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CHOLINESTERASE ACTIVITY AS A BIOINDICATOR FOR MONITORING MARINE POLLUTION IN THE BALTIC SEA AND THE MEDITERRANEAN SEA
Biomarkers in Marine Organisms: A Practical Approach. Ph. Garrigues, H. Barth, C.H. Walker and J.F. Narbonne, editors. 9 2001 Elsevier Science B.V. All rights reserved.
CHAPTER 12
CHOLINESTERASE ACTIVITY AS A BIOINDICATOR FOR MONITORING MARINE POLLUTION IN THE BALTIC SEA AND THE MEDITERRANEAN SEA H. Dizer, H. C. da Silva de Assis, and P.-D. Hansen
Berlin University of Technology, Institute for Ecological Research and Technology, Department of Ecotoxicology, D-10589 Berlin, Germany
Abstract
The inhibition of the cholinesterase (ChE) activity as a biochemical response at the cellular level of exposed organisms has been monitored and evaluated by data from some estuaries and coastal areas of the Baltic and the Mediterranean Sea. The ChE activity was measured in the gills of mussels and in muscle tissue of several fish species. The investigated monitoring invertebrate organisms were bivalves with similar morphological and physiological properties Mytilus edulis from the Baltic Sea and Mytilus galloprovincialis from the Mediterranean Sea. Beside the bivalves the flatfish species dab (Limanda limanda) and flounder (Platichthys flesus) from the Baltic Sea and Serranus cabrilla, Mullus barbatus, and Boops boops from the Mediterranean Sea were investigated by field surveys in 1996 and 1997. Because of the lag of reference site in the investigated region, the results from different sites were only compared site by site. The data for sites on the transect of estuaries in the Baltic Sea showed clearly that ChE activities in the gills of M. edulis close to the harbour sites of Kiel and Wamemtinde were slightly less inhibited in comparison to the mussels collected from the sediment at the sea bottom and mussel beds outside of the harbour areas. The laboratory exposure of mussels from the piers of the harbour to the open sea sediments led to an increase of the ChE inhibition, significantly (p<0.05). The inhibition of ChE activity in the gills of M. edulis collected from the Baltic Sea was 1.6 times higher than in the gill tissues from
331
332
the mussel species M. galloprovincialis collected from the Mediterranean Sea (p<0.01). The ChE activity was quit changing from fish species to fish species. Keywords : ChE activity, mussel Mytilus galloprovincialis, flatfish Limanda limanda, flounder Platichthys flesus, red mullet Mullus Barbatus, comber Serranus cabrilla, Boops boops, Baltic Sea, Mediterranean Sea Abbreviation
9
ChE 9Cholinesterase
333
I. INTRODUCTION Environmental contaminants influence the physiological cell reactions at different and heterogeneous basics and lead to altering in normal cell function primarily at the molecular and biochemical level (Adams, 1990). As a membrane bounded enzymes in the cholinergic area of muscle and nerve cells the ChE is highly sensitive against some toxic agents (Bocquene et al., 1990; Payne et al., 1996). The ChE activity is a powerful parameter and rapid method to evaluate the neurotoxic impacts at cellular level of marine organisms (Holland and Coppage, 1967; Coppage and Mathews 1974; Zinkl et al., 1987). The inhibition of ChE activity has been proposed as an effect parameter to detect the effects of organophosphates and carbamates. However, in the last decades some environmental contaminants such as heavy metals and some anionic surfactants were detected with anti-cholinesterase effects (Galgani and Bocquene, 1990; Payne et al., 1996; Guilhermino et al., 1998). The physiological nature of ChE and techniques and application of the protocol for investigations with different organisms have been successfully developed. Now there is a protocol available for a well known and accepted effects related parameter for the implementation in monitoring programmes (Finlayson and Rudnicki, 1985; Bocquene et al., 1990; Galgani et al., 1992). River and surface waters polluted by the ,,run off' water from agricultural fields or industrial wastewater gives clear signals for inhibition of ChE activity. These effects increase by synergetic toxic impact of organophosphates and some heavy metals such as cadmium, lead and copper (Gill et al., 1991; Payne et., al, 1996; Silva de Assis and Hansen, 1997). Fish and invertebrates living in the marine environment can be exposed to insecticide levels which ranges from acute lethal to sublethal concentrations (Day and Scott, 1990, Sturm et al. 1997). The ChE activity as a rapid biochemical response in gills of mussels and in muscle tissue of fishes gives clear signals of the environmental quality concerning neurotoxicity in organisms of the Baltic and the Mediterranean Sea.
2. MATERIALS AND METHODS The test organisms were collected during field surveys in the Baltic Sea (November 1996, and July 1997) and the Mediterranean Sea (August 1996). In the Baltic Sea, samples were taken on sampling transects from three estuaries Kiel, WamemOnde, and Peenemtinde. Four sampling sites from the harbour area of Peenemthade and Wamemiinde to the open sea constituted the gradients P1-P4 (P=PeenemOnde) and
334
W1-W4 (W=Wamemiande), respectively. The Kiel transect was formed by 5 sampling sites from the harbour towards the open sea (K1-K5). The locations of sampling sites in the Baltic Sea and the Mediterranean Sea were represented in the previous article of the same authors about genotoxicity. Sampling on the Mediterranean Sea took place on the French and Spain coastal area at the following sites" Marcella (A2), Carteau (B), Fort Brescou (C), Harbour of Leucate (D), Harbour of Vendres-inside (El), Chalut (E2), Harbour of Vendresoutside (E3), Cala Manjoy (F1), Bay of Rosas (F2), Cailela (G), Harbour of Barcelona-inside (HI), Harbour of Barcelona-outside (H2), Cap Salou (I), Cap Roig (J1), AtmeUa (J2), Bay of Santa Ponsa-inside, Majorca (K1), Bay of Santa Ponsaoutside (K2), Bay of Palma, Majorca (K3), Harbour of Soler, Majorca (L), and Bay of Pollensa, Majorca (M2). For the Baltic Sea, ChE activity was measured in the mussel species Mytilus edulis and in the flatfishes the dab Limanda limanda and the flounder Platichthys flesus. The sampled species from the Mediterranean Sea were the bivalve Mytilus galloprovincialis and the fishe species Serranus cabrilla, Mullus barbatus, and Boops boops. In average 10-20 individuals of the mussel and fish species were collected at each station. Mussels not attached to the sediment of the sea ground were only collected from the piers and rocks inshore harbour sites of Kiel, Wamemtinde and Peenemtinde (K1, W1, and P1) of the Baltic Sea. At other sampling sites mussels were taken by dredging from the sea bottom. Exposure experiments were done as well in the laboratory with mussels from the harbour sites (K1 and W1) and sediments taken from offshore site of Wamemtinde transect (W4). The laboratory exposure experiments were investigated in 5 l-glass containers with a layer of 2 kg sediment and 3 L seawater and aerated continuously for 48 h at 18 _+2 ~
Cholinesterase activity: Gills (0.2-0.3 g) of mussel and muscle (0.2 g) of fish were homogenised in phosphate buffer (0.1 M, pH 7.4) on board. After centrifugation of the homogenate at 10,000 x g for 10 min, the supematant was frozen and stored at 20~ until analysis in the laboratory. ChE activity was followed on a microtiterplate by measuring the colorimetric reaction between the hydrolysed acetylcholine analogue and the reagent dithiobisnitrobenzoate (Sigma, D-8130) (Ellman et al., 1961; Galgani et al., 1992). The reaction was monitored by absorption at 414 nm using the photometer for microtiter plate (Fa: Dynetec, SLT ATTC 340) at room temperature. Protein concentration in the extract was determined by the method of Bradford using IgG (Sigma, G-5009) as standard.
335
3. RESULTS
In gills o f mussels: The ChE activity in gills of M. edulis from sampling sites of the Baltic Sea was represented in figure 1. The average ChE activity of all measured samples ranged from 3.7 to 13.8 nmol" m i n l ' m g l protein. A relatively low ChE activity of 5.9 + 2.5 nmol'min~.mgl protein was measured in mussel samples collected from the sediment of sea bottom at the site K 2. Mussels from the sites W2 and W3 showed also a low ChE activity with mean values 5.6 + 1.3 and 8.2 + 2.9 nmol.minl.mg 1 protein, respectively. The ChE activity was less inhibited in mussel samples from the harbour sites ofKiel (K1) with mean activities of 7.1 + 2.7 and Wamemtinde (W1) 7 + 2.4 nmol.minl.mg 1 protein. These mussels were collected in November 1996 from the piers and stones of the harbour areas and there was no contact to the sediments (figure 1).
161 laboratory exposure to sediments
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Figure 1 " Cholinesterase activity (ChE) in the gills of the mussel M. edulis from estuaries of the Baltic Sea (K = Kiel, W = Warnemiinde, P = Peenemtinde) as well as laboratory experiments with sediments from the gradient site W4 (open Sea). After the laboratory exposure experiments with mussels sampled from the harbours of Wamemiinde (W 1) and exposed to the offshore sediment from the sampling site W4 in the Baltic Sea. The ChE activity decreased significantly from 7.2 + 3.9 to
336 6.1 +_ 1.7 nmol.minl.mg 1 protein (p<0.05). The ChE activity was moderately inhibited in mussels from the harbour of Kiel (K1) after an exposure to the sediment sample from the Wamemtinde site W4 (figure 1). In the Mediterranean Sea, mussels (M. galloprovincialis) were collected only at 6 sampling sites (figure 2). ChE-activity in gills of M. galloprovincialis ranged from 7.3 _+ 3.4 to 16.8 _+ 2.2 nmol.min'~.mg l protein. The lowest ChE activity was detected in gills of mussels from the site Cap Roj (J1) with 7.3 +_ 3.4 nmol.min l. mg "1 protein. The enzyme activity was significantly high at the sites Marseilles (A2), Harbour of Venders (El, E3), and Ford Brescou (C) (p<0.05). The average ChE activity was amount of 6.6 _+ 2.5 nmol.minl.mg "1 protein in all samples of M. edulis (n-80) from the Baltic Sea, and 10.5 _+ 4.3 nmol.minl.mg "1 protein in the samples of M. galloprovincialis (n=60) from the Mediterranean Sea. ChE activity was 1.6 time higher in M.edulis than in M. galloprovincialis (p
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Figure 2 9 Cholinesterase activity in gills of the mussel M. galloprovincialis fi'om French and Spanish coastal sites of the Mediterranean Sea in August, 1996 (A2: Marseille, C: Fort Brescou, E 1, E3: Inside and outside of the Harbor Vendres, H: Harbor Barcelona, J: Cap Roig)
337
Muscle tissue offishes: The flatfish L. limanda were sampled at sites K5 and W4 in November 1996. By the same survey the fish species P. flesus was caught at sites of P2, P3 and P4 on the Peenemtinde transect. In 1997, L. limanda was only sampled at sites P2 and P4 (figure 3). The mean ChE activity of L. limanda was about 68.6 _+ 22.8 nmol.min'l.mg "l protein at site K5 and 93.9 +_ 22.1 nmol.min~.mg ~ protein at site W4 (p<0.05). At the sites P2 and P4 (July 1997), ChE activity of L. Limanda were found 56.5 _+ 19.8 and 49.7 _+ 17.4 nmol.minl.mg 4 protein, respectively. 150 P. flesus
L. limanda
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Figure 3 9 Cholinesterase activity in muscle tissue of flatfish species caught on tranect stations (gradients) ofharbour sites (Baltic Sea: K = Kiel, W = Wamemfinde, P = Peenemfinde). In muscle tissue of P. flesus ChE activity in samples from sites P3 and P4 were relatively low (59.1+ 25.8 and 61.2 _+ 22.5 nmol.minl.mg 1 protein) compared to enzyme activity in samples from site P2 with a ChE activity of 102.3 _+ 20.6 nmol.minl.mg -~ protein (p<0.01). Flounder from site K5 caught in July 1997 showed also a low enzyme activity with a mean value of 24.9 + 10.6 nmol.minl.mg" 1 protein.
338
Results of ChE activity in muscle tissue of S. cabrilla, M. barbatus, and B. boops from the sampling sites of the Mediterranean Sea were represented in figure 4. The lowest ChE-activity was measured in muscle tissue of S. cabrilla from sites L (Soler, Majorca) and M (Pollensa, Majorca) with a mean value of 39.8 +_ 9.9 and 37.3 +_ 10.6 n m o l . m i n l . m g "1 protein, respectively. The ChE activity was also clearly inhibited in fish samples from sites D (Port Leucata) and K3 (Palma, Majorca). There was a significant difference between the three fish species from the station K3. The lowest ChE activity was determined in M. barbatus (21.2 _+2.3 nmol.min "1. mg 1 protein). ChE activity of M. barbatus was always lower than the activity of S. cabrilla. The highest ChE activity was found in B. boops collected at the stations L and K3 (figure 4). The mean ChE activity was determined 76.7 +_ 39.3 nmol.minl.mg "~ protein (n=75) in all muscle samples of L. limanda from the Baltic Sea and 70.8 _+ 24.8 nmol.min l.mgl protein (n=155) by fish species from the Mediterranean Sea. 150 n=5-10 S. cabrilla
M. barbatus
B. hoops
"~~ 100
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Figure 4 9 Cholinesterase activity in muscle tissues of fish species of the Mediterranean Sea in August, 1996 (MarseiUe: A2, Fort Brescou: C, Harbour of Leucate: D, Harbour of Vendresinside El, Cala Manjoy: F1, Bay of Rosas (F2), Callela (G), AtmeUa (J2), Bay of Santa Ponsaoutside (K2), Bay of Palma, Majorca (K3), Harbour of Sollen, Majorca (L), and Bay of Pollensa, Majorca (M2).
339
4. DISCUSSION
The ChE activity was formed into a protocol for monitoring and screening as a rapid cost effective acute neurotoxicity test method. The results of this study shows clearly that only certain tissues of the individual species should be used and compared in their ChE activity. The effect related parameter ChE is useful tool to predict the effects and toxic impact of marine pollution. Three fish species from the same station (K3) in the Mediterranean Sea showed different ChE activities. The ChE activity in the muscle tissue of S. cabrilla and B. hoops was 1.8 or 2.1 times higher than those of M. barbatus, respectively. Burgeot et al., (1996) detected also a high ChE activity in muscle of S. cabrilla at 4 different sites of the coastal area of France that was about approximately 2 times higher than ChE activity of M. barbatus. There was significant difference between ChE activity of Serranus cabrilla and Serranus hepatus from the same coastal stations of the Mediterranean Sea (Burgeot et al., 1996). Bocquene et al. (1990) reported on ChE activities of 8 marine fish species that distinguished either in all species or different tissues of the same organisms. The different physiological characteristics of ChE activity in the fish species does not allow comparison between fish species from the Baltic and the Mediterranean Sea. Because of similar morphology and physiology of the mussel species M. edulis from the Baltic Sea and M. galloprovincialis from the Mediterranean Sea, it was possible to compare the results of ChE activity in both organisms. A C h E activity was detected about 1.6 times lower in M. edulis than in M.galloprovincialis (p<0.01). Thus significantly low CHE activity is related to the high pollution of the Baltic Sea in comparison to the Mediterranean Sea (Baumard, 1997). For lack of a significant unpolluted sampling area as a reference site for our investigations, the data for ChE activity could only be compared to each other. Generally it has been accepted that a 20 % reduction in ChE activity in fish and invertebrates indicates an exposure to neurotoxic compounds. Depression in ChE activity more than 20 % up to 50 % indicates sublethal impact of organophosphates (Zink et al., 1987; Busby et al., 1989). Comparing samples collected on the transect from different estuaries in the Baltic Sea, no significantly differences were measured between ChE activities in muscle tissue of fish. An exception was the high difference of ChE activity reduction (> 30%) detected in samples from site P4 compared with sites P3 and P4. A high inhibition of ChE activity with more than 50 % was observed in samples of the flatfish L. limanda collected at the estuary area of the river Rhine (Hansen, 1997).
340
In the Mediterranean Sea, fish samples of S. cabrilla from sites of Majorca (K3, L, and M) resulted also in a high inhibition of ChE activity compared to sampling sites A (Marseilles) and G (Callela). Burgeot et al. (1996) reported a low inhibition of ChE activity in areas remoter from agricultural fields and industrial plants and the highest inhibition of ChE in fish samples at sites of heavy industrial and domestic waste such as Genoa and Naples in Italy, Rio Ter in Spain, Barcelona and Cortiou in France. Generally, a lower ChE activity was observed in gills of mussels from the harbour sites compared with the mussels from outside of harbour areas. The mussels at the harbour sites were picked flom the piers. These mussels were not attached to the sediment on the sea ground and not exposed to toxic pollutants of the sediment. The exposure of mussels from the piers of the harbour to different sediments also showed a significantly decrease of ChE activity. Sediments from sampling estuaries in the Baltic Sea were highly polluted by domestic, agricultural and industrial wastewater and contained a large variety of toxic agems (Perkowska and Protasowicki, 1996; Baumard, 1997). Therefore, the high inhibition of ChE activity in mussels from the sea ground could be most probably caused by higher concentration of xenobiotics in sediment than in seawater. Compared to muscle tissues of fish, the ChE activity was rather low in gills of the mussels (M. edulis and M. galloprovincialis) and did not clearly show a significant difference between samples from estuaries and the open sea. The muscle tissue has usually the highest ChE activity among a variety of fish species (Bocquene et al., 1990). In general there is a correlation between the physical activity of tissue and levels of ChE activity (Metcalf et al, 1955). The skeletal muscle of L. limanda and S. cabrilla are more active than the gills in the mussels M. edulis and M. galloprovincialis. Because of the migration of fish from polluted to clean areas and vice versa, mussels can be considered as a powerful tissue for monitoring the impact of the marine environment and to predict the environmental health status.
REFERENCES
1. S.M. Adams. Status and use of biological indicators for evaluating the effects of stress on fish, American Fisheries Society Symposium 8 (1990) 1-8. 2. G. Bocquen6, F. Galgani and P. Truquet. Characterisation and assay conditions for use of AChE activity from several marine species in pollution monitoring. Marine Environ. Research, 30 (1990) 75-89.
341 3. J.F. Payne, A. Mathieu, W. Melvin and L.L. Fancey. Acethylcholin-esterase, an old biomarker with new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Marine Pollution Bulletin, 32 (1996) 225-231. 4. H.T. Holland, D.L. Coppage and P.A. Butler. Use of fish brain acethylcholinesterase in to monitor pollution by organophosphorus poeticised. Bull. Environ. Contam. and Toxicol., 2 (1967) 156-162. 5. D.L. Coppage and E. Matthews. Short-term effects of organophosphate pesticides on cholinesterase's of estuarine fishes and pink shrimp. Bull. Environ. Contam. and Toxicol., II, 5 (1974) 438-488. 6. J.G. Zinkl, P.J. Shea, R.J. Nakamoto and J. CaUman. Technical and biological considerations for the analysis of brain cholinesterase of rainbow trout. Trans. Am. Fish. Soc. 116 (1987) 570573 7. F. Galgani and G. Bocquen6. In vitro inhibition of acethylcholinesterase from four marine species by organophosphates and carbamates. Bull. Environ. Contam. Toxicol. 45 (1990) 243249. 8. L. Guilhermino, P. Barros, M.C. Silva and A. Soares. Should the use of inhibition of cholinesterase as a specific biomarker for organophosphate and carbamate pesticides be questinod? Biomarkers, p. 7 (in press 1998) 9. B.J. Finlayson and R.A. Rudnicki. Storage and handling as a source of error in measmfng fish acethylcholinesterase activity. Bull. Environ. Contam. Toxicol. 35 (1985) 790-795. 10. F. Galgani, G. Bocquen6 and Y. Cadiou. Evidence of variation in cholinesterase activity in fish along a pollution gradient in the North Sea. Mariene Ecology Progress Series, 19 (1992) 7782 11. T.S. Gill, H. Tewari and J. Pande. In vivo and in vitro of effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius (rosy barb). Comp. Biochem. Physiol. 100C (1991) 501-505. 12. H.C. Silva de Assis and P.D. Hansen. Evaluation of neurotoxic compounds and oxidative stress along the river Elbe. Presentation on the 7. Meeting of SETAC-Europe, p. 248, Amsterdam (1997). 13. K.E. Day and I.M. Scott. Use of acethylcholinesterase activity to detect sublethal toxicity in stream invertebrates exposed to low concentrations of organophosphate insecticides. Aquatic Toxicology, 18 (1990) 101-114. 14. A. Sturrn, J. Wogram, P.D. Hansen and M. Liess. Potential use of cholinesterase in monitoring low levels of organophosphates in small streams: natural variability in three-spinned stickleback (Gasterosteus aculeatus) and relation to pollution. (in press 1998). 15. G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone. A new rapid colorimetric determination of acethylcholinesterase activity. Biochem. Pharmacol. 7 (1961) 88-95. 16. T. Burgeot, G. Bocquen6, C. Porte, J. Dimet, R.M. Santella, L.M. Garcia, A. PhfolLeszkowicz, C. Raoux and F. Galgani. Bioindicators of pollutant exposure in the northwestern Mediterranean Sea. Marine Ecology Progress Series, 131 (1996) 125-141. 17. P. Baumard. Biog6ochimie des compos6s aromatiques dans l'environnement marin. Th6se n ~ 1788. Universit6 Bordeaux I, Bordeaux, France, 168 pp. (1997). 18. D.G. Busby, L.M. White, P.A. Pearce and P. Mineau. Fenitrothion effects on forest songbirds: a critical new look. In: Environmental effects of fenitrothion use in forestry, Ed.:Ernst, W.R. et al. Environment Canada Publication, noEN40-370/1989E, p. 43-108 (1989) 19. P.D. Hansen. Bioassays on sediment toxicity. In: Sediments and toxic substances, eds." W. Calmano and U. FSrstner, Springer, Berlin, N.York, (1997) p. 179-196.
342 20. R.L. Metcalf, R.B. March and M.G. Maxon. Substrate preferences of insect cholinesterase. Ann. Entomol. Soc. Am. 48 (1955) 222-228. 21. A. Perowska and M. Protasowicki. Heavy metal contents in sediment and the blue mussel Mytilus edulis from three regions of the southern Baltic Sea. Oceanological Studies 4 (1996) 5563.
CHAPTER 12
CHOLINESTERASE ACTIVITY AS A BIOINDICATOR FOR MONITORING MARINE POLLUTION IN THE BALTIC SEA AND THE MEDITERRANEAN SEA H. Dizer, H. C. da Silva de Assis, and P.-D. Hansen
Berlin University of Technology, Institute for Ecological Research and Technology, Department of Ecotoxicology, D-10589 Berlin, Germany
Abstract
The inhibition of the cholinesterase (ChE) activity as a biochemical response at the cellular level of exposed organisms has been monitored and evaluated by data from some estuaries and coastal areas of the Baltic and the Mediterranean Sea. The ChE activity was measured in the gills of mussels and in muscle tissue of several fish species. The investigated monitoring invertebrate organisms were bivalves with similar morphological and physiological properties Mytilus edulis from the Baltic Sea and Mytilus galloprovincialis from the Mediterranean Sea. Beside the bivalves the flatfish species dab (Limanda limanda) and flounder (Platichthys flesus) from the Baltic Sea and Serranus cabrilla, Mullus barbatus, and Boops boops from the Mediterranean Sea were investigated by field surveys in 1996 and 1997. Because of the lag of reference site in the investigated region, the results from different sites were only compared site by site. The data for sites on the transect of estuaries in the Baltic Sea showed clearly that ChE activities in the gills of M. edulis close to the harbour sites of Kiel and Wamemtinde were slightly less inhibited in comparison to the mussels collected from the sediment at the sea bottom and mussel beds outside of the harbour areas. The laboratory exposure of mussels from the piers of the harbour to the open sea sediments led to an increase of the ChE inhibition, significantly (p<0.05). The inhibition of ChE activity in the gills of M. edulis collected from the Baltic Sea was 1.6 times higher than in the gill tissues from
331
332
the mussel species M. galloprovincialis collected from the Mediterranean Sea (p<0.01). The ChE activity was quit changing from fish species to fish species. Keywords : ChE activity, mussel Mytilus galloprovincialis, flatfish Limanda limanda, flounder Platichthys flesus, red mullet Mullus Barbatus, comber Serranus cabrilla, Boops boops, Baltic Sea, Mediterranean Sea Abbreviation
9
ChE 9Cholinesterase
333
I. INTRODUCTION Environmental contaminants influence the physiological cell reactions at different and heterogeneous basics and lead to altering in normal cell function primarily at the molecular and biochemical level (Adams, 1990). As a membrane bounded enzymes in the cholinergic area of muscle and nerve cells the ChE is highly sensitive against some toxic agents (Bocquene et al., 1990; Payne et al., 1996). The ChE activity is a powerful parameter and rapid method to evaluate the neurotoxic impacts at cellular level of marine organisms (Holland and Coppage, 1967; Coppage and Mathews 1974; Zinkl et al., 1987). The inhibition of ChE activity has been proposed as an effect parameter to detect the effects of organophosphates and carbamates. However, in the last decades some environmental contaminants such as heavy metals and some anionic surfactants were detected with anti-cholinesterase effects (Galgani and Bocquene, 1990; Payne et al., 1996; Guilhermino et al., 1998). The physiological nature of ChE and techniques and application of the protocol for investigations with different organisms have been successfully developed. Now there is a protocol available for a well known and accepted effects related parameter for the implementation in monitoring programmes (Finlayson and Rudnicki, 1985; Bocquene et al., 1990; Galgani et al., 1992). River and surface waters polluted by the ,,run off' water from agricultural fields or industrial wastewater gives clear signals for inhibition of ChE activity. These effects increase by synergetic toxic impact of organophosphates and some heavy metals such as cadmium, lead and copper (Gill et al., 1991; Payne et., al, 1996; Silva de Assis and Hansen, 1997). Fish and invertebrates living in the marine environment can be exposed to insecticide levels which ranges from acute lethal to sublethal concentrations (Day and Scott, 1990, Sturm et al. 1997). The ChE activity as a rapid biochemical response in gills of mussels and in muscle tissue of fishes gives clear signals of the environmental quality concerning neurotoxicity in organisms of the Baltic and the Mediterranean Sea.
2. MATERIALS AND METHODS The test organisms were collected during field surveys in the Baltic Sea (November 1996, and July 1997) and the Mediterranean Sea (August 1996). In the Baltic Sea, samples were taken on sampling transects from three estuaries Kiel, WamemOnde, and Peenemtinde. Four sampling sites from the harbour area of Peenemthade and Wamemiinde to the open sea constituted the gradients P1-P4 (P=PeenemOnde) and
334
W1-W4 (W=Wamemiande), respectively. The Kiel transect was formed by 5 sampling sites from the harbour towards the open sea (K1-K5). The locations of sampling sites in the Baltic Sea and the Mediterranean Sea were represented in the previous article of the same authors about genotoxicity. Sampling on the Mediterranean Sea took place on the French and Spain coastal area at the following sites" Marcella (A2), Carteau (B), Fort Brescou (C), Harbour of Leucate (D), Harbour of Vendres-inside (El), Chalut (E2), Harbour of Vendresoutside (E3), Cala Manjoy (F1), Bay of Rosas (F2), Cailela (G), Harbour of Barcelona-inside (HI), Harbour of Barcelona-outside (H2), Cap Salou (I), Cap Roig (J1), AtmeUa (J2), Bay of Santa Ponsa-inside, Majorca (K1), Bay of Santa Ponsaoutside (K2), Bay of Palma, Majorca (K3), Harbour of Soler, Majorca (L), and Bay of Pollensa, Majorca (M2). For the Baltic Sea, ChE activity was measured in the mussel species Mytilus edulis and in the flatfishes the dab Limanda limanda and the flounder Platichthys flesus. The sampled species from the Mediterranean Sea were the bivalve Mytilus galloprovincialis and the fishe species Serranus cabrilla, Mullus barbatus, and Boops boops. In average 10-20 individuals of the mussel and fish species were collected at each station. Mussels not attached to the sediment of the sea ground were only collected from the piers and rocks inshore harbour sites of Kiel, Wamemtinde and Peenemtinde (K1, W1, and P1) of the Baltic Sea. At other sampling sites mussels were taken by dredging from the sea bottom. Exposure experiments were done as well in the laboratory with mussels from the harbour sites (K1 and W1) and sediments taken from offshore site of Wamemtinde transect (W4). The laboratory exposure experiments were investigated in 5 l-glass containers with a layer of 2 kg sediment and 3 L seawater and aerated continuously for 48 h at 18 _+2 ~
Cholinesterase activity: Gills (0.2-0.3 g) of mussel and muscle (0.2 g) of fish were homogenised in phosphate buffer (0.1 M, pH 7.4) on board. After centrifugation of the homogenate at 10,000 x g for 10 min, the supematant was frozen and stored at 20~ until analysis in the laboratory. ChE activity was followed on a microtiterplate by measuring the colorimetric reaction between the hydrolysed acetylcholine analogue and the reagent dithiobisnitrobenzoate (Sigma, D-8130) (Ellman et al., 1961; Galgani et al., 1992). The reaction was monitored by absorption at 414 nm using the photometer for microtiter plate (Fa: Dynetec, SLT ATTC 340) at room temperature. Protein concentration in the extract was determined by the method of Bradford using IgG (Sigma, G-5009) as standard.
335
3. RESULTS
In gills o f mussels: The ChE activity in gills of M. edulis from sampling sites of the Baltic Sea was represented in figure 1. The average ChE activity of all measured samples ranged from 3.7 to 13.8 nmol" m i n l ' m g l protein. A relatively low ChE activity of 5.9 + 2.5 nmol'min~.mgl protein was measured in mussel samples collected from the sediment of sea bottom at the site K 2. Mussels from the sites W2 and W3 showed also a low ChE activity with mean values 5.6 + 1.3 and 8.2 + 2.9 nmol.minl.mg 1 protein, respectively. The ChE activity was less inhibited in mussel samples from the harbour sites ofKiel (K1) with mean activities of 7.1 + 2.7 and Wamemtinde (W1) 7 + 2.4 nmol.minl.mg 1 protein. These mussels were collected in November 1996 from the piers and stones of the harbour areas and there was no contact to the sediments (figure 1).
161 laboratory exposure to sediments
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Figure 1 " Cholinesterase activity (ChE) in the gills of the mussel M. edulis from estuaries of the Baltic Sea (K = Kiel, W = Warnemiinde, P = Peenemtinde) as well as laboratory experiments with sediments from the gradient site W4 (open Sea). After the laboratory exposure experiments with mussels sampled from the harbours of Wamemiinde (W 1) and exposed to the offshore sediment from the sampling site W4 in the Baltic Sea. The ChE activity decreased significantly from 7.2 + 3.9 to
336 6.1 +_ 1.7 nmol.minl.mg 1 protein (p<0.05). The ChE activity was moderately inhibited in mussels from the harbour of Kiel (K1) after an exposure to the sediment sample from the Wamemtinde site W4 (figure 1). In the Mediterranean Sea, mussels (M. galloprovincialis) were collected only at 6 sampling sites (figure 2). ChE-activity in gills of M. galloprovincialis ranged from 7.3 _+ 3.4 to 16.8 _+ 2.2 nmol.min'~.mg l protein. The lowest ChE activity was detected in gills of mussels from the site Cap Roj (J1) with 7.3 +_ 3.4 nmol.min l. mg "1 protein. The enzyme activity was significantly high at the sites Marseilles (A2), Harbour of Venders (El, E3), and Ford Brescou (C) (p<0.05). The average ChE activity was amount of 6.6 _+ 2.5 nmol.minl.mg "1 protein in all samples of M. edulis (n-80) from the Baltic Sea, and 10.5 _+ 4.3 nmol.minl.mg "1 protein in the samples of M. galloprovincialis (n=60) from the Mediterranean Sea. ChE activity was 1.6 time higher in M.edulis than in M. galloprovincialis (p
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Figure 2 9 Cholinesterase activity in gills of the mussel M. galloprovincialis fi'om French and Spanish coastal sites of the Mediterranean Sea in August, 1996 (A2: Marseille, C: Fort Brescou, E 1, E3: Inside and outside of the Harbor Vendres, H: Harbor Barcelona, J: Cap Roig)
337
Muscle tissue offishes: The flatfish L. limanda were sampled at sites K5 and W4 in November 1996. By the same survey the fish species P. flesus was caught at sites of P2, P3 and P4 on the Peenemtinde transect. In 1997, L. limanda was only sampled at sites P2 and P4 (figure 3). The mean ChE activity of L. limanda was about 68.6 _+ 22.8 nmol.min'l.mg "l protein at site K5 and 93.9 +_ 22.1 nmol.min~.mg ~ protein at site W4 (p<0.05). At the sites P2 and P4 (July 1997), ChE activity of L. Limanda were found 56.5 _+ 19.8 and 49.7 _+ 17.4 nmol.minl.mg 4 protein, respectively. 150 P. flesus
L. limanda
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Figure 3 9 Cholinesterase activity in muscle tissue of flatfish species caught on tranect stations (gradients) ofharbour sites (Baltic Sea: K = Kiel, W = Wamemfinde, P = Peenemfinde). In muscle tissue of P. flesus ChE activity in samples from sites P3 and P4 were relatively low (59.1+ 25.8 and 61.2 _+ 22.5 nmol.minl.mg 1 protein) compared to enzyme activity in samples from site P2 with a ChE activity of 102.3 _+ 20.6 nmol.minl.mg -~ protein (p<0.01). Flounder from site K5 caught in July 1997 showed also a low enzyme activity with a mean value of 24.9 + 10.6 nmol.minl.mg" 1 protein.
338
Results of ChE activity in muscle tissue of S. cabrilla, M. barbatus, and B. boops from the sampling sites of the Mediterranean Sea were represented in figure 4. The lowest ChE-activity was measured in muscle tissue of S. cabrilla from sites L (Soler, Majorca) and M (Pollensa, Majorca) with a mean value of 39.8 +_ 9.9 and 37.3 +_ 10.6 n m o l . m i n l . m g "1 protein, respectively. The ChE activity was also clearly inhibited in fish samples from sites D (Port Leucata) and K3 (Palma, Majorca). There was a significant difference between the three fish species from the station K3. The lowest ChE activity was determined in M. barbatus (21.2 _+2.3 nmol.min "1. mg 1 protein). ChE activity of M. barbatus was always lower than the activity of S. cabrilla. The highest ChE activity was found in B. boops collected at the stations L and K3 (figure 4). The mean ChE activity was determined 76.7 +_ 39.3 nmol.minl.mg "~ protein (n=75) in all muscle samples of L. limanda from the Baltic Sea and 70.8 _+ 24.8 nmol.min l.mgl protein (n=155) by fish species from the Mediterranean Sea. 150 n=5-10 S. cabrilla
M. barbatus
B. hoops
"~~ 100
,
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so
0
1 i l I A2 C D E1 F1 G K3 L M2
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i K3 L
Figure 4 9 Cholinesterase activity in muscle tissues of fish species of the Mediterranean Sea in August, 1996 (MarseiUe: A2, Fort Brescou: C, Harbour of Leucate: D, Harbour of Vendresinside El, Cala Manjoy: F1, Bay of Rosas (F2), Callela (G), AtmeUa (J2), Bay of Santa Ponsaoutside (K2), Bay of Palma, Majorca (K3), Harbour of Sollen, Majorca (L), and Bay of Pollensa, Majorca (M2).
339
4. DISCUSSION
The ChE activity was formed into a protocol for monitoring and screening as a rapid cost effective acute neurotoxicity test method. The results of this study shows clearly that only certain tissues of the individual species should be used and compared in their ChE activity. The effect related parameter ChE is useful tool to predict the effects and toxic impact of marine pollution. Three fish species from the same station (K3) in the Mediterranean Sea showed different ChE activities. The ChE activity in the muscle tissue of S. cabrilla and B. hoops was 1.8 or 2.1 times higher than those of M. barbatus, respectively. Burgeot et al., (1996) detected also a high ChE activity in muscle of S. cabrilla at 4 different sites of the coastal area of France that was about approximately 2 times higher than ChE activity of M. barbatus. There was significant difference between ChE activity of Serranus cabrilla and Serranus hepatus from the same coastal stations of the Mediterranean Sea (Burgeot et al., 1996). Bocquene et al. (1990) reported on ChE activities of 8 marine fish species that distinguished either in all species or different tissues of the same organisms. The different physiological characteristics of ChE activity in the fish species does not allow comparison between fish species from the Baltic and the Mediterranean Sea. Because of similar morphology and physiology of the mussel species M. edulis from the Baltic Sea and M. galloprovincialis from the Mediterranean Sea, it was possible to compare the results of ChE activity in both organisms. A C h E activity was detected about 1.6 times lower in M. edulis than in M.galloprovincialis (p<0.01). Thus significantly low CHE activity is related to the high pollution of the Baltic Sea in comparison to the Mediterranean Sea (Baumard, 1997). For lack of a significant unpolluted sampling area as a reference site for our investigations, the data for ChE activity could only be compared to each other. Generally it has been accepted that a 20 % reduction in ChE activity in fish and invertebrates indicates an exposure to neurotoxic compounds. Depression in ChE activity more than 20 % up to 50 % indicates sublethal impact of organophosphates (Zink et al., 1987; Busby et al., 1989). Comparing samples collected on the transect from different estuaries in the Baltic Sea, no significantly differences were measured between ChE activities in muscle tissue of fish. An exception was the high difference of ChE activity reduction (> 30%) detected in samples from site P4 compared with sites P3 and P4. A high inhibition of ChE activity with more than 50 % was observed in samples of the flatfish L. limanda collected at the estuary area of the river Rhine (Hansen, 1997).
340
In the Mediterranean Sea, fish samples of S. cabrilla from sites of Majorca (K3, L, and M) resulted also in a high inhibition of ChE activity compared to sampling sites A (Marseilles) and G (Callela). Burgeot et al. (1996) reported a low inhibition of ChE activity in areas remoter from agricultural fields and industrial plants and the highest inhibition of ChE in fish samples at sites of heavy industrial and domestic waste such as Genoa and Naples in Italy, Rio Ter in Spain, Barcelona and Cortiou in France. Generally, a lower ChE activity was observed in gills of mussels from the harbour sites compared with the mussels from outside of harbour areas. The mussels at the harbour sites were picked flom the piers. These mussels were not attached to the sediment on the sea ground and not exposed to toxic pollutants of the sediment. The exposure of mussels from the piers of the harbour to different sediments also showed a significantly decrease of ChE activity. Sediments from sampling estuaries in the Baltic Sea were highly polluted by domestic, agricultural and industrial wastewater and contained a large variety of toxic agems (Perkowska and Protasowicki, 1996; Baumard, 1997). Therefore, the high inhibition of ChE activity in mussels from the sea ground could be most probably caused by higher concentration of xenobiotics in sediment than in seawater. Compared to muscle tissues of fish, the ChE activity was rather low in gills of the mussels (M. edulis and M. galloprovincialis) and did not clearly show a significant difference between samples from estuaries and the open sea. The muscle tissue has usually the highest ChE activity among a variety of fish species (Bocquene et al., 1990). In general there is a correlation between the physical activity of tissue and levels of ChE activity (Metcalf et al, 1955). The skeletal muscle of L. limanda and S. cabrilla are more active than the gills in the mussels M. edulis and M. galloprovincialis. Because of the migration of fish from polluted to clean areas and vice versa, mussels can be considered as a powerful tissue for monitoring the impact of the marine environment and to predict the environmental health status.
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341 3. J.F. Payne, A. Mathieu, W. Melvin and L.L. Fancey. Acethylcholin-esterase, an old biomarker with new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Marine Pollution Bulletin, 32 (1996) 225-231. 4. H.T. Holland, D.L. Coppage and P.A. Butler. Use of fish brain acethylcholinesterase in to monitor pollution by organophosphorus poeticised. Bull. Environ. Contam. and Toxicol., 2 (1967) 156-162. 5. D.L. Coppage and E. Matthews. Short-term effects of organophosphate pesticides on cholinesterase's of estuarine fishes and pink shrimp. Bull. Environ. Contam. and Toxicol., II, 5 (1974) 438-488. 6. J.G. Zinkl, P.J. Shea, R.J. Nakamoto and J. CaUman. Technical and biological considerations for the analysis of brain cholinesterase of rainbow trout. Trans. Am. Fish. Soc. 116 (1987) 570573 7. F. Galgani and G. Bocquen6. In vitro inhibition of acethylcholinesterase from four marine species by organophosphates and carbamates. Bull. Environ. Contam. Toxicol. 45 (1990) 243249. 8. L. Guilhermino, P. Barros, M.C. Silva and A. Soares. Should the use of inhibition of cholinesterase as a specific biomarker for organophosphate and carbamate pesticides be questinod? Biomarkers, p. 7 (in press 1998) 9. B.J. Finlayson and R.A. Rudnicki. Storage and handling as a source of error in measmfng fish acethylcholinesterase activity. Bull. Environ. Contam. Toxicol. 35 (1985) 790-795. 10. F. Galgani, G. Bocquen6 and Y. Cadiou. Evidence of variation in cholinesterase activity in fish along a pollution gradient in the North Sea. Mariene Ecology Progress Series, 19 (1992) 7782 11. T.S. Gill, H. Tewari and J. Pande. In vivo and in vitro of effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius (rosy barb). Comp. Biochem. Physiol. 100C (1991) 501-505. 12. H.C. Silva de Assis and P.D. Hansen. Evaluation of neurotoxic compounds and oxidative stress along the river Elbe. Presentation on the 7. Meeting of SETAC-Europe, p. 248, Amsterdam (1997). 13. K.E. Day and I.M. Scott. Use of acethylcholinesterase activity to detect sublethal toxicity in stream invertebrates exposed to low concentrations of organophosphate insecticides. Aquatic Toxicology, 18 (1990) 101-114. 14. A. Sturrn, J. Wogram, P.D. Hansen and M. Liess. Potential use of cholinesterase in monitoring low levels of organophosphates in small streams: natural variability in three-spinned stickleback (Gasterosteus aculeatus) and relation to pollution. (in press 1998). 15. G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone. A new rapid colorimetric determination of acethylcholinesterase activity. Biochem. Pharmacol. 7 (1961) 88-95. 16. T. Burgeot, G. Bocquen6, C. Porte, J. Dimet, R.M. Santella, L.M. Garcia, A. PhfolLeszkowicz, C. Raoux and F. Galgani. Bioindicators of pollutant exposure in the northwestern Mediterranean Sea. Marine Ecology Progress Series, 131 (1996) 125-141. 17. P. Baumard. Biog6ochimie des compos6s aromatiques dans l'environnement marin. Th6se n ~ 1788. Universit6 Bordeaux I, Bordeaux, France, 168 pp. (1997). 18. D.G. Busby, L.M. White, P.A. Pearce and P. Mineau. Fenitrothion effects on forest songbirds: a critical new look. In: Environmental effects of fenitrothion use in forestry, Ed.:Ernst, W.R. et al. Environment Canada Publication, noEN40-370/1989E, p. 43-108 (1989) 19. P.D. Hansen. Bioassays on sediment toxicity. In: Sediments and toxic substances, eds." W. Calmano and U. FSrstner, Springer, Berlin, N.York, (1997) p. 179-196.
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