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

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 µ g/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 µ g/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.


Introduction
Erythromycin (ERY), a typical macrolide antibiotic, is commonly used to treat bacterial infections since the 1950 s (Berthet et al., 2010).Following the administration of ERY in humans or animals, most of the parent compound and its metabolites are excreted via feces and urine, before being received by sewage treatment plants (Schafhauser et al., 2018); however, the removal rates of ERY were approximately 30%, and it was not uncommon that the detected levels of ERY in effluents exceeded that in influents (Leung et al., 2012).Effluents from hospitals and aquaculture pounds can be another point sources of ERY (Schafhauser et al., 2018).ERY has been detected in freshwater and marine systems at the maximum concentration of 75.5 and 1.9 µg/L, respectively (Lin and Tsai, 2009;Minh et al., 2009).Moreover, exposure to ERY can introduce undesired impacts on bacteria, algae, invertebrates, fish, etc. (Ji et al., 2012;Schafhauser et al., 2018), and thus ERY is recognized as one of the prioritized pollutants in aquatic environments (see reviews in Mo et al., 2022).
It is of particular concern that ERY can impose hormetic effects in cyanobacteria including M. aeruginosa and M. flos-aquae, where the growth performance was enhanced at relatively low ERY exposure levels but reduced at higher ERY exposure levels (Wan et al., 2015;Zhang et al., 2020).This may have ecological and human health consequences such as the low-dose enhancement of harmful microorganism, including bacteria, fungi, and algae, degrading the water quality and facilitating the development and spread of diseases (Agathokleous et al., 2018(Agathokleous et al., , 2020)).Importantly, the presence of hormesis challenges the current concept of environmental risk assessment, as chemical dose-response models dominantly adopted in environmental risk assessment by regulatory agencies are either threshold models or linear non-threshold models using high exposures, both of which refute the possibility of promoted actions at low exposure levels as in hormesis (Calabrese and Baldwin, 2002;Calabrese, 2013;Agathokleous et al., 2020).
Hormesis, a biphasic dose response phenomenon, is qualitatively characterized by stimulation under low-dose treatments and inhibition under high-dose treatments.The dose response curves of hormesis are typically inverted U-shape or J-shape depending on the endpoints measured (Calabrese, 2013).Additionally, the quantitative features of hormesis can be characterized by the maximum stimulation (~130-160% compared to the control) (Calabrese and Baldwin, 2002).Antibiotics have been reported to induce hormesis in algae and high plants, but most of these studies only measured endpoints at the individual (cellular) and/or physiological (biochemical) levels, but rarely at the molecular level (Agathokleous et al., 2018).Thus, molecular mechanisms underpinning hormesis revealed by omics technologies are particularly lacking and warranted.Notably, Guo et al. (2021) recently found that ERY exposure induced hormesis in R. subcapitata, in which the growth promotion was linked to the modification of xenobiotic metabolism, DNA replication, and metabolism of porphyrin and chlorophyll at the transcriptional level.Metabolomics analysis showed that ABC transporters, metabolism of nucleosides, and energy metabolism were responsible for the observed hormesis (Mo et al., 2023).However, physiological (biochemical) apical endpoints, such as cell viability, photosynthesis, ROS production, soluble protein content, etc., were not determined in these studies, and thus the links between physiological responses and global gene expression patterns remain underexplored.
Unlike their freshwater counterparts, although ERY has been detected in coaster seawater, limited ERY toxicity information is available for marine algae, hampering the risk assessment and environmental surveillance of ERY in the marine coaster waters.For instance, exposure of P. yezoensis to high levels of ERY (e.g., 7.5 g/L) reduced Chl-a fluorescence and photochemical efficiency of PSII (Fv/Fm) while increasing the non-photochemical quenching (Oh et al., 2005).Marine diatoms (3-day EC 50 < 1000 µg/L for C. closterium, C. gracilis, and P. tricornutum) showed relatively less sensitive to ERY toxicity, and inhibited growth, reduced Fv/Fm, and elevated ROS formation were observed in a marine diatom P. tricornutum exposed to high levels of ERY (Sendra et al., 2018).However, whether and how exposure to ERY can induce dose-dependent toxicity in marine algal species remain obscure, especially at environmentally relevant concentrations.
Diatoms are a predominant group of microalgae in marine ecosystems, in which they contribute approximately 40% of the global carbon fixation and serve as food for zooplankton (Armbrust et al., 2004).Particularly, Thalassiosira weissflogii (T.weissflogii) is a commonly used model marine diatom for ecotoxicological studies and biomonitoring due to its global distribution, ecological importance, rapid growth, good sensitivity, and easy cultivation (Araújo and Souza-Santos, 2013;US EPA, 1987).
In this study, using T. weissflogii as a model marine diatom, it is hypothesized that ERY exposure causes hormetic effects in T. weissflogii through perturbing key signaling pathways and biochemical processes involved in exobiotic metabolism, photosynthesis, energy metabolism, and DNA replication-coupled repair.A holistic assessment on ERY to T. weissflogii was performed to assess the toxicological responses at the population (growth performance), physiological (photosynthesis, ROS production, and metabolomic activity), and transcriptional (de novo transcriptome) levels.

Chemical preparation and algae cultivation
A marine diatom T. weissflogii (CCMP 1587) was obtained from Provasoli-Guillard National Centre for Culture of Marine Phytoplankton, Bigelow.T. weissflogii was cultured in the f/2 medium made with artificial seawater (30 ppt salinity; filtered with a 0.45 µm membrane) under 22 ± 1 ℃ and 80 μmol photons m − 2 s − 1 with a 12 h light: 12 h dark cycle.To keep the algae in suspension and facilitate the transfer of CO 2 , algal cultures were manually shaken thrice daily.Algal stocks were sub-cultured each week.T. weissflogii in the logarithmic growth phase was used for the subsequent experiments.A stock solution of ERY (CAS# 114-07-8, HPLC ≥ 98%; Merck, Germany) at concentration of 10 mg/L was freshly prepared and used in the exposure experiments (Nie et al., 2013;Yoshinobu et al., 1983).

Growth inhibition test
Freshwater algae are usually exposed to a chemical for 3 or 4 days in the acute toxicity tests (OECD, 2011;US EPA, 2012).In the present study, the marine diatom (T.weissflogii) was exposed to ERY for 7 days to evaluate the chronic toxicity.Moreover, it allowed sufficient algal biomass to be harvested for the transcriptomic analysis.Briefly, T. weissflogii (1 × 10 5 cells/mL) was seeded in a 250 mL conical flask filled with 150 mL of f/2 medium.A calculated proportion of ERY stock solution was added into the f/2 medium within a conical flask.The ERY concentrations in the exposure medium were measured at day 0 and day 7 using HPLC-MS/MS as in our previous works (Guo et al., 2021;Ma et al., 2021).At day 7, the remained ERY were 56.2% ± 11.1%, 70.7% ± 8.0%, 76.4% ± 8.8% of their initial exposure concentrations for the 1, 750 and 2500 μg/L ERY treatment groups, respectively, while ERY was undetectable in the control group.Cell density was then estimated by counting the algal cells with a hemacytometer under a microscope (Nikon Eclipse 90i, Japan).At day 7, collected algae were either used to measure physiological parameters or transcriptomic analysis.

Determination of total soluble protein, chlorophyll-a and chlorophyll fluorescence
Algae were counted and collected through centrifugation at 3000g for 10 min.Phosphate buffer solution (pH = 7.3) supplemented with a protease inhibitor cocktail (Beyotime, Shanghai, China) was added to resuspend the algae, followed by sonication (HN-650Y, Shanghai, China) in an ice-water bath.The resulting homogenates were centrifuged (12000g) at 4 • C for 10 min.The content of total soluble protein was determined using a BCA assay kit with bovine serum albumin as a standard (Beyotime, Shanghai, China).
Chl-a of collected microalgae were extracted using 90% acetone at 4 • C in the dark for 24 h.Following the extraction, centrifugation at 5000g for 10 min was performed.The absorbances of the algal extracts were recorded using a spectrophotometer (UV-2700, Shimadzu, Japan).The content of Chl-a was calculated based on the Eq. ( 1) (Ritchie, 2006).
where, A 664 and A 630 is the absorbance measured at 664 and 630 nm, respectively.
The collected algae were transferred to a 24-well plate.As described in Gao et al. (2018), after dark adaptation for 15 min, the minimum fluorescence yield (F0) and the maximum fluorescence yield (Fm) were measured using an Imaging-PAM fluorometer (Maxi version; Heinz Walz GmbH, Germany).The maximum quantum yield of PSII was calculated as Fv/Fm = (Fm -F0)/Fm.

Transcriptomic analysis
Based on the data of growth inhibition test, algae from the control and the 1 (an environmentally realistic concentration), 750 (EC 20 ), and 2500 µg/L (EC 50 ) ERY treatments were collected at day 7 for transcriptomic analysis.Total RNA of T. weissflogii was extracted using Trizol method.RNA quality was checked using Agilent 2100 Bioanalyzer (Agilent Technologies, USA), and algal samples (RIN > 8) were used for the subsequent library preparation.In total, 12 cDNA library were prepared and sequenced using an Illumina NovaSeq platform.Reads of paired-end with a 150 bp read-length were generated.The resulting raw reads were cleared up, and high-quality reads (> Q20) were then de novo assembled using Trinity.The transcript with the longest sequence was considered as a unigene.All the assembled unigenes were compared with the databases including NR, Swiss-Prot, GO, KEGG, and eggNOG, and Pfam using NCBI BLASTX program.Based on matches in these databases, an E-value cutoff < 1 × 10 − 5 was applied to assign annotations to these unigenes.Transcript abundance was then calculated by mapping clean reads to unigene sequences using RSEM, followed by normalization using the FPKM method.Differential expression of genes was analyzed using DEseq2 package in R, and the differentially expressed genes (DEGs) were selected using an adj-p value < 0.05 and | foldchange| > 1.5.The identified DEGs were finally subjective to the enrichment analysis of GO and KEGG pathway, with a p < 0.05 considered statistically significant.

Statistical analysis
Statistical analysis was conducted using Graph Pad Prism 9 (San Diego, USA).Normality test was performed on collected data including growth, content of total soluble protein, Chl-a content and Chl-a fluorescence and ROS formation.A one-way analysis of variance (ANOVA) followed by Tukey's posthoc test was applied, and a p < 0.05 was deemed significant (*).
Fig. 1.Induction of hormesis in a model diatom T. weissflogii following erythromycin (ERY) exposure for 7 days.Cell densities of T. weissflogii were estimated at day 0, 2, 3, 4 and 7 of the ERY treatments, respectively, using a hemacytometer under a microscope.An asterisk (*) indicates significant difference (p < 0.05) between the control and ERY treatments in one-way ANONA followed by Tukey's posthoc test.Data are presented as mean ± SD (n = 3).

Discussion
Using T. weissflogii as a model marine diatom, in accordance with the hypothesis, the present study revealed that ERY exposure induced hormesis through the modification of signaling pathways and biochemical processes involved in exobiotic metabolism, energy metabolism, and photosynthesis.Notably, signaling pathways related to ribosome and protein translation, but not DNA replication and repair as in R. subcapitata (Guo et al., 2021), were shown to be highly involved in the ERY-induced hermetic effects in T. weissflogii.Novel mechanistic information was generated, in which the transcriptional alterations were linked to the physiological responses including cell viability, ROS production, Chl-a content, Fv/Fm, and soluble protein content in ERY-induced hormesis.

Genes of exobiotic metabolism
Xenobiotics in plants are metabolized in the detoxification system including phases I, II and III (Coleman et al., 1997).Redox reactions in phase I are catalyzed by cytochrome P450 monooxygenases, and through which hydrophobic compounds are typically converted into less Fig. 4. Detection of reactive oxygen species (ROS) formation in T. weissflogii following erythromycin (ERY) exposure for 7 days.Hydroxyl radical (HO • ) and Singlet oxygen ( 1 O 2 ) was determined using use 3′-(p-hydroxyphenyl) fluorescein (HPF) and Singlet Oxygen Sensor Green (SOSG) using a flow cytometer, as illustrated in A (H 2 O 2 was used as a positive control) and C, respectively.Relative production of HO • and 1 O 2 are presented in bar charts B and D, respectively.An asterisk (*) indicates significant difference (p < 0.05) between the control and ERY treatments in one-way ANONA followed by Tukey's posthoc test.Data are presented as mean ± SD (n = 3).hydrophobic metabolites (Ma et al., 2015).Notably, hydrolases and/or laccases may also play a critical role in phase I detoxification reactions in plants including algae.Subsequently, metabolites resulting from the phase I reactions are conjugated with hydrophilic moieties, including glutathione, glucuronic acid, and sulfate, catalyzed by glutathione S-transferases (GSTs), glycosyltransferases, and sulfotransferases, respectively, to further improve their solubility (Zhang and Yang, 2021).Metabolites generated from reactions of phases I and II are finally transported into vacuoles intracellularly and/or exported into extracellular matrix by ATPbinding cassette (ABC) -type transporters in phase III processes (Coleman et al., 1997).
In the present study, no genes of detoxification systems were differentially expressed in T. weissflogii exposed to 1 µg/L ERY, nor DEGs of phrase I detoxification was identified in the 750 and 2500 µg/L ERY exposure group; however, several genes related to exobiotic metabolism and transportation were highlighted in T. weissflogii exposed to 750 and 2500 µg/L ERY.Specifically, gst, large (encodes glycosyltransferase-like protein LARGE), and abcg2 were downregulated, while abca3 and abcb1
processes of ribosome recycling and translational control (Kang et al., 2010;Pisarev et al., 2010).The altered expression of ABC transporter genes may contribute to disturbed molecular signaling and biochemical processes of ribosome and translation (discussed in Section 4.2) and reduced resistance to ERY stress, amplified as growth inhibition in the 750 and 2500 µg/L ERY exposure groups.These findings are partially in agreement with ERY-induced growth inhibition in R. subcapitata, in which genes of exobiotic metabolism (encodes CYP450, hydrolases, and amine oxidases) and ABC transporter genes (encodes ABCA, ABCB, ABCI, ABCG) were exclusively downregulated following exposure to 80 or 120 µg/L of ERY (Guo et al., 2021).These molecular findings supported the hypothesis that plant defense mechanisms could be activated by exposure of antibiotics, leading to stimulation in the framework of xenohormesis to enhance tolerance to expected adverse environmental conditions (Agathokleous et al., 2018;Howitz and Sinclair, 2008).

Pathways related to ribosome and translation
Ribosomes are the cellular organelles where proteins are produced through translation (Thomson et al., 2013).In eukaryotic cells, ribosome biogenesis involves correct generation and dedicate assembly of 4 rRNAs (i.e., 18 S, 5.8 S, 25 S, and 5 S rRNA) and 79 ribosomal proteins (i.e., 46 large subunits and 33 small subunits), with the assistance of numerous factors (e.g., ATPases, GTPases, helicases, kinases, and nucleases) in multiple steps (Thomson et al., 2013).
Ribosome biogenesis starts with co-transcription of the 18 S, 5.8 S and 25 S rRNA, followed by their assembly into the 90 S pre-ribosomes with other components (e.g., UTP-C complex, t-UTP complex, UTP-B complex, and MPP10 complex) in the nucleolus (Piazzi et al., 2019;Thomson et al., 2013).The co-transcript of rRNAs is then cleaved to produce two particles, pre-40S and pre-60S.Subsequently, both pre-40S and pre-60S are matured in the nucleoplasm with multiple assembly factors (e.g., the A3 factors, dynein-like Rea1).The resulting matured pre-40S and pre-60S particles are processed into mature 40 S and mature 60 S in the cytoplasm (Fig. 5).Ribosome synthesis is a highly energy-demanding process, and all these components and associated factors are essential for ribosome biogenesis (Piazzi et al., 2019).The mature 40 S and mature 60 S are finally assembled into ribosomes functioning as protein production machines (Thomson et al., 2013).

Pathways of amino acid metabolism and nitrogen metabolism
Molecular signaling pathways related to synthesis or metabolism of amino acid were altered in T. weissflogii under ERY stress.Specifically, there are 1, 2, and 7 signaling pathways related to amino acid metabolism or biosynthesis enriched in the 1, 750 and 2500 µg/L ERY exposure groups, respectively (Table 1).Signaling pathway of arginine and proline metabolism and/or signaling pathway of arginine biosynthesis were altered in these ERY treatments, suggesting that the biosynthesis and metabolism of arginine may be a prioritized target for ERY toxicity in diatom.Notably, these data show that more types of amino acid may be affected with the increasing of ERY treatment does.Given that ribosome assembly and translation are the major targets of ERY toxicity in bacteria (Arenz et al., 2014) and potentially in T. weissflogii as discussed above, modifications on amino acid metabolism are likely to be downstream events of the disturbed ribosome biosynthesis and translation (production of proteins with various amino acids); however, further experimental analyses and data collection are required to test these hypotheses.In a previous study, metabolomics analysis shows that amino acids including glutamate, leucine, alanine, proline, isoleucine, tryptophan, glutamine were depleted but content of arginine increased in R. subcapitata treated with 80 and 120 µg/L ERY (Mo et al., 2023).These findings are in line with the transcriptomic data that signaling pathways, such as valine, leucine and isoleucine degradation, glycine, serine and threonine metabolism, valine, leucine and isoleucine degradation, and tyrosine metabolism, were altered in R. subcapitata exposed to 120 µg/L ERY (Guo et al., 2021).
Amino acids are vital for living organisms including algae, in which they can be used to produce proteins and metabolites in various biological processes, such as growth, development, reproduction, etc. (Causin, 1996;Kumar and Bera, 2020).Additionally, the biosynthesis and metabolism of amino acids are intimately linked to central carbon metabolism, nitrogen metabolism, sulfur metabolism, etc. (Chaput et al., 2020).The ERY-induced perturbations on biosynthesis and/or metabolism of amino acid may affect signaling pathways and biochemical processes related to energy metabolism, as observed in T. weissflogii treated with 750 or 2500 µg/L ERY, where signaling pathways including pentose phosphate pathway, sulfur metabolism, and nitrogen metabolism were significantly altered (Table 1).Notably, nitrogen is often a growth rate-limiting factor in plants including algae and keeping a balance between nitrogen metabolism and accumulation is vital for maintaining redox homeostasis (Chaput et al., 2020;Noctor et al., 2007;Smith et al., 2019).The consistent downregulation of nrt (encodes nitrate/nitrite transporter), nr (encodes nitrate reductase), glnA (encodes glutamine synthetase) may reduce the capacity of algae to use extracellular nitrate and nitrite as nitrogen sources to produce intermediates for the biosynthesis and/or metabolism of amino acids (e.g., arginine biosynthesis, glutamate metabolism) and disrupt the redox hemostasis, as exemplified by excessive production of ROS within the algal cells.Taken together, exposure to high levels of ERY modified the biosynthesis and metabolism of amino acid and nitrogen metabolism, altering energy metabolism, redox status, and metabolomic activity, which leads to decline of total soluble protein and ultimately growth inhibition of T. weissflogii.

Photosynthesis system
Oxygenic photosynthesis in plants including algae requires Chl to absorb and transmit light energy in the light harvesting processes (Wang and Grimm, 2021).Chl bind to antenna proteins to form the core components of photosynthesis systems I and II.Biosynthesis of Chl starts with L-glutamate to produce 5-aminolevulinate through three enzymatic reactions generating two intermediates (L-glutamyl-tRNA (Glu) and L-glutamate 1-semialdehyde) catalyzed by glutamyl-tRNA synthetase (encoded by ears), glutamyl-tRNA reductase, and glutamate-1-semialdehyde in each step, respectively (Bollivar, 2006).Subsequently, from 5-aminolevulinate, it takes six more enzymatic reactions to generate protoporphyrin IX, and these catalysis enzymes include hydroxymethylbilane synthase (encoded by hemC), uroporphyrinogen decarboxylase (encoded by hemE), coproporphyrinogen oxidative decarboxylase (encoded by hemF), etc. (Bollivar, 2006;Wang and Grimm, 2021).Down-regulation of genes including ears, hemC, hemE, and hemF were observed in algae treated with 2500 µg/L ERY, suggesting the biosynthesis of protoporphyrin IX was inhibited (Table 1).
Following the generation of protoporphyrin IX, another eight enzymatic reactions are required to finally produce Chl-a, in which eight enzymes are responsible for the catalysis (Bollivar, 2006;Wang and Grimm, 2021).Of which, magnesium chelatase (encoded by chlD, chlH, and chlI) catalyzes the insertion of magnesium into protoporphyrin IX to produce magnesium protoporphyrin.Another important intermediate, divinyl-protochlorophyllides, is converted into divinyl chlorophyllide a by protochlorophyllide reductase (encoded by por), followed by the generation of divinylchlorophyll a catalyzed by divinyl chlorophyllide a 8-vinyl-reductase (encoded by dvr) in the next step (Bollivar, 2006).Downregulation of chlH, por, and dvr was evident in the 2500 µg/L ERY treatment (Table 1), suggesting that biosynthesis of Chl-a was inhibited.Indeed, these results agree with physiological measurement data, where the contents of Chl-a declined in the 750 and 2500 µg/L ERY treatments but increased in T. weissflogii exposed to 1 µg/L ERY.Notably, protoporphyrin IX can also be converted to heme and then cytochrome c, an essential component of the mitochondrial respiratory chain, with the catalysis of protoporphyrin ferrochelatase and cytochrome c heme-lyase (encoded by hccs), respectively (Heinemann et al., 2008).The downregulation of hccs suggests that the production of cytochrome c may be reduced, causing structural and functional impairments to mitochondria in algae exposed to high levels of ERY (Nie et al., 2013;Machado and Soares, 2019a).
Pigments are assembled into antenna proteins to form the cores of light-harvesting complex I and II (LHCI and LHCII), serving as essential components embedded in the chloroplastic thylakoid membrane for the light harvesting in photosynthesis (Wang and Grimm, 2021).In this study, expression of lhca1 and lhca4 (encodes light-harvesting complex I Chl-a/b binding protein 1 and 4) were downregulated, suggesting the structure and function of LHCI may be impaired following exposure to high levels of ERY, associated with the reduced Fv/Fm and excessive production of ROS (e.g., HO • and 1 O 2 ).These findings are generally in agreement with hormesis induced in M. flos-aquae where a slight increase in ROS production and Fv/Fm, accompanied by significantly elevated Chl-a content and rETR in the low-dose stimulation.By contrast, ROS production, enzymatic activities of superoxide dismutase (SOD) and catalase (CAT), and malonaldehyde (MDA) significantly increased, while Chl-a content, Fv/Fm, rETR were significantly reduced in high-dose inhibition (Wan et al., 2015).Taken together, a slight but non-significant elevation of ROS production is likely to serve as an initial incentive for the low-dose growth stimulation in ERY-induced hormesis (Agathokleous et al., 2020), followed possibly by a slight increase in ribosome resemble and protein translation, photosynthesis, and cell viability.Exposure to high dose of ERY, on the other hand, continued to increase the ROS production, causing structural and functional impairments to T. weissflogii (e.g., ribosomes, protein translation, photosynthesis, and cell viability) and ultimately resulting in growth inhibition.

Fig. 2 .
Fig. 2. Determination of cell viability of T. weissflogii following a 7-day erythromycin (ERY) exposure.Cell viability was assessed using fluorescein diacetate (FDA) staining at day 7 of the ERY treatments with a flow cytometer.An asterisk (*) indicates significant difference (p < 0.05) between the control and ERY treatments in one-way ANONA followed by Tukey's posthoc test.Data are presented as mean ± SD (n = 3).

Fig. 3 .
Fig.3.Alterations in protein contents and photosynthesis parameters in T. weissflogii following a 7-day erythromycin (ERY) exposure.Contents of total soluble protein (A) and Chl-a (B) were assessed using a spectrophotometer, while Fv/Fm (C) was determined using an Imaging-PAM fluorometer.An asterisk (*) indicates significant difference (p < 0.05) between the control and ERY treatments in one-way ANONA followed by Tukey's posthoc test.Data are presented as mean ± SD (n = 3).