Removal efficiency for emerging contaminants in a WWTP from Madrid (Spain) after secondary and tertiary treatment and environmental impact on the Manzanares River.

The effluents from wastewater treatment plants (WWTPs) can be an important contamination source for receiving waters. In this work, a comprehensive study on the impact of a WWTP from Madrid on the aquatic environment has been performed, including a wide number of pharmaceuticals and pesticides, among them those included in the European Watch List. 24-h composite samples of influent (IWW) and effluent wastewater after secondary (EWW2) and after secondary + tertiary treatment (EWW3) were monitored along two campaigns. Average weekly concentrations in IWW and EWW2 and EWW3 allowed estimating the removal efficiency of the WWTP for pharmaceutical active substances (PhACs). In addition, the impact of EWW3 on the water quality of the Manzanares River was assessed, in terms of PhAC and pesticide concentrations, through analysis of the river water collected upstream and downstream of the discharge point. After a preliminary risk assessment, a detailed evaluation of the impact on the aquatic environment, including a toxicological study and screening of pharmaceutical metabolites, was made for the seven most relevant PhACs: sulfamethoxazole, azithromycin and clarithromycin (antibiotics), metoprolol (antihypertensive), diclofenac (anti-inflammatory/analgesic), irbesartan (antihypertensive), and the antidepressant venlafaxine. Among selected PhACs, irbesartan, clarithromycin and venlafaxine presented moderate or high risk in the river water downstream of the discharge. Albeit no acute toxicity was detected, more detailed studies should be carried out for these substances, including additional toxicological studies, to set up potential sublethal and chronic effects on aquatic organisms.

Global impact of diet and temperature over aquaculture of Octopus vulgaris paralarvae from a transcriptomic approach.

Common octopus, Octopus vulgaris, is an economically important cephalopod species. However, its rearing under captivity is currently challenged by massive mortalities previous to their juvenile stage due to nutritional and environmental factors. Dissecting the genetic basis and regulatory mechanism behind this mortality requires genomic background knowledge. A transcriptomic sequencing of 10 dph octopus paralarvae from different experimental conditions was constructed via RNA-seq. A total of 613,767,530 raw reads were filtered and de novo assembled into 363,527 contigs of which 82,513 were annotated in UniProt carrying also their GO and KEGG information. Differential gene expression analysis was carried out on paralarvae reared under different diet regimes and temperatures, also including wild paralarvae. Genes related to lipid metabolism exhibited higher transcriptional levels in individuals whose diet includes crustacean zoeas, which had an impact over their development and immune response capability. High temperature induces acclimation processes at the time that increase metabolic demands and oxidative stress. Wild individuals show an expression profile unexpectedly similar to Artemia fed individuals. Proteomic results support the hypothesis revealed by transcriptional analysis. The comparative study of the O. vulgaris transcriptomic profiles allowed the identification of genes that deserve to be further studied as candidates for biomarkers of development and health. The results obtained here on the transcriptional variations of genes caused by diet and temperature will provide new perspectives in understanding the molecular mechanisms behind nutritional and temperature requirements of common octopus that will open new opportunities to deepen in paralarvae rearing requirements.

Identification of very long-chain (>C24) fatty acid methyl esters using gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with atmospheric pressure chemical ionization source.

Gas chromatography coupled to a quadrupole time-of-flight mass analyzer (QTOF) with an atmospheric pressure chemical ionization (APCI) source has been tested to study the ionization and mass spectrometric behavior of long-chain and very long-chain polyunsaturated fatty acids (LC-PUFAs, C18-24; VLC-PUFAs, >C24). The protonated molecule ([M+H]+), measured at accurate mass, became the base peak of the spectrum for all the studied compounds and was promoted by the addition of water into the source. This fact overcame the existing difficulties for the identification of VLC-PUFAs when using an electron ionization source (EI). The extensive fragmentation of PUFAs in this source is the main drawback due to the fact that since reference standards are not commercially available, final identification relies on retention time estimation. The application of GC-APCI-QTOF to the screening of lipid extracts from the eyes of different fish species added confidence to the identification of several VLC-PUFAs. Further investigation of ion ratios allowed to predict the position of key double bonds enabling the classification of VLC-PUFAs as ω3 or ω6 compounds. VLC-PUFAs spectra found in biological samples were compared to those obtained from corresponding peaks found in heterologous expression experiments of fish's Elovl4.

Effects of ibuprofen and carbamazepine on the ion transport system and fatty acid metabolism of temperature conditioned juveniles of Solea senegalensis.

The increasing presence of pharmaceuticals in aquatic environments in the last decades, derived from human and veterinary use, has become an important environmental problem. Previous studies have shown that ibuprofen (IB) and carbamazepine (CBZ) modify physiological and biochemical processes in Senegalese sole (Solea senegalensis) in a temperature-dependent manner. In other vertebrates, there is evidence that both of these pharmaceuticals interfere with the 'arachidonic acid (AA) cascade', which is responsible for the biosynthesis of numerous enzymes that are involved in the osmoregulatory process. The present work aims to study the temperature-dependent effects of these two pharmaceuticals on several biochemical and molecular parameters in Senegalese sole. Regarding osmoregulation, Na+, K+ -ATPase enzyme activity was determined in the gills, kidney and intestine, and the expressions of both Na+, K+ -ATPase 1α-subunit isoforms (ATP1A1a and ATP1A1b) were quantified in gills. Gill prostaglandin-endoperoxide synthase-2 (PTGS2) gene expression and fatty acid composition were selected to determine the interference of both pharmaceuticals with the AA cascade. Senegalese sole juveniles, acclimatised at 15°C or 20°C, were exposed through intraperitoneal injection to IB (10mg/kg) and CBZ (1mg/kg) for 48h. Non-injected fish (Control) and those injected with the carrier (sunflower oil; S.O.), acclimated at each of the two temperatures, were used for comparison. The results show that IB directly affected the osmoregulatory mechanisms that alter gill and intestine Na+, K+ -ATPase activities. In addition, the copy number of ATP1A1a was higher at 20°C than at 15°C, which could be a direct response to the temperature variation. The gene expression of PTGS2 was affected by neither drug administration nor acclimation temperature. Nevertheless, detailed analysis of AA and eicosapentaenoic acid (EPA) percentages revealed a CBZ-derived effect in the fatty acid composition of the gills.

Drugs of environmental concern modify Solea senegalensis physiology and biochemistry in a temperature-dependent manner.

The alerted presence in recent decades of pharmaceuticals has become an issue of environmental concern, and most of the mechanisms of biotransformation and biochemical and physiological responses to them in fish are still unknown, as well as the influence of water temperature in their ability to cope with them. This study aims to detect the main effects of two of the most widespread drugs on a set of physiological and biochemical markers in Solea senegalensis. Sole juveniles acclimatized at 15 and 20 °C were administered an intraperitoneal injection of the non-steroidal anti-inflammatory drug ibuprofen (IB; 10 mg/kg) and the anti-convulsant drug carbamazepine (CBZ; 1 mg/kg). Two days after the injection, liver, muscle and plasma were sampled. Liver enzymatic activities of 15 °C acclimated fish were more responsive to pharmaceuticals than those acclimated at 20 °C, especially for CYP450-related activities (7-ethoxyresorufin (EROD), 7-methoxyresorufin (MROD), 3-cyano-7-ethoxycoumarin (CECOD) and 7-benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD)) and uridine diphosphate glucuronosyltransferase (UDPGT). Cytosolic anti-oxidant enzyme activities and glutathione S-transferase (GST) did not show a clear effect of temperature. Glucose and transferase activities in plasma were not affected by the treatments, while ammonium, osmolality and lactate were affected by both pharmaceuticals. Plasma triglycerides were affected in a temperature-dependent manner, and creatinine was only responsive to CBZ injection. HSP70 levels in muscle were only affected by CBZ injection. Some of the physiological identified responses to IB and CBZ are proposed as endpoints in further chronic studies.

Hepatic and branchial xenobiotic biomarker responses in Solea spp. from several NW Mediterranean fishing grounds.

The common sole, Solea solea and the Senegalese sole, Solea senegalensis are two important commercial benthic species that coexist in the NW Mediterranean Sea. Several common biomarkers of chemical exposure were measured in two organs (liver and gills) involved in a different degree in biotransformation and detoxification processes. These parameters were: phase I cytochrome P450 CYP1A-dependent ethoxyresorufin O-deethylase and carboxylesterase activities, phase II glutathione S-transferase activity and the enzymatic antioxidants: catalase, glutathione reductase and glutathione peroxidase. Principal Component Analysis (PCA) considering biometric variables (size and weight) and all liver and gill biomarkers discriminated at a certain extent individuals of both species collected at the different fishing grounds. Esterase inhibition by the organophosphorus pesticides dichlorvos and diazinon was also compared in vitro in muscle, liver and gill of the two species revealing a differential sensitivity. The use of benthic sole in pollution monitoring of Southern Europe is discussed as local sentinel in respect to other benthic fish from more Northern latitudes.

Aquatic pollution may favor the success of the invasive species A. franciscana.

The genus Artemia consists of several bisexual and parthenogenetic sibling species. One of them, A. franciscana, originally restricted to the New World, becomes invasive when introduced into ecosystems out of its natural range of distribution. Invasiveness is anthropically favored by the use of cryptobiotic eggs in the aquaculture and pet trade. The mechanisms of out-competition of the autochthonous Artemia by the invader are still poorly understood. Ecological fitness may play a pivotal role, but other underlying biotic and abiotic factors may contribute. Since the presence of toxicants in hypersaline aquatic ecosystems has been documented, our aim here is to study the potential role of an organophosphate pesticide, chlorpyrifos, in a congeneric mechanism of competition between the bisexual A. franciscana (AF), and one of the Old World parthenogenetic siblings, A. parthenogenetica (PD). For this purpose we carried out life table experiments with both species, under different concentrations of the toxicant (0.1, 1 and 5µg/l), and analyzed the cholinesterase inhibition at different developmental stages. The results evidence that both, AF and PD, showed an elevated tolerance to high ranges of chlorpyrifos, but AF survived better and its fecundity was less affected by the exposure to the pesticide than that of PD. The higher fecundity of AF is a selective advantage in colonization processes leading to its establishment as NIS. Besides, under the potential selective pressure of abiotic factors, such as the presence of toxicants, its higher resistance in terms of survival and biological fitness also indicates out-competitive advantages.

Comparative analysis of selected biomarkers and pesticide sensitivity in juveniles of Solea solea and Solea senegalensis.

The common sole, Solea solea (Linneus, 1758), and the Senegalese sole, Solea senegalensis (Kaup, 1858), are two important commercial species that coexist in the NW Mediterranean. In order to assess the species' ability to respond to chemical insults, a comparison of activities on enzymes involved in xenobiotic metabolism was carried out. Juveniles of both species were sampled in winter 2011 from the Ebro Delta region, and activities of selected enzymes such as acetylcholinesterase (AChE), carboxylesterase (CbE), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) were determined in several tissues. Lipid peroxidation (LP) levels in plasma were measured as a sign of oxidative stress. In vitro exposures to selected pesticides were contrasted, analysing AChE and CbE activities in several tissue homogenates. Overall, enzymatic activities were higher in S. solea except for gill GST and CbE and kidney GST, while plasmatic LP levels were similar. In vitro contrasts revealed lower IC50 values for CbE activities in S. solea, suggesting a greater buffer capacity of this enzyme to potentially reduce pesticide toxicity over AChE.

Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: a proteomic approach.

Gilthead sea bream Sparus aurata is the most commercialized Mediterranean aquacultured fish species. Ivermectin has recently (experimentally) started to be used to control ectoparasitic infestations in Mediterranean cultured marine fish. The potential hepatotoxicity of ivermectin was investigated in gilthead sea bream juveniles (35g) following oral administration at the recommended dose of 0.2 mgkg(-1) fish for 10d. Difference Gel Electrophoresis Technology (DIGE) was used to study the effect of this treatment in gilthead sea bream liver protein profile under routine culture conditions. The 2D-DIGE protein maps obtained were analyzed using the DeCyder 6.5 software. The results obtained showed significant changes in the expression of 36 proteins respect to the control group. Among these proteins, six increased in abundance, and 30 decreased. Spot showing differential expression respect to the control were analyzed by mass spectrometry and database search, which resulted in three positive identifications corresponding to hepatic proteins involved in lipid metabolism (apoA-I), oxidative stress responses and energy generation (beta-globin, ATP synthase subunit beta). These proteins have not been previously associated to invermectin effect.

Sublethal zinc exposure has a detrimental effect on reproductive performance but not on the cyst hatching success of Artemia parthenogenetica.

The sublethal zinc toxicity to Artemia parthenogenetica as regards the possibility of colonization of zinc polluted salterns by means of cysts has been assessed by a cyst hatching assay and a life table approach. Emergence and hatching at different times as well as the whole hatching profile were taken as end-points for evaluating success of development. Demographic and reproductive parameters calculated according to the Lotka equation were used as an indicator of the chronic toxicity of the population. No adverse effects of waterborne zinc were found on hatching and emergence of cysts of A. parthenogenetica at any of the concentrations tested (0.01 mg/l, 0.1 mg/l, 0.5 mg/l, 1 mg/l, 5 mg/l). Chronic zinc exposure at 0.08 mg/l had detrimental effects on A. parthenogenetica fecundity, as detected by a decrease in the percentage of fertile females, which in turn produces a decrease in r. Chronic toxicity of zinc may be a limiting step for A. parthenogenetica colonization and the establishing of permanent populations in zinc-polluted brine ponds.

Acute toxicity of dichlorvos to Aphanius iberus (Cuvier & Valenciennes, 1846) and its anti-cholinesterase effects on this species.

This study evaluates the toxic effects of the organophosphate pesticide (OP) dichlorvos to the endangered Iberian toothcarp (Aphanius iberus). To this end, the lethal toxicity of dichlorvos based on 96h-LC50 bioassays was determined in saline water (50g/L), and in vivo effects of dichlorvos on cholinesterase (ChE) activity were investigated in adult female and male specimens. The 96h-LC50 value determined by probit analysis was 3.17mg/L (95% confidence limits: 1.34-3.97). The characterisation of the ChE using different substrates and specific inhibitors was also carried out in head and muscle tissues. Acetylthiocholine was the substrate preferred by both head and muscle ChE in males and females. Eserine sulphate and BW284C51 significantly inhibited both head and muscle enzyme activity at low concentrations (muM range), and iso-OMPA had no significant effect. These results indicate that in the head and muscle the predominant ChE form is acetylcholinesterase (AChE) for both sexes. The kinetic parameters for ChE activity (Km and Vmax) were similar in both sexes. The 96h-LC50 value obtained for adult specimens of Iberian toothcarp was 3.17mg/L. ChE activity in head and body tissues of both sexes was significantly inhibited in all concentrations tested (0.5, 1, 2 and 4mg/L) after "in vivo" dichlorvos exposure. However, Iberian toothcarp was able to tolerate high concentrations of dichlorvos, and resist high levels of brain and muscle ChE inhibition without mortality. Both ChE inhibition and recovery followed a similar time-course pattern in response to sub-lethal exposure to dichlorvos (1mg/L), and the enzyme activity did not return to control levels after 96h in clean water. The results of this study show that ChE activity is a good biomarker of exposure to OP in the Iberian toothcarp adults.

Assessment of the efficacy of Artemia sp (Crustacea) cysts chorion as barrier to chlorpyrifos (organophosphorus pesticide) exposure. Effect on hatching and survival.

In order to reveal the efficacy of the Artemia cysts chorion as barrier to the organophosphorus pesticide chlorpyrifos, whole and decapsulated cysts have been exposed to 10 mg L(-1) chlorpyrifos in sea water during hydration and hatching phase, separately. The concentration of chlorpyrifos in capsulated and decapsulated cysts after exposure has been determined in order to elucidate the efficacy of chorion as protection to the embryo. The results obtained demonstrate the ability of the cysts chorion to obstruct the pass of chlorpyrifos molecules through this protection structure. Thus, the concentration of chlorpyrifos in exposed decapsulated cysts is higher than in exposed whole cysts. Moreover, after removing the chorion of exposed cysts, the concentration of chlorpyrifos in the embryo was lower than that of cysts exposed, what would demonstrate the retention of chlorpyrifos molecules by the shell. Hatching was not severely affected by exposure to the insecticide whereas survival at 44 h of the nauplii exposed to chlorpyrifos was significantly different from the controls. Survival of nauplii hatched from exposed decapsulated cysts was higher than that from those hatched from exposed whole cysts, probably because of the lower vitality of the latter, due to depletion of energy reserves during hatching.

Comparative toxicokinetics of cadmium in Artemia.

The toxicokinetics of cadmium was determined for five populations belonging to four species of Artemia (A. salina, A. parthenogenetica, A. franciscana, and A. persimilis) using a bicompartmental model. The effects of sublethal cadmium concentration on the kinetic parameters in A. parthenogenetica were determined. The BCF values are inversely related to the concentration, as is the uptake rate (ku), whilst the elimination rate (ke) constant seems to be directly related to the exposure concentration. Values corresponding to the rate of metal influx (I) remain relatively constant (0.2477 to 0.4455 microg/g.h) in the concentration range from 0.1 to 1 mg Cd/L, and are higher (1.098 microg/g.h) at an exposure to 10 mg Cd/L. The cadmium accumulation pattern seems well conserved in the genus and is characterized by a fast elimination of the metal with Ke ranging from 0.0050 to 0.0231 h(-1). A. persimilis displays a different model to that corresponding to the other studied species exposed to the same cadmium concentration, presenting a low uptake rate constant (1.0564 mL/g. h) and a low BCF (211.3 mL/g).

Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica.

In this study, the acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos to two different species of Artemia (A. salina and A. parthenogenetica) was evaluated. In addition, the in vivo effect of these two pesticides on cholinesterase (ChE) activity of both A. salina and A. parthenogenetica was also determined. The characterisation of the ChE, using different substrates and specific inhibitors, and the normal range of activity in non-exposed individuals were previously investigated for both species. The results obtained indicate that the ChE of A. salina is different from that of A. parthenogenetica and that both enzymes cannot be classified neither as acetylcholinesterase nor as butyrylcholinesterase since they show intermediary characteristics between the two vertebrate forms. The range of normal ChE activity was 2.65+/-0.15 U/mg protein for A. salina, and 3.69+/-0.17 U/mg protein for A. parthenogenetica. Significant in vivo effects of both pesticides on Artemia ChE activity were found, at concentrations between 5.38 and 9.30 mg/l for dichlorvos and between 1.85 and 3.19 mg/l for chlorpyrifos. Both Artemia species are resistant to these pesticides and they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina, with about a 95% reduction in its ChE activity respect to the control for nauplii exposed to the median lethal concentrations (LC50), without lethal effects after 24 h of exposure.

Bioaccumulation of chlorpyrifos through an experimental food chain: study of protein HSP70 as biomarker of sublethal stress in fish.

The accumulation and transfer of the organophosphorus pesticide chlorpyrifos has been studied in an experimental aquatic two-level food chain using two species of the crustacean Artemia (A. franciscana and A. parthenogenetica) and the small fish Aphanius iberus. Artemia adults contaminated by exposure to the pesticide in water were used as live prey for Aphanius, the next trophic level. During the experimental bioaccumulation phase, fish were fed chlorpyrifos-contaminated Artemia pools with concentrations between 6.5 and 14.5 ng/g fresh weight for 32 days. Both concentrations accumulated in fish, and biomagnification factor (BMF) values showed a continuous decrease during the bioaccumulation phase, probably due to the physicochemical characteristics of the organophosphorus pesticide, to the biotransformation ability of fish and to the progressive adaptation of fish metabolism to toxic exposure. The first day that fish were fed uncontaminated preys, the pesticide accumulated via food was rapidly eliminated and was not detected. The effect of chlorpyrifos exposure through the food chain on stress protein (HSP70) synthesis was measured as a general biochemical response of stress in the fish (A. iberus). The levels of HSP70 were significantly higher in fish fed on contaminated Artemia than in the control fish fed on uncontaminated Artemia. Results showed that the HSP70 induction in fish could be associated to exposure of chlorpyrifos via food.

Effect of parasitism on respiration rates of adults of different Artemia strains from Spain.

The effect of cestode parasitism on the respiration rate (MO2) of different strains of Artemia from wild populations of Spain was studied. Respiration rates (MO2) of adults from each strain were not affected by the presence of cysticercoids of Flamingolepis liguloides or Hymenolepis stellorae (Cestode, Hymenolepididae). This finding could be related to the absence of reproductive activity (parasite castration) in parasitized females.