the action of heavy metals on the gametes of the marine mussel, mytilus edulis (l.)—i....

Comp. Biochem. f’hysiol. Vol. 77C, No. 2, pp. 289-294, 1984 0306-4492/84 $3.00 + 0.00

Printed in Great Britain 80 1984 Pergamon Press Ltd

THE ACTION OF HEAVY METALS ON THE GAMETES OF THE MARINE MUSSEL, MYTKUS EDULIS (L.)--I. COPPER-INDUCED UNCOUPLING OF RESPIRATION

IN THE UNFERTILIZED EGG

H. B. AKBERALI,‘.’ M. 3. EARNSHAW’ and K. R. M. MARRIOTT’

‘Departments of Zoology and 2Botany, University of Manchester, Oxford Road, Manchester Ml3 9PL, UK. Telephone: 061-273-7121

(Received 12 July 1983)

Abstract-l. The direct addition of Cu2+ to unfertilized eggs of Mytilus edulis results in a stimulation of respiration with maximal stimulation occurring at a Cu2+ concentration of ca 0.5 mM. By contrast, the addition of Zn*+ has no effect on egg respiration. The uncoupler CCCP produces a 5/6 fold stimulation of egg respiration but the addition of ADP leads to only a small release of respiration.

2. In contrast, sperm respiration is unaffected by Cu*+, inhibited by Zn2+ and CCCP produces only a small respiratory stimulation.

3. The addition of Cu*+ to respiring Mytilus mantle tissue mitochondria produces an initial stimulation of State 4 oxidation which is then followed by a progressive inhibition.

4. It is suggested that respiration in the unfertilized egg may be inhibited by a high ATP/ADP ratio in the cytosol. Respiration can, therefore, be released by either the addition of a H+-translocating uncoupler or by Cuz+ which may act by stimulating mitochondrial K+ influx.

INTRODUCTION

The biological and toxicological effects of heavy metals have been extensively reviewed and numerous studies have suggested the possible use of marine bivalve molluscs as indicator organisms of environmental pollution (e.g. Phillips, 1977; Bryan, 1979). The initial response observed when estuarine bivalve molluscs, such as Mytilus edulis and Scrobicularia plana, are exposed to heavy metals (Cu, Zn) is siphon withdrawal followed by valve closure (Davenport, 1977; Akberali and Black, 1980; Akberali et al., 1981). This behavioural response is important to minimize the adverse environmental effects so allow- ing the animal a period of stress avoidance. However, with longer term exposure to heavy metals, it is essential for the organism to open the shell valves and interact with the environment for normal metabolic processes. The fact that bivalve molluscs can concen- trate heavy metals at very high levels in the different tissues (Phillips, 1977; Bryan, 1979) and apparently survive and reproduce normally indicates that they have evolved control or tolerance mechanisms at the cellular level. Such mechanisms can include the syn- thesis of metallothioneins (George et af., 1979) and the immobilization of the heavy metal in membrane- bound vessicles prior to its excretion by the organism (George and Pirie, 1980).

The mechanism of siphon withdrawal in Scrobicu- laria has been investigated in both in situ and isolated siphon tissue preparations. Low concentrations of Cu2+ cause reversible contractions of the isolated siphon which depend on the presence of Ca’+ in the external medium (Akberali, 1981; Akberali et al., 1982). Zinc, however, has no effect on the isolated

siphon but produced a marked contraction of the in situ siphon which may involve sensory systems (Ak- berali et al., 1981). The effects of Cu*+ and Zn*+ have been investigated further using isolated bivalve mol- lust mitochondria which possess a comparable Ca*+ transporter to that found in isolated rat liver mitochondria (Akberali and Earnshaw, 1982a). It has been shown that (i) Cu*+ inhibits Ca*+ transport although the initial respiration rate is essentially unaffected (Akberali and Earnshaw, 1982~); (ii) by contrast, Zn*+ inhibits both respiration and Ca*+ transport although respiration in marine mollusc mitochondria is more resistant to Zn*+ than is respiration in rat liver mitochondria (Akberali and Earn- shaw, 1982b,c); (iii) addition of Cu*+ to mitochondria pre-loaded with CaZf causes a rapid Ca*+ efflux, whereas Zn*+ is relatively ineffective (Akberali and Earnshaw, 1982~). Accordingly, it has been suggested that the Cu*+-induced contraction of the isolated siphon of Scrobicularia (Akberali, 1981; Akberali et al., 1982) may arise via the displacement of free Ca2+ from intracellular reservoirs resulting in a stimulation of the nerve/muscle system (Akberali and Earnshaw, 1982~).

An understanding of the effects of heavy metal pollutants on marine invertebrate gametes is equally important at organismal level because propagation of the species and maintenance of populations de- pends on the survival and fitness of both gametes and larvae. The gametogenic cycle in Myths can be affected by variation in natural environmental par- ameters, e.g. temperature, food abundance (Seed, 1976) but is also potentially subject to stress imposed by heavy metal input from past and present indus- tries.

289

290 H. B. AKBERALI et al,

Published work on the effect of heavy metals on marine bivalve mollusc reproduction has largely con- centrated on their lethal effects on the embryonic and juvenile stages (Calabrese and Nelson, 1974; Cala- brese et al., 1973, 1977). However, it has recently been shown that Cu2+ is more effective than Zn2+ in suppressing gametogenesis in Mytilus (Maung-Myint and Tyler, 1982). Little information is available con- cerning the mechanism of heavy metal action on gametogenesis or the early developmental stages of Myths. The present paper describes the uncoupling of respiration in the unfertilized egg produced by Cu2+. Further work on heavy metal uptake, localization and the respiratory consequences is in preparation.

MATERIALS AND METHODS

Gamete production

The work described here was carried out during the spawning season in the Spring of 1983 using adult ?r4yriZus edulis collected from Bangor, North Wales, UK. The shells were thoroughly cleaned of epiphytes and other adhering material The animals were then placed individually in dishes containing approx 60 ml of membrane-filtered (0.3 pm) sea water at 10°C to allow the shells to open and the animal to resume pumping. Techniques for inducing spawning in M.yt~lus have received considerable attention (Bayne, 1965). The method used in this work involved the use of an electrical stimulus as developed by Iwata (1950) and Sugiura (1962). After 30 min in filtered sea water, each animal was stimulated using a Grass S8 stimulator (100 V pulses of 10 msec duration and frequency of 12 set-‘, for 10 set) with a chlorided silver electrode held on either side of the shell gape. The release of gametes generally com- menced after a latent period of 30-60 min. Once the individuals were sexed, spawning was allowed to proceed for a further 30 min in a fresh volume of filtered sea water during which time the shed sperm and eggs were placed on ice at frequent intervals. At the end of the 30min collection period, the gametes from one or more individuals were combined and filtered through two layers of muslin to remove the small amount of associated debris. The filtered gamete suspension was then centrifuged at 10,OOOg x 10min at 2°C in the case of sperm and 5000 g x 5 min at 2°C for eggs. The resulting pellets were made up in approx 10 ml of ice-cold, filtered and buffered sea water (see later for the use of buffered sea water) and stored on ice. Preliminary experiments established that gamete respiration was relatively labile at 10°C and that this could be deiayed by storage on ice so that the initial rate of respiration was constant for approx 2; hours (data not shown).

MytiEus eduiis were collected from the population at Morecambe (Lancashire) and maintained in aquaria containing sea water (S, 31%) at 10°C. Mitochondria were isolated from the mantle essentially using the method of Zaba et al. (1978) described previously (Akberali and Earn- shaw, 1982b).

Measurement qf res~irat~on

Gamete respiration was determined by the addition of 1.3 ml of the gamete suspension to the compartment of the Rank oxygen electrode (Rank Brothers, Bottisham, Cam-

*Tris(hydroxymethyl)aminoethane. tN-[(Tris-hydroxymethyl)methyl]-2-aminoethane sulphonic

acid. $N-Tris(hydroxymethyl)methylglycine.

bridge, UK.) which was maintained at 10°C. The suspension was stirred for 3 min in order to achieve equi- libration of the oxygen con~ntration at 10°C after which the compartment was sealed and respiration recorded. The range of gamete protein concentrations used in the experiments described in this paper were 2-6mg protein ml-.’ for sperm and 3-15 mg protein ml-’ for eggs. The stock solutions (in distilled water) of heavy metals used were 100 mM Cu(NO,),-KOH, pH 7.0 and 100 mM Zn(NOJ-KOH, pH 6.0. Additions from these stock solutions to normal sea water resulted in an una~ep~ble pH shift which was minimized by increasing the buffering capacity of the sea water by the addition of 10 mM Tris*-Test, pH 7.91. The pH range of the buffered sea water after the addition of the Cu’ + and Zn*+ concentrations used in this paper was pH 7.84-7.68. The effects of carbonyl cyanide m- chlorophenylhydrazone (CCCP) and adenosine-5’- diphosphate (ADP) on gamete respiration was followed by the addition of appropriate volumes from stock solutions of 1.0 mM CCCP in absolute ethanol and 1.0 mM ADP in distilled water. Appropriate controls showed that the addition of equivalent volumes of ethanol or distilled water had no effect on gamete respiration (data not shown).

Mitochondrial respiration was determined at 25°C using a Clark oxygen electrode (Yellow Springs Instrument Co., Ohio). Respiration was measured by adding 0.15 ml of mitochondrial suspension (3.5 mg protein ml-‘) to 2.85 ml of 200 mM KCI, IO mM KH2P0,, 10 mM Tricinef-Tris (pH 7.2), 5 mg/ml bovine serum albumin (BSA), 5pM cyto- chrome c as described previously (Akberah and Eamshaw, 1982b). After I min, respiration was initiated by the addition of 6 mM succinate plus 6 mM glutamate (pH 7.2) which was followed by addition of the appropriate volume of 30 mM CuSO,,

Rates of gamete and mitochondrial respiration were calculated using published tables of oxygen solubility (Washbun, 1928) and are expressed per mg protein. Protein content was determined with the Folin reagent (Lowry et al., 1951) using BSA (Fraction V) as the standard.

RESULTS

The effects of Cu2+ and ZnZf on the respiration of M. edulis gametes are compared in Fig. 1. The addition of 0.5 mM CL? to respiring unfertilized eggs resulted in a 47% stimulation of respiration in this experiment whereas Zn2+, by contrast, had no effect. A similar, direct addition of Cu2+ to sperm produced no effect but the respiration rate was inhibited 49% by the addition of Zn*+. However, additional experiments in which the gametes were pre-incubated with the heavy metal at 0°C before measurement of respiration showed that egg respiration was inhibited by Zn2+ and sperm respiration by Cu2+ (data not shown). Presumably time-dependent permeation of the heavy metal is variable but appears to be rapid in the reaction of Cu2+ with the egg (Fig. 1).

Figure 2 shows that a concentration of ca 0.5 mM Cu2+ causes a maximal stimulation in egg respiration of 106% in this experiment. Higher Cu2+ concentrations result in a progressively smaller stimulation of respiration.

The further experiments described in this paper were designed to establish the nature of the Cu’+-induced stimulation of egg respiration. The effects of Cu2+ on the respiration of mitochondria isolated from M. edufis mantle tissue are shown in Fig. 3. The addition of 0.4mM Cu2+ to a respiring mitochondrial suspension stimulates the initial rate of

The action of heavy metals on the gametes of the marine mussel 291

2 min - -

Fig. 1. The effect of direct additions (1) of Cu2+ or ZnZ+ on egg and sperm respiration. Heavy metal ions were present at a final concentration of 0.5 mM. Numerals refer to respiration as nmoles 0, min _ ’ mg

protein I.

respiration by 30% which is then followed by a progressive inhibition of respiration to 63% of the initial rate. Similar observations on the effect of Cu*+ ions on the respiration in fish and rat liver mitochondria has been reported. Both rat liver (Zaba and Harris, 1976) and fish liver (Zaba and Harris, 1975, 1978) mitochondria take up Cu*+ in an energy- independent process. It has been shown that, although reduced Cu2+ uptake occurs in energized mitochondria, K + uptake is initiated which results in an accompanying swelling and respiratory stimulation which becomes progressively inhibited with time in the presence of high Cu*+ concentrations (Zaba and Harris, 1976, 1978). It seems reasonable to assume that the mode of action of Cu2+ on isolated M. edulis mitochondria in a KC1 reaction medium (Fig. 3) is comparable, particularly as the addition of

i

t \

l 1. 1

Fig. 2. Egg respiration as a function of Cu’+ concentration. The experiment was carried out as in Fig. 1.

Cu2+ to M. edulis mitochondria in a reaction medium containing sucrose does not produce a respiratory stimulation (Akberali and Earnshaw, 1982~). By contrast to Cu’+, Zn*+ possesses a marked inhibitory effect on the respiration of Myths digestive gland/mantle tissue (Akberali and Earnshaw, 1982b) and fish liver (Zaba and Harris, 1978) mitochondria.

The apparent similarity between the stimulation of egg respiration by Cu*+ (Figs 1, 2) and the Cu*+-induced uncoupling of State 4 respiration in isolated mitochondria (Fig. 3) suggests the possibility that respiration in the unfertilized egg of M. eduiis is not fully released. If this were the case, then egg respiration should be susceptible to release by a more traditional H+-translocating uncoupler. Indeed, the addition of CCCP to respiring unfertilized eggs rap- idly results in a dramatic stimulation of 510% of the initial respiration rate (Fig. 4). It seems likely that respiration in the unfertilized egg may be inhibited by a high ATP/ADP ratio (see Discussion). Figure 4 also shows that ADP is capable of producing a small stimulation in respiration of 21”/ As might be expected, the addition of ADP to M. edulis sperm had no effect on the rate of respiration (data not shown).

Finally, the effect of CCCP concentration on both egg and sperm respiration is shown in Fig. 5. The maximal stimulation of egg respiration is achieved at a CCCP concentration of cu 1 .O pm and was 632% of the control rate in this experiment. Higher concentrations of CCCP led to a progressive decrease in the degree of respiratory stimulation. On the other hand, sperm respiration gave the expected relatively small increase upon titration with CCCP amounting to 143% of the control rate.

DISCUSSION

The work described in this paper shows that Cu*+, but not Zn*+, stimulates respiration in the unfertilized eggs of M. edulis (Figs 1 and 2) and also leads to a transient stimulation of State 4 respiration in isolated Myths mitochondria (Fig. 3). The addi-

292 H. B. AKBERALI et al.

I 50 nmoles O2

2min -

Fig. 3. The action of Cu2+ on the respiration of isolated M. edulis mitochondria. The substrate addition (IS) was made 1 min after addition of the mitochondria to the reaction medium and consisted of 6 mM succinate plus 6 mM glutamate. Cu2 + was added (1) at a final concentration of 0.4 mM. Numerals refer

to respiration as nmoles 0, min I mg mitochondrial protein- ‘.

tion of the uncoupler CCCP causes a several-fold It has been shown that isolated fish and rat liver increase in egg respiration (Figs 4 and 5) showing that mitochondria are capable of binding Cu2+ and Zn2+ respiration is only partially released in the un- to a considerable degree (Zaba and Harris, 1976, fertilized egg. 1978). The action of Cu2+ on respiration is biphasic

Q

732

I

50 n moles O2

2min

Fig. 4. The effect of direct additions (1) of CCCP and ADP on egg respiration. Final concentrations were 1.0 p M CCCP and 100 PM ADP. Numerals refer to respiration as nmoles 0, min ’ mg protein - ‘.

The action of heavy metals on the gametes of the marine mussel 293

the egg respiration. Moreover, the percentage increase is higher in the unfertilized eggs than in fertilized eggs (Runstrom, 1935; Needham, 1942). Krahl and Clowes (1934) have shown that dinitro- cresol raised the oxygen consumption in the unfertilized and fertilized sea urchin eggs to 6-fold and 4-fold respectively. In the marine bivalve molluscs Ostrea edulis, Spisula solidissima and Mytilus edulis, the oxygen consumption rate of fertilized eggs increases during development (Cleland, 1950; Sclufer, 1951; Raven, 1972). Generally, respiration in marine bivalve molluscs continues to increase during development but the rise is not always regular and may be interrupted by plateaus or even drops at certain stages.

Fig. 5. Egg and sperm respiration as a function of CCCP concentration. The data represents the mean of two experiments in each case. Control values (100%) were 1.98 nmoles 0, min-’ mg protein’ for egg respiration and 11.1 nmoles

O,min-’ mg protein’ for sperm respiration.

giving an initial stimulation of respiration arising from enhanced K+ influx (Zaba and Harris, 1978). The ensuing respiratory inhibition is linked with K+ loss due to a further reaction of Cu2+ with the mitochondrial membranes which appears to take place at higher Cu*+ concentrations. It seems reasonable to suppose that the stimulation of respiration in the egg of M. edulis caused by Cu2+ (Figs 1 and 2) is due to a similar uncoupling of oxidative phosphorylation. However, it should be noted that. an inhibition of egg respiration caused by Cu2+ was not observed in the present study suggesting that the intracellular Cu*+ concentration was not high enough to effect respiration inhibition during the time period of our experiment. The degree of respiratory stimulation though was reduced at Cu2+ concentrations >ca 0.5 mM (Fig. 2).

The mechanism of control of respiration in the unfertilized egg appears to be unknown. Clearly, respiration is only partially released and is not subject to substrate limitation as the addition of uncouplers can result in a marked stimulation of respiration (Krahl and Clowes, 1934; Needham, 1942; this paper, Figs 4 and 5). It has been clearly shown with isolated mitochondria that the rate of oxidative phosphorylation is controlled by the phosphorylation potential (Slater et al., 1973). Moreover, a high external ATP/ADP ratio produces an inhibition of State 3 oxidation which is probably due to com- petition between ATP and ADP for entry via the adenine nucleotide translocator. It is possible that a similar situation exists in the unfertilized egg of M. edulis which may be reflected by a high energy charge (Chapman et al., 1971). Admittedly, the addition of ADP to Mytilus eggs only results in a small release of respiration (Fig. 4) but it is expected that the plasma membrane would be essentially impermeable to adenine nucleotides. Presumably, the low rate of basal respiration in the unfertilized egg ensures that the substrate reserves are not depleted prior to fertilization. Fertilization is followed by dramatic increases in, for example, nucleic acid and protein biosynthesis (Balinsky, 1981) which would be expected to reduce the cytosol ATP/ADP ratio leading to release of respiration. Clearly, though, further work is required on the post-fertilization changes in egg respiration, adenine nucleotide levels and bio- synthetic events in order to test this hypothesis.

Acknowledgements-One of us (H.B.A.) was supported by the Nuffield Foundation. We are also grateful to Dr P. D. Gabbut for helpful discussion, Mrs S. E. Hardman for typing the script and Mr L. Lackey for photographic assistance.

It is well known that the oxygen consumption of fertilized eggs increases with the development of the embryo. It has been reported that, immediately after fertilization of sea urchin eggs, there is a sharp increase in oxygen consumption of well above 400% in comparison to that of unfertilized eggs after which respiration settles down to a lower rate though still well above that of the unfertilized eggs (Warburg, 1908; Laser and Rothschild, 1939; Needham, 1942; Balinsky, 1981). However, in contrast in the eggs of some species such as the clam Cumingia tellinoides and the annelid Chaetopterus, the oxygen rate de- creases soon after fertilization (Needham, 1942; Bal- insky, 1981). Addition of compounds such as pyocyanine, nitrophenols, methylene blue, to a suspension of sea urchin eggs causes an increase in

REFERENCES

Akberali H. B. (1981) Effects of copper (Cu*+) on an isolated tissue preparation from the bivalve, Scrobicularia plana (da Costa).- J. exp. mar. Biol. Ecol. 52, 115-120.

Akberali H. B. and Black J. E. (1980) Behavioural responses of the bivalve Scrobiculuria plana (da Costa) subjected to short-term (Cu II) concentrations. Mar. enuir. Res. 4, 97-107.

Akberali H. B. and Eamshaw M. J. (1982a) The mechanism of calcium transport in mitochondria isolated from the marine mussel, Mytilus edulis (L.). Cell Calcium 3, 55-66.

Akberali H. B. and Eamshaw M. J. (1982b) Studies on the effects of zinc on the respiration of mitochondria from

294 H. B. AKBERALI et al.

different tissues in the bivalve mollusc Mytilus edulis (L.). Comp. Biochem. Physiol. 72C, 149-152.

Akberali H. B. and Earnshaw M. J. (1982~) A possible role for mitochondrial Car+ in the contraction of the mollusc siphon induced by Cu’+. Comp. Biochem. Physiol. 73C, 395-398.

Akberali H. B., Trueman E. R. and Earnshaw M. J. (1982) The influence of external calcium in facilitating copper induced contraction in the isolated siphon of Scrobicula- ria plana (Bivalvia). J. mall. Stud 48, 9697.

Akberali H. B., Wong T. M. and Trueman E. R. (1981) Behavioural and siphonal tissue responses of Scrobiculu- ria plana (Bivalvia) to zinc. Mar. enoir. Res. 5, 251-264.

Balinsky B. I. (1981) Fertilization and the beginning of embryogenesis. In An Introduction to Embryology, 5th edn, pp. 768. Saunders College Publishing, Philadelphia, New York.

Bayne B. L. (1965) Growth and the delay of metamorphosis of the larvae of Mytilus edulis (L.). Ophelia 2, 147.

Bryan G. W. (1979) Bioaccumulation of marine pollutants. Phil. Trans. R. Sot. Land. B 286, 4833505.

Calabrese A. and Nelson D. A. (1974) Inhibition of the embryonic development of the hard clam, Mercenaria mercenaria, by heavy metals. Bull. envir. Contam. Toxicol. 11, 92-97.

H. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.

Maung-Myint U. and Tyler P. A. (1982) Effects of temperature, nutritive and metal stressors on the reproductive biology of Mytilus edulis. Mar. Biol. 67, 2099223.

Needham J. (1942) In Biochemistry and Morphogenesis, p. 785, University Press, Cambridge.

Phillips D. J. H. (1977) The use of biological indicator organisms to monitor trace metal pollution in marine and estuarine environments-a review. Envir. Pollut. 13, 281-317.

Raven C. P. (1972) Chemical embryology of mollusca. In Chemical Zoology Vol. VII (edited by Florkin M. and Sheer T.) pp. 155-185. Academic Press, New York.

Runstrom J. (1935) On the influence of pyocyanine on the respiration of the sea urchin eggs. Biol. Bull. 68, 327-334.

Scluffer E. (195 1) The respiration of Spisula eggs. Biol. Bull. 109, 113-122.

Calabrese A., Collier R. S., Nelson D. A. and MacInnes J. R. (1973) The toxicity of heavy metals to embryos of the American Oyster, Crassostrea virginica. Mar. Biol. 18, 1622166.

Seed R. (1976) Ecology. In Marine Mussels (edited by Bayne B. L.) pp. 13-65. International Biological Programme 10, . .

Calabrese A., MacInnes J. R., Nelson D. A. and Miller J. E. (1977) Survival and growth of bivalve larvae under heavy-metal stress. Mar. Biol. 41, 179-184.

Chapman A. G., Full L. and Atkinson D. E. (1971) Adenylate energy charge in Escherichia colt during growth and starvation. J. Bact. 108, 1072-1086.

Cleland K. W. (1950) Respiration and cell division in developing oyster eggs. Proc. Linn. Sot. N.S. W. 75, 282-295.

Cambridge University Press, Cambridge. Slater E. C., Rosrig J. and Mol A. (1973) The phos-

phorylation potential generated by respiring mitochondria. Biochim. biophys. Acta 292, 534553.

Sugiura Y. (1962) Electrical induction of spawning in two marine invertebrates (Urechis unicinctus, hermaphrodite Mytilus edulis). Biol. Bull. 123, 2033206.

Warburg 0. (1908) Beobachtungen iiber die oxy- dationsprozesse im Seeigelei. Hoppe-Seyler’s Z. physiol. Chem. 57, l-16.

Davenport J. (1977) A study of the effects of copper applied continuously and discontinuously to specimens of M. edulis (L.) exposed to steady and fluctuating salinity levels. J. mar. biol. Ass. U.K. 57, 63-74.

George S. G. and Pirie B. J. S. (1980) Metabolism of zinc in the mussel, Mytilus edulis (L.). J. mar. biol. Ass. U.K. 60, 573-590.

Washbun E. W. (1928) International Critical Tables, Vol. 3, p. 258. McGraw-Hill, New York.

Zaba B. N. and Harris E. J. (1975) The interaction of cod liver mitochondria with copper ions. Biochem. Sot. Trans. 3, 494-950.

Zaba B. N. and Harris E. J. (1976) Uptake and effects of copper in rat liver mitochondria. Biochem. J. 160, 707-714.

George S. G., Carpene E., Coombs T. L., Overnell J. and Zaba B. N. and Harris E. J. (1978) Accumulation and effects Youngson A. V. (1979) Characterisation of cadmium- of trace metal ions in fish liver mitochondria. Camp. binding proteins from mussels, Mytilus edulis (L.), ex- Biochem. Physiol. 61C, 89-93. posed to cadmium. Biochim. biophys. Acta 580, 225-233. Zaba B. N., de Bont A. M. and de Zwaan A. (1978)

Iwata K. S. (1950) Spawning of Mytilus edulis. (2) Discharge by electrical sttmulation. Bull. Jap. Sot. scient. Fish. 15,

Respiration and properties of mitochondria from tissues of the sea mussel Mytilus edulis L. Int. J. Biochem. 9,

443446. 191-197.

Krahl M. E. and Clowes G. H. A. (1934) Action of dinitro-cresol on respiration and cell division in Arbacia eggs. Biol. Bull. 67, 332.

Laser H. and Rothschild V. (1939) The metabolism of the eggs of Psammechinus milaris during the fertilization reaction. Proc. R. Sot. Lond. B 126, 539-556.

Lowrv 0. H.. Rosebroueh N. J.. Farr A. L. and Randall

the action of heavy metals on the gametes of the marine mussel, mytilus edulis (l.)—i....

Documents