Valeska Contardo-Jara

Exposure to human pharmaceuticals Carbamazepine, Ibuprofen and Bezafibrate causes molecular effects in Dreissena polymorpha

Carbamazepine (CBZ), Ibuprofen (IBU) and Bezafibrate (BEZ) were tested for their potential to bioaccumulate and provoke molecular changes in the non-target organism Dreissena polymorpha. mRNA changes of enzymes and other proteins involved in the prevention from protein damage (heat shock protein 70, hsp70) and oxidative stress (superoxide dismutase, SOD; catalase, CAT; metallothionein, MT), biotransformation (pi-class glutathione S-transferase, piGST; aryl hydrocarbon receptor, AH-R), elimination (P-glycoprotein, P-gp) and reversible protein posttranslational modification (protein phosphatase 2A, PP2A) served as molecular biomarkers. Mussels were exposed in a flow-through system to increasing concentrations of the three substances (1, 10, 100 and 1000 nM). The two lower concentrations correspond to environmentally relevant concentrations detected in surface and effluent waters, respectively. Measuring tissue concentration after one, four and seven days the uptake of CBZ and IBU by the mussels could be evidenced, whereas no accumulation data could be achieved for BEZ. The bioconcentration factor was highest for mussels exposed to the lowest CBZ and IBU concentrations, with 90 and 460-fold higher tissue concentration, respectively, after seven days. CBZ was the only substance tested which caused a significant increase in gill mRNA level of hsp70 after only one day exposure, evidencing the potential of CBZ to immediately provoke a stress condition and assumingly protein damage in gills. After longer exposure, mussels displayed down-regulated mRNA levels of hsp70 and SOD in gills, as well as of MT and P-gp in the digestive gland, hinting on an inhibitory character of CBZ. In IBU exposed mussels increased oxidant stress conditions were evidenced by induced mRNA levels in the digestive gland of CAT and MT, as well as SOD after one and four days, respectively. A concentration as found at sewage treatment plant effluents provoked an increase in transcript levels of piGST, suggesting enhanced need for biotransformation of IBU or by-products derived from oxidative stress. Also exposure to an environmentally relevant BEZ concentration provoked an immediate increase in piGST transcript level in the digestive gland followed by up-regulated hsp70 after four and seven days evidencing a chronic stress condition for the mussels.

Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes

The bioaccumulation potential of glyphosate and the formulation Roundup Ultra, as well as possible effects on biotransformation and antioxidant enzymes in Lumbriculus variegatus were compared by four days exposure to concentrations between 0.05 and 5 mg L(-1) pure glyphosate and its formulation. Bioaccumulation was determined using (14)C labeled glyphosate. The bioaccumulation factor (BCF) varied between 1.4 and 5.9 for the different concentrations, and was higher than estimated from logP(ow). Glyphosate and its surfactant POEA caused elevation of biotransformation enzyme soluble glutathione S-transferase at non-toxic concentrations. Membrane bound glutathione S-transferase activity was significantly elevated in Roundup Ultra exposed worms, compared to treatment with equal glyphosate concentrations, but did not significantly differ from the control. Antioxidant enzyme superoxide dismutase was significantly increased by glyphosate but in particular by Roundup Ultra exposure indicating oxidative stress. The results show that the formulation Roundup Ultra is of more ecotoxicological relevance than the glyphosate itself.

Multi-xenobiotic-resistance a possible explanation for the insensitivity of bivalves towards cyanobacterial toxins.

Filterfeeders, such as bivalves, are highly affected during toxic cyanobacterial blooms, as they are non-selective and may use the cyanobacteria as main nutrition source. The freshwater mussel Dreissena polymorpha, living in lakes and rivers coexisting with cyanobacteria, was exposed to 100 microg L(-1) microcystin-LR (MC-LR) for up to three days. MC-LR concentration in mussel tissue and surrounding media was quantified by HPLC-PDA during uptake and depuration phase, revealing an immediate, continuous uptake, and release of non-metabolized toxin, and occurrence of reincorporation. The involvement of multi-xenobiotic-resistance protein (P-glycoprotein, P-gp) on the excretion of MC-LR was evidenced by efflux and accumulation version of the Rhodamine Assay as well as on P-gp gene expression. P-gp expression was enhanced after 1 h exposure but no changes were detected after longer (72 h) exposure. P-gp enzyme activity showed a significant increase with exposure time, supporting the hypothesis that P-gp is involved in the excretion of MC-LR. Induction of biotransformation enzyme such as pi-class glutathione S-transferase (piGST) and antioxidant enzyme catalase (CAT) was immediately inhibited and returned to control values only after more than 72 h expose time. Heat shock protein 70 (hsp70) and protein phosphatase 2A (PP2A) gene expression was not changed due to the treatment with cyanobacterial toxin MC-LR.

The β-receptor blocker metoprolol alters detoxification processes in the non-target organism Dreissena polymorpha.

Due to increasing amounts of pharmaceutically active compounds (PhACs) in the aquatic environment, their largely unknown effects to non-target organisms need to be assessed. This study examined physiological changes in the freshwater mussel Dreissena polymorpha exposed to increasing concentrations (0.534, 5.34, 53.4 and 534 microg L(-1)) of the beta-blocker metoprolol in a flow-through system for seven days. The two lower concentrations represent the environmentally relevant range. Surprisingly, metallothionein mRNA was immediately up-regulated in all treatments. For the two higher concentrations mRNA up-regulation in gills was found for P-glycoprotein after one day, and after four days for pi class glutathione S-transferase, demonstrating elimination and biotransformation processes, respectively. Additionally, catalase and superoxide dismutase were up-regulated in the digestive gland indicating oxidative stress. In all treated mussels a significant up-regulation of heat shock protein mRNA was observed in gills after four days, which suggests protein damage and the requirement for repair processes. Metoprolol was 20-fold bioaccumulated for environmentally relevant concentrations.

Biotransformation and antioxidant enzymes of Limnoperna fortunei detect site impact in watercourses of Córdoba, Argentina.

The golden mussel Limnoperna fortunei was used as a biomonitor of environmental pollution in the Suquía River basin around Córdoba City (Argentina). The sampling sites along the river were chosen according to their increasing levels of pollutants (e.g. heavy metals) as well as biological oxygen demand (BOD) and chemical oxygen demand (COD). A water quality index (WQI) was constructed from the interaction of several normalized factors that affect the aquatic environment, such as the mentioned pollutants and physico-chemical characteristics of the sampling sites. Activity changes of biotransformation enzyme glutathione S-transferase (GST) and the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT), after exposure to pollutants, served as biomarkers. Membrane bound GST and antioxidant enzymes responded at the most polluted sampling site within 1 day showing increased activities lasting for 4 days. Further sampling was restricted due to no survival of the animals. Antioxidant enzymes GPx, GR and CAT were sensitive responding to the different pollution scenarios, showing good correlation to the chemical characterization.

The Synthetic Gestagen Levonorgestrel Impairs Metamorphosis in Xenopus laevis by Disruption of the Thyroid System

Synthetic gestagens, including levonorgestrel (LNG), are active compounds in contraceptives, and several studies report their occurrence in surface waters. However, information about endocrine-disrupting effects in nontarget organisms is scarce. The present study investigated effects of LNG exposure on thyroid hormone-dependent metamorphosis of Xenopus laevis. Premetamorphic X. laevis tadpoles at Nieuwkoop and Faber (NF) stage 48 were exposed in a flow-through culture system to four LNG concentrations (10(-11), 10(-10), 10(-9), and 10(-8)M) over the period of metamorphosis. At NF 58 and 66, tadpoles were examined sex specifically. Developmental time and organismal responses were recorded and correlated with molecular and histopathological endpoints. Exposure to 10(-8)M LNG caused an inhibition of metamorphosis resulting in developmental arrest at early climax stages as giant tadpoles or tailed frogs. In brain-pituitary tissue of NF 58 tadpoles, gene expression of thyroid-stimulating hormone (β-subunit; TSHβ), TH receptor β (TRβ), and deiodinase type 3 (D3) was not changed. Instead, prolactin (PRL) messenger RNA (mRNA) was significantly increased by 10(-9)M LNG in females and by 10(-8)M LNG in both sexes. In NF 66 tadpoles, mRNA levels of TSHβ mRNA were significantly increased in the 10(-9) and 10(-8)M LNG treatment groups indicating a hypothyroid state. No changes of TRβ, D3, and PRL gene expression were detected. Histopathological evaluation of thyroid gland sections revealed no typical sign of hypothyroidism but rather an inactivated appearance of the thyroid. In conclusion, our data demonstrate for the first time a completely new aspect of thyroid system disruption caused by synthetic gestagens in developing amphibians.

The Synthetic Gestagen Levonorgestrel Disrupts Sexual Development in Xenopus laevis by Affecting Gene Expression of Pituitary Gonadotropins and Gonadal Steroidogenic Enzymes

In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10(-11), 10(-10), 10(-9), and 10(-8)M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10(-9) and 10(-8)M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) β in both genders. This decrease was persistent after further treatment until NF 66 in the 10(-8)M LNG treatment. Expression of follicle-stimulating hormone (FSH) β was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10(-9) and 10(-8)M significantly increased FSHβ mRNA levels in males. In NF 66 females, 10(-9)M LNG treatment increased FSHβ gene expression, whereas a decrease was observed in NF 66 males exposed to 10(-8)M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10(-8)M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10(-8)M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHβ expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.

Biotransformation and antioxidant enzymes of Dreissena polymorpha for detection of site impact in watercourses of Berlin

This study investigated whether biotransformation and antioxidant enzymes of an organism tolerant to pollution such as the zebra mussel Dreissena polymorpha can be employed to evaluate the extent of urban water pollution. Activity changes of soluble and membrane bound glutathione S-transferase (s- and mGST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) to environmental stress were explored in precultured mussels exposed for one day to one week at selected sites with reasonable anthropogenic impact. The enzymatic response of whole mussel tissue was compared to gill tissue. Changes in enzyme activity were accordant with the different pollution scenarios, as charges with polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT) and other pesticides, as well as contamination with metals. Whereas in whole mussel tissue all analyzed enzymes responded with elevation, in gills inhibition took place with the exception of mGST. The results confirm the high sensitivity of gills, nevertheless enzymatic changes measured in whole mussel tissue provided the clearest results. Significant changes in GST, CAT and GPx enzyme activity were only observed at water temperatures above 20 degrees C. SOD activity was not restricted by temperatures and serves for this reason as a biomarker for oxidative stress at lower water temperatures. Discrepancies between biological and chemical evaluation of the sampling sites are presented and biomarker responses appraised.

Uptake, distribution in different tissues, and genotoxicity of imidacloprid in the freshwater fish Australoheros facetus

The neonicotinoid imidacloprid is under re-evaluation by regulatory agencies because of the poor current information available regarding its potential effects. One of the goals of the present study was to determine imidacloprid uptake and distribution in the freshwater fish Australoheros facetus experimentally exposed for 24 h and 48 h to 100 μg/L, 300 μg/L, and 2500 μg/L. The toxicity of imidacloprid to fish reported in the literature is in the milligrams per liter or gram per liter range, but sublethal effects at micrograms per liter in some groups other than fish have been described. Another goal of the present study was to evaluate imidacloprids potential genotoxicity and to compare it between the individual compound and a commercial formulation. Concentrations of imidacloprid were measured in water, brain, muscle, gills, gut, liver, and blood by liquid chromatography-tandem mass spectrometry. Imidacloprid was detected in all the tissues tested. Concentrations were higher after 48 h than after 24 h in liver, gills, gut, and muscle, whereas in brain and blood they were similar at both exposure times. Although there was no accumulation, only uptake, of imidacloprid, genotoxicity was observed. In fish exposed to IMIDA NOVA 35® , increased micronucleus frequency at 100 µg/L and 1000 µg/L was detected, whereas in the imidacloprid active ingredient bioassay it increased only at 1000 µg/L imidacloprid. The present findings warn of the possible consequences that fish living in freshwater ecosystems can suffer. Environ Toxicol Chem 2017;36:699-708.

Contamination of Urban Surface and Ground Water Resources and Impact on Aquatic Species

Since people have been settling on flood plains, they influenced the freshwater resources more or less intensely. Historically, the process of population expansion and industrial development in urban areas has proved disastrous to the quantity and quality of both surface and ground waters (Ellis 1999). Thus, for an appropriate assessment and development of urban areas, the integrated surface and subsurface water systems, including their ecological functioning, are playing an important role (Zaadnoordijk et al. 2004). Stability criteria for both quantity and quality of urban water resources are on the one hand the “natural” discharges and recharges of rivers and groundwater, which are mainly controlled by anthropogenic and climate change effects. On the other hand, diffuse and direct pollutions with innumerable complex chemical compounds determine serious challenges for a sustainable urban water resources management (Lerner 2004). Whereas climate change is primarily influencing the water volumes of the hydrological cycle in all components and its effect has been quantified continuously, the surface and subsurface water pollution and the circulation of these substances represent qualitative parameters, which could have a large impact on the urban water resources again (Zhang et al. 2004).