Chemosphere 104 (2014) 258–261

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Chemosphere

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Short Communication

Neutralization of the antimicrobial effect of glyphosate by humic acidin vitro

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.10.064

⇑ Corresponding author at: Institute of Bacteriology and Mycology, Faculty ofVeterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig,Germany. Tel.: +49 03419738183; fax: +49 03419738199.

E-mail addresses: shehata@vetmed.uni-leipzig.de, dr_awadali_1@yahoo.com(A.A. Shehata).

Awad A. Shehata a,b,c,⇑, Manfred Kühnert d, Svent Haufe d, Monika Krüger a

a Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germanyb Albrecht Daniel Thaer-Institute of Agronomy at the University Leipzig, Gustav-Kuhn Straße 8, 04159 Leipzig, Germanyc Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egyptd WHPharmawerk Weinböhla GmbH, Poststr. 58, 01689 Weinböhla, Germany

h i g h l i g h t s

� Glyphosate has an antimicrobial effect on intestinal microorganisms.� Glyphosate can disturb the bacterial community due to differences in sensitivity between microorganisms.� Humic acids neutralized the antimicrobial effect of glyphosate.

a r t i c l e i n f o

Article history:Received 1 July 2013Received in revised form 26 September 2013Accepted 16 October 2013Available online 20 November 2013

Keywords:GlyphosateHumic acidsMicrobiotaAntimicrobial

a b s t r a c t

In the present study, the neutralization ability of the antimicrobial effect of glyphosate by different humicacids was investigated. The minimal inhibitory concentrations of glyphosate for different bacteria such asBacillus badius, Bifidobacterium adolescentis, Escherichia coli, E. coli 1917 strain Nissle, Enterococcus faecalis,Enterococcus faecium, Salmonella enteritidis and Salmonella typhimurium were determined in the presenceor absence of different concentrations of humic acid (0.25, 0.5 and 1.0 mg mL�1). Our findings indicatedthat humic acids inhibited the antimicrobial effect of glyphosate on different bacteria. This informationcan help overcome the negative impact of glyphosate residues in feed and water.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Glyphosate (N-(phosphonomethyl) glycine) is a highly effectiveherbicide because of its potent inhibition of 5-enolpyruvyl shikim-ate 3-phosphate synthase (EPSPS), enzymes of the shikimate path-way that govern the synthesis of aromatic amino compounds inhigher plants, algae, bacteria and fungi (Banta et al., 2009). Its her-bicidal action is generated by chelating manganese required in thereduction of the flavin mononucleotide (FMN) co-factor for EPSPS(Cerdeira and Duke, 2006). Glyphosate and its metabolite, amin-omethylphosphonate (AMPA), have been detected in green imma-ture seed (Lorenzatti et al., 2004), harvested seeds (Benachouret al., 2007) and in ground water (Sanchís et al., 2012). Glyphosateresidues differ from country to country (in some countries

glyphosate is sprayed out of control) and even within a countryindiscriminately depending on the quantity and frequency of gly-phosate application. Glyphosate treatment for desiccation shortlybefore crop desiccation harvest assures an increased glyphosateresidue in food sources as well (Baig et al., 2003). The maximumdaily intake (MDI) of glyphosate depends on the ration composi-tion and the percent of each contaminated component in the ra-tion. Glyphosate also has been found in water samples from atransgenic soybean cultivation area ranging from 0.10 to0.70 mg L�1 (Peruzzo et al., 2008). Some poultry and cattle feedsamples in Germany were found to have 0.4–0.9 mg glyphos-ate kg�1 (Data not published). Different opinions exist about thesafety of this herbicide in the literature. Some reports have as-sumed there is no any significant risk to human and animal healthby the use of glyphosate since the EPSPS enzyme is absent in ani-mals. However, other inhibition pathways are involved, such ascytochrom P450 aromatase inhibition (Samsel and Seneff, 2013).

It has been shown that glyphosate in vertebrates is genotoxic(Poletta et al., 2009) and teratogenic (Paganelli et al., 2010), cyto-toxic (Isolauri et al., 2001; Duke et al., 2003; Gasnier et al., 2009)

A.A. Shehata et al. / Chemosphere 104 (2014) 258–261 259

and causes apoptosis (Clair et al., 2012a). Glyphosate showed acytotoxic effect to human placental, embryonic kidney and livercell lines at very low sub-agricultural dilutions (Havenaar et al.,1992; Duke et al., 2003; Gasnier et al., 2009). Glyphosate inducesnecrosis and apoptosis in mature rat testicular cells in vitro and de-creases testosterone at low concentrations (1 ppm) (Clair et al.,2012a,b). Recently, it has been proposed that glyphosate may bea significant factor in the observed increased risk to Clostridiumbotulinum infection in cattle in Germany over the past 10–15 years(Krüger et al., 2013). Glyphosates toxicity to Enterococcus spp. leadsto an imbalance in the gut favoring overgrowth of Clostridium spp.(Krüger et al., 2013; Shehata et al., 2013a) because common bene-ficial bacterium, Enterococcus spp. suppresses Clostridium (Shehataet al., 2013b).

Humic acids are classified among the polyvalent weak organicacids and possess the ability to chemical form complexes. Theuse of humic acids and their sodium salt for the oral treatmentof all animals on food production farms is currently permitted.Although many studies have been reported that glyphosate canbe sorbed to humic acids (Piccolo et al., 1995, 1996; Albers et al.,2009; Banta et al., 2009; Mazzei and Piccolo, 2012), there is no dataabout the effect of humic acids on the antimicrobial effect of gly-phosate. Accordingly, the present study was initiated to investigatethe ability of humic acids to neutralize the antimicrobial effect ofglyphosate in vitro.

2. Materials and methods

2.1. Indicator microorganisms

The following bacteria were used in the present study: Bacillusbadius, Bifidobacterium adolescentis, Escherichia coli, E. coli 1917strain Nissle, Enterococcus faecalis, Enterococcus faecium, Salmonellaenteritidis and Salmonella typhimurium. Cultural conditions and theorigin of these strains are shown in Table 1.

2.2. Minimal inhibitory concentration

The minimal inhibitory concentrations (MIC) of glyphosate(Roundup UltraMax�, Monsanto, USA) for these pathogens weretested according to the National Committee for Clinical LaboratoryStandards (NCCLS). Briefly, the lowest concentration of glyphosatewhich showed bactericidal or bacteriostatic effects was deter-mined in a 24-well micro-titre plate. Serial dilutions of glyphosate(10, 5, 2.5, 1.2, 0.6, 0.3, and 0.15, 0.075 mg mL�1) were made inbroth culture media. After the addition of bacteria (105 cfu mL�1)to the plates, they were incubated at 37 �C. The MIC value was

Table 1Target strains used, their origin and medium used for glyphosate experiments.

Genus/species Reference/source

Bacillus badius Shehata et al. (2013a)Bifidobacter adolescentis Shehata et al. (2013a)E. coli Shehata et al. (2013a)E. coli 1917 strain Nissle Ardeypharm GmbH, Herdecke, GermanyEnterococcus faecalis Shehata et al. (2013a)Enterococcus faecium Shehata et al. (2013a)Salmonella enteritidis Shehata et al. (2013a)Salmonella typhimurium Shehata et al. (2013a)

a Blood agar (Fluka, Germany).b RCM: reinforced clostridial medium (RCM, SIFIN, Germany).c MRS: deMan, Rogosa and Sharpe Lactobacillus Agar (Oxoid, Germany).d CATC: citric-acid-Tween-carbonate agar (Oxoid, Germany).e Caso agar (3.5% casein–soya, 0.3% yeast extract, 0.1% glucose, 1.5% agar–agar).

evaluated by quantitative analysis of bacterial growth on suitableagar media (Table 1).

2.3. Neutralization of antibacterial activity of glyphosate using humicacids

The MIC value of glyphosate (10, 5, 2.5, 1.2, 0.6, 0.3, 0.15,0.075 mg mL�1) on E. faecalis, B. badius and B. adolescentis in thepresence of 1 mg mL�1 each of the following humic acids; WH67/1, WH67/2, WH67/3, WH67/4, WH67/5, WH67/6.1, WH67/6.2,WH67/7, WH67/8.1, WH67/8.2 and WH67/9 (WH PharmawerkWeinböhla GmbH, Weinböhla, Germany) was evaluated. Differentconcentrations (1 mg, 0.5 and 0.25 mg mL�1) of the most effectivehumic acid (WH67/4) were tested with B. badius, B. adolescentis, E.coli, E. coli 1917 strain Nissle, E. faecalis, E. faecium, S. enteritidis andS. typhimurium in the presence of different glyphosate concentra-tions (10, 5, 2.5, 1.2, 0.6, 0.3, 0.15, 0.075 mg mL�1).

3. Results and discussion

The MIC value of glyphosate for E. faecalis, B. badius and B. adole-scentis were 0.3, 0.3 and 0.15 mg mL�1, respectively. Humic acidsneutralized the antimicrobial effect of glyphosate in different pat-terns. The WH67/2, WH67/4/3, and WH67/4 humic acids at1 mg mL�1 showed the highest neutralization of the antimicrobialeffect of glyphosate. The MIC-value of glyphosate for E. faecalis, B.badius and B. adolescentis in the presence of WH67/2, WH67/3,and WH67/4 humic acids (1 mg mL�1) were more than2.4 mg mL�1. The other humic acids had less neutralizing activityand MIC values ranged from 0.3–0.6 mg mL�1. This indicates thatglyphosate adsorption on humic substances varies considerablydepending on their macromolecular structure. These data supportthe results of previous studies that indicated humic acids could ad-sorb glyphosate and lead to the formation of relatively stable com-plexes (Albers et al., 2009; Banta et al., 2009; Mazzei and Piccolo,2012).

The WH67/4 humic acid neutralized the antimicrobial effect ofglyphosate on B. badius, B. adolescentis, E. coli, E. coli 1917 strainNissle, E. faecalis, E. faecium in a dose dependent manner (Fig. 1).Even at a low concentration of WH67/4 (0.25 mg mL�1), glyphos-ate-sensitive Enterococci, Bifidobacteria and B. badius bacteria growwell in a culture medium containing 0.6 mg glyphosate (Fig. 1). Incontrast, WH67/4 in a concentration of 1 mg mL�1 could not neu-tralize the antimicrobial effect on glyphosate-resistant Salmonellaspp. The neutralization of high concentrations of glyphosate areof no value since they have no biological relevance and exceedby many fold the expected glyphosate residues in the feed and

Lab. No. Culture media Incubation conditions

Agar Broth

1/12 Blood agara RCMb 37 �C/24 h aerobic4/12 MRSc MRS 37 �C/48 h anaerobic5/12 Blood agar RCM 37 �C/24 h aerobicNissle Blood agar RCM 37 �C/24 h aerobic6/12 CATCd RCM 37 �C/24 h aerobic7/12 CATC RCM 37 �C/24 h aerobic9/12 Caso agare RCM 37 �C/24 h aerobic10/12 Caso agar RCM 37 �C/24 h aerobic

Fig. 1. Neutralization of the antimicrobial effect of glyphosate to different bacteria bacteria with WH67/4 humic acid (HA). Bacterial counts were expressed as meanlog10 CFU mL�1.

260 A.A. Shehata et al. / Chemosphere 104 (2014) 258–261

environment. It is worthy to mention that glyphosate also has aninhibitory effect on microbial growth and antibiotics effect at low-er concentrations than those found in agriculture (Clair et al.,

2012b). Glyphosate could disrupt the bacterial community due todifferences in sensitivity between microorganisms (Clair et al.,2012; Shehata et al., 2013a; Krüger et al., 2013). Any modification

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of the environment which leads to a response by living organismsmay be considered as a stress (Missous et al., 2007). The bioticstress observed in biology, is considered a global phenomenon,and can be extended to anthropogenic pressure such as geneticengineering or xenobiotic (including glyphosate) pollution (Tham-mavongs et al., 2008). Protection of Enterococci and Bifidobacteriafrom glyphosate residues by humic acids could help reduce theincidence of glyphosate- induced dysbiose and to reduce the riskof C. botulinum infection hence an antagonistic effect ofEnterococcus spp. on Clostridia was proved (Shehata et al., 2013b).Bifidobacteria is sensitive to glyphosate and thought to create con-ditions unfavourable to the growth of pathogens such as Salmonella(Isolauri et al., 2001). The tested humic acids showed no directsignificant effect on the growth of the tested bacteria (Fig. 1).

In conclusion supplementation of humic acids in feed not onlysubstantially reduces mycotoxicoses (Sabater-Vilar et al., 2007)and improves the performance, carcass, gastrointestinal tract andmeat quality traits (Ozturk et al., 2011); but also neutralize theantimicrobial effect of glyphosate and reduces glyphosate accumu-lation in animal products. Moreover, the use of humic acids inenvironmental clean-up also serves to promote the microbialdiversity in ecosystems.

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