Ecotoxicological effects of ketoprofen and fluoxetine and their mixture in an aquatic microcosm.

The effects of fluoxetine (antidepressant) and ketoprofen (analgesic) on aquatic ecosystems are largely unknown, particularly as a mixture. This work aimed at determining the effect of sublethal concentrations of both compounds individually (0.050 mg/L) and their mixture (0.025 mg/L each) on aquatic communities at a microcosm scale for a period of 14 d. Several physicochemical parameters were monitored to estimate functional alterations in the ecosystem, while model organisms (Daphnia magna, Lemna sp., Raphidocelis subcapitata) and the sequencing of 16S/18S rRNA genes permitted to determine effects on specific populations and changes in community composition, respectively. Disturbances were more clearly observed after 14 d, and overall, the microcosms containing fluoxetine (alone or in combination with ketoprofen) produced larger alterations on most physicochemical and biological variables, compared to the microcosm containing only ketoprofen, which suffered less severe changes. Differences in nitrogen species suggest alterations in the N-cycle due to the presence of fluoxetine; similarly, all pharmaceutical-containing systems decreased the brood rate of D. magna, while individual compounds inhibited the growth of Lemna sp. No clear trends were observed regarding R. subcapitata, as indirectly determined by chlorophyll quantification. The structure of micro-eukaryotic communities was altered in the fluoxetine-containing systems, whereas the structure of bacterial communities was affected to a greater extent by the mixture. The disruptions to the equilibrium of the microcosm demonstrate the ecological risk these compounds pose to aquatic ecosystems.

Occurrence and risk assessment of pharmaceuticals in hospital wastewater in Costa Rica.

This work aims to determine the occurrence, hazard and prioritization of pharmaceuticals from hospital wastewater in Costa Rica through the monitoring of 70 compounds and assessing their environmental risk through a hazard quotient approach (HQ). Moreover, the quantification of selected antibiotic resistance genes (ARGs) was conducted for the first time in this matrix in this geographical location. Thirty-four pharmaceuticals were detected, being caffeine, 1,7-dimethylxanthine, acetaminophen, ibuprofen, naproxen, ciprofloxacin and ketoprofen the most frequent (>50% of the samples). Eighteen pharmaceuticals exhibited high hazard (HQ ≥ 1), while five more showed medium hazard (1 > HQ ≥ 0.1). Prioritization, which also included frequency parameters, revealed caffeine, lovastatin, diphenhydramine, acetaminophen, ibuprofen, ciprofloxacin, and sildenafil as the compounds of major concern. Similarly, cumulative hazard per sample (ΣHQ) estimated high hazard towards aquatic organisms in every sample. All selected ARGs, except mcr-1 (polymyxin resistance), were detected. Among genes conferring resistance to beta-lactams, blaCTX-M and blaKPC were the most abundant, related to resistance to cephalosporins and carbapenems. Ecotoxicological evaluation showed mostly low toxicity towards Daphnia magna and Vibrio fischeri, contrary to the marked effect observed towards Lactuca sativa. These findings provide relevant and novel information on the risk posed by hospital wastewater and their pharmaceutical content in the Latin American environmental context.

Phenolic variation among Chamaecrista nictitans subspecies and varieties revealed through UPLC-ESI(-)-MS/MS chemical fingerprinting.

INTRODUCTION: Comparative analysis of metabolic features of plants has a high potential for determination of quality control of active ingredients, ecological or chemotaxonomic purposes. Specifically, the development of efficient and rapid analytical tools that allow the differentiation among species, subspecies and varieties of plants is a relevant issue. Here we describe a multivariate model based on LC-MS/MS fingerprinting capable of discriminating between subspecies and varieties of the medicinal plant Chamaecrista nictitans, a rare distributed species in Costa Rica. METHODS: Determination of the chemical fingerprint was carried out on a LC-MS (ESI-QTOF) in negative ionization mode, main detected and putatively identified compounds included proanthocyanidin oligomers, several flavonoid C- and O-glycosides, and flavonoid acetates. Principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and cluster analysis of chemical profiles were performed. RESULTS: Our method showed a clear discrimination between the subspecies and varieties of Chamaecrista nictitans, separating the samples into four fair differentiated groups: M1 = C. nictitans ssp. patellaria; M2 = C. nictitans ssp. disadena; M3 = C. nictitans ssp. nictitans var. jaliscensis and M4 = C. nictitans ssp. disadena var. pilosa. LC-MS/MS fingerprint data was validated using both morphological characters and DNA barcoding with ITS2 region. The comparison of the morphological characters against the chemical profiles and DNA barcoding shows a 63% coincidence, evidencing the morphological similarity in C. nictitans. On the other hand, genetic data and chemical profiles grouped all samples in a similar pattern, validating the functionality of our metabolomic approach. CONCLUSION: The metabolomic method described in this study allows a reliably differentiation between subspecies and varieties of C. nictitans using a straightforward protocol that lacks extensive purification steps.

Phylogenetic analyses of antibiotic-producing Streptomyces sp. isolates obtained from the stingless-bee Tetragonisca angustula (Apidae: Meliponini).

Many insects have been associated with actinobacteria in protective symbiosis where antimicrobial metabolites inhibit host pathogens. However, the microbiota of neotropical insects such as the stingless-bee Tetragonisca angustula is poorly explored. T. angustula is a meliponid bee widely distributed in Latin America, its honey is traditionally exploited because of its ethno-pharmacological properties and its antimicrobial activity has been demonstrated. Also, the well-structured nest of this species allows exploration of the microbiota of its different components. Even though Streptomyces spp. have been cultured from stingless-bees, little is known about their role in this insect-microbe relationship. In this study, we examined the association between culturable actinobacteria and T. angustula, and evaluated the isolates' potential as antimicrobial producers. We isolated 51 actinobacteria from adult bees and different substrates of the hive of T. angustula (pollen and honey storage, garbage pellets and cerumen). We then performed a 16S rRNA phylogenetic analysis that clusters the bacteria to previously described lineages of host-associated Streptomyces. In addition, all the isolates were classified according to their antibacterial activity against human pathogens, measured by a growth inhibition test based on diffusion in agar. More than 50 % of our isolates exhibit antimicrobial activity, mainly to Gram-positive bacteria and fungi and only two against Gram-negative bacteria. Additionally, we obtained electron micrographs of adult bees with what appears to be patches of hyphae with Streptomyces-like cell morphology on their body surface. Our results suggest that T. angustula possibly uptakes and transfers actinobacteria from the environment, acting as vectors for these potentially beneficial organisms. This research provides new insights regarding the microbiota associated with T. angustula and justify future studies exploring the full diversity of the microbial community associated with the hive and the possible exchange of microbes with the crops they pollinate.