Elio Arias

Sister chromatid exchange induction by the herbicide 2,4-dichlorophenoxyacetic acid in chick embryos.

As genetic damage may result from exposure to agricultural chemicals, it seemed appropriate to assess the genotoxic potential of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used broad-leaf herbicide, using a test system that may provide some indications on the genetic risk to animal species in the wild. In the present study, sister chromatid exchange (SCE) induction and cell cycle kinetics alterations by 2,4-D in 4-day old chick embryos were evaluated. Both a commercial herbicide formulation containing 37% 2,4-D isooctyl ester as active ingredient and pure 2,4-D were tested. Chick embryos were treated with 0, 0.5, 1, 2, or 4 mg 2,4-D. Test solutions were applied to the inner shell membrane on day 0 of incubation. Either commercial formulation or pure 2,4-D induced a dose-related increase in SCE frequency over the concentration range from 0 to 4 mg/embryo. Significantly higher SCE frequency was seen for the 4-mg group of embryos treated with the commercial product. A slightly higher SCE value was observed for the vehicle group (acetone-treated embryos) compared with the negative controls (untreated embryos). Significant inhibition of cell cycle progression was evident in both experimental groups and was generally dose related. The extent of changes in cell kinetics was similar in both groups, although somewhat more marked in the group treated with pure 2,4-D. The present findings corroborate the positive results from recent in vivo rodent studies.

Teratogenic effects of the fungicide maneb on chick embryos

The teratogenicity of a commercial formulation of the fungicide maneb (Maneb 80, containing 80% manganese ethylenebisdithiocarbamate and 20% inert ingredients) in chick embryos was evaluated. Unincubated eggs (157-207 per treatment group) were immersed in 0.5, 1.5, 4.5, or 13.5 g/liter maneb aqueous solutions for 30 sec. Two control groups were used: one group of 200 eggs was immersed in tap water and a second group of 205 eggs was immersed in a solution of the inert ingredients (sodium lignin sulfonate and n-butylnaphthalene sulfonate) at the concentration present in the 13.5 g/liter maneb solution. Eggs were then incubated for 19 days. A single treatment with maneb was teratogenic at all concentrations tested, producing mainly unilateral lower limb deformities. No adverse effects on development were noticed after exposure to the inert ingredients.

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.

Sister-chromatid exchanges and chromosomal aberrations in chick embryos after treatment with the fungicide maneb

The genotoxic potential of a commercial formulation of the fungicide maneb (Maneb 80, containing 80% manganese ethylenebisdithiocarbamate as active ingredient) in chick embryos was evaluated, using as genetic end-points the frequency of sister-chromatid exchanges (SCE) and of chromosomal aberrations. Unincubated eggs were dipped in 0, 0.5, 1.5, 4.5, 13.5 or 27 g/l maneb aqueous solutions for 30 sec. Eggs were then incubated for 4 days. Maneb significantly increased SCE values at 13.5 g/l and at 27 g/l (means 1.33 and 1.74) over the control value (mean 0.87). The concentration of 27 g/l, which corresponds to 10.8 times the recommended maximum application level for use in the field, also resulted in a high mortality rate. No clastogenic effects following exposure to maneb were observed.

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.

Toxicity and distribution of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in developing chick embryos

The toxicity of the herbicide Erbitox E30, a commercial formulation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) containing 28% MCPA as sodium-potassium salt and 72% of unknown ingredients, was tested on chick embryos. Sterile aqueous solutions of MCPA were injected into the air chamber at doses of 0, 1.5, 3.0, 6.0, 9.0, or 10.5 mg/egg on day 0 or on day 4 of incubation. The mortality rate for the embryos treated on day 0 of incubation was high in the first 5 days, low from 5-12 days and again increased by 15 days. The 15-day LD50 was 4.4 mg/egg (95% C.I. 3.7-5.3 mg/egg). HPLC analysis of albumen and yolk showed that concentrations of MCPA in the albumen were detectable at 5 min, highest at 7 days and markedly diminished by 14 days of incubation; a significantly lower concentration of MCPA was found in the yolk throughout the incubation period, except at 14 days when the yolk concentration was 4 times higher than the albumen concentration. At 15 days of incubation, MCPA was evenly distributed in the tissues of the embryo. MCPA was more toxic to 4-day embryos; concentrations above 6.0 mg/egg were lethal to all embryos within the first week of incubation. The 15-day LD50 for treatment on day 4 of incubation was 2.8 mg/egg (95% C.I. 2.5-3.2 mg/egg). The liver was affected by treatment with MCPA, being green in treated embryos. However, histological examination revealed few changes in the liver parenchyma.

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.