Claudia Rivetti

Attenuation of emerging organic contaminants in a hybrid constructed wetland system under different hydraulic loading rates and their associated toxicological effects in wastewater

The capacity of a hybrid constructed wetland (CW) system consisting of two vertical flow (VF) CWs working alternatively (3m(2)), one horizontal flow (HF) CW (2m(2)) and one surface flow (FWS) CW (2m(2)) in series to eliminate 13 emerging organic contaminants (EOCs) under three different hydraulic loading rates (HLRs) (0.06, 0.13 and 0.18 m d(-1) considering the area of the two VF beds) was studied through a continuous injection experiment. General toxicity, dioxin-like activity, antimicrobial activity and estrogenicity were also measured under the highest hydraulic loading rate. The hybrid system was highly efficient on the removal of total injected EOCs (except for antibiotics, 43 ± 32%) at all three HLRs (87 ± 10%). The removal efficiency in the hybrid CW system showed to decrease as the HLR increased for most compounds. The VF wetlands removed most of the injected EOCs more efficiently than the other two CWs, which was attributable to the predominant aerobic degradation pathways of the VF beds (70 ± 21%). General toxicity was reduced up to 90% by the VF beds. Estrogenicity and dioxin-like activity were similarly reduced by the VF and the HF wetlands, whereas antimicrobial activity was mainly removed by the FWS wetland. Bearing this in mind, this injection study has demonstrated that the use of hybrid CW systems is a suitable wastewater technology for removing EOCs and toxicity even at high HLRs.

Effects of nanoparticles of TiO2 on food depletion and life-history responses of Daphnia magna.

The extent to which different forms of nanoparticles of titanium dioxide (nano-TiO2) aggregated with microalgae, decreased food levels and hence impaired growth, reproduction and fitness of Daphnia magna individuals were studied. Treatments included three different types of nano-TiO2 differing in their coating or crystalline structure but of similar primary size (20 nm) plus a micron-sized bulk material, two exposure levels (1, 10mg/l) and two food ration levels of the microalgae Chlorella vulgaris that included a non limiting (1.5 μgC/ml) and a limiting one (0.3 μgC/ml). Effects were assessed using standardized chronic tests and assays that maximized food depletion in the water column under semi-static and re-suspension conditions. Results indicated that the high ion levels in culture medium lead to the aggregation of nanoparticles followed by particle destabilization. Nanoparticle aggregates interacted with the algae cells, forming clusters. Large TiO2-algae agglomerates settled readily dramatically depleting the concentration of available food for D. magna. At limiting food rations food depletion by nanoparticle aggregation had dramatic effects on reproduction and fitness of exposed D. magna at 1mg/l irrespectively of the particle form. At high food rations effects were only observed for one of the nano-TiO2, P-25, at high exposure levels (10 mg/l) under both semi-static and particle re-suspension conditions, which suggest that P-25 effects were mediated by clogging the gut and hence diminishing food acquisition. These results indicate that nano-TiO2 may affect the transfer of energy throughout the planktonic aquatic food webs increasing the settlement of edible particles from the water column.

Identification of Metabolic Pathways in Daphnia magna Explaining Hormetic Effects of Selective Serotonin Reuptake Inhibitors and 4-Nonylphenol Using Transcriptomic and Phenotypic Responses

The molecular mechanisms explaining hormetic effects of selective serotonin reuptake inhibitors (SSRIs) and 4-nonylphenol in Daphnia magna reproduction were studied in juveniles and adults. Transcriptome analyses showed changes in mRNA levels for 1796 genes in juveniles and 1214 genes in adults (out of 15000 total probes) exposed to two SSRIs (fluoxetine and fluvoxamine) or to 4-nonylphenol. Functional annotation of affected genes was improved by assuming the annotations of putatively homologous Drosophila genes. Self-organizing map analysis and partial least-square regression coupled with selectivity ratio procedures analyses allowed to define groups of genes with specific responses to the different treatments. Differentially expressed genes were analyzed for functional enrichment using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes databases. Serotonin metabolism, neuronal developmental processes, and carbohydrates and lipid metabolism functional categories appeared as selectively affected by SSRI treatment, whereas 4-nonylphenol deregulated genes from the carbohydrate metabolism and the ecdysone regulatory pathway. These changes in functional and metabolic pathways are consistent with previously reported SSRIs and 4-nonylphenol hormetic effects in D. magna, including a decrease in reserve carbohydrates and an increase in respiratory metabolism.

Low environmental levels of neuro-active pharmaceuticals alter phototactic behaviour and reproduction in Daphnia magna

Assessing the risks of emerging contaminants, such as pharmaceuticals in the environment requires an understanding of their exposure regime and their effects at environmentally relevant concentrations across species. Daphnia magna represents an excellent invertebrate model species to study the mode of action of emerging pollutants, allowing the assessment of effects at different biological levels. The present study aims to test the hypothesis that different families of neuro-active pharmaceuticals at low environmentally relevant concentrations may lead to similar phenotypic responses in D. magna. Phenotypic traits included reproduction and behavioural responses. Selected pharmaceuticals were carbamazepine, diazepam and propranolol, three widely prescribed compounds, already detected at considerable levels in the environment (ng to few μg/L). Fluoxetine was also included in behavioural assays. The three tested neuro-active pharmaceuticals were able to enhance reproduction at 1ng/L of propranolol, 0.1μg/L of diazepam and 1μg/L of carbamazepine. Fluoxetine, carbamazepine and diazepam increased positive phototactic behaviour at concentrations ranging from 1, 10 and 100ng/L, respectively. Reported responses were nonmonotonic, which means that eco-toxicity testing of pharmaceuticals need to assess effects at the ng/L range.

Bacteria-mediated effects of antibiotics on Daphnia nutrition.

In polluted environments, contaminant effects may be manifested via both direct toxicity to the host and changes in its microbiota, affecting bacteria-host interactions. In this context, particularly relevant is exposure to antibiotics released into environment. We examined effects of the antibiotic trimethoprim on microbiota of Daphnia magna and concomitant changes in the host feeding. In daphnids exposed to 0.25 mg L(-1) trimethoprim for 24 h, the microbiota was strongly affected, with (1) up to 21-fold decrease in 16S rRNA gene abundance and (2) a shift from balanced communities dominated by Curvibacter, Aquabacterium, and Limnohabitans in controls to significantly lower diversity under dominance of Pelomonas in the exposed animals. Moreover, decreased feeding and digestion was observed in the animals exposed to 0.25-2 mg L(-1) trimethoprim for 48 h and then fed 14C-labeled algae. Whereas the proportion of intact algal cells in the guts increased with increased trimethoprim concentration, ingestion and incorporation rates as well as digestion and incorporation efficiencies decreased significantly. Thus, antibiotics may impact nontarget species via changes in their microbiota leading to compromised nutrition and, ultimately, growth. These bacteria-mediated effects in nontarget organisms may not be unique for antibiotics, but also relevant for environmental pollutants of various nature.

Depressing Antidepressant: Fluoxetine Affects Serotonin Neurons Causing Adverse Reproductive Responses in Daphnia magna

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants. As endocrine disruptive contaminants in the environment, SSRIs affect reproduction in aquatic organisms. In the water flea Daphnia magna, SSRIs increase offspring production in a food ration-dependent manner. At limiting food conditions, females exposed to SSRIs produce more but smaller offspring, which is a maladaptive life-history strategy. We asked whether increased serotonin levels in newly identified serotonin-neurons in the Daphnia brain mediate these effects. We provide strong evidence that exogenous SSRI fluoxetine selectively increases serotonin-immunoreactivity in identified brain neurons under limiting food conditions thereby leading to maladaptive offspring production. Fluoxetine increases serotonin-immunoreactivity at low food conditions to similar maximal levels as observed under high food conditions and concomitantly enhances offspring production. Sublethal amounts of the neurotoxin 5,7-dihydroxytryptamine known to specifically ablate serotonin-neurons markedly decrease serotonin-immunoreactivity and offspring production, strongly supporting the effect to be serotonin-specific by reversing the reproductive phenotype attained under fluoxetine. Thus, SSRIs impair serotonin-regulation of reproductive investment in a planktonic key organism causing inappropriately increased reproduction with potentially severe ecological impact.

Transcriptomic, biochemical and individual markers in transplanted Daphnia magna to characterize impacts in the field

Daphnia magna individuals were transplanted across 12 sites from three Spanish river basins (Llobregat, Ebro, Jucar) showing different sources of pollution. Gene transcription, feeding and biochemical responses in the field were assessed and compared with those obtained in re-constituted water treatments spiked with organic eluates obtained from water samples collected at the same locations and sampling periods. Up to 166 trace contaminants were detected in water and classified by their mode of action into 45 groups that included metals, pharmaceuticals, pesticides, illicit drugs, and other industrial compounds. Physicochemical water parameters differentiated the three river basins with Llobregat having the highest levels of conductivity, metals and pharmaceuticals, followed by Ebro, whereas the Jucar river had the greatest levels of illicit drugs. D. magna grazing rates and cholinesterase activity responded similarly than the diversity of riparian benthic communities. Transcription patterns of 13 different genes encoding for general stress, metabolism and energy processes, molting and xenobiotic transporters corroborate phenotypic responses differentiated sites within and across river basins. Principal Component Analysis and Partial Least Square Projections to Latent Structures regression analyses indicated that measured in situ responses of most genes and biomarkers and that of benthic macroinvertebrate diversity indexes were affected by distinct environmental factors. Conductivity, suspended solids and fungicides were negatively related with the diversity of macroinvertebrates cholinesterase, and feeding responses. Gene transcripts of heat shock protein and metallothionein were positively related with 11 classes of organic contaminants and 6 metals. Gene transcripts related with signaling paths of molting and reproduction, sugar, protein and xenobiotic metabolism responded similarly in field and lab exposures and were related with high residue concentrations of analgesics, diuretics, psychiatric drugs, β blockers, illicit drugs, trizoles, bisphenol A, caffeine and pesticides. These results indicate that application of omic technologies in the field is a promising subject in water management.

Identification of compounds bound to suspended solids causing sub-lethal toxic effects in Daphnia magna. A field study on re-suspended particles during river floods in Ebro River.

Identifying chemicals causing adverse effects in organisms present in water remains a challenge in environmental risk assessment. This study aimed to assess and identify toxic compounds bound to suspended solids re-suspended during a prolonged period of flushing flows in the lower part of Ebro River (NE, Spain). This area is contaminated with high amounts of organochlorine and mercury sediment wastes. Chemical characterization of suspended material was performed by solid phase extraction using a battery of non-polar and polar solvents and analyzed by GC-MS/MS and LC-MS/MS. Mercury content was also determined for all sites. Post-exposure feeding rates of Daphnia magna were used to assess toxic effects of whole and filtered water samples and of re-constituted laboratory water with re-suspended solid fractions. Organochlorine and mercury residues in the water samples increased from upstream to downstream locations. Conversely, toxic effects were greater at the upstream site than downstream of the superfund Flix reservoir. A further analysis of the suspended solid fraction identified a toxic component eluted within the 80:20 methanol:water fraction. Characterization of that toxic component fraction by LC-MS/MS identified the phytotoxin anatoxin-a, whose residue levels were correlated with observed feeding inhibition responses. Further feeding inhibition assays conducted in the lab using anatoxin-a produced from Planktothrix agardhii, a filamentous cyanobacteria, confirmed field results. This study provides evidence that in real field situation measured contaminant residues do not always agree with toxic effects.

Two‐generational effects of contaminants in Daphnia magna: Effects of offspring quality

The authors set up a protocol to perform a 2-generational ring test using the existing guidelines for the Daphnia magna reproduction test. It is well known in ecology that size and quality of offspring vary across the first clutches in D. magna and that certain chemicals affect offspring quality. Therefore, the origin of the second generation is an important factor to consider. Two-generational effects across first, second, and third clutches were evaluated using 4-nonylphenol; those across first and third clutches were evaluated using tributyltin, and those across the third clutch were evaluated using piperonyl butoxide. The compound showing the greatest aggravation of toxic effects between the parental and second generations was piperonyl butoxide, followed by 4-nonylphenol, whereas intergenerational effects of tributyltin varied across experiments. The studied chemicals affected the quantity and quality of the offspring produced by exposed females of the parental generation, those effects being greater in third-clutch neonates. Therefore, when third-clutch offspring were further exposed, they turned out to be more sensitive than the parental generation. The results are in line with those obtained in multigenerational studies using mammalian tests, which showed that, in many cases, effects on the second generation can be predicted by evaluating the quality of the offspring produced. Environ Toxicol Chem 2016;35:1470-1477.

Integrated environmental risk assessment of chemical pollution in a Mediterranean floodplain by combining chemical and biological methods

The Tablas de Daimiel National Park (TDNP) is a unique floodplain ecosystem in central Spain, serving as permanent resting and breeding areas for many waterbird species. In the last decades, this biodiversity hotspot has been severely endangered by poorly treated wastewater discharges from upstream urban communities arriving through its two major contributors, the Cigüela and Guadiana rivers. In this work, we analysed the potential risk of this constant input of micropollutants (estrogens, dioxin-like compounds and other endocrine disruptors) for the resident wildlife. We sampled 12 locations in TDNP and in the nearby Navaseca Pond during 2013, and performed a series of in-vivo and in-vitro bioassays, including Daphnia magna post-exposure feeding inhibition and recombinant yeast-based assays for dioxin-like and estrogenic activities. These results were then compared with the chemical composition of the samples, analysed by GC-MS/MS and LC-MS/MS, and evaluated according to their toxic potential as toxic equivalents or TEQ. The Navaseca Pond, heavily impacted by wastewater from the town of Daimiel, showed the highest levels of toxic compounds, estrogenic activity, and Daphnia toxicity. Conversely, the less impacted TDNP sites showed low residue levels of contaminants, low estrogenicity and dioxin-like activity and negligible toxicity. The results indicates that the current good chemical status of TDNP is menaced by both the inflow of wastewater treatment plants effluents from Guadiana and Cigüela rivers into TDNP tributaries and, as it occurs in the Navaseca Pond, by direct sewage discharges.