Short Communication

Neutralization of the antimicrobial effect of glyphosate by humic acid

in vitro
Awad A. Shehata
a,b,c,
, Manfred Khnert
d
, Svent Haufe
d
, Monika Krger
a
a
Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germany
b
Albrecht Daniel Thaer-Institute of Agronomy at the University Leipzig, Gustav-Kuhn Strae 8, 04159 Leipzig, Germany
c
Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egypt
d
WHPharmawerk Weinbhla GmbH, Poststr. 58, 01689 Weinbhla, 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 2013
Received in revised form26 September 2013
Accepted 16 October 2013
Available online 20 November 2013
Keywords:
Glyphosate
Humic acids
Microbiota
Antimicrobial
a b s t r a c t
In the present study, the neutralization ability of the antimicrobial effect of glyphosate by different humic
acids was investigated. The minimal inhibitory concentrations of glyphosate for different bacteria such as
Bacillus badius, Bidobacterium adolescentis, Escherichia coli, E. coli 1917 strain Nissle, Enterococcus faecalis,
Enterococcus faecium, Salmonella enteritidis and Salmonella typhimurium were determined in the presence
or absence of different concentrations of humic acid (0.25, 0.5 and 1.0 mg mL
1
). Our ndings indicated
that humic acids inhibited the antimicrobial effect of glyphosate on different bacteria. This information
can 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 effective
herbicide 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 in
higher plants, algae, bacteria and fungi (Banta et al., 2009). Its her-
bicidal action is generated by chelating manganese required in the
reduction of the avin 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 (Benachour
et al., 2007) and in ground water (Sanchs et al., 2012). Glyphosate
residues differ from country to country (in some countries
glyphosate is sprayed out of control) and even within a country
indiscriminately depending on the quantity and frequency of gly-
phosate application. Glyphosate treatment for desiccation shortly
before crop desiccation harvest assures an increased glyphosate
residue in food sources as well (Baig et al., 2003). The maximum
daily 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 a
transgenic soybean cultivation area ranging from 0.10 to
0.70 mg L
1
(Peruzzo et al., 2008). Some poultry and cattle feed
samples in Germany were found to have 0.40.9 mg glyphos-
ate kg
1
(Data not published). Different opinions exist about the
safety of this herbicide in the literature. Some reports have as-
sumed there is no any signicant risk to human and animal health
by the use of glyphosate since the EPSPS enzyme is absent in ani-
mals. However, other inhibition pathways are involved, such as
cytochrom 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)
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 of

Veterinary 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).
Chemosphere 104 (2014) 258261
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and causes apoptosis (Clair et al., 2012a). Glyphosate showed a
cytotoxic effect to human placental, embryonic kidney and liver
cell lines at very low sub-agricultural dilutions (Havenaar et al.,
1992; Duke et al., 2003; Gasnier et al., 2009). Glyphosate induces
necrosis 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 be
a signicant factor in the observed increased risk to Clostridium
botulinum infection in cattle in Germany over the past 1015 years
(Krger et al., 2013). Glyphosates toxicity to Enterococcus spp. leads
to an imbalance in the gut favoring overgrowth of Clostridium spp.
(Krger et al., 2013; Shehata et al., 2013a) because common bene-
cial bacterium, Enterococcus spp. suppresses Clostridium (Shehata
et al., 2013b).
Humic acids are classied among the polyvalent weak organic
acids and possess the ability to chemical form complexes. The
use of humic acids and their sodium salt for the oral treatment
of all animals on food production farms is currently permitted.
Although many studies have been reported that glyphosate can
be sorbed to humic acids (Piccolo et al., 1995, 1996; Albers et al.,
2009; Banta et al., 2009; Mazzei and Piccolo, 2012), there is no data
about the effect of humic acids on the antimicrobial effect of gly-
phosate. Accordingly, the present study was initiated to investigate
the ability of humic acids to neutralize the antimicrobial effect of
glyphosate in vitro.
2. Materials and methods
2.1. Indicator microorganisms
The following bacteria were used in the present study: Bacillus
badius, Bidobacterium adolescentis, Escherichia coli, E. coli 1917
strain Nissle, Enterococcus faecalis, Enterococcus faecium, Salmonella
enteritidis and Salmonella typhimurium. Cultural conditions and the
origin 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 were

tested according to the National Committee for Clinical Laboratory
Standards (NCCLS). Briey, the lowest concentration of glyphosate
which 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 in
broth culture media. After the addition of bacteria (10
5
cfu mL
1
)
to the plates, they were incubated at 37 C. The MIC value was
evaluated by quantitative analysis of bacterial growth on suitable
agar media (Table 1).
2.3. Neutralization of antibacterial activity of glyphosate using humic
acids
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 the
presence 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 Pharmawerk
Weinbhla GmbH, Weinbhla, Germany) was evaluated. Different
concentrations (1 mg, 0.5 and 0.25 mg mL
1
) of the most effective
humic acid (WH67/4) were tested with B. badius, B. adolescentis, E.
coli, E. coli 1917 strain Nissle, E. faecalis, E. faecium, S. enteritidis and
S. 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 acids
neutralized the antimicrobial effect of glyphosate in different pat-
terns. The WH67/2, WH67/4/3, and WH67/4 humic acids at
1 mg mL
1
showed the highest neutralization of the antimicrobial
effect 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 than
2.4 mg mL
1
. The other humic acids had less neutralizing activity
and MIC values ranged from 0.30.6 mg mL
1
. This indicates that
glyphosate adsorption on humic substances varies considerably
depending on their macromolecular structure. These data support
the 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 of
glyphosate on B. badius, B. adolescentis, E. coli, E. coli 1917 strain
Nissle, 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, Bidobacteria and B. badius bacteria grow
well in a culture medium containing 0.6 mg glyphosate (Fig. 1). In
contrast, WH67/4 in a concentration of 1 mg mL
1
could not neu-
tralize the antimicrobial effect on glyphosate-resistant Salmonella
spp. The neutralization of high concentrations of glyphosate are
of no value since they have no biological relevance and exceed
by many fold the expected glyphosate residues in the feed and
Table 1
Target strains used, their origin and medium used for glyphosate experiments.
Genus/species Reference/source Lab. No. Culture media Incubation conditions
Agar Broth
Bacillus badius Shehata et al. (2013a) 1/12 Blood agar
a
RCM
b
37 C/24 h aerobic
Bidobacter adolescentis Shehata et al. (2013a) 4/12 MRS
c
MRS 37 C/48 h anaerobic
E. coli Shehata et al. (2013a) 5/12 Blood agar RCM 37 C/24 h aerobic
E. coli 1917 strain Nissle Ardeypharm GmbH, Herdecke, Germany Nissle Blood agar RCM 37 C/24 h aerobic
Enterococcus faecalis Shehata et al. (2013a) 6/12 CATC
d
RCM 37 C/24 h aerobic
Enterococcus faecium Shehata et al. (2013a) 7/12 CATC RCM 37 C/24 h aerobic
Salmonella enteritidis Shehata et al. (2013a) 9/12 Caso agar
e
RCM 37 C/24 h aerobic
Salmonella typhimurium Shehata et al. (2013a) 10/12 Caso agar RCM 37 C/24 h aerobic
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% caseinsoya, 0.3% yeast extract, 0.1% glucose, 1.5% agaragar).
A.A. Shehata et al. / Chemosphere 104 (2014) 258261 259
environment. It is worthy to mention that glyphosate also has an
inhibitory 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 to
differences in sensitivity between microorganisms (Clair et al.,
2012; Shehata et al., 2013a; Krger et al., 2013). Any modication
Fig. 1. Neutralization of the antimicrobial effect of glyphosate to different bacteria bacteria with WH67/4 humic acid (HA). Bacterial counts were expressed as mean
log
10
CFU mL
1
.
260 A.A. Shehata et al. / Chemosphere 104 (2014) 258261
of the environment which leads to a response by living organisms
may be considered as a stress (Missous et al., 2007). The biotic
stress observed in biology, is considered a global phenomenon,
and can be extended to anthropogenic pressure such as genetic
engineering or xenobiotic (including glyphosate) pollution (Tham-
mavongs et al., 2008). Protection of Enterococci and Bidobacteria
from glyphosate residues by humic acids could help reduce the
incidence of glyphosate- induced dysbiose and to reduce the risk
of C. botulinum infection hence an antagonistic effect of
Enterococcus spp. on Clostridia was proved (Shehata et al., 2013b).
Bidobacteria 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 direct
signicant effect on the growth of the tested bacteria (Fig. 1).
In conclusion supplementation of humic acids in feed not only
substantially reduces mycotoxicoses (Sabater-Vilar et al., 2007)
and improves the performance, carcass, gastrointestinal tract and
meat quality traits (Ozturk et al., 2011); but also neutralize the
antimicrobial effect of glyphosate and reduces glyphosate accumu-
lation in animal products. Moreover, the use of humic acids in
environmental clean-up also serves to promote the microbial
diversity in ecosystems.
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