Tissue:
(3) <24.3 i 6.2 106 2 17 (5) (range: 54-145)
5.6 + 3.4 (4) 138270 (4) (range: 31-340)
~~_-..
(4) N.D. (5) 63.0 2 47.3 (range: ND.-249) _.__... .-....- .l_l.,._....- .___ -._.“_ ..
.
Antenna1 gland N.D. 2.2 f 1.8
_...
N.D. 1.9 i 1.1
(4) <1.0*0.1 (4) <0.9+0.1
-‘-’ --
Tail muscle
__“. _...._ I..._ ____.____“__“_~
(2) (5)
(3) <0.7 + 0.1 13) 2.0 2 1.3
(2) (7)
. ---.-
Wepatopancreas
_l_--i_-
(4) < 24.8 rf:8.5 (5) (20.8 i 3.5
Heart
Tissue concentrations of lead (pg g- ‘, wet wt) in lobsters exposed 30 days to 50 pg Pb I- ’ in flowing sea water k2 subsequent days in clean sea water at ambient (27’&,)or low (17Y,,)salinity: analyses were not performed on heart and tail muscle from the 30-day exposure or from the 2-day low-salinity treatment: sample numbers are in parentheses.
TABLEIII
LEAD-EXPOSED
LOBSTERS
AT LOW SALINITY
293
suggests a very active excretory mechanism for this metal. After only 2 days of clearing, levels of lead had dropped by about one-third (Table III) and the only metal effect in that tissue, lowered G6PDH activity, had disappeared. The mechanism by which lead is so efficiently accumulated in and excreted by the antennal gland remains an object for future study. LOW-SALINITY
RESPONSE
Low salinity effects in the lead-study control lobsters were greatest in the tail muscle (Table I), as in the earlier cadmium study (Table II). In the cadmium-study control lobsters held at low salinity for 7 days, the effects were primarily depressed activities, which was not the case in the lead study (a 2-day holding period). Both heart AAT and muscle PK were elevated in the low-salinity controls of the lead study, indicating an increased amino acid catabolism and expenditure of metabolic energy in response to the 2-day osmotic stress. These same enzyme activities were depressed, by contrast, in the 7-day low-salinity controls on the cadmium study. These observations suggest an oscillatory reaction: an initial stimulus in response to the low-salinity stress, followed by depressed activity and a gradually damped oscillation. They also conform to the perception of the lobster as an osmoconformer that adapts only slowly to hyposalinity (e.g. Dall, 1970). The only low-salinity responses common to both studies were elevated MDH and LDH in muscle and lowered LDH in male gonad. Gilles observed (1973) that the drop in amino-acid concentrations seen during hyposmotic stress is associated with increased catabolism of amino acids, which leads to the formation of reducing equivalents and consequently elevated levels of reduced pyridine nucleotides. Thus in the low-salinity controls of both metal-exposure studies, the continued increase in redox activity (MDH) served to regenerate the oxidized NAD necessary for the increased citric acid cycle activity (IDH). In lobster tissues, apparently, MDH is a more effective redox regulator than LDH. METAL-EXPOSURE
RESPONSE
Cadmium, even at one-fifth the concentration of lead used in the study reported here, and with 7 rather than 2 days of clearing, induced far more metabolic perturbation (Table II) than did lead. Moreover, the observed cadmium effects were entirely hermetic and predominantly in the heart, whereas lead exposure alone produced only a single effect, a lowered G6PDH (Table I) in the lead-burdened antennal gland (Table III) an effect that disappeared after only 2 days of clearing at both ambient and low salinity. Of the two metals, cadmium clearly places greater stress on the lobster by a continuing strong demand for metabolic energy. Lead, at five times the cadmium concentration, induced no such energy expenditure. A less direct effect attributable to lead exposure alone was the abolishing of several adjustments to low salinity seen in the control animals (Table I). Similarly attenuated
294
EGOULDAND
R.A.GREIG
biochemical activity has been found in teleosts (Gould, 1977) and bivalve moliuscs (Gould, unpubl. data), as well as other crustaceans (Gould et al., 1976) exposed to sublethal concentrations of cadmium. Such weakening of normal physioiogic~ adaptive mechanisms argues a metabolism fess capable both of adjusting to changing environmental conditions and of channeling tissue energy toward reproductive needs. The animal is also, under continuing stress, less likely to survive.
ACKNOWLEDGEMENTS
We thank M.T. Grojean, J. S. Rancourt, and S. Schurman for expert technical assistance, and M. R. Cox for preparing the graphs.
REFERENCES BAYNE, B. L., M. N. MOORE, D. R. LIVINGSTONE& P. SALKELD,1979. Measurement of the responses of individuals to environmental stress and pollution: studies with bivalve molluscs. Phil. Trans. R. Ser. London Ser. B, Vol. 286,pp. 563-681.
CAMPBELL,J.A. & D. H. LORING, 1980. Baseline levels of heavy metals in the waters and sediments of Ba%n Bay. Mar. Pollur. Bull., Vol. 11, pp. 257-261. CARMICHAEL,N.G. & B.A. FOWLER,1981. Cadmium accumulation and toxicity in the kidney of the bay scallop Argapecten irradians. Mar. Biol., Vol. 65, pp. 35-43. DALL, W., 1970. Osmoregulation in the lobster Homarus americanus. 1. Fish. Res. Board Can., Vol. 27, pp. 1123-I 130. FOWLEK,B.A., N.G. CARMICHAEL& KS. SQUISB, 1981. Factors affecting trace-metal uptake and toxicity to marine organisms. II. Cellular mechanisms. In, Bja~o~a6 monjto~ng of mar~ep~l~utanfs, edited by F. 3. Vernberg, A. Calabrese, F. P. Thurberg & W.B. Vernberg, Academic Press, New York, pp. 145-163. GEORGE,S.G., B.J. S. PIRIE & T. L. COOMBS,1980. Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.). J. Exp. Mar. Biol. EC&, Vol. 42, pp. 143-156. GIL.LES,R., 1973. Oxygen consumption as related to the amino acid metabolism during osmoregulation in the blue crab, Callinectes sapidus. Neth. J. Sea Res., Vol. 7, pp. 280-289. Gour.r>, E., 1977. Alteration of enzymes in winter flounder. Pseudop(euranectes americanus, exposed to sublethal amounts o~cad~~u~ chloride. In, P~y~~~~og~~u~ resp#~ses oft7~ar~nebiura to pa~~utan~~, edited by F .I. Vernberg, A. Calabrese. F. P. Thurberg & W. B. Vernberg, Academic Press, New York, pp. 209-224. Got I.I), E.. IY80. Low-salinity stress in the American lobster, ffomarus americanus, after chronic sublethal exposure to cadmium: biochemical effects. He&al. Meeresunters., Vol. 33, pp. 36-46. GOUI D, E., R. S. COLLIER,J.J. KAROLUS& S. GIVENS, 1976. Heart transaminase in the rock crab, Cancer irroratus, exposed to cadmium salts. Bull. Environ. Contam. Toxicol., Vol. 15. pp. 635-643. GREIG, R.A., T.K. SAWYER,E. J. LEWIS & M.E. GALASSO, 1982. A study of metal concentrations in relation to gill cofor and pathology in the rock crab, Cancer irroratus. Arch. Environ. Contum. Taxicol.. Vol. 11) pp. 539-545. KING, E. N., 1966.Oxidative activity of crab gill mitochondria as a function ofosmotic concentration. Conz~~. Biochem. Physiol., Vol. 17, pp, 245-258. KLEIN, L. A., M. LANG, N. NASH & S.L. KIRSCHNEK,1974. Sources of metals in Nets York City wastewater. Department of Water Resources, City of New York, 18pp. MOUNT. D. I. & W.A. BRUNGS,1967. A simplified dosing apparatus for fish toxicology studies. WaterRes.. Vol. 1. pp. 21-29. MUNDAY,K. A. & P.C. POAI, 1971. Respiration and energ meta~lism in Crustacea. In, ~~~enzi~f7f zootogv. Vat. IV, Arthropoda, Part B, edited by M. Florkin & B.T. Scheer, Academic Press, New York, pp. 191-211.
LEAD-EXPOSED
LOBSTERS
AT LOW SALINITY
295
POPHAM,J. D., D. C. JOHNSON& J.M. D’AURIA, 1980. Mussels (Myrilus edulis) as “point source” indicators of trace metal pollution. Mar. POW. Bull., Vol. 11, pp. 261-263. PROSSER, C.L., 1973. Inorganic ions. In, Comparative animal physiology, edited by CL. Prosser, W.B. Saunders Co., Philadelphia, pp. 79-l 10. RAY, S., D.W. MCLEESE & L.E. BURRIDGE,1981. Cadmium in tissues of lobsters captured near a lead smelter. Mar. Pollut. BUN., Vol. 12, pp. 383-386. SERGEANT,D. B. & G. F. WESTLAKE,1980. Industrial discharges of cadmium at Belledune. In, Cadmium pollution of Beliedune Harbour, New Brunswick, Canada, edited by J. F. Uthe & V. Zitko, Can. Tech. Rep., Fisheries and Aquatic Sciences, No. 963, pp. 3-10. WALDICHUK,M., 1980. Lead in the marine environment. Mar. Pollut. Bull., Vol. 11, pp. 241-242. Wools, J.M., 1974. Biological cycles for toxic elements in the environment. Science, Vol. 183, pp. 1049-1052.
283
Elsevier
SHORT-TERM LOW-SALINITY LOBSTERS, HOMARUS
RESPONSE IN LEAD-EXPOSED
AMERZCANUS
E. GOULD and R.A.
(Milne Edwards)
GREIG
National Marine Fisheries Service, Northeast Fisheries Center, Mivord Laboratory, Milford. CT 06460, U.S.A. Abstract: The only effect attributable solely to lead exposure (50 ng .I-‘, 30 days) in lobsters, Homarus americanus (Milne Edwards), was decreased pentose shunt activity (G6PDH) in the antenna1 gland, which
contained the greatest concentration of lead taken up by the tissues analyzed. Similarly-exposed lobsters were subsequently held for 2 days in clean, aerated sea water at either ambient or low salinity (27 or 17x., respectively), after which time the lead concentration in the antennal gland had dropped by one-third and G6PDH had returned to control values. Low-salinity effects far outnumbered lead-induced effects and were found primarily in the tail muscle, corroborating findings in an earlier study with cadmium. In that earlier study, cadmium produced more metabolic disturbance than did the lead exposure reported here. Lead exposure at low salinity abolished several normal hyposaline reactions seen in control animals.
In a continuing study of biological effects in marine animals after long-term exposure to low concentrations of heavy metals, this laboratory has begun to examine subsequent challenge by extremes of a normal environmental variable for the purpose of learning how such sublethal exposures may affect an animal’s capacity to adapt to its surroundings. Salinity was the variable chosen for the first exposure series. The test animal was the American lobster, Homarus americanus (Milne Edwards), a good subject for metabolic study at low salinity because considerable hyperosmoregulation has been observed at the lower end of its salinity-tolerance range (Dall, 1970). The stronger the adaptive response, the more likely is any detection of stress-induced change in that response. In an initial study, lobsters were exposed for 1 month to low concentrations of cadmium and subsequently held for 1 wk in clean sea water of either ambient or low salinity (Gould, 1980). Low-salinity effects were more prominent than the cadmium effects, probably because the 7-day holding (clearing) period at the different salinities may have diminished or eliminated some of the metal effects. We report here the results of an analogous study with lead, another element of environmental concern, and compare the effects of the two metals. Like cadmium or mercury, lead is bioaccumulative, toxic, and has no known physiological role as a trace metal (Wood, 1974; Waldichuk, 1980). In the marine environment it is found primarily in sediments (Campbell & Loring, 1980) although the continuous input to oceanic 0022-0981/83/$03.00 0 1983 Elsevier Science Publishers B.V
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E. GOULD
AND
K.A. GREIG
waters by rivers and by atmospheric fallout ensures the presence of lead in the water column; as high as 140 pg. 1-i has been reported for waters of the New York Bight Apex (Klein &al., 1974). Edible species of moliuscs and crustacean shellfish in contaminated estuaries of the U.K. have been found to have high concentrations of lead in their tissues, averaging 3.5 to 5.2 mg* kg-’ (Popham et al., 1980). Marine animals, in particular the invertebrates, have a variety of mechanisms for metabolizing or sequestering toxic metals (Fowler et al., 1981), which may differ from tissue to tissue. Moreover, different tissues in the same animal react differently to osmotic variation, and whole-animal respiration rates are the resultant of several different organs (Munday & Poat, 1971). These rates individually may be greater or lesser than the sum of all; in hyposmotic gill mitochondria from marine crabs, for example, King (1966) found that activity of citric acid cycle enzymes increased > 200-fold, whereas overall respiration increased only 35 Ye. It is at the subceilular level that one finds the earliest, most sensitive response to stress, and where the first adaptive mechanisms appear. Bayne et al. (1979) have pointed out that “Cytological and biochemical responses are likely to prove more sensitive to environmental changes than some physiological [or ‘whole-animal’ response] indices.” In the study reported here, the holding period at different salinities was shortened to 2 days to minimize clearing. The lead exposure concentration was set at 50 pg. 1~ ’ rather than the 10 pg. I- ’ used for cadmium, because we have found that lead produces fewer metabolic effects than does cadmium in adult marine animals such as the winter flounder, Pseudopleuronectes americanus (Gould, 1977). In crustaceans, the primary repository organs for metals are the hepatopancreas and the antenna1 gland, which functions largely as a kidney (e.g. Prosser, 1973); these tissues and gills were analyzed for the presence of lead. Heart and tail muscle from lobsters held at ambient salinity were also analyzed for lead content, although the muscle samples were not expected to accumulate significant amounts of the metal. To examine metabolic reaction in the lobster to the imposed stresses of lead exposure and low salinity, and for comparison with data from the cadmium study, we looked at the antennal gland, heart, tail muscle, and male gonad. Measurement was made of enzyme activities that changed significantly after cadmium exposure and low salinity in that study (Gould, 1980). These were enzymes involved in the mobilization or expenditure of energy (pyruvate kinase, PK, EC 2.7.1.40; isocitrate dehydrogenase, IDH, EC I. 1.1.42), in biosynthetic (glucose-6-phosphate dehydrogenase, G6PDH, EC 1.1.1.49) and redox (malate and lactate dehydrogenases, MDH and LDH, EC 1.1.1.37 and 1.1.1.27) activities, and in amino-acid metabolism (aspartate aminotransferase, AAT, EC 2.6.1. I).
LEAD-EXPOSED
LOBSTERS
METHODS
ANIMAL
AND
AT LOW SALINITY
285
MATERIALS
EXPOSURE
Lobsters were collected by trap from the waters of Long Island Sound near Milford, Connecticut (U.S.A.), and acclimated in the laboratory in flowing, filtered sea water for 2 wk before experimental exposure in the summer of 1979. They were fed chopped clams (Spisula solidissima (Say)) daily during acclimation and throughout the exposure period. In duplicate experiments, the lobsters were exposed to 50 pg * l- ’ lead as Pb(NO,)z in a flow-through diluter system (Mount & Brungs, 1967) providing the intermittent delivery of either metal-containing or control water at a flow rate of 1.5 1. min- ’ to each tank throughout the test exposure. Sixteen lobsters served as controls (four per 285-l fiberglass tank) and the same number for lead exposure in each of the duplicate test periods. Salinity and temperature ranges during the two 30-day exposures were 25.9-27.3x, and 21-25 “C, respectively. At the end of each 30-day exposure, half of the controls and half of the lead-exposed lobsters were removed and their tissues dissected, packaged, and frozen-stored at - 80 ‘C to await testing. The remaining lobsters for each exposure were placed in two large fiberglass tanks, one for controls and the other for metal-exposed animals, containing aerated sea water at 21 “C of either ambient (26.9x,) or low (17%,) salinity. At the end of each 2-day holding period, the lobsters were removed and handled as above. TISSUE
PREPARATION
AND
ENZYME
ASSAYS
Tissues were homogenized and centrifuged and assays were performed with reaction media providing maximal activity, all as specifically described in Gould (1980). The supernatants represented tissue dilutions of 10 x for antennal gland (AG) and male gonad (MG), 5 x for tail muscle (M), and 20 x for heart (H). Amounts of these tissue preparations or further dilutions thereof for each 3-ml reaction mixture were: MDH 0.05 ml 100 x (AG and H) and 0.1 ml 100 x (MG), and 0.1 ml 50 x (M); LDH - 0.1 ml 10 x (AG and MG), 0.1 ml 40 x (H), and 0.1 ml 50 x (M); PK - 0.05 ml 5 x (M) and 0.1 ml 20 x (H); AAT - 0.1 ml 20 x (H); and G6PDH - 0.1 ml 10 x (AC). CHEMICAL
ANALYSIS
Tissue analyses for lead employed wet (acid) digestion followed by atomic absorption using a graphite furnace (Greig etal., 1982). This procedure was employed in a collaborative study on fish tissue (involving about 20 laboratories in various countries) conducted by the International Council for the Exploration of the Sea (Copenhagen, Denmark), in which our results compared favorably with the majority of the participating laboratories. In the lead-exposure study reported here, analytical precision could not be determined on the small, individual samples; in other, pooled samples for which
2X6
E.GOULDAND
R.A.GRElG
replicate analyses could be made, however, relative standard deviation was 15-20 “/, Reagent blanks analyzed with these lobster tissue samples were no more than one-tenth of the lowest observed concentration of lead found in the tissue samples.
RESULTS
In lobster tail muscle, no effects attributable solely to lead exposure were seen. The 2-day low-salinity stress alone, however, produced a significantly increased energy expenditure in this tissue. Fig. la, b, and c, respectively, illustrate the elevated redox activity (MDH, P >O.OOl)and glycolytic rates (PK, P > 0.05, IDH, P > 0.01) in unexposed lobsters held at low salinity. In the lead-exposed lobsters, on the other hand, these low-salinity effects were abolished for MDH and PK. LDH, which was not affected by either lead or low salinity alone, was synergistically depressed (P> 0.001) in the lead-exposed lobsters at low salinity (Fig. Id). In heart muscle, also, no lead effects were observed at ambient salinity. At low salinity, AAT increased in the controls (P> 0.01) but not in the lead-exposed animals (Fig. 2a). IDH activity was unchanged by either low salinity or lead exposure alone, but the two stresses acted synergistically to elevate IDH (P> 0.02, Fig. 2b). Although the data for MDH looked like synergistic induction (3999 vs. 4862), the range of variation was too great to indicate statistical significance. Neither LDH nor PK changed under any of the test conditions. The antenna1 gland provided the only clear-cut effect due to lead exposure alone, a depression of G6PDH (P> 0.025) in the lead-exposed lobsters at ambient salinity (Fig. 3b). Low-salinity effects were limited to a synergistic depression of MDH (P> 0.05) in the lead-exposed animals (Fig. 3a). A greater sample number (antenna1 glands were pooled) might have shown both MDH and G6PDH to be elevated in the low-salinity controls; in both cases, however, the apparent increase was not statistically significant. Neither low salinity nor lead exposure alone affected LDH. No effects due to lead exposure alone were observed in the male gonad. LDH dropped significantly (P> 0.05) in controls at low salinity, but increased (P> 0.02) in lead-exposed lobsters at low salinity (Fig. 4b). A synergistic effect in the lead-exposed lobsters at low salinity was seen in the elevated MDH (P> 0.01).
LEAD-EXPOSED
LOBSTERS AT LOW SALINITY
A8 CONTROLS
Pb-EXPOSED
287
La
CONTROLS
a 26 -
looO-
7
~
P
a
li A8
600 -
La
‘b-EXPOW
Fig. 1. Effects of sublethal lead exposure (50 pg. I-‘, 30 days) and of low salinity, indi~dual~y and combined, on enzyme activities in lobster tail muscle: enzyme activity units are ymol substrate turnover. min- ’ . mg- ’ biuret protein; in Fig. a the bar graphs are identified as either 3O-day exposure alone or + 2-day holding period at either ambient (27x,, AS) or low (17x,, LS) salinity; bar height represents arithmetic mean, and the ticked vertical lines, standard error; numbers atop the SE line are sample number; level of confidence, derived from Student’s t-test, is inserted in each arrow connecting a pair of bars representing compared data sets; these conventions are also used in Fig. 2-4.
IDH 1.1.1.42 250-
AAT 15
2.8.1.1 800-
7
11
i
f
1506
500-
[’ C
50-
k
-I
AS
CONTROLS
ofsublethal lead
Fig. 2. Effects
] LSf
and low salinity, indiwdually in lobster heart
I K.37 1200
1000-
on
enzyme activities
0.05 t 4\
t
t
and combined,
%PDH 1.1.1.4s
P
@
Pb-EXPOSED
CONTROLS
Pb-EXPOSED
exposure
LS -
AS -
01
T
/-I
I i
4 -i 5
I+
800
-
CONTROLS
Fig. 3. Effects of sublethal
‘b-EXPOSED
lead exposure
CONTROLS
and low salinity. individually in lobster antenna1 gland.
and combined,
Pb-EXF
on enzyme activities
LEAD-EXPOSED
289
LOBSTERS AT LOW SALINITY
NS 2SOr
(880-1271)
5
1.1.1.27
I
_
NS
\ \
3_w35-291) I-
(157-256)+
T
r i
750
-
500
-
250
-
/
5
1! !(16,QQ)
st ILS LO! 5 AS o-
CONTR
1
IO1
Pb-EXPOSE
,
1
rn0L.s
AS 0
Pb-EXPOSED
Fig. 4. Effects of sublethal lead exposure and low salinity, individually and combined, on enzyme activities in lobster male gonad.
DISCUSSION
A summary of the effects of lead exposure and low salinity on enzyme activity in lobster tissues is presented in Table I. For ease in comparing the effects of lead and of cadmium in the presence and absence of low salinity, Table II shows an analogous summary of the earlier study with cadmium-exposed lobsters (Gould, 1980). Lowsalinity effects outnumbered lead-induced effects and were found primarily in the tail muscle, as was true for the cadmium-exposed lobsters. In contrast to the numerous cadmium effects reported in the earlier study (Table II), however, the only metal effect produced by lead exposure alone was the lowered G6PDH activity in antennal gland, where the greatest bulk of lead taken up by the various tissues was located.
290
E. GOULD
AND R.A. GREIG TABLF
Classification
of effects
of lead exposure combination.
I
(50 fig. I- ‘, 30 days) and low salinity on enzyme activities in lobster tissues.
( 17?;,, 2 days), alone and m
3. Effect of Pb + low salinity (LS) combined
I. Low-salinity
effect alone (in controls)
Response type:
2. Pb effect alone (at ambient salinity, 27%,)
Elevated MDH IDH
.4ntagonism Synergism
LS > Pb
Pb > LS
.4bolition of depressed activity
activity IDH MDH
(M) (M)
(H) (MG)
G6PDH
(AG)
LDH
(MG)
PK (M) AAT (H) Response type:
Depressed LDH
TISSUE
(MG)
CONCENTRATIONS
.Abolition of cievated activity
activity
G6PDH
(AG)
LDH MDH
(M) (AC)
MDH
(M)
PK (M) .4AT (H)
OF METAL
The relative distributions of lead and cadmium in lobster antenna1 gland and hepatopancreas apparently differ. Ray et al. (1981) reported that the hepatopancreas ( < 5 “/, of the total wet weight of the lobster) contained 93-99 y0 of the total cadmium in lobsters taken from cadmium-polluted waters near a lead smelter, which produces cadmium-laden dust (Sergeant & Westlake, 1980). Ray’s group (198 1) found the antennal or green gland to rank second or third in total cadmium content, although it is only 0.01 y0 of the animal’s total wet weight. By contrast, in the lead-exposed lobsters reported here, the antennal gland contained the highest concentration of lead of those tissues analyzed (Table III). Moreover, of the seven hepatopancreas specimens from lead-exposed lobsters analyzed for metal uptake, five had no detectable lead. Concentration of cadmium in the hepatopancreas suggests an active sequestering mechanism for this metal in the lobster: intracellular calcium-phosphorus concretions in the crustacean hepatopancreas, as well as in bivalve kidneys, have been shown to contain high concentrations of metals (Fowler et al., 1981). In bivalve kidneys, especially, these concretions have the major role in the accumulation, detoxification, and excretion of cadmium (Carmichael & Fowler, 1981) and other metals (George et al., 1980). For marine invertebrates in general, lysosomes and metallothionein-like proteins are also important in compartmentalizing or sequestering heavy metals (Fowler et al., 1981). The prominent concentration of lead in lobster antenna1 gland, on the other hand,
TABLE II
Response type:
Response type:
LDH GPI PK LDH AAT LDH
MDH IDH MDH MDH
(M) (M) (M) (H) (H) (MG)
(M) (M) (AG) (MG)
1. Low-salinity effect alone (in controls)
(M) (H) (H) (H) (AG)
Depressed activity
MDH MDH LDH GPI MDH
Elevated activity
2. Cd effect alone (at ambient salinity, 27%,J
MDH (M) IDH (M)
Additive effect
MDH (AG) LDH (AG)
GPI (MG)
Synergism
Cd>LS
GPI (H) LDH (H)
Abolition of elevated activity
GPI (M) LDH (MG)
Abolition of depressed activity
LS > Cd
Antagonism
3. Effect of Cd + low salinity (LS) combined
Classification of effects of cadmium exposure (10 fig. I-‘, 30 days) and low salinity (17x,,, 7 days), alone and in combination, on enzyme activities in lobster tissues (from Gould, 1980).
M
N.D.h 2.9 + 1.0
(3) (5)
---.--..-
N.D. 8.8 f 6.1
(4) < I.0 i 0.3 2.5 & 0.9 (5)
(4)” (5)
..-- _...
Gills
” ND., not detectable.
” Unrts: (sample _VJ arnhmcttc mean 2 sk
+ 7 days low satinity: Controls Exposed
+ 2 days ambient salinity: Controls Exposed
Exposed
30 days: Controls
Tissue:
(3) <24.3 i 6.2 106 2 17 (5) (range: 54-145)
5.6 + 3.4 (4) 138270 (4) (range: 31-340)
~~_-..
(4) N.D. (5) 63.0 2 47.3 (range: ND.-249) _.__... .-....- .l_l.,._....- .___ -._.“_ ..
.
Antenna1 gland N.D. 2.2 f 1.8
_...
N.D. 1.9 i 1.1
(4) <1.0*0.1 (4) <0.9+0.1
-‘-’ --
Tail muscle
__“. _...._ I..._ ____.____“__“_~
(2) (5)
(3) <0.7 + 0.1 13) 2.0 2 1.3
(2) (7)
. ---.-
Wepatopancreas
_l_--i_-
(4) < 24.8 rf:8.5 (5) (20.8 i 3.5
Heart
Tissue concentrations of lead (pg g- ‘, wet wt) in lobsters exposed 30 days to 50 pg Pb I- ’ in flowing sea water k2 subsequent days in clean sea water at ambient (27’&,)or low (17Y,,)salinity: analyses were not performed on heart and tail muscle from the 30-day exposure or from the 2-day low-salinity treatment: sample numbers are in parentheses.
TABLEIII
LEAD-EXPOSED
LOBSTERS
AT LOW SALINITY
293
suggests a very active excretory mechanism for this metal. After only 2 days of clearing, levels of lead had dropped by about one-third (Table III) and the only metal effect in that tissue, lowered G6PDH activity, had disappeared. The mechanism by which lead is so efficiently accumulated in and excreted by the antennal gland remains an object for future study. LOW-SALINITY
RESPONSE
Low salinity effects in the lead-study control lobsters were greatest in the tail muscle (Table I), as in the earlier cadmium study (Table II). In the cadmium-study control lobsters held at low salinity for 7 days, the effects were primarily depressed activities, which was not the case in the lead study (a 2-day holding period). Both heart AAT and muscle PK were elevated in the low-salinity controls of the lead study, indicating an increased amino acid catabolism and expenditure of metabolic energy in response to the 2-day osmotic stress. These same enzyme activities were depressed, by contrast, in the 7-day low-salinity controls on the cadmium study. These observations suggest an oscillatory reaction: an initial stimulus in response to the low-salinity stress, followed by depressed activity and a gradually damped oscillation. They also conform to the perception of the lobster as an osmoconformer that adapts only slowly to hyposalinity (e.g. Dall, 1970). The only low-salinity responses common to both studies were elevated MDH and LDH in muscle and lowered LDH in male gonad. Gilles observed (1973) that the drop in amino-acid concentrations seen during hyposmotic stress is associated with increased catabolism of amino acids, which leads to the formation of reducing equivalents and consequently elevated levels of reduced pyridine nucleotides. Thus in the low-salinity controls of both metal-exposure studies, the continued increase in redox activity (MDH) served to regenerate the oxidized NAD necessary for the increased citric acid cycle activity (IDH). In lobster tissues, apparently, MDH is a more effective redox regulator than LDH. METAL-EXPOSURE
RESPONSE
Cadmium, even at one-fifth the concentration of lead used in the study reported here, and with 7 rather than 2 days of clearing, induced far more metabolic perturbation (Table II) than did lead. Moreover, the observed cadmium effects were entirely hermetic and predominantly in the heart, whereas lead exposure alone produced only a single effect, a lowered G6PDH (Table I) in the lead-burdened antennal gland (Table III) an effect that disappeared after only 2 days of clearing at both ambient and low salinity. Of the two metals, cadmium clearly places greater stress on the lobster by a continuing strong demand for metabolic energy. Lead, at five times the cadmium concentration, induced no such energy expenditure. A less direct effect attributable to lead exposure alone was the abolishing of several adjustments to low salinity seen in the control animals (Table I). Similarly attenuated
294
EGOULDAND
R.A.GREIG
biochemical activity has been found in teleosts (Gould, 1977) and bivalve moliuscs (Gould, unpubl. data), as well as other crustaceans (Gould et al., 1976) exposed to sublethal concentrations of cadmium. Such weakening of normal physioiogic~ adaptive mechanisms argues a metabolism fess capable both of adjusting to changing environmental conditions and of channeling tissue energy toward reproductive needs. The animal is also, under continuing stress, less likely to survive.
ACKNOWLEDGEMENTS
We thank M.T. Grojean, J. S. Rancourt, and S. Schurman for expert technical assistance, and M. R. Cox for preparing the graphs.
REFERENCES BAYNE, B. L., M. N. MOORE, D. R. LIVINGSTONE& P. SALKELD,1979. Measurement of the responses of individuals to environmental stress and pollution: studies with bivalve molluscs. Phil. Trans. R. Ser. London Ser. B, Vol. 286,pp. 563-681.
CAMPBELL,J.A. & D. H. LORING, 1980. Baseline levels of heavy metals in the waters and sediments of Ba%n Bay. Mar. Pollur. Bull., Vol. 11, pp. 257-261. CARMICHAEL,N.G. & B.A. FOWLER,1981. Cadmium accumulation and toxicity in the kidney of the bay scallop Argapecten irradians. Mar. Biol., Vol. 65, pp. 35-43. DALL, W., 1970. Osmoregulation in the lobster Homarus americanus. 1. Fish. Res. Board Can., Vol. 27, pp. 1123-I 130. FOWLEK,B.A., N.G. CARMICHAEL& KS. SQUISB, 1981. Factors affecting trace-metal uptake and toxicity to marine organisms. II. Cellular mechanisms. In, Bja~o~a6 monjto~ng of mar~ep~l~utanfs, edited by F. 3. Vernberg, A. Calabrese, F. P. Thurberg & W.B. Vernberg, Academic Press, New York, pp. 145-163. GEORGE,S.G., B.J. S. PIRIE & T. L. COOMBS,1980. Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.). J. Exp. Mar. Biol. EC&, Vol. 42, pp. 143-156. GIL.LES,R., 1973. Oxygen consumption as related to the amino acid metabolism during osmoregulation in the blue crab, Callinectes sapidus. Neth. J. Sea Res., Vol. 7, pp. 280-289. Gour.r>, E., 1977. Alteration of enzymes in winter flounder. Pseudop(euranectes americanus, exposed to sublethal amounts o~cad~~u~ chloride. In, P~y~~~~og~~u~ resp#~ses oft7~ar~nebiura to pa~~utan~~, edited by F .I. Vernberg, A. Calabrese. F. P. Thurberg & W. B. Vernberg, Academic Press, New York, pp. 209-224. Got I.I), E.. IY80. Low-salinity stress in the American lobster, ffomarus americanus, after chronic sublethal exposure to cadmium: biochemical effects. He&al. Meeresunters., Vol. 33, pp. 36-46. GOUI D, E., R. S. COLLIER,J.J. KAROLUS& S. GIVENS, 1976. Heart transaminase in the rock crab, Cancer irroratus, exposed to cadmium salts. Bull. Environ. Contam. Toxicol., Vol. 15. pp. 635-643. GREIG, R.A., T.K. SAWYER,E. J. LEWIS & M.E. GALASSO, 1982. A study of metal concentrations in relation to gill cofor and pathology in the rock crab, Cancer irroratus. Arch. Environ. Contum. Taxicol.. Vol. 11) pp. 539-545. KING, E. N., 1966.Oxidative activity of crab gill mitochondria as a function ofosmotic concentration. Conz~~. Biochem. Physiol., Vol. 17, pp, 245-258. KLEIN, L. A., M. LANG, N. NASH & S.L. KIRSCHNEK,1974. Sources of metals in Nets York City wastewater. Department of Water Resources, City of New York, 18pp. MOUNT. D. I. & W.A. BRUNGS,1967. A simplified dosing apparatus for fish toxicology studies. WaterRes.. Vol. 1. pp. 21-29. MUNDAY,K. A. & P.C. POAI, 1971. Respiration and energ meta~lism in Crustacea. In, ~~~enzi~f7f zootogv. Vat. IV, Arthropoda, Part B, edited by M. Florkin & B.T. Scheer, Academic Press, New York, pp. 191-211.
LEAD-EXPOSED
LOBSTERS
AT LOW SALINITY
295
POPHAM,J. D., D. C. JOHNSON& J.M. D’AURIA, 1980. Mussels (Myrilus edulis) as “point source” indicators of trace metal pollution. Mar. POW. Bull., Vol. 11, pp. 261-263. PROSSER, C.L., 1973. Inorganic ions. In, Comparative animal physiology, edited by CL. Prosser, W.B. Saunders Co., Philadelphia, pp. 79-l 10. RAY, S., D.W. MCLEESE & L.E. BURRIDGE,1981. Cadmium in tissues of lobsters captured near a lead smelter. Mar. Pollut. BUN., Vol. 12, pp. 383-386. SERGEANT,D. B. & G. F. WESTLAKE,1980. Industrial discharges of cadmium at Belledune. In, Cadmium pollution of Beliedune Harbour, New Brunswick, Canada, edited by J. F. Uthe & V. Zitko, Can. Tech. Rep., Fisheries and Aquatic Sciences, No. 963, pp. 3-10. WALDICHUK,M., 1980. Lead in the marine environment. Mar. Pollut. Bull., Vol. 11, pp. 241-242. Wools, J.M., 1974. Biological cycles for toxic elements in the environment. Science, Vol. 183, pp. 1049-1052.