Safety Assessment of a Novel Intravenous Diclofenac Sodium Formulation Following 28-Day Repeated Administration in Wistar Rats.

These toxicity studies aimed to assess the safety and tolerability of a novel intravenous diclofenac sodium (37.5 mg/mL) formulation containing povidone K12 (80 mg/mL) as the key excipient in Wistar rats. This formulation was tested at doses of 3, 7, and 15 mg/kg/day and was administered daily for 28 days by intravenous route. Toxicokinetic estimation revealed a dose-proportional increase in plasma exposure to diclofenac. The formulation was well tolerated in males; however, mortality was observed in females (2/15) at the highest dose (15 mg/kg/day). Adverse gastrointestinal events related to NSAIDS and a few other treatment-related effects on clinical and anatomic pathology were noted at the 15 mg/kg/day dose, which normalized at the end of the 2-week recovery period. In addition, the excipient povidone K12 was present in a higher amount than the approved Inactive Ingredient Database (IID) limit in the proposed novel formulation. It was qualified through a separate 28-day repeated dose toxicity study by intravenous route in Wistar rats. Povidone K12 was found to be well tolerated and safe up to a dose of 165 mg/kg/day. No treatment-related adverse effects were observed in this study. In conclusion, repeated administration of a novel intravenous formulation containing diclofenac sodium was found to be safe up to the dose of 7 mg/kg/day in female rats and 15 mg/kg/day in male rats.

Electrochemical degradation of diclofenac generates unexpected thyroidogenic transformation products: Implications for environmental risk assessment.

Diclofenac (DCF) is an environmentally persistent, nonsteroidal anti-inflammatory drug (NSAID) with thyroid disrupting properties. Electrochemical advanced oxidation processes (eAOPs) can efficiently remove NSAIDs from wastewater. However, eAOPs can generate transformation products (TPs) with unknown chemical and biological characteristics. In this study, DCF was electrochemically degraded using a boron-doped diamond anode. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was used to analyze the TPs of DCF and elucidate its potential degradation pathways. The biological impact of DCF and its TPs was evaluated using the Xenopus Eleutheroembryo Thyroid Assay, employing a transgenic amphibian model to assess thyroid axis activity. As DCF degradation progressed, in vivo thyroid activity transitioned from anti-thyroid in non-treated samples to pro-thyroid in intermediately treated samples, implying the emergence of thyroid-active TPs with distinct modes of action compared to DCF. Molecular docking analysis revealed that certain TPs bind to the thyroid receptor, potentially triggering thyroid hormone-like responses. Moreover, acute toxicity occurred in intermediately degraded samples, indicating the generation of TPs exhibiting higher toxicity than DCF. Both acute toxicity and thyroid effects were mitigated with a prolonged degradation time. This study highlights the importance of integrating in vivo bioassays in the environmental risk assessment of novel degradation processes.

Towards Sustainable Wastewater Treatment: Bioindication as a Technique for Supporting Treatment Efficiency Assessment.

Constructed wetlands (CWs) are a promising alternative for conventional methods of wastewater treatment. However, the biggest challenge in wastewater treatment is the improvement of the technology used so that it is possible to remove micropollutants without additional costs. The impact of wastewater treatment in CWs on toxicity towards Aliivibrio fischeri, Daphnia magna and Lemna minor was investigated. The effects of feeding regime (wastewater fed in five batches per week at a batch volume of 1 L, or twice per week at a batch volume of 2.5 L) and the presence of pharmaceuticals (diclofenac and sulfamethoxazole), as well as the presence of Miscantus giganteus plants in CW columns (twelve of the 24 columns that were planted) were analyzed. A reduction in toxicity was observed in all experimental setups. The effluents from constructed wetlands were classified as moderately toxic (average TU for A. fischeri, D. magna and L. minor was 0.9, 2.5 and 5.5, respectively). The feeding regime of 5 days of feeding/2 days of resting resulted in a positive impact on the ecotoxicological and chemical parameters of wastewater (removal of TOC, N-NH4 and pharmaceuticals). Extended exposure of Miscantus giganteus to the wastewater containing pharmaceuticals resulted in elevated activity of antioxidant enzymes (catalase and superoxide dismutase) in leaf material.

Diclofenac biotransformation and toxicity assessment of laccase from Pleurotus floridanus.

Laccase producing fungus Pleurotus floridanus was isolated from Siruvani forest, Tamil Nadu, India. The potential of P. floridanus to produce laccase by using various lignocellulosic substrates was screened under submerged fermentation. Laccase production in the presence of lignocellulosic substrates such as rice, wheat and maize bran as a sole source of carbon as well as an additional supplement was examined. Laccase activity of P. floridanus using varied substrates was observed in the order of rice bran > wheat bran > maize bran. The isolate showed maximum laccase activity of 13.29±0.01 U/mL using rice bran as a carbon source within 11 days. This was 18 fold higher than the control media that lacks lignocellulosic substrates. The diclofenac tolerance was assessed in solid media at various concentrations and the results showed that the mycelia growth is not significantly affected by the drug. Finally, the laccase mediated degradation of diclofenac at a concentration of 10 mg/L showed 98% degradation in 2 h. The phytotoxicity of the crude laccase treated diclofenac was lower than the untreated diclofenac. In conclusion, findings suggested direct application of crude laccase produced from P. floridanus using agro-residues as ideal substrate for environmental applications.

Synergistic adsorption and degradation of diclofenac by zero-valent iron modified spent bleaching earth carbon: Mechanism and toxicity assessment.

Diclofenac (DCF) is a drug compound that exists widely in water bodies, which may pose a threat to the ecological environment. In this study, spent bleaching earth (SBE) was pyrolyzed, modified with cetyltrimethylammonium bromide (CTAB) and loaded with zero-valent iron (nZVI) to obtain CTAB-SBE@C-nZVI. The effects of CTAB concentration, Fe0 loading, CTAB-SBE@C-nZVI dosage, and initial pH value on the removal efficiency of DCF were studied. The results showed that the DCF removal efficiency could reach a maximum of 87.0% with 2.0 g/L dosage of the optimal material, which was prepared under the conditions of 30 mmol/L CTAB concentration, 25% Fe0 loading, and initial pH 5. It indicated that the strong adsorption of the material and the reduction effect of nZVI can achieve high-efficiency removal of DCF. Based on the detected reaction intermediate products, four possible degradation paths were inferred. The toxicity assessment of DCF and its intermediates manifested that the degradation of DCF by CTAB-SBE@C-nZVI was a process of gradual dechlorination and toxicity reduction. CTAB-SBE@C-nZVI displayed excellent DCF removal efficiency, good stability and environmental friendliness, achieving wastes treat wastes and exhibiting good prospects.

Occurrence of carbamazepine, diclofenac, and their related metabolites and transformation products in a French aquatic environment and preliminary risk assessment.

With questions emerging on the presence and risks associated with metabolites and transformation products (TPs) of organic contaminants in the aquatic environment, progress has been made in terms of monitoring and regulation of pesticide metabolites. However, less interest is shown for pharmaceutical residues, although their pseudo-persistence and adverse effects on non-target organisms are proven. This study provides original knowledge about the contamination of ten sites located along three French rivers (water, sediments, biofilms, clams) by pharmaceutical metabolites and TPs, as well as a preliminary environmental risk assessment. Studied compounds included carbamazepine with five metabolites and TPs, and diclofenac with three metabolites and TPs. Results show that metabolites and TPs are present in all studied compartments, with mean concentrations up to 0.52 µg L-1 in water, 229 ng g-1 in sediments, 2153 ng g-1 in biofilms, and 1149 ng g-1 in clams. QSAR estimations (OECD toolbox) were involved to predict the studied compounds ecotoxicities. QSAR models showed that diclofenac and its metabolites and TPs could be more toxic than carbamazepine and its metabolites and TPs to three aquatic species representing green algae, invertebrates, and fish. However, real ecotoxicological effects are still to be determined. The environmental risk assessment showed that hydroxydiclofenac, 2-[(2-chlorophenyl)-amino]-benzaldehyde and dibenzazepine could present a greater risk than other studied compounds for aquatic organisms. In addition, the risk associated with a mixture of diclofenac and its related metabolites and TPs has been found to be greater than that of the compounds considered individually.

Ecotoxicological assessment of micropollutant Diclofenac biosorption on magnetic sawdust: Phyto, Microbial and Fish toxicity studies.

Diclofenac (DCF), a persistent pharmaceutical micropollutant which occurs in the ecosystems causing adverse effects on aquatic as well as terrestrial organisms. In this study, magnetic sawdust (MSD) was prepared using co-precipitation method for biosorptive removal of DCF from water. The MSD was characterized using various analytical techniques like microscopic and spectroscopic analysis. Magnetometer study confirms the ferromagnetic behavior of the biosorbent which is a key advantage in the separation of MSD after biosorption. The effect of experimental parameters was optimized in batch mode with evaluated maximum efficiency of 86.12 % at pH 6, biosorbent dosage 25 mg for 50 mg/L of DCF. Ecotoxicological assessment has been performed for the treated and untreated sample using plant seeds, microbes and zebra fish to check the adverse effects of DCF on these organisms. Evaluation of toxicity studies revealed that inhibition concentration of DCF for various seeds (60.91 mg/L to 43.11 mg/L), E. coli (48.82 µg/mL) and B. subtilis (31.55 µg/mL). The lethal concentration of DCF on the Danio rerio was found to be 156.99 mg/L. In contrast, significant increase in both the concentration measures of DCF after biosorption was observed making this biosorbent a potent alternative to other available treatment measures.

Photocatalytic-persulfate- oxidation for diclofenac removal from aqueous solutions: Modeling, optimization and biotoxicity test assessment.

In this paper, the influence of several aquatic factors (the nature of catalyst, the initial pH and the initial concentration of the pollutant) on the photocatalytic degradation of diclofenac (DFC), one of the most widely prescribed anti-inflammatory non-steroidal drug, was studied. Also, in order to examine the intensification process, the variation of the photocatalytic DFC degradation in the presence of sodium persulfate (PPS) was analyzed. It was found that, compared to titanium dioxide (TiO2), the zinc oxide (ZnO) photocatalyst performed exceptionally well, with a 96.13% DFC degradation efficiency after 150 min. The photodegradation of DFC by ZnO catalyst fitted well the Langmuir-Hinshelwood kinetic model. The maximum efficiency is 97.27% for simulated solar-UVA/ZnO/PPS and 77% for simulated solar-UVA/ZnO. In order to determine the optimal conditions leading to the maximization of DFC removal, an artificial neural network (ANN) modeling approach combined with genetic algorithm (GA) was applied. The best ANN determined had a correlation of 0.999 and it was further used in the process optimization where a 99.7% degradation efficiency was identified as the optimum under the following conditions: DFC initial concentration 37,9 mg L-1, pH 5,88 and PPS initial concentration 500 mg L-1. The effectiveness of the process and the toxicity of the pharmaceutical pollutants and their by-products were also evaluated and confirmed by the biological tests using liver and kidney of Mus musculus mice.

Perspectives on Relevancy Assessment for Non-Standard Ecotoxicity Data in Environment Quality Standard derivation: Examples for Diclofenac.

A key step in deriving an Environmental Quality Standard (EQS) is assessing the reliability and relevance of the underpinning ecotoxicity data. While the assessment of data reliability is relatively well established, the detailed evaluation of data relevancy is a more recent development. We applied broadly accepted relevancy criteria to a series of non-standard ecotoxicity studies on diclofenac, focusing on some aspects that should be accounted for in studies used in EQS derivation. Specific relevancy issues include potential experimental bias, claimed 'significant effects' that are indistinguishable from controls, or within the range of normal, and lack of environmental applicability. We highlight that rigorous, comprehensive and, where necessary, specialist assessment of data relevancy for studies potentially applicable for EQS setting is critical if studies are to be appropriately used regulatory decision-making. We provide recommendations for researchers and environmental practitioners to ensure robust accounting of relevancy in non-standard studies is undertaken.

Ozonation of diclofenac in the aqueous solution: Mechanism, kinetics and ecotoxicity assessment.

The pharmaceutical and personal care products (PPCPs) in aquatic environment have aroused more interest recently. Many of them are hard to degrade by the typical biological treatments. Diclofenac (DCF), as a significant anti-inflammatory drug, is a typical PPCP and widely existed in water environment. It is reported that DCF has adverse effects on aquatic organisms. This work aims to investigate the mechanism, kinetics and ecotoxicity assessment of DCF transformation initiated by O3 in aqueous solution, and provide a solution to the degradation of DCF. The O3-initiated oxidative degradations of DCF were performed by quantum chemical calculations, including thirteen primary reaction pathways and subsequent reactions of the Criegee intermediates with H2O, NO and O3. Based on the thermodynamic data, the kinetic parameters were calculated by the transition state theory (TST). The total reaction rate constant of DCF initiated by O3 is 2.57 × 103 M-1 s-1 at 298 K and 1 atm. The results show that the reaction rate constants have a good correlation with temperature. The acute and chronic toxicities of DCF and its degradation products were evaluated at three different trophic levels by the ECOSAR program. Most products are converted into less toxic or harmless products. Oxalaldehyde (P3) and N-(2,6-dichlorophenyl)-2-oxoacetamide (P6) are still harmful to the three aquatic organisms, which should be paid more attention in the future.

Assessment of phytotoxic effects, uptake and translocation of diclofenac in chicory (Cichorium intybus).

Pharmaceuticals in the environment have been an increasing research topic over the past decade, since they can be found in both natural and drinking water, including irrigation of crops and edible plants with contaminated water. Our main goal was to evaluate the phytotoxic effect of diclofenac (DCF), a widely used pharmaceutical, on chicory (Cichorium intybus) seedlings. Additionally, we verified the uptake, bioconcentration and translocation of DCF from soil to chicory tissues. Results show that DCF induces different physiological changes in chicory seedlings. On the other hand, the soil-chicory experiment showed the activation of the detoxification system in plants treated with DCF (1 mg L-1). Finally, we found the migration of DCF from the irrigation water to the soil, followed by its uptake through the root, and its translocation to the aerial part of the chicory. However, DCF does not bioaccumulate in chicory leaves, being scarcely translocated from roots to aerial parts. This last result, along with the estimation of a daily intake of chicory, show that irrigation with water containing DCF (≤1 mg L-1) does not represent a threat to human health. To our knowledge, this is the first report on the effect of DCF on chicory seedlings, including the evaluation of its uptake and translocation.

Biotoxicity of diclofenac on two larval amphibians: Assessment of development, growth, cardiac function and rhythm, behavior and antioxidant system.

The non-steroidal anti-inflammatory drug diclofenac (DCF) threatens the health of aquatic animals and ecosystems. In the present study, different biological endpoints (mortality, development and growth, abnormalities, cardiotoxicity, neurotoxicity and antioxidant system) were used to characterize the acute and chronic effects of DCF (at concentrations ranging between 125 and 4000 µg L-1) on two amphibian species from Argentina (Trachycephalus typhonius and Physalaemus albonotatus). Results showed that the larval developmental, growth rates, and body condition of DCF-exposed individuals of both species were significantly reduced. DCF-exposed individuals also showed several morphological abnormalities, including significantly altered body axis, chondrocranium and hyobranchial skeleton, and organ and visceral abnormalities including cardiac hypoplasia, malrotated guts, asymmetrically inverted guts, and cholecystitis. DCF also had a significant effect on the swimming performance of both species: at low concentrations (125 and 250 µg L-1), swimming distance, velocity and global activity decreased, whereas, at high concentrations (1000 and 2000 µg L-1), these behavioral responses increased. Regarding cardiac function and rhythm, at DCF concentrations higher than 1000 µg L-1, the heart frequency and ventricular systole interval of both species were significantly reduced. Regarding the antioxidant system, the activity of acetylcholinesterase indicated that DCF is neurotoxic and thus related to the changes in behavioral performance. The DCF concentrations studied produced a biochemical imbalance between radical oxygen species production and antioxidant systems. The sensitivities to sublethal and chronic DCF exposure in both anuran species were similar, thus indicating the inherent complexity involved in understanding the biotoxic effects of DCF.

Assessment of dose-dependent reproductive toxicity of diclofenac sodium in male rats.

Diclofenac sodium is widely used in the non-steroidal anti-inflammatory drug in the treatment of pain and inflammation. It is also particularly associated with its adverse effects on avian fauna and linked to environmental issues. The present study was aimed to assess the dose-dependent toxicity of diclofenac sodium on a male reproductive system of rats. Four groups of healthy adult fertile male rats were administered with saline (control) or 0.25 mg/kg, 0.50 mg/kg and 1.0 mg/kg of diclofenac sodium, respectively for 30 days. Alterations in body and organ weight, sperm and testicular cell population dynamics, serum biochemistry, histopathology, and hematology were investigated as per aimed objectives. Diclofenac sodium treatment significantly (p ≤ 0.001) reduced weights of testis, epididymis, ventral prostate and seminal vesicle. Sperm count, sperm density (in epididymis and testis), sperm motility and testicular cell population dynamics were lowered in a dose-dependent manner. Administration of diclofenac exhibited varying degrees of degeneration testis, abnormal histo-architectures, and shrinkages in seminiferous tubules, particularly in higher doses. Diclofenac sodium treatments also altered hepatic and renal function parameters significantly. In conclusion, it may claim that diclofenac sodium treatment altered reproductive metabolic status, androgenic activities and histo-architecture of the testis of male rats and induced hepatotoxicity and renal toxicity.

Mechanism-based risk assessment strategy for drug-induced cholestasis using the transcriptional benchmark dose derived by toxicogenomics.

Cholestasis is one of the major causes of drug-induced liver injury (DILI), which can result in withdrawal of approved drugs from the market. Early identification of cholestatic drugs is difficult due to the complex mechanisms involved. In order to develop a strategy for mechanism-based risk assessment of cholestatic drugs, we analyzed gene expression data obtained from the livers of rats that had been orally administered with 12 known cholestatic compounds repeatedly for 28 days at three dose levels. Qualitative analyses were performed using two statistical approaches (hierarchical clustering and principle component analysis), in addition to pathway analysis. The transcriptional benchmark dose (tBMD) and tBMD 95% lower limit (tBMDL) were used for quantitative analyses, which revealed three compound sub-groups that produced different types of differential gene expression; these groups of genes were mainly involved in inflammation, cholesterol biosynthesis, and oxidative stress. Furthermore, the tBMDL values for each test compound were in good agreement with the relevant no observed adverse effect level. These results indicate that our novel strategy for drug safety evaluation using mechanism-based classification and tBMDL would facilitate the application of toxicogenomics for risk assessment of cholestatic DILI.

Hydroxyl radical-mediated degradation of diclofenac revisited: a computational approach to assessment of reaction mechanisms and by-products.

Advanced oxidation processes (AOPs) are based on the in situ production of hydroxyl radicals (•OH) and reactive oxygen species (ROS) in water upon irradiation of the sample by UV light, ultrasound, electromagnetic radiation, and/or the addition of ozone or a semiconductor. Diclofenac (DCF), one of the emerging organic contaminants (EOC), is of environmental concern due to its abundancy in water and is known to be subjected to AOPs. The current study uses density functional theory (DFT) to elucidate the mechanisms of the reactions between •OH and DCF leading to degradation by-products, P1-P9. The initial encounter of DCF with •OH is proposed to lead to either the abstraction of a hydrogen or the addition of the hydroxyl radical to the molecule. The results showed that OH addition radicals (R add) are both kinetically and thermodynamically favored over H abstraction radicals (R abs). The intermediate radicals give degradation by-products by subsequent reactions. The by-products P7 and P8 are easily formed in agreement with experimental findings. Finally, acute toxicities at three trophic levels are estimated with the Ecological Structure Activity Relationships program. DCF and most of the by-products were found to be harmful to aquatic organisms, P9 being the only by-product that is not harmful at all three trophic levels.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System.

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1ß, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1ß, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.

Assessment of in vitro antiovulatory activities of nonsteroidal anti-inflammatory drugs and comparison with in vivo reproductive toxicities of medaka (Oryzias latipes).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used therapeutic agents; however, their pharmacological actions raise concerns about potential risks to the reproductive health of aquatic vertebrates. In the present study, a medaka ovulation assay was applied as an in vitro model to evaluate NSAID-induced antiovulatory activity. We first tested five NSAIDs, including diclofenac sodium (DCF), ketoprofen (KP), salicylic acid (SA), mefenamic acid (MA), and acetylsalicylic acid (ASA) for their antiovulatory activities toward the follicles isolated from the ovaries of spawning females. Of all the chemicals tested, DCF had the highest antiovulatory activity, with the concentration that caused 50% inhibition (IC50) (101 µM). MA was the second most potent inhibitor following DCF, but KP, SA, or ASA had little inhibitory effect on the ovulation of the follicles. The in vitro antiovulatory activity of five NSAIDs showed good correlation with data published on the inhibitory activity on human COX-2. Second, we selected DCF and SA as the most and least potent NSAIDs, respectively, and examined the effects on reproduction of intact fish in order to evaluate whether the ovulation assay was a reasonable predictor of potential reproductive effects in fish. Females exposed to DCF showed a concentration-dependent decrease in the number of spawned eggs and an increment in the gonadosomatic index (GSI), possibly due to an anovulation in the females. In contrast, neither fecundity nor the GSI of females decreased at up to 20 mg/L of SA, at which acute lethality to medaka was induced. In conclusion, the medaka ovulation assay reflected the potency of NSAID-induced antiovulatory activity and may thus serve as an in vitro model for the prediction of NSAID-induced reproductive toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1710-1719, 2016.

Ecotoxicogenomic assessment of diclofenac toxicity in soil.

Diclofenac is widely used as nonsteroidal anti-inflammatory drug leaving residues in the environment. To investigate effects on terrestrial ecosystems, we measured dissipation rate in soil and investigated ecotoxicological and transcriptome-wide responses in Folsomia candida. Exposure for 4 weeks to diclofenac reduced both survival and reproduction of F. candida in a dose-dependent manner. At concentrations ≥ 200 mg/kg soil diclofenac remained stable in the soil during a 21-day incubation period. Microarrays examined transcriptional changes at low and high diclofenac exposure concentrations. The results indicated that development and growth were severely hampered and immunity-related genes, mainly directed against bacteria and fungi, were significantly up-regulated. Furthermore, neural metabolic processes were significantly affected only at the high concentration. We conclude that diclofenac is toxic to non-target soil invertebrates, although its mode of action is different from the mammalian toxicity. The genetic markers proposed in this study may be promising early markers for diclofenac ecotoxicity.

Molecular and microscopic assessment of the effects of caffeine, acetaminophen, diclofenac, and their mixtures on river biofilm communities.

The authors examined effects of three common contaminants, caffeine (CF), acetaminophen (AC), and diclofenac (DF), as well as their mixtures on the development, functioning, and biodiversity of river biofilm communities. Biofilms were cultivated in rotating annular reactors. Treatments included AC, CF, DF, AC + CF, AC + DF, CF + DF, AC + CF + DF at 5 µg/L, and their molar equivalent as carbon and nutrients. Incubations using ¹4C-labeled AC, DF, and CF indicated that 90% of the CF, 80% of the AC, and less than 2% of the DF were converted to CO2. Digital imaging revealed a variety of effects on algal, cyanobacterial, and bacterial biomass. Algal biomass was unaffected by AC or CF in combination with DF but significantly reduced by all other treatments. Cyanobacterial biomass was influenced only by the AC + DF application. All treatments other than AC resulted in a significant decrease in bacterial biomass. Diclofenac or DF + CF and DF + AC resulted in increases in micrometazoan grazing. The denaturing gradient gel electrophoresis of Eubacterial community DNA, evaluated by principal component analysis and analysis of similarity, indicated that relative to the control, all treatments had effects on microbial community structure (r = 0.47, p < 0.001). However, the AC + CF + DF treatment was not significantly different from its molar equivalent carbon and nutrient additions. The Archaeal community differed significantly in its response to these exposures based on community analyses, confirming a need to integrate these organisms into ecotoxicological studies.

Biomarker assessment of toxicity with miniaturised bioassays: diclofenac as a case study.

The development of suitable biomarker-based microbioassays with model species with ecological relevance would help increase the cost-efficiency of routine environmental monitoring and chemical toxicity testing. The anti-inflammatory drug diclofenac has been widely reported in the environment but ecotoxicological data are scarce. The aim of this work is to assess the acute and chronic sublethal toxicity of diclofenac in relevant taxa of aquatic and riparian ecosystems (the fish Danio rerio and the fern Polystichum setiferum). Reliable biomarkers of cell viability (mitochondrial activity), plant physiology (chlorophyll), growth (DNA content) or oxidative damage (lipid peroxidation) were assessed as sensitive endpoints of toxicity. DNA quantification shows that diclofenac induces acute lethal phytotoxicity at 24 and 48 h (LOECs 30 and 0.3 µg l(-1), respectively). Hormetic effects in mitochondrial activity in spores of Polystichum setiferum mask lethality, and adverse effects are only observed at 48 h (LOEC 0.3 µg l(-1)). In chronic exposure (1 week) LOEC for DNA is 0.03 µg l(-1). Mitochondrial activity shows a strong hormetic stimulation of the surviving spore population (LOEC 0.3 µg l(-1)). Little changes are observed in chlorophyll autofluorescence (LOEC 0.3 µg l(-1)). A very short exposure (90 min) of zebrafish embryos induces a reduction of lipid peroxidation at 0.03 µg l(-1). Environmental concentrations of diclofenac can be deleterious for the development of significant populations of sensitive individuals in aquatic and riparian ecosystems.