TOXICITY AND INTERACTIVE EFFECTS OF PENTACHLOROPHENOL, ORGANOTINS, NICKEL AND CYANIDE ON THE SEAWATER BRINE SHRIMP ARTEMIA FRANCISCANA S.HADJISPYROU 1 M.K.NIMFOPOULOS 2 A.KUNGOLOS 3 ΣΥΝΟΨΗ Μελετήθηκε η τοξικότητα πέντε ουσιών, της πενταχλωροφαινόλης,τού τριμέθυλοκασσιτέρου(ιv), του διβούτυλο-κασσιτέρου(ιv),νικελίου(ιι) και κυανιούχων(-ι) πάνω στη γαρίδα Artemia franciscana.ο τριμέθυλο-κασσίτερος αποδείχθηκε ότι ήταν η πιό τοξική ουσία γιά τα εφίππια της Artemia, ενώ η λιγότερο τοξική ουσία ήταν το νικέλιο.τα κυανιούχα παρουσίασαν μέτρια τοξικότητα, ενώ η τοξικότητα της πενταχλωροφαινόλης ήταν συγκρίσιμη με την αντίστοιχη του διβούτυλο-κασσιτέρου.στα πειράματα συνδυαστικής δράσης, βρέθηκε ότι υπάρχει συνεργισμός μεταξύ πενταχλωροφαινόλης και καθενός απο τα δύο οργανομεταλλικά παράγωγα του κασσιτέρου, ενώ μεταξύ των δύο αυτών παραγώγων βρέθηκε ότι υπήρχε ανταγωνιστική δράση.τέλος, συνεργιστικό ήταν το αποτέλεσμα της ταυτόχρονης επίδρασης τριών τοξικών ουσιών. ABSTRACT The effects of five toxicants, namely pentachlorophenol, trimethyltin(iv), dibutyltin(iv), nickel(ii) and cyanide(-i) on the brine shrimp Artemia franciscana were investigated. Trimethyltin proved to be the most toxic compound towards Artemia nauplii, whereas nickel was the least toxic one.cyanides were moderately toxic, whereas the toxicity of pentachlorophenol was comparable to that of dibutyltin.the interactive effect between each of organotins and pentachlorophenol on survival of Artemia was synergistic, whereas between organotins antagonistic action was observed. Finally, the combined effect of three toxicants, acting simultaneously, was synergistic. Key Words Artemia franciscana, interactive effects, pentachlorophenol, organotins, nickel, cyanide. 1 Lecturer, Laboratory of Inorganic Chemistry, Department of Chemical Engineering, Faculty of Technology, Aristotle University, 540 06 Thessaloniki, Greece. 2 Research Manager, Geochemistry Division, Institute of Geology and Mineral Exploration, 1 Frangon str., 546 26 Thessaloniki, Greece. 3 Assistant Professor, Department of Planning and Regional Development, University of Thessaly, 38 334 Volos, Greece.
INTRODUCTION It is now generally accepted, that environmental pollution, although an old problem,is far from finding realistic and effective solutions. Not only that, but the situation of the biosphere deteriorates every day even further,despite the efforts of many scientists, working on this framework. The increase of environmental contamination with various kinds of compounds, originating from many sources, has established today the use of model systems, which are organisms-indicators of pollution. These organisms serve as models, to assay the impact of toxicants on higher animals and human beings. Among marine invertebrates, Artemia franciscana is one of the most commonly used species. There are several advantages for the selection of brine shrimp Artemia as a test organism, including the continuous availability, low cost, ease of culture and the extensive literature describing their morphological, biochemical and molecular characteristics (Mc Rae et al.,1986, Mc Rae and Pandey 1991a, Warner et al.,1989). The special feture of larval and adult Artemia species, is that they are relatively insensitive to metals in general. The joint effects of trimethyltin, dimethyltin, chromium and cadmium on Artemia, and also the bioaccumulation of the two organotins on this organism have been investigated(s.hadjispyrou et al.,2000, S.Hadjispyrou et al., 2001).The high tolerance of Artemia adults and larvae to metals, coupled with the fact that Artemia are well suited for the bioassay not only for metals, but also for oils and oildispersants(verriopoulos and Dimas., 1988), has prompted us to consider earlier life steges of Artemia in our study, and to test an organic substance, as well. The aim of our work was to evaluate the interactive toxic effects of five toxicants on Artemia, namely trimethyltin(iv), dibutyltin(iv), pentachlorophenol, cyanide and nickel(ii). MATERIALS AND METHODS Larvae of brine shrimp Artemia franciscana, were hatched from cysts using the Artoxkit M (Persoone and Wells, 1987) along with an Algaltoxkit incubator, equipped with illumination unit.cyst hatching was initiated 48 h prior to the start of the toxicity tests.only neonates (younger than 24 h ) were used in the tests. Prior to use Artemia, organisms were kept in the refrigerator in the form of dormant eggs(nauplii).artificial seawater was used as hatching medium.it was prepared by dissolving the following substances in distilled and deionised water (g/l) : Sodium chloride 26.4, potassium chloride 0.84, calcium chloride 1.26, magnesium chloride 2.15, magnesium sulphate 2.72, sodium bicarbonate 0.17 and boric acid 0.03. Encysted Artemia were hydrated in distilled water at 4 0 C overnight, followed by washing to separate the cysts that float from those that sink. The sinkers were collected in a Buchner funnel and washed with cold, distilled water, followed by hatch medium (Mc Raeand Pandey., 1991b).Emergence of Artemia was started 24 h prior to the onset of the toxicity tests. Toxicity tests Solutions with concentrations of 100, 10,1, 0.1, and 0.01 mg/l (ppm) were prepared of the following substances : Trimethyltin chloride (TMT), dibutyltin diacetate (DBT), pentachlorophenol (PCP), potassium cyanide KCN and nickel chloride NiCl 2.6H 2 O.These solutions were tested for the toxicity range determination tests. For toxicity tests, 6-10 replicate samples of 20-30 encysted embryos each were added to 12 ml of artificial seawater in 50mm diameter petri dishes.these samples were examined for all concentrations of each metal.the metal compounds were added from stock solutions prepared in hatch medium.the plates were then incubated at 25 0 C for 24h.A reference solution, with artificial seawater was everytime used. After the incubation was completed, the number of dead nauplii for each metal concentration was counted and the % mortality was determined. Toxic action was expressed in terms of LC 50 values, that is the concentration of the toxicant killing the half of the total number of the initial organisms(table 1). The LC 50 determination was done by means of the specific computer program BASIC (Trevors, 1986).The program BASIC
has been developed for estimation of LD 50 values in toxicity tests on laboratory animals and in a similar way, one can also estimate the LC 50 values of various substances.in fig.1, is depicted the mortality vs. concentration curve for TMT. Experiments for interactive toxicity assessment Four substances were examined for their interactive toxic effects on Artemia. These were trimethyltin chloride (TMT), dibutyltin diacetate (DBT), pentachlorophenol (PCP) and potassium cyanide, KCN.The conditions of artificial seawater (as hatching medium) preparation, the hatching procedure, the emergence and development of Artemia nauplii was the same as in LC 50 prediction experiments. What was different, were the amounts and concentrations of the toxicants(see fig.2).the interactive effect of chemicals can be antagonistic, additive or synergistic. There are various mopdels predicting the theoretically expected interactive effect of toxicants(gowing 1960, Tammes 1964, Sprague 1970).The model used in this work, is based on the theory of probabilities and has been presented on details elsewhere(kungolos and Aoyama, 1998).The results are depicted in figures 2,3 and 4. RESULTS AND DISCUSSION The LC 50 values for the toxicants examined, were as follows(in mg/l, ppm) :0.22 for TMT, 89.4 for DBT, 92.5 for PCP, 6.15 for CN - and 980 for Ni(II)(Table 1).Ni(II) was excluded from the interactive tests due to its negligible toxicity.the other four toxicants were tested in combinations of two, and finally the three of them. In all cases, the combined effect of every other substance, tested with cyanide was proved to be antagonistic. For this reason, cyanide was not included in the final interactive experiments, of more than two substances, acting simultaneously. The interactive effect of every organotin with pentachlorophenol was synergistic, whereas antagonistic was the interactive effect of two organotins applied together(fig.2,3). Synergism was also observed after the application of TMT, DBT and PCP acting together on Artemia.This result is very important, because is an indication, that pentachlorophenol, an organic substance, stimulates the synergism between itself and each orgaotin compound in such extent, resulting in overcoming the antagonism between the two organotins(fig.4) Further experiments are necessary, in terms of bioaccumulation studies, with the above and also with a great number of compounds- both organic and inorganic-in order to construct a more precise picture and to make extrapolations for the behaviour of Artemia in the interactions with its ecosystem, in general. REFERENCES Gowing D. (1960) Comments on tests of herbicide mixtures, Weeds, 8, pp.379-391. Hadjispyrou S., Kungolos A., Topis S. and Sortsis A.(2000) Interactive toxic effects and bioaccumulation of trimethyltin, dimethyltin, chromium and cadmium on the brine shrimp Artemia salina, Proc.5 th Intern. Conf. Env. Poll. Thessaloniki, Greece,Aug.28-Sep.1, pp.399-405. Hadjispyrou S., Kungolos A., Anagnostopoulos A. (2001) Bioaccumulation and interactive effects of organotin, cadmium and chromium on Artemia franciscana. Ecotoxicol.Environ.Saf.49, pp.179-186. Kungolos A., Aoyama I.(1998) Interactive effects of heavy metals on Saccaromyces cerevisiae, Submitted to : Environ.Toxic. Wat. Qual. Mc Rae T., Pandey A.S.(1991a) Effects of metals on early life stages of the brine shrimp, Artemia : A developmental toxicity assay, Arch.Environ.Contam.Toxicol.20, pp.247-252.
Mac Rae T., Pandey A.S.(1991b) Toxicity of organic mercury compounds to the developing brine shrimp, Artemia.Ecoxicol.Environ.Saf.21, pp.68-79. Mac Rae T., Bagshaw J.C., Rafiee P.,Matthews C.O. (1986)Cadmium and zinc reversibly arrest development of Artemia larvae Bull.Environ.Contam.Toxicol.37,pp.289-296. Rersoone G., Wells P.G. (1987) Artemia in aquatic toxicology:a review. In : Artemia research and its applications(p.sorgeloos, D.A.Bengtson, W.Decleir and E.Jaspers, Eds.) Universa Press, Wetteren, Belgium, pp. 259-275. Sprague J.B.(1970) Measurement of pollutant toxicity to fish. II.Utilising and applying bioassay results.wat.res. 4, pp.3-32. Tammes P.M.L. (1964) Isoboles, a graphic representation of synergism in pesticides.neth.j.plant Path.,70,pp.73-80. Trevors J.T. (1986). A BASIC program for estimating LD 50 values using the IBM-PC. Bull.Environ.Contam.Toxicol.37, pp.18-26. Verriopoulos G., Dimas S., ( 1988) Combined toxicity of copper, cadmium, zinc, lead, nickel and chrome to the copepod Tisbe holothuriae. Bull.Environ.Contam.Toxicol.41, pp.378-384. Warner A.H., Mac Rae T., Bagshaw J.C. (1989) Cell and molecular biology of Artemia development.plenum Press, N.Y., pp.17-28. CAPTIONS Table 1 : LC 50 s for the effect of various compounds on Artemia franciscana Πίνακας 1: LC 50 για την επίδραση διαφόρων ενώσεων στην Artemia franciscana Fig.1 : Toxicity of trimethyltin(iv) to Artemia franciscana Διάγ.1 : Τοξικότητα τού τριμέθυλο-κασσιτέρου στην Artemia franciscana Fig.2 : Comparison between theoretically expected and observed mortalities for the combined effect of trimethyltin(iv) and pentachlorophenol on Artemia Διάγ.2 : Σύγκριση μεταξύ θεωρητικά αναμενόμενων και παρατηρούμενων τιμών θνησιμότητας κατά την συνδυασμένη επίδραση τριμέθυλο-κασσιτέρου και πεντάχλωροφαινόλης στην Artemia Fig.3 : Comparison between theoretically expected and observed mortalities for the combined effect of trimethyltin(iv) and potassium cyanide on Artemia
Διάγ.3 : Σύγκριση μεταξύ θεωρητικά αναμενόμενων και παρατηρούμενων τιμών θνησιμότητας κατά την συνδυασμένη επίδραση τριμέθυλο-κασσιτέρου και κυνιούχου καλίου στην Artemia Fig.4 : Comparison between theoretically expected and observed mortalities for the combined effect of TMT,DBT and PCP, applied together on Artemia Διάγ.4 : Συγκριση μεταξύ θεωρητικά αναμενόμενων και παρατηρούμενων τιμών θνησιμότητας κατά την συνδυασμένη επίδραση ταυτόχρονα των TMT, DBT και PCP στην Artemia Table 1. LC 50 s for the effect of various compounds on Artemia franciscana Artemia, 24h LC 50 95% Confidence Limits Trimethyltin chloride (mg/l Sn) 0.22 0.20-0.24 Pentachlorophenol,PCP mg/l 92.5 90.0-94.3 Dibutyltin diacetate (mg/l Sn) 89.4 87.3-91.7 Potassium cyanide (mg/l CN - ) 6.15 5.0-7.6 Nickel chloride (mg/l Ni) 980 888-1082
trimeth 100% 90% 80% 70% Mortality(%) 60% 50% 40% 30% 20% 10% 0% 0.01 0.1 1 10 100 Concentration(ppm) 0.01 0 0.1 0.043 0.15 0.25 0.2 0.368 0.22 0.5 0.25 0.7 0.3 0.9 0.382825 0.993 10 1 100 1
120 PCP Sn A: 90 mg/l 0.1 mg/l B: 90 0.18 C: 90 0.24 D: 92 0.1 E: 92 0.24 F: 92.5 0.22 Expected Observed 100 Mortality, % 80 60 40 20 0 A B C D E F Concentration combinations
80 CN Sn A: 0.22 mg/l 0.1 mg/l B: 0.22 0.18 C: 0.22 0.24 D: 5 0.1 E: 5 0.24 F: 6 0.22 Expected Observed 60 Mortality, % 40 20 0 A B C D E F Concentration combinations
120 DBT PCP TMT A: 80 mg/l 80mg/L 0.1 mg/l B: 85 85 0.18 C: 89 90 0.24 D: 89 92 0.1 E: 89.4 92.5 0.22 Expected Observed 100 80 Mortality, % 60 40 20 0 A B C D E Concentration combinations