Phytoremediation of diclofenac using the Green Liver System: Macrophyte screening to system optimization.

Green Liver Systems employ the ability of macrophytes to take up, detoxify (biotransform), and bioaccumulate pollutants; however, these systems require optimization to target specific pollutants. In the present study, the aim was to test the applicability of the Green Liver System for diclofenac remediation considering the effects of selected variables. As a starting point, 42 macrophyte life forms were evaluated for diclofenac uptake. With the three best performing macrophytes, the system efficiency was evaluated at two diclofenac concentrations, one environmentally relevant and that other significantly higher (10 µg/L and 150 µg/L) and in two system sizes (60 L and 1000 L) as well as at three flow rates (3, 7, and 15 L/min). The effect of single species and combinations on removal efficiency was also considered. The highest internalization percentage was recorded in Ceratophyllum spp., Myriophyllum spp., and Egeria densa. Phytoremediation efficiency with species combinations was far superior to utilizing only a single macrophyte type. Furthermore, the results indicate that the flow rate significantly affected the removal efficiency of the pharmaceutical tested, with the highest remediation efficiency obtained with the highest flow rate. System size did not significantly affect phytoremediation; however, increase diclofenac concentration reduced the systems performance significantly. When planning the setup of a Green Liver System for wastewater remediation, basic knowledge about the water, i.e., pollutant types and flow, must be utilized during planning to optimize remediation. Various macrophytes show diverse uptake efficiencies for different contaminants and should be selected based on the pollutant composition of the wastewater.

Titanium (IV) oxide anatase nanoparticles as vectors for diclofenac: assessing the antioxidative responses to single and combined exposures in the aquatic macrophyte Egeria densa.

Titanium dioxide, frequently used in commonplace products, is now regularly detected in aquatic environments. Understanding its toxic effects on native biota is essential. However, combined toxicity with commonly occurring pollutants, such as the pharmaceutical diclofenac, may provide more insight into environmental situations. Therefore, the present study aimed to evaluate the effects of titanium dioxide and diclofenac, individually and combined, on the macrophyte Egeria densa. Diclofenac uptake and removal by the macrophyte were assessed. Diclofenac and titanium dioxide were mixed prior to exposure to allow binding, which was assessed. Toxicity of the individual compounds and the combination was evaluated by assaying enzymes as bioindicators of biotransformation and the antioxidative system. Cytosolic glutathione S-transferase and glutathione reductase activities were increased by diclofenac, titanium dioxide, and the combination. Both enzymes' activities were more significantly elevated by diclofenac and the combination than nanoparticles alone. Microsomal glutathione S-transferase was unaffected by diclofenac exposure but inhibited with titanium dioxide and the mixture. Diclofenac elicited the most significant response. Based on the data, the cytosolic enzymes effectively prevented damage.

Ecotoxicological implications of leachates from concrete demolition debris on oligochaetes: survival and oxidative stress status.

Urbanization and population growth demand the construction of structures to facilitate the need for space, and old infrastructures must make space for new ones leading to demolition and concrete debris. In addition to demolition, aging and weather are factors leading to concrete deterioration and, thus, a new challenge as an environmental pollutant. Studies on how concrete debris and leachate affect biota in the environment are limited. The present study aimed to understand the effects of leachate from various sizes of concrete debris on the three oligochaete species Enchytraeus crypticus, Tubifex, and Lumbriculus variegatus. Acute toxicity testing was carried out to determine the adverse effects over time. The oligochaetes' survival was monitored as well as the activity of the biotransformation enzyme glutathione S-transferase and the antioxidative enzyme catalase as indicators of the oxidative stress status. Leachate from the smallest concrete particle size (<1 mm) was found to be the most toxic as it caused, on average, 6-fold increased oligochaete mortality compared to the larger pieces (2-5 cm) after 96 h of exposure, potentially due to the larger surface area facilitating the release of toxicants. Substrate buffered the toxic effect of the leachate with 42 ± 12% fewer mortalities and reduced adverse effects on the enzymes. Of the three oligochaetes, E. crypticus was the most resilient to the concrete leachate. The study is the first to investigate the effects of concrete leachate on oligochaetes.

In vivo oxidative stress responses of the freshwater basket clam Corbicula javanicus to microplastic fibres and particles.

Microplastics have been detected in several aquatic organisms, especially bivalves such as clams, oysters, and mussels. To understand the ecotoxicological implication of microplastic accumulation in biota, it is crucial to investigate effects at the physiological level to identify knowledge gaps regarding the threat posed to the environment and assist decision-makers to set the necessary priorities. Typically, xenobiotics elicit an overproduction of reactive oxygen species in organisms, resulting in oxidative stress and cellular damage when not combated by the antioxidative system. Therefore, the present study aimed to establish the impacts of microplastic particles and fibres on the freshwater basket clam Corbicula javanicus. We measured the oxidative stress responses following microplastic exposure as the specific activities of the antioxidative enzymes glutathione S-transferase and catalase. When exposed to polyester fibres from the fleece jackets, the enzyme activities increased in the clams, while the enzyme activities decreased with high-density polyethylene microplastic fragments from bottle caps. All the exposures showed that the adverse effects on the antioxidative response system were elicited, indicating the negative ecotoxicological implications of microplastic pollution.

Reviewing Interspecies Interactions as a Driving Force Affecting the Community Structure in Lakes via Cyanotoxins.

The escalating occurrence of toxic cyanobacterial blooms worldwide is a matter of concern. Global warming and eutrophication play a major role in the regularity of cyanobacterial blooms, which has noticeably shifted towards the predomination of toxic populations. Therefore, understanding the effects of cyanobacterial toxins in aquatic ecosystems and their advantages to the producers are of growing interest. In this paper, the current literature is critically reviewed to provide further insights into the ecological contribution of cyanotoxins in the variation of the lake community diversity and structure through interspecies interplay. The most commonly detected and studied cyanobacterial toxins, namely the microcystins, anatoxins, saxitoxins, cylindrospermopsins and ß-N-methylamino-L-alanine, and their ecotoxicity on various trophic levels are discussed. This work addresses the environmental characterization of pure toxins, toxin-containing crude extracts and filtrates of single and mixed cultures in interspecies interactions by inducing different physiological and metabolic responses. More data on these interactions under natural conditions and laboratory-based studies using direct co-cultivation approaches will provide more substantial information on the consequences of cyanotoxins in the natural ecosystem. This review is beneficial for understanding cyanotoxin-mediated interspecies interactions, developing bloom mitigation technologies and robustly assessing the hazards posed by toxin-producing cyanobacteria to humans and other organisms.

Ageing affects microplastic toxicity over time: Effects of aged polycarbonate on germination, growth, and oxidative stress of Lepidium sativum.

Plastic has been an environmental pollutant far longer than claimed by the first reports surfacing in 1979, meaning some plastic materials have been decaying in nature for decades. Nevertheless, the threat posed to biota is not fully understood, especially from aged microplastic. The question considered in this study was whether the adverse effects of new plastic differ from those of old plastic material. Therefore, the morphological and physiological effects on Lepidium sativum with exposure to both new and aged polycarbonate were considered against a known stressor leaching from polycarbonate with time, bisphenol-A. Exposure to new and short-term aged polycarbonate (up to 80 days) elicited the most severe effects such as germination inhibition, reduced seedling growth, decreased chlorophyll concentrations, and increased catalase activity. These adverse effects in L. sativum associated with polycarbonate exposure were reduced as a function of the ageing time applied to the polycarbonate. The chemical substances that lend new polycarbonate material its toxicity were likely leached with time during the ageing process. Based on the results obtained, temperature and humidity based artificial ageing significantly reduced the phytotoxicity of the microplastic particles.

Testing of Eluates from Waterproof Building Materials for Potential Environmental Effects Due to the Behavior of Enchytraeus albidus.

In order to determine the potential environmental impact of construction products, it is necessary to evaluate their influence on organisms exposed to them or their eluates under environmental conditions. The behavior of the white worm Enchytraeus albidus is a useful tool for assessing the potential environmental impact of construction products in contact with water and soil. This study investigates the environmental effects of eluates from two construction products, a reactive waterproofing product, and an injection resin, on the reproduction and avoidance behavior of E. albidus. The eluates were prepared according to existing guidelines. The soil used for the tests was moistened with the eluates of the construction products. The reproduction results of the worms were collected after six weeks of exposure. Offsprings were counted under the microscope and statistically analyzed. Results from the avoidance behavior were collected after 48 h of exposure, and results were compared with the reproduction results. The eluates from both construction products induced significant changes in the reproduction behavior of E. albidus. Undiluted or only slightly diluted eluates of the injection resin drastically reduced the reproduction of the worms, whereas the leaches of the reactive waterproofing product only had a minor effect. The avoidance results for the injection resin indicates that its presence in the habitat is clearly detrimental to the survival of E. albidus, while the avoidance results for the waterproofing resin showed an initial avoidance of the eluates, but no harmful effects were observed. The avoidance test is a way of rapid toxicity screening of environmental samples when time is a critical parameter to measure possible environmental effects. This study shows that ecotoxicological tests using Enchytraeids are a valuable and important tool for understanding the mode of action of eluates from construction products in the environment.

Large-Scale Green Liver System for Sustainable Purification of Aquacultural Wastewater: Construction and Case Study in a Semiarid Area of Brazil (Itacuruba, Pernambuco) Using the Naturally Occurring Cyanotoxin Microcystin as Efficiency Indicator.

The aquaculture industry in Brazil has grown immensely resulting in the production of inefficiently discarded wastewater, which causes adverse effects on the aquatic ecosystem. The efficient treatment of aquaculture wastewater is vital in reaching a sustainable and ecological way of fish farming. Bioremediation in the form of the Green Liver System employing macrophytes was considered as wastewater treatment for a tilapia farm, COOPVALE, in Itacuruba, Brazil, based on previously demonstrated success. A large-scale system was constructed, and the macrophytes Azolla caroliniana, Egeria densa, Myriophyllum aquaticum, and Eichhornia crassipes were selected for phytoremediation. As cyanobacterial blooms persisted in the eutrophic wastewater, two microcystin congeners (MC-LR and -RR) were used as indicator contaminants for system efficiency and monitored by liquid-chromatography-tandem-mass-spectrometry. Two trial studies were conducted to decide on the final macrophyte selection and layout of the Green Liver System. In the first trial, 58% MC-LR and 66% MC-RR were removed and up to 32% MC-LR and 100% MC-RR were removed in the second trial. Additional risks that were overcome included animals grazing on the macrophytes and tilapia were spilling over from the hatchery. The implementation of the Green Liver System significantly contributed to the bioremediation of contaminants from the fish farm.

The Influence of New and Artificial Aged Microplastic and Leachates on the Germination of Lepidium sativum L.

With the increase in environmental monitoring and assessing, we are gaining insight into the extent of microplastic pollution in our environment. The threat posed by microplastics to biota could come, e.g., from leached substances. As some plastic materials have been decaying in nature for extended periods already, the toxic effects of leaching compounds need to be investigated. It is furthermore essential to understand the adverse effects of new plastic and how these effects differ from the effects elicited by old plastic material. Therefore, in the present study, the effects of exposure to leachates from new and artificial aged polycarbonate as well as new and aged polycarbonate granules on various germination parameters of Lepidium sativum were studied. Germination, root, and shoot length, as well as the calculated germination rate index as a measure for germination speed, was negatively influenced in substrate-free and substrate containing exposures. From an ecological and agricultural point of view, this implies possible yield losses with less germinating seeds, slower plant germination speed, and smaller seedlings in general.

Microplastics Exposure Causes Negligible Effects on the Oxidative Response Enzymes Glutathione Reductase and Peroxidase in the Oligochaete Tubifex tubifex.

Microplastics (MPs) are emerging pollutants, which are considered ubiquitous in aquatic ecosystems. The effects of MPs on aquatic biota are still poorly understood, and consequently, there is a need to understand the impacts that MPs may pose to organisms. In the present study, Tubifex tubifex, a freshwater oligochaete commonly used as a bioindicator of the aquatic environment, was exposed to fluorescent polyethylene microspheres (up to 10 µm in size) to test whether the oxidative stress status was affected. The mortality rate of T. tubifex, as well as the activities of the oxidative stress status biomarker enzymes glutathione reductase and peroxidase, were assessed. In terms of oxidative stress, no significant differences between the exposure organisms and the corresponding controls were detected. Even though the data suggest that polyethylene MPs and the selected concentrations did not pose a critical risk to T. tubifex, the previously reported tolerance of T. tubifex to environmental pollution should be taken into account and thus MPs as aquatic pollutants could still represent a threat to more sensitive oligochetes.

Enchytraeus crypticus Avoid Soil Spiked with Microplastic.

Microplastics (MPs) of varying sizes are widespread pollutants in our environment. The general opinion is that the smaller the size, the more dangerous the MPs are due to enhanced uptake possibilities. It would be of considerably ecological significance to understand the response of biota to microplastic contamination both physically and physiologically. Here, we report on an area choice experiment (avoidance test) using Enchytraeus crypticus, in which we mixed different amounts of high-density polyethylene microplastic particles into the soil. In all experimental scenarios, more Enchytraeids moved to the unspiked sections or chose a lower MP-concentration. Worms in contact with MP exhibited an enhanced oxidative stress status, measured as the induced activity of the antioxidative enzymes catalase and glutathione S-transferase. As plastic polymers per se are nontoxic, the exposure time employed was too short for chemicals to leach from the microplastic, and as the microplastic particles used in these experiments were too large (4 mm) to be consumed by the Enchytraeids, the likely cause for the avoidance and oxidative stress could be linked to altered soil properties.

Self-contamination from clothing in microplastics research.

Self-contamination should not be underestimated when quantifying microplastics (MPs) in environmental matrices. Standardised and validated methodologies for MP sampling, extraction, and analysis are lacking. The various applications of plastics in our society have made them ubiquitous, even in clothing, rendering MP self-contamination inevitable. In the present study, we sampled lake sediment, snow, and ice, purposefully wearing red overalls composed of cotton; fibres from which we could quantify using Fourier-Transform Infrared Spectroscopy (FTIR), serving as an indication of possible self-contamination from clothes. The suitability of cotton as a representation of MP contamination was also evaluated. For all detected fibres, 25 ± 1%, 20 ± 7%, and 8 ± 6% for snow, ice, and sediment, respectively, originated from sampling attire. These findings demonstrate that self-contamination can play a significant role when quantifying MP pollution, highlighting that sampling conducted to date might have overestimated the presence of MP or even contaminated MP-free samples.

Uptake, Growth, and Pigment Changes in Lemna minor L. Exposed to Environmental Concentrations of Cylindrospermopsin.

Cylindrospermopsin (CYN)-producing cyanobacterial blooms such as Raphidiopsis, Aphanizomenon, Anabaena, Umezakia, and Lyngbya spp. are occurring more commonly and frequently worldwide. CYN is an environmentally stable extracellular toxin, which inhibits protein synthesis, and, therefore, can potentially affect a wide variety of aquatic biota. Submerged and floating macrophytes, as primary producers in oligotrophic habitats, are at risk of exposure and information on the effects of CYN exposure at environmentally relevant concentrations is limited. In the present study, we investigated CYN uptake in the floating macrophyte Lemna minor with exposure to reported environmental concentrations. The effects were evaluated in terms of bioaccumulation, relative plant growth, and number of fronds per day. Variations in the concentrations and ratios of the chlorophylls as stress markers and carotenoids as markers of oxidative stress defense were measured. With exposure to 25 µg/L, L. minor could remove 43% of CYN within 24 h but CYN was not bioaccumulated. Generally, the pigment concentrations were elevated with exposure to 0.025, 0.25, and 2.5 µg/L CYN after 24 h, but normalized quickly thereafter. Changes in relative plant growth were observed with exposure to 0.25 and 2.5 µg/L CYN. Adverse effects were seen with these environmentally realistic concentrations within 24 h; however, L. minor successfully recovered within the next 48-96 h.

Imidacloprid modifies the mitotic kinetics and causes both aneugenic and clastogenic effects in the macrophyte Bidens laevis L.

Imidacloprid (IMI) is a neonicotinoid insecticide widely used in agricultural activities all around the world. This compound is transported from croplands to surrounding freshwater ecosystems, producing adverse effects on non-target organisms. Because of the relevance of aquatic macrophytes in the above-mentioned environments and the lack of studies of potential effects of IMI on them, this work aimed to assess the mitotic process and potential genotoxicity in the aquatic macrophyte Bidens laevis L. Although the analysis of the Mitotic Index (MI) showed that IMI was not cytotoxic, the Cell Proliferation Kinetics (CPK) frequencies evidenced modifications in the kinetics of the mitotic process. Indeed, the anaphases ratio decreased at 10 and 100 µg/L IMI, while at 1000 µg/L an increase of prophases ratio and a decrease of metaphases ratio were observed. Regarding genotoxicity, IMI produced an increase of the abnormal metaphases frequency from 10 µg/L to 1000 µg/L as well as an increase in clastogenic anaphases-telophases frequency at 100 and 1000 µg/L. In addition, aneugenic anaphases-telophases and C-mitosis frequencies also increased at 1000 µg/L, confirming the effects on the mitotic spindle. Considering the genotoxic effects on B. laevis through two different mechanisms (aneugenic and clastogenic) and the wide spread use of IMI in agriculture, these mechanisms of toxicity on macrophytes should be considered among other recognized effects of this insecticide on aquatic biota.

Translocation of the cyanobacterial toxin microcystin-LR into guttation drops of Triticum aestivum and remaining toxicity.

Uptake of the commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) into crop plants via spray irrigation has been demonstrated. As other hazardous compounds such as pesticides were shown to be transported within plants, it was essential to understand the transport and fate of MC-LR in plants and the risks posed to grazers and other consumers. Of specific interest was to investigate if MC-LR could be detected in guttation drops and the toxicity thereof. Triticum aestivum (wheat) seedlings were exposed to 100 µg L-1 MC-LR in two separate experiments during which guttation drops were collected at various time points. The plants of one experiment were sectioned to investigate MC-LR distribution to the various plant appendages via liquid chromatography-tandem mass spectrometry analysis. After exposure, MC-LR could be detected in the roots, stems, leaves, and the guttation drops. However, the guttation drops were not toxic to Daphnia. As the environmentally relevant toxin concentration used was not sufficient to promote mortality in Daphnia, the physiological effect in insects, which rely on guttation drops as a water source, remains unknown. Combined with other contaminants that insects may be exposed to, the additional MC-LR exposure could contribute to the overall toxicity through the "tears of death".

Desmodesmus subspicatus co-cultured with microcystin producing (PCC 7806) and the non-producing (PCC 7005) strains of Microcystis aeruginosa.

Although microcystins (MCs) are the most commonly studied cyanotoxins, their significance to the producing organisms remains unclear. MCs are known as endotoxins, but they can be found in the surrounding environment due to cell lysis, designated as extracellular MCs. In the present study, the interactions between MC producing and the non-producing strains of Microcystis aeruginosa, PCC 7806 and PCC 7005, respectively, and a green alga, Desmodesmus subspicatus, were studied to better understand the probable ecological importance of MCs at the collapse phase of cyanobacterial blooms. We applied a dialysis co-cultivation system where M. aeruginosa was grown inside dialysis tubing for one month. Then, D. subspicatus was added to the culture system on the outside of the membrane. Consequently, the growth of D. subspicatus and MC contents were measured over a 14-day co-exposure period. The results showed that Microcystis negatively affected the green alga as the growth of D. subspicatus was significantly inhibited in co-cultivation with both the MC-producing and -deficient strains. However, the inhibitory effect of the MC-producing strain was greater and observed earlier compared to the MC-deficient strain. Thus, MCs might be considered as an assistant factor that, in combination with other secondary metabolites of Microcystis, reinforce the ability to outcompete co-existing species.

Interspecies interactions between Microcystis aeruginosa PCC 7806 and Desmodesmus subspicatus SAG 86.81 in a co-cultivation system at various growth phases.

In lakes, cyanobacterial blooms are frequently associated with green algae and dominate the phytoplankton community in successive waves. In the present study, the interactions between Microcystis aeruginosa PCC 7806 and Desmodesmus subspicatus were studied to clarify the probable ecological significance of algal secondary metabolites; focusing on the role of cyanotoxin 'microcystin-LR' (MC-LR). A dialysis co-cultivation technique was applied where M. aeruginosa was grown inside and D. subspicatus was cultured outside of the dialysis tubing. The concentration of the intra- and extracellular MC-LR and the growth of two species were measured at different time points over a period of one month. Additionally, the growth of the two species in the culture filtrate of one another and the effect of the purified MC-LR on the growth of the green alga were studied. The results indicated that the co-existing species could affect each other depending on the growth phases. Despite the early dominance of D. subspicatus during the logarithmic phase, M. aeruginosa suppressed the growth of the green alga at the stationary phase, which coincided with increased MC production and release. However, the inhibitory effects of Microcystis might be related to its other extracellular metabolites rather than, or possibly in addition to, MC.

Phytoremediation: green technology for the removal of mixed contaminants of a water supply reservoir.

The Iraí Reservoir, a water supply in Brazil, is constantly impacted by anthropogenic activities such as waste inputs from agriculture, hospitals and urbanization, resulting toxic cyanobacterial blooms causing economic, social and environmental problems. This study assessed the concentration of some common contaminants of the Iraí Reservoir, namely paracetamol, diclofenac and microcystin-LR and tested whether a laboratory scale Green Liver System® would serve as a suitable technology to remove these contaminants. Further, the study investigated whether the pollutants caused adverse effects to the macrophytes using catalase as a biomarker for oxidative stress and investigated whether biotransformation (glutathione S-transferase) was a main route for detoxification. Egeria densa, Ceratophyllum demersum and Myriophyllum aquaticum were exposed to a mixture of the three contaminants for 14 days in a concentration range similar to those detected in the reservoir. The plants removed 93% of diclofenac and 100% of MC-LR after 14 days. Paracetamol could not be detected. Catalase and glutathione S-transferase enzyme activities remained unaltered after the 14-day exposure, indicating that the mixture did not cause oxidative stress. The study showed that the aquatic macrophytes used are suitable tools to apply in a Green Liver System® for the remediation of mixed pollutants.

Vegetables cultivated with exposure to pure and naturally occurring ß-N-methylamino-L-alanine (BMAA) via irrigation.

Bioaccumulation and biomagnification of ß-N-methylamino-L-alanine (BMAA), a potent neurotoxin, has been demonstrated in various food webs. It is alarming as this intensification of BMAA will result in exposure to higher concentrations from a direct cyanobacterial source. As more food items are being identified as a source of BMAA and with the large variations in BMAA content, the aim of the present study was to evaluate BMAA uptake by, and accumulation in, two commonly consumed vegetables, Lactuca sativa and Allium fistulosum. Plants exposed to pure BMAA in controlled laboratory experiments, as well as vegetables naturally irrigated with water containing a BMAA producing cyanobacterial bloom were evaluated during growth and ripening. In the laboratory exposures, free BMAA was detected in both the edible ripe parts of L. sativa and A. fistulosum after 60 days of exposure to a total of 4.5 µg BMAA. However, in the bloom exposure samples no BMAA could be detected in the ripe vegetables of A. fistulosum, Cucurbita pepo, or Brassica rapa chinensis. The study emphasises the need to further screen items for BMAA to understand the human exposure risk as well as the difference between BMAA uptake patterns with free BMAA and that contained in cyanobacterial cells.

Genotoxicity and pharmacokinetic characterization of Cereus jamacaru ethanolic extract in rats.

The effect of Cereus jamacaru ethanolic extract in rats was analyzed using genotoxicity assays and liver ABCB1 and CYP2D4 gene expression. The lyophilized extract of C. jamacaru cladodes was analyzed with LC-MS/MS. Male Wistar rats (n=36) were equally distributed into six groups that did (+) or did not (-) receive cyclophosphamide treatments: Control (-); Control (+); EXP 1 (-), and EXP 1 (+), both treated with 210 mg/kg of ethanolic extract; and EXP 2 (-) and EXP 2 (+), both treated with 420 mg/kg of ethanolic extract. After 30 d of treatment, body weight and food and water intake were monitored. Right femur of the rats and spinal canal fluid were harvested and used for genotoxicity assays, and the liver samples were used for gene expression studies. The phytochemical analysis identified novel compounds. Animals treated with C. jamacaru showed lower body weight and food ingestion compared to controls (P<0.05). The genotoxicity assay showed an absence of ethanolic extract cytotoxicity. CYP2D4 expression was higher in EXP 2 groups compared with EXP 1 (-) group (P<0.05). ABCB1A expression was higher in negative groups compared with the positive groups. These results indicated a new phytochemical characterization of C. jamacaru and its effect on food ingestion and body weight gain. Moreover, the genotoxicity assay suggested that C. jamacaru ethanolic extract treatment presents significant intrinsic genotoxic potential and the enhanced expression of ABCB1 and CYP2D4 on C. jamacaru extract treatment suggests a role of the efflux transporter and microsomal enzyme, respectively, in C. jamacaru pharmacokinetics.