Combined cytotoxicity of ZnO nanoparticles and chlorpyrifos in the rainbow trout, Oncorhynchus mikyss, gonadal cell line RTG-2.

The toxicity of ZnO nanoparticles (ZnO NPs) in aquatic organisms has been extensively studied, but little information is available on the effects associated with their interaction with other contaminants. In this context, the in vitro effects of co-exposure of chlorpyrifos (CPF) and ZnO NPs on fish-derived cells were investigated. A selection of concentrations was tested in single and binary exposures: CPF (0.312 - 75 mg/L) and ZnO NPs (10 - 100 mg/L). Cytotoxicity was measured using commonly used cellular endpoints: Alamar Blue/CFDA-AM for viability and plasma membrane integrity, NRU for lysosomal disruption and MTT for mitochondrial function. In addition, specific mechanisms of toxicity for CPF and ZnO NPs were tested: acetylcholinesterase (AChE) activity and ROS generation, respectively. AChE was by far the most sensitive assay for single exposure to CPF. There was no concentration-response relationship for ROS after single exposure to ZnO NPs, but 10 mg/L produced significant effects only for this cellular endpoint. Co-exposure of CPF with 10 m/L of ZnO NPs produced significant effects in almost all endpoints tested, which were enhanced by co-exposure with 100 mg/L of ZnO NPs. AChE testing of additional co-exposures with bulk ZnO, together with the application of the Independent Action (IA) prediction model, which allowed us to draw more in-depth conclusions on the toxicological behavior of the mixture. Synergism was observed at 0.625 mg/L CPF concentration and antagonism at 5 mg/L CPF in mixtures containing 100 mg/L of both ZnO NPs and bulk ZnO. However, more cases of synergism between CPF and ZnO NPs occurred at intermediate CPF concentrations, demonstrating that nano-sized particles have a more toxic interaction with CPF than bulk ZnO. Therefore it can be argued that in vitro assays allow the identification of interaction profiles of NP-containing mixtures by achieving multiple endpoints with a large number of concentration combinations.

Developmental/reproductive effects and gene expression variations in Chironomus riparius after exposure to reclaimed water and its fortification with carbamazepine and triclosan.

The potential benefits of reclaimed water (RW) uses for environmental enhancement and restoration could become adverse impacts if RW does not meet the quality criteria that ensure wildlife preservation. RW can contain complex mixtures of micropollutants that may accumulate in sediment after environmental uses and affect benthic fauna. Therefore, we designed this study to assess the effects of RW on a sediment insect species used mainly in ecotoxicology (Chironomus riparius). Whole organism effects and gene expression were measured in a water sediment system after spiking RW as overlying water, which was renewed 3 times during the test. Development rate, emergence rate and fecundity were monitored after the 21-day exposure. Endocrine-related genes (EcR, ERR, E75, Vtg), cellular stress genes (hsp70, hsc70, hsp24, hsp10) and biotransformation genes (gp93, GSTd3, GPx, cyp4g) were assessed in larvae after the 10-day exposure. The experimental design also included single or binary fortifications of both test medium and RW, obtained by adding two emerging pollutants: carbamazepine (100 µg/L CBZ) and triclosan (20 µg/L TCS). The chemical characterisation of RW showed that 20 of the 23 screened emerging pollutants fell within the detection limit, 10 exceeded 0.01 µg/L (including CBZ) and three exceeded 0.1 µg/L (hydrochlorothiazide, atenolol, ibuprofen). The analytical measures of sediment (day 21) and overlying water (days 7, 14 and 21) were taken to know the water-sediment distribution of CBZ and TCS added to fortifications. CBZ distributed mainly in overlying water (110-164 µg/L and 73-100 µg/kg), while TCS showed a higher affinity to sediment (2.8-5.1 µg/L and 36-55 µg/kg). RW had significant effects in molecular terms (Vtg, hsp70, hsc70), but had no significant effects on the whole organism. Nevertheless, the single RW fortifications impaired both the development rate and fecundity, while the binary RW fortification impaired only fecundity. The most marked increase in EcR expression was observed for the binary RW fortification. Hsps, GSTd3 and cyp4g showed a similar tendency to that observed for EcR and Vtg in the binary and single RW fortifications. The binary mixture (CBZ and TCS together) in RW was toxic, but not in the medium tests. Therefore, the major concern of RW uses is apparently related to the interactivity between this complex matrix and any other pollutants possibly present in the environment where RW is applied. Our results underscore the need for raising awareness about RW effects, which can be achieved by ecotoxicological testing.

Effects on tadpole snail gene expression after exposure to vinclozolin.

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests.

Combining the assessment of apical endpoints and gene expression in the freshwater snail Physa acuta after exposure to reclaimed water.

Post-treatment wastewater reuses are diverse. Recreational and environmental restoration uses of reclaimed water (RW) can be potentially harmful to aquatic organisms. In this work the freshwater snail Physa acuta was exposed to RW (100%) and its dilution (RW 50%). A simple laboratory mixture of three emerging pollutants was used to address the complex problem of mixture toxicity of RW. Hence fortified reclaimed water (FRW), obtained by adding fluoxetine (400 µg FLX/L), perfluorooctane sulphonic acid (90 µg PFOS/L) and methylparaben (9 µg MP/L), was tested at two dilution percentages: 100% and 50%. The effects of the laboratory mixture of FLX, PFOS and MP on the test medium were also studied. Long-lasting effects, together with early molecular responses, were assessed. Fecundity (cumulative egg production) over 21 days and the hatching of produced eggs (F1) after another 21-day embryonic exposure were monitored. The gene expression of three genes was analysed after 24 h of exposure: two endocrine-related nuclear receptors (ERR and RXR) and one stress protein gene (Hsp70). This reproduction test, with additional assessments of the F1 recovered eggs' hatching success, showed that both RW and FRW significantly reduced fecundity. F1 hatching was affected only by FRW. The gene expression results showed that the RXR response was strikingly similar to the fecundity response, which suggests that this nuclear receptor is involved in the reproductive pathways of gastropods. ERR remained virtually unaltered. Hsp70 was overexpressed by the laboratory mixture in the test medium, but no effect was observed in the fortification of RW. This opposite effect and lack of response for F1 hatching produced by the laboratory mixture in the test medium highlighted the difficulty of predicting mixture effects. The experimental approach allowed us to test the effects caused by RW on P. acuta at different biological organisation levels. Thus, the combination of molecular biomarkers and ecological relevant endpoints is a good strategy to test complex mixtures like RW as it provides a framework to link mechanisms of action and whole organism effects when it is almost impossible to detect the pollutant(s) that cause toxic effects.

The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae).

Bisphenol S (BPS) is an industrial alternative to the endocrine disruptor bisphenol A (BPA), and can be found in many products labeled "BPA-free". Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1) crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3) that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13) which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 µg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control) were EcR (3.8), ERR (2), E74 (2.4), cyp18a1 (2.5), hsp70 (1.7), hsp40 (2.5), cyp4g (6.4), GPx (1.8), and GST (2.1), while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.

Bisphenol A (BPA) modulates the expression of endocrine and stress response genes in the freshwater snail Physa acuta.

Bisphenol A (BPA), a known endocrine disrupting chemical (EDC) that can mimic the action of oestrogens by interacting with hormone receptors, is potentially able to influence reproductive functions in vertebrates and invertebrates. The freshwater pulmonate Physa acuta is a sensitive organism to xenobiotics appropriate for aquatic toxicity testing in environmental studies. This study was conducted to explore the effects of BPA on the Gastropoda endocrine system. The effects following a range of exposure times (5-96h) to BPA in P. acuta were evaluated at the molecular level by analysing changes in the transcriptional activity of the endocrine-related genes oestrogen receptor (ER), oestrogen-related receptor (ERR), and retinoid X receptor (RXR), as well as in genes involved in the stress response, such as hsp70 and hsp90. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis showed that BPA induced a significant increase in the mRNA levels of ER, ERR, and RXR, suggesting that these receptors could be involved in similar pathways or regulation events in the endocrine disruptor activity of this chemical at the molecular level in Gastropoda. Additionally, the hsp70 expression was upregulated after 5 and 72h of BPA exposures, but hsp90 was only upregulated after 5h of BPA exposure. Finally, we assessed the glutathione-S-transferase (GST) activity after BPA treatment and found that it was affected after 48h. In conclusion, these data provide, for the first time, evidences of molecular effects produced by BPA in the endocrine system of Gastropoda, supporting the potential of ER, ERR and RXR as biomarkers to analyse putative EDCs in ecotoxicological studies. Moreover, our results suggest that P. acuta is an appropriate sentinel organism to evaluate the effect of EDCs in the freshwater environment.

Genotoxic effects of vinclozolin on the aquatic insect Chironomus riparius (Diptera, Chironomidae).

Vinclozolin (Vz) is a pollutant found in aquatic environments whose antiandrogenic effects in reproduction are well known in mammals. Although its reproductive effects have been less studied in invertebrates, other effects, including genotoxicity, have been described. Therefore, in this work, we studied the genotoxic effects of Vz in the freshwater benthic invertebrate Chironomus riparius. DNA damage was evaluated with the comet assay (tail area, olive moment, tail moment and % DNA in tail), and the transcriptional levels of different genes involved in DNA repair (ATM, NLK and XRCC1) and apoptosis (DECAY) were measured by RT-PCR. Fourth instar larvae of C. riparius, were exposed to Vz for 24 h at 20 and 200 µg/L. The Vz exposures affected the DNA integrity in this organism, since a dose-response relationship occurred, with DNA strand breaks significantly increased with increased dose for tail area, olive moment and tail moment parameters. Additionally, the lower concentration of Vz produced a significant induction of the transcripts of three genes under study (ATM, NLK and XRCC1) showing the activation of the cellular repair mechanism. In contrast, the expression of these genes with the highest concentration were downregulated, indicating failure of the cellular repair mechanism, which would explain the higher DNA damage. These data report for the first time the alterations of Vz on gene transcription of an insect and confirm the potential genotoxicity of this compound on freshwater invertebrates.

Effects of exposure to pharmaceuticals (diclofenac and carbamazepine) spiked sediments in the midge, Chironomus riparius (Diptera, Chironomidae).

Human and veterinary pharmaceuticals and degradation products are continuously introduced into the environment. To date, there is a lack of information about the effects of pharmaceuticals in spiked toxicity tests with non-target organisms. In this study, we have evaluated the effects of exposure to two common pharmaceuticals in the midge Chironomus riparius in spiked sediment experiments. The selected pharmaceuticals are the nonsteroidal anti-inflammatory drug (NSAID): diclofenac (DF) and the anti-depressant drug carbamazepine (CBZ). In order to assess the effects of the pharmaceuticals, a chronic toxicity test with the midge was carried out. The endpoints survival, growth and developmental stage by means of biomass, were measured after 10days, and emergence rates and sex-ratio (male/female) were measured after 21days of exposure. Significant mortality was observed in organisms at day 10 with a 40% of larvae surviving in the highest exposure concentration of CBZ. DF decreased the emergence ratio with respect to the controls in organisms exposed at concentrations of 34.0µg·g-1 whereas CBZ reduced the growth of the midges (30,6% with respect to the control) and induced a significant change in sex-ratio at concentrations of 31.4µg·g-1. The results obtained in the present study indicate possible adverse effects on aquatic invertebrates, which should be taken into account for environmental risk assessment of pharmaceutical compounds in sediments.

Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism.

The freshwater snail Physa acuta is a sensitive organism to xenobiotics that is appropriate for toxicity testing. Cadmium (Cd) is a heavy metal with known toxic effects on several organisms, which include endocrine disruption and activation of the cellular stress responses. There is scarce genomic information on P. acuta; hence, in this work, we identify several genes related to the hormonal system, the stress response and the detoxification system to evaluate the effects of Cd. The transcriptional activity of the endocrine-related genes oestrogen receptor (ER), oestrogen-related receptor (ERR), and retinoid X receptor (RXR), the heat shock proteins genes hsp70 and hsp90 and a metallothionein (MT) gene was analysed in P. acuta exposed to Cd. In addition, the hsp70 and hsp90 genes were also evaluated after heat shock treatment. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis showed that Cd presence induced a significant increase in the mRNA levels of ER, ERR and RXR, suggesting a putative mode of action that could explain the endocrine disruptor activity of this heavy metal at the molecular level on Gastropoda. Moreover, the hsp70 gene was upregulated after 24-h Cd treatment, but the hsp90 gene expression was not affected. In contrast, the hsp70 and hsp90 genes were strongly upregulated during heat shock response. Finally, the MT gene expression showed a non-significant variability after Cd exposure. In conclusion, this study provides, for the first time, information about the effects of Cd on the endocrine system of Gastropoda at the molecular level and offers new putative biomarker genes that could be useful in ecotoxicological studies, risk assessment and bioremediation.

Vinclozolin alters the expression of hormonal and stress genes in the midge Chironomus riparius.

Vinclozolin is a fungicide used in agriculture that can reach aquatic ecosystems and affect the organisms living there. Its effects have been intensively studied in vertebrates, where it acts as an antiandrogen, but there is a lack of information about its mechanistic effects on invertebrates. In this work, we analyzed the response of genes related to the endocrine system, the stress response, and the detoxification mechanisms of Chironomus riparius fourth instar larvae after 24h and 48h exposures to 20 (69.9nM), 200 (699nM), and 2000µg/L (6.99µM) of Vinclozolin. Survival analysis showed that this compound has low toxicity, as it was not lethal for this organism at the concentrations used. However, this fungicide was shown to modify the transcriptional activity of the ecdysone response pathway genes EcR, E74, and Kr-h1 by increasing their mRNA levels. While no changes were observed in disembodied, a gene related with the ecdysone synthesis metabolic pathway, Cyp18A1, which is involved in the inactivation of the active form of ecdysone, was upregulated. Additionally, the expression of two genes related to other hormones, FOXO and MAPR, did not show any changes when Vinclozolin was present. The analysis of stress response genes showed significant changes in the mRNA levels of Hsp70, Hsp24, and Gp93, indicating that Vinclozolin activates the cellular stress mechanisms. Finally, the expressions of the genes Cyp4G and GstD3, which encode enzymes involved in phase I and phase II detoxification, respectively, were analyzed. It was found that their mRNA levels were altered by Vinclozolin, suggesting their involvement in the degradation of this compound. For the first time, these results show evidence that Vinclozolin can modulate gene expression, leading to possible significant endocrine alterations of the insect endocrine system. These results also offer new clues about the mode of action of this compound in invertebrates.

Linking embryo toxicity with genotoxic responses in the freshwater snail Physa acuta: single exposure to benzo(a)pyrene, fluoxetine, bisphenol A, vinclozolin and exposure to binary mixtures with benzo(a)pyrene.

Genotoxic effects on fauna after waterborne pollutant exposure have been demonstrated by numerous research programmes. Less effort has been focused on establishing relationship between genotoxicity and long-term responses at higher levels of biological organization. Taking into account that embryos may be more sensitive indicators of reproductive impairment than alterations in fertility, we have developed two assays in multiwell plates to address correlations between embryo toxicity and genotoxicity. The potential teratogenicity was assessed by analyzing abnormal development and mortality of Physa acuta at embryonic stage. Genotoxicity was measured by the micronucleus (MN) test using embryonic cells. Our results showed that linkage between genotoxicity and embryo toxicity depends on mechanisms of action of compounds under study. Embryo toxic responses showed a clear dose-related tendency whereas no clear dose-dependent effect was observed in micronucleus induction. The higher embryo toxicity was produced by benzo(a)pyrene exposure followed by fluoxetine and bisphenol A. Vinclozolin was the lower embryo toxic compound. Binary mixtures with BaP always resulted in higher embryo toxicity than single exposures but antagonistic effects were observed for MN induction. Benzo(a)pyrene produced the higher MN induction at 0.04 mg/L, which also produced clear embryo toxic effects. Fluoxetine did not induce cytogenetic effects but 0.25mg/L altered embryonic development. Bisphenol A significantly reduced hatchability at 0.5mg/L while MN induction appeared with higher treatments than those that start causing teratogenicity. Much higher concentration of vinclozolin (5mg/L) reduced hatchability and induced maximum MN formation. In conclusion, while validating one biomarker of genotoxicity and employing one ecologically relevant effect, we have evaluated the relative sensitivity of a freshwater mollusc for a range of chemicals. The embryo toxicity test is a starting point for the development of a life cycle test with freshwater snails even for undertaking multigeneration studies focused on transgenerational effects.

Environmental risk assessment for the serotonin re-uptake inhibitor fluoxetine: Case study using the European risk assessment framework.

The serotonin re-uptake inhibitor fluoxetine was selected for an environmental risk assessment, using the most recent European guideline (EMEA 2006) within the European Union (EU)-funded Environmental Risk Assessment of Pharmaceuticals (ERAPharm) project due to its environmental persistence, acute toxicity to nontarget organisms, and unique pharmacokinetics associated with a readily ionizable compound. As a widely prescribed psychotropic drug, fluoxetine is frequently detected in surface waters adjacent to urban areas because municipal wastewater effluents are the primary route of entry to aquatic environments. In Phase I of the assessment, the initial predicted environmental concentration of fluoxetine in surface water (initial PEC(SW)) reached or exceeded the action limit of 10 ng/L, when using both a default market penetration factor and prescription data for Sweden, Germany, and the United Kingdom. Consequently, a Phase II risk assessment was conducted in which green algae were identified as the most sensitive species with a NOEC of <0.6 microg/L. From this value, a predicted no effect concentration for surface waters (PNEC(SW)) of 0.012 microg/L was derived. The PEC/PNEC ratio was above the trigger value of 1 in worst-case exposure scenarios indicating a potential risk to the aquatic compartment. Similarly, risks of fluoxetine for sediment-dwelling organisms could not be excluded. No risk assessment was conducted for the terrestrial compartment due to a lack of data on effects of fluoxetine on soil organisms. The need for a separate risk assessment for the main metabolite of fluoxetine, norfluoxetine, was not conducted because of a lack of fate and effect studies. Based on published data, fluoxetine and norfluoxetine appeared to have a low to moderate bioaccumulation potential, which should be confirmed in formal studies according to OECD guidelines. Exposure assessments for fluoxetine according to the current framework rely heavily on K(OC) and K(OW) values. This approach is problematic, because fluoxetine is predominantly a cationic substance at environmental pH values. Consequently, the fate of fluoxetine (and other ionic substances) cannot be predicted using partition coefficients established for nonionic compounds. Further, published estimates for partition coefficients of fluoxetine vary, resulting in considerable uncertainties in both the exposure and environmental risk assessments of fluoxetine.

Recommendations on the environmental risk assessment of pharmaceuticals: Effect characterization.

The effects testing of pharmaceuticals consists of a tiered investigation of ecotoxicological endpoints. However, effects testing has to be performed only when the predicted environmental concentrations (PECs) of pharmaceuticals are above certain action limits. To study the appropriateness of these action limits, a literature search was performed for pharmaceuticals with predicted no-effect concentrations (PNECs) close to or below the action limits. Some human pharmaceuticals showed effects at concentrations ≤100 ng/L, mostly in nonstandard fish or invertebrate tests. In addition, antibiotics and parasiticides sometimes had effects at concentrations <10 mg/kg soil. To help in identifying pharmaceuticals that should undergo effects testing although their PECs are below the action limits, "however clauses" are postulated for pharmaceuticals that are potentially persistent, bioaccumulative, carcinogenic, mutagenic, or reproductively toxic. Effects testing should also be performed for pharmaceuticals that 1) affect target structures that are conserved across species, 2) have a high potency or a small therapeutic margin, 3) are from a new therapeutic class, and 4) are structurally similar to compounds with known effects. Furthermore, suggestions for improving the effects testing of pharmaceuticals are made. These include inter alia chronic effects testing as a general approach, the use of invertebrate tests including sexual reproduction, the application of endpoints reflecting the mode of action of the drug or known side effects, and the simulation of more realistic exposure conditions in terrestrial laboratory tests.

Assessing the effects of fluoxetine on Physa acuta (Gastropoda, Pulmonata) and Chironomus riparius (Insecta, Diptera) using a two-species water-sediment test.

Fluoxetine has been tested in a two-species water-sediment system, which allowed a two-generation study with Chironomus riparius and a partial life-cycle with the freshwater snail Physa acuta to be performed at the same time. The design considered the continuous application of fluoxetine to overlaying water for nominal concentrations of 31.25, 62.5, 125 and 250 microg/L. A fifth treatment (87.5 microg/L) level consisted of pulse applications once a week. Measures of water and sediment concentrations were determined once a week and at the end of experiment (day 44), respectively. The fate study demonstrated that water dissipation can be explained by partitioning of fluoxetine to sediment. At the end of experiment, the percentage of detected fluoxetine was up to 10-fold higher in sediment than in overlaying water. The employed two-species test allowed distinguishing, in the same exposure conditions, effects due to waterborne exposure together ingestion at the sediment surface (freshwater grazing snail P. acuta) and exposure by burrowing activities (sediment-dwelling insect larvae C. riparius). The effect assessment showed a stimulation of P. acuta reproduction at lower concentrations (31.25 and 62.5 microg/L), while the opposite effect was observed at the highest treatment (250 microg/L). Additional studies should be conducted to establish if the statistically significant differences observed in F0 sex ratio at the 62.5 microg/L and F1 adult emergence at 31.25 microg/L of C. riparius have a toxicological significance. This study showed that fluoxetine can affect reproduction of freshwater molluscs. The results of the present study may contribute to knowledge on ecotoxicology of pharmaceuticals, about which little data is available. The possible consequences and implications for targeting the environmental risk assessment of fluoxetine are discussed.