Arsenic in Argentina: Technologies for arsenic removal from groundwater sources, investment costs and waste management practices.

An overview about the presence of arsenic (As) in groundwaters of Argentina, made by a transdisciplinary group of experts is presented. In this second part, the conventional and emerging technologies for As removal, management of wastes, and the initial investment costs of the proposed technologies, with emphasis on developments of local groups are described. Successful examples of real application of conventional and emerging technologies for As removal in waters for human consumption, for medium, small and rural and periurban communities are reported. In the country, the two most applied technologies for arsenic removal at a real scale are reverse osmosis and coagulation-adsorption-filtration processes using iron or aluminum salts or polyelectrolytes as coagulants. A decision tree to evaluate the possible technologies to be applied, based on the population size, the quality of the water and its intended use, is presented, including preliminary and indicative investment costs. Finally, a section discussing the treatment and final disposal of the liquid, semiliquid and solid wastes, generated by the application of the most used technologies, is included. Conclusions and recommendations, especially for isolated rural and periurban regions, have been added.

Arsenic in Argentina: Occurrence, human health, legislation and determination.

An overview about the presence of arsenic (As) in groundwaters of Argentina, made by a transdisciplinary group of experts is presented. Aspects on As occurrence, effects of As on human health, regulations regarding the maximum allowable amount of As in drinking water as well as bottled water, and analytical techniques for As determination are presented. The most affected region in Argentina is the Chaco-Pampean plain, covering around 10 million km2, where approximately 88% of 86 groundwater samples collected in 2007 exceeded the World Health Organization (WHO) guideline value. In the Salí river basin, As concentrations ranged from 11.4 to 1660 µg/L, with 100% of the samples above the WHO guideline value. In the Argentine Altiplano (Puna) and Subandean valleys, 61% of 62 samples collected from surface and groundwaters exceeded the WHO limit. Thus, it can be estimated that, at present, the population at risk in Argentina reaches around four million people. Pathologies derived from the chronic consumption of As, the metabolism of As in the human body and the effects of the different As chemical forms, gathered under the name HACRE (hidroarsenicismo crónico regional endémico in Spanish, for chronic regional endemic hydroarsenicism) are described. Regarding the regulations, the 10 µg/L limit recommended by the WHO and the United States Environmental Protection Agency has been incorporated in the Argentine Food Code, but the application is still on hold. In addition, there is disparity regarding the maximal admitted values in several provinces. Considerations about the As concentrations in bottled water are also presented. A survey indicates that there are several Argentine laboratories with the suitable equipment for As determination at 10 µg/L, although 66% of them are concentrated in Buenos Aires City, and in the Santa Fe, Córdoba and Buenos Aires provinces. Conclusions and recommendations of this first part are provided.

Living organisms influence on environmental conditions: pH modulation by amphibian embryos versus aluminum toxicity.

The LC10, 50 and 90/24h of aluminum for Rhinella arenarum embryos at complete operculum stage were 0.55, 0.75 and 1mgAl(3+)/L respectively. Those values did not change significantly by expanding the exposure period till 168h. The aluminum toxicity was evaluated in different pH conditions by means of a citrate buffer resulting for instance, 1mgAl(3+)/L at pH 4, 4.1, 5 and 6 in 100%, 70%, 35% and 0% of lethality respectively. As an outstanding feature, the embryos changed the pH of the maintaining media both in the case of Al(3+) or citrate buffer treatments toward neutral. 10 embryos in 40mL of AMPHITOX solution were able to increase the pH from 4.2 to 7.05, a fact related with a metabolic shift resulting in an increase in nitrogen loss as ammonia. Our study point out the natural selection of the most resistant amphibian embryos both for pH or aluminum as well as the capacity of living organisms (as a population) to alter their chemical environment toward optimal conditions for their survival. As these facts occur at early life stages, it expand the concept that living organisms at ontogenic stages are biomarker of environmental signatures of the evolutionary process (Herkovits, 2006) to a global Onto-Evo concept which imply also the feedback mechanisms from living organisms to shape environmental conditions in a way that benefits them.

Stage-dependent toxicity of bisphenol a on Rhinella arenarum (anura, bufonidae) embryos and larvae.

The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L(-1) , in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L(-1) BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L(-1) onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels.

Sublethal effects of atrazine on embryo-larval development of Rhinella arenarum (Anura: Bufonidae).

Atrazine (ATR), one of the most widely used herbicides in the world, affects not only target organisms but also the biota in general. Here, the teratogenic and neurotoxic effects of ATR on Rhinella arenarum (South American toad) embryos, and larvae were evaluated by means of standardized bioassays during acute and chronic exposures. The herbicide had a significant incidence of malformations, with a Teratogenic Index (TI) of 3.28. The main effects were delayed development, reduced body size, microcephaly, axial flexures, wavy tail and edema. In addition, delayed development, reduced development of forelimbs, and edema were recorded at metamorphosis stages. Scanning electron microscopy allowed observing different degrees of cellular dissociation and persistent cilliar cells in specific regions like the adhesive structure and tail fin. Results obtained by ATR 24 h pulse exposures at six developmental stages pointed out blastula as the most susceptible developmental stage both for immediate and delayed adverse effects. A noteworthy recovery capacity from acute toxic effects was recorded from the neural plate stage onwards. Regarding neurotoxic effects, abnormal, and erratic swimming and spasmodic contractions were recorded. Both the teratogenic and neurotoxic effects reported in this study demonstrate the importance of evaluating sublethal effects in non-target organisms as they could imply reduced fitness of individuals and eventually a population decline. The Hazard Quotients (HQ) for ATR ranged from 0.14 to 10.80, and the fact that some of these values are above USEPA's level of concern indicate that ATR is likely a risk to R. arenarum.

Stage-dependent susceptibility to copper in Rhinella arenarum embryos and larvae.

Copper toxicity in different embryonic and larval stages of the common South American toad Rhinella arenarum was evaluated by means of continuous and 24-h pulse treatments in 12 different developmental stages. Lethal concentrations (LC) of 10, 50, and 90% of continuous treatment with Cu from early blastula (S.4), complete operculum (S.25), and hind limb bud (S.28) stages were plotted from 24 to 168 h, resulting from S.4 in a 24-h LC50 of 137 µg Cu(2+) /L and a 168-h LC50 of 19.5 µg Cu(2+) /L. This result was in agreement with pulse treatments that showed a high resistance to Cu at blastula and gastrula stages, whereas the organogenic period, between muscular response (S.18) and open mouth (S.21), was very susceptible to this metal. Continuous treatments from S.25 showed no significant differences along exposure time (168-h LC50 = 51 µg Cu(2+) /L), but in the case of S.28 toxicity increased slightly from a 24-h LC50 of 138.6 µg Cu(2+) /L to a 168-h LC50 of 104 µg Cu(2+) /L, pointing out that, although the larval period was significantly more resistant to Cu, there was also a remarkable stage-dependent susceptibility to this metal. Copper teratogenic potential was approximately two, and main adverse effects were reduced body size, axial flexure, microcephaly, acephaly, mouth malformations, agenesis of or underdeveloped gills, agenesis of or underdeveloped tail, and hydropsy. The results are discussed considering Cu toxicity mechanisms, an evolutionary perspective, and environmental protection.

Nickel toxicity in embryos and larvae of the South American toad: effects on cell differentiation, morphogenesis, and oxygen consumption.

Nickel, a widely distributed heavy metal in the biosphere, produces systemic, carcinogenic, and teratogenic effects. The objectives of the present study are to report the acute, short-term chronic, and chronic toxicity of Ni in Rhinella arenarum embryos as well as the stage-dependent susceptibility to this heavy metal, including oxygen consumption, teratogenesis, and adverse effects on cell differentiation processes. The stages evaluated were blastula (S.7), gastrula (S.11), tail bud (S.17), fin circulation (S.22), and complete operculum (S.25), in this last case by means of toxicity profile curves. Nickel increases its adverse effects gradually, with a maximum value after 96 h. The 50% lethal concentrations (LC50s) for 96, 168, and 240 h at S.25 were 1.14, 0.60, and 0.48 mg Ni²(+) /L, respectively; S.11 and S.22 were the least and most susceptible to Ni with, LC50s 96 h of 6.12 and 0.19 mg Ni²(+) /L, respectively. A reduction of approximately 25% in oxygen consumption anticipates lethal effects from S.17 onward. The main teratogenic effects were retarded growth and development, extremely severe axis incurvations, persistent yolk plug, asymmetry, microcephaly and mouth and gill agenesia, and limited neuromuscular activity. Ciliated cells were not functional. The possibility of associating the remarkable stage-dependent susceptibility to Ni with environmental changes during the evolutionary process is also considered.

Stage-dependent toxicity of 2,4-dichlorophenoxyacetic on the embryonic development of a South American toad, Rhinella arenarum.

The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenoxyacetic acid (2,4-D) and a commercial formulation (CF) were evaluated on Rhinella (= Bufo) arenarum embryos at different developmental stages. Adverse effects were analyzed by means of the isotoxicity curves for lethality, malformations, stage-dependent susceptibility, and ultrastructural features. For all experimental conditions, the CF was more toxic, up to 10 times, than the active ingredient, being the open mouth stage (S.21) the most susceptible to the herbicide. For continuous treatment conditions, the early embryonic development was the most susceptible to 2,4-D and the LC50s for 96 and 168 h were 9.06 and 7.76 mg L(-1) respectively. In addition, both the active ingredient and the CF were highly teratogenic, resulting in reduced body size, delayed development, microcephaly, agenesis of gills, and abnormal cellular proliferation processes as the main adverse effects. According to US EPA, 2,4-D in agricultural scenarios may be up to three times higher than the NOEC values for teratogenic effects reported in this study. Therefore, they might represent a risk for amphibians. This study also points out the relevance of reporting the susceptibility of embryos at different developmental stages to both the active ingredient and the CF of agrochemicals in order to protect nontarget organisms.

Acute and subchronic toxicity of arsenite and zinc to tadpoles of Rhinella arenarum both alone and in combination.

The current study evaluated acute and subchronic toxicity of arsenite (As(3+)) and zinc (Zn(2+)) to stage 25 tadpoles of Rhinella arenarum in both single and joint laboratory exposures. LC50 values obtained for As(3+) were elevated and remained within the range of 46 to 50 mg/L of As(3+) between 4 and 17 d of exposure. Growth of tadpoles was completely inhibited with 30 mg/L of As(3+), demonstrating the presence of ecologically relevant sublethal effects at concentrations lower than those resulting in lethality. With respect to Zn(2+), a 96-h LC50 value of 2.49 mg/L was calculated in soft water. Contrary to results obtained for As(3+), LC50 values of Zn(2+) gradually decreased with increasing exposure duration, from 2.49 mg/L at 96 h to 1.30 mg/L after 21 d. In joint exposures to both metals, the type of interaction observed between As(3+) and Zn(2+) was concentration dependent. Lethal effects of As(3+) were mitigated, unaffected, or potentiated by 0.01, 0.1, and 1-2 mg/L of Zn(2+), respectively. However, although 0.01 mg/L of Zn(2+) significantly reduced lethality of As(3+)-exposed tadpoles, the same concentration of Zn(2+) did not help to reverse the stunt growth of these animals. Further studies need to examine which are the lowest concentrations As(3+) required to reduce growth and whether Zn(2+) serves to antagonize growth effects in this range of concentrations.

Stage-dependent teratogenic and lethal effects exerted by ultraviolet B radiation on Rhinella (Bufo) arenarum embryos.

The adverse effects of ultraviolet B radiation from 547.2 to 30,096 J/m2 on morphogenesis, cell differentiation, and lethality of amphibian embryos at six developmental stages were evaluated from 24 up to 168 h postexposure. The ultraviolet B radiation lethal dose 10, 50, and 90 values were obtained for all developmental stages evaluated. The lethal dose 50 values, considered as the dose causing lethality in the 50% of the organisms exposed, in J/m2 at 168 h postexposure, ranged from 2,307 to 18,930; gill circulation and blastula were the most susceptible and resistant stages, respectively. Ultraviolet B radiation caused malformations in all developmental stages but was significantly more teratogenic at the gill circulation and complete operculum stages. Moreover, at the gill circulation stage, even the lowest dose (547.2 J/m2) resulted in malformations to 100% of embryos. The most common malformations were persistent yolk plug, bifid spine, reduced body size, delayed development, asymmetry, microcephaly and anencephaly, tail and body flexures toward the irradiated side, agenesia or partial gill development, abnormal pigment distribution, and hypermotility. The stage-dependent susceptibility to ultraviolet B radiation during amphibian embryogenesis could be explained in the framework of evoecotoxicology, considering ontogenic features as biomarkers of environmental signatures of living forms ancestors during the evolutionary process. The stage-dependent susceptibility to ultraviolet B radiation on Rhinella (Bufo) arenarum embryos for both lethal and teratogenic effects could contribute to a better understanding of the role of the increased ultraviolet B radiation on worldwide amphibian populations decline.

Comparative susceptibility to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis: non-monotonous acceleration of the time to climax and delayed tail resorption.

Acute and subchronic toxicity of atrazine was evaluated in embryos (stage 4) and in premetamorphosis (stage 25) and prometamorphosis (stage 38-39) larvae of the common South American toad Rhinella arenarum (Anura: bufonidae). The influence of atrazine on the last stages of metamorphosis was also examined by exposing prometamorphosis larvae until completion of metamorphosis. Results obtained revealed that larvae in premetamorphosis are more sensitive than larvae in prometamorphosis and that these are, in turn, more sensitive than embryonic stages. Indeed, concentrations of atrazine as high as 30 mg/L had little effects on embryonic stages, the embryos surviving and developing in a similar manner as controls. LC50s of premetamorphosis larvae equaled 27.16, 7.03 and 2.32 mg/L of atrazine after 4, 14 and 21 days of exposure, respectively, compared to LC50s values of 18.27 and 14.43 mg/L after 14 and 21 days of exposure for larvae in prometamorphosis. In experiments with premetamorphosis larvae, the range of tested concentrations was extended to very low concentrations (down to 0.0001 mg/L) to examine whether recent findings of greater mortality at lower doses than at higher doses were also observed in R. arenarum but no such pattern was found. Exposure of prometamorphosis larvae to concentrations of atrazine of 10 mg/L and above widely prevented completion of metamorphosis and caused important mortality. Alternatively, whereas all animals eventually completed metamorphosis when exposed to concentrations of atrazine between 0.1 and 5 mg/L, the timings of metamorphosis were altered starting from 0.1 mg/L, the lowest concentration tested. Indeed, a significant decrease in the time needed for 50% of the larvae to reach the metamorphic climax (stage 42) was observed within this range of atrazine concentrations, the response presenting a U-shaped non-monotonic dose-response curve. Larvae exposed to these concentrations of atrazine also needed significantly more time for completing tail resorption, this effect being equivalent at all concentrations. Overall, the combination of these two different facets of atrazine influence on metamorphosis resulted in a significant acceleration of metamorphosis at 1 mg/L and a significant increase in the duration of metamorphosis at 5 mg/L, whereas no significant difference was observed with 0.1 mg/L.

Nickel tissue residue as a biomarker of sub-toxic exposure and susceptibility in amphibian embryos.

Although low level exposure to physicochemical agents is the most common environmental scenario, their effects on living organisms are very controversial. However, there is an increasing need to integrate low level exposures from risk assessment to remediation purposes. This study focus on the possibility to employ Ni tissue residue values as biomarkers of sub-toxic exposure and susceptibility to this metal in a range of almost pristine to sub-toxic concentrations for Rhinella arenarum embryos. For that purpose, three batches of amphibian embryos were pretreated during 10 days with three increasing concentrations of Ni starting in 2, 8 and 20 microg Ni(2+) L(-1) and ending in 16, 64 and 160 microg Ni(2+) L(-1) (in natural fresh waters this value ranges from 2 to 10 microgL(-1); the LC(50)-24h for R. arenarum is 26.2mg Ni(2+) L(-1)). For the experimental conditions, the Ni tissue residue values at 360 h post exposure were 0.5, 2.1 and 3.6 microg Ni g(-1) embryo w/w, respectively, corresponding to BCFs of 31, 33 and 23. The susceptibility to Ni in those experimental embryos was evaluated by means of challenge exposures to three lethal concentrations of this metal (10, 20 and 30 mg Ni(2+) L(-1)), registering survival during the following 10 days of treatment. As a general pattern, the lower, intermediate and higher pretreatments with Ni resulted in enhanced, neutral and adverse effects on embryonic survival, respectively. Thus, sub-toxic exposure to Ni could modify the resistance of the amphibian embryo to this metal and Ni tissue residue values could be considered as biomarkers of both, exposure and susceptibility.

Acclimation to low level exposure of copper in Bufo arenarum embryos: linkage of effects to tissue residues.

The acclimation possibilities to copper in Bufo arenarum embryos was evaluated by means of three different low level copper exposure conditions during 14 days. By the end of the acclimation period the copper content in control embryos was 1.04 +/- 0.09 microg g(-1) (wet weight) while in all the acclimated embryos a reduction of about 25% of copper was found. Thus copper content could be considered as a biomarker of low level exposure conditions. Batches of 10 embryos (by triplicate) from each acclimation condition were challenged with three different toxic concentrations of copper. As a general pattern, the acclimation protocol to copper exerted a transient beneficial effect on the survival of the Bufo arenarum embryos. The acclimation phenomenon could be related to the selection of pollution tolerant organisms within an adaptive process and therefore the persistence of information within an ecological system following a toxicological stressor.

Synergistic effects of copper and butylic ester of 2,4-Dichlorophenoxyacetic acid (Esternon Ultra) on amphibian embryos.

Cu2+ and butylic ester of 2,4-Dichlorophenoxyacetic acid as Esternon Ultra (2,4-D) toxicity on Bufo arenarum embryos were evaluated by means of a short-term chronic toxicity test (AMPHITOX). The NOEC values for Cu and 2,4-D were 0.02 mg/L and 2 mg/L respectively. The toxicity profile curves for Cu and 2,4-D were reported. The interactions of the metal and the herbicide were evaluated by combined treatments with different concentrations of Cu and 2,4-D. Although in all cases, a synergistic effect between these chemicals was observed, the combination of concentrations exerting low level effects in isolated treatments resulted in more adverse embryonic survival. Considering that both products are extensively used in agroecosystems, this fact could be of concern for non target species like amphibians.

Evoecotoxicology: environmental changes and life features development during the evolutionary process-the record of the past at developmental stages of living organisms.

For most of evolutionary history, scientific understanding of the environment and life forms is extremely limited. In this commentary I discuss the hypothesis that ontogenetic features of living organisms can be considered biomarkers of coevolution between organisms and physicochemical agents during Earth's history. I provide a new vision of evolution based on correlations between metabolic features and stage-dependent susceptibility of organisms to physicochemical agents with well-known environmental signatures. Thus, developmental features potentially reflect environmental changes during evolution. From this perspective, early multicellular life forms would have flourished in the anoxic Earth more than 2 billion years ago, which is at least 1.2 billion years in advance of available fossil evidence. The remarkable transition to aerobic metabolism in gastrula-stage embryos potentially reflects evolution toward tridermic organisms by 2 billion years ago. Noteworthy changes in embryonic resistance to physicochemical agents at different developmental stages that can be observed in living organisms potentially reflect the influence of environmental stress conditions during different periods of evolutionary history. Evoecotoxicology, as a multidisciplinary and transdisciplinary approach, can enhance our understanding of evolution, including the phylogenetic significance of differences in susceptibility/resistance to physicochemical agents in different organisms.

Estradiol uptake, toxicity, metabolism, and adverse effects on cadmium-treated amphibian embryos.

The exposure of Bufo arenarum embryos to 25 micromol/L 17beta-estradiol (E2) resulted in 100% lethality within 48 hr, whereas 10 micromol//L E2 was the no observed effect concentration value for short-term chronic (7 days) exposure. The toxicity profile curves show that lethal effects were proportional to the E2 concentration and the time of exposure. The E2 uptake resulted in 20.1 ng E2/mg embryo at 8 hr posttreatment, but 67.3% of this value was achieved during the first 30 min of incubation with this estrogen. Regarding metabolism, the embryos synthesize estrone (E1) from E2 by means of 17beta-hydroxysteroid dehydrogenase. Simultaneous treatments of Bufo arenarum embryos with 1 mg/L Cd2+ and 0.1, 1, or 10 micromol/L E2 enhanced the lethality exerted by cadmium in 76.7, 80, and 83.3% of embryos, respectively. The results indicate that estrogenic endocrine disruptors could have an adverse effect on amphibian embryos and enhance the toxic effect of Cd on amphibian embryos. This study points to the possibility of using the AMPHITOX test as a screening method for potential endocrine disruption as well as the combined effects of chemical mixtures.

Photodynamic toxicity and its prevention by antioxidative agents in Bufo arenarum embryos.

In this work we describe an experimental model to evaluate the photodynamic toxicity on amphibian embryos, as well as the protective effect of antioxidants against the lethal oxidative stress induced by photosensitization. Bufo arenarum embryos were treated with 10 mg/l methylene blue (MB) in AMPHITOX solution for 72 h and then irradiated with a red laser or white light for variable times. Both light sources affected the survival of MB-treated animals and lethal effects occurred within the initial 12 h post-irradiation. For white light irradiation, the most effective phototoxic condition in our study, the LD10, 50 and 90 at 6 h post-irradiation corresponded to 13.57, 19.87 and 29.10 J/cm2, respectively. To explore the action of antioxidants against the photogenerated oxidative stress, MB-treated embryos were incubated with 1mM glutathione (GSH) or ascorbic acid (AA) during 48 h before irradiation. For GSH and 21.6 J/cm2 irradiation, the survival increased from 20 to 90%, whereas 100% survival was achieved with AA even after 43.2 J/cm2 irradiation. These results indicate that both the lethal photodynamic effect and its prevention by antioxidants can be evaluated by means of a simple toxicity test employing amphibian embryos.

Toxicología y química ambiental: contribuciones para un desarrollo sustentable

La Serie de Publicaciones SETAC LA de Trabajos Presentados en Congresos es útil no solo para los científicos y profesionales que desarrollan actividades en los sectores gubernamental, empresario y académico en relación con objetivos de cuidado ambiental y de la salud humana, sino también y muy especialmente para los estudiantes en Ciencias Ambientales. El libro contiene unas 100 contribuciones agrupadas en 10 capítulos que son representativos de la creciente capacidad científica en temas de toxicología y química ambiental en América Latina