The use of unicellular algae to remove xenobiotics (including drugs) from wastewaters is one of the rapidly developing areas of environmental protection. Numerous data indicate that for efficient phycoremediation three processes are important, i.e. biosorption, bioaccumulation, and biotransformation. Although biosorption and bioaccumulation do not raise any serious doubts, biotransformation is more problematic since its products can be potentially more toxic than the parent compounds posing a threat to organisms living in a given environment, including organisms that made this transformation. Thus, two questions need to be answered before the proper algae strain is chosen for phycoremediation, namely what metabolites are produced during biotransformation, and how resistant is the analyzed strain to a mixture of parent compound and metabolites that appear over the course of culture? In this work, we evaluated the remediation potential of the model green alga Chlamydomonas reinhardtii in relation to non-steroidal anti-inflammatory drugs (NSAIDs), as exemplified by diclofenac. To achieve this, we analysed the susceptibility of C. reinhardtii to diclofenac as well as its capability to biosorption, bioaccumulation, and biotransformation of the drug. We have found that even at a relatively high concentration of diclofenac the algae maintained their vitality and were able to remove (37.7%) DCF from the environment. A wide range of phase I and II metabolites of diclofenac (38 transformation products) was discovered, with many of them characteristic rather for animal and bacterial biochemical pathways than for plant metabolism. Due to such a large number of detected products, 18 of which were not previously reported, the proposed scheme of diclofenac transformation by C. reinhardtii not only significantly contributes to broadening the knowledge in this field, but also allows to suggest possible pathways of degradation of xenobiotics with a similar structure. It is worth pointing out that a decrease in the level of diclofenac in the media observed in this study cannot be fully explained by biotransformation (8.4%). The mass balance analysis indicates that other processes (total 22%), such as biosorption, a non-extractable residue formation, or complete decomposition in metabolic cycles can be involved in the diclofenac disappearance, and those findings open the prospects of further research.
- MeSH
- antiflogistika nesteroidní analýza MeSH
- biotransformace MeSH
- chemické látky znečišťující vodu * analýza MeSH
- Chlamydomonas reinhardtii * metabolismus MeSH
- diklofenak toxicita metabolismus MeSH
- voda MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Diclofenac is a drug commonly used in human and veterinary medicine for the treatment of diseases associated with inflammation and pain. Medicinal products enter waste and surface waters on an everyday basis and contaminate the aquatic environment. Fish are therefore permanently exposed to these chemicals dissolved in their aquatic environment. To simulate variable environmental conditions, the aim of our study was to examine adverse effects of diclofenac under different temperatures of cell incubation (18, 21, 24, 27 and 30 °C). Cyto-toxic and -static effects of diclofenac in concentrations of 0.001 mcg/ml, 0.01 microg/ml, 0.1 mcg/ml, 1 mcg/ml, 10 mcg/ml and 100 mcg/ml for the carp (Cyprinuscarpio) cultured leukocytes were quantified using detection of lactate dehydrogenase released from damaged cells. Overall DCF cytotoxicity was relatively low and its impact was pronounced at higher temperature and DCF concentration. Cells growth inhibition is changing more rapidly but it is high mainly at the highest concentration from low temperature. DNA fragmentation was not detected in tested leukocyte cell line. CYP450 increased diclofenac cytotoxicity only at the highest concentration but at incubation temperatures 18 and 27 °C. Leukocyte viability is essential for immune functions and any change can lead to reduction of resistance against pathogens, mainly in cold year seasons, when the immune system is naturally suppressed.
- MeSH
- antiflogistika nesteroidní toxicita MeSH
- diklofenak toxicita MeSH
- kapři imunologie metabolismus MeSH
- kultivované buňky MeSH
- leukocyty účinky léků imunologie metabolismus patologie MeSH
- proliferace buněk účinky léků MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Early stages of ontogenesis determining subsequent growth, development, and productivity of crops can be affected by wastewater and sludge contaminated with pharmaceuticals. Diclofenac (DCF) and paracetamol (PCT; both 0.0001 to 10 mg/L) did not affect seed germination and primary root length of onion, lettuce, pea, and tomato. Conversely, 20-day-old pea and maize plants exhibited decrease in biomass production, leaf area (by approx. 40% in pea and 70% in maize under 10 mg/L DCF), or content of photosynthetic pigments (by 10% and 60% under 10 mg/L PCT). Quantum yields of photosystem II were reduced only in maize (FV/FM and ΦII by more than 40% under 10 mg/L of both pharmaceuticals). Contents of H2O2 and superoxide increased in roots of both species (more than four times under 10 mg/L PCT in pea). Activities of antioxidant enzymes were elevated in pea under DCF treatments, but decreased in maize under both pharmaceuticals. Oxidative injury of root cells expressed as lowered oxidoreductase activity (MTT assay, by 40% in pea and 80% in maize) and increase in malondialdehyde content (by 60% and 100%) together with the membrane integrity disruption (higher Evans Blue accumulation, by 100% in pea and 300% in maize) confirmed higher sensitivity of maize as a C4 monocot plant to both pharmaceuticals.
- MeSH
- antioxidancia analýza MeSH
- chemické látky znečišťující vodu toxicita MeSH
- diklofenak analýza toxicita MeSH
- fotosyntéza účinky léků MeSH
- klíčení účinky léků MeSH
- kořeny rostlin účinky léků metabolismus MeSH
- listy rostlin účinky léků MeSH
- malondialdehyd analýza MeSH
- odpadní voda chemie MeSH
- paracetamol analýza toxicita MeSH
- peroxid vodíku analýza metabolismus MeSH
- semena rostlinná účinky léků fyziologie MeSH
- zemědělské plodiny účinky léků růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
Growth and developmental changes in plants induced by pharmaceuticals reflect changes in processes at the cellular and subcellular levels. Due to their growth and cellular characteristics, plant cell suspension cultures can be a suitable model for assessing toxicity. In this study, 10-1000 μg/L of the non-steroidal anti-inflammatory drug diclofenac (DCF) decreased the viability of Nicotiana tabacum BY-2 cells after 24 h of treatment. Further, 0.1-10 mg/L DCF diminished the density of the cell suspension by 9-46% after 96 h of treatment, but at 1 and 10 μg/L, DCF increased the density by 13% and 5%, respectively, after 120 h. These changes were accompanied by increased production of total reactive oxygen species (ROS) and mitochondrial superoxide (up to 17-fold and 5-fold, respectively), and a decrease in the mitochondrial membrane potential (by ∼64%) especially at 1000 μg/L DCF. The increased ROS production was accompanied by decrease in level of reactive nitrogen species (RNS; by 36%) and total thiols (by 61%). Damage to BY-2 cells was evidenced by accumulation of neutral red in acidic compartments (up to 10-fold at 1000 μg/L DCF), and increase of autophagic vacuole formation (up to 8-fold at 1000 μg/L DCF). Furthermore, irregular or stretched nuclei were observed in nearly 27% and 50% of cells at 100 and 1000 μg/L DCF, respectively. Highest levels of chromatin condensation (11% of cells) and apoptotic DNA fragmentation (7%) were found at 10 μg/L DCF. The results revealed a significant effect of DCF on BY-2 cells after 24 h of exposure. Changes in the growth and viability parameters were indisputably related to ROS and RNS production, changes in mitochondrial function, and possible activation of processes leading to cell death.
Mechanisms of pharmaceuticals action on biochemical and physiological processes in plants that determine plant growth and development are still mostly unknown. This study deals with the effects of non-steroidal anti-inflammatory drug diclofenac (DCF) on photosynthesis as an essential anabolic process. Changes in primary and secondary photosynthetic processes were assessed in chloroplasts isolated from Lemna minor exposed to 1, 10, 100, and 1000 μM DCF. Decreases in the potential and effective quantum yields of photosystem II (FV/FM by 21%, ΦII by 44% compared to control), changes in non-photochemical fluorescence quenching (NPQ), and a substantial drop in Hill reaction activity (by 73%), especially under 1000 μM DCF, were found. Limitation of electron transport through photosystem II was confirmed by increased fluorescence signals in steps J and I (by 50% and 23%, respectively, under 1000 μM DCF) in OJIP fluorescence transient. Photosystem I exhibited changes only in the redox state of P700 reaction centres (decrease in Pm by 10%, increase in reduced P700 by 5% under 1000 μM DCF). Similarly, RuBisCO activity was only lowered by 30% under 1000 μM DCF. In contrast, a significant increase in reactive oxygen and nitrogen species (by 116% and 157%, respectively) was observed under 10 μM DCF, and lipid peroxidation increased even at 1 μM DCF (by nearly seven times compared to the control). Results demonstrate the ability of environmentally relevant DCF concentrations to induce oxidative stress in isolated duckweed chloroplasts; however, photosynthetic processes were affected considerably only by the highest DCF treatments.
- MeSH
- Araceae účinky léků růst a vývoj ultrastruktura MeSH
- chloroplasty účinky léků metabolismus MeSH
- diklofenak farmakologie toxicita MeSH
- fotosyntéza účinky léků MeSH
- fotosystém I - proteinový komplex MeSH
- fotosystém II - proteinový komplex účinky léků metabolismus MeSH
- oxidační stres účinky léků MeSH
- peroxidace lipidů účinky léků MeSH
- transport elektronů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
The bacterial strain KDF8 capable of growth in the presence of diclofenac and codeine analgesics was obtained after chemical mutagenesis of nature isolates from polluted soils. The strain KDF8 was identified as Raoultella sp. based on its morphology, biochemical properties, and 16S rRNA gene sequence. It was deposited in the Czech Collection of Microorganisms under the number CCM 8678. A growing culture efficiently removed diclofenac (92% removal) and partially also codeine (about 30% degradation) from culture supernatants within 72 h at 28 °C. The degradation of six analgesics by the whole cell catalyst was investigated in detail. The maximum degradation of diclofenac (91%) by the catalyst was achieved at pHINI of 7 (1 g/L diclofenac). The specific removal rate at high concentrations of diclofenac and codeine increased up to 16.5 mg/gCDW per h and 5.1 mg/gCDW per h, respectively. HPLC analysis identified 4'-hydroxydiclofenac as a major metabolite of diclofenac transformation and 14-hydroxycodeinone as codeine transformation product. The analgesics ibuprofen and ketoprofen were also removed, albeit to a lower extent of 3.2 and 2.0 mg/gCDW per h, respectively. Naproxen and mefenamic acid were not degraded.
- MeSH
- analgetika metabolismus toxicita MeSH
- chemické látky znečišťující vodu metabolismus MeSH
- diklofenak metabolismus toxicita MeSH
- DNA bakterií genetika MeSH
- Enterobacteriaceae klasifikace účinky léků metabolismus MeSH
- fylogeneze MeSH
- kodein metabolismus toxicita MeSH
- koncentrace vodíkových iontů MeSH
- mikrobiální viabilita účinky léků MeSH
- půdní mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Lemna minor is often used in environmental risk assessment and it can be supposed that usually evaluated parameters will be reliable even for assessing the risk of pharmaceuticals. Subtle changes in duckweed plant number, biomass production, and leaf area size induced by 10-day-exposure to diclofenac (DCF) and paracetamol (PCT) (0.1, 10, and 100 μg/L), excepting 100 μg/L DCF, are in contrast with considerable changes on biochemical and histochemical level. Both drugs caused a decrease in content of photosynthetic pigments (by up to 50%), an increase in non-photochemical quenching (by 65%) and decrease in relative chlorophyll fluorescence decay values (by up to 90% with DCF). Both DCF and especially PCT increased amount of reactive nitrogen and oxygen species in roots. DCF-induced effects included mainly increased lipid peroxidation (by 78%), disturbation in membrane integrity and lowering both oxidoreductase and dehydrogenase activities (by 30%). PCT increased the content of soluble proteins and phenolics. Higher concentrations of both DCF and PCT increased the levels of oxidised ascorbate (by 30%) and oxidised thiols (by up to 84% with DCF). Glutathion-reductase activity was elevated by both pharmaceuticals (nearly by 90%), glutathion-S-transferase activity increased mainly with PCT (by 22%). The early and sensitive indicators of DCF and PCT phytotoxicity stress in duckweed are mainly the changes in biochemical processes, connected with activation of defense mechanisms against oxidative stress.
- MeSH
- antioxidancia metabolismus MeSH
- biologické markery metabolismus MeSH
- diklofenak metabolismus toxicita MeSH
- fotosyntéza účinky léků MeSH
- fyziologický stres MeSH
- hodnocení rizik MeSH
- Magnoliopsida účinky léků růst a vývoj metabolismus MeSH
- paracetamol metabolismus toxicita MeSH
- peroxidace lipidů účinky léků MeSH
- reaktivní formy dusíku metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this study was to investigate effects of subchronic exposure to sublethal levels of diclofenac on growth, oxidative stress, and histopathological changes in Danio rerio. The juvenile growth tests were performed on Danio rerio according to OECD method number 215. Fish at the age of 20 days were exposed to the diclofenac environmental concentration commonly detected in the Czech rivers (0.02 mg L(-1)) and the range of sublethal concentrations of diclofenac (5, 15, 30, and 60 mg L(-1)) for 28 days. A significant decrease (P < 0.01) in the fish growth caused by diclofenac was observed in the concentrations of 30 and 60 mg L(-1). The identified value of LOEC (lowest observed effect concentration) was 15 mg L(-1) of diclofenac and NOEC (no observed effect concentration) value was 5 mg L(-1) of diclofenac. We did not find histopathological changes and changes of selected parameters of oxidative stress (glutathione S-transferase, glutathione reductase) in tested fish. The environmental concentration of diclofenac in Czech rivers did not have any effect on growth, selected oxidative stress parameters (glutathione S-transferase, glutathione reductase), or histopathological changes in Danio rerio but it could have an influence on lipid peroxidation.
- MeSH
- chemické látky znečišťující vodu toxicita MeSH
- dánio pruhované anatomie a histologie růst a vývoj metabolismus MeSH
- diklofenak toxicita MeSH
- oxidační stres účinky léků MeSH
- velikost těla účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ubiquitous occurrence of pharmaceuticals in aquatic environment results in concern about potential adverse the effects on nontarget organisms. In water, drugs are present in complex mixtures, in which complicated interactions affect toxicity of single components. The purpose of this study was to examine effect of 35-day-long exposure to mixture of ibuprofen, diclofenac, and carbamazepine on the mortality, growth, early ontogeny, and histopathological changes in tench (Tinca tinca). Early life stage toxicity test was carried out using a modified protocol according to OECD guideline 210. Exposure to mixture of pharmaceuticals at concentration of 60 μg · L(-1) for each substance was associated with significant increase in mortality, as well as significant increase in growth and elevated incidence of malformations. Any of the tested concentrations resulted in histopathological changes of liver, kidney, skin, or gill. After fourteen days of exposure there was short-term delay of development related to increased concentrations of pharmaceuticals in the mixture (2, 20, and 60 μg · L(-1)). Environmentally relevant concentrations (0.02; and 0.2 μg · L(-1)) used in this experiment did not result in toxic impairment of tench.
- MeSH
- chemické látky znečišťující vodu toxicita MeSH
- Cyprinidae růst a vývoj MeSH
- diklofenak toxicita MeSH
- karbamazepin toxicita MeSH
- testy toxicity MeSH
- vodní organismy účinky léků růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Diclofenac residues have been found in surface water, and thus could present a potential risk to aquatic species. The aim of this study was to assess the impact of diclofenac on the mortality, growth, and development of fish, as well as the impact of the drug on histological changes and selected parameters of oxidative stress in the fish. Subchronic toxic effects of diclofenac at concentrations of 0.015, 0.03, 1, and 3mg/L on embryos and larvae of common carp (Cyprinus carpio) were investigated during a 30-day toxicity test under experimental conditions. Exposure to diclofenac at 3mg/L was associated with increased mortality, increased activity of glutathione S-transferase, and decreased activity of glutathione reductase. Decreases in the levels of thiobarbituric-acid-reactive substances were associated with concentrations ≥ 0.03 mg/L. Based on these results a no observed effect concentration (NOEC)=0.015 mg/L and lowest observed effect concentration (LOEC)=0.03 mg/L were generated.
- MeSH
- antiflogistika nesteroidní toxicita MeSH
- chemické látky znečišťující vodu toxicita MeSH
- dávka bez pozorovaného nepříznivého účinku MeSH
- diklofenak toxicita MeSH
- glutathionperoxidasa metabolismus MeSH
- glutathionreduktasa metabolismus MeSH
- glutathiontransferasa metabolismus MeSH
- játra anatomie a histologie účinky léků MeSH
- kapři * embryologie růst a vývoj metabolismus MeSH
- kůže anatomie a histologie účinky léků MeSH
- látky reagující s kyselinou thiobarbiturovou metabolismus MeSH
- tělesná hmotnost účinky léků MeSH
- žábry anatomie a histologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
