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Some aspects of lead ecotoxicology in the marine environment
Aquatic Toxicology, 26 (1993) 163-170 © 1993 Elsevier SciencePublishers B.V. All rights reserved 0166-445X/93/$06.00
163
AQTOX 00594
Minireview
Some aspects of lead ecotoxicology in the marine environment M. Gnassia-Barelli and M. Romeo I.N.S.E.R.M. U.303 "Mer et Santk", Facult~ de M~decine, Laboratoire de Toxicologie Marine, Nice, France
(Received 3 August 1992; revision received 1 March 1993; accepted 9 March 1993)
While lead emissionshave been reduced in Western Europe, they remain high in Eastern Europe and may enter the marine environment through rainfall and dry deposits. Thus, it appears necessary to understand the ecotoxicology of lead in the marine environment. Induction of metallothionein-like protein synthesis by lead in marine animals was not found in the literature, whereas the immobilization of lead in a chemically inert form might be a general detoxification process. Few results have been published on lead processes of intracellular toxicity in marine organisms. Unstable tetraethyl lead can be rapidly taken up by marine organisms probably through controlled diffusion processes. Once inside the organisms, this compound can be dealkylated into an ionized chemical species and react at the molecular level within the cell. Lead may affect some biochemical functions. A destabilization of the membranes of tysosomes has been reported in invertebrates contaminated by lead. Some effects on populations and ecosystems are reviewed. In controlled ecosystems, tetraethyl lead, although unstable, can persist in sedimentsplaced at a depth of 5 m and exert a toxic influenceon the fauna.
Key words: Lead; Intracellular toxicity; Detoxification; Lysosome; Metal-containing granule
INTRODUCTION As o p p o s e d to other trace elements whose presence is essential for p r o p e r b o d y f u n c t i o n i n g (iron, m a g n e s i u m , copper, zinc, etc.), lead is n o t involved in a n y biological m e c h a n i s m . Its toxicity to h u m a n beings, at high c o n c e n t r a t i o n s , is well-known. T h e s y m p t o m s o f acute i n t o x i c a t i o n associated with m i n i n g have been k n o w n since a n t i q u i t y ( C a p l u n et al., 1984). I n the m o s t severe cases, it c a n p r o v o k e peripheral a n d encephalic polyneuritis.
Correspondence to: M. Gnassia-Barelli, I.N.S.E.R.M.U.303 "Mer et Sant6", Facult6 de M6decine, Laboratoire de Toxicologie Marine, F-06107 Nice Cedex 2, France.
164 Based on measurements of lead in cores of ice and polar snow, Boutron (1988) indicated that lead contamination is worldwide and is primarily due to human activities. A low but significant concentration of lead is present in laboratory air (Boutron and Patterson, 1987; Patterson and Settle, 1987). Pacyna (1984) demonstrated that among the numerous trace elements from nonferrous metal production which are released into the air, lead is the one with the highest emission factor, i.e., its emission rate is equal to 6360 g per ton of metal produced. While lead emissions have been reduced in Western Europe due primarily to the utilization of unleaded gasoline, they remain high in Eastern Europe, according to Pacyna et al. (1991). These authors evaluated the emission rate into the atmosphere at 89.5 tons of Pb/year in Europe in 1982 and predicted a rate of 30.8 to 52.1 t of Pb/year by the year 2000. It must be emphasized that lead emissions may enter the oceans through both rainfall and dry deposits. Lead is ranked on the grey list (annex II) of pollutants by the Oslo and Barcelona Convention and the Barcelona protocol prohibits dumping toxic substances into the marine environment. The US EPA (Environmental Protection Agency) has no criteria for seawater quality in terms of lead content. Thus, it appears necessary to understand the ecotoxicology of lead in the marine environment. This article is a brief review of work published over the past few years on the mechanisms of sequestration, of possible detoxification and of cellular toxicity of lead. Some effects of lead on populations and ecosystems are also given. Data on in situ or experimental lead accumulation by different marine organisms is not included. Marine organisms from metal contaminated environments are capable of accumulating very high levels of metals in their tissues with no obvious biological effects. This tolerance may be due, in part, to homeostasis and detoxification mechanisms. Under conditions of high metal uptake when the sequestration capacity of the detoxification systems is exceeded, toxicity will occur. HOMEOSTASISAND DETOXIFICATIONMECHANISMS Three processes of heavy metal cation homeostasis have been identified in marine invertebrates cells (Viarengo and Nott, 1993): (1) binding to specific soluble ligands, the most important of which are metallothioneins; (2) compartmentalization within membrane-limited vesicles mostly recognised as lysosomes; (3) formation of insoluble precipitates such as Ca/Mg concretions or Ca/S granules. These processes are schematized in Fig. 1. Numerous studies refer to the role of specific proteins that are implicated in detoxification mechanisms. Animals respond to heavy metal stress by induction of metallothionein gene expression, while plants respond by phytochelatin formation through polymerization of peptide precursors (Gekeler et al., 1988). Gekeler et al. (1988) reported that the unicellular algae Scenedesmus and Chlorella increase production of phytochelatins when exposed to the toxic metal ions Cd 2+,
165 f
Granule
)--~r~k
/( [~ - - ~, ^
,~
~
~
~
Protein synthesis
" @Storage
Fig. 1. Metabolismof metal ions by cells (accordingto Simkiss, 1979).Within the cell, metal ions (M+)may be regarded as existing in a labile pool which may be involved in stimulating specificprotein synthesis (which is not a common process for Pb). The role of storage vesicles or inorganic granules is outlined together with the functions of lysosomesin recyclingmetal ions (particularlyPb).
Pb 2+, Zn 2÷, Ag +, Cu 2÷ and Hg 2+. However, it should be noted that these are both freshwater algae. Talbot and Magee (1978) looked for metal complexing proteins naturally present in the mussel Mytilus edulis living in a metal polluted bay and showed that these proteins bind cadmium, but not lead. Further, Rainbow and Scott (1979) found that lead present at a rate of 2 ¢tg.g -l (dry wt.) in the midgut gland of the crab Carcinus maenas sampled in a contaminated zone was not associated with these proteins. The mussel Perna viridis can tolerate and accumulate high lead concentrations (up to 4.46 mg.l-l; Tan and Lim, 1984; accumulation of 3.62 ¢tg Pb g-1 per day, Chan, 1988). According to these authors, the animal appears to possess mechanisms which inhibit the interaction of the toxic metal with essential enzymes. Most of the articles examine metallothioneins and/or phytochelatins chelating copper, zinc or cadmium; only a few focus on lead. Roesijadi (1980/81) gives no examples of this type of protein binding to lead in a review of metallothionein-like proteins in fifteen marine invertebrates. In a more recent review, Roesijadi (1992) gave no reference to metallothioneins binding lead. It is likely that lead homeostasis is realized by the two latter mechanisms identified by Viarengo and Nott (1993). Immobilization of metals by sequestration in lysosomes represents a major nonspecific mechanism for detoxification of many metals in animals exposed to excessively high levels. Lead, in colloidal or particulate form, is taken into invertebrate epithelial cells (particularly the gills and mantle) by endocytosis and after fusion of these endo-
166
cytic vesicles with lysosomes, this metal becomes immobilized (Coombs and George, 1979). Metal-containing granules represent a well-documented means by which cation concentration is regulated in the cells. Insoluble granules are present in the cells of animals from virtually every invertebrate phylum (Nott, 1991). Coombs and George (1979) found granules containing both iron and lead bound within membranes in the gill cells, digestive glands and mantle of contaminated Mytilus edulis, along with granules containing high concentrations of Zn, Fe and Pb in the liver cells. In the gills of Mytilus edulis, Pb is associated with calcium in an equimolar ratio in the form of extracellular crystalline deposits (Marshall and Talbot, 1979; George, 1982). Pullen and Rainbow (1991) reported that pyrophosphate granules isolated from the barnacle (crustacean) Elminius modestus can bind not only Zn, Fe, Ca and K, but also Cu, Mn, Pb, Ag and Cd under natural environmental conditions. The granules in the R cells of the hepatopancreas of Carcinus maenas, studied by X-ray microanalysis, always contain calcium and phosphorus together with magnesium and in some animals these granules contain lead (Hopkin and Nott, 1979). It is concluded that the capability of Carcinus rnaenas to detoxify lead by incorporating it in these granules may be directly related to the number of granules which are immobilizing calcium in the hepatopancreas. In Mytilus edulis, Schulz-Baldes (1977) demonstrated that lead is taken up at the gills and viscera, distributed by the blood and is finally stored as a phosphorus or sulfur-rich complex in membrane-bound vesicles (i.e., in intracellular storage sites) within the excretory cells of the kidney. He suggested that uptake into the cells occurs by pinocytosis. Chassard-Bouchaud et al. (1985) found lead in the oyster Crassostrea gigas and in the mussel Mytilus edulis collected from French coastal waters. Uptake and storage occurred in gills and digestive gland, while excretion occurred in the kidney; macrophage-like haemocytes played an important role in these processes. INTRACELLULAR PROCESS OF TOXICITY
Wood (1980) underlined the instability of tetraethyl and tetramethyl lead compounds in the marine environment, which dealkylate to yield corresponding trialkyls and dialkyls. The latter are highly stable in seawater, probably due to their ionic nature, and do not bioaccumulate in the marine environment. Tetraethyls and, to a lesser degree, tetramethyls are non-polar compounds which can be rapidly taken up by marine organisms, probably through controlled diffusion processes. Once inside the organisms, these compounds can be dealkylated into an ionized chemical species and react at the molecular level within the cell. Authors (Amiard-Triquet et al., 1981; Maddock and Taylor, 1980) generally recognized that the acute toxicity of organic lead is much higher than that of inorganic lead. Some examples of intracellular processes of lead toxicity are given below. Somero et al. (1977) found that when the fish Gillichthys mirabilis was exposed to
167 2650/.tg'1-1 of Pb, oxygen consumption of the whole organism was higher than in controls. However, respiration rate of gills in vitro was the same in both contaminated and uncontaminated fish. The metabolic changes caused by Pb would appear to be due more to its effect on the central nervous system than to a direct effect on the enzymatic metabolism of each cell. Chaisemartin et al. (1978) noted a decrease in the respiratory metabolism of the crab Macropodia rostrata when exposed to 15 and 100 /tg. 1-1 of Pb. They attributed this decrease to an inhibition of the energy consuming mechanisms. A few studies have been made on the effect of lead on biochemical functions. The high glycine absorption of the oligochaetous annelid Enchytraeus albidus is not affected by the presence of 10 mg. 1-l of lead in the environment, although it is reduced by the presence of mercury, cadmium or copper at much lower concentrations (Siebers and Ehlers, 1979). According to Thomas and Juedes (1985), lead increases the glutathione level in the intestines and liver of the fish Micropogonias undulatus, but not in the kidneys or brain tissues. These authors suggest that the interactions between glutathione and lead are indirect and imply a stimulating effect of the metal on the activity of enzymes leading to the synthesis of glutathione. Suszkiw et al. (1984) studied the effects of Pb 2÷ on acetylcholine release and on voltage dependent calcium channels in synaptic vesicles isolated from Torpedo fish electric organs and noted a blocking of Ca 2÷ entry through the calcium channels of synaptosomes and the release of acetylcholine linked to the entry of calcium through the channels. Heavy metals can interact with cell membranes and alter their function. Viarengo (1989) emphasized the destabilization of the membranes of lysosomes, the cellular organelles primarily devoted to macromolecular catabolism. Regoli (1992) studied the lysosomal responses in digestive cells of mussels Mytilus galloprovincialis, collected from a relatively clean area and a heavy metal (particularly Pb)-polluted one. Organisms exposed to high environmental levels of heavy metals showed a reduced lysosomal membrane stability and an enhanced production of lipofuscin. Lysosomal responses, as an early warning system for detection of environmental disturbances, could represent a useful tool for biomonitoring studies (Viarengo, 1989; Regoli, 1992; Viarengo and Nott, 1993). EFFECTS ON POPULATIONSAND ECOSYSTEMS Using a natural population of algae in enriched seawater sampled in a CEPEX ecosystem (Controlled Ecosystem Pollution Experiment, Saanich Bay, Western Canada), Hollibaugh et al. (1980) examined specific diversity as a function of lead concentration in the environment. Concentrations at which toxic effects were seen varied from 60 to 200/~g-1-1. Skeletonema costatum was always the dominant species, while Thalassiosira sp. was present, but not always abundant and Chaetoceros sp. was present except above 200/lg. 1-1 of lead.
168 A r n o u x et al. (1988) studied the b e h a v i o u r o f e x p e r i m e n t a l m o d u l e s c o n t a i n i n g sediments, p l a c e d in sea w a t e r at a d e p t h o f 5 m. These sediments, where f a u n a was r e m o v e d , were c o n t a m i n a t e d (1 g o f P b p e r kg) with either m i n e r a l o r t e t r a e t h y l lead. The r e c o l o n i z a t i o n o f biological c o m p a r t m e n t s (microflora, m e i o f a u n a , m a c r o f a u n a ) was s t u d i e d in the m o d u l e s over a 2-year cycle. T h e m o d u l e s c o n t a i n i n g m i n e r a l l e a d revealed a b e h a v i o u r similar to reference m o d u l e s . S e d i m e n t p o p u l a t i o n was essentially c o m p o s e d o f n e m a t o d e s , h a r p a c t i c o i d c o p e p o d s a n d polychaetes. T h e a d d i t i o n o f o r g a n i c lead, however, resulted in t o t a l s e d i m e n t inertia for 16 m o n t h s . A f t e r this p e r i o d , lead b e g a n to be e l i m i n a t e d f r o m the m o d u l e s a n d a process similar to t h a t o c c u r r i n g with m i n e r a l l e a d was o b s e r v e d ( p o p u l a t i o n c o m p o s e d o f n e m a t o d e s after 16 m o n t h s , h a r p a c t i c o i d c o p e p o d s a n d p o l y c h a e t e s a p p e a r e d after 18 m o n t h s ) .
CONCLUSION This s t u d y r e p o r t s the w o r k d o n e b y v a r i o u s a u t h o r s on lead h o m e o s t a s i s a n d d e t o x i f i c a t i o n in m a r i n e organisms. S o m e sublethal effects a n d i n t r a c e l l u l a r processes o f toxicity are also given. W h i l e the a u t h o r s a d m i t t h a t the overall risks to m a r i n e o r g a n i s m s due to c o n t a m i n a t i o n b y i n o r g a n i c l e a d are low, those d u e to o r g a n i c l e a d a p p e a r to be difficult to estimate as the o r g a n o - l e a d cycle in the m a r i n e e n v i r o n m e n t is as yet p o o r l y u n d e r s t o o d .
REFERENCES Amiard-Triquet, C., P. Lassus, J.C. Amiard, J. Devineau et C. Denuit, 1981. Influence des procrdures exprrimentales sur la drtermination de la toxicit6 aigu~ de quelques polluants h l'rgard de divers organismes marins et euryhalins. Les colloques de L'INSERM: les tests de toxicit6 aigu~ en milieu aquatique, 106, 485496. Arnoux, A., C. Diana, Th. Schembri, L. Favre, G. Stora, R. Elkaim, M. Galas, E. Vacelet, P. Vitiello et G. Bellan, 1988. Etude exprrimentale dans le milieu naturel, de s~diments artificiellement contaminrs par diff~rentes formes chimiques d'un m+tal (plomb): 6volution chimique et biologique du s~diment. Rapport PIREN-ATP Ecotoxicologie. Contrat No. 1294. Boutron, C. and C.C. Patterson, 1987. Relative levels of natural and industrial lead in recent Antartic snow. J. Geophys. Res. 92, 8454-8464. Boutron, C., 1988. Le plomb dans l'atmosph+re. La Recherche 19, 446-455. Caplun, E., D. Petit, et E. Piciotto, 1984. Le plomb dans l'essence. La Recherche, 15, 270-280. Chaisemartin, C., R.A. Chaisemartin et J.C. Breton, 1978. Aspect de la drtresse m~tabolique chez Macropodia: bioconcentration du plomb et activit6 de raspartate amino-transfrrase. C.R. Soc. Biol. 172, 11801187. Chan, H.M., 1988. Accumulation and tolerance to cadmium, copper, lead and zinc by the green mussel Perna viridis. Mar. Ecol. Prog. Ser. 48, 295-303. Chassard-Bouchaud, C., P. Galle and F. Escaig, 1985. Mise en 6vidence d'une contamination par l'argent et le plomb de l'Hu~tre Crassostrea gigas et de la Moule Mytilus edulis dans les eaux crti+res franqaises. Etude microanalytique par 6mission ionique secondaire. C.R. Acad. Sci. Paris 300, III, 1, 3-8. Coombs, T.L. and S.G. George, 1979 Mechanisms of immobilisation and detoxication of metals in marine
169 organisms. In: Physiology and behaviour of marine organisms. Proc. 12th Eur. Symp. Mar. Biol., edited by D.S. McLusky and A.J. Berry, Pergamon Press, Oxford, pp. 179 187. Gekeler, W., E. Grill, E.L. Winnacker and M.H. Zenk, 1988. Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch. Microbiol. 150, 197-202. George, S.G., 1982. Subcellular accumulation and detoxication of metals in aquatic animals. In: Physiological mechanisms of marine pollutant toxicity, edited by W.B. Vernberg, A. Calabrese, F.P. Thurberg and J.F. Vernberg. Academic Press, New York, pp. 3-52. Hollibaugh, J.T., D.L.R. Seibert and W.H. Thomas, 1980. A comparison of the acute toxicities of ten heavy metals to phytoplankton from Saanich Inlet, B.C., Canada. Estuarine Coastal Mar. Sci. 10, 93-105. Hopkin, S.P. and J.A. Nott, 1979. Some observations on concentrically structured, intracellular granules in the hepatopancreas of the shore crab Carcinus maenas (L.). J. Mar. Biol. Assoc. UK 59, 867-877. Maddock, B.G. and D. Taylor, 1980. The acute toxicity and bioaccumulation of some lead alkyl compounds in marine animals. In: Lead in the marine environment, edited by M. Branica and Z. Konrad. Pergamon Press, Oxford, pp. 345-361. Marshall, A.T. and U. Talbot, 1979. Accumulation of cadmium and lead in the gills of Mytilus edulis: X-ray microanalysis and chemical analysis. Chem.-Biol. Interact. 27, 111 123. Nott, J.A., 1991. Cytology of pollutant metals in marine invertebrates: a review of microanalytical applications. Scanning Microscopy 5, 1,191-205. Pacyna, J.M., 1984. Estimation of the atmospheric emissions of trace elements from anthropogenic sources in Europe. Atmos. Environ. 18, 41 50. Pacyna, J.M., J. Mtinch, J. Alcamo and S. Anderberg, 1991. Emission trends for heavy metals in Europe. In: Heavy Metals in the Environment, edited by J.G. Farmer. CEP Consultants, Edinburgh, Vol. I, pp. 314-317. Patterson, C.C. and D.M. Settle, 1987. Review of data on eolian fluxes of industrial and natural lead to the lands and seas in remote regions on a global scale. Mar. Chem. 22, 137-162. Pullen, J.S.H. and P.S. Rainbow, 1991. The composition of pyrophosphate heavy metal detoxification granules in barnacles. J. Exp. Mar. Biol. Ecol. 150, 249-266. Rainbow, P.S. and A.G. Scott, 1979. Two heavy metal-binding proteins in the midgut-gland of the crab Carcinus maenas. Mar. Biol. 55, 143 150. Regoli, F., 1992. Lysosomal responses as a sensitive stress index in biomonitoring heavy metal pollution. Mar. Ecol. Prog. Ser. 84, 63-69. Roesijadi, G., 1980/1981. The significance of low molecular weight, metallothionein-like proteins in marine invertebrates: current status. Mar. Environ. Res. 4, 167 179. Roesijadi, G., 1992. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22, 81-114. Schulz-Baldes, M., 1977. Lead transport in the common mussel Mytilus edulis. In: Physiology and behaviour of marine organisms edited by D.S. McLusky and A.J. Berry. Pergamon Press, Oxford, pp. 211-218. Siebers, D. and U. Ehlers, 1979. Heavy metal action on transintegumentary absorption of glycine in two annelid species. Mar. Biol. 50, 175-179. Simkiss, K., 1979. Metal ions in cells. Endeavour, New Series, 3, 1, 2-6. Somero, G.N., P.H. Yancey, T.J. Chow and C.B. Snyder, 1977. Lead effects on tissue and whole organism respiration of the estuarine teleost fish Gillichthys mirabilis. Arch. Environ. Contain. Toxicol. 6, 349-354. Suszkiw, J., G. Toth, M. Murawsky and G.P. Cooper, 1984. Effects of Pb 2÷ and Cd 2÷ on acetylcholine release and Ca 2÷ movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ. Brain Res. 323, 31-46. Talbot, V. and R.J. Magee, 1978. Naturally-occurring heavy metal binding proteins in invertebrates. Arch. Environ. Contam. Toxicol. 7, 73-81. Tan, W.H. and L.H. Lim, 1984. The tolerance to and uptake of lead in the green mussel, Perna viridis (L.). Aquaculture 42, 317-332.
170 Thomas, P. and M.J. Juedes, 1985. Altered glutathione status in Atlantic Croaker (Micropogonias undulatus) tissues exposed to lead. Mar. Environ. Res. 17, 192-195. Viarengo, A., 1989. Heavy metals in marine invertebrates: mechanisms of regulation and toxicity at the cellular level. Rev. Aquat. Sci. 1,295-317. Viarengo, A. and J.A. Nott, 1993. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp. Biochem. Physiol. C, in press. Wood, J.M., 1980. Lead in the marine environment. Some biochemical considerations. In: Lead in the Marine Environment, edited by M. Branica and Z. Konrad. Pergamon Press, Oxford, pp. 299-303.
163
AQTOX 00594
Minireview
Some aspects of lead ecotoxicology in the marine environment M. Gnassia-Barelli and M. Romeo I.N.S.E.R.M. U.303 "Mer et Santk", Facult~ de M~decine, Laboratoire de Toxicologie Marine, Nice, France
(Received 3 August 1992; revision received 1 March 1993; accepted 9 March 1993)
While lead emissionshave been reduced in Western Europe, they remain high in Eastern Europe and may enter the marine environment through rainfall and dry deposits. Thus, it appears necessary to understand the ecotoxicology of lead in the marine environment. Induction of metallothionein-like protein synthesis by lead in marine animals was not found in the literature, whereas the immobilization of lead in a chemically inert form might be a general detoxification process. Few results have been published on lead processes of intracellular toxicity in marine organisms. Unstable tetraethyl lead can be rapidly taken up by marine organisms probably through controlled diffusion processes. Once inside the organisms, this compound can be dealkylated into an ionized chemical species and react at the molecular level within the cell. Lead may affect some biochemical functions. A destabilization of the membranes of tysosomes has been reported in invertebrates contaminated by lead. Some effects on populations and ecosystems are reviewed. In controlled ecosystems, tetraethyl lead, although unstable, can persist in sedimentsplaced at a depth of 5 m and exert a toxic influenceon the fauna.
Key words: Lead; Intracellular toxicity; Detoxification; Lysosome; Metal-containing granule
INTRODUCTION As o p p o s e d to other trace elements whose presence is essential for p r o p e r b o d y f u n c t i o n i n g (iron, m a g n e s i u m , copper, zinc, etc.), lead is n o t involved in a n y biological m e c h a n i s m . Its toxicity to h u m a n beings, at high c o n c e n t r a t i o n s , is well-known. T h e s y m p t o m s o f acute i n t o x i c a t i o n associated with m i n i n g have been k n o w n since a n t i q u i t y ( C a p l u n et al., 1984). I n the m o s t severe cases, it c a n p r o v o k e peripheral a n d encephalic polyneuritis.
Correspondence to: M. Gnassia-Barelli, I.N.S.E.R.M.U.303 "Mer et Sant6", Facult6 de M6decine, Laboratoire de Toxicologie Marine, F-06107 Nice Cedex 2, France.
164 Based on measurements of lead in cores of ice and polar snow, Boutron (1988) indicated that lead contamination is worldwide and is primarily due to human activities. A low but significant concentration of lead is present in laboratory air (Boutron and Patterson, 1987; Patterson and Settle, 1987). Pacyna (1984) demonstrated that among the numerous trace elements from nonferrous metal production which are released into the air, lead is the one with the highest emission factor, i.e., its emission rate is equal to 6360 g per ton of metal produced. While lead emissions have been reduced in Western Europe due primarily to the utilization of unleaded gasoline, they remain high in Eastern Europe, according to Pacyna et al. (1991). These authors evaluated the emission rate into the atmosphere at 89.5 tons of Pb/year in Europe in 1982 and predicted a rate of 30.8 to 52.1 t of Pb/year by the year 2000. It must be emphasized that lead emissions may enter the oceans through both rainfall and dry deposits. Lead is ranked on the grey list (annex II) of pollutants by the Oslo and Barcelona Convention and the Barcelona protocol prohibits dumping toxic substances into the marine environment. The US EPA (Environmental Protection Agency) has no criteria for seawater quality in terms of lead content. Thus, it appears necessary to understand the ecotoxicology of lead in the marine environment. This article is a brief review of work published over the past few years on the mechanisms of sequestration, of possible detoxification and of cellular toxicity of lead. Some effects of lead on populations and ecosystems are also given. Data on in situ or experimental lead accumulation by different marine organisms is not included. Marine organisms from metal contaminated environments are capable of accumulating very high levels of metals in their tissues with no obvious biological effects. This tolerance may be due, in part, to homeostasis and detoxification mechanisms. Under conditions of high metal uptake when the sequestration capacity of the detoxification systems is exceeded, toxicity will occur. HOMEOSTASISAND DETOXIFICATIONMECHANISMS Three processes of heavy metal cation homeostasis have been identified in marine invertebrates cells (Viarengo and Nott, 1993): (1) binding to specific soluble ligands, the most important of which are metallothioneins; (2) compartmentalization within membrane-limited vesicles mostly recognised as lysosomes; (3) formation of insoluble precipitates such as Ca/Mg concretions or Ca/S granules. These processes are schematized in Fig. 1. Numerous studies refer to the role of specific proteins that are implicated in detoxification mechanisms. Animals respond to heavy metal stress by induction of metallothionein gene expression, while plants respond by phytochelatin formation through polymerization of peptide precursors (Gekeler et al., 1988). Gekeler et al. (1988) reported that the unicellular algae Scenedesmus and Chlorella increase production of phytochelatins when exposed to the toxic metal ions Cd 2+,
165 f
Granule
)--~r~k
/( [~ - - ~, ^
,~
~
~
~
Protein synthesis
" @Storage
Fig. 1. Metabolismof metal ions by cells (accordingto Simkiss, 1979).Within the cell, metal ions (M+)may be regarded as existing in a labile pool which may be involved in stimulating specificprotein synthesis (which is not a common process for Pb). The role of storage vesicles or inorganic granules is outlined together with the functions of lysosomesin recyclingmetal ions (particularlyPb).
Pb 2+, Zn 2÷, Ag +, Cu 2÷ and Hg 2+. However, it should be noted that these are both freshwater algae. Talbot and Magee (1978) looked for metal complexing proteins naturally present in the mussel Mytilus edulis living in a metal polluted bay and showed that these proteins bind cadmium, but not lead. Further, Rainbow and Scott (1979) found that lead present at a rate of 2 ¢tg.g -l (dry wt.) in the midgut gland of the crab Carcinus maenas sampled in a contaminated zone was not associated with these proteins. The mussel Perna viridis can tolerate and accumulate high lead concentrations (up to 4.46 mg.l-l; Tan and Lim, 1984; accumulation of 3.62 ¢tg Pb g-1 per day, Chan, 1988). According to these authors, the animal appears to possess mechanisms which inhibit the interaction of the toxic metal with essential enzymes. Most of the articles examine metallothioneins and/or phytochelatins chelating copper, zinc or cadmium; only a few focus on lead. Roesijadi (1980/81) gives no examples of this type of protein binding to lead in a review of metallothionein-like proteins in fifteen marine invertebrates. In a more recent review, Roesijadi (1992) gave no reference to metallothioneins binding lead. It is likely that lead homeostasis is realized by the two latter mechanisms identified by Viarengo and Nott (1993). Immobilization of metals by sequestration in lysosomes represents a major nonspecific mechanism for detoxification of many metals in animals exposed to excessively high levels. Lead, in colloidal or particulate form, is taken into invertebrate epithelial cells (particularly the gills and mantle) by endocytosis and after fusion of these endo-
166
cytic vesicles with lysosomes, this metal becomes immobilized (Coombs and George, 1979). Metal-containing granules represent a well-documented means by which cation concentration is regulated in the cells. Insoluble granules are present in the cells of animals from virtually every invertebrate phylum (Nott, 1991). Coombs and George (1979) found granules containing both iron and lead bound within membranes in the gill cells, digestive glands and mantle of contaminated Mytilus edulis, along with granules containing high concentrations of Zn, Fe and Pb in the liver cells. In the gills of Mytilus edulis, Pb is associated with calcium in an equimolar ratio in the form of extracellular crystalline deposits (Marshall and Talbot, 1979; George, 1982). Pullen and Rainbow (1991) reported that pyrophosphate granules isolated from the barnacle (crustacean) Elminius modestus can bind not only Zn, Fe, Ca and K, but also Cu, Mn, Pb, Ag and Cd under natural environmental conditions. The granules in the R cells of the hepatopancreas of Carcinus maenas, studied by X-ray microanalysis, always contain calcium and phosphorus together with magnesium and in some animals these granules contain lead (Hopkin and Nott, 1979). It is concluded that the capability of Carcinus rnaenas to detoxify lead by incorporating it in these granules may be directly related to the number of granules which are immobilizing calcium in the hepatopancreas. In Mytilus edulis, Schulz-Baldes (1977) demonstrated that lead is taken up at the gills and viscera, distributed by the blood and is finally stored as a phosphorus or sulfur-rich complex in membrane-bound vesicles (i.e., in intracellular storage sites) within the excretory cells of the kidney. He suggested that uptake into the cells occurs by pinocytosis. Chassard-Bouchaud et al. (1985) found lead in the oyster Crassostrea gigas and in the mussel Mytilus edulis collected from French coastal waters. Uptake and storage occurred in gills and digestive gland, while excretion occurred in the kidney; macrophage-like haemocytes played an important role in these processes. INTRACELLULAR PROCESS OF TOXICITY
Wood (1980) underlined the instability of tetraethyl and tetramethyl lead compounds in the marine environment, which dealkylate to yield corresponding trialkyls and dialkyls. The latter are highly stable in seawater, probably due to their ionic nature, and do not bioaccumulate in the marine environment. Tetraethyls and, to a lesser degree, tetramethyls are non-polar compounds which can be rapidly taken up by marine organisms, probably through controlled diffusion processes. Once inside the organisms, these compounds can be dealkylated into an ionized chemical species and react at the molecular level within the cell. Authors (Amiard-Triquet et al., 1981; Maddock and Taylor, 1980) generally recognized that the acute toxicity of organic lead is much higher than that of inorganic lead. Some examples of intracellular processes of lead toxicity are given below. Somero et al. (1977) found that when the fish Gillichthys mirabilis was exposed to
167 2650/.tg'1-1 of Pb, oxygen consumption of the whole organism was higher than in controls. However, respiration rate of gills in vitro was the same in both contaminated and uncontaminated fish. The metabolic changes caused by Pb would appear to be due more to its effect on the central nervous system than to a direct effect on the enzymatic metabolism of each cell. Chaisemartin et al. (1978) noted a decrease in the respiratory metabolism of the crab Macropodia rostrata when exposed to 15 and 100 /tg. 1-1 of Pb. They attributed this decrease to an inhibition of the energy consuming mechanisms. A few studies have been made on the effect of lead on biochemical functions. The high glycine absorption of the oligochaetous annelid Enchytraeus albidus is not affected by the presence of 10 mg. 1-l of lead in the environment, although it is reduced by the presence of mercury, cadmium or copper at much lower concentrations (Siebers and Ehlers, 1979). According to Thomas and Juedes (1985), lead increases the glutathione level in the intestines and liver of the fish Micropogonias undulatus, but not in the kidneys or brain tissues. These authors suggest that the interactions between glutathione and lead are indirect and imply a stimulating effect of the metal on the activity of enzymes leading to the synthesis of glutathione. Suszkiw et al. (1984) studied the effects of Pb 2÷ on acetylcholine release and on voltage dependent calcium channels in synaptic vesicles isolated from Torpedo fish electric organs and noted a blocking of Ca 2÷ entry through the calcium channels of synaptosomes and the release of acetylcholine linked to the entry of calcium through the channels. Heavy metals can interact with cell membranes and alter their function. Viarengo (1989) emphasized the destabilization of the membranes of lysosomes, the cellular organelles primarily devoted to macromolecular catabolism. Regoli (1992) studied the lysosomal responses in digestive cells of mussels Mytilus galloprovincialis, collected from a relatively clean area and a heavy metal (particularly Pb)-polluted one. Organisms exposed to high environmental levels of heavy metals showed a reduced lysosomal membrane stability and an enhanced production of lipofuscin. Lysosomal responses, as an early warning system for detection of environmental disturbances, could represent a useful tool for biomonitoring studies (Viarengo, 1989; Regoli, 1992; Viarengo and Nott, 1993). EFFECTS ON POPULATIONSAND ECOSYSTEMS Using a natural population of algae in enriched seawater sampled in a CEPEX ecosystem (Controlled Ecosystem Pollution Experiment, Saanich Bay, Western Canada), Hollibaugh et al. (1980) examined specific diversity as a function of lead concentration in the environment. Concentrations at which toxic effects were seen varied from 60 to 200/~g-1-1. Skeletonema costatum was always the dominant species, while Thalassiosira sp. was present, but not always abundant and Chaetoceros sp. was present except above 200/lg. 1-1 of lead.
168 A r n o u x et al. (1988) studied the b e h a v i o u r o f e x p e r i m e n t a l m o d u l e s c o n t a i n i n g sediments, p l a c e d in sea w a t e r at a d e p t h o f 5 m. These sediments, where f a u n a was r e m o v e d , were c o n t a m i n a t e d (1 g o f P b p e r kg) with either m i n e r a l o r t e t r a e t h y l lead. The r e c o l o n i z a t i o n o f biological c o m p a r t m e n t s (microflora, m e i o f a u n a , m a c r o f a u n a ) was s t u d i e d in the m o d u l e s over a 2-year cycle. T h e m o d u l e s c o n t a i n i n g m i n e r a l l e a d revealed a b e h a v i o u r similar to reference m o d u l e s . S e d i m e n t p o p u l a t i o n was essentially c o m p o s e d o f n e m a t o d e s , h a r p a c t i c o i d c o p e p o d s a n d polychaetes. T h e a d d i t i o n o f o r g a n i c lead, however, resulted in t o t a l s e d i m e n t inertia for 16 m o n t h s . A f t e r this p e r i o d , lead b e g a n to be e l i m i n a t e d f r o m the m o d u l e s a n d a process similar to t h a t o c c u r r i n g with m i n e r a l l e a d was o b s e r v e d ( p o p u l a t i o n c o m p o s e d o f n e m a t o d e s after 16 m o n t h s , h a r p a c t i c o i d c o p e p o d s a n d p o l y c h a e t e s a p p e a r e d after 18 m o n t h s ) .
CONCLUSION This s t u d y r e p o r t s the w o r k d o n e b y v a r i o u s a u t h o r s on lead h o m e o s t a s i s a n d d e t o x i f i c a t i o n in m a r i n e organisms. S o m e sublethal effects a n d i n t r a c e l l u l a r processes o f toxicity are also given. W h i l e the a u t h o r s a d m i t t h a t the overall risks to m a r i n e o r g a n i s m s due to c o n t a m i n a t i o n b y i n o r g a n i c l e a d are low, those d u e to o r g a n i c l e a d a p p e a r to be difficult to estimate as the o r g a n o - l e a d cycle in the m a r i n e e n v i r o n m e n t is as yet p o o r l y u n d e r s t o o d .
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