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Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential early indicator of health status of the Mediterranean “whale sanctuary”
Marine Environmental Research 54 (2002) 761–767 www.elsevier.com/locate/marenvrev
Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential early indicator of health status of the Mediterranean ‘‘whale sanctuary’’ M.C. Fossia,c,*, J.F. Borsanib, R. Di Mentob, L. Marsilic, S. Casinic, G. Neric, G. Moric, S. Ancorac, C. Leonzioc, R. Minutolia, G. Notarbartolo di Sciarab a
Department of Animal Biology and Marine Ecology, University of Messina, Italy Istituto Centrale per la Ricerca Applicata al Mare, Via di Casalotti 300, 00166 Rome, Italy c Department of Environmental Science, University of Siena, Italy
b
Abstract The aim of this study was to propose a suite of biomarkers (BPMO activity, NADPHcytocrome c reductase, NADH-ferricyanide reductase, esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multi-disciplinary diagnostic tool for assessment of the health status of the Mediterranean ‘‘whale sanctuary’’. Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h. On the other hand larger differences between sites were found for reductase activities. Esterases (AChE), porphyrins (Copro-, Uro-, Proto-porphyrins) vitellogenin and zona radiata proteins were also detectable in this zooplanctonic species. Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w. Total PAHs ranged from 860.7 to 5037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w., HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Meganyctiphanes norvegica; Zooplancton; Biomarkers; Heavy metals; Organochlorines; PAHs
Zooplankton is an essential component of the marine food chain. The ecotoxicological risk of zooplanktonic communities, estimated by a multi-trial biomarker * Corresponding author. Tel.: +39-0577-232-913; fax: +39-0571-652-818. E-mail address: [email protected] (M.C. Fossi). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00148-4
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approach, can be used as an early warning signal of pelagic marine ecosystem ecotoxicological status (Fossi, Minutoli, & Guglielmo, 2001). The aim of this project was to propose a suite of biomarkers (BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase, esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multidisciplinary diagnostic tool for assessment of the health status of the Mediterranean ‘‘whale sanctuary’’, situated between the coasts of Provence, Monaco, Liguria, Tuscany, Corsica and Sardinia (Fig. 1 A). In November 1999, Environment Ministers from Italy, Monaco and France signed a treaty to establish a protected area in the Mediterranean Sea. This is the first time in the Northern Hemisphere that several countries have established a marine protected area which includes international waters. The area was chosen because it plays a significant role in providing rich biological diversity.
Fig. 1. (A) Sampling stations of Meganyctiphanes norvegica in the Mediterranean ‘‘whale sanctuary’’. (B) Samples of M. norvegica were collected, during the NATO Sound, Oceanography and Living Marine Resources (SOLMAR) 2000 Project, in five stations (in circle) in the Ligurian Sea in summer 2000, using an Isaacs-Kidd Midwater Trawl (IKMT) towed day and night from a depth of 200 m upwards. Numbers indicate planned profile stations. IKMT tows were made in order to obtain a coverage for the very same station at noon and at midnight with a 36 h temporal spacing. Stations were made at these times according to the predefined shiptrack without previous knowledge on the potential presence of krill. Therefore catches of krill (five circle stations) were totally incidental.
M.C. Fossi et al. / Marine Environmental Research 54 (2002) 761–767
763
The fin whale (Balaenoptera physalus) is a common cetacean species in this area and the euphausiid M. norvegica is its main food item. Although fin whale has long been known to frequent the Ligurian Sea, it was only recently realised that congregations of this whale in limited areas of the western sector in summer were correlated with blooms of the euphausiid M. norvegica (Relini, Tartaglia, Torchia, & Zanboni, 1994). A statistically significant relation was found between quantity of M. norvegica fished with a large IKMT net and sightings of B. physalus. M. norvegica is the most abundant euphausiid in the western Mediterranean, especially north of the 42nd parallel and is fundamental in the food chains, being the main constituent of Mediterranean krill. It has a varied diet, consisting principally of copepods at deeper levels by day and phytoplankton near the surface at night. The total number of specimens captured in the water column between 965 m and the surface is much greater at night than during the day. Samples of M. norvegica were collected, during the NATO Sound, Oceanography and Living Marine Resources (SOLMAR) 2000 Project, in five stations in the Ligurian Sea (Fig. 1B) in summer 2000, using an Isaacs-Kidd Midwater Trawl (IKMT) towed day and night from a depth of 200 m upwards. The main yield was obtained at night. The ultimate goal of the SOLMAR Lower Trophic Level Oceanographic Measurement Project is to examine the correlation between the distribution of whales in parts of the Mediterranean Sea and that of their prey, as well as that of lower trophic levels and basic oceanographic parameters. BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase, esterases (AChE), porphyrins (Uro-, Copro- and Proto-porphyrins) vitellogenin and zona radiata proteins, organochlorines, PAHs and heavy metals were evaluated in pooled samples in order to test the applicability of a multi-trial biomarker approach in this species as a potential early indicator of the ‘‘health status’’ of the Mediterranean ‘‘whale sanctuary’’. These biomarkers of contaminants exposure and effects have not been studied in this species. This information is of potential importance for environmental management and risk assessment. For the detection of MFO activities, the microsomal fraction was isolated by adding an aliquot of homogenisation buffer (pH 7.5) to a pool of whole organisms at 4 C and homogenising in a Potter blender. The homogenate was centrifuged at 9000g for 20 min and again at 10,0000g for 1 h. Before analysis, the microsomes were re-suspended in 2.6 ml re-suspension buffer per gram of tissue. MFO activity was evaluated in the microsomal fraction by assaying benzo[a]pyrene monooxygenase (BPMO) activities. BPMO activity was measured by the method of Kurelec, Britvic, Rijavec, Muller, and Zahn (1977), using 400 ml of microsomes as source of enzymes and incubating the reaction mixture for 1 h. Fluorimetric assay was carried out using a Perkin Elmer LS 50B luminescence spectrometer. NAD(P)H cytochrome c reductase, NADH cytochrome c reductase and NADH ferricyanide reductase were assayed by the method of Livingstone and Farrar (1984). All the tests were carried out at 30 C. Acetylcholinesterase (AChE) activities were determined in whole tissue homogenate, by the method of Westlake, Bunyan, Martin, Stanley, and Steel (1981).
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Porphyrin (Copro-, Uro-, Proto-porphyrins) concentrations were determined in pools of whole organisms; 0.2 ml of homogenate in water was spiked with 1.6 ml of 50:50 methanol/perchloric acid mixture. After vortex-mixing, the samples were kept in the dark for 10 min and then centrifuged for 5 min at a low speed. The porphyrin extract in the upper layer was used for spectrofluorimetric separation. Quantitative determination of porphyrins was performed by the method of Grandchamp, Deybach, Grelier, Deverneuil, and Nordman (1980). Immunochemical analysis of Vtg and Zrp was performed in homogenate of whole organisms by indirect ELISA (Goksoyr, 1991). A 96-well microplate was used for the test, each sample was tested in triplicate, adding 10 mg of protein to each well. Dilution of the primary antibodies was 1:1000 for anti-Vtg (PO-2) and 1:3000 for anti-Zrp (O-173). Results were expressed as adsorbance at 492 nm. Lyophilised organisms were digested with HNO3 in a teflon bomb (Stoeppler & Backhaus, 1978). Levels of metals were determined by atomic emission ICP/AES (Zn, Fe, Cu, Mn) and atomic absorption spectrometry with a transverse heated graphite furnace equipped with Zeeman background correction (Pb, Cd) and flow injection mercury system (Hg) techniques. The method of additions was used and the analytical procedure was checked with Certified Reference Material (Mussel Tissues CSM 278, Community Bureau of Reference) and blanks. Freeze-dried samples were extracted in a Soxlhet apparatus for analysis of chlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Extraction of OCs was carried out according to Marsili (2000) by high resolution capillary gas chromatography with a Perkin-Elmer Series 8700 GC and a 63Ni ECD, revealing op0 - and pp0 isomers of DDT and its derivatives DDD and DDE, and about 30 PCB congeners. PAHs were analysed by HPLC/fluorescence system. Extraction was carried out according to Houlobek et al. (1990) with some modifications (Marsili, 2000). M. norvegica is a zooplantonic species well studied in the Mediterranean sea for ecology and distribution (Relini et al., 1992). On the other hand very little information has been published on the biochemistry of this species (Mulkiewicz, Zietara, Stromberg, & Skarkowski, 2001; Salomon, Mayzaud, & Buchholz, 2000) and nothing exists on the ecotoxicological biomarker aspect. For this reason reference comparison can be done only with other zooplantonic species or other groups of crustacea. Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h (Table 1). The values are one order of magnitude lower than in the crustacea decapod species Carcinus aestuari. (Fossi et al. 2000). Larger differences between sites were found for reductase activities (Table 1), with the higher values found in station No. 19 (NAD(P)H cytochrome c reductase) and No. 34 (NADH ferricyanide reductase). Interestingly the highest values of total porphyrins are found in the same sampling station (No. 19, No. 34), the closest to the Ligurian coast (Italy). Esterase (AChE) activity values are in the same range of values found in the copepods species Acartia margalefi and Acartia latisetosa, but one order of magnitude lower than in the mysid species Siriella clausi (Fossi et al., 2001). Vitellogenin and zona radiata proteins were detectable in this zooplanctonic species (Table 1).
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Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w (Table 2). Total PAHs ranged from 860.7 to 5037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w. with the highest values detected again in the station N 34. HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w, hence again the highest value are detected in the station No. 34 one of the closest to the Ligurian coast (Table 2). Finally, it is interesting to explore the relationship between ‘‘prey’’ (M. norvegica) and ‘‘predator’’ (B. physalus) in the bioaccumulation of lipophilic contaminants (Marsili, 2000). The rate between contaminant levels in B. physalus and M. norvegica Table 1 Biomarker responses in pooled samples of Meganyctiphanes norvegica collected in the Mediterranean ‘‘whale sanctuary’’ Biomarker
Station No. 11
BPMO (A.F.U./mg prot/h) NADPH-cytochrome c reductase (nmol/mg prot/min) NADH-ferricyanide reductase (nmol/mg prot/min) AChE (mmol/g zoop/min) Uroporphyrin (pmol/g zoop) Coproporphyrin (pmol/g zoop) Protoporphyrin (pmol/g zoop) Total Porphyrins (pmol/g zoop) Vitellogenin (U.A.A./490 nm) Zona radiata proteins (U.A.A./490 nm)
Station No. 19
Station No. 26
Station No. 34
Station No. 43
Mean (S.D.)
0.50 3.69
2.89 4.69
3.20 3.41
2.08 2.44
0.47 4.23
1.83 (1.29) 3.69 (0.86)
0.81
1.20
3.40
8.76
2.96
3.43 (3.18)
7.60 29.0 88.0 133.0 251.0 0.115 0.069
7.60 19.0 163.0 199.0 382.0 0.124 0.066
9.25 18.0 92.5 126.0 238.0 0.127 0.080
5.40 29.0 116.0 139.0 284.0 0.108 0.054
7.85 7.5 135.0 123.5 266.5 0.133 0.150
7.54 (1.36) 20.5 (8.97) 118.8 (31.19) 144.1 (31.29) 284.3 (57.26) 0.12 (0.01) 0.08 (0.04)
Table 2 PAHs, OCs and heavy metals in pooled samples of Meganyctiphanes norvegica collected in the Mediterranean ‘‘whale sanctuary’’
Total PAHs (ng/g d.w.) Cancerogenic PAHs (ng/g d.w.) HCB (ng/g d.w.) DDTs (ng/g d.w.) PCBs (ng/g d.w.) Hg (mg/g f.w.) Cd (mg/g f.w.) Zn (mg/g f.w.) Mn (mg/g f.w.) Fe (mg/g f.w.) Cu (mg/g f.w.) Zn (mg/g f.w.)
Station No. 11
Station No.19
Station No. 26
Station No. 34
Station No. 43
3547 92.96
2030 141.7
1539 61.84
5038 81.82
963.8 63.61
3.890 163.2 195.9 – – – – – – –
3.500 35.29 99.64 – – – – – – –
6.700 50.53 136.6 0.030 0.031 25.68 0.600 35.95 13.90 0.158
11.56 74.10 210.2 – – – – – – –
4.760 41.02 84.60 0.033 0.023 17.04 0.540 12.52 11.43 0.065
Mean (S.D.) 2624 (1658) 88.39 (32.50) 6.082 (3.302) 72.83 (52.65) 145.4 (56.16) 0.032 (0.002) 0.027 (0.005) 21.36 (6.112) 0.570 (0.045) 24.23 (16.57) 12.67 (1.742) 0.111 (0.066)
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is 23.1 for PCBs and only 3.4 for total PAHs confirming the higher biomagnifications capacity of PCBs with respect to PAHs. In conclusion these preliminary results suggest the potential application of this multi-disciplinary ecotoxicological approach in M. norvegica as an early indicator of the health status of the Mediterranean ‘‘whale sanctuary’’ and particularly of the B. physalus population.
Acknowledgements We thank the personnel of ICRAM (Istituto Centrale per la Ricerca Scientifica e Tecnologica Associata al Mare) and the personnnel of ‘‘ITS Ammiraglio Magnaghi’’ (Italian Navy, Hydrographic Vessel) for technical assistance in the sampling activities.
References Fossi, M. C., Casini, S., Savelli, C., Franchi, E., Mattei, N., Sanchez-Hernandez, J. C., Corsi, I., Bamber, S., & Depledge, M. H. (2000). Biomarker responses at different levels of biological organization in crab (Carcinus aestuarii) experimentally exposed to benzo (a)pyrene. Chemosphere, 40, 861–874. Fossi, M. C., Minutoli, R., & Guglielmo, L. (2001). Preliminary results of biomarker responses in zooplankton of brackish environments. Marine Pollution Bulletin, 22(9), 745–748. Goksoyr, A. (1991). A semi-quantitative cytochrome P450IA1 ELISA: a simple method for studing the monooxygenase induction response in environmental monitoring and ecotoxicological testing of fish. Science of the Total Environment, 101, 255–262. Grandchamp, B., Deybach, J. C., Grelier, M., Deverneuil, H., & Nordmann, Y. (1980). Studies of porphyrin synthesis in fibroblasts of patients with congenital erythropoietic porphyria and one patient with homozygous coproporphyria. Biochemica Biophysica Acta, 629, 577–586. Mulkiewicz, E., Zietara, M. S., Stromberg, J. O., & Skorkowski, E. F. (2001). Lactate dehydrogenase from the northen krill Meganyctiphanes norvegica: comparison with LDH from the antartic krill Euphasia superba. Comparative Biochemisty & Physiology B—Biochemistry & Molecular Biology, 128(2), 233–245. Houlobek, I., Paasivirta, J., Maatela, P., Lahtipera¨, M., Holoubkova`, I., Korı´nek, P., Boha`cek, Z., & Ca`slavsky´, J. (1990). Comparison of extraction methods for polycyclic aromatic hydrocarbon determination in sediments. Toxicological and Environmental Chemistry, 25, 137–154. Kurelec, B., Britvic, S., Rijavec, M., Muller, W. E. G., & Zahn, R. K. (1977). Benzo(a)pyrene monooxygenase induction in marine fish- Molecular response to oil pollution. Marine Biology, 44, 211–216. Livingstone, D. R., & Farrar, S. (1984). Tissue and subcellular distribution of enzyme activity of mixedfunction and benzo(a)pyrene metabolism in the common mussel Mytilus edulis. Science of the Total Environment, 39, 209–235. Marsili, L. (2000). Lipophilic contaminants in marine mammals: review of the results of ten years work at the Department of Environmental Biology, Siena University (Italy). ‘‘The Control of Marine Pollution: Current Status and Future Trends’’ (special issue). Special Issue International Journal of the Environment and Pollution, 13, 416–452. Relini, M., Tartaglia, M. P., Torchia, G., & Zanboni, A. (1992). Macroplancton, M. norvegica and Balaenoptera physalus along some transects in the Ligurian Sea. European Research on Cetaceans, 6, 134–137. Salomon, M., Mayzaud, P., & Buchholz, F. (2000). Studies on metabolic properties in the Northen Krill, Meganyctiphanes norvegica (Crustacea, euphasiacea): influence of nutrition and season on pyruvate
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kinase. Comparative Biochemistry and Physiology A—Molecular & Integrative Physiology, 127(4), 505– 514. Stoeppler, M., & Backhaus, F. (1978). Pre-treatment studied with biological and environmental materials I. System of pressurised multisample decomposition. Fresenius Journal of Analytical Chemistry, 292, 116–200. Westlake, G. E., Bunyan, P. J., Martin, A. D., Stanley, P. L., & Steel, L. C. (1981). Organophosphorus poisoning: effects of selected organophosphorus pesticides on plasma enzymes and brain esterases of Japanese quail (Coturnix coturnix japonica). Journal of Agricultural and Food Chemistry, 29, 772–777.
Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential early indicator of health status of the Mediterranean ‘‘whale sanctuary’’ M.C. Fossia,c,*, J.F. Borsanib, R. Di Mentob, L. Marsilic, S. Casinic, G. Neric, G. Moric, S. Ancorac, C. Leonzioc, R. Minutolia, G. Notarbartolo di Sciarab a
Department of Animal Biology and Marine Ecology, University of Messina, Italy Istituto Centrale per la Ricerca Applicata al Mare, Via di Casalotti 300, 00166 Rome, Italy c Department of Environmental Science, University of Siena, Italy
b
Abstract The aim of this study was to propose a suite of biomarkers (BPMO activity, NADPHcytocrome c reductase, NADH-ferricyanide reductase, esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multi-disciplinary diagnostic tool for assessment of the health status of the Mediterranean ‘‘whale sanctuary’’. Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h. On the other hand larger differences between sites were found for reductase activities. Esterases (AChE), porphyrins (Copro-, Uro-, Proto-porphyrins) vitellogenin and zona radiata proteins were also detectable in this zooplanctonic species. Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w. Total PAHs ranged from 860.7 to 5037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w., HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Meganyctiphanes norvegica; Zooplancton; Biomarkers; Heavy metals; Organochlorines; PAHs
Zooplankton is an essential component of the marine food chain. The ecotoxicological risk of zooplanktonic communities, estimated by a multi-trial biomarker * Corresponding author. Tel.: +39-0577-232-913; fax: +39-0571-652-818. E-mail address: [email protected] (M.C. Fossi). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00148-4
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approach, can be used as an early warning signal of pelagic marine ecosystem ecotoxicological status (Fossi, Minutoli, & Guglielmo, 2001). The aim of this project was to propose a suite of biomarkers (BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase, esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multidisciplinary diagnostic tool for assessment of the health status of the Mediterranean ‘‘whale sanctuary’’, situated between the coasts of Provence, Monaco, Liguria, Tuscany, Corsica and Sardinia (Fig. 1 A). In November 1999, Environment Ministers from Italy, Monaco and France signed a treaty to establish a protected area in the Mediterranean Sea. This is the first time in the Northern Hemisphere that several countries have established a marine protected area which includes international waters. The area was chosen because it plays a significant role in providing rich biological diversity.
Fig. 1. (A) Sampling stations of Meganyctiphanes norvegica in the Mediterranean ‘‘whale sanctuary’’. (B) Samples of M. norvegica were collected, during the NATO Sound, Oceanography and Living Marine Resources (SOLMAR) 2000 Project, in five stations (in circle) in the Ligurian Sea in summer 2000, using an Isaacs-Kidd Midwater Trawl (IKMT) towed day and night from a depth of 200 m upwards. Numbers indicate planned profile stations. IKMT tows were made in order to obtain a coverage for the very same station at noon and at midnight with a 36 h temporal spacing. Stations were made at these times according to the predefined shiptrack without previous knowledge on the potential presence of krill. Therefore catches of krill (five circle stations) were totally incidental.
M.C. Fossi et al. / Marine Environmental Research 54 (2002) 761–767
763
The fin whale (Balaenoptera physalus) is a common cetacean species in this area and the euphausiid M. norvegica is its main food item. Although fin whale has long been known to frequent the Ligurian Sea, it was only recently realised that congregations of this whale in limited areas of the western sector in summer were correlated with blooms of the euphausiid M. norvegica (Relini, Tartaglia, Torchia, & Zanboni, 1994). A statistically significant relation was found between quantity of M. norvegica fished with a large IKMT net and sightings of B. physalus. M. norvegica is the most abundant euphausiid in the western Mediterranean, especially north of the 42nd parallel and is fundamental in the food chains, being the main constituent of Mediterranean krill. It has a varied diet, consisting principally of copepods at deeper levels by day and phytoplankton near the surface at night. The total number of specimens captured in the water column between 965 m and the surface is much greater at night than during the day. Samples of M. norvegica were collected, during the NATO Sound, Oceanography and Living Marine Resources (SOLMAR) 2000 Project, in five stations in the Ligurian Sea (Fig. 1B) in summer 2000, using an Isaacs-Kidd Midwater Trawl (IKMT) towed day and night from a depth of 200 m upwards. The main yield was obtained at night. The ultimate goal of the SOLMAR Lower Trophic Level Oceanographic Measurement Project is to examine the correlation between the distribution of whales in parts of the Mediterranean Sea and that of their prey, as well as that of lower trophic levels and basic oceanographic parameters. BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase, esterases (AChE), porphyrins (Uro-, Copro- and Proto-porphyrins) vitellogenin and zona radiata proteins, organochlorines, PAHs and heavy metals were evaluated in pooled samples in order to test the applicability of a multi-trial biomarker approach in this species as a potential early indicator of the ‘‘health status’’ of the Mediterranean ‘‘whale sanctuary’’. These biomarkers of contaminants exposure and effects have not been studied in this species. This information is of potential importance for environmental management and risk assessment. For the detection of MFO activities, the microsomal fraction was isolated by adding an aliquot of homogenisation buffer (pH 7.5) to a pool of whole organisms at 4 C and homogenising in a Potter blender. The homogenate was centrifuged at 9000g for 20 min and again at 10,0000g for 1 h. Before analysis, the microsomes were re-suspended in 2.6 ml re-suspension buffer per gram of tissue. MFO activity was evaluated in the microsomal fraction by assaying benzo[a]pyrene monooxygenase (BPMO) activities. BPMO activity was measured by the method of Kurelec, Britvic, Rijavec, Muller, and Zahn (1977), using 400 ml of microsomes as source of enzymes and incubating the reaction mixture for 1 h. Fluorimetric assay was carried out using a Perkin Elmer LS 50B luminescence spectrometer. NAD(P)H cytochrome c reductase, NADH cytochrome c reductase and NADH ferricyanide reductase were assayed by the method of Livingstone and Farrar (1984). All the tests were carried out at 30 C. Acetylcholinesterase (AChE) activities were determined in whole tissue homogenate, by the method of Westlake, Bunyan, Martin, Stanley, and Steel (1981).
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Porphyrin (Copro-, Uro-, Proto-porphyrins) concentrations were determined in pools of whole organisms; 0.2 ml of homogenate in water was spiked with 1.6 ml of 50:50 methanol/perchloric acid mixture. After vortex-mixing, the samples were kept in the dark for 10 min and then centrifuged for 5 min at a low speed. The porphyrin extract in the upper layer was used for spectrofluorimetric separation. Quantitative determination of porphyrins was performed by the method of Grandchamp, Deybach, Grelier, Deverneuil, and Nordman (1980). Immunochemical analysis of Vtg and Zrp was performed in homogenate of whole organisms by indirect ELISA (Goksoyr, 1991). A 96-well microplate was used for the test, each sample was tested in triplicate, adding 10 mg of protein to each well. Dilution of the primary antibodies was 1:1000 for anti-Vtg (PO-2) and 1:3000 for anti-Zrp (O-173). Results were expressed as adsorbance at 492 nm. Lyophilised organisms were digested with HNO3 in a teflon bomb (Stoeppler & Backhaus, 1978). Levels of metals were determined by atomic emission ICP/AES (Zn, Fe, Cu, Mn) and atomic absorption spectrometry with a transverse heated graphite furnace equipped with Zeeman background correction (Pb, Cd) and flow injection mercury system (Hg) techniques. The method of additions was used and the analytical procedure was checked with Certified Reference Material (Mussel Tissues CSM 278, Community Bureau of Reference) and blanks. Freeze-dried samples were extracted in a Soxlhet apparatus for analysis of chlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Extraction of OCs was carried out according to Marsili (2000) by high resolution capillary gas chromatography with a Perkin-Elmer Series 8700 GC and a 63Ni ECD, revealing op0 - and pp0 isomers of DDT and its derivatives DDD and DDE, and about 30 PCB congeners. PAHs were analysed by HPLC/fluorescence system. Extraction was carried out according to Houlobek et al. (1990) with some modifications (Marsili, 2000). M. norvegica is a zooplantonic species well studied in the Mediterranean sea for ecology and distribution (Relini et al., 1992). On the other hand very little information has been published on the biochemistry of this species (Mulkiewicz, Zietara, Stromberg, & Skarkowski, 2001; Salomon, Mayzaud, & Buchholz, 2000) and nothing exists on the ecotoxicological biomarker aspect. For this reason reference comparison can be done only with other zooplantonic species or other groups of crustacea. Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h (Table 1). The values are one order of magnitude lower than in the crustacea decapod species Carcinus aestuari. (Fossi et al. 2000). Larger differences between sites were found for reductase activities (Table 1), with the higher values found in station No. 19 (NAD(P)H cytochrome c reductase) and No. 34 (NADH ferricyanide reductase). Interestingly the highest values of total porphyrins are found in the same sampling station (No. 19, No. 34), the closest to the Ligurian coast (Italy). Esterase (AChE) activity values are in the same range of values found in the copepods species Acartia margalefi and Acartia latisetosa, but one order of magnitude lower than in the mysid species Siriella clausi (Fossi et al., 2001). Vitellogenin and zona radiata proteins were detectable in this zooplanctonic species (Table 1).
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Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w (Table 2). Total PAHs ranged from 860.7 to 5037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w. with the highest values detected again in the station N 34. HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w, hence again the highest value are detected in the station No. 34 one of the closest to the Ligurian coast (Table 2). Finally, it is interesting to explore the relationship between ‘‘prey’’ (M. norvegica) and ‘‘predator’’ (B. physalus) in the bioaccumulation of lipophilic contaminants (Marsili, 2000). The rate between contaminant levels in B. physalus and M. norvegica Table 1 Biomarker responses in pooled samples of Meganyctiphanes norvegica collected in the Mediterranean ‘‘whale sanctuary’’ Biomarker
Station No. 11
BPMO (A.F.U./mg prot/h) NADPH-cytochrome c reductase (nmol/mg prot/min) NADH-ferricyanide reductase (nmol/mg prot/min) AChE (mmol/g zoop/min) Uroporphyrin (pmol/g zoop) Coproporphyrin (pmol/g zoop) Protoporphyrin (pmol/g zoop) Total Porphyrins (pmol/g zoop) Vitellogenin (U.A.A./490 nm) Zona radiata proteins (U.A.A./490 nm)
Station No. 19
Station No. 26
Station No. 34
Station No. 43
Mean (S.D.)
0.50 3.69
2.89 4.69
3.20 3.41
2.08 2.44
0.47 4.23
1.83 (1.29) 3.69 (0.86)
0.81
1.20
3.40
8.76
2.96
3.43 (3.18)
7.60 29.0 88.0 133.0 251.0 0.115 0.069
7.60 19.0 163.0 199.0 382.0 0.124 0.066
9.25 18.0 92.5 126.0 238.0 0.127 0.080
5.40 29.0 116.0 139.0 284.0 0.108 0.054
7.85 7.5 135.0 123.5 266.5 0.133 0.150
7.54 (1.36) 20.5 (8.97) 118.8 (31.19) 144.1 (31.29) 284.3 (57.26) 0.12 (0.01) 0.08 (0.04)
Table 2 PAHs, OCs and heavy metals in pooled samples of Meganyctiphanes norvegica collected in the Mediterranean ‘‘whale sanctuary’’
Total PAHs (ng/g d.w.) Cancerogenic PAHs (ng/g d.w.) HCB (ng/g d.w.) DDTs (ng/g d.w.) PCBs (ng/g d.w.) Hg (mg/g f.w.) Cd (mg/g f.w.) Zn (mg/g f.w.) Mn (mg/g f.w.) Fe (mg/g f.w.) Cu (mg/g f.w.) Zn (mg/g f.w.)
Station No. 11
Station No.19
Station No. 26
Station No. 34
Station No. 43
3547 92.96
2030 141.7
1539 61.84
5038 81.82
963.8 63.61
3.890 163.2 195.9 – – – – – – –
3.500 35.29 99.64 – – – – – – –
6.700 50.53 136.6 0.030 0.031 25.68 0.600 35.95 13.90 0.158
11.56 74.10 210.2 – – – – – – –
4.760 41.02 84.60 0.033 0.023 17.04 0.540 12.52 11.43 0.065
Mean (S.D.) 2624 (1658) 88.39 (32.50) 6.082 (3.302) 72.83 (52.65) 145.4 (56.16) 0.032 (0.002) 0.027 (0.005) 21.36 (6.112) 0.570 (0.045) 24.23 (16.57) 12.67 (1.742) 0.111 (0.066)
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is 23.1 for PCBs and only 3.4 for total PAHs confirming the higher biomagnifications capacity of PCBs with respect to PAHs. In conclusion these preliminary results suggest the potential application of this multi-disciplinary ecotoxicological approach in M. norvegica as an early indicator of the health status of the Mediterranean ‘‘whale sanctuary’’ and particularly of the B. physalus population.
Acknowledgements We thank the personnel of ICRAM (Istituto Centrale per la Ricerca Scientifica e Tecnologica Associata al Mare) and the personnnel of ‘‘ITS Ammiraglio Magnaghi’’ (Italian Navy, Hydrographic Vessel) for technical assistance in the sampling activities.
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