Nicoletta Pacces Zaffaroni

Natural variation in the limb skeletal pattern of the crested newt, Triturus carnifex (amphibia: Salamandridae)

The forelimb skeletal patterns of two geographically isolated populations of Triturus carnifex, one from northern Italy (Rosate, Milano) and one from central Italy (Bagnaia, Perugia) were studied. A total of 1,018 limbs were examined. Limb skeletal variants, generally consisting either of a reduced number of carpals and/or of an alternate phalangeal formula, were commonly observed, as has been reported for other amphibian species. The population from northern Italy showed a greater variability than that from central Italy. Skeletal variants were present in 34% and 12% of the limbs, respectively (i.e., in 48% and 20% of the newts). In the Rosate population, the majority of variants consisted of fusions of radiale and prepollicis and of the phalangeal formula 1‐2‐3‐2. Severe skeletal defects were also found in 3% of the limbs of the newts from Rosate and in 1% of the limbs of those from Bagnaia. At present, no conclusion can be drawn about the evolutionary significance of the limb skeletal variation observed in the two populations, because of the paucity of data on intra‐ and interpopulation variations in other species of the genus Triturus.

Abnormal limb regeneration in adult newts exposed to the fungicide maneb 80. A histological study

The effects of the fungicide Maneb 80 (manganese ethylenebisdithiocarbamate, 80% active ingredient) on the regenerating limb of the adult crested newt, Triturus cristatus carnifex, was studied. Female newts were exposed percutaneously to 5 ppm Maneb 80. One group of control newts was exposed to the inert ingredients of Maneb 80 (sodium lignin sulfonate and n-butylnaphthalene sulfonate), and another control group was kept in tap water. The limbs were examined histologically at weekly intervals throughout the regeneration period and at the end of the experiment (10-12 wk postamputation). The regenerating limbs of all the animals exposed to Maneb 80 showed growth retardation and skeletal abnormalities. Histological examination provided evidence that vascular disturbances are important for the genesis of the developmental abnormalities induced by Maneb 80. The inert ingredients had a promoting effect on limb growth and had no teratogenic effects under our experimental conditions. There were no histological differences between the two control groups.

The toxicity of manganese ethylenebisdithiocarbamate to the adult newt, Triturus cristatus.

SummaryThe toxicity of ethylenebisdithiocarbamate (maneb), a widely used agricultural fungicide, to the adult newt (Triturus cristatus carnifex) was evaluated after percutaneous exposure. Maneb was lethal to all the animals at the 50 ppm level within fifteen days. Male newts seem to be less resistant to the toxicant than females. At concentrations as low as 25 ppm all males died within 25 days, whereas some of the females were still alive after 5 months. Microscopic examination showed that the skin and the kidney were the most severely affected organs. It has been hypothesized that an osmoregulatory breakdown and an impairment of cutaneous respiratory exchanges play a role in the death of the animals. Renal failure must also be taken into account. However, additional mechanisms can not be ruled out at present.

Toxicity of 2-methyl-4-chlorophenoxyacetic acid to the adult crested newt

The toxicity of the herbicide Agroxone 3, a commercial formulation of the sodium salt of 2-methyl-4-chlorophenoxyacetic acid (MCPA), in the adult newt (Triturus cristatus carnifex) was tested after percutaneous exposure. At the concentration of 3200 ppm, the LT50 values were 17 and 21 hr for male and female newts. At 1600 ppm, the LT50 values were 35 and 45.5 hr. At 800 ppm, most of the animals were still alive after the 3 months of experimentation. There were no deaths at 400 and 200 ppm. Severe neuromuscular disorders were observed in animals exposed to lethal concentrations.

Teratological evaluation of the phenoxyacid herbicide MCPA in the regenerating forelimb of the adult newt

The effects of a commercial formulation of the phenoxyacid herbicide 2-methyl-4-chlorophenoxyacetic acid sodium salt (MCPA) on the regenerating forelimb of the adult crested newt were studied. The animals were exposed percutaneously to 0, 200, 400, or 800 ppm of MCPA for 4 days a week throughout the regeneration period and were sacrificed 11 weeks after amputation. The concentrations tested did not cause histologically detectable toxic lesions of the examined organs. Exposure to MCPA resulted in a significant retardation of the morphogenetic process as evidenced by skeletal examination. At the highest concentration tested, a significant growth delay was also evident during the regeneration period. No significant differences in the frequency of skeletal malformations were observed between control and treated newts, although the frequency was higher in the latter.

The toxicity of 2,4-dichlorophenoxyacetic acid to the adult crested newt

The toxicity of the herbicide Agroxone 5, a commercial formulation of the iso-octyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D), in the adult crested newt (Triturus cristatus carnifex) was tested after percutaneous exposure. The compound was lethal to all the animals within 72 hr at concentrations of 100, 125, and 150 ppm. Males appeared to be more susceptible than females to the toxic effects of 2,4-D, since the LT50 values at 75 ppm were 102 and 132 hr for male and female newts, respectively. At 50 ppm, all the males died within 31 days of exposure, whereas the females were still alive at the end of the 3-month experiment. Only one male died at 25 ppm after 21 days. Vacuolar degeneration of liver parenchyma and necrosis of kidney tubules were found at lethal concentrations of 2,4-D. The severity of the lesions was related to the duration of exposure more than to the concentration of the herbicide.

Testing of the fungicide maneb for carcinogenicity in two populations of the European crested newt.

The carcinogenic effects of the fungicide manganese ethylenebisdithiocarbamate (maneb) have been investigated in 2 populations of adult crested newt, which have different prevalences of spontaneous melanoma. Animals were exposed percutaneously to a 5 ppm solution of maneb 4 days a week for 37 weeks. No differences in tumor incidence were found between control and maneb-treated animals of either population. Most of the control and treated animals from Settala (Milan) died within 7 months because of the development of widely disseminated melanomas. Tumor incidence was 94% in controls and 98% in maneb-treated newts. In the population from Bagnaia (Perugia) the incidences of melanoma were 7% and 2%, respectively.

Preliminary study on the carcinogenic activity of the fungicide manganese ethylenebisdithiocarbamate in the adult newt. Triturus cristatus carnifex.

Percutaneous exposure of adult newts to the fungicide manganese ethylenebisdithiocarbamate (maneb) at 3 concentration levels (0.5, 2.5 and 5.0 ppm) for a total of 19-23 weeks did not result in the induction of any neoplasms.

Natural variation in the appendicular skeleton of Triturus carnifex (Amphibia: Salamandridae)

Intraspecific variation in the appendicular skeleton of two geographically isolated populations of Triturus carnifex, one from northern Italy (Rosate, Milano) and one from central Italy (Bagnaia, Perugia), has been studied. A total of 1,746 forelimbs and 830 hindlimbs were examined. Forelimb skeletal variability was much greater in the Rosate than the Bagnaia population. Skeletal variants were present in 36.3% and 13.5% of the forelimbs, respectively, or in 54.7% and 22.7% of the netws (P < 0.0001). There were no predominant skeletal variants in Bagnaia, while in the Rosate population, the majority of the variants consisted of fusion of radiale and prepollicis and of phalangeal formula 1‐2‐3‐2. Hindlimb skeletal variability was similar in the two populations and appeared to be much lower than that of the forelimb, with highly significant differences in the frequency of basipodium variants within the Rosate population and in the frequency of acropodium variants in both populations. Skeletal variants were present in about 9% of the hindlimbs, or in about 12% of the newts from either population. At present, no conclusion can be drawn about the mechanisms, genetic and/or epigenetic, underlying the skeletal variability observed in the Triturus carnifex from northern and central Italy.

Evaluation of the carcinogenic risk of the phenoxyherbicide MCPA to an urodele amphibian

To evaluate the carcinogenic potential of the phenoxyherbicide 2-methyl-4-chlorophenoxy-acetic acid (MCPA) in the crested newt, Triturus cristatus carnifex, a long-term study has been carried out exposing the animals by the percutaneous route. Two hundred adult newts were divided into one control and three experimental groups of 20 females and 30 males each. The control group was kept in tap water and the experimental groups were kept for 4 days a week in an aqueous solution of Agroxone 3, a commercial formulation of MCPA, at concentrations equivalent to 100, 200, and 400 ppm of the active ingredient. Treatment was continued for 1 year, after which all the animals were kept under observation for approximately another year. Surviving female newts were killed 22-24 months after the beginning of experimentation, whereas the male newts were killed after 28 months, at the end of 18 weeks of exposure to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate. Under experimental conditions, there was no carcinogenic activity of MCPA. Putative preneoplastic nodules of the liver and tumor-like lesions of the lower jaw were occasionally observed among the animals that survived more than 22 months after the beginning of experimentation. However, no significant differences in frequency between control and experimental groups were found.