Aquatic Toxicology 91 (2009) 151–160 Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Exposure to heptachlor: Evaluation of the effects on the larval and adult epidermis of Rana kl. esculenta Carla Fenoglio a,∗ , Amelia Grosso a , Eleonora Boncompagni a , Carlo Gandini b , Gloria Milanesi a , Sergio Barni a a Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 10, I-27100 Pavia, Italy b Dipartimento di Chimica Farmaceutica, Via Taramelli, Università di Pavia, 27100 Pavia, Italy article info Article history: Received 3 March 2008 Received in revised form 8 July 2008 Accepted 9 July 2008 Keywords: Heptachlor Rana kl. esculenta Tadpoles Survival Epidermis ultrastructure Enzyme histochemistry abstract Widely used in the past against termites and soil insects, the chlorinated insecticide heptachlor (H) is a toxic contaminant which represents a risk for both terrestrial and aquatic organisms. Like many organochlorine pesticides, heptachlor and heptachlor epoxide (HE), with oxidation products synthesized by many plant and animal species, degrade slowly since many of the derived compounds are persistent. This increases the status of heptachlor as a hazardous pollutant. In the present experimental study we exposed specimens of Rana kl. esculenta, from the tadpole stage through to their complete metamorphosis, to three different concentrations of heptachlor (4, 40 and 400 ppb). Mortality and HE bioaccumulation were evaluated on all the experimental groups. Since amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the toxic effects on the ventral epidermis of both tadpole and adult samples by employing such histo-cytopathological biomarkers as ultrastructural morphology, certain enzyme activities (acid and alkaline phosphatases, AcPase, and AlkPase; succinic dehydrogenase, SDH; -naphtyl butyrate esterase, ANBE; nitric oxide synthase/NADPH diaphorase, NOS/NADPHd). Also, the levels of reactive oxygen species (ROS) in the different conditions were evaluated. The results obtained were of ecological relevance, in particular as regards the effects of this environmen- tal toxicant on the samples of tadpole epidermis. Severe morphological alterations were observed in the larval epidermal cells (apical and skein cells), whereas the cell epidermis (keratinocytes and mitochondria- rich cells) of the adult survivors showed changes in enzyme activities, particularly those involved in the protective response to xenobiotic injury. In general, morpho-histochemical studies, analysis of HE bioaccumulation and mortality showed a relation to the H doses employed. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Heptachlor (H) is a chlorinated insecticide which was widely used in the developed world for about 30 years, mainly against ter- mites and soil insects. It has been banned in the U.S. and in other developed countries since 1978 due to its proven toxicity to animals and humans. Its use has eventually been restricted to the control of fire ants in power transformers (ATSDR, 1993). However, heptachlor is still produced and employed in many other countries as a con- stituent of chlordane, a closely related organochlorine insecticide (WHO, 2006). ∗ Corresponding author at: Dipartimento di Biologia Animale, Lab. Anatomia Comparata, Università di Pavia, Piazza Botta 10, I-27100 Pavia, Italy. Tel.: +39 0382 506316; fax: +39 0382 506406. E-mail address: fenoglio@unipv.it (C. Fenoglio). Due to its chemical properties, H is considered a persistent pol- lutant like many organochlorine pesticides. Its levels are likely to remain in the soil for long periods of time (Huber, 1993), albeit at relatively low concentrations (parts per billion). Its reported repre- sentative field half-life is 250 days (Augustijn-Beckers et al., 1994). However, traces of H have been detected in the soil even 14 and 16 years after application. This continuing presence, despite the low mobility of the substance, may result in appreciable movement over time, posing a risk for ground water contamination (WHO, 2006). The main metabolite of H is heptachlor epoxide (HE), an oxi- dation product in many plant and animal species. It is considered more toxic than its parent compound due to its slower degradation and consequentially greater persistence (ATSDR, 1993). The scarce solubility in water of both H and HE result in their strong adsorption to sediments and bioaccumulation in terrestrial and aquatic organisms, mainly fish and molluscs, probably the most 0166-445X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2008.07.005