Toxicity of shredded tire leachate to sheepshead minnow (Cyprinodon variegatus): A morphological and cytological evaluation

Abstract

Whole and shredded tires are frequently discarded or purposely used with little or no knowledge of the toxicity of tire components that are leached into aquatic environments. No information is available on the toxicological pathology in fish exposed to tire leachate nor on the in vitro cytotoxicity to fish cells. Histopathological, ultrastructural and immunohistochemical (Proliferating Cell Nuclear Antigen, PCNA) analyses of larval sheepshead minnows (Cyprinodon variegatus) exposed to sequential shredded tire leachate extractions at 5, 15 and 25 ppt salinities indicated that tire leachate acted as a central nervous system neurotoxin. Cell death, both necrosis and apoptosis, plasma membrane alterations and edematous changes were observed in all of the major brain divisions and photoreceptor and nuclear layers of the retina in larvae exposed to leachate from the first extraction at 5 and 15 parts per thousand salinity. Lesions and decreases in DNA synthesis were most severe after 1 and 2 days of exposure to 2 and 3 day post hatch larvae. The extent of cellular deterioration decreased with subsequent leachate extractions and increased salinity. Morphological visualization and in vitro bioassays; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),5-(3-carboxylmeth-oxyphenyl)-2-({dollar}4, 5{dollar}-dimethylthiazoyl)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS), lactate dehydrogenase (LDH), bromodeoxyuridine (BrdU), neutral red and trypan blue, showed that sheepshead minnow and tilapia (Oreochromis nilotica) primary cultures of brain cells were targets of toxicity. Cytotoxicity was not seen in fathead minnow (Pimephales promelas) epithelial cell cultures. Significant decreases in cell viability and DNA synthesis, increases in LDH release and morphological injury to nuclear and plasma membranes were demonstrated in brain cells exposed to 50 and 100% tire leachate concentrations at 24 h. Decreases in DNA synthesis in tilapia brain cells persisted after 4 days of exposure. Excellent correlations existed between in vitro cytotoxicity and in vivo mortality. Cytotoxicity assays coupled with morphological examination may be applicable to in vivo mortality data and useful predictors of aquatic animal pollutants. These findings will hopefully foster the routine use of toxicological pathology and cytotoxicity assays and the examination of the fish central nervous system in the assessment of known and unknown toxicants.