Integrating transcriptomics and biochemical analysis to understand the interactive mechanisms of the coexisting exposure of nanoplastics and erythromycin on Chlorella pyrenoidosa.

Nanoplastics and antibiotics frequently co-exist in water polluted by algal blooms, but little information is available about interaction between substances. Erythromycin, as a representative of antibiotics, has been frequently detected in aquatic environments. This investigation attempted to reveal the interaction mechanism of nanoplastics and erythromycin on Chlorella pyrenoidosa. Results demonstrated that the joint toxicity of erythromycin and nanoplastics was dynamic and depended on nanoplastics concentration. Antagonistic effects of 1/2 or 1 EC50 erythromycin and nanoplastic concentration (10 mg/L) on the growth of C. pyrenoidosa was observed. The joint toxicity of 1/2 or 1 EC50 erythromycin and nanoplastic concentration (50 mg/L) was initially synergistic during 24-48 h and then turned to antagonistic during 72-96 h. Consequently, antagonistic effect was the endpoint for joint toxicity. Integration of transcriptomics and physiological biochemical analysis indicated that the co-existence of nanoplastics and erythromycin affected the signal transduction and molecular transport of algal cell membrane, induced intracellular oxidative stress, and hindered photosynthetic efficiency. Overall, this study provided a theoretical basis for evaluating the interactive mechanisms of nanoplastics and antibiotics.

Mechanistic insights into hormesis induced by erythromycin in the marine alga Thalassiosira weissflogii.

Erythromycin (ERY) is a typical macrolide antibiotic with large production and extensive use on a global scale. Detection of ERY in both freshwaters and coaster seawaters, as well as relatively high ecotoxicity of ERY have been documented. Notably, hormesis has been reported on several freshwater algae under ERY stress, where growth was promoted at relatively lower exposures but inhibited at higher treatment levels. On the contrary, there is limited information of ERY toxicity in marine algae, hampering the risk assessment on ERY in the coaster waters. The presence of hormesis may challenge the current concept of dose-response adopted in chemical risk assessment. Whether and how exposure to ERY can induce dose-dependent toxicity in marine algae remain virtually unknown, especially at environmentally relevant concentrations. The present study used a model marine diatom Thalassiosira weissflogii (T. weissflogii) to reveal its toxicological responses to ERY at different biological levels and decipher the underlying mechanisms. Assessment of multiple apical endpoints shows an evident growth promotion following ERY exposure at an environmentally relevant concentration (1 µg/L), associated with increased contents reactive oxygen species (ROS) and chlorophyll-a (Chl-a), activated signaling pathways related to ribosome biosynthesis and translation, and production of total soluble protein. By contrast, growth inhibition in the 750 and 2500 µg/L treatments was attributed to reduced viability, increased ROS formation, reduced content of total soluble protein, inhibited photosynthesis, and perturbed signaling pathways involved in xenobiotic metabolism, ribosome, metabolism of amino acid, and nitrogen metabolism. Measurements of multiple apical endpoints coupled with de novo transcriptomics analysis applied in the present study, a systems biology approach, can generate detailed mechanistic information of chemical toxicity including dose-response and species sensitivity difference used in environmental risk assessment.

Combined toxicity of erythromycin and roxithromycin and their removal by Chlorella pyrenoidosa.

The ecological effects of antibiotics in surface water have attracted increasing research attention. In this study, we investigated the combined ecotoxicity of erythromycin (ERY) and roxithromycin (ROX) on the microalgae, Chlorella pyrenoidosa, and the removal of ERY and ROX during the exposure. The calculated 96-h median effect concentration (EC50) values of ERY, ROX, and their mixture (2:1 w/w) were 7.37, 3.54, and 7.91 mg∙L-1, respectively. However, the predicted EC50 values of ERY+ROX mixture were 5.42 and 1.51 mg∙L-1, based on the concentration addition and independent action models, respectively. This demonstrated the combined toxicity of ERY+ ROX mixture showed an antagonistic effect on Chlorella pyrenoidosa. During the 14-d culture, low-concentration (EC10) treatments with ERY, ROX, and their mixture caused the growth inhibition rate to decrease during the first 12 d and increase slightly at 14 d. In contrast, high-concentration (EC50) treatments significantly inhibited microalgae growth (p < 0.05). Changes in the total chlorophyll contents, SOD and CAT activities, and MDA contents of microalgae suggested that individual treatments with ERY and ROX induced higher oxidative stress than combined treatments. After the 14-d culture time, residual Ery in low and high concentration Ery treatments were 17.75% and 74.43%, and the residual Rox were 76.54% and 87.99%, but the residuals were 8.03% and 73.53% in ERY+ ROX combined treatment. These indicated that antibiotic removal efficiency was higher in combined treatments than that in individual treatments, especially at low concentrations (EC10). Correlation analysis suggested that there was a significant negative correlation between the antibiotic removal efficiency of C. pyrenoidosa and their SOD activity and MDA content, and the enhanced antibiotic removal ability of microalgae benefited from increased cell growth and chlorophyll content. Findings in this study contribute to predicting ecological risk of coexisting antibiotics in aquatic environment, and to improving biological treatment technology of antibiotics in wastewater.

Effects of erythromycin and roxithromycin on river periphyton: Structure, functions and metabolic pathways.

Macrolides have been frequently detected in the surface waters worldwide, posing a threat to the aquatic microbes. Several studies have evaluated the ecotoxicological effects of macrolides on single algal and bacterial strains. However, without considering the species interaction in the aquatic microbial community, these results cannot be extrapolated to the field. Thus, the present study aimed to evaluate the effects of two macrolides (erythromycin and roxithromycin) on the structure, photosynthetic process, carbon utilization capacity, and the antibiotic metabolic pathways in river periphyton. The colonized periphyton was exposed to the graded concentration (0 µg/L (control), 0.5 µg/L (low), 5 µg/L (medium), 50 µg/L (high)) of ERY and ROX, respectively, for 7 days. Herein, high levels of ERY and ROX altered the community composition by reducing the relative abundance of Chlorophyta in the eukaryotic community. Also, the Shannon and Simpson diversity indexes of prokaryotes were reduced, although similar effects were seldomly detected in the low and medium groups. In contrast to the unchanged carbon utilization capacity, the PSII reaction center involved in the periphytic photosynthesis was significantly inhibited by macrolides at high levels. In addition, both antibiotics had been degraded by periphyton, with the removal rate of 51.63-66.87% and 41.85-48.27% for ERY and ROX, respectively, wherein the side chain and ring cleavage were the main degradation pathways. Overall, this study provides an insight into the structural and functional toxicity and degradation processes of macrolides in river periphyton.

Metabolomic profiles in a green alga (Raphidocelis subcapitata) following erythromycin treatment: ABC transporters and energy metabolism.

A recent study showed that erythromycin (ERY) exposure caused hormesis in a model alga (Raphidocelis subcapitata) where the growth was promoted at an environmentally realistic concentration (4 µg/L) but inhibited at two higher concentrations (80 and 120 µg/L), associated with opposite actions of certain signaling pathways (e.g., xenobiotic metabolism, DNA replication). However, these transcriptional alterations remain to be investigated and verified at the metabolomic level. This study uncovered metabolomic profiles and detailed toxic mechanisms of ERY in R. subcapitata using untargeted metabolomics. The metabolomic analysis showed that metabolomic pathways including ABC transporters, fatty acid biosynthesis and purine metabolism were associated with growth promotion in algae treated with 4 µg/L ERY. An overcompensation was possibly activated by the low level of ERY in algae where more resources were reallocated to efficiently restore the temporary impairments, ultimately leading to the outperformance of growth. By contrast, algal growth inhibition in the 80 and 120 µg/L ERY treatments was likely attributed to the dysfunction of metabolomic pathways related to ABC transporters, energy metabolism and metabolism of nucleosides. Apart from binding of ERY to the 50S subunit of ribosomes to inhibit protein translation as in bacteria, the data presented here indicate that inhibition of protein translation and growth performance of algae by ERY may also result from the suppression of amino acid biosynthesis and aminoacyl-tRNA biosynthesis. This study provides novel insights into the dose-dependent toxicity of ERY on R. subcapitata.

Responses of zebrafish (Danio rerio) cells to antibiotic erythromycin stress at the subcellular levels.

Erythromycin (ERY) is one of the most used antibiotics frequently detected in different aquatic environments and may bring burdens to aquatic ecosystems. However, the impacts of antibiotics on aquatic systems other than the antibiotic resistance genes remain largely unknown. In the present study, the responses to ERY exposure at the subcellular-organelle levels were for the first time investigated and imaged over 24 h. Exposure to ERY hampered the zebrafish (Danio rerio) cell growth and decreased the cell viability in a time-dependent mode. Meanwhile, exposure to a low concentration of ERY (73.4 µg L-1) induced reactive oxygen species (ROS) overproduction and lysosomal damage following lysosomal alkalization and swelling. In turn, the lysosomal stress was the major driver of altering the ROS level, superoxide dismutase (SOD) activity, and glutathione (GSH) content. Subsequently, mitochondria displayed dysfunction such as increased mitochondrial ROS, impaired mitophagy, and induced mitochondria-driven apoptosis, as well as impaired mitochondrial electron transport chain and loss of membrane potential. These results collectively demonstrated the subcellular sensitive machinery responses to ERY stress at environmentally relevant and slightly higher sub-lethal concentrations. ERY may induce switching from autophagy to apoptosis with corresponding changes in lysosomal activity, antioxidant activity, and mitochondrial activity. The findings provided important information on the physiological and subcellular responses of fish cells to ERY.

Removal mechanisms of erythromycin by microalgae Chlorella pyrenoidosa and toxicity assessment during the treatment process.

Microalgae-based biotechnology for antibiotic removal has received increasing attention as an economical and green method. This study investigated the removal mechanism of erythromycin by Chlorella pyrenoidosa and its correlation with the ecotoxic responses of microalgae. The degradation products (DPs) were identified, and their toxicity was predicted. The results indicated that only 4.04 %, 6.28 % and 23.53 % of erythromycin were left after 21-day microalgae treatment in 0.1, 1.0 and 10 mg/L treatments, respectively. Biodegradation contributed 48.62-67.01 %, 16.67-52.32 % and 6.42-24.82 %, while abiotic degradation contributed 8.76-29.61 %, 5.19-41.39 %, and 16.55-51.22 % to erythromycin attenuation in 0.1, 1.0, and 10 mg/L treatments, respectively. The growth and physiological-biochemical parameters of microalgae were slightly affected in low concentration treatment, which may be the main reason that biodegradation was the prominent removal mechanism. By contrast, oxidative damage in high concentration treatment inhibited the cell growth and chlorophyll content of microalgae, which hindered erythromycin biodegradation. In addition, eleven erythromycin degradation products (DPs) were identified during microalgae treatment of 21 days. Seven DPs including DP717, DP715, DP701A, DP701B, DP657, DP643, and DP557, represented higher toxicity to aquatic organisms than erythromycin.

Three critical regions of the erythromycin resistance methyltransferase, ErmE, are required for function supporting a model for the interaction of Erm family enzymes with substrate rRNA.

6-Methyladenosine modification of DNA and RNA is widespread throughout the three domains of life and often accomplished by a Rossmann-fold methyltransferase domain which contains conserved sequence elements directing S-adenosylmethionine cofactor binding and placement of the target adenosine residue into the active site. Elaborations to the conserved Rossman-fold and appended domains direct methylation to diverse DNA and RNA sequences and structures. Recently, the first atomic-resolution structure of a ribosomal RNA adenine dimethylase (RRAD) family member bound to rRNA was solved, TFB1M bound to helix 45 of 12S rRNA. Since erythromycin resistance methyltransferases are also members of the RRAD family, and understanding how these enzymes recognize rRNA could be used to combat their role in antibiotic resistance, we constructed a model of ErmE bound to a 23S rRNA fragment based on the TFB1M-rRNA structure. We designed site-directed mutants of ErmE based on this model and assayed the mutants by in vivo phenotypic assays and in vitro assays with purified protein. Our results and additional bioinformatic analyses suggest our structural model captures key ErmE-rRNA interactions and indicate three regions of Erm proteins play a critical role in methylation: the target adenosine binding pocket, the basic ridge, and the α4-cleft.

Ecotoxicological effects of erythromycin on a multispecies biofilm model, revealed by metagenomic and metabolomic approaches.

The presence of antibiotics such as erythromycin, even in trace amounts, has long been acknowledged for negatively impacting ecosystems in freshwater environments. Although many studies have focused on the impact of antibiotic pollution at a macroecological level, the impact of erythromycin on microecosystems, such as freshwater biofilms, is still not fully understood. This knowledge gap may be attributed to the lack of robust multispecies biofilm models for fundamental investigations. Here, we used a lab-cultured multispecies biofilm model to elucidate the holistic response of a microbial community to erythromycin exposure using metagenomic and metabolomic approaches. Metagenomic analyses revealed that biofilm microbial diversity did not alter following erythromycin exposure. Notably, certain predicted metabolic pathways such as cell-cell communication pathways, amino acid metabolism, and peptidoglycan biosynthesis, mainly by the phyla Actinobacteria, Alpha/Beta-proteobacteria, Bacteroidetes, and Verrucomicrobia, were found to be involved in the maintenance of homeostasis-like balance in the freshwater biofilm. Further untargeted metabolomics data highlighted changes in lipid metabolism and linoleic acid metabolism and their related molecules as a direct consequence of erythromycin exposure. Overall, the study presented a unique picture of how multispecies biofilms respond to single environmental stress exposures. Moreover, the study demonstrated the feasibility of using lab simulated multispecies biofilms for investigating their interaction and reactivity of specific bioactive compounds or pollutants at a fundamental level.

Erythromycin in the aquatic environment: deleterious effects on the initial development of zebrafish.

Due to the large volume of erythromycin continuously reaching waterbodies and its high persistence, this antibiotic drug has been detected in the aquatic environment at elevated concentrations. Although the problem of the presence of erythromycin in the environment is evident due to its influence in development of antimicrobial resistance, the toxicological consequences on non-target organisms remain to be determined. There are no apparent data on the impact of environmentally relevant concentrations of erythromycin on developing fish. Data on toxic effects during development are essential for evaluation of environmental risk to organisms. Therefore, the aim of this study was to investigate the effects of exposure to erythromycin on certain parameters including hatchability, survival rate, heart rate, and behavior in developing zebrafish. Zebrafish were exposed to a range of environmentally relevant concentrations of antibiotic (0.001, 0.01, 0.1, 1 µg/L) and one concentration 10-fold higher (10 µg/L). Exposure to erythromycin at 0.1 µg/L delayed hatching and decreased survival rate. Exposure to all tested concentrations increased heart rate. Further, exposure to erythromycin at 1 or 10 µg/L enhanced swimming activity. Our results indicated that erythromycin present in the aquatic environment might lead to disabling consequences in developing fish organisms and subsequently may result in ecological imbalance in the natural environment.

Effects of erythromycin and sulfamethoxazole on Microcystis aeruginosa: Cytotoxic endpoints, production and release of microcystin-LR.

Antibiotics can cause severe ecological problems for aquatic ecosystems due to their wide use and incomplete removal. Microcystis aeruginosa was exposed to different levels of erythromycin (ERY) and sulfamethoxazole (SMX) separately to assess their cytotoxic effects on harmful cyanobacteria. The production and release of the toxin MC-LR was measured, and several endpoints were investigated using flow cytometry (FCM) for 7 d. ERY resulted in cell membrane hyperpolarization and a hormesis effect on growth rate and chlorophyll a fluorescence at environmentally relevant concentrations (0.5 and 5 µg/L). Microcystis exhibited elevated photosynthesis and hyperpolarization at 50 and 125 µg/L of SMX. An increase of metabolically non-active cells was observed in either ERY or SMX cultures while stimulation of esterase activity was also found at 7 d. ERY and SMX caused damage of membrane integrity due to the overproduction of ROS, which led to increased release of MC-LR. MC-LR production apparently was induced by ERY (0.5-500 µg/L) and SMX (50 and 125 µg/L). In conclusion, ERY and SMX can disrupt the physiological status of Microcystis cells and stimulate the production and release of MC-LR, which can exacerbate potential risks to water systems.

The Effect of Acute Erythromycin Exposure on the Swimming Ability of Zebrafish (Danio rerio) and Medaka (Oryzias latipes).

Erythromycin is a widely used antibiotic, and erythromycin contamination may pose a threat to aquatic organisms. However, little is known about the adverse effects of erythromycin on swimming ability. To quantify erythromycin-induced damage to fish swimming ability, Oryzias latipes and Danio rerio were acutely exposed to erythromycin. The swimming ability of the experimental fish was measured after exposure to varying doses of erythromycin (2 µg/L, 20 µg/L, 200 µg/L, and 2 mg/L) for 96 h. Burst speed (Uburst) and critical swimming speed (Ucrit) of experimental fish significantly decreased. In addition, gene expression analysis of O. latipes and D. rerio under erythromycin treatment (2 mg/L) showed that the expression of genes related to energy metabolism in the muscle was significantly reduced in both species of fish. However, the gene expression pattern in the head of the two species was differentially impacted; D. rerio showed endocrine disruption, while phototransduction was impacted in O. latipes. The results of our study may be used as a reference to control erythromycin pollution in natural rivers.

1,25-dihydroxyvitamin D3 prevents deleterious effects of erythromycin on mitochondrial function in rat heart isolated mitochondria.

Erythromycin (ERY) is a risk factor for cardiotoxicity through the mitochondria pathway. In the current study, we tested the hypothesis that erythromycin could impair mitochondrial function and oxidative stress and 1,25-dihydroxivitamin D3 (calcitriol) treatment could prevent these effects in rat heart isolated mitochondria. Rat heart mitochondria were isolated with mechanical lysis and differential centrifugation. Then isolated mitochondria were first pretreated with three different concentrations of 1,25-dihydroxivitamin D3 (2.5, 5 and 10 µmol/L) for 5 minutes at 37°C, after which erythromycin (10 µmol/L) was added to promote deleterious effects on mitochondria. During 1 hour of incubation, using by flow cytometry and biochemical evaluations, the parameters of mitochondrial toxicity were evaluated, including: succinate dehydrogenase (SDH) activity, mitochondrial swelling, mitochondrial membrane potential (MMP) collapse, reactive oxygen species (ROS) formation and lipid peroxidation (LP). The results showed that erythromycin (10 µmol/L) caused a significant change in mitochondrial function, ROS formation, mitochondrial swelling, MMP collapse, increasing lipid peroxidation and oxidative stress. 1,25-dihydroxivitamin D3 (10 µmol/L) reverted the effect of erythromycin on the tested parameters . In this study, we showed that erythromycin impairs mitochondrial function and induces mitochondrial toxicity in rat heart isolated mitochondria, which were reverted by calcitriol. These findings suggest that 1,25-dihydroxivitamin D3 may be a preventive/therapeutic strategy for cardiotoxicity complications caused by erythromycin.

Resistance of cyanobacteria Microcystis aeruginosa to erythromycin with multiple exposure.

Here we report a set of experiments in which water blooming cyanobacteria Microcystis aeruginosa was repeatedly exposed to erythromycin. Growth inhibition increased with increasing erythromycin concentration (1-150 µg/L) upon first exposure. Maximum inhibition rate (76.06%), occurred under 150 µg/L erythromycin. Moreover, 96-h 50% effective concentration (EC50) was 22.97 µg/L, indicating that the growth of M. aeruginosa was affected by erythromycin under common environmental concentrations. Photosynthesis was hindered by chlorophyll and photosystem II limitations. Malondialdehyde, reactive oxygen species, and superoxide dismutase contents increased significantly under certain concentrations of erythromycin, but superoxide dismutase was suppressed by 150 µg/L erythromycin. Synthesis of intracellular and extracellular microcystins was promoted by 10-60 and by 20-60 µg/L erythromycin, respectively, but both were inhibited by 100-150 µg/L. Principal component analysis and Pearson's correlation revealed the accumulation of reactive oxygen species as the dominant mechanism of erythromycin toxicity to cells. M. aeruginosa repeatedly subjected to erythromycin exposure showed obvious resistance against the antibiotic, especially when treated twice with 60 µg/L erythromycin. The 96-h EC50 was 81.29 µg/L. As compared to the first exposure to erythromycin, photosynthetic and antioxidant activities increased, while growth inhibition and oxidation stress decreased upon multiple exposures. Production and release of microcystins were enhanced by repeated exposure to the antibiotic. Thus, erythromycin persistence in water should be examined, as repeated exposure may lead to serious environmental and human health hazards.

Antioxidant defenses and metabolic responses of blue mussels (Mytilus edulis) exposed to various concentrations of erythromycin.

Erythromycin, one of the most widely used macrolide antibiotics, has been detected in various aquatic environments, so erythromycin ecotoxicity should deserve more attention. In this study, blue mussels (Mytilus edulis) were exposed to erythromycin to explore its potential physiological toxicity. After 2d acute and 7d sub-acute exposure to erythromycin, blue mussel glutathione S-transferase (GST) and catalase (CAT) activities were determined with microplate methods and metabolic responses were analyzed using 1H nuclear magnetic resonance (1H NMR). The results revealed that GST was approximately 1.6 times higher in exposed mussels at 200 mg/L and higher concentrations. CAT was about 1.9 times higher in exposed mussels at 200 mg/L, indicating that erythromycin exposure led that blue mussels enhanced antioxidant responses. Low doses of erythromycin exposure had a relatively small impact on the metabolism, while high doses of erythromycin exposure (200 and 400 mg/L) disturbed metabolic balance. With the increase of erythromycin concentrations, the individual metabolic differences within the same treatment groups also increased. The significant increase in alanine, glutamate, taurine, glycine and betaine were observed after acute and subacute exposure. Betaine played an important role in protecting antioxidant enzyme activities through adjusting osmotic pressure. The metabolomic results also showed the modes of erythromycin acted on the energy metabolism, osmoregulation, nerve activities and amino acid metabolism. This study highlighted how metabolomics can provide a comprehensive picture of metabolic responses, although significant antioxidant and metabolic responses were observed at high exposure concentrations.

The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing.

Erythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5-100 µM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study, the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 µM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12 ± 9% at concentrations of 15-45 µM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28 ± 12% (p < 0.01). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a "window" of concentrations of the drug (15-30 µM) at which the probability of re-entry increased.

Histopathological effects of the antibiotic erythromycin on the freshwater fish species Oncorhynchus mykiss.

Pharmaceuticals are found in the aquatic compartment due to their continuous release in wastewater effluents or direct dispersal in aquaculture practices raising serious threats to human and environmental health. Erythromycin (ERY) is a macrolide antibiotic widely prescribed in human and veterinary medicine to threat a number of bacterial infections, being consequently found in the aquatic environment. The present work intends to evaluate the sub-lethal effects of ERY on juveniles of rainbow trout (Oncorhynchus mykiss) in terms of tissue damage using histochemical staining procedures. Individuals were exposed for 96 h (acute exposure: 0.001-10 mg/L) and 28 days (chronic exposure: 0.05-0.8 µg/L) to environmentally realistic concentrations of ERY. Qualitative and quantitative approaches were used to assess O. mykiss gills and liver tissue alterations after exposure to ERY. For both exposures the most common gill changes recorded were progressive (e.g. hypertrophy of mucous cells and hyperplasia of the epithelial cells). However, circulatory (e.g. aneurysms and oedemas) and regressive (e.g. epithelial lifting of lamellae and lamellar fusion) changes were also observed in the acute assay. Gill morphometric analysis revealed to be a good indicator of subtle alterations in gill architecture in agreement with the qualitative scoring system. In liver, regressive (e.g. cytoplasmic vacuolization, pyknotic nucleus and hepatocellular degeneration) and circulatory disturbances (e.g. hemorrhage and increase of sinusoidal space) were the most frequently observed alterations, but only for the acute assay. Furthermore, all histological changes observed contributed to a significant increase in the pathological index for both organs. The current data demonstrate the existence of a direct dose-effect relationship between the exposure to this specific macrolide antibiotic and the histological disorders recorded in different tissues of the exposed fish. The histopathological findings observed in this study may have been the result of several physio-metabolic dysfunctions. However, the observed tissue lesions were of minimal or moderate pathological importance, non-specific and reversible. Further investigation into the cellular mechanism of action of ERY is needed.

Combined effects of erythromycin and enrofloxacin on antioxidant enzymes and photosynthesis-related gene transcription in Chlorella vulgaris.

Multiple antibiotics are simultaneously detected in aquatic environment, so it is extremely important to study the combined effects of their mixtures. In this study, we investigated the toxic effects of erythromycin (ERY) and enrofloxacin (ENR), added individually or in combination, on Chlorella vulgaris and explored the toxic mechanisms. Results showed that the 96 h-EC50 values of ERY, ENR and ERY-ENR mixture to C. vulgaris were 85.7, 124.5 and 39.9 µg L-1 respectively, and combined toxicity assessment found that joint effect of the two antibiotics was synergism, which was proven by the chlorophyll content in algae. Antioxidant defense system and photosynthesis were involved in toxic mechanisms and the results revealed that both the activities of antioxidant enzymes, and the malondialdehyde (MDA) and glutathione (GSH) contents increased in antibiotic treatments. In addition, the increase was more significant in joint exposure treatment, which implied that the antioxidant defense system was synergistically affected. RT-PCR showed that ERY and ENR upregulated the transcript abundance of psaB, psbC and chlB at low concentrations and the transcription abundance was synergistically increased in combined treatment. Therefore, the risk of the toxicity of antibiotics to aquatic organisms in real environment both at organismal and molecular level increases as a result of their combined presence.

Histopathological effects in gills and liver of Sparus aurata following acute and chronic exposures to erythromycin and oxytetracycline.

Due to their worldwide use and environmental persistence, antibiotics are frequently detected in various aquatic compartments. Their toxic properties raise environmental concerns to non-target organisms. Histopathology data is frequently applied in ecotoxicology studies to assess the effects of different classes of environmental stressors in fish, including antibiotics. Tissue alterations in gills and liver of gilthead seabream (Sparus aurata) individuals acutely (96 h) and chronically (28 days) exposed to environmentally relevant concentrations of the antibiotics erythromycin (ERY: 0.0002-200 µg/L) and oxytetracycline (OTC: 0.0004-400 µg/L), including a control non-exposed group, were evaluated. Several disorders (circulatory, regressive, progressive, and inflammatory) were observed in both organs of all exposed animals. The hereby obtained data showed a higher and significant increase in gill histopathological index of organisms acutely exposed to ERY and of those chronically exposed to OTC. In terms of categorical lesions, only a significant increase of regressive and progressive alterations occurred in gills after chronic exposure to OTC. For the liver, a significant increase in pathological index was also detected, as well as regressive changes, after chronic exposure to OTC. Furthermore, the present study indicates that most of the changes observed in gills and liver were of mild to moderate severity, which might be adaptive or protective, non-specific, and mostly reversible. Despite being observed, irreversible lesions were not significant in any of the fish organs analyzed. Although there were histological changes, gill apparatus was considered still functionally normal, as well as liver tissue, not supporting the occurrence of severe toxicity. In general, the observed histological changes were not stressor-specific, and toxicological mechanistic explanations for the alterations observed in gills and liver are presented. The obtained data showed that histopathological biomarkers can be successfully applied in ecotoxicological studies, evidencing their relevance, responsivity, and complementarity to other biochemical biomarker-based approaches.

Impact of erythromycin on a non-target organism: Cellular effects on the freshwater microalga Pseudokirchneriella subcapitata.

The increasing and indiscriminate use of antibiotics is the origin of their introduction in aquatic systems through domestic and livestock effluents. The occurrence of erythromycin (ERY), a macrolide antibiotic, in water bodies raises serious concerns about its potential toxic effect in aquatic biota (non-target organisms), particularly in microalgae, the first organisms in contact with aquatic contaminants. This study aimed to evaluate the possible toxic effects of ERY on relevant cell targets of the freshwater microalga Pseudokirchneriella subcapitata. Algal cells incubated with significant environmental ERY concentrations presented disturbance of the photosynthetic apparatus (increased algal autofluorescence and reduction of chlorophyll a content) and mitochondrial function (hyperpolarization of mitochondrial membrane). These perturbations can apparently be attributed to the similarity of the translational machinery of these organelles (chloroplasts and mitochondria) with the prokaryotic cells. P. subcapitata cells treated with ERY showed a modification of metabolic activity (increased esterase activity) and redox state (alteration of intracellular levels of reactive oxygen species and reduced glutathione content) and an increased biovolume. ERY induced an algistatic effect: reduction of growth rate without loss of cell viability (plasma membrane integrity). The present study shows that chronic exposure (72 h), at low (µg L-1) ERY concentrations (within the range of concentrations detected in surface and ground waters), induce disturbances in the physiological state of the alga P. subcapitata. Additionally, this work alerts to the possible negative impact of the uncontrolled use of ERY on the aquatic systems.