Gessica Gorbi

Toxic effect of heavy metals on the activated sludge protozoan community

Abstract The acute toxicity of five heavy metals to the protozoan community inhabiting the activated sludge of a waste treatment plant, was determined on the basis of reduction in both cell density and species richness. The activated sludge mixed-liquor was treated with different concentrations of cadmium, copper, chromium (VI), lead, and zinc for a period of 24-h. The experimental results enabled to determine the relative toxicity of the tested metals, and indicated that the order of toxicity of the five metals to the studied microbial community was generally: Cd, Cu > Pb > Zn > Cr. For nine out of sixteen protozoan species constituting the studied community was possible to determine the 24-h median lethal concentration (24-h LC 50 ) for each tested metal. Large differences appeared in sensitivities of the nine species to the metals. Ciliated protozoa such as Chilodonella uncinata and Trochilia minuta showed the highest sensitivity to all the studied metals, while Opercularia coarctata and O. minima , were the most tolerant species.

Is Cr(VI) toxicity to Daphnia magna modified by food availability or algal exudates? The hypothesis of a specific chromium/algae/exudates interaction

The long-term effects of Cr(VI) on life history traits of Daphnia magna Straus were studied in relation to different feeding conditions or to the presence of Scenedesmus acutus exudates, which had proved to reduce chromium toxicity to the alga. In high feeding conditions the concentration of 14 microg Cr(VI)/L had negative effects on survival, growth and fecundity. In low feeding conditions the effects on growth were not evident, but daphnids changed the reproductive strategy normally adopted in case of food shortage and showed a daily rate of newborn production higher than control; however, newborn were of a smaller size and less resistant to starvation. The most relevant effect of Cr was a shortening of the daphnid life span, which was independent of the feeding conditions and occurred also when growth and daily rate of neonate production were not affected. The algal exudates resulted ineffective in reducing Cr toxicity to daphnids, but caused a strong decrease in daphnid fecundity. Further experiments on S. acutus demonstrated that the detoxifying effect observed in the algae might involve a specific Cr/algae/exudates interaction, which occurs only when the algae are previously subjected to a short-time stress by chromium.

Standardized methods for acute and semichronic toxicity tests with the copepod Acartia tonsa

The availability of standardized protocols for both organism culture and bioassay with ecologically relevant species is of great concern in ecotoxicology. Acartia tonsa represents an important, often dominant, member of zooplankton communities and meets all the practical criteria suggested for model species. New standardized procedures for laboratory culturing of the copepod A. tonsa and standardized methods for acute (24- and 48-h) and semichronic (7-d, static-renewal) toxicity tests with the nauplius stage are described. In both cases, eggs are the starting stage, and nauplius immobilization is the endpoint. The methods were the object of an intercomparison test involving nine laboratories, and nickel was the reference toxicant. Relative reproducibility was 24, 25, and 34% for 24-h, 48-h, and 7-d tests, respectively.

Prediction of mortality in chronic toxicity tests on Daphnia magna

Abstract The present study deals with the analysis of mortality of Daphnia magna in chronic toxicity tests. Linear regression models were used to assess the possibility of predicting the probabilities of death over the long term on the basis of those recorded early in the toxicity test. The results based on data from toxicity tests with cadmium and pyridine, which yielded different mortality patterns over time, indicate that such predictions are practicable. These results suggest the feasibility of performing empirical relationships which would enable toxicologists to predict mortality over 21 days (usual length of the Daphnia magna chronic toxicity tests) and/or longer using the information obtained from shorter exposure to toxicants.

Comet Assay on Daphnia magna in eco-genotoxicity testing.

Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species.

Identification of S2-T A63: a cDNA fragment corresponding to a gene differentially expressed in a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus.

The gene expression of the wild type (S2-N) and a Cr-tolerant strain (S2-T) of the unicellular green alga Scenedesmus acutus has been compared in order to get more insight on their different chromium sensitivity. The RNA of the two strains was extracted after 4 days of culture in standard medium without chromium and analyzed by means of RNA differential display. The two strains showed differential gene transcription even in the absence of the heavy metal and six putatively differential amplicons were evidenced in the Cr-tolerant strain. Among the isolated amplicons, S2-T A63 was much more pronouncedly transcribed in the tolerant than in the wild type strain and was further characterized. S2-T A63 corresponding gene is present with the same copy number in the wild type and tolerant genomes and corresponds to an mRNA of about 2000 nt. The corresponding transcript is overexpressed in the Cr-tolerant strain after a 4-day culture and is not up-regulated by chromium exposure. The S2-T A63 sequence, obtained up to now, does not show significant homologies with any known gene. However, the analysis of the putative translation product reveals the presence of an interrupted fasciclin domain. This extracellular domain has been found in proteins from mammals, insects, echinoderms, plants, yeast and bacteria and is usually involved in cell adhesion. This finding suggests that the product for the S2-T A63 translation has an extracellular collocation, maybe as surface or secreted protein involved in external chromium detoxification.

Increase of chromium tolerance in Scenedesmus acutus after sulfur starvation: Chromium uptake and compartmentalization in two strains with different sensitivities to Cr(VI).

In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys. Hence, sulfur availability can modulate the capacity to cope with environmental stresses, a phenomenon known as SIR/SED (Sulfur Induced Resistance or Sulfur Enhanced Defence). Since chromate may compete for sulfate transport into the cells, in this study chromium accumulation and tolerance were investigated in relation to sulfur availability in two strains of the unicellular green alga Scenedesmus acutus with different Cr-sensitivities. Paradoxically, sulfur deprivation has been demonstrated to induce a transient increase of Cr-tolerance in both strains. Sulfur deprivation is known to enhance the sulfate uptake/assimilation pathway leading to important consequences on Cr-tolerance: (i) reduced chromate uptake due to the induction of high affinity sulfate transporters (ii) higher production of cysteine and GSH which can play a role both through the formation of unsoluble complexes and their sequestration in inert compartments. To investigate the role of the above mentioned mechanisms, Cr accumulation in total cells and in different cell compartments (cell wall, membranes, soluble and miscellaneous fractions) was analyzed in both sulfur-starved and unstarved cells. Both strains mainly accumulated chromium in the soluble fraction, but the uptake was higher in the wild-type. In this type a short period of sulfur starvation before Cr(VI) treatment lowered chromium accumulation to the level observed in the unstarved Cr-tolerant strain, in which Cr uptake seems instead less influenced by S-starvation, since no significant decrease was observed. The increase in Cr-tolerance following S-starvation seems thus to rely on different mechanisms in the two strains, suggesting the induction of a mechanism constitutively active in the Cr-tolerant strain, maybe a high affinity sulfate transporter also in the wild-type. Changes observed in the cell wall and membrane fractions suggest a strong involvement of these compartments in Cr-tolerance increase following S-starvation.

Acartia tonsa eggs as a biomonitor to evaluate bioavailability/toxicity of persistent contaminants in anoxic/sulfidic conditions: The case of cadmium and nickel

The evaluation of toxicity due to persistent pollutants in anoxic aquatic environments has met with various problems, as most test organisms can not withstand oxygen lack and exposure to free sulfide. We evaluated the suitability of the eggs of the brackish copepod Acartia tonsa for bioassays in anoxic/sulfidic conditions: when exposed to deep hypoxia and free sulfide, the eggs become quiescent and are able to resume hatching after restoring normoxic conditions. Tests with cadmium and nickel were performed in normoxic and deeply hypoxic conditions and in anoxic water containing H2S or H2S+FeSO4 on an equimolar basis. Active and quiescent eggs showed equivalent sensitivity to the metals, both suffering significant reductions in hatching success at 89μM Cd and 17μM Ni. As expected on the basis of the SEM/AVS model, Cd toxicity was almost completely suppressed in presence of sulfides. Dissolved Cd concentration drastically dropped and hatching success was generally >80%, as against values <6% observed in sulfide-free water, indicating that the applied experimental procedure can simulate metal-sulfide interaction. Ni toxicity was only slightly reduced by the presence of sulfides. High dissolved Ni concentrations were detected and mean hatching percentages were ≤32%, suggesting that Ni bioavailability/toxicity was only partially controlled by excess reactive sulfides. The results suggest that A. tonsa eggs could be a useful biomonitor to evaluate toxicity due persistent contaminants in anoxic conditions and the role of sulfides in reducing metal bioavailability/toxicity.