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Contents lists available atScienceDirect

Ecotoxicology and Environmental Safety

journal homepage:www.elsevier.com/locate/ecoenv

Antibiotic e

ffects on seed germination and root development of tomato

(Solanum lycopersicum L.)

Alessandro Bellino

a

, Giusy Lofrano

a

, Maurizio Carotenuto

a

, Giovanni Libralato

b

,

Daniela Baldantoni

a,⁎

aDepartment of Chemistry and Biology“Adolfo Zambelli”, University of Salerno, Via Giovanni Paolo II, 132 - 84084 Fisciano (SA), Italy bDepartment of Biology, University of Naples Federico II, Via Cinthia, ed. 7, 80126 Naples, Italy

A R T I C L E I N F O

Keywords: Chloramphenicol Spectinomycin Spiramycin Vancomycin Cell division Phytotoxicity

A B S T R A C T

Antibiotics are emerging pollutants released into the environment through wastewater and manure or effluents from livestock plants. Compared to the wide literature on the effects of antibiotics on the development of drug-resistant bacteria and on the adverse effects on animals and human beings, the effects on plants are less in-vestigated. Here we evaluated the effects of four antibiotics (cloramphenicol: CAP, spiramycin: SPR, spectino-mycin: SPT, vancospectino-mycin: VAN) belonging to different chemical groups, on seed germination and root devel-opment of tomato (Solanum lycopersicum L. cv. San Marzano). Specifically, seed germination and root elongation kinetics, as well as the number of mithoticfigures in root apical meristem, were studied in relation to different concentrations of each antibiotic (0, 0.1, 1, 10, 100, 1000 mg L−1) for 10 and 7 days, respectively. Results showed that seed germination was not affected, but root development (root elongation kinetics and cell division) was impaired at concentrations from 10 mg L−1(SPT) and 100 mg L−1(CAP) to 1000 mg L−1(SPR and VAN).

1. Introduction

Antibiotics constitute a new class of contaminants of emerging concern with adverse effects on the ecosystems and potential risks for human health, related to non-voluntary exposure or selection of anti-biotic-resistent bacteria (Halling-Sorensen et al., 1998; Martinez, 2009; Du and Liu, 2012; Roose-Amsaleg and Laverman, 2016). These com-pounds are extensively used for the treatment of both human and ve-terinary infections, as well as in agriculture and aquaculture, with production and consumes increasing worldwide (Sarmah et al., 2006; Pan and Chu, 2016a; Roose-Amsaleg and Laverman, 2016). As bioactive molecules, their half-life in animals is usually short, ranging from days to few weeks, therefore most of them are excreted through urines and feces (Sarmah et al., 2006; Lofrano et al., 2017) in metabolized and non-metabolized forms. Thus, they can enter soil, surface and ground-water through soil manure amendments and effluents from wastewater treatment plants originally not designed to deal with these micro-pollutants (Sarmah et al., 2006; Li, 2014; Bondarczuk et al., 2016; Kaczala and Blum, 2016; Lofrano et al., 2017). Considering that anti-biotics are administered to livestock not only to treat diseases, but also

as growth promoters through food (Phillips et al., 2004; Landers et al., 2012), theirfinal concentrations in soils and water can be exceedingly high, ranging between μg kg−1– mg kg−1and μg L−1– mg L−1, re-spectively (Xie et al., 2011; Yang et al., 2011; Yan et al., 2013).

The number of studies focusing on the toxicological assessment of antibiotics in the environment is constantly increasing, with the aim to bridge the various knowledge gaps (i.e. relevant endpoints to be con-sidered) associated with these issues. Boxall (2004) and Kümmerer (2009)represent two comprehensive reviews on the ecotoxicity of an-tibiotics. Conversely, little is known about the effects of antibiotics, and generally pharmaceuticals, in relation to plants (Bártíková et al., 2016). Only few studies investigated the toxic effects on plants, involving growth reduction and metabolic disorders (Migliore et al., 1995; Hillis et al., 2011; Chen et al., 2011; Bártíková et al., 2016; Pan and Chu, 2016b; Pino et al., 2016). The effects vary in relation to the species, the molecule and the end-points (Martí et al., 2011; Corbel et al., 2015; Pino et al., 2016).Li et al. (2011) reported that in wheat (Triticum aestivum) exposed to oxytetracycline (10, 20, 40, 80 mM) root number was more affected than chlorophyll content and photosynthesis, with IC50= 7.1 mM.Hillis et al. (2011)studied the effects of 10 antibiotics,

http://dx.doi.org/10.1016/j.ecoenv.2017.10.006

Received 17 May 2017; Received in revised form 28 September 2017; Accepted 3 October 2017

Corresponding author.

E-mail address:dbaldantoni@unisa.it(D. Baldantoni).

Abbreviation: CAP, Cloramphenicol; SPR, Spiramycin; SPT, Spectinomycin; VAN, Vancomycin; ANOVA, Analysis of variance; MANOVA, Multivariate analysis of variance; H0, Null

hypothesis; λg, Length of lag phase - seed germination; λe, Length of lag phase - root elongation; μg, Growth rate - seed germination; μe, Growth rate - root elongation; Ag, Maximal

growth - seed germination; Ae, Maximal growth - root elongation; MI , Mithotic Index; IC50, Half maximal inhibitory concentration

Available online 06 November 2017

0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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at concentration ranging from 1 to 10,000 μ g L−1, on seed germination and root elongation of lettuce (Lactuca sativa), alfalfa (Medicago sativa) and carrot (Daucus carota), proving the absence of toxic effects on germination up to the highest concentrations and, conversely, the usefulness of root elongation as a sensitive endpoint, due to root phy-sical interaction with soil contaminants (Klaine et al., 2003). Similarly, Chen et al. (2011) reported inhibition of root elongation in wheat, Chinese cabbage (Brassica campestris) and corn (Zea mays) in relation to chloramphenicol concentrations. Only one study highlighted effects on seed germination even at low concentrations of penicillin (0.004 mg L−1), enrofloxacin (1 mg L−1) and oxytetracycline (10 mg L−1) on soybean (Glycine max), of enrofloxacin (0.1 mg L−1) on sunflower (Helianthus annuus), and of penicillin (4 mg L−1) and oxyte-tracycline (100 mg L−1) on corn (Eluk et al., 2016).

The potential effects of antibiotics on plant growth and develop-ment still need to be fully assessed. Such studies would be of paramount importance considering the possible effects of antibiotics on food webs, ecosystem dynamics and human health. Tomato (Solanum lycopersicum L.) is one of the most important crops worldwide (Baldantoni et al., 2016) and is considered a model species for ecotoxicological tests (Kapustka and Reporter, 1998). The plant can uptake and accumulate tetracyclines and sulfonamides, which impair its growth (Ahmed et al., 2015), and is sensitive, in terms of root elongation, to quinolones, 14-ring macrolides and phenicols (Pan and Chu, 2016b). However, phy-totoxicity or resistance toward other widely employed antibiotics still need to be evaluated. To this aim, the effects of four antibiotics be-longing to different chemical groups, glycopeptides (vancomycin, VAN), 16-ring macrolides (spiramycin, SPR), aminogylcosides (specti-nomycin, SPT) and phenicols (cloramphenicol, CAP), were evaluated on S. lycopersicum cv. San Marzano through seed germination and root development (elongation and cell division in apical meristem) analyses.

2. Materials and methods

2.1. Experimental design and laboratory analyses

Seed germination, root elongation and cell division were evaluated according to internal protocols.

The antibioticsTable 1, supplied by Sigma-Aldrich (Milan, Italy), were dissolved in distilled and sterile water and then diluted to the desired concentrations, nominally 0, 0.1, 1, 10, 100, 1000 mg L−1. The concentrations of CAP, SPR and VAN were evaluated spectro-photometrically using a Cary 50 UV–Vis spectrophotometer (Varian, Palo Alto, USA), whereas those of SPT using a Focus GC equipped with a Flame Ionization Detector (Thermo Fisher Scientific, Waltham, USA). Tomato seeds were exposed to 5 mL of antibiotic solution in Petri dishes during 10 and 7 days for the seed germination and the root elon-gation tests, respectively. Seed germination was evaluated on batches of 100 seeds (subdivided in 5 Petri dishes with 20 seeds each) for each concentration and antibiotic, kept at25±0.5 C in the dark. Germinated° seeds were counted twice per day, early in the morning and in the evening.

Root elongation was evaluated on 20 seeds (1 per Petri dish) for each concentration and antibiotic, kept at25±0.5 C in the dark. Seeds were° imaged once per day with a Coolpix S8300 camera (Nikon, Tokyo, Japan) and root length was derived through image analysis using the Fiji software (Schindelin et al., 2012). After 7 days, roots were excised 1.5 cm from the apex,fixed in cold MeOH:acetic acid 3:1 v:v and kept at −18 C. Mithotic° cells were counted on the Feulgen stained (10' at 60 °C hydrolisis, 60' Feulgen reaction) root apices, squashed in a drop of 50% acetic acid, using a Dialux 20 microscope (Leitz, Germany) at 250x and 400x magnification using phase contrast. The Mithotic Index(MI)was defined as the number of mithotic cells per root meristem.

2.2. Data analysis

Seed germination and root elongation kinetics were described using the logistic, the Gompertz or the Richards models, choosing the one providing the bestfit in terms of Akaike Information Criterium. The set of free parameters shared by the three models, namely the length of lag phase λ( gfor seed germination, λefor root elongation), the growth rate

μ

( g for seed germination, μe for root elongation) and the maximum growth A( gfor seed germination,Aefor root elongation), were derived through nonlinear least-squares using the Gauss-Newton algorithm and employed as descriptors of the kinetics for the following analyses. The functions of the“grofit” package (Kahm et al., 2010) for the R pro-gramming language (R Core Team, 2016) were employed in describing the seed germination and root elongation kinetics.

The overall differences in seed germination and root elongation kinetics among the concentrations of each antibiotic were evaluated, according to Rencher and Christensen (2012), through multivariate analyses of variance (MANOVAs) usingλ, μ and A as dependent vari-ables and the antibiotic concentration asfixed factor. Post hoc evalua-tion of the differences among the concentraevalua-tions of each antibiotic was performed, following MANOVA rejection of the null hypothesis , through linear discriminant analysis with the superimposition of 95% confidence circles for each antibiotic dose. In addition, the differences in each parameter among the doses of each antibiotic were evaluated through one-way analyses of variance (ANOVAs) or Kruskal-Wallis H tests, according to the homoscedasticity or heteroscedasticity of the groups, respectively, evaluated through Breuch-Pagan tests. In the case ofH0rejection, post hoc tests of Tukey HSD, following ANOVA, or Dunn,

following Kruskal-Wallis H test, were employed. Finally, the differences in MI among the concentrations of each antibiotic were evaluated through Kruskal-Wallis H tests, followed by the Dunn post hoc test.

IC50values for the seed germination, root elongation and cell

divi-sion end points were calculated byfitting the concentration-response curves using log-logistic functions with lower asymptote equal to 0. In the case of theMI for CAP and SPR, the concentration-response curve wasfitted using a Cedergreen-Ritz-Streibig model withα=1and lower asymptote equal to 0.

Multivariate analyses were performed using the functions of the “stats” (R Core Team, 2016) and“candisc” (Friendly and Fox, 2016) packages for the R programming language, whereas the univariate analyses with the functions of the“stats”, “lmtest” and “drc” (Zeileis and Hothorn, 2002) packages.

3. Results and discussion

In relation to the three germination parameters λ( g, μg, A )g , no significant difference (forα=0.05)was found among the concentra-tions of each antibioticFig. 1.

Conversely, in relation to the root elongation parameters(λe, μe, A )e , MANOVAs highlighted significant differences, primarily related to μe and Ae Fig. 2, among the concentrations of CAP (Pillai's trace = 0.642,P<0.001), SPT (Pillai's trace=0.656,P<0.001)and VAN (Pil-lai's trace = 0.382,P<0.001). According to the ANOVAs or Kruskal-Wallis H tests, CAP, SPT and VAN determined significant differences in

Table 1

Concentrations (mg L−1), as mean ± s.d. of n = 3 replicate measures, of chloramphenicol (CAP), spiramycin (SPR), spectinomycin (SPT) and vancomycin (VAN) employed in seed germination and root development analyses. Solubility in water at 25 °C is also reported.

Nominal concentration CAP SPR SPT VAN

0 0.0 0.0 0.0 0.0 0.1 0.10 ± 0.04 0.04 ± 0.12 0.09 ± 0.12 0.5 ± 0.2 1 0.89 ± 0.08 0.90 ± 0.02 0.99 ± 0.03 1.5 ± 0.4 10 9.7 ± 0.2 8.53 ± 0.06 9.9 ± 0.2 11 ± 0.2 100 100 ± 2 68.0 ± 0.8 98 ± 2 102 ± 2 1000 1013 ± 24 748 ± 6 992 ± 11 1131 ± 16 Solubility in H2O 2.5·103 <1·103 1.5·105 2.25·102

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μeandAeamong the concentrations, but did not elicit differences in λe. In particular, roots treated with SPT at 10, 100 and 1000 mg L−1had significantly lowerμeandAewith respect to the other concentrations, CAP treated roots showed reducedμe andAe at the concentrations of 100 and 1000 mg L−1in respect to the others and VAN treated roots showed reducedμeandAeonly at 1000 mg L−1(Fig. 3). The differences observed in μe and Ae among the concentrations for each antibiotic were observed also in relation to MI which, in addition, highlighted differences also between the roots treated with 1000 mg L−1

of SPR and the other concentrationsFig. 4.

TheIC50values for the four antibiotics in relation to the germination

and elongation parameters, as well as the mithotic index, are reported inTable 2.

CAP, SPR, SPT and VAN had negative effects on root development, but did not affect seed germination. This is consistent with most of the studies on the topic, which demonstrated weak effects of antibiotics on germination of seeds from several crops, including wheat, rice,

cucumber, lettuce, carrots, alfalfa and tomato (Jin et al., 2009; Liu et al., 2009; Hillis et al., 2011; Pan and Chu, 2016b; Pino et al., 2016). No reduction in the percentage of seed germination was observed in tomato up to concentrations of 300 mg L−1 of tetracycline, sulfa-methazine, norfloxacin, erythromycin and chloramphenicol (Pan and Chu, 2016b) or up to 120 mg kg−1of sulfadiazine sodium, sulfamo-nomethoxine sodium and enrofloxacin (Jin et al., 2009). Our results, considering also germination rate and lag, extend this limit up to con-centrations of 1000 mg L−1of CAP, SPR, SPT and VAN, demonstrating an exceptional tolerance of tomato seed germination toward antibiotics belonging to different chemical groups. The reduced permeation of antibiotics through the seed coat is usually thought to protect seeds from the toxic effects exerted by these molecules (An et al., 2009; Pan and Chu, 2016b). However, considering the differences in seed coat permeability among species and in relation to different molecules (Taylor and Salanenka, 2012), also detoxification or resistance me-chanisms are likely to be involved.

Fig. 1. Barplots of mean ± s.e.m. of μg, Agand λgfor

chlor-amphenicol (CAP), spiramycin (SPR), spectinomycin (SPT) and vancomycin (VAN) in relation to the 0.1, 1, 10, 100, 1000 mg L−1 concentrations, normalized to the 0 mg L−1concentration.

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Apart from λe, the parameters describing root elongation kinetics highlighted significant reductions both in the rate of root elongation and in thefinal root length, as expected considering, on the one hand, the results of the germination experiment and, on the other hand, lit-erature data. Indeed, it is widely accepted that elongation-related parameters are sensitive endpoints to evaluate phytotoxic effects due to the roots physical interaction with the antibiotics, and more generally with soil contaminants (Klaine et al., 2003). Significant decreases in root length were observed at 1000 mg L−1sulfamethazine treatment in carrot, at 10000 mg L−1treatment in lettuce and alfalfa (Hillis et al., 2011), and at 1 mg L−1of oxytetracycline, 10 mg L−1of enrofloxacin, 15 mg L−1of kanamycin and 0.4 mg L−1of penicillin in wheat (Eluk et al., 2016).

In our study, tomato showed wide differences in sensitivity toward the fours antibiotics. According to the IC50, SPT was the most toxic,

followed by CAP, VAN andfinally SPR. Among the used antibiotics, SPT and SPR are the most hydrophilic and the most hydrophobic, respec-tively, confirming the indication of Pan and Chu (2016b)about the importance of thekow in predicting the toxicity of these molecules. However, these considerations cannot be applied to CAP and VAN, the latter more hydrophilic than thefirst, but less toxic toward tomato root elongation. Therefore, it is probable that other chemical or physical properties, like molecular mass, can be involved in determining the phytotoxicity of different antibiotics.

TheMI proved to be a sensitive endpoint, providing even clearer responses than the root elongation parameters and demonstrating toxic

Fig. 2. Linear Discriminant Analysis biplots for chloramphenicol (CAP), spiramycin (SPR), spectinomycin (SPT) and vancomycin (VAN) in relation to μe, Aeand λe. The confidence circles

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effects of SPR in addition to those elicited by the other three antibiotics. Despite its usefulness, this parameter was rarely used in evaluating phytotoxicity of antibiotics, the most notable exception being the study ofXie et al. (2011), who demonstrated the toxic effects of tetracycline on wheat. Moreover, during the analyses, developmental aberrations like lignified vessels and root hair proximal to meristem were observed (Supplementary Fig. 1) in the roots exposed to the highest concentra-tions of all of the antibiotics. This occurrence could indicate hormone-like behaviour of CAP, SPR, SPT and VAN, and deserves further in-vestigations. Sulfadimethoxine- and sulfamethazine-induced develop-ment of root hair and lateral roots in a region proximal to the root tip of barley was previously reported byMichelini et al. (2013), as well as the alteration of willow root morphology induced by sulfadimethoxine (Michelini et al., 2012).

Despite the adverse effects of the four antibiotics on root elongation and cell division in root apical meristem of tomato, the concentrations

at which the effects become appreciable are greater than those usually reported for antibiotics in the environment (Xie et al., 2011; Yang et al., 2011; Yan et al., 2013). Acute phytotoxic effects may thus be rare in the environment, but the presence of low concentrations of such pollutants does not exclude the possibility of chronic effects, nor the presence of acute effects related to the additive or synergistic interactions of dif-ferent antibiotics.

4. Conclusions

Chloramphenicol, spiramycin, spectinomycin and vancomycin, be-longing to four different chemical groups, have phytotoxic effects on tomato root development but not on seed germination. In spite of the observed effects, it has to be considered that their environmental con-centrations are usually lower than the lowest doses eliciting toxic ef-fects in our study, so the effective ecological relevance of their

Fig. 3. Barplots of mean ± s.e.m. of μe, Aeand λefor

chlor-amphenicol (CAP), spiramycin (SPR), spectinomycin (SPT) and vancomycin (VAN) in relation to the 0.1, 1, 10, 100, 1000 mg L−1 concentrations, normalized to the 0 mg L−1 concentration. Different letters, if present, indicate significant differences among the doses of each antibiotic.

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phytotoxicity may be low. However, considering the capability of sev-eral species, including tomato, to accumulate antibiotics, the under-standing of how these compounds can be transferred along trophic webs and affect population dynamics, community assemblages or eco-system processes represents a novel challenge in ecotoxicology. Acknowledgments

The work was carried out using facilities at the Department of Chemistry and Biology of the University of Salerno“Adolfo Zambelli”. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Appendix A. Supplementary data

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Fig. 4. Barplots of mean ± s.e.m. of MI for chloramphenicol (CAP), spiramycin (SPR), spectinomycin (SPT) and vancomycin (VAN) in relation to the 0.1, 1, 10, 100, 1000 mg L−1 con-centrations, normalized to the 0 mg L−1concentration. Different letters, if present, indicate significant differences among the doses of each antibiotic.

Table 2

IC50values (mg L−1) ± 95% confidence level for chloramphenicol (CAP), spiramycin (SPR), spectinomycin (SPT) and vancomycin (VAN) in relation to seed germination and root

elongation parameters, as well as to the mithotic index.

Germination Elongation Mithotic index

λg μg Ag λe μe Ae MI

CAP >1000 >1000 >1000 >1000 221.96±95.16 306.28 ± 127.32 22.73±5.84 SPR >1000 >1000 >1000 >1000 >1000 >1000 250.91±145.98 SPT >1000 >1000 >1000 >1000 83.10±38.45 79.07±39.22 5.03±3.68 VAN >1000 >1000 >1000 >1000 >1000 >1000 294.89±202.86

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