Factors affecting R6 fungicide toxicity on sea urchin fertilization and early development: Roles of exposure routes and mixture components

Abstract

A technical fungicide mixture, R6 and its components, cymoxanil (CYM) and cupric oxychloride (Cu-OCl), were tested by sea urchin bioassays (Paracentrotus lividus and Sphaerechinus granularis). A set of toxicity endpoints was evaluated including both lethal and sublethal effects with the following endpoints: (a) acute embryotoxicity, (b) developmental defects, (c) changes in sperm fertilization success, (d) transmissible damage from sperm to the offspring, and (e) cytogenetic abnormalities. Acute effects on developing embryos were observed as early (prehatch) mortality at R6 levels 25 g/ml. The pesticide mixture R6 was tested at realistic concentrations, ranging from 25 ng/ ml to 2.5 g/ml, and the two components, CYM and CuOCl, were tested, either alone or in mixture, at concentrations equal to their levels in the corresponding R6 solutions. R6 was either dissolved in filtered seawater (water only, W-O), or spiked in ‘‘pristine’’ silt–clay sediment or soil samples before bioassays. Developmental toxicity of R6, following W-O dissolution, displayed a significant dose-related increase of larval malformations and differentiation arrest at concentrations of 750 ng/ml to 2.5 g/ml both in P. lividus and in S. granularis larvae. Developmental toxicity was removed in spiked sediment up to R6 nominal levels (25 g/ml), 10-fold above the embryotoxic R6 levels in W-O exposure. No significant developmental toxicity was exerted by CYM or Cu-OCl (W-O exposure) up to their concentrations equivalent to 2.5 g/ml R6. The laboratory-prepared mixture of CYM and Cu-OCl, in the same concentration range, only resulted in minor effects, as larval retardation, suggesting the presence of toxic impurities (or additional components) in the R6 formulation. When sperm from either P. lividus or S. granularis were exposed to R6 before fertilization, a W-O exposure resulted in a dose-related decrease in fertilization of P. lividus sperm (up to 250 g/ml R6), whereas S. granularis sperm underwent a significant increase of fertilization rate at the highest R6 nominal levels (up to 25 g/ml). Equivalent CYM or Cu-OCl levels were ineffective on sperm fertilization success in both species. The offspring of S. granularis sperm exposed to 25 g/ml R6 showed a significant increase in larval malformations, which were not detected in the offspring of R6-exposed P. lividus sperm. Again, CYM or Cu-OCl was unable to exert any transmissible damage from sperm to the offspring in either species. The present study raises the case of possible discrepancies between toxicity of a technical mixture and of its analytical-grade components, also providing evidence for a loss of pesticide toxicity following dispersion in an environmental matrix such as sediment or soil.

Keywords

fungicides technical mixtures sea urchins early development fertilization environmental availability water-only exposure ediment/soil spiking formulation ingredients additives

References

Somerville L . The metabolism of fungicides . Xenobiotica 1986; 16: 1017–1030 .

US Environmental Protection Agency (USEPA) . Cymoxanil; pesticide tolerance. 40 CFR Part 180 . Fed Regist 1998; 63 (87): 24941–24949 .

Helling B , Reinecke SA , Reinecke AJ . Effects of the fungicide copper oxychloride on the growth reproduction of Eisenia fetida (Oligochaeta) . Ecotoxicol Environ Saf 2000; 46: 108–116 .

Snyman RG , Reinecke SA , Reinecke AJ . Hemocytic lysosome response in the snail Helix aspersa after exposure to the fungicide copper oxychloride . Arch Environ Contam Toxicol 2000; 39: 480–485 .

Pagano G , Cipollaro M , Corsale G , Esposito A , Ragucci E , Giordano GG , Trieff NM . The sea urchin: Bioassay for the assessment of damage from environmental contaminants. In: Cairns J Jr (Ed), Community Toxicity Testing: ASTM STP 920. American Society for Testing and Materials, Philadelphia, PA , 1986, pp. 67–92.

Pagano G , Korkina LG , Iaccarino M , De Biase A , Deeva IB , Doronin YK , Guida M , Melluso G , Meriç S , Oral R , Trieff NM , Warnau M . Developmental, cytogenetic and biochemical effects of spiked or environmentally polluted sediments in sea urchin bioassays. In: Garrigues P , Walker CH , Barth H (Eds), Biomarkers in Marine Ecosystems: A Practical Approach. Elsevier, Amsterdam (in press).

Kobayashi N . Marine pollution bioassay by using sea urchin eggs in the inland sea of Japan (The SetoNaikai) . Publ Seto Mar Biol Lab 1972; 19: 359–381 .

Pagano G , Corsale G , Dinnel PA , Esposito A , Romaña LA . Use of sea urchin sperm and embryo bioassay in testing the sublethal toxicity of realistic pollutant levels . In: Grandjean E (Ed), Carcinogenic, Mutagenic, and Teratogenic Marine Pollutants: Impact on Human Health and Environment. WHO EURO/UNEP. Adv Appl Biotechnol Ser 1989; 5: 153–164 . Gulf, Houston, TX.

Romaña L-A , Brisset P , Pagano G , Arnoux A , Martin Y , Caillot A , Loarer R . Use of radioactive tracing techniques to identify particulate deposits and biological effects of urban effluents discharges from sewage outfalls . Water Sci Technol 1992; 25: 115–122 .

10.

Graillet C , Pagano G , Girard JP . Stage-specific effects of teratogens on sea urchin embryogenesis . Teratog Carcinog Mutagen 1993; 13: 1–14 .

11.

Pagano G , Anselmi B , Dinnel PA , Esposito A , Guida M , Iaccarino M , Melluso G , Pascale M , Trieff NM . Effects on sea urchin fertilization and embryogenesis of water and sediment from two rivers in Campania, Italy . Arch Environ Contam Toxicol 1993; 25: 20–26 .

12.

Trieff NM , Romaña L-A , Esposito A , Oral R , Quiniou F , Iaccarino M , Alcock N , Ramanujam VMS , Pagano G . Effluent from bauxite factory induces developmental and reproductive damage in sea urchins . Arch Environ Contam Toxicol 1995; 28: 173–177 .

13.

Korkina LG , Deeva IB , Iaccarino M , De Biase A , Oral R , Warnau M , Pagano G . Redox-dependent toxicity of diepoxybutane and mitomycin C in sea urchin embryogenesis . Carcinogenesis 2000; 21: 213–220

14.

Ingersoll CG . Sediments test. In: Rand GM (Ed), Fundamentals of Aquatic Toxicology. Effects, Environmental Fate, and Risk Assessment, 2nd ed. Taylor and Francis, Bristol, PA, 1995, pp. 231–255.

15.

Suedel BC , Rodgers JH . Toxicity of fluoranthene to Daphnia magna, Hylella azteca, Chironomus tentans, and Stylaria lacustris in water-only and whole sediment exposure . Bull Environ Contam Toxicol 1996; 57: 132–138 .

16.

Cohen Y , Levy Y . Joint action of fungicides in mixtures theory and practice . Phytoparasitica 1990; 18: 159–170 .

17.

Gisi U . Synergistic interaction of fungicides in mixtures . Phytopathology 1996; 86: 1273–1279 .

18.

Thompson HM . Interactions between pesticides: A review of reported effects and their implications for wildlife risk assessment . Ecotoxicology 1996; 5: 59–81 .

19.

Pagano G , Esposito A , Giordano GG . Fertilization and larval development in sea urchins following exposure of gametes and embryos to cadmium . Arch Environ Contam Toxicol 1982; 11: 47–55 .

20.

Pagano G , Esposito A , Bove P , de Angelis M , Rota A , Giordano GG . The effects of hexavalent and trivalent chromium on fertilization and development in sea urchins . Environ Res 1983; 30: 442–452 .

21.

Whorton EB . Some experimental design and analysis considerations for cytogenetics studies . Environ Mutagen 1985; 7: 9–15 .

22.

Godfrey K . Comparing the means of several groups. In: Bailar JC III , Mosteller F (Eds), Medical Uses of Statistics. NEJM Books, Boston, MA , 1992, pp. 233–257.

23.

Vallini G , Pera A , De Bartoldi M . Genotoxic effects of some agricultural pesticides in vitro tested with Aspergillus nidulans . Environ Pollut Ser A 1983; 30: 39-39 .

24.

Leroux P . Effect of pH, amino acids and various organic compounds on the fungitoxicity of pyrimethanil, glufosinate, captafol, cymoxanil and fenpiclonil in Botrytis cinerea . Agronomie (Paris) 1994; 14: 541–554 .

25.

Biles RW , Connor TH , Trieff NM , Legator MS . The influence of contamination on the mutagenic activity of dibromochloropropane (DBCP) . J Environ Pathol Toxicol 1978; 2: 301–312 .

26.

Sittig M . Handbook of Toxic and Hazardous Chemicals and Carcinogens, 3rd ed. Vol. 2 (G–Z). Noyes Publications, Park Ridge, NJ , 1991, pp. 1570-1570, 1659-1659.

27.

Hathaway G , Proctor NH , Hughes JP . Chemical Hazards in the Workplace. 4th ed. Wiley, New York, NY , 1996, p. 605 (Toluene); p. 662 (Xylenes).

28.

De Simone C , Piccolo A , De Marco A . Evaluation of genotoxic effects induced by herbicide maleic hydrazide in Vicia faba root tips, regarding the different organic matter content of the sowing soils. In: Senesi N , Miano TM (Eds), Humic Substances in the Global Environment and Implications on Human Health. Elsevier, Amsterdam , 1994, pp. 1113–1118.

29.

Abdel-Wahhab MA , Nada SA , Amra HA . Effect of aluminosilicates and bentonite on aflatoxin-induced developmental toxicity in rat . J Appl Toxicol 1999; 19: 199–204 .

30.

Pagano G , Cipollaro M , Corsale G , Esposito A , Mineo A , Ragucci E , Giordano GG , Kobayashi N , Trieff NM . Effects of sodium azide on sea urchin embryos and gametes . Teratog Carcinog Mutagen 1988; 8: 363–376 .

31.

Calabrese EJ , Baldwin LA . Chemical hormesis: its historical foundations as a biological hypothesis. Hum Exp Toxicol 2000; 2–31.

32.

Warnau M , Pagano G . Developmental toxicity of PbCl2in the sea urchin Paracentrotus lividus . Bull Environ Contam Toxicol 1994; 53: 434–441 .

33.

Pagano G , Cipollaro M , Corsale G , Esposito A , Giordano GG , Ragucci E , Trieff NM . Comparative toxicities of benzene, chlorobenzene and dichlorobenzenes on sea urchin embryos and sperm . Bull Environ Contam Toxicol 1988; 40: 481–488 .