Acetylcholinesterase Levels in Marine Organisms ...

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monitoring limitations (distribution, availability, ecology); and to present original results relative to. AChE activity levels in several marine species of the.
Volume 26/Number 2/February 1993

Edited by D. J. H. Phillips The objective of BASELINE is to publish short communications on different aspects of pollution of the marine environment. Only those papers which clearly identify the quality of the data will be considered for publication. Contributors to Baseline should refer to 'Baseline--The New Format and Content' (Mar. Pollut. Bull. 24, 124). M~rine Pollution Bulletin, Volume 26, No. 2, pp. 101 106, 1993. Printed in Great Britain.

0025-326X/93 $6.00+0.00 © 1993 Pergamon Press Ltd

Acetylcholinesterase Levels in Marine Organisms along French Coasts GILLES BOCQUENI~, FRANCOIS GALGANI, THIERRY BURGEOT, LOIC LE DEAN and PHILLIPE TRUQUET

IFREMER, Del/Ecotoxicologie, B.P. 1049, 44037Nantes Cedex, France The preservation of the quality of the marine environment has become a priority on a European scale. In France, a National Observation Network for the Marine Environment was created in 1972 to ensure the monitoring of metallic and organic pollutant concentrations in water, living matter and sediment. Since the 1980s, at the initiative of various study groups within the International Council for the Exploration of the Sea (ICES) and the UNESCO Intergovernmental Oceanographic Commission (IOC), a need has been recognized for new techniques to monitor the biological effects of contaminants. Several international workshops have been held (Charlottenhund, Denmark, 1978; Oslo, Norway, 1986; Bremerhaven, Germany, 1990) which have advocated the development of biochemical and biological methods for pollutant monitoring programmes (ICES/IOC, 1978, 1990; UNESCO, 1986). Among the biochemical tools studied, acetylcholinesterase (ACHE; EC 3.1.1.7) and its inhibition by organophosphate and carbamate insecticides have been proposed and used to measure the impact of these contaminants on the aquatic environment (Weiss & Gakstatter, 1964; Weiss, 1965; Williams & Sova, 1966; Holland et al., 1967; Coppage & Matthews, 1974; Coppage & Braidech, 1976; Zinckl et al., 1987; Day & Scott, 1990). A few works have been published concerning the characterization of AChE activity in various marine species (Coppage, 1971; Gelman & Herzberg, 1979; Bocquen8 et al., 1990). Information concerning the preparation, storage and handling of samples is available (Gibson et al., 1969; Finlayson & Rudnicki, 1985; Bocquen6 et at., 1990). Finally, measurements along a pollution gradient in the North

Sea have revealed great variations in AChE activity levels in the dab, Limanda limanda (Galgani et al., 1992). The purpose of the present work was twofold: to distinguish among the various species captured those which are the most suitable in the context of the monitoring limitations (distribution, availability, ecology); and to present original results relative to AChE activity levels in several marine species of the French Atlantic and Mediterranean coasts. These data can be considered as a baseline survey measured in precise geographical and temporal conditions, preliminary to the establishment of a monitoring network to determine the trends in organophosphate and carbamate effects on marine fauna. Fish samples were obtained during two scientific cruises along the French Atlantic coast (17 August and 5 September 1991) aboard the oceanograhic vessel N/O Gwen-Drez, and two along the Mediterranean coasts of mainland France and Corsica (21 and 30 October 1991) aboard the research vessel N/O Roselys II. Animals were captured by trawling at a depth of less than 20 m. In addition, measurements were performed on 49 samples of mussels (Mytilus edulis) from all areas of the French coasts provided by the National Observation Network in May 1990. The choice of fish species was closely dependent on the availability of a particular animal at the time of the cruises. The choice of stations reflected an effort to cover most French coastal areas. However, trawling was impossible in certain areas where the sea bottom is irregular (eastern Mediterranean). As the presence of dab (L. limanda) was limited to seven stations in the English Channel and that of sole (Solea solea) to four stations, data on these two species are relatively sparse. In the absence of these flatfish, we chose the dragonet (Callionymus lyra) which is present along all English Channel and Atlantic coasts (a total of 27 stations). Analyses were performed initially on the hepatic tissue of this species (all 27 stations) and subsequently on muscle tissue in fish from areas south of the Loire River estuary (! 1 stations). For the western Mediterranean (the Gulf of Lions), the red mullet (Mullus barbatus) present at 10 stations, was chosen as the most common species. However, data were also obtained for a flatfish, the leaf (Citharus macrolepidotus) (three stations). Around Corsica, measurements of AChE activity were performed on the comber (Serranus cabrilla) at nine stations and the gurnard (Trigloporus lastoviza) at 5 stations. In all cases, a few grams of tissue (muscle) were removed immediately from the living animal. When sea conditions allowed, the measurements were performed directly onboard; otherwise, the organs were preserved in liquid nitrogen. Measurements of AChE activity in mussels were performed on the adductor muscle. Extraction was 101

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performed on fresh muscle (1 g was sufficient) using TRIS buffer 0.1 M, pH 7.8 (Bocquen6 et al., 1990). Tissues were homogenized (1/4 w/v) and then centrifuged at 15 0 0 0 x g for 30 min. Crude supernatants were used as the enzyme source. The colorimetric method of Ellman et al. (1961) was applied to all samples. Activity was determined by measuring the hydrolytic product of acetylthiocholine with a crude tissue extract. Specific enzymatic activity was expressed after total protein assay of the extract by the method of Bradford (1976). All measurements were transposed to allow use of a microplate reader (Galgani & Bocquen6, 1988). All assays were completed in quadruplicate. Results are given as units/min/mg of protein, one unit of AChE activity being the variation of 0.001 optical density. Data are expressed as the mean values + S.D. obtained in 10 animals per station. The AChE activity levels measured in the different species are summarized in Figs 1-9 in the form of histograms associated with protein values. Dab (L. limanda) fished in the English Channel had scattered AChE activity values (Fig. 1). Three of the four stations in the eastern part of the Channel (6, 7 and 8) showed the highest activities (mean 4761 U_+290 for the three stations), whereas animals captured near the Channel Islands (station 10) had an appreciably lower value (2954 U_+440). This variability in dab data was also apparent for the sole (S. so&a) which exhibited values ranging from 1060 U ± l 1 8 in Douarnenez Bay (station 16) to 2225 U +_700 in Vilane Bay (station 22) as shown in Fig. 2. Callionymus lyra (Fig. 3) had more uniform activity levels when measurements were performed on muscle

I Protein

0 = 10 mg/ml

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extracts. The mean for 10 stations in the Bay of Biscay was 1152 U + 1 4 9 . Only station 28 showed higher activities (1944 U + 4 8 8 ) . The activity values for

Protein

~ = 10 mg/ml

AChE activity I = 2000 U.mg -1 P Fig. 2 AChE activity levels in muscle extracts from the sole (Solea solea) captured in the English Channel and Atlantic Ocean (August 1991).

Protein

~ = 10 mg/ml

AChE activity I = 2000 U.mg -I P

AChE activity I = 2000 U.mg -1 P Fig. 1 AChE activity levels in muscle extracts from the dab (Limanda limanda) captured in the English Channel (August 1991).

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Fig. 3 AChE activity levels in muscle extracts from the dragonet (Callionymus lyra) captured in the Atlantic Ocean (August 1991).

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hepatic extracts (Fig. 4) were quite scattered, ranging any of the 25 Mediterranean sites, where the red mullet from 336 U + 121 (station 26) to 919 U + 289 (station and comber had the widest distribution. Other species 22). The elevated standard deviation indicates a great were only present in limited geographical areas. Experts disparity between animals at the same station. are in agreement regarding the usefulness of benthic or The highest activities were found for S. cabrilla (Fig. demersal species in monitoring biochemical parameters 5). Moreover, the study of values measured for this (Lundin, 1962; Bayne, 1985; Giam & Ray, 1987). species showed a significant decrease in the AChE Dragonets, which are in permanent contact with the activity of animals captured off Fos sur Mer (7430 sediment, present numerous advantages over other U + 4 0 2 ) compared to the mean values of those species. These are sedentary territorial fish (Wilson, captured off Corsica (10 210 U + 769 for nine stations). 1978) representative of the milieu in which they are Measurements for the red mullet (M. barbatus see Fig. 6) gave lower mean values (4229 U + 2 2 2 for eight stations), although AChE activity was appreciably increased at stations 3 (Valras Beach) and 20 (South Corsica). Station 19, near station 20, also had higher values for the gurnard (T. lastoviza), whereas the mean value for this species in four stations was 2767 U + 47 (Fig. 7). The leaf (C. macrolepidotis), present only in three stations near the Fos sur Mer Gulf, had a mean activity of 3185 U + 87 (Fig. 8). AChE activities measured in the adductor muscle of the mussel (M. edulis) were considerably lower than those for vertebrates. The mean activity level for all 49 samples was 228 U + 58 (Fig. 9). Mussels from stations in the north of France (North English Channel and the Straits of Dover) showed the highest activities, whereas those from the coves, bays and estuaries of Brittany (Benoit Aber, Brest Harbour, Douarnenez Bay) as well as the Leucate lagoon along the western Mediterranean coast had significantly lower AChE activities. The choice of species suitable for use in a network monitoring the biological effects of pollutants depends on a variety of criteria, with availability being the most restrictive limitation. Thus, the dab, a fish recommended by the North Sea Task Force in the context of a Protein ~ = 10 mg/rnl [ biological monitoring programme in the North Sea, is AChE activity I = 2000 U.mg "1 P not only available in the eastern English Channel. Along the French Atlantic coasts, only the dragonet is Fig. 4 AChE activity levels in liver extracts from the dragonet present in 80% of stations (27/34 fishing stations). (Callionymus lyra) captured in the English Channel and Atlantic Ocean (August 1991). However, no member of this species was captured in

I Serranuscabrilla I

Fig. 5 AChE activity levels in muscle extracts from the comber (Serranus

cabrilla) captured in the Mediterranean Sea (October 1991).

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Mullus barbatus I

J AChE activity I = 2000 U.mg-1 P Protein ~ = 10 mg.m1-1

Fig. 6 AChE activity levels in muscle extracts from the red mullet (Mullus barbatus) captured in the Mediterranean Sea (October 1991).

Fig. 7 AChE activity levels in muscle extracts from the gurnard (Trigloporus lastoviza) captured in the Mediterranean Sea (October 1991).

l Citharusmacrolepidotus i

Fig. 8 AChE activity levels in muscle extracts from the leaf (Citharus macrolepidotus) captured in the Mediterranean Sea (October 1991 ). 104

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related to age, sex or reproduction period in most fishes (Galgani et al., 1992), the temperature of the environment and the assay have a determinant effect (Hogan, 1970; Bocquen~ et al., 1990). Constant attention should be paid to these two parameters. In the field, samples should be obtained synchronously within a time-scale of several days and in comparable climatic conditions. For this reason, only stations in the same geographical area should be compared. Many further studies are required concerning, in particular, the presence of inhibitory compounds (organophosphates, carbamates) in the marine environment. Data on these substances are currently almost non-existent, but the inhibition of AChE activity could rapidly become a reliable tool for monitoring pollutant effects. The authors thank Mr. Gray for translating the text, Miss Guesselin for secretarial assistance and Mr. Giboire for doing the graphs.

Acetylcholinesterase ~ = 1000 U/mg Protein (mean value * SD, P > .95) Protein [j = 5 rag/ml (mean value)

Fig. 9 AChE activity levels in adductor muscle extracts from the mussel (Mytilus edulis) obtained in the English Channel, Atlantic Ocean and Mediterranean Sea (May 1990).

captured. Abundant in estuary and coastal areas, this species is quite satisfactory for biological monitoring along the Atlantic and English Channel coasts. The red mullet, for the same reasons of abundance, sedentary habits and contact with sediment, would seem to be the best target species for monitoring the Mediterranean environment. All the organisms studied exhibited detectable AChE activity, but this activity was much higher in fish. Numerous studies have demonstrated the great sensitivity of fish and crustaceans to organophosphates and carbamates (Galgani & Bocquen6, 1990; Coppage & Matthews, 1974; Olson & Christensen, 1980). Mussel adductor muscles show low activities compared to fish, although the facility of collecting these molluscs and their wide geographical distribution along coasts are valuable aids to monitoring the marine environment (e.g. as in the French National Observation Network or American Mussel Watch). In general, the AChE activity of the different organisms studied was rather uniform when measured in muscle tissue. With the exception of the stations off Fos sur Mer and in certain Breton bays where activities were significantly lower, the mean values per species for all of the stations showed only slight variability within the same geographical area. Measurements in nine Corsican stations gave a variation coefficient of 7.53% for comber and 5.24% for red mullet (eight stations), whereas the coefficient was 12.9% for Atlantic dragonet (10 stations). Conversely, the data relative to hepatic extracts were quite scattered. In fact, if AChE is the major cholinesterase in fish muscle (Van der Wel & Welling, 1989), the liver is the site of many other non-specific cholinesterases (pseudocholinesterases) which hydrolyse acetylthiocholine nonpreferentially (Froede & Wilson, 1969). If the variability of AChE activity is not directly

Bayne, B. L. (1985). The effects of stress and pollution on marine animals. Praeger Scientific, New York. Bocquen6, G., Galgani, E & Truquet, P. (1990). Characterisation and assay conditions for use of AChE activity from several marine species in pollution monitoring. Mar. Environ. Res. 30, 75-89, Bradford, M. (1976). A rapid and sensitive assay of protein utilizing the principle of dye binding. Analyt. Biochem. 772,248-264. Coppage, D. L. (1971). Characterization of fish brain acetylcholinesterase with an automated pH for inhibition studies. Bull. Environ. Contam. Toxicol. 6, 304-310. Coppage, D. L. & Bradeich, J. (1976). River pollution by anticholinesterase agents. WaterRes. 10, 19-24. Coppage, D. L. & Matthews, E. (1974). Short-term effects of organophosphate insecticides on cholinesterases of estuarine fishes and pink shrimp. Bull. Environ. Contain. Toxicol. 11,438-488. Day, K. E. & Scott, 1. M. (1990). Use of acetylcholinesterase activity to detect sublethal toxicity in stream invertebrates exposed to low concentrations of organophosphate insecticides. Aquatic Toxicol. 18, 101-114. Ellman, G. L., Courtney, K. O., Andres, V. & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Finlayson, B. L. & Rudnicki, R. A. (1985). Storage and handling as a source of error in measuring fish acetylcholinesterase activity. Bull. Environ. Contam. Toxicol. 35,790-795. Froede, H. C. & Wilson, I. B. (1969). Acetylcholinesterase. In Enzymes (P. D. Boyer, ed.). Academic Press, New York. Galgani, E & Bocquen6, G. (1988). A method for routine detection of organophosphates and carbamates in sea water. Environ. Technolo~ Letters 10,311-322. Galgani, E & Bocquen~, G. (1990). In vitro inhibition of acetylcholinesterase from four marine species by organophosphates and carbamates. Bull. Environ. Contam. Toxicol. 45,243-249. Galgani, E, Bocquen6, G. & Cadiou, Y. (1992). Evidence of variation in cholinesterase activity in fish along a pollution gradient in the North Sea. Mar. Ecol. Prog. Ser. (in press). Gelman, A. & Herzberg, A. (1979). A field method to certify whether fish died fro poisoning by acetylcholinesterase inhibition. Bamidgeh

31(1). Giam, C. S. & Ray, L. E. (1987). Pollutant studies" in marine animals'. CRC Press Inc., Boca Ratom Florida. Gibson, J. R., Ludke, J. L. & Fergusson, D. E. (1969). Sources of error in the use of fish brain acetylcholinesterase activity as a monitor for pollution. Bull. Environ. Contam. Toxicol. 4, 17-23. Hogan, J. W. (1970). Water temperature as a source of variation in the specific activity of brain cholinesterase of Bluegills. Bull. Environ. Contam. and Toxicol. 5,347-354. Holland, H. T., Coppage, D. L. & Butler, P. A. (1967). Use offish brain acetylcholinesterase to monitor pollution by organophosphorous pesticides. Bull. Environ. Contain. Toxicol. 2, 156-162. ICES/IOC (1978). Cooperative research report No. 75. On the feasibility of effects monitoring. Charlottenhund, Denmark. ICES/IOC (1990). Workshop on the Biological Effects of Contaminants in the North Sea. Bremerhaven, Germany. ICES, Copenhagen. Lundin, S. J. (1962). Comparative studies of cholinesterases in body muscles of fishes. J. Cellular Cornp. PhysioL 59, 93-105.

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Marine Pollution Bulletin Olson, D. L. & Christensen, G. M. (1980). Effects of water pollutants and other chemicals on fish acetylcholinesterase inhibition. Bull. Environ. contain. Toxicol. 21,502-506. UNESCO (1986). A report on the IOC/GEEP workshop on the biological effects of pollutants held at t h e University of Oslo, Norway. Van Der Wel, H. & Welling, W. (1989). Inhibition of acetylcholinesterase in guppies (Poecilia reticulata) by chlorpyrifos at sublethal concentrations: methodological aspects. Exotoxicol. Environ. Safety 17,205-219. Weiss, C. M. (1965). Use of fish to detect organic insecticides in water.

Journal WPCF 37,647-658. Weiss, C. M. & Gakstatter, J. H. (19641. Detection of pesticides in water by biochemical assay. Journal WPCF 36, 240-252. Wilson, D. P. (1978). Territorial behaviour of male dragonets ( Callionymus lyra). J. Mar. Biol. Ass. UK 58,731-734. Williams, A. K. & Sova, R. C. (19661. Acetylcholinesterase levels in brains of fish from polluted waters. Bull. Environ. Contam. Toxicol. 1,198-204. Zinkl, J. G., Shea, P. J. Nakamoto, R. J. & Callman, J. (1987). Brain cholinesterase of rainbow trout poisoned by carbaryl. Bull Environ. Contam. Toxicol. 38, 29-35.

Marine Pollution Bulletin,

Bay. Both the port area and the central part of the inner Bay are dredged to improve navigation. Pollution by nutrients is a problem, and red tide occurrences are reported to have increased in frequency in recent years (Kocata~ et al., 1988). Surveys were carried out in 1989 and 1990 to measure the concentrations of selected trace elements in surface sediments of the Bay. Sediment samples were collected at 11 sampling stations using a Van-Veen grab (Fig. 1). All samples were stored in clean polyethylene bags. In the laboratory, samples were oven-dried at 40°C, ground in an agate mortar and wet-digested in teflon flasks using concentrated nitric acid. Blank digestions were also carried out. Trace metals from each subsample were extracted according to the method of Sinex et al. (1980). The acid extracts were filtered, made up to a fixed volume, and placed in acid-washed polyethylene sample bottles. The metal extracts were analysed by atomic absorption spectrophotometry using a Pye Unicam SP9 Model. Chromium, copper and lead were determined by direct aspiration using an air acetylene flame, while mercury was analysed by the cold vapour technique. The accuracy of the method was established using reference sediment material, from the International Atomic Energy Agency laboratory in Monaco, coded SD-N-1/2. The analysis of reference material gave the following results:

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Heavy Metals in Sediments from lzmir Bay, Turkey AHMET BALCI and MUHAMMET TORKOGLU Institute of Marine Sciences and Technology, Dokuz Eylu'l University, Iskele-Urla, Izmir, Turkey Izmir Bay is located in the eastern part of the Aegean Sea, lying between latitudes 38020 ' and 38042 ' and longitudes 26025 ' and 27010 '. The port of Izmir city and several industries are located in the Inner Bay. Untreated or partially treated industrial and domestic wastewaters are discharged directly or carried by several streams into the Bay. izmir, with a population of over 1.5 million, is one of the fastest growing cities in Turkey. The main sources of pollution in Izmir Bay include domestic and industrial wastes; rainfall and associated pollutants from run-off; shipping; and agricultural sources. From the topographic and hydrographic points of view, the Bay is divided into inner and outer regions. Its average depth is about 20-25 m. The inner Bay, which is shallower, reaches a maximum depth of about 20 m and exhibits a limited water exchange with the outer

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