An Efficient Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Method for the Analysis of Methyl Farnesoate Released in Growth Medium by Daphnia pulex.

Methyl farnesoate (MF), a juvenile hormone, can influence phenotypic traits and stimulates male production in daphnids. MF is produced endogenously in response to stressful conditions, but it is not known whether this hormone can also be released into the environment to mediate stress signaling. In the present study, for the first time, a reliable solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed and validated for the ultra-trace analysis of MF released in growth medium by Daphnia pulex maintained in presence of crowding w/o MK801, a putative upstream inhibitor of MF endogenous production. Two different clonal lineages, I and S clones, which differ in the sensitivity to the stimuli leading to male production, were also compared. A detection limit of 1.3 ng/L was achieved, along with good precision and trueness, thus enabling the quantitation of MF at ultra-trace level. The achieved results demonstrated the release of MF by both clones at the 20 ng/L level in control conditions, whereas a significant decrease in the presence of crowding was assessed. As expected, a further reduction was obtained in the presence of MK801. These findings strengthen the link between environmental stimuli and the MF signaling pathway. Daphnia pulex, by releasing the juvenile hormone MF in the medium, could regulate population dynamics by means of an autoregulatory feedback loop that controls the intra- and extra-individual-level release of MF produced by endogenous biosynthesis.

An integrated approach for chemical water quality assessment of an urban river stretch through Effect-Based Methods and emerging pollutants analysis with a focus on genotoxicity.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.

DNA damage detection by Comet Assay on Daphnia magna: Application in freshwater biomonitoring.

Monitoring of water genotoxicity still remains underexploited in risk assessment. The present study aimed at standardizing and evaluating the sensitivity and applicability of the Comet Assay adapted for Daphnia magna in genotoxicological investigations in freshwater environments. Two sampling campaigns (2014-2015) were performed in the watercourses of a pilot basin located in the Parma district (Italy). Fourteen sampling stations with different Ecological Status and/or EBI values were selected, all with a good Chemical Status according to the EU-Water Framework Directive 2000/60. The Alkaline Comet Assay was performed on 48 h-aged daphnids exposed (24 h) to 23 water samples. In parallel, the acute toxicity test was carried out. Daphnids exposed to samples, collected upstream the main watercourses in non-impacted areas, showed low DNA migration (Tail Intensity percentage - TI% - in the range 2.97-13.21), similar to laboratory controls. An increase in genotoxicity (TI% in the range 20-40) proceeding from the mountain towards the plain area was observed, in agreement with the land uses and the ES/EBI values of the stations. The highest genotoxic damage was observed after exposure to samples from watercourses of the minor hydrographic network in the plain area and waterbodies receiving wastewater treatment plant outflows. A modified version of the Comet Assay able to identify the presence of genotoxins inducing DNA oxidative damage, after standardization, was applied to daphnids treated with waters from 4 selected monitoring stations. The presence of oxidative contaminants was detected downstream a wastewater treatment plant outflow. The Comet Assay on D. magna has proven to be sensitive and able to discriminate among differently impacted areas and might be applied routinely. The FPG-Comet proved to be able to highlight the presence of contaminants causing oxidative stress. In our knowledge, this is the first time that Comet Assay on Daphnia magna is successfully applied for freshwater monitoring.

The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase.

Sulfur availability and the end products of its metabolism, cysteine, glutathione and phytochelatins, play an important role in heavy metal tolerance, chromium included. Sulfate and chromate not only compete for the transporters but also for assimilation enzymes and chromium tolerance in various organisms has been associated to differences in this pathway. We investigated the mechanisms of Cr(VI)-tolerance increase induced by S-starvation focusing on the role of ATP sulfurylase (ATS) in two strains of Scenedesmus acutus with different chromium sensitivity. S-starvation enhances the defence potential by increasing sulfate uptake/assimilation and decreasing chromium uptake, thus suggesting a change in the transport system. We isolated two isoforms of the enzyme, SaATS1 and SaATS2, with different sensitivity to sulfur availability, and analysed them in S-sufficient and S-replete condition both in standard and in chromium supplemented medium. SaATS2 expression is different in the two strains and presumably marks a different sulfur perception/exploitation in the Cr-tolerant. Its induction and silencing are compatible with a role in the transient tolerance increase induced by S-starvation. This enzyme can however hardly be responsible for the large cysteine production of the Cr-tolerant strain after starvation, suggesting that cytosolic rather than chloroplastic cysteine production is differently regulated in the two strains.

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.

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.

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.

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.

Sulfur starvation and chromium tolerance in Scenedesmus acutus: a possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes.

In a laboratory-selected Cr-tolerant strain of the unicellular green alga Scenedesmus acutus, the capacity to synthesize higher amounts of cysteine (Cys) and reduced glutathione (GSH) than the wild-type was demonstrated to underlie tolerance to Cd and Cr(VI). In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys, hence it was hypothesized that the sensitivity of the two strains to Cr(VI) could be modified after culturing in sulfate-deprived medium. Both strains were grown in the presence of different concentrations or in the absence of sulfate (sulfur-starved) and then assayed for Cr(VI) tolerance in standard medium. Unstarved, sulfur-starved and sulfur-replete cells (cells maintained in standard medium after S-starvation) were analysed for Cys, GSH and sulfur content. Sulfur-starved cells showed a greater tolerance to Cr(VI) than unstarved ones. The increased tolerance was ascribable to a transient physiological change and can be considered as specifically due to sulfur deprivation, since it was lost after a 3-day culture in standard medium and was not exhibited by nitrogen-starved cells. The comparison between Cys, GSH and sulfur content in sulfur-starved and sulfur-replete cells of the two strains suggests that the higher tolerance to Cr(VI) after S-starvation could depend on the up-regulation of sulfate uptake mechanisms, and that the primary reason for the higher tolerance to chromium in the selected strain could be due to greater sensitivity to the decrease in negative intracellular end-products (free Cys and GSH) leading to an earlier up-regulation of sulfate assimilation processes.

Differential responses to Cr(VI)-induced oxidative stress between Cr-tolerant and wild-type strains of Scenedesmus acutus (Chlorophyceae).

A wild-type and a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus were used to investigate if the difference in tolerance to Cr(VI) could depend on a different response to oxidative stress in terms of free cysteine (Cys) and reduced glutathione (GSH), and in preventing membrane lipid peroxidation. The growth of the two strains in standard medium in the presence of Cr(VI) 9.6, 19.2 or 38.4microM was compared, and the content of thiobarbituric acid reactive substances (TBARS) after a 4-day treatment was determined as marker of lipid peroxidation. The Cys and GSH contents were determined in both strains treated with Cr(VI) for 24h in HEPES buffer both enriched and not with sulphate. The treated algae were also subjected to recovery test in standard medium. The growth of wild type was inhibited at all Cr(VI) concentrations, while that of the Cr-tolerant strain only at the highest one. After exposure to 19.2microM Cr(VI), TBARS levels ranging from 0.091 to 0.263micromol/g d.w. were detected in the wild type, while only a slight increase (0.090+/-0.014micromol/g d.w.) was observed in the Cr-tolerant strain. Following treatment with 38.4microM Cr(VI), TBARS levels rose significantly and were similar in the two strains. The Cys content did not vary significantly in the cells exposed to Cr(VI) in either sulphate-lacking or sulphate-enriched buffer, and the differences between the two strains were insignificant. In the wild-type strain, the GSH content showed a significant downward trend with the increase in Cr(VI) concentration in the sulphate-lacking buffer, while it remained as high as the one of control cells in the sulphate-enriched buffer. In the Cr-tolerant strain, the GSH content did not vary significantly when the treatment took place in the sulphate-lacking buffer, while it showed a significant rise with the increase in Cr(VI) concentration in the sulphate-enriched buffer. The growth of both strains during recovery was significantly faster after treatment in the sulphate-enriched than in the sulphate-lacking buffer, the Cr-tolerant strain showing a much higher recovery capacity than the wild type. It appears that the Cr-tolerant strain, when exposed to Cr(VI) in the presence of a sulphur source, can increase GSH pool to levels not achievable by the wild type, and is thus able to recover better. This first report on the role of thiol compounds in Cr tolerance in algae suggests that tolerance to Cr(VI) in S. acutus could depend on a prompt up-regulation of the pathways leading to GSH synthesis.

Bioavailability, bioaccumulation and tolerance of chromium: consequences in the food chain of freshwater ecosystems.

Many abiotic and biotic factors can influence the partitioning equilibrium of heavy metals, thus influencing metal impact on aquatic environments. Unicellular algal species release soluble organic substances able to complex metals. In our laboratory a Cr-tolerant strain was selected and isolated from a wild type strain of Scenedesmus acutus. The exudates released by the two strains counteracted the growth inhibition caused by Cr(VI) and the exudates of the Cr-tolerant strain were more effective. On the contrary, the exudates did not reduce chromium toxicity to the cladoceran Daphnia magna. The reduction of chromium effect on algae seems the consequence of an algae-specific interaction among Cr(VI), exudates and algal cells. Chromium uptake resulted to be energy-dependent since bioaccumulation rate in subdued light condition was lower than at high light intensity. The effect of Cr(VI) on algae changed depending on metabolism of the cells and in particular it seemed to be related to the bioaccumulation rate. Tolerance in the selected strain could not be ascribed to a lower uptake of chromium. The difference in sensitivity to chromium between the two strains was exploited to evaluate if tolerance acquired by algae could have consequences for Daphnia. After treatment with Cr(VI), the two strains of S. acutus were used as food source for D. magna. The results indicate that chromium is accumulated by algae in a form not available for daphnids and that Cr tolerance acquired by the algae can be of some advantage to the consumer organism.

Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae).

Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 microM). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one.

Use of a mathematical model in the analysis of survival curves of Daphnia magna exposed to toxicants.

Chronic toxicity tests carried out on species of the genus Daphnia (Crustacea: Cladocera) are bioassays commonly used in ecotoxicology. Mortality in the cohorts exposed to toxicants can be examined by the analysis of survival curves. The shape of these curves may be very different because of inter-individual heterogeneity: the less rectangular is the shape the more different is the probability of dying of daphnids in the cohort. Aim of this paper is to analyze the characteristics-in particular the shape-of survival curves of cohorts of Daphnia magna exposed to heavy metals in chronic toxicity tests. Experimental curves were fitted by a mathematical model recently proposed, which puts emphasis just on this heterogeneity, described by the parameter of the model S(0). The information contained in S(0) is very useful for the present purposes: the higher the value of S(0) the less rectangular the shape of the curve and, as a consequence, the higher the age-at-death heterogeneity of the cohort is thought to be. The model contains a second parameter, omega, representing the maximum potential ability of the individuals to survive in a specific environment and is related with the maximal life span. The model fitted well the survival curves in most cases and both S(0) and omega showed statistically different values between treatments, useful for comparisons. It was concluded that S(0) provides a quantitative estimation of curve "rectangularization", useful to check different sensitivities to a specific toxicant concentration among daphnids belonging to the same cohort, while omega provides an estimate of maximal life span.

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.