Microplastics in feed cause sublethal changes in the intestinal microbiota and a non-specific immune response indicator of the freshwater crayfish Procambarus clarkii (Decapoda: Cambaridae).

Microplastics (MP) are a hazardous pollutant of global concern that threatens aquatic ecosystems and public health. We used the invasive, cosmopolitan, and environmentally versatile red swamp crayfish Procambarus clarkii as a model to study the effects of MP on the intestinal microbiome. Crayfish collected from the environment were compared with specimens exposed to recycled Polyethylene terephthalate (rPET) MP in feed (30%) for 96 h in the laboratory and a control group. We analyzed the 16S rRNA of the intestinal bacteria by PCR-DGGE and high-throughput sequencing. MP exposure caused dysbiosis of the intestinal microbiota, with an increase in Alphaproteobacteria and Actinobacteria. We detected higher abundance of opportunistic genera such as Klebsiella, Acinetobacter, Hydromonas, Pseudomonas, Gemmobacter, and Enterobacter on MP fed organisms. Moreover, MP exposure reduced the abundance of Clostridia and Bateroidetes, which are important for immune system development and pathogen prevention. Furthermore, MP exposure decreased the phenoloxidase (PO) immune response in crayfish. There was a significant difference in the richness of intestinal bacterial communities after consumption of food contaminated with MP, likely increasing the abundance of opportunistic bacteria in the intestinal microbiota. Our results suggest that MP alter the gut microbial composition and impair the health of P. clarkii.

Pesticide use in banana plantations in Costa Rica - A review of environmental and human exposure, effects and potential risks.

Biodiversity is declining on a global scale. Especially tropical ecosystems, containing most of the planetary biodiversity, are at risk. Agricultural monocrop systems contribute to this decline as they replace original habitats and depend on extensive use of synthetic pesticides that impact ecosystems. In this review we use large-scale banana production for export purposes in Costa Rica as an example for pesticide impacts, as it is in production for over a century and uses pesticides extensively for more than fifty years. We summarise the research on pesticide exposure, effects and risks for aquatic and terrestrial environment, as well as for human health. We show that exposure to pesticides is high and relatively well-studied for aquatic systems and humans, but hardly any data are available for the terrestrial compartment including adjacent non target ecosystems such as rainforest fragments. Ecological effects are demonstrated on an organismic level for various aquatic species and processes but are not available at the population and community level. For human health studies exposure evaluation is crucial and recognised effects include various types of cancer and neurobiological dysfunctions particularly in children. With the many synthetic pesticides involved in banana production, the focus on insecticides, revealing highest aquatic risks, and partly herbicides should be extended to fungicides, which are applied aerially over larger areas. The risk assessment and regulation of pesticides so far relies on temperate models and test species and is therefore likely underestimating the risk of pesticide use in tropical ecosystems, with crops such as banana. We highlight further research approaches to improve risk assessment and, in parallel, urge to follow other strategies to reduce pesticides use and especially hazardous substances.

El impacto de la resistencia a los antibióticos en el desarrollo sostenible / The impact of the antibiotic resistance on the sustainable development

Resumen La resistencia a los antimicrobianos (RAM) es un problema de salud pública cada vez más complejo y se considera como una de las mayores amenazas en todo el mundo. El desarrollo de la RA en los patógenos bacterianos es una consecuencia esperada de la adaptación evolutiva, debido a la presencia de este tipo de contaminantes, los antibióticos, en variedad de nichos ecológicos. Además, hay múltiples factores asociados con su origen y diseminación, entre ellos, el uso desmedido y poco regulado de los antibióticos en la medicina humana y veterinaria, así como en la agricultura, la ganadería y la industria. De hecho, recientemente se ha indicado el papel del ambiente como reservorio para genes de RA y bacterias resistentes a antibióticos. En este sentido, el enfoque para contener y controlar este problema tan complejo involucra de forma necesaria a diversas áreas como la medicina, la veterinaria, las ciencias ambientales y sectores de la industria y la economía. En este artículo, se realiza una descripción tanto del problema de la RA y sus elementos causales, como del enfoque multidisciplinario que ha sido propuesto para su manejo en el ámbito global. Se detalla también cómo la RA afecta el desarrollo humano sostenible conforme a la Agenda 2030 formulada por la ONU, en el cumplimiento de algunos de los objetivos del desarrollo sostenible (ODS).

Abstract Introduction. Antibiotic resistance (AR) is an increasingly complex public health problem, considered one of the greatest threats globally. The development of AR in bacterial pathogens is an expected consequence of evolutionary adaptation due to antimicrobials contamination in the environment. However, multiple factors are associated with the emergence and dissemination of AR, including excessive and poorly regulated use of antibiotics in human and animal medicine, agriculture, livestock, and industry, among other fields. The role of the environment as a reservoir for the generation and dissemination of AR genes and AR bacteria has recently been indicated. The approach to contain and control this problem involves multiple disciplines such as human and animal medicine, the environment, the industry, and the economy. This article describes the AR problem, the factors associated with its origin, and the multidisciplinary approach proposed for its management at a global level. Also, it will be described how AR affects sustainable development according to the UN 2030 Agenda, in compliance with some of the sustainable development goals.

Short-term exposure to benzalkonium chloride in bacteria from activated sludge alters the community diversity and the antibiotic resistance profile.

The continuous introduction of cleaning products containing benzalkonium chloride (BAC) from household discharges can mold the microbial communities in wastewater treatment plants (WWTPs) in a way still poorly understood. In this study, we performed an in vitro exposure of activated sludge from a WWTP in Costa Rica to BAC, quantified the changes in intI1, sul2, and qacE/qacEΔ1 gene profiles, and determined alterations in the bacterial community composition. The analysis of the qPCR data revealed elevated charges of antibiotic resistance genes in the microbial community; after BAC's exposure, a significant increase in the qacE/qacEΔ1 gene, which is related to ammonium quaternary resistance, was observed. The 16S rRNA gene sequences' analysis showed pronounced variations in the structure of the bacterial communities, including reduction of the alpha diversity values and an increase of the relative abundance of Alphaproteobacteria, particularly of Rhodospseudomonas and Rhodobacter. We confirmed that the microbial communities presented high resilience to BAC at the mg/mL concentration, probably due to constant exposure to this pollutant. They also presented antibiotic resistance-related genes with similar mechanisms to tolerate this substance. These mechanisms should be explored more thoroughly, especially in the context of high use of disinfectant.

Who is where in the Plastisphere, and why does it matter?

To fully understand how plastic is affecting the ocean, we need to understand how marine life interacts directly with it. Besides their ecological relevance, microbes can affect the distribution, degradation and transfer of plastics to the rest of the marine food web. From amplicon sequencing and scanning electron microscopy, we know that a diverse array of microorganisms rapidly associate with plastic marine debris in the form of biofouling and biofilms, also known as the "Plastisphere." However, observation of multiple microbial interactions in situ, at small spatial scales in the Plastisphere, has been a challenge. In this issue of Molecular Ecology Resources, Schlundt et al. apply the combination labelling and spectral imaging - fluorescence in situ hybridization to study microbial communities on plastic marine debris. The images demonstrate the colocalization of abundant bacterial groups on plastic marine debris at a relatively high taxonomic and spatial resolution while also visualizing biofouling of eukaryotes, such as diatoms and bryozoans. This modern imaging technology provides new possibilities to address questions regarding the ecology of marine microbes on plastic marine debris and describe more specific impacts of plastic pollution in the marine food webs.

Effect of the veterinary ionophore monensin on the structure and activity of a tropical soil bacterial community.

Monensin (MON) is a coccidiostat used as a growth promoter that can reach the environment through fertilization with manure from farm animals. To verify whether field-relevant concentrations of this drug negatively influence the structure and activity of tropical soil bacteria, plate counts, CO2 efflux measurements, phospholipid fatty acids (PLFA) and community-level physiological profiling (CLPP) profiles were obtained for soil microcosms exposed to 1 or 10 mg kg-1 of MON across 11 days. Although 53% (1 mg kg-1) to 40% (10 mg kg-1) of the MON concentrations added to the microcosms dissipated within 5 days, a subtle concentration-dependent decrease in the number of culturable bacteria (<1 log CFU g-1), reduced (-20 to -30%) or exacerbated (+25%) soil CO2 effluxes, a marked shift of non-bacterial fatty acids, and altered respiration of amines (1.22-fold decrease) and polymers (1.70-fold increase) were noted in some of the treatments. These results suggest that MON quickly killed some microorganisms and that the surviving populations were selected and metabolically stimulated. Consequently, MON should be monitored in agronomic and environmental systems as part of One Health efforts.

Microplastics: New substrates for heterotrophic activity contribute to altering organic matter cycles in aquatic ecosystems.

Heterotrophic microbes with the capability to process considerable amounts of organic matter can colonize microplastic particles (MP) in aquatic ecosystems. Weather colonization of microorganisms on MP will alter ecological niche and functioning of microbial communities remains still unanswered. Therefore, we compared the functional diversity of biofilms on microplastics when incubated in three lakes in northeastern Germany differing in trophy and limnological features. For all lakes, we compared heterotrophic activities of MP biofilms with those of microorganisms in the surrounding water by using Biolog® EcoPlates and assessed their oxygen consumption in microcosm assays with and without MP. The present study found that the total biofilm biomass was higher in the oligo-mesotrophic and dystrophic lakes than in the eutrophic lake. In all lakes, functional diversity profiles of MP biofilms consistently differed from those in the surrounding water. However, solely in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. These results demonstrate that the functionality and hence the ecological role of MP-associated microbial communities are context-dependent, i.e. different environments lead to substantial changes in biomass build up and heterotrophic activities of MP biofilms. We propose that MP surfaces act as new niches for aquatic microorganisms and that the constantly increasing MP pollution has the potential to globally impact carbon dynamics of pelagic environments by altering heterotrophic activities.

Microplastic pollution increases gene exchange in aquatic ecosystems.

Pollution by microplastics in aquatic ecosystems is accumulating at an unprecedented scale, emerging as a new surface for biofilm formation and gene exchange. In this study, we determined the permissiveness of aquatic bacteria towards a model antibiotic resistance plasmid, comparing communities that form biofilms on microplastics vs. those that are free-living. We used an exogenous and red-fluorescent E. coli donor strain to introduce the green-fluorescent broad-host-range plasmid pKJK5 which encodes for trimethoprim resistance. We demonstrate an increased frequency of plasmid transfer in bacteria associated with microplastics compared to bacteria that are free-living or in natural aggregates. Moreover, comparison of communities grown on polycarbonate filters showed that increased gene exchange occurs in a broad range of phylogenetically-diverse bacteria. Our results indicate horizontal gene transfer in this habitat could distinctly affect the ecology of aquatic microbial communities on a global scale. The spread of antibiotic resistance through microplastics could also have profound consequences for the evolution of aquatic bacteria and poses a neglected hazard for human health.

Microplastics increase impact of treated wastewater on freshwater microbial community.

Plastic pollution is a major global concern with several million microplastic particles entering every day freshwater ecosystems via wastewater discharge. Microplastic particles stimulate biofilm formation (plastisphere) throughout the water column and have the potential to affect microbial community structure if they accumulate in pelagic waters, especially enhancing the proliferation of biohazardous bacteria. To test this scenario, we simulated the inflow of treated wastewater into a temperate lake using a continuous culture system with a gradient of concentration of microplastic particles. We followed the effect of microplastics on the microbial community structure and on the occurrence of integrase 1 (int1), a marker associated with mobile genetic elements known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. The abundance of int1 increased in the plastisphere with increasing microplastic particle concentration, but not in the water surrounding the microplastic particles. Likewise, the microbial community on microplastic was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of typical indicators of microbial anthropogenic pollution in natural waters, and substantiate that their removal from treated wastewater should be prioritised.

In situ toxicity and ecological risk assessment of agro-pesticide runoff in the Madre de Dios River in Costa Rica.

The River Madre de Dios (RMD) and its lagoon is a biodiversity rich watershed formed by a system of streams, rivers, channels, and a coastal lagoon communicating with the Caribbean Sea. This basin sustains a large area of agricultural activity (mostly banana, rice, and pineapple) with intensive use of pesticides, continually detected in water samples. We investigated in situ the toxicological effects caused by pesticide runoff from agriculture and the relation of pesticide concentrations with different biological organization levels: early responses in fish biomarkers (sub-organismal), acute toxicity to Daphnia magna (organismal), and aquatic macroinvertebrate community structure. The evaluation was carried out between October 2011 and November 2012 at five sites along the RMD influenced by agricultural discharges and a reference site in a stream outside the RMD that receives less pesticides. Acute toxicity to D. magna was observed only once in a sample from the RMD (Caño Azul); the index of biomarker responses in fish exposed in situ was higher than controls at the same site and at the RMD-Freeman. However, only macroinvertebrates were statistically related to the presence of pesticides, combined with both physical-chemical parameters and habitat degradation. All three groups of variables determined the distribution of macroinvertebrate taxa through the study sites.

Risk assessment of agriculture impact on the Frío River watershed and Caño Negro Ramsar wetland, Costa Rica.

The Caño Negro Ramsar wetland is a conservation area of great natural and societal value, located in the lower part of the Frío River watershed in the north of Costa Rica. Its aquatic ecosystems may be considered vulnerable to pollution due to recent changes in land use toward agriculture. In 2011 and 2012, quarterly sampling was done at ten sites located in the middle and lower sections of the Frío River Basin that pass through crop areas and later drain into Caño Negro wetland. Pesticide residues, nitrates, sediment concentrations, and diversity of benthic macroinvertebrates and fish biomarkers were studied in the selected sites. Additionally, risk of toxicity was calculated in two different ways: (1) by using a ratio of MEC to hazard concentrations threshold for 5% of species (HC5) to calculate a risk quotient (RQ), and (2) by using a ratio of MEC to available ecotoxicity data of native fish and cladocera for diazinon and ethoprophos, to obtain a risk quotient for native species (RQns). Results indicated that three out of the ten sites (rivers Thiales, Mónico, and Sabogal) showed variable levels of pollution including six different active ingredients (a.i.) of pesticide formulations (herbicides ametryn, bromacil, and diuron; insecticides cypermethrin, diazinon, and ethoprophos). Moreover, potential adverse effects on fishes in Thiales and Mónico rivers were indicated by cholinesterase (ChE) inhibition and glutathione S-transferase (GST) enhancement. Risk evaluations indicated pesticide residues of ametryn, bromacil, and ethoprophos to be exceeding the limits set by MTR, also RQ was high (>1) in 70% of the positive samples for diuron (most frequently found pesticide in water samples), cypermethrin, diazinon, and ethoprophos, and RQns was high for diazinon. Therefore, these substances might be of major concern for the ecological health of aquatic ecosystems in the middle basin of the Frío River. The most critical site was Mónico River, which had the highest pollution (75% detection samples with 3-5 a.i.) and highest calculated risk (RQ > 1 in 75% of the samples). This is also the river that most directly drains into the protected wetland. Even though pesticide pollution in this area is not as severe as in other parts of Costa Rica, it is imperative that measures are taken, particularly in the surroundings of Mónico River, in order to diminish and mitigate possible detrimental effects to biota in Caño Negro Ramsar Site.

Sensitivity of Costa Rica's native cladoceran Daphnia ambigua and Simocephalus serrulatus to the organophosphate pesticide ethoprophos.

The study of pesticide toxicity in aquatic environments is assessed with ecotoxicological tests and most research has been performed using species from temperate regions. In the present study, series of acute (48 hrs) toxicity tests to compare the sensibility of two indigenous cladocera of Costa Rica and two reference species were used in temperate regions to the organophosphate pesticide, Ethoprophos. Additionally, reproduction tests using S. serrulatus with sub lethal concentrations of ethoprophos and a control were assayed to check its sensitivity over a longer period exposure. The sensitivity of Costa Rica's native species Daphnia ambigua (EC50 48 hr: 12.9 +/- 3.0 microg(l(-1)) and Simocephalus serrulatus (10.6 +/- 2.1 microg l(-1)) to ethoprophos were higher (p < 0.05) when compared to the exotic species Daphnia magna (289.8 +/- 77.4 microg l(-1)), and were comparable to that of the more widely distributed species, Ceriodaphnia dubia (18.2 +/- 5.2 microg l(-1)). No effect on S. serrulatus reproduction was observed at concentrations between 1 and 4 microg l(-1). This study provides information that can be considered in the selection of species for ecosystem studies of pesticide toxicity in neotropical regions.

Ecotoxicological evaluation of aquaculture and agriculture sediments with biochemical biomarkers and bioassays: antimicrobial potential exposure.

Inappropriate practices and lack of regulations regarding antimicrobial use in agricultural production of developing countries increase the risk of exposure to aquatic ecosystems. Sediments may act as sink of antimicrobial compounds and can provide a historical record of pollution. In the present study, toxic potential of sediments receiving effluents from a fish farm (TIL1), rice farm (AZ) and swine farm (RD2) and from a reference natural wetland (PV) in a tropical dry region was evaluated. According to local surveys of antimicrobials and national product registries, sites were classified from highest to lowest potential exposure as following: RD2>TIL1>AZ>PV. Both, whole sediment and interstitial water tests, showed a high toxicity of pig farm sediments to the behavior of Anodontites luteola and the survival of Daphnia magna (EC50 -48hrs: 2.4 -11.8%) (ANOVA, p < 0.05). Integrated responses from Cholinesterase activity (ChE), Gluthathion-S-transferase (GST) and Lipoperoxidation (LPO) measured in A. luteola tissue pointed at the pig and rice farms as sites influenced by activities with an intensive use of xenobiotic substances. The assessment of toxicity pointed at the need of more research on sub-lethal effects of antimicrobials on aquatic invertebrates. With this purpose, we analyzed biomarker response of A. luteola to oxytetracycline in vitro and found a decrease of ChE and GST in concentrations of 100 microg I(-1).

Tetracycline and 4-epitetracycline modified the in vitro catabolic activity and structure of a sediment microbial community from a tropical tilapia farm idiosyncratically.

Aquaculture farmers commonly add tetracycline to fish feed or to their ponds to prevent or treat bacterial infections in their crops. To assess the short-term effect of tetracycline (TET) and of one of its reversible epimers, 4-epitetracycline (ETC), on the function and structure of a sediment microbial community from a tropical tilapia farm, we contrasted community-level physiological profiles (CLPP) and phospholipid fatty acid profiles (PLFA) obtained from microcosms exposed for 12 days to 5, 10, 50, or 75 mg kg(-1)of these antibiotics. Notwithstanding that the concentration of the antibiotics during the experiment decreased between 13-100% (TET) or 16-61% (ETC), both compounds provoked opposing metabolic responses that did not revert. TET displayed a tendency to inhibit respiration at concentrations < 50 mg kg(-1), whereas ETC showed the opposite effect. As revealed by the finding of the fatty acids 11:0 iso 3OH, 16:1w6c, and 18:1w6c, the sediment analyzed was predominantly colonized by Gram-negative bacteria. A marked decrease in fatty acid diversity accompanied the aforementioned metabolic responses, with TET concentrations > 50 mg kg(-1)leading to an enrichment of yeast and fungal biomarkers and both antibiotics at concentrations < 10 mg kg(-1)selecting for microorganisms with 11:0 iso 3OH. In agreement with CLPP data, differences between the PLFA profiles of control and treated microcosms were more pronounced for TET than for ETC. We conclude that high, yet field-relevant, concentrations of TET and ETC have the potential to modify the composition, and to a lesser extent, the functioning of a sediment microbial community. This study highlights the importance of considering antibiotic degradation products in ecotoxicological research.

Acute toxicity and cholinesterase inhibition of the nematicide ethoprophos in larvae of gar Atractosteus tropicus (Semionotiformes: Lepisosteidae).

Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1 microg/L to 1 500 microg/L. The 96hr LC50 calculated was 859.7 microg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50 microg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10 microg/L. Ethoprophos concentration of 400 microg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.

Acute toxicity and cholinesterase inhibition of the nematicide ethoprophos in larvae of gar Atractosteus tropicus (Semionotiformes: Lepisosteidae)

Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1μg/L to 1 500μg/L. The 96hr LC50 calculated was 859.7μg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50μg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10μg/L. Ethoprophos concentration of 400μg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.

El proceso de reproducción inducida de Atractosteus tropicus es útil para la acuicultura y la reintroducción en zonas donde las poblaciones silvestres se han reducido considerablemente. En larvas de esta especie se evaluó la toxicidad aguda, así como la respuesta de tres biomarcadores: actividad colinesterasa (ChE), actividad de Glutation S-transferasa (GST) y peroxidación de lípidos (LPO). Asimismo, se realizaron exposiciones agudas (96hr) a etoprofos (nematicida organofosforado), en donde se utilizaron concentraciones entre 0.1μg/L y 1 500μg/L del nematicida. La concentración letal 50 (LC50) calculada fue de 859.7μg/L; la máxima concentración sin efecto en los organismos (NOEC) 10μg/L y la concentración más baja en la cual se observó algún efecto (LOEC) 50μg/L. A esa concentración, el efecto observado fue una reducción significativa (p<0.05) en la actividad de la ChE. Una concetración de etoprofos de 400μg/L causó una inhibición del 79% en la actividad ChE. La actividad GST y la LPO no mostraron una respuesta significativa (p>0.05) luego de la exposición de los organismos a etoprofos.