S. Sánchez-Fortún

Influence of water hardening of the chorion on cadmium accumulation in medaka (Oryzias latipes) eggs

This report describes a study in which in vitro fertilization methods were used to expose medaka (Oryzias latipes) eggs to cadmium (Cd(2+)). This approach was applied to address the differential sensitivity and cumulative potential of Cd(2+) when exposure was initiated early (before fertilization and water hardening of the chorion) versus later during embryo development (i.e., well after the chorion has undergone water hardening). Following range finding exposures (2.5, 10, 20, 40 or 80 mg/l) under artificially controlled experimental procedures, results from hatching success and embryo malformations showed the earlier exposure interval more sensitive than the assay involving only the embryonated egg. Subsequent accumulation studies have shown that the exposure initiated before fertilization apparently led to more Cd(2+) deposition in the chorion compared to the exposure during embryonated stages of the eggs. Similarly, values for total Cd(2+) indicated higher concentrations in those eggs exposed prior to--and during--water hardening. Results suggest an alteration of the properties of the zona radiata in the early-stage eggs, making it more permeable to the potential exit or entrance of waterborne agents even after water hardening. Ongoing studies must now address the development of more realistic exposure conditions of the gametes by using incubation media with osmolarities similar to surface waters, and by shortening duration for gamete exposure. Also, sensitive methods to localize Cd(2+) and to delineate the transfer from the chorion to the embryo are needed.

Toxicity and adaptation of Dictyosphaerium chlorelloides to extreme chromium contamination

Metals are often spilled by industries into inland water environments, with adverse consequences. Numerous papers have reported that heavy metals produce massive destruction of algae. Nevertheless, algal populations seem to become tolerant when they have had previous exposures to heavy metals. Because the mechanisms allowing heavy metal tolerance of algae are not yet known, the present study analyzed the effect of hexavalent chromium on growth and photosynthetic performance of Dictyosphaerium chlorelloides, stressing on the adaptation mechanisms to chromium contamination. Growth and photosynthetic performance of algal cells were inhibited by Cr(VI) at 10 mg/L, and the 72-h median inhibition concentration was established as 1.64 and 1.54 mg/L, respectively. However, after further incubation for a three month period in an environment with 25 mg/L of chromium, some rare, chromium-resistant cells occasionally were found. A Luria-Delbrück fluctuation analysis was performed to distinguish between resistant algae arising from rare, spontaneous mutations and resistant algae arising from physiological adaptation and other adaptive mechanisms. Resistant cells arose only by spontaneous mutations before the addition of chromium, with a rate of 1.77 x 10(-6) mutants per cell division. From a practical point of view, the use of both chromium-sensitive and chromium-resistant genotypes could make possible a specific algal biosensor for chromium.

Cytotoxicity and genotoxicity of sewage treatment plant effluents in rainbow trout cells (RTG-2).

The cytotoxicity and genotoxicity of 11 organic fractions from sewage treatment plant (STP) effluents were tested using the RTG-2 rainbow trout permanent cell line. An automated in vitro micronucleus assay developed for RTG-2 cells was used to test the genotoxicity, whereas neutral red uptake, kenacid blue protein assay and ATP content were used to evaluate cytotoxicity. The induction of micronuclei (MN) and alterations in the cell cycle were analysed in these cells by flow cytometry after exposure to the organic fractions for 72 h. More than half of the organic extracts tested demonstrated a significant increase in the MN frequency, thus indicating that most of them can be considered to be genotoxic. The extracts were analysed chemically by GC/MS. Although the most frequently detected compounds in the effluents were bisphenol A (BPA), octylphenol (OP), di-2-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), as well as other possible mutagens, the concentrations cannot explain the genotoxicity of the individual chemicals, thereby suggesting a mixture effect. The results obtained support the need to apply effect-based tests to monitor complex mixtures as the most accurate means of assessing the genotoxicity of environmental samples.

Inhibition of growth and photosynthesis of selected green microalgae as tools to evaluate toxicity of dodecylethyldimethyl-ammonium bromide

The effect of dodecylethyldimethyl-ammonium bromide (DEAB), a quaternary ammonium, compound widely used as disinfectant, on phytoplankton of inland water systems was analysed by using an experimental model. A toxicity test was based on inhibition of photosynthesis performances (effective quantum yield from photosystem II, ΦPSII and O2 production) of the phytoplanktonic species Scenedesmusintermedius and Dictiosphaerium chlorelloides (Chlorophyceae) under growing doses of DEAB. A concentration-dependent toxic response was obtained in both parameters analysed. In addition, this response was almost immediate. Consequently, the measurement of both parameters (ΦPSII and O2 production) allows to assess DEAB toxicity with higher standards of precision and repeatability. We propose that this procedure could be used to detect presence of quaternary ammonium pollutants in freshwater.

Bioadsorption and bioaccumulation of chromium trivalent in Cr(III)-tolerant microalgae: a mechanisms for chromium resistance.

Anthropogenic activity constantly releases heavy metals into the environment. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. While hexavalent chromium uptake in plant cells has been reported that an active process by carrying essential anions, the cation Cr(III) appears to be taken up inactively. Dictyosphaerium chlorelloides (Dc1M), an unicellular green alga is a well-studied cell biological model organism. The present study was carried out to investigate the toxic effect of chromium exposures on wild-type Cr(III)-sensitive (Dc1M(wt)) and Cr(III)-tolerant (Dc1M(Cr(III)R30)) strains of these green algae, and to determine the potential mechanism of chromium resistance. Using cell growth as endpoint to determine Cr(III)-sensitivity, the IC₅₀(₇₂) values obtained show significant differences of sensitivity between wild type and Cr(III)-tolerant cells. Scanning electron microscopy (SEM) showed significant morphological differences between both strains, such as decrease in cell size or reducing the coefficient of form; and transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization and cell wall thickening in the Cr(III)-tolerant strain with respect to the wild-type strain. Energy dispersive X-ray analysis (SEM/XEDS) revealed that Cr(III)-tolerant D. chlorelloides cells are able to accumulate considerable amounts of chromium distributed in cell wall (bioadsorption) as well as in cytoplasm, vacuoles, and chloroplast (bio-accumulation). Morphological changes of Cr(III)-tolerant D. chlorelloides cells and the presence of these electron-dense bodies in their cell structures can be understood as a Cr(III) detoxification mechanism.

Toxic effects and specific chromium acquired resistance in selected strains of Dyctiosphaerium chlorelloides.

Due to its various uses, chromium contamination has become widespread in a diverse array of environments. The present study was carried out to investigate the toxic effect of chromium exposures on sensitive and resistant strains of the green algae Dyctiosphaerium chlorelloides, and to determine the nature and mechanism of chromium-resistant cells that arise. The toxic effect on the photosynthetic performance of chromium exposures in both cell populations, and the sensitive differences due to chromium oxidation state, were estimated, and the results indicate that although the photosynthetic performance in both strains were inhibited, there are not significant differences among IC(50(72)) values obtained in toxicity assays with both chromium oxidation states in wild-type cells, and however these differences are very significant when the assays were performed with Cr(VI) resistant cells. The 72-h 50% inhibitory concentration values obtained with Cr(III) exposures were similar for both strains. Additionally, by means of the SEM/EDX and TEM microscopic techniques, the occurrence of rapid morphological evolution in the microalgal cells and the possible detoxificant mechanisms was observed after exposure of the wild strain to chromium hexavalent. Moreover, the different response in photosynthetic activity observed between sensitive and resistant cells of D. chlorelloides in the presence of Cr(VI) and Cr(III) could be used to obtain a chromium-specific eukaryotic microalgal biosensor.

GENETIC ADAPTATION AND ACCLIMATION OF PHYTOPLANKTON ALONG A STRESS GRADIENT IN THE EXTREME WATERS OF THE AGRIO RIVER–CAVIAHUE LAKE (ARGENTINA)1

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komárek et Perman (Chlorophyta) and Microcystis aeruginosa (Kütz.) Kütz. (Cyanobacteria) to different water samples (from extremely acid, metal‐rich water to moderate stressful conditions) of the Agrio River–Caviahue Lake system (Neuquén, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria‐Delbrück fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal‐rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10−7 resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.

Assessment of genotoxic effects induced by selected pesticides on RTG‐2 fish cells by means of a modified fast micromethod assay

The cytotoxic and genotoxic effects induced by alachlor and dichlorvos, two pesticides broadly used, were studied on RTG‐2 fish cell line. As measure of cytotoxicity, neutral red assay was used to determine the cellular viability. Toxicity ranking based on IC50 values found that alachlor was more cytotoxic than dichlorvos. DNA damage has been evaluated on RTG‐2 cultures by means of an in vitro assay based on the ability of PicoGreen® fluorochrome to interact preferentially with double‐stranded DNA (dsDNA), and the results indicated that alachlor induced DNA strand breaks at concentrations above 1.52 μg/mL, equivalent to 1/50‐EC50(48), whereas exposures to dichlorvos induced DNA damage only at the maximal concentrations tested 25 μg/mL (1/10‐EC50(48)). These results confirm the suitability of this method for the screening of genotoxic effects of this type of aquatic pollutants, and we suggest their use in hazard assessment for environmental risk procedures.

Effects of selected biocides used in the disinfection of cooling towers on toxicity and bioaccumulation in Artemia larvae

This paper reports the acute toxicity of three biocides used in the disinfection of cooling towers, tetrakis(hydroxymethyl) phosphonium chloride (THPC), trichloroisocyanuric acid (TCIC), and sodium bromide (NaBr), on Artemia larvae, their effect on the phototactic response of this organism, and the potential of bioaccumulation in this species. The 24-h median lethal concentration (LC50) values for these biocides with respect to 24-, 48-, and 72-h-old Artemia, determined by static bioassays, showed the following rank order of toxicity: THPC < TCIC < NaBr. An age-dependent increase in sensitivity was seen for each compound. All three biocides reduced the phototactic response of 24-h-old Artemia larvae in 24-h static bioassays; the median inhibitory concentration ratios obtained were 30 to 40 times lower than their respective 24-h LC50 values. The results suggest that phototaxis bioassays could provide the speed and simplicity required for screening many potential pollutants for harmful effects. The bioconcentration factors obtained for Artemia larvae exposed to 10% LC50 for 168 h in renewal assays were 93.75, 1.67, and 0.23 for THPC, TCIC, and NaBr, respectively. This shows these biocides pose no bioaccumulation risk in this organism, although the value of 93.75 obtained for THPC is close to the threshold above which such a risk is considered to exist.

Toxicity and characterization of cholinesterase-inhibition induced by diisopropyl fluorophosphate in Artemia salina larvae.

The acute toxicity of diisopropyl fluorophosphate (DFP) on three age classes of Artemia salina was evaluated. An increase in toxicity of this organophosphorous (OP) compound was found following longer development of A. salina larvae. The effects of pretreatment with the non-selective muscarinic antagonist atropine, the two reversible acetylcholinesterase inhibitors physostigmine and pyridostigmine, and the cholinesterase-reactivating oxime 2-pyridine aldoxime methoiodide (2-PAM), as individual and combined pretreatments, on DFP-induced lethality in 24h Artemia were also investigated. The lethal action of DFP was not prevented by pretreatment of 24h Artemia with atropine, physostigmine, and pyridostigmine, while 2-PAM proved effective against intoxication with this OP compound. The inhibitory effects of combinations of atropine (10(-5)M) plus 2-PAM or physostigmine were greater than those elicited by either drug alone, with the maximum protection afforded being 100%. Pretreatment with 2-PAM (10(-6)M) plus physostigmine or pyridostigmine was ineffective. These results suggest that the combinations of atropine plus 2-PAM or physostigmine are effective in the prevention of the lethal effects induced by DFP in A. salina larvae.