Physical and chemical impacts on marine organisms
When standing on the calm seashore, we look at the beautiful color of the sea and enjoy listening to the sound of dialogues between wind and water, and smell the sea breeze. Marine fauna and flora have been attracting not only divers, who spend some time under water, but also those who do not dive but take pleasure from the contemplation of nature. Life once started in the sea, and the sea has been stabilizing the global environment and keeping all the life on the earth. It is everyone’s wish to leave the clean, beautiful and productive sea to our descendants. The sea, where life started, is now the terminus where all effluents resulted from human activities accumulate. It is now widely recognized that the global environment is endangered by anthropogenic pollution.
It has long been believed that the marine resources are inexhaustible without any human efforts. In fact, even Huxley (1883) had no doubt on the eternity of marine resources. In the twentieth century, however, we recognized that marine resources are limited. It has been pointed out and well known that human activities endanger global environment and also the existence of human beings themselves. Marine organisms are affected by marine pollution, synthetic chemicals, rise in water temperature, increase of UV radiation, and others. The effects of chemical pollutants have been of major interest for long time. It is clear now that chemical pollutants increase the susceptibility of organisms to infections, thermal changes, diseases and radiation. When acute effects of pollutants are apparent, counter-actions against them are carried out conveniently. However, as chronic effects of pollutants do not appear clearly, their damage may be significant only when we recognize their noxious effects. Thus, chronic toxicity caused by pollutants generally results in malformation of sexual organs, followed by a decrease in the reproductive functions of the organism.
Although organic synthetic compounds are generally produced and consumed on land, they flow in the sea through effluents and sewage. Since major industrial zones are located on the oceanfront, a variety of pollutants tend to flow into the sea. In addition, major global transport is sea-borne. Then, the elution of chemicals from ships and release of their ballast waters may cause marine pollution. Most of marine organisms, which are exploited as fishery objects, inhabit the littoral zone, the conditions of which are easily influenced by anthropogenic activities. Thus, marine organisms are easily exposed to the influence of artificial organic chemicals.
A major part of our energy sources currently consumed is petroleum. Transport of petroleum often causes accidental outflow of oil. Spill of oil from tankers is significant to marine pollution, as crude oil contains a variety of hazardous organic compounds. The quantities of oil spilt from tankers were 1,176, 1,140 and 146 kt in 1980s, 1990s and 2000–2004, respectively, except the spillage resulting from acts of wars. Recently, two major accidents of oil tankers occurred also in the Mediterranean Sea (Haven in Genova, 1991, 144 kt spilt) and in the Sea of Japan (Nakhodka in Shimane, 1997, 6.2 kt).
Another major concern on global environment is the increase of UV radiation reaching the earth due to ozone depletion; this causes at first a reduced growth of phytoplankton and consequently a decrease in zooplankton (Smith et al. 1992). Most marine invertebrates undergo planktonic larval stages; thus malformations consequent to the exposure to UV radiations have been described (Lesser and Barry 2003; Bonaventura et al. 2005; Schröder et al. 2005)
Finally, release of chlorofluorocarbons into the environment results in the decrease in the reproduction of marine fauna, which is one of our important protein sources, especially true for Japanese people.
Today many marine species are endangered and even their existence is questionable. We cannot pronounce about the possibility that others will not follow the same fate. The limit for the authorized amount of chemical substances found in marine waters has been estimated based on human safety. In the case of PCBs, the average concentrations in human and Orcius orca lipids are approximately 1 and 500 ppm, respectively, although PCB contamination is higher in the terrestrial environment than in the marine environment. Since Cetacea genetically lack P450, PCBs are significantly accumulated in their bodies. However, the maximum permitted amount should be indicated taking into consideration the general ecosystem risk, as the catalytic activity of organisms changes dependently on species and as biological accumulation of chemical substances is also a significant concern to our environmental management.
Last but not least, since in our countries, as already remembered, the consumption of seafood is pretty high, the accumulation of toxic compounds in our bodies can generate devastating diseases. For example, the “itai-itai” disease, which occurred in the 1950s, was caused by cadmium and mercury intoxication due to the ingestion of heavy metals-contaminated seafood. Serious toxic effects are still present in the population and are being studied in many victims living in Minamata (Japan) (Jouany 1998; Kondo 2000).
A long-lasting friendship
It must be remembered that Italy and Japan have a long history of collaboration in the field of biological studies. It was 1881 when Dr. Kakichi Mitsukuri visited the Stazione Zoologica in Napoli (SZN). A close friendship between Anton Dohrn, Director of the SZN at that time, and Mitsukuri lasted for their lives. Mitsukuri asked Dohrn his advice on the establishment of the first marine biological station in Japan. Later, he wrote to Dohrn a letter, dated March 29, 1887, announcing that the Misaki Marine Biological Station was founded on December 13, 1886. Since then, a number of Japanese scientists who played important roles in the biology field in Japan were appointed at the SZN as visiting researchers. In addition, a long-lasting collaboration between the research group, which Alberto Monroy founded in Palermo, and Japanese biologists was established in the middle 1950s and it is still maintained these days. This collaboration greatly contributed to the development of the biological science of both Italy and Japan. It must be also remembered here that a cycle of Japan–Italy Joint Seminars on Molecular Biology started in 1985, promoted by Prof. G. Giudice and Prof. E. Nakano, and were held afterwards in 1988, 1990, 1993, 1995, 1997 and 1999.
Italy and Japan are also linked by some common geographical and cultural characteristics; for example, they have an extended coastline and a large consumption of seafood. In addition, aquaculture is very active in both Italy and Japan. Therefore, sustainable productivity and marine pollution are of great interest. For these reasons, one of the major interests for both countries is to find an appropriate way to preserve marine environment.
Based on this scientific and cultural analysis and linked by a sincere friendship, the Japanese and the Italian organizers planned the Joint Seminar which materialized after sometime. The meeting was aimed at the promotion of interdisciplinary research on various aspects of pollution, monitoring and assessment of sea quality, with intention of promoting the collaboration between Italy and Japan in these scientific fields.
Field and laboratory studies
The first Italy–Japan seminar on “Physical and Chemical Impacts on Marine Organisms” was officially opened on November 16, 2004 at the Conference Center, Mielpearl-Ise Shima, Japan, by Prof. Yukio Yokota, Aichi Prefectural University. The co-organizer, Dr. Valeria Matranga, CNR Palermo, introduced the Italian participants, Prof. Daniela Candia-Carnevali, Dr. Ezio Amato, Dr. Marco Faimali and herself to the Japanese colleagues: Prof. Yoshitaka Nagahama, Prof. Akihiko Hara, Prof. Junichi Nishikawa, Prof. Kiyoshi Soyano, Prof. Makoto Kakinuma, Prof. Masato Kiyomoto, Dr. Takeshi Kitano, Dr. Tatsuya Unuma, Prof. Hajime Watanabe and Prof. Yukio Yokota. An informal welcome party was offered by the local organizer to the participants.
On November 17, after brief “opening remarks” by Prof. Yokota, the first scientific session was opened and chaired by Prof. Daniela Candia-Carnevali on “Impacts and Damages”. The first presenting author, Prof. Soyano, described the effects of persistent organic pollutants (POPs) on fishes collected along the Japanese costal waters. Data were presented on the determination of serum vitellogenin concentrations in wild and exposed animals. It was concluded that, since the gray mullet Mugil cephalus used for the studies is widely distributed all over the world and the mudskipper Periophthalmus modestus is found in tidal flats of South-East Asia, it would be feasible to survey estrogenic activity by the use of these sentinel organisms.
The first Italian speaker, Dr. M. Faimali, gave a talk on two interesting topics, namely: (1) the use of gasteropods imposex in monitoring studies meant to assess tributyltin (TBT) contamination along the Northern Adriatic coasts and (2) the description of a newly formulated biocide usable to fight against contamination by ballast waters. In the first case the effects of TBTs in two areas, Croatian coastline and Venice lagoon, were analyzed and compared. Paradoxically, no correlation between organic tin compounds (OTCs) levels and imposex was found in Croatia, possibly due to the lack in TBT use regulation. On the contrary, a correlation between chemical and biological data was found in samples from Venice lagoon. In addition the presenting author illustrated a newly developed acute toxicity test used on bacteria, microalgae and zooplanktonic larvae. New generation biocides were tested for their ability to kill the aforementioned organisms, which usually proliferate in ballast waters, and their use was proposed for future ballast water treatments (see Faimali et al., this issue).
Shifting to field studies and in particular to the chemical pollution caused by the massive release of Second World War weapons in the Mediterranean Sea, Dr. Amato described a series of oceanographic campaigns (1999–2004) which took place in the Southern Adriatic Sea, aimed at the survey of the sea floor and to determine distribution, characteristics and state of the dumped ordnance. Samplings of sediments, demersal fish (Conger conger) and benthic invetebrates (Paracentrotus lividus) were done for the analysis of contaminants and markers of stress. The described studies indicated that the leakage of chemical weapons from the rusted bombshells was likely to produce negative effects on the related benthic ecosystem. The need of a multidisciplinary approach for the assessment of chemical pollution in the aquatic environment caused by weapons’ dumping was also stressed (see Amato et al., this issue).
The last speaker of the morning section, Dr. Matranga, illustrated the use of a newly introduced bioindicator of chemical and physical pollution by the description of P. lividus coelomocytes. The latter are immune cells contained in the coelomic cavity of the adult sea urchin, whose capability to sense environmental changes and/or pollution and activate the expression of specific stress proteins has been recently shown (Matranga et al. 2000, 2002, 2005; Angelini et al. 2003). An extensive review on their use in laboratory experiments and field studies was given at the meeting. New data were reported on culture conditions, also in relation to their proper classification into different cellular subtypes and apoptotic behavior (see Matranga et al., this issue).
Overall, the data presented in this session supported the notion that it is essential to measure at the same time the concentration levels and biological endpoint effects of toxicant dispersed in the sea.
Defined by the International Program for Chemical Safety (IPCS) of the World Health Organization (WHO) as “exogenous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub)populations”, Endocrine Disruptors (ED) are recognized as important toxicants whose effects can be dangerous at different levels of the animal kingdom and whose concentrations in the sea need to be constantly monitored. Appropriately, the afternoon session on “Impacts on the Reproduction”, chaired by Dr. Valeria Matranga, dealt with the effects of ED on sex determination and plasticity as well as their influence in endocrine-specific gene expression and development growth control.
There is growing evidence that, in analogy with high vertebrate model systems, vitellogenin can be used as biomarker of estrogen-like contaminants dissolved in the environment. In fact its presence in the blood of male fishes can be used as an indicator of exposure to xenobiotics. As first speaker of the section Prof. Hara reported on fish vitellogenesis in general, and in particular he proposed the application of immunoassays for vitellogenin detection as biomarker. Laboratory and field studies on a few fish species were reported. Interestingly, it is possible to detect vitellogenin using immunoassays having different degrees of sensitivity (from milligrams to picograms) and working times (from days to minutes). Concerns on the use of vitellogenin as biomarker were raised based on the extent of vitellogenin multiplicity in the target species of choice (see Hiramatsu et al., this issue).
The sexual plasticity as possible target for the action of ED has been well described by Prof. Nagahama, who gave an extensive lecture on gonadal sex differentiation and gametogenesis in the medaka fish in relation to other vertebrates, including mammals. The identification of the sex-determining gene and its expression during sex differentiation were described. Its role in the establishment of sex in medaka fish was assessed by sophisticated molecular biology and genetic studies: loss of function (mutants and knock down) and gain of function (over-expression by transgenic). The gene has been mapped on Y chromosome which in this species, contrary to human and mouse Y sex chromosome, is almost identical in size to chromosome X. It was emphasized that among vertebrates the true sex-determining genes are known only in mouse (Sry) and medaka fish (DMY) (see review by Nagahama et al. 2004). Moreover, given the fact that in nature are present gonochoristic species, with ovarian and testicular tissues, as well as hermaphroditic species, that can change their sex, it was concluded that fishes are excellent vertebrate models to study the basic mechanisms involved in sex determination/differentiation and gender plasticity.
The following talk was given by Dr. Kitano who described the effects of hormones and endocrine-disrupting chemicals on the Japanese flounder Paralichthys olivaceus, whose sex determination is apparently dependent on the seawater temperature. The expression of sex-differentiation-related genes in response to aromatase inhibitor (fadrozole), anti-estrogen (tamoxifen) and steroids was measured. Finally, an in vitro gene reporter assay was described that provides an excellent system for elucidating the action mechanism of estrogenic chemicals in fishes (see Kitano et al., this issue).
The last speaker of the section, Prof. M. Kyomoto, introduced the Japanese sea urchin, from both the species Hemicentrotus pulcherrimus and Strongylocentrotus nudus, as an invertebrate model system that could be used to test the effects of exposure to steroid compounds on embryogenesis, larva development, juvenile growth, gametogenesis and yolk protein gene expression. He showed that, depending on the chemical and the species used, embryogenesis as well as adult growth are drastically impaired. Little effects were observed on larva growth, and no apparent differences were found in gene expression. In conclusion it seems that the sensitivity and response to ED changes markedly throughout the sea urchins’ ontogeny (see Kyomoto et al., this issue).
Biomarkers of environmental stress
The “Biomarkers of Impacts” was the topic of the November 18 morning session, chaired by Prof. Kiyoshi Soyano, where two Italian and two Japanese scientists proposed diverse species, namely crinoids, echinoids, barnacles and macroalgae, as sentinels of the health state of the sea by the molecular analysis of mechanisms activated upon chemical or physical contamination. The first speaker, Prof. Candia-Carnevali, illustrated some of the results achieved in the frame of a EU target project on endocrine disruptors (COMPRENDO). She studied the effects of exposure to estrogen disruptors on arm regeneration in the crinoid Antedon mediterranea. The spectacular regenerative capabilities of crinoids were tested in the laboratory following a severe exposure protocol. Results showed that the organotipic and cellular parameters affected are compatible with a pattern of serious steroid dysfunction (see Barbaglio et al., this issue).
The next speaker, Dr. Matranga, discussed on the developmental perturbations and the stress markers activated in response to physical (UV-B) and chemical (cadmium) contamination of seawaters using as the embryos of the model organism P. lividus. Variations in the expression of genes and proteins were correlated to the amount of developmental abnormalities found upon exposure to heavy metals and/or UV-B radiation. Interestingly, the developmental malformations observed followed the activation of early molecular markers of stress. In addition new results were discussed on the effects of UV-B on early cleavage embryos, both at the morphological and molecular levels (see Bonaventura et al., this issue).
The research purpose of the following speaker, Dr. Faimali, was to investigate on the feasibility of developing bioassays for acute and chronic toxicity using larvae of the crustacean species Balanus amphitrite. A new methodology was proposed, the swimming speed alteration test (SSA test), which measures the swimming speed by means of video-graphic techniques. The author claimed that if compared to the already acknowledged mortality and settlement inhibition tests, the SSA test is more sensible, easily interpretable and representative of a broad range of toxic compounds, including antifouling biocides, paints, neurotoxic pesticides and heavy metals (see Faimali et al., this issue).
The last speaker of the morning session, Prof. Kakinuma, described the molecular analysis of responses to environmental changes, such as temperature and salinity, in the marine macroalga Ulva pertusa. Effects at the gene expression level were measured after the identification of candidate genes involved in the pathways was achieved. The author concluded that thermal stress affected the photosynthetic, respiratory and molecular chaperone (hsp70) system whereas the salinity stress influenced the intracellular transport system (see Kakinuma, this issue).
The concluding section of the Italy–Japan Seminar which took place in the afternoon of November 18, chaired by Prof. Yokota, was titled “Molecular Tools for Evaluation of Chemical Contamination”. The first speaker, Prof. Watanabe, discussed on the evaluation of chemical contaminants by gene expression profiling of animal planktons. A list of candidate maker genes, which are induced or repressed under environmental pollutants (ED for example), were obtained by screening of a DNA microarray of Daphnia magna. Interestingly, the authors propose an easy but high-tech assay which gives multiple endpoints in a short period of time (see Watanabe and Iguchi, this issue).
Last but not least, Prof. Nishikawa, gave a talk on the effects of suspected ED on genes of the nuclear receptor family in the gastropod Thais clavigera. In synthesis he reported on the effects of organotins on retinoid-responsive gene expression, showing that several chemicals exhibit agonistic activity on various kinds of nuclear receptors. In addition it was demonstrated that TBT and triphenyltin (TPT) possess strong agonistic activities for both peroxisome proliferator-activated gamma receptor (PPARγ) and retinoic X receptor (RXR), and promote adipocyte differentiation through PPARγ/RXR pathway. The conclusion was that the nuclear RXR receptor plays an important role in inducing the development of imposex by organotin compounds (see Nishikawa, this issue).
Perspectives and future directions
We must always remember that marine pollution is borderless. Although it takes a very long time for a bottle of ink spilt in Japanese coast to arrive at the Pacific Coast of USA, pollutants in the state of aerosols and mists are able to diffuse unexpectedly fast to unexpectedly distant areas, depending on the air stream and turbulence. Data on Benzenehexachloride (BHC) levels suggest that pollutants accumulate in areas distant from the location of their actual use. Production and consumption of BHC is now prohibited in developed nations, but its consumption has increased in developing countries of the tropical zone. Interestingly, the highest environmental concentration of BHC found in the Pacific Ocean is detected in the Arctic Sea and the Bering Sea areas where the molecule is not consumed.
What scientists have to do is to analyze the current situation, fix rigid regulations which could guarantee the sustainability of human society and establish procedures to predict the spreading of hazardous factors. As previously emphasized, the fact that marine pollution is borderless stress the importance of international collaboration. Frequent exchange of information on the matters of pollution is crucial in order to make our earth more safe and environmentally protected. Italian and Japanese scientists working on marine biology met at the Bilateral Seminar, Italy and Japan, named “Physical and Chemical Impacts on Marine Organisms” which took place from November 16 to 19, 2004 at Mielpearl Ise Shima, Mie, Japan, to exchange their knowledge and discuss current subjects. Investigations using algae, zooplankton, mollusk, echinoderm, fish and others as experimental organisms were reported. The accent was put on the need for the establishment of appropriate model system(s) for the prediction and assessment of pollutants’ toxicity. In addition, since the development of new synthetic compounds as well as the probability of their accidental and/or guilty spill out is not avoidable, the possibility of testing their putative toxic effects is essential. Thus, to obtain easy and low cost tests for screening of substances capable of endocrine disturbance and malformations in the progeny is very important for the safety of our environment.
It is to say, the work of scientists is not limited to the scientific society, but the enlightenment to stake holders and common people is also of great importance. When we hear news about mass mortality of wild animals supposed due to pollution, we have to realize that the tragedy is not limited to wild animals but it expands also to us. Human life and our society are dependent on the productivity of healthy terrestrial and marine organisms; our actions are crucial for whether or not sustainable development of human society is possible. Then, the human being has to be responsible for what he does through his activities to the natural environment.
Finally, after concluding remarks by Prof. Yokota and Dr. Matranga, everybody congratulated on the success of the event and stressed the need for successive meetings. All participants confirmed their wish to exchange information on the environmentally related projects ongoing at their Institutions and to promote scientific collaborations between laboratories of the two countries. The organizers expressed their determination in finding a support for the continuation of the Italy–Japan Joint Seminar activities and their availability in organizing the next meeting, possibly in 2006 at the CNR in Palermo, Italy.
The Seminar would not have been possible without the sponsorships of the Consiglio Nazionale delle Ricerche (CNR), the Japan Society for Promotion of Science (JSPS) and the Istituto Centrale per la Ricerca Scientifica e Tecnologica Applicata al Mare (ICRAM). We are certainly indebted to the sponsors. We express special thanks to Aichi Prefectural University for the assistance.
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