Comparative effects of technical-grade and a commercial formulation of glyphosate on the pigment content of periphytic algae.

We investigated the potentially different effects of one of the most commonly used glyphosate formulations in Argentina, Glifosato Atanor(®), and the technical-grade glyphosate on the pigment content, as biomass indicators of the algal fraction in a freshwater periphytic community. A laboratory bioassay was carried out in 250-ml beakers. Two treatments were used: technical-grade glyphosate acid and Glifosato Atanor(®) (isopropylamine salt of glyphosate 48 % w/v), which were at a concentration of 3 mg active ingredient per liter. Treatments and the control (without herbicide) were replicated in triplicate. The concentrations of chlorophyll a and b and carotenes were determined at 0, 2, 6, 10, 24, 48, 96 and 192 h after herbicide addition. A significant increase in pigment content was observed for both herbicides after a 2-day exposure. Moreover, the formulation had little or no effect compared to the active ingredient, suggesting that the additives of Glifosato Atanor(®) may not enhance glyphosate toxicity.

Oxidative stress induced by a commercial glyphosate formulation in a tolerant strain of Chlorella kessleri.

We studied the toxicity of a glyphosate formulation and provide evidence of metabolic alterations due to oxidative stress caused in a Chlorella kessleri tolerant strain by exposure to the herbicide. After 96 h of exposure to increasing concentrations of the herbicide (0-70 mg L(-1)) with alkylaryl polyglycol ether surfactant, growth was inhibited (EC50-96 h 55.62 mg L(-1)). Glyphosate increased protein and malondialdehyde content which was significantly higher than in the control at 50-70 mg L(-1). Superoxide dismutase and catalase activities and reduced glutathione levels increased in a concentration-dependant manner. Morphological studies showed increases in vacuolisation and in cell and sporangia sizes. The glyphosate formulation studied has a cytotoxic effect on C. kessleri through a mechanism that would involve the induction of oxidative stress. Upon glyphosate exposure, oxidative stress parameters such as SOD and CAT activities and MDA level could be more sensitive biomarkers than usually tested growth parameters in C. kessleri.

Oxidative stress and antioxidant defenses in two green microalgae exposed to copper.

The aim of this work was to assess the effects of 1 week copper exposure (6.2, 108, 210 and 414microM) on Scenedesmus vacuolatus and Chlorella kessleri. The strains showed different susceptibility to copper. Copper content was determined in both strains by total X-ray reflection fluorescence analysis (TXRF). In S. vacuolatus, the increase of medium copper concentration induced an augmentation of protein and MDA content, and a significant decrease in the chlorophyll a/chlorophyll b ratio. S. vacuolatus showed a significant increase of catalase activity in 210 and 414microM of copper, and a significant increment of SOD activity and GSH content only in 414microM of copper. On the contrary, C. kessleri did not show significant differences in these parameters between 6.2 and 108microM of copper. Increased copper in the environment evokes oxidative stress and an increase in the antioxidant defenses of S. vacuolatus.

Phytotoxicity and genotoxicity assessment of imazethapyr herbicide using a battery of bioassays.

The imazethapyr herbicide (formulation Verosil(®)) was evaluated for phytotoxicity and genotoxicity using a battery of bioassays: (1) the growth inhibition of the green alga Pseudokirchneriella subcapitata, (2) the root growth and germination of the higher plant Lactuca sativa, (3) the genetic damage using the Salmonella/microsome test, and (4) the aneugenic and clastogenic effects on Allium cepa. The Verosil(®) formulation was highly toxic to the non-target green alga (median effective concentration (EC50) = 1.05 ± 0.05 mg active ingredient (a.i.) L(-1)), and concentrations above 10 mg a.i. L(-1) inhibited root elongation in lettuce: relative growth index (RGI) between 0.28 ± 0.01 and 0.66 ± 0.10. No genotoxic effect was observed in S almonella typhimurium at 100 mg a.i. L(-1), either with or without the microsomal fraction. However, significant differences in the frequency of chromosomal aberrations in anaphases and telophases (bridges, chromosome fragments, and vagrants) were observed in A. cepa at concentrations between 0.01 and 1 mg a.i. L(-1) with respect to the control. The frequencies of micronuclei showed significant differences with respect to the control at concentrations between 0.001 and 0.1 mg a.i. L(-1). A very high mitotic index (MI = 93.8 ± 5.8) was observed associated with a high number of cells in the prophase stage at 100 mg a.i. L(-1), indicating cytotoxicity. These results showed that imazethapyr is toxic to the non-target populations in both aquatic and terrestrial ecosystems. This herbicide might also exert clastogenic and aneugenic mitotic damage in higher plants. Therefore, the imazethapyr formulation may constitute an environmental risk to plants.

Dietary copper effects in the estuarine crab, Neohelice (Chasmagnathus) granulata, maintained at two different salinities.

We analyzed the dietary copper effects in the estuarine crab Neohelice (Chasmagnathus) granulata and its interaction with water salinity. Crabs were maintained at 2 per thousand and 30 per thousand salinity for 5 weeks and they were fed with commercial food supplemented with the green alga Scenedesmus vacuolatus previously exposed to copper. No mortalities were observed, but crabs maintained at 2 per thousand salinity accumulated on average 40% more copper compared to animals maintained at 30 per thousand salinity. At 2 per thousand salinity, superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels were increased at the first and second weeks, respectively, while lipid peroxidation and protein oxidation were evident after 4 weeks of copper exposure. At 30 per thousand salinity, all measured variables increased progressively but were significantly higher only at the end of the assay (5th week), except for protein oxidation that remained unchanged throughout the experiment. The hepatosomatic index (HSI) was significantly decreased in response to copper exposure, but only in crabs acclimated to 2 per thousand. These findings have suggested that dietary copper exposure induces greater metal accumulation and larger oxidative stress responses in crabs maintained at 2 per thousand salinity.

Studies on uroporphyrinogen decarboxylase from Chlorella kessleri (Trebouxiophyceae, Chlorophyta).

Uroporphyrinogen decarboxylase (UroD) (EC 4.1.1.37) is an enzyme from the tetrapyrrole biosynthetic pathway, in which chlorophyll is the main final product in algae. This is the first time that a study on UroD activity has been performed in a green alga (Chlorella). We isolated and partially purified the enzyme from a Chlorella kessleri (Trebouxiophyceae, Chlorophyta) strain (Copahue, Neuquén, Argentina), and describe for the first time some of its properties. In C. kessleri, the decarboxylation of uroporphyrinogen III occurs in two stages, via 7 COOH and then 6 and 5 COOH intermediates, with the decarboxylation of the 7 COOH compound being the rate-limiting step for the reaction. Cultures in the exponential growth phase showed the highest specific activity values. The most suitable conditions to measure UroD activity in C. kessleri were as follows: 0.23-0.3 mg protein/mL, approximately 6-8 micromol/L uroporphyrinogen III, and 20 min incubation time. Gel filtration chromatography and Western blot assays indicated that UroD from C. kessleri is a dimer of approximately 90 kDa formed by species of lower molecular mass, which conserves enzymatic activity.

Evaluación ecotóxica y genotóxica de aguas residuales hospitalarias / Ecotoxicological and genotoxic evaluation of hospital wastewaters

Los líquidos residuales provenientes de hospitales constituyen un riesgo potencial para los ecosistemas y la salud humana debido a la presencia de compuestos tóxicos y genotóxicos. El objetivo de este trabajo fue analizar la toxicidad y la genotoxicidad de los efluentes provenientes del Hospital de Clínicas José de San Martín (Buenos Aires). Las muestras del efluente se tomaron durante los días y horarios de mayor actividad del hospital y se separaron en dos fracciones: acuosa y orgánica (extractos). Los ensayos de toxicidad se realizaron en la fracción acuosa utilizando dos especies de algas verdes: Pseudokirchneriella subcapitata y Chlorella vulgaris. La genotoxicidad se evaluó en las dos fracciones mediante el ensayo de Salmonella/ microsomas en ausencia y presencia de mezcla S9, utilizando las cepas TA98 y TA100. Veintinueve muestras de un total de 53 muestras analizadas resultaron tóxicas para P. subcapitata (entre 18 y 55 % de inhibición), mientras que sólo 8 muestras lo fueron para C. vulgaris (entre 21 y 50 % de inhibición). Ninguna de las muestras resultó genotóxica para Salmonella, ni en los extractos ni en las fracciones acuosas. De los tres ensayos utilizados, P. subcapitata fue el más sensible, siendo el ensayo más apropiado para el monitoreo de estos efluentes.

Wastewaters from hospitals constitute a potential risk to the ecosystems and human health due to the presence of toxic and genotoxic chemical compounds. The objective of this work was to analyze the toxicity and genotoxicity of wastewaters from the "Hospital de Clínicas José de San Martín" (Buenos Aires). Wastewater samples were obtained during the days and hours of major hospital activities and they were separated into two fractions: aqueous and organic (extracts). The toxicity assays were performed for the aqueous fraction using the green algae species: Pseudokirchneriella subcapitata and Chlorella vulgaris. Genotoxicity was assessed for the two fraction samples using the Salmonella/microsome assay in presence and in absence of S9 mix, with the strains TA98 and TA100. Twenty nine of the 53 total analyzed samples were toxic to P. subcapitata (between 18 and 55 % inhibition), whereas only 8 samples were toxic to C. vulgaris (between 21 and 50 % inhibition). None of the samples resulted genotoxic to Salmonella. Of the three tests used, P. subcapitata was the most sensible, resulting in the most suitable species to be used in hospital wastewaters monitoring.