Helgol Mar Res (1999) 53:219–243 © Springer-Verlag and AWI 1999 ORIGINAL ARTICLE Vladimir Bresler · Vera Bissinger · Avigdor Abelson Halim Dizer · Armin Sturm · Renate Kratke Lev Fishelson · Peter-Diedrich Hansen Marine molluscs and fish as biomarkers of pollution stress in littoral regions of the Red Sea, Mediterranean Sea and North Sea Received: 2 March 1999 / Received in revised form: 2 August 1999 / Accepted: 3 August 1999 Abstract The intensive development of industry and ur- higher in the surface epithelia of molluscs from a pollut- ban structures along the seashores of the world, as well ed site than that of the same species from control, unpol- as the immense increase in marine transportation and luted stations, providing clear evidence of response to other activities, has resulted in the deposition of thou- stress. Enhanced frequency of DNA lesions (alkaline and sands of new chemicals and organic compounds, endan- acidic DNA unwinding) and micronucleus-containing gering the existence of organisms and ecosystems. The cells was significantly higher in samples from polluted conventional single biomarker methods used in ecologi- sites in comparison to those from the clean sites that ex- cal assessment studies cannot provide an adequate base hibited genotoxic and clastogenic activity of the pollu- for environmental health assessment, management and tants. In all the studied molluscs a negative correlation sustainability planning. The present study uses a set of was found between the MXRtr levels of activity and the novel biochemical, physiological, cytogenetic and mor- frequency of micronucleus-containing hemocytes. The phological methods to characterize the state of health of expression of this was in accordance with the level of selected molluscs and fish along the shores of the Ger- pollution. The complete histopathological examination man North Sea, as well as the Israeli Mediterranean and demonstrates significantly higher frequencies of patho- Red Sea. The methods include measurement of activity logical alterations in organs of animals from polluted of multixenobiotic resistance-mediated transporter sites. A strong negative correlation was found between (MXRtr) and the system of active transport of organic the frequency of these alterations and MXRtr activity in anions (SATOA) as indicators of antixenobiotic defence; the same specimens. In addition to these parameters, a glutathione S-transferase (GST) activity as an indicator decrease in the viability was noted in molluscs from the of biotransformation of xenobiotics; DNA unwinding as polluted sites, but ChE activities remained similar at a marker of genotoxicity; micronucleus test for clastoge- most sites. The methods applied in our study unmasked nicity; levels of phagocytosis for immunotoxicity; cho- numerous early cryptic responses and negative altera- linesterase (ChE) activity and level of catecholamines as tions of health in populations of marine biota sampled indicators of neurotoxicity; permeability of external epi- from the polluted sites. This demonstrates that genotox- thelia to anionic hydrophilic probe, intralysosomal accu- ic, clastogenic and pathogenic xenobiotics are present mulation of cationic amphiphilic probe and activity of and act in the studied sites and this knowledge can pro- non-specific esterases as indicators of cell/tissue viabili- vide a reliable base for consideration for sustainable de- ty. Complete histopathological examination was used for velopment. diagnostics of environmental pathology. The obtained da- ta show that the activity of the defensive pumps, MXRtr Key words Molluscs · Fish · Red Sea · Mediterranean and SATOA in the studied organisms was significantly Sea · North Sea V. Bissinger · H. Dizer · A. Sturm · R. Kratke · P.-D. Hansen Institute for Ecological Research and Technology, Introduction Department of Ecotoxicology, Berlin Technical University, Berlin, Germany The enormous urbanization and industrial development V. Bresler (✉) · A. Abelson · L. Fishelson along riverbanks and seashores have introduced into the Institute for Nature Conservation Research water masses large and diverse quantities of biologically and Department of Zoology, The George S. Wise Faculty of Life Sciences, active substances, including thousands of organic and in- Tel Aviv University, Ramat Aviv 69978, Israel organic man-made chemical compounds (xenobiotics). e-mail: [email protected] These xenobiotics stress the natural ecosystems, causing 220 diseases of selected biota, enhanced lethality and often mediated detoxification and conjugation of xenobiotics, leading to elimination (extinction) of the more sensitive reflects early defensive responses of organisms to envi- taxa and the dominance of more resistant and opportu- ronmental polycyclic aromatic hydrocarbons (PAHs), nistic organisms, such as various algal and bacterial spe- polychlorinated biphenyls (PCBs) and dioxins (Yawetz cies. Such degradation is well documented in the north- et al. 1992; Bresler and Fishelson 1994; Bresler et al. ern seas as well as in coral reefs in the tropics, concomi- 1998; Fishelson et al. 1999). Altered glutathione S-trans- tant with the decline in economically important fish ferase (GST) activity also indicates defensive response stocks in the more northern global regions due to over- of the organism to enhanced pollution stress (Roy and fishing and environmental pollution. Alarmed by these Hänninen 1993). Numerous methods employing molecu- facts, decision-makers have started to ask ecologists for lar biology, biochemistry, cytochemistry and cytogenet- solutions offering a more reasonable and sustainable de- ics are also used to detect various genotoxic (DNA ad- velopment. The conventional ecological practice and sin- ducts formation, single-stranded DNA breaks, muta- gle biomarker methods cannot provide, in fact, the basic tions) and clastogenic (chromosome breaks, chromo- answers as to what is the actual state of health in the nat- some aberrations, aneuploidy, enhanced sister chroma- ural populations and, owing to this, cannot predict the tide exchange) effects of pollution (Jensen and Leary future of such biota. The frequency of sick animals in a 1990; Scarpato et al. 1990; Erbes et al. 1997; Nepomu- population can increase dramatically in a polluted envi- ceno et al. 1997; Ralph and Petras 1997; Torous, et al. ronment. To detect and assess early responses and the 1998; Wakata et al. 1998). Recently, DNA-unwinding state of health of populations, it is necessary to introduce assays and micronucleus tests have been developed as in ecology a special new set of methods derived from cy- prospective methods for mass monitoring of genotoxicity tochemistry, cytophysiology, cytogenetics and pathology and clastogenicity (Darzynkiewicz 1993, 1994; Hansen (Fishelson 1995; Bresler et al. 1998; Fishelson et al. et al. 1996; Bresler et al. 1998; Herbert and Hansen 1999). These novel biochemical, cytochemical, cyto- 1998). The inhibition of cholinesterase activity has been physiological, cytogenetic and cytopathological methods also suggested as an effective parameter to detect the enable us to detect cryptic early responses and early pre- toxic influence of organophosphates, carbamates, some pathological alterations in exposed organisms before en- heavy metals and surfactants (Galgani et al. 1992; Silva hanced disease and mortality rates occur or population de Assis 1998). Finally, cyto- and histopathological ex- shifts take place (Adams 1990; Roy and Hänninen 1993; amination permits us to detect early pre-pathological and Bresler and Fishelson 1994; Hansen 1995; Bresler et al. pathological alterations, and to unmask target species, 1998; Fishelson et al. 1999). Such methods can therefore specimens, organs, cells and subcellular structures as offer an effective early warning system in monitoring well as to assess etiology and pathogenesis of these alter- biota of aquatic environments. For example, altered ac- ations (Cotran et al. 1989; Schwaiger et al. 1997). tivity of several antixenobiotic defence mechanisms, Taking these facts into consideration and using a nov- such as membrane export pumps, multixenobiotic resis- el methodology, our study focused on the assessment of tance-mediated transporter (MXRtr), the system of ac- the health in littoral biota of marine environments affect- tive transport of organic anions (SATOA) and enzyme- ed by anthropogenic activities. The state of health was Table 1 Sites of collection, dates of sampling and species collected along the Israeli Mediterranean shore Sites of collection Collected species Dates of collection Frutarom Main: Donax trunculus (374) 12.11.95; 17.04.96; 3.07.96; 19.12.96; 2.01.97; 17.09.97; 4.03.98 Additional: Mactra corallina Na’aman River Main: Donax trunculus (212) 12.11.95; 17.04.96; 2.01.97 Additional: Mactra corallina Maagan Michael Main: Donax trunculus (212), 17.04.96; 3.07.96; 19.12.96; 2.01.97; 4.03.97; 14.04.97; 8.05.98 (Taninim River) Patella coerulea (75) Additional: Mactra corallina, Monodonta turbinata Shemen Beach Main: Patella coerulea (107) 12.11.95; 17.04.96; 3.07.96; 19.12.96; 14.04.97; 30.04.97; 4.03.98; 8.05.98 Achziv Main: Patella coerulea (55) 17.04.96; 3.07.96; 4.03.98; 8.05.98 Additional: Monodonta turbinata Akko Main: Patella coerulea (85) 17.04.96; 14.04.97; 30.04.97; 4.03.98; 8.05.98 Additional: Monodonta turbinata Sdot Yam Main: Patella coerulea (70) 3.07.96; 14.04.97; 4.03.98; 8.05.98 Additional: Monodonta turbinata Tel Shikmona Main: Patella coerulea (25) 14.04.97; 30.04.97;