Charlotte Lynggaard Katholm Master Thesis, Agrobiology – Health and Welfare May 2015 – January 2016 60 ECTS

Matriculation number: 20095408 Department: Department of Animal Science, Research Centre Foulum, Aarhus University Principal supervisor: Senior scientist Ole Højberg Co – supervisor: Senior scientist Martin Tang Sørensen

Effects of Roundup (glyphosate) on gut microorganisms of farm animals

Effekter af Roundup (glyfosat) på mikroorganismer fra husdyr

Preface and Acknowledgement This master thesis is completed as a final part of the master degree, Agrobiology – Animal Health and Welfare,

at Aarhus University.

The thesis corresponds to 60 ECTS and consists of a literature review combined with an experimental part.

The experiments were conducted in the laboratory facilities at Research Centre Foulum, Aarhus University.

The focus is whether Roundup (glyphosate) affects gut microorganisms of farm animals. First and foremost, special thanks goes to Senior Scientist, Ole Højberg, Department of Animal Science –

Immunology and Microbiology and Senior Scientist Martin Tang Sørensen, Department of Animal Science –

Animal Health, Welfare and Nutrition, both Research Centre Foulum, Aarhus University. They have both been

of great help and guidance throughout the process; making themselves available, answering questions all the

way from the beginning, and right until the end.

Thanks also goes to the Immunology and Microbiology group, and especially to the laboratory staff; Trine

Poulsen, Thomas Rebsdorf and Karin Durup. I could not have done the laboratory work without their help.

Furthermore, thanks to Nuria Canibe, Department of Animal Science – Immunology and Microbiology and

Leslie Foldager, Department of Animal Science – Behaviour and Streesbiology, for statistical guidance.

Last, but definitely not least, thanks to Ann-Sofie Riis Poulsen for always making herself available and to

Rasmus Krarup, Katrine Bjørn and Christina Katholm, for help proofreading the thesis.

Aarhus University, January 31th 2016

Charlotte Lynggaard Katholm

Abstract

Glyphosate, is a broad-spectrum, nonselective, systemic herbicide, existing in the form of the acid itself or

formulated as a salt. Roundup consists of the glyphosate isopropylamine (IPA) salt, water and a surfactant,

often a polyethoxylated tallowamine, POEA.

Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate-pathway, which

leads to formation of aromatic amino acids. As the shikimate-pathway is present in plants, fungi and bacteria,

and not in animals, usually glyphosate has been considered as non-toxic to animals. However, recent studies

have raised concerns about the effects of glyphosate on gut microbiota, indirectly affecting farm animals,

when fed feed, containing residues of glyphosate. It has been proposed that glyphosate has a potential

inhibiting effect on growth of commensal bacteria, normally occupying the gut of farm animals, whereas

potential pathogens in general, should be more tolerant.

The present study was conducted to investigate effects of glyphosate on growth of selected commensals and

potential pathogens, in lab media. In addition, effects on the overall activity of microbiota in stomach, cecum

and colon contents from pigs, as well as in rumen contents from cows, were investigated. Glyphosate acid,

glyphosate IPA salt, Roundup and POEA, were included to differentiate eventual effects between different

compounds, included in commercial glyphosate formulations.

We were able to show that glyphosate suppressed growth of both commensals and potential pathogens, and

that inhibition differed between gram-positive and gram-negative bacteria; gram-negative being more

tolerant towards all treatments. In addition, glyphosate changed fermentation pattern in the gut of pigs and

cows and overall, the effect of glyphosate depended on the chemical formulation (acid, salt, mixture and

surfactant) used. In general, glyphosate acid were the least toxic of the treatments, whereas toxicity levels

of glyphosate IPA salt, Roundup and POEA were higher and more similar. The concentrations, at which we

observed an effect on gut content, were much higher than the concentrations affecting individual bacteria,

indicating that gut microbiota is complex.

As pigs and cows are not fed glyphosate directly, feeding trials should be performed, to give a better

indication of how, and if, the animals are affected by residues of glyphosate in feed.

Sammendrag

Glyfosat er et bredspektret, ikke-selektivt, systemisk herbicid, der findes som en syre eller formuleret som et

salt. Roundup består af glyfosat isopropylamin (IPA) saltet, vand og et overfladeaktivt middel, ofte et

polyethoxyleret amin, POEA.

Glyfosat inhiberer 5-enolpyruvylshikimat-3-phosphat-syntasen (EPSPS) i shikimate-pathway’en, som fører til

dannelsen af aromatiske aminosyrer. Shikimate pathway’en er tilstede i planter, svampe og bakterier, men

ikke i dyr, og derfor er glyfosat normalt blevet betragtet som ikke-toksisk overfor dyr. Imidlertid har nyere

undersøgelser udtrykt bekymring overfor virkningerne af glyfosat på tarmens mikroflora, som indirekte kan

have indflydelse på husdyrenes ve og vel, hvis de får foder, der indeholder rester af glyfosat. Det er blevet

foreslået, at glyfosat har en potentiel hæmmende effekt på væksten af kommensale bakterier mens

potentielle patogener overordnet er mere tolerante.

Den foreliggende undersøgelse blev udført for at undersøge virkningerne af glyfosat på væksten af udvalgte

kommensale og potentielle patogener i laboratorie medier. Derudover blev virkninger på den samlede

aktivitet af mikrofloraen i mave -, blindtarms - og tyktarmsindhold fra svin, såvel som i vomindhold fra køer,

undersøgt. Både glyfosat syren, glyfosat IPA saltet, Roundup og POEA, blev inkluderet for at skelne mellem

eventuelle virkninger mellem de forbindelser, der normalt indgår i kommercielle glyfosat-produkter.

Vi var i stand til at vise, at glyfosat (Roundup) hæmmede væksten af både kommensale og potentielle

patogene bakterier, og at hæmningen var forskellig for gram-positive og gram-negative bakterier; gram-

negative var mere tolerante overfor alle behandlingerne. Derudover ændrede glyfosat (Roundup)

forgæringsmønster i mave, blind – og tyktarm hos grise og i vommen hos køer. Overordnet afhang virkningen

af, hvilke formuleringer (syre, salt, Roundup og POEA) der blev anvendt. Generelt var glyfosat syren den

mindst toksiske af behandlingerne, mens toksisiteten af glyfosat IPA saltet, Roundup og POEA var større, og

generelt mere ens. De koncentrationer, hvor vi observerede en effekt på mave -, tarm – og vomindhold, var

meget højere end de koncentrationer, der påvirkede de enkelte bakterier, hvilket indikerer, at den

mikrobielle flora er en kompleks størrelse.

Da grise og køer ikke fodres direkte med glyfosat, bør der udføres fodringforsøg, for at give en bedre

indikation af, hvordan, og hvis, dyrene påvirkes af rester af glyfosat i foder.

Content Theory ................................................................................................................................................................ 1

Introduction ................................................................................................................................................... 1

Working hypotheses .................................................................................................................................. 2

Objectives .................................................................................................................................................. 2

Glyphosate formulations ............................................................................................................................... 3

The chemical properties of glyphosate ..................................................................................................... 3

Surfactants in Roundup ............................................................................................................................. 4

Herbicidal mechanism of glyphosate ............................................................................................................ 5

5-enolpyruvylshikimate-3-phosphate synthase ........................................................................................ 6

The main target of glyphosate – the plant .................................................................................................... 7

Degradation of glyphosate and subsequent effects on soil .......................................................................... 8

Production of glyphosate resistant (GR) crops .............................................................................................. 9

The success of glyphosate ........................................................................................................................... 11

Possible pesticide residues in crops treated with glyphosate ..................................................................... 13

Toxicity of glyphosate .................................................................................................................................. 15

Glyphosate as a carcinogen? ................................................................................................................... 15

Toxicity of POEA ....................................................................................................................................... 16

Toxicity of glyphosate towards microorganisms ......................................................................................... 16

Changes in gut microbiota with respect to glyphosate ............................................................................... 17

Potential levels of glyphosate in gut ....................................................................................................... 17

The effect of glyphosate on poultry microbiota ...................................................................................... 18

The effect of glyphosate on dairy cow microbiota .................................................................................. 19

Microbial fermentation in the gut of pigs and dairy cows .......................................................................... 20

Experimental setup ...................................................................................................................................... 21

Materials and Methods ................................................................................................................................... 22

Chemicals ..................................................................................................................................................... 22

Bacteria used in the experiment ................................................................................................................. 22

Media ........................................................................................................................................................... 22

Techniques used .......................................................................................................................................... 22

Determination of viable counts by use of Drop Plate Procedure............................................................ 22

Quantification of VFA analysis ................................................................................................................. 23

Gas measurements .................................................................................................................................. 23

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial cultures ... 23

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows ................... 25

Slaughter pigs .......................................................................................................................................... 25

Dairy cows ............................................................................................................................................... 26

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs ............................ 27

Statistical analyses ....................................................................................................................................... 28

Results ............................................................................................................................................................. 29

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial cultures ... 29

Commensal bacteria ................................................................................................................................ 29

Potential pathogens ................................................................................................................................ 30

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows ................... 32

Pigs ........................................................................................................................................................... 32

Cows ........................................................................................................................................................ 35

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs ............................ 37

Discussion ........................................................................................................................................................ 38

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial cultures ... 38

Objective 1 ............................................................................................................................................... 38

Objective 2 ............................................................................................................................................... 39

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows ................... 41

Objective 3 ............................................................................................................................................... 41

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs ............................ 45

Objective 4 ............................................................................................................................................... 45

Comparisons of the three different experiments ....................................................................................... 45

Additional work, not included in the thesis ................................................................................................ 46

Conclusion ....................................................................................................................................................... 47

Perspectives and future considerations .......................................................................................................... 49

References ....................................................................................................................................................... 50

1

Theory

Introduction

N-(phosphonomethyl)glycine, commonly known as Glyphosate, is a broad-spectrum, nonselective, systemic

herbicide (Franz et al., 1997). As an analogue of the amino acid glycine, glyphosate can exist in different ionic

states, depending on pH (Chamberlain et al., 1996; Herold et al., 2013). In addition, it can exist in the form of

the acid itself (CAS number 1071-83-6, C3H8NO5P, M = 169.1 g/mol) or formulated as a salt (Budavari, 1996;

Giesy et al., 2000), as isopropylamine (IPA) (CAS number 38641-94-0, C6H17N2O5P, M = 228 g/mol), which is

the one, found in original Roundup products (Malik et al., 1989). In addition to glyphosate IPA salt, Roundup

is made up of water and a surfactant, referred to as inert (Giesy et al., 2000). The surfactant most often used,

is a polyethoxylated tallowamine, POEA (CAS number 61791-26-2)(Giesy et al., 2000).

The herbicidal properties of glyphosate was discovered in the beginning of the 1970’s (Franz, 1974) and since

the commercial introduction of glyphosate formulations in 1974 (Franz et al., 1997; Monsanto, 2015),

glyphosate has become the most dominant herbicide in the world (Grube et al., 2011). The main reasons for

this progress are its broad-spectrum characters and the introduction of glyphosate resistant (GR) crops,

which entered the market in 1996, under the brand ‘Roundup Ready®’ (Monsanto, 2015). The expiry of the

last patent protection for glyphosate took place in 2000, and since, multiple products with glyphosate, as the

active ingredient, have entered the market. This has, in addition to increased use of Roundup Ready crops,

led to an even more widespread use of glyphosate, throughout the years (Duke and Powles, 2008).

Crops treated with glyphosate, either by pre-harvest applications or treatment of GR-crops, during the

growing season, can contain residues that end up in feed (Arregui et al., 2004; Bøhn et al., 2014;

Miljøstyrelsen, 2014; Plantedirektoratet, 2010). As glyphosate exerts the same mechanisms on some

microorganisms, as it does on non-GR-crops (plants), glyphosate residues in feed may have the capacity to

affect gut microorganisms when ingested by livestock and, potentially, reaching critical levels in the gut.

Previous studies have found that glyphosate may affect the growth characteristics of dominating

microorganisms in gut of monogastrics and ruminants, in potential favor of pathogenic bacteria, affecting

health parameters negatively (Ackermann et al., 2015; Krüger et al., 2013b; Shehata et al., 2013a; Shehata et

al., 2013b). These studies have typically worked either with glyphosate acid or with Roundup.

In the beginning of the present study, we overlooked the fact that commercial Roundup formulations

typically contain glyphosate formulated as an IPA salt, instead of the acid itself; a mistake, we have observed

other researchers to have made as well. However, do to this misunderstanding, both glyphosate acid and

glyphosate IPA salt have been included and tested in the present study.

The aim of the project was to compare the effects of different chemical formulations of glyphosate

(glyphosate acid, glyphosate IPA salt and Roundup) as well as the POEA surfactant, and to investigate, at

which concentrations of these formulations, in vitro growth of gut bacteria was affected, if affected at all. In

addition, effects of glyphosate IPA salt, Roundup and POEA, added to stomach, cecum and colon contents

from slaughter pigs and rumen contents from dairy cows, were investigated. Here, the possible in situ effects

on the microbiota composition and activity were measured, by analyzing changes in bacterial cell counts,

production of volatile fatty acids (VFA), changes in pH and production of methane (rumen content only).

Finally, effects of glyphosate IPA salt on growth of a Lactobacillus sobrius strain, inoculated into stomach

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content (pH=5), was studied. This last part was included to investigate whether glyphosate IPA salt had any

effects on bacterial growth, independent of pH in solution.

The first part of this thesis comprises a literature review. I have intended to give an overview of the working

mechanisms behind glyphosate; how and why it has succeeded to be the most used herbicide in the world

and which challenges, spraying possibly can create. Subsequently, the experimental part follows, and even

though the focus here is narrower, compared to the focus in the literature review, I chose to include a

relatively broad spectrum of literature, to put my research objectives into a broader context.

Working hypotheses

1. Glyphosate (Roundup) will suppress growth of gut bacteria

2. Different gut bacteria will have different susceptibilities towards glyphosate (Roundup)

3. Glyphosate (Roundup) will change fermentation pattern in the gut of pigs and cows

4. The effect of glyphosate will depend on the chemical formulation (acid, salt, mixture and surfactant)

Objectives

To investigate if glyphosate acid, glyphosate IPA salt, Roundup and POEA inhibits bacterial growth.

To investigate if inhibiting effects of glyphosate acid, glyphosate IPA salt, Roundup and POEA differs between

commensal - and potential pathogenic bacteria.

To investigate if glyphosate IPA salt, Roundup and POEA show any effects on the composition and activity of

gut microbiota, when added to stomach, cecum and colon content from slaughter pigs and to rumen content

from dairy cows.

To investigate if growth of a strain of Lactobacillus sobrius in stomach content is affected by glyphosate IPA

salt, when pH is held constant (pH=5).

3

Glyphosate formulations

Glyphosate exists as an acid or in formulation with different salts (Budavari, 1996; Giesy et al., 2000) and

glyphosate IPA salt is the one, primarily used in formulations (Malik et al., 1989). In glyphosate acid,

intermolecular hydrogen bonding between -OH and phosphono oxygens, and between -NH and phosphono

oxygens, creates a strong crystalline matrix, leaving the compound relatively insoluble in water (Knuuttila

and Knuuttila, 1985; Shoval and Yariv, 1981). At 25° the solubility is only 12 mg/ml (acid) (Budavari, 1996)

whereas glyphosate IPA salt has a solubility of 900 mg/ml. Here, hydrogen bonding only forms between three

IPA groups (NH4+) and the phosphono oxygens (PO3

2-), leaving the compound less resistible to breakage and

therefore more soluble in water (Shoval and Yariv, 1981). This is the main reason why, glyphosate is

formulated as the IPA salt in Roundup.

Glyphosate is the active ingredient (a.i) in Roundup (Franz et al., 1997). When comparing the effect of

different glyphosate-products, acid equivalent (a.e.) is more useful, than a.i. Active ingredient includes the

weight of the salt, formulated with glyphosate acid, and as the salt does not have herbicidal activity, active

equivalent only includes the amount of glyphosate, present as acid. EQ1, outlines the relationship between

glyphosate acid and glyphosate IPA salt:

EQ1: a. i. = Molecular mass of the acid−1

Molecular mass of the salt∗ 100 =

169−1

228∗ 100 = 74 %

As an example (EQ2), it is given on the product sheet of Roundup® 2000 that the product contains 400 g/L of

glyphosate acid and 541 g/L of glyphosate IPA salt. This is consistent with EQ1, as the amount of glyphosate

acid can be calculated based on the amount of glyphosate IPA salt:

EQ2: 541g/L ∗ 0.74 = 400 g/L

In general, the formulations contains different concentrations of the IPA salt, even though 360 g/L (a.e.) is

one of the most frequently used (Giesy et al., 2000).

When I refer to glyphosate concentration, throughout the rest of the literature review, as well as the

experimental part, concentrations are given as mg/ml glyphosate acid equivalent, no matter which

compound, glyphosate acid originates from. This eases comparisons.

The chemical properties of glyphosate

Glyphosate is an amphoteric molecule, with a complex ionization pattern. It has four ionizable functional

groups; pKa1 = 0.8 (1st phosphoric), pKa2 = 2.3 (carboxylate), pKa3 = 6 (2nd phosphoric) and pKa4 = 11 (amine),

see Figure 1 (Chamberlain et al., 1996; Herold et al., 2013).

4

Figure 1. Structures and pKa values of glyphosate, modified after Chamberlain et al. (1996); Sprankle et al. (1975)

In its solid, crystalline state, glyphosate exists as a zwitterion (Knuuttila and Knuuttila, 1985). Here, one

proton from the 1st phosphonic group is dissociated and will associate with the extra proton on the amine

group, creating a dipolar molecule. This is evident between pKa 0.8 and 2.3 and as the overall charge of the

molecule equals zero, this is the most stable form, in which glyphosate can exist, see Figure 2.

Figure 2. Structure formula of glyphosate acid (zwitterion). Modified after Chamberlain et al. (1996).

In contrast to the acid, the most stable form of glyphosate IPA salt is present between pKa 2.3 and 6. Here,

the overall charge is zero, and the amine group in IPA will associate with the phosphono group in glyphosate,

see Figure 3.

Figure 3. Structure formula of glyphosate IPA salt. Modified after Chamberlain et al. (1996); Shoval and Yariv (1981)

Surfactants in Roundup

As mentioned, POEA is the surfactant, most used in Roundup, Figure 4 (Giesy et al., 2000). POEA is composed

of tallowamine and two chains of ethoxylate (di-ethoxylates) and is characterized based on the average

oxide/tallowamine ratio (Brausch and Smith, 2007; Mesnage et al., 2013). Tallowamine is a derivative of

tallow, containing a complex mixtures of different fatty acids. These include oleic acid (37-43%), palmitic acid

(24-32%), stearic acid (20-25%), myristic acid (3-6%) and linoleic acid (2-3%) (Diamond and Durkin, 1997). The

5

solubility properties of POEA depends on the length of the polyethylene segment, and the compound is

soluble in water when 12-15 ethylene units are added (Budavari, 1996).

Figure 4. Structure of POEA. The sum of x+y gives 15, and R is a mix of C16 and C18 alkyl and alkenyl chains. After Ahle (1985); Graham

et al. (2006)

Usually, POEA constitutes 15 %, or less, of the formulations, corresponding to 150 g/L (Giesy et al., 2000;

Sawada et al., 1988).

Herbicidal mechanism of glyphosate

In 1980, (Steinrucken and Amrhein) discovered that glyphosate is a potent inhibitor of 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS) (EC 2.5.1.19), Figure 5. EPSPS, catalyses the reversible

formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate from

phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) (Levin and Sprinson, 1964). In plants, EPSPS is

a nuclear-encoded enzyme, located in the chloroplasts (Della-Cioppa et al., 1986) where it catalyses the sixth

(penultimate) step in the shikimate-pathway, leading to the formation of chorismate (Bentley, 1990).

Chorismate is the precursor of the aromatic amino acids; phenylalanine, tyrosine and tryptophane, Figure 5

(Doy and Gibson, 1961; Gibson and Jackman, 1963; Gibson and Gibson, 1962; Gibson et al., 1962), but also

of vitamins K1 and B9 and the plant hormone salicylic acid, as reviewed by Maeda and Dudareva (2012). In

higher plants, the aromatic amino acids are used as precursors of secondary metabolites, which makes up a

substantial part of the total dry weight of the plant (Herrmann, 1995). According to Maeda and Dudareva

(2012), tryptophan is a precursor of alkolids, phytoalexins, indole glucosinolates and auxin. Isoquinoline

alkaloids, pigment betalains and quinones can be synthesized from tyrosine, and phenylalanine is the

common precursor for phenolic compounds, as flavonoids, condensed tannins and lignin. By inhibiting EPSPS,

the plant will no longer be able to synthesize the aromatic amino acids and secondary metabolites, all used

for growth and, consequently, the plant will die.

The shikimate-pathway is only present in plants, fungi and bacteria and not in animals and insects (Bentley,

1990; Franz et al., 1997; Herrmann, 1995; Kishore and Shah, 1988; Padgette et al., 1995a). In addition there

is evidence that apicomplexan parasites, like Toxoplasma gondii, Plasmodium falciparum (malaria) and

Cryptosporidium parvum (Roberts et al., 1998) and Archaea also possesses the shikimate pathway (Bult et

al., 1996; Daugherty et al., 2001; Graham et al., 2001). As the pathway is absent in animals, they need to

obtain the essential aromatic amino acids through their diet (Franz et al., 1997; Herrmann, 1995; Kishore and

Shah, 1988; Padgette et al., 1995a), although tyrosine can be synthesized from phenylalanine (Herrmann,

1995).

6

Figure 5. The shikimate pathway that leads to the formation of aromatic amino acids and the inhibition of EPSPS by glyphosate

(Pollegioni et al., 2011).

In addition to the inhibition of EPSPS, glyphosate has also been shown to target other plant enzymes,

indicating that it might have several sites of action (Bode et al., 1984; Ganson and Jensen, 1988; Lee, 1980;

Rubin et al., 1982). However, glyphosate is not shown to inhibit other PEP dependent reactions (Anton et al.,

1983; Steinrucken and Amrhein, 1984).

5-enolpyruvylshikimate-3-phosphate synthase

To understand how glyphosate exerts its effect on EPSPS, the three dimensional structure of the enzyme,

with and without ligand, have been studied. Stallings et al. (1991) were the first to elucidate the three

dimensional crystal structure of unliganded EPSPS (from E. coli). In short, they revealed a unique protein fold

with two globular hemispheric domains, connected to each other by two crossover chain segments. Some

years later, Schönbrunn et al. (2001) were able to identify the structure of E. coli EPSPS in its liganded form,

especially investigating the binding of S3P and glyphosate. They found, as already stated, that EPSPS is a two-

domain enzyme, but that binding of the ligand S3P, induces a conformational change in the enzyme, turning

it into a closed state. The active site is formed in the interdomain cleft, indicative of an induced-fit

mechanism. The closure leads to an accumulation of positive charges in the cleft, which will attract negatively

7

charged molecules to the active site, and glyphosate can bind. This makes glyphosate an uncompetitive

inhibitor with respect to S3P (Boocock and Coggins, 1983), as binding of S3P is a prerequisite for binding of

glyphosate (Schönbrunn et al., 2001). Schönbrunn et al. (2001) also concluded that glyphosate and PEP share

the same binding site, which is supported by Eschenburg et al. (2002). This makes glyphosate a competitive

inhibitor with respect to PEP (Anton et al., 1983; Boocock and Coggins, 1983).

The main target of glyphosate – the plant

As the main target of glyphosate is located intracellularly, glyphosate has to enter the cells of the plant, to

exert its mechanisms. In general, glyphosate is a polar, hydrophilic molecule, as reflected in its low log Kow

values, which Chamberlain et al. (1996) found to range between -3.39 and -4.85, depending on pH level.

Before spraying, a tank mix is prepared. As mentioned, glyphosate is mainly formed as the IPA salt in

formulations, due to its higher solubility in water (Shoval and Yariv, 1981). Glyphosate IPA salt is most stable

between pH 2.3 and pH 6 (see Figure 3), as the overall charge of the compound is zero. Here, glyphosate IPA

salt exist as two separate ions, weakly held together by hydrogen bonds (Shoval and Yariv, 1981). Therefore,

acidic conditions are most suitable in tank mixes.

Plant cells are covered by a cell wall, composed of cellulose, hemicellulose, pectin and, in many cases, also

lignin. Beneath is a single plasma membrane, surrounding the cytoplasm. The plasma membrane is a lipid

bilayer, embedded with proteins. It works as a semipermeable barrier between inner and outer surface of

the cell, and thereby regulate entry and exit of cells (Raven et al., 2005). The outermost cells of the plant

(upper epidermis) are covered with a waxy protection layer (cuticle). As mentioned, surfactants are a part of

Roundup and they aid in penetration of the waxy cuticle (Franz et al., 1997; Giesy et al., 2000). They reduce

surface tension of spray droplets on the plant (Relyea, 2005), enlarging the area of pesticide coverage and

thereby the penetration of glyphosate, through the plant surface. By improving retention of spray droplets,

they also minimize run-off (McCloskey). These properties enhances the effect of glyphosate, as its

penetration of the cuticle otherwise will be inadequate (Tsui and Chu, 2003).

After penetration of the cuticle, glyphosate is translocated via the vascular tissue (Raven et al., 2005)

throughout stems, leaves and roots of the entire plant; therefore the definition as a systemic herbicide (Franz

et al., 1997). Pline et al. (2002) suggested an increased sensitivity to glyphosate in reproductive tissue over

vegetative, as the shikimate pathway is most active in the growing parts of the plant. Therefore translocation

of glyphosate to the growing points is vital, as glyphosate blocks amino acids synthesis in the fast growing

parts of the plant, followed by inhibition in the other tissues (Pline et al., 2002).

Before glyphosate can inhibit the shikimate pathway, it has to enter the cells, by crossing cell wall and plasma

membrane. Until now, both passive diffusion and active transport have been suggested to be of importance

(Hetherington et al., 1998). A phosphate carrier is expected to be involved in the active transport, and

especially at low concentrations, the phosphate carrier might play a crucial role (Hetherington et al., 1998).

In addition, passive diffusion becomes more prominent at higher glyphosate concentrations (Denis and

Delrot, 1993).

Even though, glyphosate IPA salt exist as two separate ions in aqueous solution, they form an ion-pair, when

entering a lipohilic environment, as the plasma membrane. This facilitates an easier entrance, due to the

overall neutral charge (Krogh, 2016). When glyphosate IPA salt has entered the cell, it must be assumed that

the ion-pair splits again. In higher plants, the cytoplasm is slightly alkaline (7.4 – 7.5) (Gout et al., 1992),

8

leaving glyphosate IPA salt more unstable, due to the anionic character (see Figure 1). In addition to

glyphosate IPA salt, some glyphosate might also enter as the acid.

When glyphosate has exerted its effect, the plant dies slowly, and the process takes several days (Kishore

and Shah, 1988).

Degradation of glyphosate and subsequent effects on soil

One of the reasons why glyphosate is toxic to plants is that they have a limited ability to neutralize or degrade

glyphosate, compared to the soil environment (Franz et al., 1997).

When glyphosate enters soil, it can either be bound to soil particles, leached to subsoil or be degraded

through two principal pathways, mainly by microorganisms, Figure 6 (Borggaard and Gimsing, 2008). In the

first pathway, glyphosate is cleaved by glyphosate oxidoreductase, yielding glyoxylate and AMPA

(aminomethylphosphonic acid). AMPA can further be metabolized to inorganic phosphate and methylamine,

which ultimately is converted to CO2 and NH3, supporting growth of microorganisms. The second pathway

leads to the cleavage of inorganic phosphate from glyphosate, by C-P lyase, and sarcosine is formed.

Sarcosine can be further degraded, yielding formaldehyde and glycine, and both these products can also be

converted, and eventually utilized, by microorganisms (Barry et al., 1992; Borggaard and Gimsing, 2008; Duke

et al., 2012).

Figure 6. Microbial degradation of glyphosate. Modified after Borggaard and Gimsing (2008); Duke et al. (2012).

Even though glyphosate can be used as a nutrient source for some microorganisms (Partoazar et al., 2011;

Sviridov et al., 2015), additon of varying rates influences the soil microflora in different ways, as glyphosate

have shown toxicity towards some groups of microorganisms. Temporary effects were reported by Mijangos

et al. (2009), who observed increased activity and functional diversity within the culturable portion of

heterotrophic soil bacteria, after single glyphosate additions. Those short term effects might be due to a

rapid metabolization of glyphosate (Mijangos et al., 2009; Ratcliff et al., 2006). Lane et al. (2012) found

increased microbial activity in soils with a history of glyphosate applications, with additional glyphosate

applications, opposed to microbial activity in soils, with no prior glyphosate history. This was confirmed by

Dick and Quinn (1995), who compared a soil with no history of glyphosate applications, with a soil treated

with glyphosate, the three previous years. The latter contained approximately a 10 times higher microbial

9

population, even though it had a lower diversity of colonies. In contrast, Hart and Brookes (1996), observed

no long term differences in microbial biomass and activity in a soil, with 19 years of annual glyphosate

applications prior to the experiment. Mekwatanakarn and Sivasithamparam (1987) found glyphosate to

reduce bacterial numbers, at the glyphosate rate recommended for field use. This is supported by Kremer et

al. (2005), which observed a decrease in bacterial growth in root exudates from glyphosate-treated soybean

plants. However, Stratton and Stewart (1992) did not find any negative effects on numbers of bacteria, fungi

and actinomycetes, when applied at recommended rates.

Production of glyphosate resistant (GR) crops

To overcome the herbicidal effects of glyphosate on plants, different mechanisms for introduction of

glyphosate resistance (GR) has been explored; overexpression of EPSPS, detoxification of glyphosate and the

introduction of an insensitive form of EPSPS (Dill, 2005).

So far, overexpression of EPSPS has not generated any commercially available GR plants (Dill, 2005), but Shah

et al. (1986) succeeded in producing a Petunia plant, expressing higher levels of EPSPS, compared to normal,

which were able to tolerate increased levels of glyphosate. However, the growth rate was impaired and the

modified plant was not competitive compared to those, exhibiting the wild type.

Detoxification of glyphosate can be achieved by insertion of genes, which degrades glyphosate (Dill, 2005).

In combination with glyphosate insensitive EPSPS, the insertion of GOX (glyphosate oxidoreducatase) into

rape seed, has produced a commercial GR line. The detoxification of glyphosate itself, cannot provide

adequate resistance (Dill, 2005).

10

The method used to create the majority of GR crops on the market, is the insertion of a gene coding for a

naturally GR EPSPS (Dill, 2005). There have been several attempts to produce such, and some are summarized

in box below.

Agrobacterium sp. strain CP4 is naturally resistant towards glyphosate, and the majority of all commercially

GR crops, are produced from the insertion of EPSPS derived from Agrobacterium sp. strain CP4 (Barry et al.,

1997; Padgette et al., 1995a). EPSPS from Agrobacterium sp. strain CP4 was found from a screen of

microorganisms, and it was observed that the enzyme had favorable kinetics with respect to glyphosate and

PEP, indicating that EPSPS binds well to PEP (Padgette et al., 1995b). The reason for the insensitivity of CP4

EPSPS to glyphosate, is attributed to a single substitution from glycine to alanine in position 100 (Funke et

al., 2006). The methyl group of alanine interferes with one of the oxygen atoms in the phosphonate group of

glyphosate, which will result in a conformation change of glyphosate (Eschenburg et al., 2002; Funke et al.,

2006). Only the extended form of glyphosate has an inhibitory effect on EPSPS (Funke et al., 2006; Park et

al., 2004; Schönbrunn et al., 2001).

Sost and Amrhein (1990) found that a Gly96 to Ala substitution in the EPSPS from Klebsiella pneumonia,

reduced its affinity towards glyphosate. Later Eschenburg et al. (2002) revealed that it is the interference

between glyphosate and the additional methyl group of Ala96, protruding into the active site of the

enzyme, which confers the resistance, as glyphosate only interacts efficiently with EPSPS in its extended

conformation (Funke et al., 2006). As PEP is a smaller molecule than glyphosate, it can still fit in the

narrowed binding site. However, its translational freedom to S3P and the residues in the binding site

becomes limited, and therefore the affinity towards PEP also decreases (Eschenburg et al., 2002). The

affinity towards S3P remains unaltered (Eschenburg et al., 2002). In addition, one single site mutations

have conferred resistance towards glyphosate in Salmonella enterica Typhimurium. As in Klebsiella

pneumonia, it is one amino acid substitution, which is responsible for the resistance, although it is a

change from Pro to Ser at position 101, instead (Stalker et al., 1985). Pro101 is not located close to the

active site, but the alteration to Ser will change the orientation of Gly96 and Thr97 in the active site,

narrowing the binding site for glyphosate (Healy-Fried et al., 2007). Since PEP and glyphosate share the

same binding site, it is difficult to obtain an enzyme, which will bind PEP and exclude glyphosate too such

an extent that it will result in commercial levels of glyphosate resistance in normal developing plants.

According to Dill (2005) none of the singlesite mutations renders the enzyme with adequate kinetic

properties, even though they are advantageous. However, more favorable kinetic characteristics, were

observed for multisite mutations. One multisite mutation is used for the production of the commercially

available GR maize line (Lebrun et al., 2003), known as GA21 (Sidhu et al., 2000). The maize EPSPS is a

transgene, with the substitution of threonine with isoleucine at position 102 and proline with serine at

position 106 (Lebrun et al., 2003). Funke et al. (2009) studied the kinetic and structural effects of this

transgene EPSPS, using E. coli as a model. The two mutations induced the conformational changes

needed, to impair efficient glyphosate binding. As with the single site mutation P101S, the orientation of

Gly96 changes, interfering with binding of glyphosate. Ile97 (Thr97 before) will point away from the

binding site, facilitating the effective binding of PEP (Funke et al., 2009). The mutated EPSPS from E. coli

showed high resistance towards glyphosate while still maintaining high affinity for both PEP and S3P

(Funke et al., 2009), which is the reason for its commercial use.

11

The success of glyphosate

As mentioned above, glyphosate is a broad-spectrum systemic herbicide (Franz et al., 1997). It is used post-

harvest to target annual and perennial weeds in subsequent crops, but can also be used pre-harvest, as a

desiccating agent. Here, it acts to secure a uniform ripening of the crop, due to uneven soil conditions and

differences in microclimate (Miljøstyrelsen, 2014). According to Knowledge Center for Agriculture (SEGES)

and Institute for Agroecology, Aarhus University, the amount of glyphosate, used as a pre-harvest agent, has

increased since 2002, but to an extent which is not exactly known (Miljøstyrelsen, 2014). However, Djursing

(2013) estimates that about 10 % of the Danish areal with cereals are treated with glyphosate before harvest,

while it is 25 % of the areal with rapeseed. When used as a pre-harvest agent, glyphosate cannot be used

later than 10 days before harvest.

In addition to the use of glyphosate, in the traditional manner, introduction of glyphosate resistant (GR) crops

have changed the market considerably. One of the main reasons why adoption of GR crops have been faster,

than any other technology seen in agriculture (Sankula, 2006), is attributed to the substitution of several

different herbicides with glyphosate. Planting GR crops allow glyphosate to be spread during most of the

growing season, instead of limiting the application to pre-plant – and post-harvest periods. This makes it

possible to target weeds, as soon as they emerge from the soil and allows several applications throughout

the growing season (Benbrook, 2012; Waltz, 2010). In addition, less or no tillage is needed (Gianessi, 2005),

and combined, these factors create greater flexibility in timing of applications, improves soil

structure/moisture, reduces the risk of runoff and generally makes the control of a broad range of weeds,

including both annual grasses, broadleaves and perennials, more effective (Gianessi, 2005). Moreover,

adopting a less – or none tillage procedure is a less expensive alternative for weed control in comparison

with conventional tillage (e.g. reduces fuel and labor costs) (Sankula, 2006).

Even though growing GR crops have some advantages, compared to growing conventional crops, the strong

reliance on glyphosate have increased the resistant weed population (Benbrook, 2012). Until now,

glyphosate resistance have been reported in 32 weed species throughout the world, all emerging from 1996

and onwards, where GR crops were introduced (Heap, 2015). Unfortunately, this has led to a further increase

in the use of glyphosate worldwide, both in the context of more frequent spreading, but also with higher

doses, which further accelerates the emergence of resistant weeds, creating a vicious circle (Benbrook, 2012;

Binimelis et al., 2009).

Table 1 shows the consumption of glyphosate in the US, from 1987 and onwards. Since 2001, glyphosate has

been the most used active ingredient in agriculture (Grube et al., 2011). This is attributable to both the

invention and use of GR crops, but also to the increasing incidences of resistant weeds, and a steady decline

in cost of glyphosate, since the patent expired in 2000 (Duke and Powles, 2008).

12

Table 1. The consumption of glyphosate in the US, measured by million pounds of active ingredient, from 1987 to 2007. Modified

after Grube et al. (2011) and Kiely et al. (2004) (estimates).

The relationship between the use of glyphosate and GR crops becomes apparent, when looking at Figure 7,

which deals with one of the most widespread GR crop in the world, soybeans. Only few percentages of all

soybeans planted in 1996, were glyphosate resistant. The amount increased to almost 70% in 2001 and has

remained stable since 2007, representing approximately 93 % of all soybeans planted. The numbers

presented for the U.S. in Fejl! Henvisningskilde ikke fundet., corresponds to GM soybeans, and not directly

to GR. However, according to several references, including Duke and Powles (2009), this is practically the

same. As also presented in Fejl! Henvisningskilde ikke fundet., it is not only in the U.S., GR soybeans play a

crucial role. In Argentina, the first GM crop to be adopted was GR soybeans. The event took place in 1996

and since then, the area planted with GR soybeans has expanded dramatically - the increase has even

exceeded the increase observed in the U.S. Now, almost all the soybeans grown in Argentina are GR

soybeans. The same trend applies for GR soybeans in Brazil, as they occupied 92,4 % of the total soybean

area in 2013 (ISAAA, 2014). At the same time, these three countries are the leading producers of soybeans

in the world, as almost 80 % of the world’s production originates from here (FAO, 2015).

Figure 7. Adoption rate of GM soybeans in the US (according to Duke and Powles (2009), this corresponds to GR soybeans)

and adoption rate of GR soybeans in Argentina. Data from Duke and Powles (2009); USDA (2014) and ArgenBio (2015).

The EU has strict GM regulations (Davison, 2010) and only one GM maize line (MON810) and one GM potato

line (Amflora) are approved for commercial cultivation (Commission, 2011). Therefore, the use of glyphosate

Year Glyphosate (million pounds of a. i.)

1987 -8

1997 34-38

1999 67-73

2001 85-90

2003 128-133

2005 155-160

2007 180-185

13

in Denmark cannot be ascribed to the cultivation of GR crops, but agriculture is still the main responsible for

the consumption. Glyphosate is mainly used for weed killing, but a substantial amount is also used pre-

harvest, as a desiccating agent (Djursing, 2013). According to Figure 8, there has been a steady increase in

the sale of glyphosate in Denmark since 1993, with few exceptions, e.g. 2009. According to Miljøstyrelsen

(2013), the enhanced regulation of other herbicides has led to this relative abrupt increase in the sale of

glyphosate. In 2012, glyphosate constituted 23% of the total amount of active ingredients sold for agricultural

production, only surpassed by prosulfocarb with 34% (Miljøstyrelsen, 2013).

Figure 8. The amount of glyphosate, as an active ingredient, sold from 1991 to 2012. Modified after Middeldatabasen/SEGES (2015);

Miljøstyrelsen (2008, 2013)

Even though no GM crops are produced in Denmark, the adoption of, especially, GR soybeans in the rest of

the world, is of importance for Danish agriculture. Since 2000, the Danish soy import has exceeded 1,400,000

tons each year, with an import of 1,560,000 tons in 2014. The vast majority originates from countries, using

GR soybeans (Statistikbanken, 2015), which, together with glyphosate as a desiccating agent, leaves the

discussion of glyphosate residues in feed important in Danish agriculture.

Possible pesticide residues in crops treated with glyphosate

When applying glyphosate to crops, there is a risk that residues can end up in the feed. A pesticide residue is

defined as the combination of a pesticide and its metabolites, degradates and other transformation products

(FAO, 2009). To secure the lowest possible consumer and animal exposure to pesticides, Maximum Residue

Levels (MRL) are established. MRL is defined as the upper legal limits of a pesticide, allowed in food or feed,

expressed as mg/kg (EFSA, 2015b). MRL are based on toxicology data from animals, and toxicity on

microorganisms has not been included. MRL for glyphosate, determined by the EU and FAO/WHO, appears

from Table 2.

14

Table 2. MRL levels of glyphosate in different cereals and oilseeds (Codex Alimentarius, 2012; Commission, 2015)

As seen in Table 2Fejl! Henvisningskilde ikke fundet., the allowed MRLs differs between the different

feedstuffs and in addition the allowed limits differs between EU and FAO/WHO. Generally, EU are more

cautious in their determination than FAO/WHO.

Several investigations have sought to examine, whether MRL are met in different feedstuffs. Unpublished

data from Germany found glyphosate levels of 0.4 – 0.9 mg/kg in poultry and cattle feed samples (Shehata

et al., 2014), which is well below the MRL (Table 2). Arregui et al. (2004) detected AMPA residues in both

leaves and grains from GR soybeans and reported glyphosate residues ranging from 1,9 to 4,4 mg/kg in stems

and leaves, and from 0,1 to 1,8 mg/kg in grains, all below the MRL. The highest concentrations were obtained

after several glyphosate treatments, during the growing season, but a relationship between application rate

and residue level could not be established (Arregui et al., 2004). Bøhn et al. (2014) compared 31 soybean

batches, almost equally distributed between GR soybeans (10), conventional soybeans (10) and organic

soybeans (11), with respect to glyphosate and AMPA content. Bøhn et al. (2014) were able to show that only

GR soybeans contained residue levels of AMPA and glyphosate, with a mean value of 3.3 and 5.7 mg/kg,

respectively.

In a Danish survey from 2009, 20 batches of imported soy products were analyzed for glyphosate content.

16 were manufactured from GM soy, and all shipments originated from South America, with different export

countries involved (Plantedirektoratet, 2010). The batches were distributed between soy hulls, soybeans and

soybean meal, and glyphosate residues were only detected in GM - soy hull batches (4). In three of the four

batches, the residue levels exceeded the MRL of 20 mg/kg for soybeans (24,7 – 26,7 mg/kg) (Commission,

2015). However, MRL is set for soybeans and not for the processed products (Commission, 2015) and it

cannot directly be transferred to apply for soy-hulls (Plantedirektoratet, 2010).

Spraying pre-harvest can also influence the residues levels of glyphosate, found in seeds, after harvesting. As

application has to take place when the crop is mature, almost no translocation will occur in the plant.

Therefore residues, if any, will be present on the surface of the plant (Cessna et al., 1994). Miljøstyrelsen

(2014) have conducted a survey, investigating different feed samples where glyphosate has been used pre-

harvest. The results are presented in Table 3 and even though glyphosate was found in a large percentage of

the samples, none of these exceeded the MRL. However there is a tendency that pre-harvest treatment of,

especially barley, has increased during the last years, with the maximum residue level found to be 13 mg/kg.

MRL of glyphosate (mg/kg) (EU) MRL of glyphosate (mg/kg) (FAO/WHO)

Barley 20 30

Oat 20 30

Rye 10 30

Wheat 10 30

Maize/corn 1 5

Rapeseeds/canola seeds 10 30

Soybeans 20 20

15

Table 3. Overview of glyphosate residues in random samples from Danish produced barley and wheat (spelt and triticale included)

for animal feed. The quantification limit was set to be approx. 0,1 mg/kg. The survey is conducted by the Danish Veterinary and

Food Administration, Modified after Miljøstyrelsen (2014).

Summarizing the abovementioned results, glyphosate residues can be present in feed, regardless of whether

glyphosate is used in GR crops or as a desiccation agent in non-GR crops, even though the levels differs

between different investigations.

Toxicity of glyphosate The absence of the shikimate pathway in mammals, might explain the relatively low toxicology of glyphosate

towards mammals (Kier, 2015; Smith and Oehme, 1992; Williams et al., 2012; Williams et al., 2000). However,

glyphosate might target other pathways and toxicity of POEA has also gained more interest.

In a review by Samsel and Seneff (2013) it is stated that glyphosate has the ability to suppress cytochrome

P450 enzymes. It is a large group of enzymes playing crucial roles in primary and secondary metabolism

(Nelson et al., 1996). Lamb et al. (1998) discovered that glyphosate can inhibit plant cytochrome P450

(CYP71B1) and several studies, focusing on human cell lines, found glyphosate, and especially glyphosate

formulations, to be the disruptor of Aromatase cytochrome P450 activity (Gasnier et al., 2009; Richard et al.,

2005; Simpson et al., 2002; Simpson et al., 1994). Glyphosate decreased the hepatic levels of cytochrome

P450 in a study where rats were treated with glyphosate for two weeks (Hietanen et al., 1983) and Paganelli

et al. (2010) hypothesized that morphological changes in frog embryos after glyphosate addition, were

associated with inhibition of CYP26 enzymes (belongs to P450 superfamily).

Glyphosate is also patented as a broad-spectrum chelator, complexing divalent cationic nutrients (Fon and

Uhing, 1964), enabling it to bind a wide range of minerals. The very low levels of Mn and Co, found in the

urine samples from dairy cows, containing varying amounts of glyphosate, could possibly be ascribed to this

chelating effect. However, Cu and Se were within the reference level, not influenced by the presence of

glyphosate (Krüger et al., 2013a).

Glyphosate as a carcinogen?

In addition to the questioning about which components lead to the toxicity of glyphosate formulations, if

toxicity is observed at all, there has been some controversy about the carcinogenic potential of glyphosate.

In 1985, The United States Environmental Protection Agency (U.S. EPA) classified glyphosate as possibly

carcinogenic to humans (Group C), based on tumor studies in mice (EPA, 1985). However, when the study

was reevaluated by US EPA in 1991, the classification of glyphosate was changed to evidence of non-

carcinogenity in humans, instead (Group E) (IARC, 2015). In 2015, the International Agency for Research

Against Cancer (IARC), reclassified glyphosate as probably carcinogenic to humans (Group 2A) (Guyton et al.,

Crops Determination of glyphosate 2008 2009 2010 2011 2012 2013

Barley Number of samples 14 20 22 21 33 23

Samples with glyphosate present (%) 7 15 45 48 30 77

Highest level measured (mg/kg) 0.4 1.6 7.1 4.6 8.1 13

Average level (mg/kg) 0.03 0.17 0.79 0.5 0.85 1.86

Wheat, triticale, spelt Number of samples 13 15 22 12 22 15

Samples with glyphosate present (%) 23 27 36 58 32 47

Highest level measured (mg/kg) 0.9 0,8 3,6 1.2 4.1 2

Average level (mg/kg) 0.15 0.1 0.28 0.3 0.51 0.26

16

2015). This category is used for pesticides showing limited evidence of carcinogenity in humans (non-Hodgkin

lymphoma) but sufficient evidence in animals (Guyton et al., 2015; IARC, 2015). After a peer-review of the

carcinogenic potential of glyphosate, the European Food Safety Authority (EFSA) concluded that glyphosate

is unlikely to pose a carcinogenic threat to humans, in contrast to the evaluation by IARC (EFSA, 2015a).

However, IARC evaluated both glyphosate and glyphosate-formulations, whereas EFSA only included studies,

concerning the active substance, which might influence the outcome of the reviews (EFSA, 2015a).

Toxicity of POEA

The information regarding content of surfactants in Roundup, are usually kept confidential by the

manufacturer. This makes it difficult to determine exactly what is used in Roundup – and thereby to create

sufficient risk assessments. Mesnage et al. (2013) concluded that it was the ethoxylated surfactants of

glyphosate-based formulations that were the active principles of human cell (embryonic, placental and

hepatic) toxicity, with POE-15 displaying the greatest effect. In addition to this finding, all formulations were

more toxic than glyphosate itself. Results from Mesnage et al. (2014) supported these findings, as they found

Roundup to be 125 times more toxic to human cells (embryonic, placental and hepatic), than glyphosate was.

Benachour and Seralini (2009) concluded that surfactants, like POEA, changed the human cell permeability

of three different cell types (umbilical, embryonic and placental) and amplified the toxicity, already induced

by glyphosate, through apoptosis and necrosis. Other experiments sought to test the toxicities on aquatic

organisms. In a study by Folmar et al. (1979), POE-15 had approximately the same acute toxicity towards fish

and aquatic invertebrates, as Roundup had. However, the contribution of glyphosate to the toxicity of

Roundup, only ranged from 29% to 33% (Folmar et al., 1979). Other studies focusing on the aquatic habitat

(different fish species) also concluded that it were the surfactants, which where main responsible for the

toxicity observed (Mitchell et al., 1987; Servizi et al., 1987).

Toxicity of glyphosate towards microorganisms

Like plant cells, almost all prokaryotic cells, are surrounded by a cell wall, and most bacteria are divided based

on a gram – reaction. Gram-positive bacteria are covered by a thick layer of peptidoglycan, which constitutes

up to 90 % of the cell wall. In gram-negative bacteria, only approximately 10 % of the cell wall consists of

peptidoglycan, with the rest being composed of an outer membrane. For all bacteria, a plasma membrane is

located beneath the cell wall (Madigan et al., 2009). As well as for plants, glyphosate has to enter the cells to

exert its effects. To my knowledge, details about transport of glyphosate into bacterial cells are unclear, even

though some of the entrance mechanisms exerted in plants, might be evident. No matter of entrance

method, some studies have found inhibiting effect of glyphosate, towards different kinds of microorganisms.

Three food microorganisms, a fungus (Geotrichum candidum) and two bacteria (Lactococcus lactis subsp.

cremoris and Lactobacillus delbrueckii subsp. bulgaris) are used as starters in the dairy technology. Clair et

al. (2012) found all three microorganisms to be more sensitive to Roundup than to glyphosate alone. In an in

vitro study, the nitrogen fixating bacteria, Azotobacter vinelandii, showed a higher degree of sensitivity

towards glyphosate than Azotobacter chroococcum did (Santos and Flores, 1995). In soil samples, Busse et

al. (2000) observed an increase in total and viable bacteria after glyphosate addition, and especially

Arthrobacter, Pseudomonas, Xanthomonas and Bacillus spp. increased in population. Difference in

glyphosate tolerance between bacteria cultured in soil and in soil-free media might be explained by the

chemical properties of glyphosate. Glyphosate binds easily to soil particles, which leaves the compound

unable to exert its herbicidal effects (Borggaard and Gimsing, 2008; Duke et al., 2012).

17

Tsui and Chu (2003) investigated whether POEA also accounted for the toxicity of Roundup to

microorganisms. Bacteria (Vibrio fischeri) and protozoa (Tetrahymena pyriformis and Euplotes vannus) had

more or less similar sensitivities towards Roundup. For the same species, POEA showed considerably lower

toxicity levels, and generally, glyphosate IPA salt was least toxic.

Based on these findings it become obvious, how important it is to investigate whole Roundup formulations.

In formulations, it is always important to know the individual toxicities of the different compounds – but also

their combined effects, as they might influence each other.

Changes in gut microbiota with respect to glyphosate

Potential levels of glyphosate in gut

As the overall objectives of this study are to investigating possible effects of glyphosate on microorganisms

in livestock, it is useful to calculate, which levels it is likely to obtain in the gut. Two scenarios are presented

below, one for pigs and one for cows.

Numbers presented in Table 4Fejl! Henvisningskilde ikke fundet., are calculated based on feeding pigs a diet

containing only barley or soybean meal. If the soybean meal and barley contain MRL of glyphosate allowed

in the EU (20 mg/kg), the residues in the gut would be between 0.005 and 0.019 mg/ml, based on the

assumptions made.

Table 4. Estimated glyphosate concentrations in the gut of pigs, fed a diet containing the MRL of glyphosate for barley and soybean

meal (20 mg/kg). Calculated by Martin Tang Sørensen.

In addition to Table 4, Table 5 shows which glyphosate concentrations it might be able to obtain in the gut of

cows, assumed that they are fed a diet, where 50 % is composed of soybean meal and barley, and MRL of

glyphosate are present in these feedstuffs. Compared to pigs, lower levels of glyphosate (from 0.0022-0.003

mg/ml) can be present in the different segments in cows, mainly because of the lower percentage of

concentrate in the feed ration.

Table 5. Estimated glyphosate concentrations in the gut of cows. Only half the diet originates from barley and soybean meal,

assumed to contain 20 mg glyphosate/kg. This means that the feed in average will contain 10 mg/kg when ingested. Calculated by

Martin Tang Sørensen

Feed DM1 Glyphosate content3

(pr. kg ingested feed) (pr. kg ingested feed)

Stomach 1.0 kg 4 liter (~25 % DM) 20 mg ~ 0.005 mg/ml

Small intestine (distal part) 0.3 kg 3 liter (~10 % DM) 14 mg ~ 0.005 mg/ml

Colon (feces) 0.15 kg 0.75 liter (~20 % DM) 14 mg ~ 0.019 mg/ml

Gut segmentGut content volume2

(pr. kg ingested feed)

Concentration of glyphosate

acid equivalents (mg/ml)

1Feed DM disappears through the GIT because of degradation and absorption (Anguita et al., 2006; Canibe and Knudsen, 2002)2The DM content in the gut segments is calculated based on results found in Canibe et al. (2005); Canibe and Jensen (2003)3In general it is assumed that glyphosate is not degraded in the GIT, but 30% is absorbed to the blood from the small intestine (Commission,

2002)

Feed DM1 Glyphosate content3

(pr. kg ingested feed) (pr. kg ingested feed)

Forestomach (at the end of the omasum) 0,65 kg 3.4 liter (~19.4 % DM) 10 mg ~ 0.003 mg/ml

Small intestine (distal part) 0.35 kg 3.2 liter (~10.8 % DM) 7 mg ~ 0.002 mg/ml

Colon (feces) 0.30 kg 2.3 liter (~13.3 % DM) 7 mg ~ 0.003 mg/ml

Gut segmentGut content volume2

(pr. kg ingested feed)

Concentration of glyphosate

acid equivalents (mg/ml)

1Feed DM disappears through the GIT because of degradation and absorption (Storm and Kristensen, 2010) 2The DM content in the gut segments is calculated based on results for cows in early lactation (63 days postpartum) found in Andrew et al. (1995)3In general it is assumed that glyphosate is not degraded in the GIT, but 30% is absorbed to the blood from the small intestine (Commission, 2002)

18

Overall, the assumptions made for the scenarios presented in Table 4 and Table 5, are based on feed rations

containing MRL of glyphosate. According to the findings of glyphosate residues in crops, presented earlier,

such levels are uncommon. However, some exceedances are observed (Plantedirektoratet, 2010), but the

risk that all feed ingredients in the ration has the same high level, might be (even) less plausible. In addition,

toxicity margins are included in MRL, meaning that it is not toxic in itself. Another important assumption used

for the calculation, which might be questioned, is that degradation of glyphosate does not take place and

that only 30 % is absorbed (Commission, 2002). Glyphosate might be degraded to AMPA in animals, as

observed in plants (Arregui et al., 2004; Bøhn et al., 2014). If that is the case, the residue levels will change.

So far, the toxicity of AMPA it is not known and therefore this also has to be taken into consideration, when

evaluating the toxic effects of glyphosate residues in livestock.

The effect of glyphosate on poultry microbiota

Shehata et al. (2013b) investigated the impact of glyphosate on pathogenic and commensal microorganisms,

in the gut of poultry, representative of a monogastric animal. The results are presented as the Minimum

Inhibitory Concentration (MIC), which is the lowest concentration of an inhibitory compound needed, to

completely block the growth of a microorganism. As seen in Table 6, most of the pathogenic bacteria were

highly tolerant to glyphosate (Roundup), with MIC values at 5.000 mg/ml (Clostridium perfringens,

Salmonella enterica Enteritidis, Salmonella enterica Gallinarium and Salmonella enterica Typhimurium),

while most of the commensal bacteria were moderate to highly sensitive (Bacillus badius, Bacillus cereus,

Bifidobacterium adolescentis, Enterococcus faecalis and Enterococcus faecium), with MIC values ranging from

0.075 to 0.300 mg/ml. Only few bacteria did not fit this characterization, as Campylobacter coli and

Campylobacter jejuni were highly sensitive (MIC = 0.150 mg/ml), while three Lactobacillus strains were more

resistant than the other beneficials (MIC = 0.600 mg/ml). In addition, Shehata et al. (2014) found MIC values

of glyphosate for B. adolescentis, B. badius and E. faecilis on 0.150, 0.300, and 0.300 mg/ml, respectively,

which all are slightly higher than the numbers presented in Table 6. Based on the two studies, referred to

above, glyphosate has the capability to suppress growth of Bifidobacteria substantially. Bifidobacteria can

create unfavorable conditions for pathogens, as Salmonella in the gut (Bielecka et al., 1998), and, therefore,

a decrease in the population of Bifidobacteria can indirectly increase growth of Salmonella and other

pathogens. The result is a disturbance in the gut microbiota, which possibly can lead to health issues.

19

Table 6. The MIC value of glyphosate on different pathogenic and beneficial bacteria. Based on Shehata et al. (2013b)

Minimum Effect Concentration (MEC) is the lowest concentration of an inhibitor needed to initiate the

inhibition of a microorganism (Arikan et al., 2001) and from data presented by Fredborg et al. (2013) it is

suggested that MEC can be calculated as 1/10 of the MIC value. This means, that the commensal bacteria,

found to be sensitive by Shehata et al. (2013b), already could be negatively affected by concentrations of

glyphosate, ranging from 0.0075 to 0.030 mg/ml, corresponding to a tenth of the MIC values.

The effect of glyphosate on dairy cow microbiota

To investigate the effect of glyphosate on the microbiota in dairy cattle, Ackermann et al. (2015) compared

two diets, one rich in crude fibers and one low in crude-fibers. Glyphosate, at very low concentrations (0.001

mg/ml), decreased the growth of Ruminococcus albus and Ruminococcus flavefaciens substantially (Leschine,

1995). In addition, cell counts of Euryarchaeota were significantly decreased at glyphosate concentrations of

0.01 and 0.1 mg/ml and for Streptococcus spp., a reduction was observed at 0.1 mg/ml. In both diets, cell

counts of the Clostridium histolyticum group, consisting of many species with pathogenic potential, increased

significantly at 0.1 mg/ml. The same effect was observed for Lactobacilli and Enterococci (Ackermann et al.,

2015). These results show that glyphosate causes a change in the microbiota, with a pronounced effect on

fiber degrading species. Fermentation of fibers enable other microbial species to exist and perform their role,

by providing important nutrients as VFA’s, lactate, gasses e.g. (Cunningham and Klein, 2007). Therefore, the

whole balance can change, even though not all species are affected directly. The authors suggest that the

significant reduction of Streptococcus spp. could be due to the inhibition of the cellulolytic species, and not

necessarily to glyphosate itself. It could also be the case for the reduction of Euryarchaeota, as especially

methanogens are associated with the abundance of some protozoal species (Lange et al., 2005), which also

were negatively affected by glyphosate (Ackermann et al., 2015).

Genus/species MIC value of glyphosate (mg/ml)

Pathogens Campylobacter coli 0.15

Campylobacter jejuni 0.15

Clostridium perfringens 5

Clostridium botulinum (type A and B) 1.2

Eschericia coli 1.2

Eschericia coli 1917 strain Nissle 1.2

Salmonella Enteritidis 5

Salmonella Gallinarium 5

Salmonella Typhimurium 5

Commensals Bacillus badius 0.15

Bacillus cereus 0.3

Bifidobacterium adolescentis 0.075

Enterococcus faecalis 0.15

Enterococcus faecium 0.15

Lactobacillus buchneri 0.6

Lactobacillus casei 0.6

Lactobacillus harbinensis 0.6

20

Compared to Table 5, most of the microorganisms described above, will not be negatively affected by the

glyphosate levels, expected in the gut of dairy cows. However, the growth of R. albus and R. flavefaciens

(found in rumen) decreased at 0.001 mg/ml. This does not directly refer to the MIC value, as few cells still

were able to grow, but if we assume it does, an inhibition would start at 0.0001 mg/ml, representing the MEC

value (Fredborg et al., 2013). This is lower than the concentration, which can be found in the rumen (0.003

mg/ml), and a possible inhibition of the two Ruminococcus strains could occur.

The effect of glyphosate on increased incidences of botulism in dairy cows

During the last years, there has been an increase in the incidences of C. botulinum associated diseases in

cattle. Strains of C. botulinum (spore forming, obligate anaerobic bacteria) generates neurotoxins (BoNT, A-

G) that blocks the release of acetylcholine in the neuromuscular junction, leading to botulism in cattle (Böhnel

et al., 2001). Lactic acid producing bacteria (LAB), as Lactobacilli, Lactococci and Enterococci are able to

produce bacteriocines, which are effective against Clostridium spp. and both E. faecalis and E. faecium

inhibited BoNT production by all C. botulinum strains, in a study by Krüger et al. (2013b). Also Shehata et al.

(2013a) discovered that E. faecalis, E. faecium and B. badius inhibited BoNT production while also reducing

the growth of all C. botulinum types tested (A, B, D and E).

Even though the reason for the increased incidences of botulism is unknown, a possible explanation is

proposed by Krüger et al. (2013b). They found that the inhibitory concentrations of glyphosate to E. faecalis

was 10-100 times lower than those, inhibiting the growth of C. Botulimum type B. The possible explanation

for the outbreaks of botulism might therefore refer to the loss of the antagonistic potential of Enterococci

towards C. botulinum, when glyphosate reaches the GIT of the cows. Unpublished data confirmed this

hypothesis; faeces samples from cows with C. botulinum associated diseases, were poorly colonized by

Enterococci, while the opposite was the case for cows, rarely showing any symptoms (of C. botulinum

associated diseases) (Krüger et al., 2013b).

The effect of glyphosate on fungi in dairy cows

Schrödl et al. (2014) studied the relationship between glyphosate excretion in dairy cows and ruminal fungi

of the order Mucorales (Lichtheimia corymbifera, Lichtheimia ramosa, Rhizopus and Mucor – all belonging to

the family Mucoraceae), as fungi are important members of the ruminal microbial community in cattle ().

Glyphosate concentrations in the urine ranged between 0.0 ng/ml and 164 ng/ml, and cows with the highest

glyphosate excretion (˃40 ng/ml) had significantly lower levels of total Mucorales, L. corymbifera and L.

ramosa, compared to cows with a lower concentration. In addition, unpublished data revealed that

Lichtheimia spp, Mucor spp. and Rhicopus spp. are highly resistant to glyphosate in in vitro trials (Schrödl et

al., 2014). The reason why Lichtheimia spp. are depressed in this trial could therefore reflect other imbalances

in the gut, not taken into account here.

Microbial fermentation in the gut of pigs and dairy cows

Microbial fermentation of organic compounds, occurs in cecum and colon of all farm animals, whether in

ruminants, most of the fermentation, takes place in the rumen (Sjaastad et al., 2010). Compared to rumen,

cecum and colon; stomach, and the beginning of the small intestine, only contains low numbers of bacteria,

ranging from 103 to 104 cells/ml, which compete with the animal for the most easily degradable nutrients

(Cunningham and Klein, 2007). The relatively low number is due to a less favorable environment in these

21

compartments, comprising a low pH in the stomach (Hao and Lee, 2004). In comparison, bacterial numbers

in rumen, cecum and colon, rages between 1010 and 1011 cells/ml (Cunningham and Klein, 2007).

Fermentation leads to production of VFA’s, the primary ones being acetate, propionate and butyrate

(Cunningham and Klein, 2007; McDonald et al., 2011). Acetate, propionate and butyrate mainly originates

from fermentation of cellulose, hemicellulose and pectin (Bergman, 1990) but can also be produced by

fermentation of proteins (Nery et al., 2012). The relative proportions of acetate, propionate and butyrate,

can change a lot, ranging between 75:15:10 and 40:40:20, depending on diet (Bergman, 1990). In addition to

acetate, propionate and butyrate, branched VFA’s can only be formed by fermentation of branched amino

acids; isobutyrate and isovalerate, are produced from fermentation of valine and leucine, respectively

(Blachier et al., 2007; McDonald et al., 2011). Valerate can be formed by fermentation of proline (McDonald

et al., 2011; Rasmussen et al., 1988) and by the condensation of acetate and propionate (Bergman, 1990).

Experimental setup

The experimental part of the thesis, comprises three different parts. First, the effect of glyphosate acid,

glyphosate IPA salt, Roundup and POEA on selected bacterial growth are investigated, and whether possible

effects differs between commensal - and potential pathogenic bacteria. In the second part, we investigate if

glyphosate IPA salt, Roundup and POEA show any effects on the composition and activity of gut microbiota,

when added to stomach, cecum and colon content from slaughter pigs and to rumen content from dairy

cows. In the last part, it is investigated, if growth of a strain of Lactobacillus sobrius in stomach content is

affected by glyphosate IPA salt, when pH is held constant (pH=5).

22

Materials and Methods

Chemicals

Jablo Glyfosat (glyphosate acid concentration 360 g/l, corresponding to a glyphosate IPA salt concentration

of 480 g/l), equivalent to Roundup, manufactured by Monsanto Crop Sciences, Denmark. Jablo is referred to

as Roundup, throughout the rest of the thesis. Glyphosate, both formulated as the acid, N-

(phosphonomethyl)glycine (CAS number 1071-83-6) and as the salt, N-(Phosphonomethyl)glycine,

monoisopropylamine salt solution (CAS number 38641-94-0) were purchased from SIGMA-ALDRICH® and

POEA (Tallowamine polyethoxylated, E17136000) from Dr. Ehrenstorfer GmbH.

Bacteria used in the experiment

Table 7 show a list of bacteria, used in these experiments. As the objective was to investigate the effect on

microbes, normally present in the GIT of pigs and cows, different culturable bacteria from the major genera

were chosen. Both the effects on selected commensals and potential pathogens were investigated.

Table 7. Information about bacterial species used in the experiment.

Media Table 7 lists the media used for preparation of overnight cultures of different bacteria. For detailed

information about these, and other media, used during the experiments, see Appendix 2.

Techniques used

Determination of viable counts by use of Drop Plate Procedure

In 96-well microtiterplates, 250 µl of sample (ex. glyphosate IPA salt, 10 mg/ml, T0) was loaded into the first

well in the first column (i), see Figure 9. 25 µl from column i was transferred to 225 µl medium (0.1 M sodium

phosphate buffer, pH 6.5) in column (i+1), mixed 10 times and the process repeated, until the wanted

dilutions were obtained. For each sample, 10 µl, from each of the dilutions of interest, were plated on MRS

agar and incubated anaerobically overnight, at 37°C. The plates were counted the following day and the

colony forming units (CFU) calculated.

Genus/Species Catalog number Origin Culture media Incubation conditions GramCommensals Bifidobacterium adolescentis DSM 20083 Adult intestine Anaerobic BHI 37°C/anaerobic +

Bifidobacterium longum DSM 20219 Adult intestine BHI 37°C/anaerobic +

Bifidobacterium longum DSM 20211 Pig faeces BHI 37°C/anaerobic +

Enterococcus faecalis O3 MRS 37°C/anaerobic +

Enterococcus faecium O4 MRS 37°C/anaerobic +

Escherichia coli K12   BHI 37°C/anaerobic -

Lactobacillus reuteri DSM 20016 Adult intestine MRS 37°C/anaerobic +

Lactobacillus sobrius DSM 16698 Feces, piglet MRS 37°C/anaerobic +Lactobacillus salivarius DSM 20555 Saliva MRS 37°C/anaerobic +

Ruminococcus albus DSM 20455 Ruminococcus media 37°C/anaerobic +

Ruminococcus flavefaciens DSM 25089 Ruminococcus media 37°C/anaerobic +

Streptococcus alactolyticus O10 MRS/colon 37°C/anaerobic +

Streptococcus hyointestinalis O18 MRS/colon 37°C/anaerobic +

Campylobacter coli Microaerophilic BHI 41°C/microaerophilic -

Campylobacter jejuni Microaerophilic BHI 41°C/microaerophilic -

Clostridium perfringens Anaerobic basal broth 37°C/anaerobic +

Escherichia coli K88   DSM 498 BHI 37°C/anaerobic -Salmonella enterica Typhimurium Anaerobic BHI 37°C/anaerobic -Salmonella enterica Entritidis Anaerobic BHI 37°C/anaerobic -

Staphylococcus aureus Clinical isolate 102480 – from OUH Anaerobic BHI 37°C/anaerobic +

Potential

pathogens

23

Figure 9. Microtiterplate used for dilution in preparation of drop plating. Modified after (Chen et al., 2003).

Quantification of VFA analysis

To quantify volatile fatty acids (VFA), 1 ml samples were extracted. To each sample (+ 1 ml standard mix and

1 ml blind sample), 100 μl intern standard was added. The acids were extracted by addition of 0.5 ml HCl and

addition of ether separated the acids from other components, leaving them in the ether phase. After

centrifugation at 2000 g for 10 min (5°C), 50 μl of the supernatant was transferred to microvials, containing

10 μl MTBSTFA, capped and mixed. After heating at 80°C for 20 min, microvials were stored in room

temperature for 48 hours, to ensure complete derivatization. The samples were run on the Gas

Chromatograph (HP 6890 Series GC System) (Richardson et al., 1989).

Gas measurements

For gas measurements, samples were analyzed by Gas Chromatography, using a ML GC82-22 (Mikrolab,

Aarhus, Denmark) with helium as carrier gas. Data were analyzed using the appurtenant PEAK359 software.

N2 gas in the bottles were used as internal standard for calculation of H2, CO2 and CH4 production (Poulsen

et al., 2013).

For further details, see Appendix 1.

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial

cultures

To study the growth susceptibilities of selected gastrointestinal bacteria (Fejl! Henvisningskilde ikke fundet.)

to glyphosate acid, glyphosate IPA salt, Roundup and POEA, pure bacterial cultures were incubated in 96-

well mikrotiterplates, in the presence of different concentrations of the four treatments.

Samples from -80°C stocks of each bacterial culture were inoculated to broth and incubated overnight. The

overnight cultures were investigated in the microscope to check for purity, and diluted 100 times in broth, if

the growth was found sufficient (density of approx. 109 cells per ml). In a few cases, the overnight cultures

were less dense and only diluted 10 times. For preparation of the microtiterplates, stock solutions of

24

glyphosate acid, glyphosate IPA salt and Roundup were made. Each solution had a start concentration of 10

mg /ml glyphosate acid equivalents, independent of treatments, to ease comparisons (Appendix 2). For each

treatment, 325 µl of the solution was added to the first well in three columns, to make a replicate of three

(Figure 10). For POEA, another procedure was performed. 325 µl growth media was added to the first well in

three columns, as for the other treatments, but POEA was first added in the wells. Experimental

concentrations of POEA were calculated based on its expected concentrations in Roundup, compared to

glyphosate acid equivalents. It was expected that POEA constituted 15 % of Roundup (Giesy et al., 2000;

Sawada et al., 1988) leaving the ratio between glyphosate acid equivalents and POEA to be 2.4 to 1. This

corresponded to addition of 1,4 µl POEA in the first well, when the start concentration of glyphosate acid

equivalent was 10 mg/ml. However, due to a mistake, only 65 % of the concentrations, we expected to be

the pure product POEA, actually consisted of POEA. This left us with an expected proportion of POEA in

Roundup at 9.4 %, instead of 15%, thereby decreasing its proportions towards glyphosate acid equivalents.

In all other wells (row B-H), 225 µl pure media were added. To reach different concentrations of the

treatments, throughout the columns, serial 3.5 dilutions were made. The last well in all columns, served as

control. In each well, dilutions were followed by application of 25 µl diluted bacterial overnight culture,

ending with a total bacterial dilution of 1000 in the wells. After mixing bacteria and media, 50 µl sterilized

paraffin oil was added to each well, to prevent evaporation and limit exchange of oxygen.

Figure 10. Experimental setup of mikrotiterplates and the concentrations used, of the four different treatments

The microtiterplates were incubated in an ELISA spectrophotometer at 37°C and run on the ELISA

spectrophotometer until cultures reached stationary phase (18-24 h). Cell density (growth) was monitored

by measuring optical density at 650 nm (OD650). Bacterial growth curves were obtained by plotting OD650 data

against time. An output from the ELISA spectrophotometer is depicted on the left part of Figure 11. It shows

growth curves of a bacterial strain, in the presence of, glyphosate acid, glyphosate IPA salt, Roundup and

POEA. The concentrations of the individual treatments corresponds to the concentrations displayed in Figure

10. At the right part of Figure 11, growth curves, at different concentrations, are collected in one graph,

facilitating easier interpretation of the data.

25

To determine µmax (maximum growth rate), absorbance data were log-transformed, to investigate where

growth was logarithmic (Figure 12). For all bacteria, at each treatment, at each concentration, µmax was

calculated from the log-transformed data, and the values for the triplicates, averaged.

Figure 12. Draft of absorbance and log(absorbance) plotted against time.

For strict anaerobes, as R. albus and R. flavefaciens, mikrotiterplates were prepared in an anaerobic chamber.

For the potential pathogens, addition of bacteria to plates, were performed in a LAF bench.

For further details, see Appendix 2.

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows

To study the effect of glyphosate IPA salt, Roundup and POEA, on microbiota in gut of slaughter pigs and

dairy cows, a total of four pig - and three cow studies were made.

Slaughter pigs

For pigs, the effects of Roundup and POEA were investigated in stomach (n=1) and colon content (n=1). In

addition, the effect of glyphosate IPA salt, Roundup and POEA have been investigated in stomach (n=2),

cecum (n=2) and colon content (n=2).

GIT content from 60 kg slaughter pigs, fed finely ground feed, were taken at slaughter (Research Center

Foulum), placed on ice, and immediately taken to the laboratory. With a cut-off syringe, 10 ml content were

added to 125 ml bottles, sealed with butyl rubber stoppers, containing 35 ml 0.1 M sterile anaerobic sodium-

Figure 11. Output from ELISA spectroscopy – example of growth curves of a bacterial strain in the presence of POEA, glyphosate acid and Roundup. You can choose to get the output as absorbance or as log absorbance on the y-axis. The figure at right, is a draft from the bachelor thesis of Camilla Koed.

26

phosphate buffer (pH=6.5), to reach a 20 % wt/vol suspension. Colon content were weighed out (10 g in each

bottle), as it was too solid to be handled with a syringe. In each bottle, 5 ml of glyphosate acid/glyphosate

IPA salt/Roundup/POEA solutions were added with a syringe, according to the desired concentrations (Table

8, Table 9Fejl! Henvisningskilde ikke fundet.). Due to the mistake, explained above, POEA concentrations

were underestimated, leaving the ratio of glyphosate acid to POEA, higher than expected. Final POEA

concentrations ended at 2.4, 0.24, 0.024 and 0.00024 mg/ml. For all treatments, 0 mg/ml served as control.

Table 8. Overview of experimental setup for glyphosate IPA salt and Roundup

Table 9. Overview of experimental setup for POEA

The gas phase of each serum bottle was changed to CO2 by three successive cycles, where the bottles were

evacuated and refilled, using a manifold, fitted to a vacuum pump and a gas tank. The bottles were removed

from the manifold under a slight overpressure and the pressure adjusted to atmospheric, using a needle. The

bottles were placed in a 37°C shaking water bath for 24 hours and samples were taken at 0, 3/4 and 24 hours.

With a syringe, 1 ml was withdrawn from each serum bottle for measurements of VFA (stored at -18°C until

analysis), 1 ml for DNA analysis (snap frozen in liquid nitrogen and stored at -80°C), 1 ml for immediately pH

measurements and 0.5 ml for Drop Plating Procedure.

Dairy cows

For cows, the effects of glyphosate IPA salt, Roundup and POEA were investigated in ruminal content (n=2).

Ruminal content were taken from normally fed, fistulated dairy cows, at Research Center Foulum. The

content was sieved through a cheesecloth to remove big particles, placed on ice, and immediately taken to

the laboratory. With a syringe, 10 ml content were added to each bottle, containing 35 ml 0.1 M sterile

anaerobic sodium-phosphate buffer (pH=6.5) to reach a 20 % wt/vol suspension. In each bottle, 5 ml of

glyphosate acid/glyphosate IPA salt/Roundup/POEA solutions were added with a syringe, according to the

desired concentrations (Table 8 and Table 9). Due to the mistake, explained above, POEA concentrations

were underestimated, leaving the ratio of glyphosate acid to POEA higher than expected. Final POEA

concentrations ended at 2.4, 0.24, 0.024 and 0.00024 mg/ml. For all treatments, 0 mg/ml served as control.

The gas phase of each serum bottle was changed to a gas mix consisting of 10% H2, 10% CO2 and 80% N2 by

three successive cycles, where the bottles were evacuated and refilled, using a manifold fitted to a vacuum

10 50 10 35 5 (100 mg/ml a.e.)

1 50 10 35 5 (10 mg/ml a.e.)

0.1 50 10 35 5 (1 mg/ml a.e.)

0.001 50 10 35 5 (0.01 mg/ml a.e.)

0 50 10 35 5 (0.00 mg/ml a.e.)

Concentration of glyphosate acid

equivalents (mg/ml)Total volume (ml) Content (ml) 0.1 M sodium-phosphate buffer (ml) Glyphosate IPA salt/Roundup (ml)

POEA concentration

(mg/ml)Total volume (ml) Content (ml) 0.1 M sodium-phosphate buffer (ml) POEA (ml)

2.6 50 10 35 5 (26 mg/ml)

0.26 50 10 35 5 (2.6 mg/ml)

0.026 50 10 35 5 (0.26 mg/ml)

0.00026 50 10 35 5 (0.0026 mg/ml)

0 50 10 35 5 (0.00 mg/ml)

27

pump and a gas tank. The bottles were removed from the manifold under a slight overpressure and the

pressure adjusted to atmospheric, using a needle. The bottles were placed in a 37°C shaking water bath for

24 hours and samples were taken at 0, 2, 4 and 24 hours. With a syringe, 1 ml was withdrawn from each

serum bottle for measurements of VFA (stored at -18°C until analysis), 1 ml for DNA analysis (snap frozen in

liquid nitrogen and stored at -80°C) and 1 ml for immediately pH measurements. 1 ml gas samples were taken

at 0, 2, 4, 6 and 24 hours.

For further details, see Appendix 3.

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs

The effect of glyphosate IPA salt on the growth of L. sobrius in stomach content, at pH 5, was studied in vitro.

As the pH was held constant, the effect on growth of L. sobrius was attributed to glyphosate IPA salt.

A batch culture system was used, simulating the major environmental conditions in the porcine stomach. The

batch culture system involved 4 bioreactors, each with a working volume of 600-3000 ml. Stomach content

was collected and pooled from four 60 kg pigs, two fed a finely ground ration and two fed a coarse. The

content was stored at -20°C until use, and after thawing, the pooled content was diluted (1:1) in a sterile

anaerobic salt medium. For incubation, 1000 ml of diluted digesta was added to the vessel of each bioreactor.

The suspensions were stirred and kept under anoxic conditions by flushing with N2 gas in the headspace. pH

was maintained at the desired value (pH 5), using a pH-controller, regulated by 1M NaOH and 5M HCl.

Incubation temperature was kept at 37°C, by a circulating water bath. The suspension was inoculated with

10 ml of an overnight culture of L. sobrius, to reach a density of approximately 107 CFU/ml. Different amounts

of glyphosate IPA salt were added, to reach final glyphosate acid concentrations of 10, 1.0, 0.1 and 0.0 mg/ml,

Table 10

Table 10. Overview of experimental setup in bioreactors

During the incubation period, samples were removed from the bioreactors at 0, 2, 4 and 24 hours. 1 ml was

used for VFA determination, and stored at -18°C, until analyses began. Another 10 ml was used for

enumeration of bacteria and were transferred to sterile 125 ml serum bottles, with rubber stoppers,

containing 85 ml broth and 5 ml R. Each suspension was poured into a plastic bag and homogenized in a

stomacher for 2 minutes. Subsequently, enumeration of lactic acid bacteria were performed, using the Drop

Plate Procedure (Chen et al., 2003) (Figure 9).

For further details, see Appendix 4.

Bioreactor Glyphosate acid concentration (mg/ml) Glyphosate IPA salt (ml)

1 0 0

2 0.1 0.3375

3 1 3.375

4 10 33.75

28

Statistical analyses

Data were statistically analyzed using RStudio (Version 0.99.489 – © 2009-2015 RStudio, Inc.).

Growth of pure bacterial cultures were analyzed using the linear mixed effects model, with concentrations

and treatments considered as fixed effects and plate as random. Data from stomach, cecum and colon were

also analyzed using the linear mixed-effects model. Again, treatments and concentrations were considered

as fixed effects, as well as time, and individual was treated as random. In addition, data from rumen were

analyzed using the linear mixed-effects model and concentrations, treatments and time were treated as fixed

effects. Here, both day and cow were included as random effects. In all models, the interaction between

concentrations and treatments were included, as we otherwise assume that differences in responses

between concentrations, are the same between all treatments.

Following descriptions are used to demonstrate different significance levels: Non significant (NS) P>0.1,

†P≤0.1, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

For further details about the statistical models, see Appendix 5.

29

Results To ease comparisons between treatments, all concentrations of glyphosate acid, glyphosate IPA salt and

Roundup, are presented in mg/ml of glyphosate acid equivalents (a.e.) throughout the text. However, even

though concentrations of POEA are lower than concentrations of glyphosate acid equivalents, see Table 9

and Figure 10, I also refer to glyphosate acid concentrations, when POEA is mentioned, as amount of POEA

originally was calculated to correspond to glyphosate acid equivalents.

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial

cultures

Commensal bacteria

To investigate at which concentrations, the four treatments starts to affect growth of bacteria, if they affect

growth at all, all growth rates were compared to the control,

Growth of B. adolescentis decreased in the presence of glyphosate IPA salt (P ≤ 0.001) and POEA (P ≤ 0.01),

starting at 0.019 mg/ml. Roundup (P ≤ 0.001) decreased growth at 0.067 mg/ml, and in the presence of

glyphosate acid (P ≤ 0.001), growth decreased at 2.86 mg/ml, see Table 11. The same tendency was observed

for S. hyointestinalis, even though the decrease in growth, in the presence of glyphosate IPA salt (P ≤ 0.001),

Roundup (P ≤ 0.001) and POEA (P ≤ 0.001), started at 0.067 mg/ml. Glyphosate acid (P ≤ 0.001) decreased

growth at 10 mg/ml. Glyphosate IPA salt (P ≤ 0.001) and glyphosate acid (P ≤ 0.001) decreased growth of S.

alactolyticus at 0.067 mg/ml and 10 mg/ml, respectively. The growth of L. salivarius and L. reuteri decreased

at 0.82 mg/ml in the presence of glyphosate IPA salt (P ≤ 0.001), while decrease, in the presence of Roundup

(P ≤ 0.001) and POEA (P ≤ 0.001) started at 2.86 mg/ml. Glyphosate acid (P ≤ 0.001) decreased growth at 10

mg/ml. In the presence of glyphosate IPA salt (P ≤ 0.001), growth of L. sobrius decreased at 0.230 mg/ml, and

for Roundup (P ≤ 0.001) and POEA (P ≤ 0.001) decreases were observed at 0.820 mg/ml. Glyphosate acid (P

≤ 0.001) decreased growth at 10 mg/ml. Growth of E. faecium decreased at 0.820 mg/ml, in the presence of

glyphosate acid (P ≤ 0.05). Both glyphosate IPA salt (P ≤ 0.05) and POEA (P ≤ 0.05) decreased growth at 2.86

mg/ml, even though no effects were observed at 10 mg/ml. Roundup (P ≤ 0.05) decreased growth at 10

mg/ml. In general, only few significant effects were observed for R. albus and R. flavefaciens. Glyphosate acid

decreased growth of R albus (P ≤ 0.01) and R. flavefaciens (P>0.05) at 10 mg/ml. The same was evident for

Roundup, but only for R. albus (P ≤ 0.01).

Graphs are presented in Appendix 6.

30

Table 11. Growth rates of commensal bacteria in the presence of glyphosate acid, glyphosate IPA salt, Roundup and POEA.

Potential pathogens

Glyphosate IPA salt (P ≤ 0.001) decreased growth of C. perfringens at 0.067 mg/ml, while decreases in the

presence of Roundup (P ≤ 0.001) and POEA (P ≤ 0.001) were observed at 0.23 mg/ml, see Table 12.

Glyphosate acid (P ≤ 0.001) decreased growth at 2.86 mg/ml. Decrease in growth of S. aureus, in the presence

of glyphosate IPA salt (P ≤ 0.05), was observed at 0.019 mg/ml. Roundup (P ≤ 0.001) and POEA (P ≤ 0.001)

decreased growth from 0.067 mg/ml while glyphosate acid (P ≤ 0.001) decreased growth from 0.82 mg/ml.

For E. coli K88, S. enterica Enteritidis and S. enterica Typhimurium, glyphosate acid (P ≤ 0.001) decreased

growth at 2.86 mg/ml. In the presence of glyphosate IPA salt (P ≤ 0.001) growth of E. coli K88 and S. enterica

Typhimurium decreased at 10 mg/ml while decrease in growth of S. enterica Enteritidis was observed at 2.86

mg/ml, in the presence of glyphosate IPA salt (P ≤ 0.05). For all three strains, Roundup (P ≤ 0.001) decreased

growth at 10 mg/ml and no effects were found on growth, in the presence of POEA.

Graphs are presented in Appendix 7.

0 0.005 0.019 0.067 0.23 0.82 2.86 10 SEM n

B. adolescentis Glyphosate acid 1.74 1.71 1.83 1.82 1.79 1.6 0.55*** 0.28*** 0.12 4

Glyphosate IPA salt 1.77 1.35 0.14*** 0.11*** 0.11*** 0.13*** 0.26*** 0.05*** 0.16 2

Roundup 1.7 1.33 1.29 0.19*** 0.27*** 0.25*** 0.25*** 0.21*** 0.16 2

POEA 1.7 1.35 0.68** 0.33*** 0.26*** 0.26*** 0.24*** 0.25*** 0.16 2

E. faecium Glyphosate acid 0.47 0.46 0.44 0.42 0.36 0.28* 0.20*** 0.25** 0.04 5

Glyphosate IPA salt 0.42 0.38 0.33 0.33 0.31 0.21 0.20* 0.23 0.06 2

Roundup 0.5 0.47 0.49 0.43 0.43 0.43 0.27 0.24* 0.05 3

POEA 0.5 0.44 0.42 0.4 0.36 0.29 0.24* 0.27 0.05 3

L. reuteri Glyphosate acid 2.42 2.34 2.43 2.36 2.38 2.27 2.07 1.33*** 0.13 4

Glyphosate IPA salt 2.5 2.42 2.3 2.24 1.32*** 0.06*** -0.11*** 0.17 2

Roundup 2.34 2.35 2.34 2.27 2.27 2.09 0.40*** 0.23*** 0.17 2

POEA 2.34 2.31 2.29 2.23 2.24 1.6 0.28*** 0.20*** 0.17 2

L. sobrius Glyphosate acid 1.31 1.32 1.36 1.32 1.31 1.26 1.15 0.37*** 0.09 4

Glyphosate IPA salt 1.28 1.32 1.41 1.27 0.66*** 0.24*** 0.23*** 0.23*** 0.1 2

Roundup 1.34 1.36 1.43 1.42 1.4 0.19*** 0.19*** 0.21*** 0.1 2

POEA 1.34 1.35 1.42 1.4 1.4 0.18*** 0.19*** 0.23*** 0.1 2

L. salivarius Glyphosate acid 2.9 2.82 2.84 2.73 2.77 2.63 2.34 1.35*** 0.37 4

Glyphosate IPA salt 2.95 2.86 2.89 2.82 2.83 0.51*** 0.26*** 0.31*** 0.42 2

Roundup 2.84 2.82 2.86 2.84 2.81 2.58 0.26*** 0.35*** 0.42 2

POEA 2.84 2.96 3.01 2.99 2.96 2.64 0.19*** 0.45*** 0.42 2

S. alactolyticus Glyphosate acid 0.81 0.81 0.79 0.75 0.71 0.62 0.43 0.25*** 0.14 4

Glyphosate IPA salt 0.82 0.46 -0.08*** -0.08*** -0.07*** -0.10*** 0.03*** 0.16 2

Roundup 0.79 0.92 0.89 0.8 0.47 0.42 0.48 0.58 0.16 2

POEA 0.79 0.72 0.71 0.67 0.4 0.5 0.44 0.42 0.16 2

S. hyointestinalis Glyphosate acid 3.06 2.89 2.9 2.92 2.95 2.44 0.21*** 0.17 4

Glyphosate IPA salt 3 2.84 0.21*** 0.52*** 0.28*** 0.29*** 0.25*** 0.23 2

Roundup 3.12 2.62 0.12*** 0.16*** 0.19*** 0.09*** 0.05*** 0.23 2

POEA 3.12 2.83 0.10*** 0.14*** 0.13*** 0.21*** 0.18*** 0.23 2

R. albus Glyphosate acid 1.89 2.04 1.61 1.61 1.61 1.63 0.98 0.28** 0.28 4

Glyphosate IPA salt 1.33 1.99 1.39 2.11 1.69 1.4 1.56 0.52 0.36 2

Roundup 2.46 2.37 2.19 2.2 1.6 1.42 1.48 0.29** 0.36 2

POEA 2.46 2.36 3.08 2.73 2.56 1.76 2.1 2.82 0.36 2

R. flavefaciens Glyphosate acid 2.03 1.81 1.82 1.85 1.71 1.56 0.93 0.24* 0.43 4

Glyphosate IPA salt 1.53 1.54 2.11 2.04 1.69 1.48 1.66 0.64 0.53 2

Roundup 2.54 3.02 2.98 3.26 3.03 3.45 1.75 0.66 0.53 2

POEA 2.54 2.5 2.65 2.29 2.53 2.31 2.37 3.44 0.53 2

Concentration of glyphosate acid equivalents (mg/ml)

31

Table 12. Growth rates of potential pathogenic bacteria in the presence of glyphosate acid, glyphosate IPA salt, Roundup and POEA.

In general, commensals seem to be more tolerant to glyphosate acid than to any of the other treatments,

especially glyphosate IPA salt, see Table 13. In contrast, sensitivity of pathogens towards glyphosate acid

seems to be more pronounced. Values for glyphosate IPA salt, Roundup and POEA are widely dispersed. For

both commensals and potential pathogens, the levels of Roundup and POEA, where decreases are observed,

are very uniform.

Table 13. Overview of concentrations, where growth significantly decreased, in presence of glyphosate acid, glyphosate IPA salt,

Roundup and POEA for all tested bacteria.

The graphs, showing the growth rates for the individual bacteria, are placed in Appendix X. To ease the

interpretation, a non-linear x-axis are used. To explain the choice, two graphs with non-linear and linear x –

0 0.005 0.019 0.067 0.23 0.82 2.86 10 SEM n

C. perfringens Glyphosate acid 5.11 5 4.96 4.9 5.14 4.98 0.13*** 0.01*** 0.32 4

Glyphosate IPA salt 5.17 5.37 4.64 1.08*** 0.00*** 0.73*** -0.20*** -0.18*** 0.43 2

Roundup 5.04 4.79 4.85 4.8 0.29*** 0.43*** 0.33*** 0.35*** 0.43 2

POEA 5.04 5.15 5.19 3.55 0.32*** 0.69*** 0.58*** 0.53*** 0.43 2

E. coli K88 Glyphosate acid 4.63 4.8 4.77 4.72 4.58 4.29 2.89*** 0.20*** 0.31 4

Glyphosate IPA salt 4.72 4.95 4.54 4.86 4.78 4.49 3.78 1.30*** 0.38 2

Roundup 4.54 4.58 4.36 4.38 4.2 4.17 3.89 0.75*** 0.38 2

POEA 4.54 4.82 4.84 4.75 4.43 4.68 4.58 4.07 0.38 2

S. enterica Enteritidis Glyphosate acid 3.92 3.97 3.99 3.96 3.88 3.65 2.09*** 0.19*** 0.12 4

Glyphosate IPA salt 3.91 3.87 3.99 4.03 3.87 3.74 3.28* 1.76*** 0.14 2

Roundup 3.94 3.89 4.00 3.62 3.99 3.58 3.6 2.50*** 0.14 2

POEA 3.94 3.97 4.01 3.82 3.95 3.81 3.99 4.06 0.14 2

S. enterica Typhimurium Glyphosate acid 3.78 3.73 3.75 3.64 3.66 3.52 2.20*** 0.19*** 0.23 4

Glyphosate IPA salt 3.87 3.8 3.86 3.75 3.75 3.62 3.33 2.03*** 0.26 2

Roundup 3.7 3.68 3.63 3.36 3.47 3.6 3.39 2.47*** 0.26 2

POEA 3.7 3.64 3.62 3.42 3.67 3.43 3.71 3.76 0.26 2

S. aureus Glyphosate acid 2.69 2.67 2.72 2.69 2.6 2.09*** 0.61*** 0.20*** 0.09 4

Glyphosate IPA salt 2.73 2.79 2.11* 0.12*** 0.12*** 0.11*** 0.13*** 0.19*** 0.11 2

Roundup 2.64 2.74 2.49 1.79*** 0.96*** 0.37*** 0.27*** 0.52*** 0.11 2

POEA 2.64 2.71 2.28 1.27*** 0.15*** 0.17*** 0.19*** 0.20*** 0.11 2

Concentration of glyphosate acid equivalents (mg/ml)

Concentration of glyphosate acid equivalents (mg/ml)

Glyphosate acid Glyphosate IPA salt Roundup POEA

Commensals B. adolescentis 2.86 0.019 0.067 0.019

E. faecium 0.82 2.86 10 2.86

L. reuteri 10 0.82 2.86 2.86

L. salivarius 10 0.82 2.86 2.86

L. sobrius 10 0.23 0.82 0.82

S. alactolyticus 10 0.067 > 10 > 10

S. hyointestinalis 10 0.067 0.067 0.067

R. albus 10 > 10 10 > 10

R. flavefaciens 10 > 10 > 10 > 10

C. perfringens 2.86 0.067 0.23 0.23

E. coli 2.86 10 10 > 10

S. enterica Enteritidis 2.86 2.86 10 > 10

S. enterica Typhimurium 2.86 10 10 > 10

S. aureus 0.82 0.019 0.067 0.067

Pontential

pathogens

32

axes, respectively, are in Figure 13. When using the linear x-axis, all the results, at the low concentrations,

are aggregated to the left. This makes it difficult to see, at which concentrations the different treatments

have an effect, and therefore, a non-linear x-axis is used for all bacteria.

Figure 13. Growth rates of S. hyointestinalis. The graph to the left has a non-linear x-axis, and the graph to the right has a linear x –

axis.

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows

Pigs

Stomach

For glyphosate acid, two replicates were made, while three were conducted for Roundup and POEA. This

leaves the values observed at 0 mg/ml the same for both Roundup and POEA.

There were no significant effects of glyphosate IPA salt and POEA on pH, even though the levels were lower

when glyphosate IPA salt was added, see Table 14. Roundup (P ≤ 0.05) decreased pH from 5.91 at 0 mg/ml

to 5.31 at 10 mg/ml.

No effects were observed on overall VFA concentrations, relative levels of acetate and propionate and the

lactate concentrations, in the presence of any of the three treatments. In addition, no effects were observed

on the numbers of lactic acid bacteria.

Graphs are presented in Appendix 8.

33

Table 14. Effects of glyphosate IPA salt, Roundup and POEA on selected parameters in stomach

Cecum

Two replicates were made for both glyphosate IPA salt, Roundup and POEA, leaving the values for 0 mg/ml

the same for all treatments.

Glyphosate IPA salt (P ≤ 0.001) decreased pH, from 5.80 at 0 mg/ml to 5.41 at 10 mg/ml, see Table 15. The

same was evident for Roundup (P ≤ 0.001), with a decrease to 5.47 at 10 mg/ml. No effects were observed

for POEA.

Overall VFA concentrations decreased at 10 mg/ml for glyphosate IPA salt (P ≤ 0.01) and Roundup (P ≤ 0.01),

with no effect of POEA. Relative levels of acetate were not affected of any of the treatments, but levels of

propionate were. Glyphosate IPA salt (P ≤ 0.001) and Roundup (P ≤ 0.05) increased relative propionate levels

at 1 mg/ml, while POEA (P ≤ 0.001) increased propionate level at 10 mg/ml. Roundup (P ≤ 0.05) decreased

relative butyrate level at 10 mg/ml, while glyphosate IPA salt (P ≤ 0.1) and POEA (P ≤ 0.1) only showed a

tendency to decrease butyrate levels. Both glyphosate IPA salt (P ≤ 0.05) and Roundup (P ≤ 0.05) increased

relative levels of isobutyrate at 10 mg/ml, while POEA did not have any effect. Relative valerate levels were

only affected by glyphosate IPA salt (P ≤ 0.1) to a certain extent and there were no effects of Roundup and

POEA. Both glyphosate IPA salt (P ≤ 0.01) and Roundup (P ≤ 0.01) increased relative levels of isovalerate at

10 mg/ml while POEA did not have any effect.

Graphs are presented in Appendix 9.

0 0.001 0.1 1 10 SEM n

pH Glyphosate IPA salt 6.03 6.05 6.04 5.97 5.45 0.22 2

Roundup 5.91 5.93 5.93 5.84 5.31* 0.19 3

POEA 5.91 5.96 5.93 5.95 5.94 0.19 3

VFA all Glyphosate IPA salt 4.83 1.78 1.78 2.04 1.86 5.34 2

Roundup 8 6.32 6.8 6.91 6.01 4.78 3

POEA 8 5.78 5.88 6.2 6.29 4.78 3

Acetate Glyphosate IPA salt 0.97 0.97 0.94 0.88 0.93 0.05 2

Roundup 0.94 0.98 0.98 0.98 0.92 0.04 3

POEA 0.94 0.96 0.98 0.91 0.92 0.04 3

Propionate Glyphosate IPA salt 0.03 0.03 0.06 0.12 0.07 0.05 2

Roundup 0.06 0.02 0.02 0.02 0.08 0.04 3

POEA 0.06 0.04 0.02 0.09 0.08 0.04 3

Lactate Glyphosate IPA salt 10.59 12.02 10 11.82 7.71 31.2 2

Roundup 34.6 35.45 33.72 34.01 28.7 28.1 3

POEA 34.6 36.48 37.17 36.75 33.52 28.1 3

Lactic acid bacteria Glyphosate IPA salt 7.12 7.14 7.22 7.11 6.93 0.55 2

Roundup 7.34 7.32 7.32 7.26 7.16 0.48 3

POEA 7.34 7.35 7.44 7.48 7.43 0.48 3

Concentration of glyphosate acid equivalents (mg/ml)

34

Table 15. Effects of glyphosate IPA salt, Roundup and POEA on selected parameters in cecum

Colon

For both Roundup and POEA, two replicates were made, leaving all the values for 0 mg/ml the same.

Roundup (P ≤ 0.001) decreased pH from 5.97 at 0 mg/ml to 5.65 at 10 mg/ml and no effects were observed

for POEA.

Overall VFA concentrations were not affected by either Roundup or POEA, but relative acetate levels

decreased at 10 mg/ml in the presence of Roundup (P ≤ 0.05), see Table 16. POEA did not have any effect.

Relative propionate levels increased at 10 mg/ml for Roundup (P ≤ 0.01) and POEA (P ≤ 0.05) and no effects

were observed on relative butyrate levels. Roundup (P ≤ 0.001) increased relative isobutyrate, valerate and

isovalerate levels at 10 mg/ml, while no effects were observed for POEA.

Graphs are presented in Appendix 10.

0 0.001 0.1 1 10 SEM n

pH Glyphosate IPA salt 5.8 5.79 5.78 5.74 5.41*** 0.06 2

Roundup 5.8 5.84 5.85 5.78 5.47*** 0.06 2

POEA 5.8 5.82 5.8 5.82 5.8 0.06 2

VFA all Glyphosate IPA salt 241 225 232 232 206** 9.93 2

Roundup 241 232 229 240 209** 9.93 2

POEA 241 223 229 222 240 9.93 2

Acetate Glyphosate IPA salt 0.615 0.625 0.617 0.601 0.583 0.035 2

Roundup 0.615 0.626 0.635 0.617 0.603 0.035 2

POEA 0.615 0.628 0.629 0.633 0.608 0.035 2

Propionate Glyphosate IPA salt 0.227 0.226 0.228 0.256*** 0.266*** 0.018 2

Roundup 0.227 0.225 0.222 0.249* 0.262*** 0.018 2

POEA 0.227 0.225 0.224 0.224 0.263*** 0.018 2

Butyrate Glyphosate IPA salt 0.128 0.12 0.126 0.116 0.101† 0.019 2

Roundup 0.128 0.121 0.114 0.107 0.090* 0.019 2

POEA 0.128 0.118 0.118 0.115 0.100† 0.019 2

Isobutyrate Glyphosate IPA salt 0.0047 0.0046 0.0048 0.0049 0.0075* 0.001 2

Roundup 0.0047 0.0045 0.0048 0.0048 0.0075* 0.001 2

POEA 0.0047 0.0045 0.0046 0.0048 0.005 0.001 2

Valerate Glyphosate IPA salt 0.0217 0.0208 0.0213 0.0195 0.0353† 0.0035 2

Roundup 0.0217 0.0211 0.0206 0.0187 0.0299 0.0035 2

POEA 0.0217 0.0209 0.021 0.0197 0.0193 0.0035 2

Isovalerate Glyphosate IPA salt 0.0035 0.0034 0.0035 0.0035 0.0070** 0.0009 2

Roundup 0.0035 0.0034 0.0036 0.0034 0.0068** 0.0009 2

POEA 0.0035 0.0034 0.0034 0.0036 0.0038 0.0009 2

Concentration of glyphosate acid equivalents (mg/ml)

35

Table 16. Effects of Roundup and POEA on selected parameters in colon

Cows

Rumen

Two replicates were made for both glyphosate IPA salt, Roundup and POEA, leaving the values for 0 mg/ml

the same for all treatments

Glyphosate IPA salt (P ≤ 0.001) decreased pH from 6.47 at 0 mg/ml to 6.37 at 1 mg/ml and further to 5.68 at

10 mg/ml, see Table 17. The same was observed for Roundup (P ≤ 0.001) where pH decreased to 6.40 and

5.79, at 1 mg/ml and 10 mg/ml, respectively. POEA (P ≤ 0.05) increased pH to 6.50 at 10 mg/ml.

Glyphosate IPA salt (P ≤ 0.05) increased overall VFA concentration at 10 mg/ml while Roundup (P ≤ 0.05)

increased overall VFA concentration at 1 mg/ml. POEA did not have any effect. Glyphosate IPA salt (P ≤ 0.01)

and Roundup (P ≤ 0.001) decreased relative levels of acetate at 1 mg/ml and POEA (P ≤ 0.001) decreased

relative acetate level at 10 mg/ml. Glyphosate IPA salt (P ≤ 0.01) and Roundup (P ≤ 0.01) increased relative

levels of propionate at 1 mg/ml and POEA (P ≤ 0.01) increased relative propionate level at 10 mg/ml. None

of the treatments had any effects on the relative levels of butyrate, isobutyrate, valerate and isovalerate.

In addition, methane measurements were performed in all rumen samples. However, due to technical

problems, the results showed unexplainable variations, and no statistical analysis were conducted. Even

though the results of methane production varied considerable, one thing became clear: at 10 mg/ml, no

methane were produced, independently of the treatments. In addition, no production was observed at 1

mg/ml for Roundup.

Graphs are presented in Appendix 11.

0 0.001 0.1 1 10 SEM n

pH Roundup 5.97 5.98 5.98 5.94 5.65*** 0.06 2

POEA 5.97 5.97 5.97 5.98 6 0.06 2

VFA all Roundup 242 235 241 235 238 8.94 2

POEA 242 234 240 243 250 8.94 2

Acetate Roundup 0.59 0.6 0.59 0.59 0.55* 0.03 2

POEA 0.59 0.59 0.58 0.58 0.58 0.03 2

Propionate Roundup 0.228 0.226 0.228 0.237 0.258** 0.005 2

POEA 0.228 0.228 0.232 0.233 0.249* 0.005 2

Butyrate Roundup 0.13 0.129 0.133 0.128 0.123 0.022 2

POEA 0.13 0.133 0.138 0.136 0.119 0.022 2

Isobutyrate Roundup 0.0088 0.0089 0.009 0.0095 0.0127*** 0.0032 2

POEA 0.0088 0.0086 0.0089 0.009 0.0097 0.0032 2

Valerate Roundup 0.0347 0.0338 0.0344 0.0343 0.0485*** 0.0022 2

POEA 0.0347 0.0344 0.0349 0.0349 0.0331 0.0022 2

Isovalerate Roundup 0.0064 0.0065 0.0066 0.0058 0.0113*** 0.0021 2

POEA 0.0064 0.0063 0.0065 0.0067 0.0073 0.0021 2

Concentration of glyphosate acid equivalents (mg/ml)

36

Table 17. Effects of glyphosate IPA salt, Roundup and POEA on selected parameters in rumen

To give an overview of the effects of glyphosate IPA salt, Roundup and POEA in the different compartments,

Table 18 has been prepared. In general, VFA concentrations show varying results, pH, relative acetate - and

butyrate levels decrease while relative propionate-, isobutyrate-, valerate- and isovalerate levels increase.

Overall, effects in rumen start earlier than in stomach, cecum and colon and, in general, POEA is less toxic

than glyphosate IPA salt and Roundup.

0 0.001 0.1 1 10 SEM n

pH Glyphosate IPA salt 6.47 6.47 6.45 6.37*** 5.68*** 0.01 2

Roundup 6.47 6.48 6.46 6.40*** 5.79*** 0.01 2

POEA 6.47 6.47 6.47 6.47 6.50* 0.01 2

VFA all Glyphosate IPA salt 110 113 112 112 116* 1.8 2

Roundup 110 107 111 115* 115* 1.8 2

POEA 110 109 110 112 110 1.8 2

Acetate Glyphosate IPA salt 0.667 0.668 0.665 0.651** 0.643*** 0.003 2

Roundup 0.667 0.669 0.669 0.650*** 0.643*** 0.003 2

POEA 0.667 0.665 0.667 0.664 0.650*** 0.003 2

Propionate Glyphosate IPA salt 0.184 0.181 0.184 0.201** 0.202** 0.005 2

Roundup 0.184 0.182 0.183 0.203** 0.202** 0.005 2

POEA 0.184 0.183 0.184 0.187 0.203** 0.005 2

Butyrate Glyphosate IPA salt 0.119 0.119 0.12 0.119 0.126 0.004 2

Roundup 0.119 0.119 0.117 0.119 0.126 0.004 2

POEA 0.119 0.12 0.118 0.118 0.119 0.004 2

Isobutyrate Glyphosate IPA salt 0.0091 0.0095 0.0093 0.0084 0.0087 0.001 2

Roundup 0.0091 0.0091 0.0092 0.0083 0.0086 0.001 2

POEA 0.0091 0.0093 0.0092 0.0092 0.0084 0.001 2

Valerate Glyphosate IPA salt 0.0176 0.0176 0.0173 0.016 0.0165 0.0009 2

Roundup 0.0176 0.0172 0.0171 0.0157 0.0165 0.0009 2

POEA 0.0176 0.0178 0.0176 0.0171 0.0162 0.0009 2

Isovalerate Glyphosate IPA salt 0.0042 0.0049 0.0047 0.0035 0.0037 0.0021 2

Roundup 0.0042 0.0042 0.0044 0.0035 0.0036 0.0021 2

POEA 0.0042 0.0046 0.0045 0.0045 0.0035 0.0021 2

Concentration of glyphosate acid equivalents (mg/ml)

37

Table 18. Overview of concentrations, where selected parameters are significantly affected by glyphosate IPA salt, Roundup and POEA

in stomach, cecum, colon and rumen

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs The experiment was performed as a pilot study, and no statistical analyses were conducted due to lack of

replicates. However, the results for lactic acid bacteria indicate that the cell count was lower at 10 mg/ml,

both after 4 and 24 hours, even though no statistical tests were performed. Due to a mistake, values for 0

and 2 hours are missing, but only for enumeration of lactic acid bacteria. The VFA results showed diverging

results, and no tendencies were visualized.

Graphs are presented in Appendix 12.

Glyphosate IPA salt Roundup POEA

Stomach pH - 10 -

VFA all - - -

Acetate - - -

Propionate - - -

Lactate - - -

Lactic acid

bacteria- - -

Cecum pH 10 10 -

VFA all 10 10 -

Acetate - - -

Propionate 1 1 10

Butyrate 10† 10 10†

Isobutyrate 10 10 -

Valerate 10† - -

Isovalerate 10 10 -

Colon pH 10 -

VFA all - -

Acetate 10 -

Propionate 10 10

Butyrate - -

Isobutyrate 10 -

Valerate 10 -

Isovalerate 10 -

Rumen pH 1 1 10

VFA all 10 1 -

Acetate 1 1 10

Propionate 1 1 10

Butyrate - - -

Isobutyrate - - -

Valerate - - -

Isovalerate - - -

Concentration of glyphosate acid equivalents (mg/ml)

38

Discussion N-(phosphonomethyl)glycine, commonly known as Glyphosate, is a broad-spectrum, nonselective, systemic

herbicide (Franz et al., 1997), existing as an acid or formulated as a salt. Normally, glyphosate isopropylamine

salt is the form of glyphosate, primarily used in Roundup formulations(Malik et al., 1989). In addition,

Roundup also contains water and a surfactant, usually a polyethoxylated tallowamine (POEA) (Giesy et al.,

2000).

Glyphosate, is the active ingredient in Roundup, inhibiting 5-enolpyruvylshikimate-3-phosphate synthase

(EPSPS) in the shikimate-pathway, which leads to formation of aromatic amino acids (Levin and Sprinson,

1964). The shikimate-pathway is present in plants, fungi and bacteria, but not in animals (Bentley, 1990; Franz

et al., 1997; Herrmann, 1995; Kishore and Shah, 1988; Padgette et al., 1995a).

Recently, studies have raised concerns about the effects of glyphosate on gut microbiota (Ackermann et al.,

2015; Krüger et al., 2013b; Shehata et al., 2013a; Shehata et al., 2013b). Results have shown that glyphosate

has a potential inhibiting effect on growth of commensal bacteria, normally occupying the gut of farm

animals. Opposed to the effect on commensals, potential pathogens were, in general, more tolerant towards

glyphosate. If these effects can be recovered in vivo, changes in microbiota, in favor of potential pathogens,

can possibly lead to negative effects of health and productivity of farm animals.

The present study was conducted to investigate effects of glyphosate on the microbiota, found in gut of

slaughter pigs and dairy cows. In the present experiment, glyphosate acid, glyphosate IPA salt, Roundup and

POEA, were included to differentiate eventual effects on microbiota between the different compounds,

included in commercial glyphosate formulations.

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial

cultures

Objective 1

The first objective was to investigate if glyphosate acid, glyphosate IPA salt, Roundup and POEA inhibited

bacterial growth. We hypothesized that glyphosate (Roundup) would suppress growth of gut bacteria and

that gut bacteria would have different susceptibilities towards glyphosate (Roundup). In addition, we

hypothesized that the effect of glyphosate would depend on the chemical formulation (acid, salt, mixture

and surfactant). Throughout the rest of the thesis, glyphosate acid, glyphosate IPA salt, Roundup and POEA

are referred to as ‘treatments’.

Overall, growth of most bacteria were inhibited by one or more of the four treatments, even though the

effect of treatments were not similar, between different strains, confirmed by Shehata et al. (2013b). In

addition, different treatments also showed differences in the degree of inhibition. However, we had

difficulties in culturing Enterococcus faecium, Ruminococcus albus and Ruminococcus flavefaciens, and

therefore, the inhibition of treatments, or lack hereof, should be questioned. In addition Enterococcus

faecalis, Bifidobacterium longum, Campylobacter jejuni and Campylobacter coli were cultured, but because

of inadequate growth, which we expected were due to poor overall growth conditions, culturing did not

succeed, and results are not included.

The lowest concentration, completely inhibiting bacterial growth, is defined as the Minimum Inhibitory

Concentration (MIC). We found MIC values in the range from 0.019 mg/ml to 10 mg/ml, depending on

39

bacteria and treatments used, indicative of considerable changes in sensitivities. However, even more

interestingly than MIC, Minimum Effect Concentration (MEC), calculated as 1/10 of MIC (Fredborg et al.,

2013), is the lowest concentration of an inhibitor needed, to initiate inhibition of a microorganism (Arikan et

al., 2001). If MIC values range between 0.019 mg/ml and 10 mg/ml, MEC values will range from 0.0019 mg/ml

to 1 mg/ml. To get an estimate of whether it is possible to observe MEC values of glyphosate acid equivalents

in vivo, we sat up two different scenarios, calculating possible concentrations of glyphosate acid equivalents

in the gut. Bifidobacterium adolescentis had a MIC value of 0.019 mg/ml, corresponding to the level possible

to find in the colon of pigs (Table 4). However, the inhibiting effect of B. adolescentis would already start at

0.0019 mg/ml, meaning, that it could also be affected in both stomach and small intestine, if present, as

concentrations of 0.005 mg/ml glyphosate acid equivalents could be present here. In addition to B.

adolescentis, Staphylococcus aureus also had the potential to be affected in colon of pigs, as to a MEC value

of 0.0067 mg/ml. However, calculations of the different possible concentrations of glyphosate acid

equivalents are based on different assumptions, which can be discussed. They are based on feed rations

containing maximum residue levels (MRL) of glyphosate, and according to the findings of glyphosate residues

in plants/crops, presented earlier, such levels are uncommon (Arregui et al., 2004; Bøhn et al., 2014;

Miljøstyrelsen, 2014). However, some exceedances have been observed (Plantedirektoratet, 2010), but the

risk that all feed ingredients in the ration has the same high level, is unlikely. Another important assumption,

which might be questioned, is that degradation of glyphosate does not take place in the gut and that only 30

% is absorbed (Commission, 2002). Glyphosate might be degraded to AMPA in the animals, like observed in

plants (Arregui et al., 2004; Bøhn et al., 2014) and if that is the case, the residue levels will change. So far, the

toxicity of AMPA it is not known and therefore it is difficult to include it in the evaluation of the overall toxicity

of glyphosate residues in farm animals. Due to these assumptions, lower concentrations than the ones

presented in Table 4 and Table 5 might be more realistic to find. However, as to MEC values, bacteria still

have the potential to be affected, even though lower levels of glyphosate acid equivalents are found in vivo.

To further examine exact MIC values, and thereby possible MEC values, different concentrations for each

bacteria, according to the results found in our study, should be further investigated. For instance, growth of

Streptococcus hyointestinalis, in the presence of Roundup, are non-existing at a concentration from 0.067

mg/ml to 10 mg/ml. Therefore, it is more interesting to investigate several concentrations from 0.019 mg/ml

to 0.067 mg/ml, as it is here, inhibition occurs. For other bacteria, as Salmonella enterica Typhimurium, MIC

value for glyphosate acid was not yet determined at 10 mg/ml, and therefore it could be an advantage to

further increase concentrations.

Objective 2

In addition to the investigation, of whether glyphosate acid, glyphosate IPA salt, Roundup and POEA inhibited

bacterial growth, we were also interested in investigating if inhibition differed between commensal - and

potential pathogenic bacteria. We hypothesized that gut bacteria would have different susceptibilities

towards glyphosate (Roundup) and that the effect of glyphosate would depend on the chemical formulation

(acid, salt, mixture and surfactant).

Results for nine commensal - and five potential pathogenic bacteria are included. Important to note is that

independent of bacteria, there were big differences in the effects of glyphosate acid, glyphosate IPA salt,

Roundup and Jablo.

40

Overall differences between commensals and potential pathogens

In general, commensal bacteria were tolerant to glyphosate acid, sensitive towards glyphosate IPA salt, and

sensitive - to moderately tolerant towards Roundup and POEA. As already mentioned, we had difficulties in

culturing E. faecium, R. albus and R. flavefaciens. In general, growth rates of E. faecium were low, even in

control wells, compared to growth rates for other bacteria. We might assume that before an inhibitor can

affect growth, bacteria need to be physiologically active, as growth only can be affected, if growth is present.

This could explain missing effects on E. faecium, and as Shehata et al. (2013b) found E. faecium to be highly

sensitive to Roundup, this further underpin the growth-theory. Not mentioned above, Streptococcus

alactolyticus, also had a lower growth rate. Even though effects of glyphosate acid and glyphosate IPA salt

were evident, it is noteworthy that S. hyointestinalis were sensitive towards both Roundup and POEA, not

affecting S. alactolyticus at all. Overall growth rates of R. albus and R. flavefaciens did not differ considerably

from the others, but as they are strictly anaerobic, even a very small amount of air, entering the well, can

possibly impair growth. Opposed to our findings, Ackermann et al. (2015) reported a high sensitivity (0.001

mg/ml) of R. albus and R. flavefaciens, towards glyphosate acid whereas both strains were highly tolerant

towards glyphosate acid in our study.

Compared to commensals, potential pathogens were less tolerant to glyphosate acid. Clostridium perfringens

and Staphylococcus aureus were both sensitive towards glyphosate IPA salt, Roundup and POEA, whereas

Escherichia coli K88, Salmonella enterica Enteritidis and S. enterica Typhimurium were tolerant towards all

treatments. In contrast, Shehata et al. (2013b), found E. coli K88 to be sensitive towards Roundup, but, as we

reported, they observed a high tolerance of S. enterica Enteritidis and S. enterica Typhimurium towards

Roundup, with MIC values of 5.00 mg/ml (Shehata et al., 2013b). Escherichia and Salmonella are closely

related, showing a genomic hybridization of about 50 % (Madigan et al., 2009). This could explain the

relatively similar results found in our study. Further, species belonging to Escherichia and Salmonella are

gram-negative, and in addition, E. coli K88, S. enterica Enteritidis and S. enterica Typhimurium are the only

gram-negative bacteria, tested in our study. Even though we do not know the exact details of how glyphosate

enter bacterial cells, these results indicate that gram-negative cell wall is more protective, than gram-

positive, and therefore considerably more difficult to penetrate. In general, most pathogens are gram-

negative bacteria (Madigan et al., 2009).

Interactions between commensals and potential pathogens

Adhesion of bacteria to intestinal mucosa is usually a prerequisite for colonization in the gut, as to the

continuously moving environment. Lactic acid bacteria are able to compete for adhesion with many

pathogenic species and their production of different antimicrobial substances is observed as mechanisms to

suppress growth of pathogens (Hao and Lee, 2004) including Salmonella, E. coli and Clostridia (Blomberg et

al., 1993; Bomba et al., 1997; Coconnier-Polter et al., 2005; Naber et al., 2004; Yun et al., 2009). Therefore,

an increased colonization of pathogens can indirectly be due to a decrease in the proliferation of Lactic acid

bacteria. Enterococcus faecalis, E. faecium and Bifidobacterium badius has the ability to inhibit BoNT

production, while reducing growth of Clostridium botulinum (A, B, D and E) and Krüger et al. (2013b) observed

that both glyphosate acid and Roundup were inhibitory to E. faecalis, at considerably lower concentrations,

by which they inhibited growth of C. botulinum Type B. Even though we did not succeed in culturing E.

faecalis, and therefore not were able to confirm or invalidate the results by Krüger et al. (2013b), a higher

sensitivity of glyphosate towards E. faecalis, could lead to increased proliferation of C. botulinum. Both due

41

to the loss of antagonistic effect of E. faecalis, against C. botulinum and to the tolerance of glyphosate

observed by C. botulinum itself.

Also, bifidobacteria can create unfavorable conditions for the colonization of Salmonella in the gut (Bielecka

et al., 1998), and therefore, sensitivity of B. adolescentis, towards glyphosate IPA salt, Roundup and POEA, in

our study, has the ability to increase proliferation of Salmonella, indirectly. As Salmonella strains themselves,

were tolerant towards all treatments in our study, addition of glyphosate has the possibility to create

disturbances in gut microbiota, in potential favor of some tolerant pathogens.

Overall effects of glyphosate depending on chemical formulation

In general, all bacteria displayed highest tolerance towards glyphosate acid. However, highest sensitivities

were observed for all gram-positive bacteria, when glyphosate was formulated as the IPA salt. To my

knowledge, no other studies on bacterial cultures have been conducted, investigated effects of glyphosate

IPA salt. However, in a study on cell cultures from the frog, Xenopus laevis, glyphosate IPA salt showed the

most severe effects, and at much lower levels, than the addition of glyphosate acid or Roundup did (Hedberg

and Wallin, 2010). Different toxicities could be explained by the fact, that more glyphosate enters plant cells,

when formulated as the IPA salt (Nalewaja et al., 1996). High tolerance observed for glyphosate acid,

compared to glyphosate IPA salt, might be attributed to its difficulties in crossing cell membranes, due to its

anionic character above pH 2.3 (Figure 1). Even though, glyphosate IPA salt exist as two separate ions in

aqueous solution, they form an ion-pair, when crossing a lipohilic environment, as a plasma membrane in

plant cells (Krogh, 2016). This facilitates an easier entrance, due to the resulting overall, neutral charge

(Krogh, 2016). As far as I am concerned, the exact entrance mechanisms into bacterial cells for glyphosate

acid and glyphosate IPA, have not yet been revealed. However, some of the mechanisms observed in plant

cells, might be applicable, suggesting that, it is the chemistry of the two different glyphosate forms, which

explains difference in toxicity, as to difference in how easy they enter cells.

If we choose to disregard the effect of Roundup on E. faecium and R. albus, Roundup and POEA were almost

equally toxic, towards the rest of the gram-positive bacteria. As Roundup is made up of glyphosate IPA salt

and POEA, the sensitivity towards Roundup should be equal or, eventually, lower than sensitivity observed

for glyphosate IPA salt and POEA, independently. All concentration levels are calculated to correspond to

each other and, subsequently, to the content of Roundup, in order to facilitate comparisons. Even though we

ended up investigating a lower amount of POEA, than the one originally intended, it might not have led to

any misleading results, as POEA also can constitute less than 15 % of Roundup (Giesy et al., 2000; Sawada et

al., 1988). The reason, why glyphosate IPA salt were more toxic than Roundup, in most cases, could be

explained by the fact that some of the glyphosate, in Roundup, could exist as the acid, even though the

majority exist as the salt. Toxicity of glyphosate acid were low compared to glyphosate IPA salt, leaving the

sensitivity of Roundup a bit lower that the sensitivity of glyphosate IPA salt.

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows

Objective 3

In addition to in vitro effects on individual bacteria, we investigated whether glyphosate IPA salt, Roundup

and POEA showed any effects on the composition and activity of gut microbiota, when added to stomach,

cecum and colon content from slaughter pigs and to rumen content from dairy cows. Glyphosate acid was

not included, as only a certain amount of samples could be handled. As glyphosate IPA salt is the form found

in Roundup, we chose to use that instead.

42

Stomach

Compared to cecum, colon and rumen, a lower fermentation rate was found in stomach, in our study.

However, VFA concentrations differed considerably between the stomach replicates, leading to exclusion of

results for 24 hours, due to unexplainable variation, between this time and the two remaining time points.

Theoretically, of the gut segments investigated, least fermentation takes place in stomach, due to acidic

conditions (Cunningham and Klein, 2007). pH is low usually lower than 3.6 (Yen, 2001), keeping bacterial

numbers low (Cunningham and Klein, 2007; McDonald et al., 2011). Most of the bacteria, inhabiting stomach,

are lactic acid bacteria (Cunningham and Klein, 2007; McDonald et al., 2011), as they, in general, are more

acid tolerant than other bacteria (Madigan et al., 2009). We were not able to detect any effects of glyphosate

IPA salt, Roundup or POEA, independent of concentrations, on levels of acetate and propionate, as well as

on production of lactate and on the cell count of lactic acid bacteria. The only significant effect was observed

for Roundup, which decreased pH, at 10 mg/ml. As no other effects was evident for Roundup, at the same

concentration, there was no evidence that lowering pH influenced any of the other parameters, measured in

stomach. As stomach content already is acidic and lactic acid bacteria are the main inhabitants, it makes

sense that lactate production, and lactic acid cell count, were not affected by a decrease in pH, due to their

high tolerance towards acid.

Cecum and colon

The main fermentation sites in pigs are cecum and colon, both compartments maintaining a big, complex

microbial population, composed of both aerobic and anaerobic bacteria (Bergman, 1990; Cunningham and

Klein, 2007).

As a starting point, both cecum and colon had the same production of total VFA’s, approximately 240

mmol/kg sample each. VFAs in cecum and colon have been measured in the range between 30-240 mmol/L,

but according to Bergman (1990), an average of 70 – 120 mmol/L is more common. However, we measured

VFA production over 24 hours, possibly explaining the higher level.

In cecum, the overall fermentation pattern changed after addition of glyphosate IPA salt and Roundup. Total

VFA concentrations decreased, while propionate levels increased, for both treatments. Whether changes

observed, were due to direct effects of glyphosate IPA salt and Roundup, or whether the effects were

mediated through a decrease in pH, can be discussed. Normally, pH is close to neutral in cecum and colon

(Cunningham and Klein, 2007) and tolerance towards fluctuations in pH, differs between bacterial groups. As

cellulolytic bacteria, preferentially producing acetate (McDonald et al., 2011; Sjaastad et al., 2010), are more

sensitive than amylolytic, mostly producing lactate and thereby propionate (McDonald et al., 2011; Sjaastad

et al., 2010), fermentation leading to a higher production of propionate would not be unlikely, if pH drops

(Sjaastad et al., 2010). However, decreased pH was probably not the reason for the changes we observed in

propionate levels, as the effects on propionate were evident before pH dropped, and as pH were below six,

independent of all concentrations. Results for POEA supports this theory, as it did not affect pH, while still

increasing relative levels of propionate.

Even though, only low levels of isobutyrate, valerate and isovalerate were present in cecum, levels increased

by glyphosate IPA salt and Roundup. As branched VFA’s only are produced from amino acids, they are

representatives of protein fermentation (Nery et al., 2012). Therefore, we might propose that glyphosate IPA

salt and Roundup, had the ability to stimulate activity of proteolytic bacteria, or that pH level indirectly

affected it. Another parameter, possibly changing fermentations patterns, could be degradation of

43

glyphosate by gut bacteria. Some soil microorganisms are able to degrade glyphosate, releasing nutrients

available for growth (Barry et al., 1992; Borggaard and Gimsing, 2008; Duke et al., 2012). Even though,

degradation has only been investigated in soils, it seems plausible that some gut bacteria, also have the

abilities to degrade glyphosate, releasing nutrients, possibly changing composition of microbiota.

In colon, only results for Roundup and POEA were included, as glyphosate acid and glyphosate IPA salt, only

were tested once, and therefore had to be excluded from the final dataset. Roundup did not have any effect

on total VFA concentration, but decreased acetate levels and increased propionate levels, meaning that

acetate levels were counterbalanced by the increase in propionate levels. As for cecum, Roundup decreased

pH and even though increase in propionate probably were independent of pH level, it cannot be rejected

that decrease in acetate correlates with a decrease in pH, as cellulolytic bacteria are pH sensitive (Sjaastad

et al., 2010). Even though relative proportions of isobutyrate, valerate and isovalerate were low, significant

increases were evident in presence of Roundup. Usually, the sum of isobutyrate, valerate and isovalerate

constitute less than 10 % of the VFA’s produced in colon, as most protein degradation takes place in stomach

and small intestine, and only a small amount escapes to colon, compared to undigestible carbohydrates

(Rasmussen et al., 1988). As for cecum, there might be an increase in proliferation of proteolytic bacteria as

to an increased fermentation of amino acids. In addition, some bacteria might be able to degrade glyphosate,

thereby changing fermentation patterns, as mentioned for cecum.

In addition to glyphosate IPA salt and Roundup, POEA only increased relative propionate levels. In several

experiments it has been concluded that POEA was main responsible for the toxicity observed for Roundup

(Benachour and Seralini, 2009; Folmar et al., 1979; Mesnage et al., 2013; Mesnage et al., 2014; Mitchell et

al., 1987; Servizi et al., 1987) but we were not able to reproduce these results. However, many of these

studies tested the effect on single cells, and here we have to do with a more complex system. In addition,

even though we are aware that POEA facilitates penetration of glyphosate to plants, we do not know how

POEA penetrate cells and how it exerts its mechanism, which would be on interest when explaining the

effects, present or missing, we observed here. The effects of glyphosate IPA salt and Roundup were observed

at same concentrations, in most cases, which implies that POEA did not contribute considerably to the effect

exerted by Roundup on cecum and colon content.

Other than propionate levels in cecum, which were affected at 1 mg/ml, the rest of the significant differences,

were not observed before addition of 10 mg/ml. Both concentrations are way above the calculated

theoretical, concentrations we presented in Table 4, meaning that fermentation patterns will not be affected

in vivo, based on our results.

Rumen

The rumen is the main fermentation site in cows, maintaining a big, complex microbial population, mostly

composed of anaerobic bacteria (Bergman, 1990; Cunningham and Klein, 2007).

In a feeding study, Huther et al. (2005) investigated the effect of Roundup on ruminal fermentation patterns

and they found total VFA concentrations ranging between 64 and 92 mmol/L, depending on time after

feeding. Our total VFA concentrations were slightly higher, increasing in the presence of glyphosate IPA salt

and Roundup. Acetate levels decreased for all treatments, even though effects of glyphosate IPA salt and

Roundup, were evident before POEA. No matter which mechanisms are involved in the changes, more

propionate were produced, at the expense of acetate, indicative of change in the overall fermentation

pattern, from cellulolytic - to amylolytic bacteria (McDonald et al., 2011; Sjaastad et al., 2010). Also in rumen,

44

we observed pH effects, but as mentioned in both cecum and colon, pH effects did not have any direct effects

on neither acetate – nor propionate levels. Normally, pH in rumen is close to neutral (Cunningham and Klein,

2007), but glyphosate IPA salt and Roundup both decreased pH, starting at 1 mg/ml. A drop in pH (below 6)

can inhibit the activity of cellulolytic bacteria, degrading structural carbohydrates as cellulose, hemicellulose

and pectin (McDonald et al., 2011; Sjaastad et al., 2010). However, even though pH decreased at 1 mg/ml, it

only decreased slightly, still ranging between 6.37 and 6.40. This pH level were too high to inhibit cellulolytic

bacteria, and as pH increased (even though only 0.03) in the presence of POEA, while acetate levels

decreased, the effect of pH on acetate levels, probably can be rejected. Levels of propionate also increased,

independent of pH, as observed in cecum and colon. As for cecum and colon, rumen might inhabit bacteria,

which are able to degrade glyphosate, possibly explaining some of the changes in fermentation patterns.

However, further studies needs to highlight this.

As no changes were observed for isobutyrate, valerate and isovalerate, fermentation of proteins were

unchanged, irrespective of treatment. In rumen, these three acids usually constitute less than 5 % of the total

VFA pool (Bergman, 1990), which also corresponds to our findings.

To my knowledge, few studies have been conducted, investigating the effect of glyphosate acid, on rumen

fermentation patterns. Reuter et al. (2007) were able to show that addition of more than 8.45 mg/ml

inhibited fermentation, and that 10.14 mg/ml decreased acetate, butyrate and total VFA concentrations,

compared to control. Concentrations of propionate fluctuated more, but after incubation for 48 hours,

propionate concentrations were higher than control, both at 1.69 mg/ml and at 10.14 mg/ml (Reuter et al.,

2007). Even though we used another form of glyphosate, we also observed decreases in acetate levels, as

well as increase in propionate levels. In addition, Huther et al. (2005) observed no changes in ruminal

fermentation pattern after addition of glyphosate acid at 1.67 mg/ml (Huther et al., 2005). Even though

rumen were the component where significant effects were evident at lowest concentrations, these

concentrations were considerably higher than the theoretical concentrations, we calculated in Table 5.

Overall, the effect of POEA was observed later than the effect of glyphosate IPA salt and Roundup, but the

effects were also greater here, than in cecum and colon. Again, several experiments concluded that POEA

was main responsible for the toxicity observed for Roundup, but we were not able to reproduce these results.

In addition to the missing effects of POEA, the effects of glyphosate IPA salt and Roundup were similar, in

most cases, which implies that POEA did not contribute considerably to the effect of Roundup.

Methane

Even though the results of methane production varied considerable, and no statistical analyses were

performed, one thing became clear: at 10 mg/ml, no methane were produced, independent of the

treatments. In addition, no production was observed at 1 mg/ml for Roundup. It has been suggested that a

feed ration, mainly composed of starch, lowers pH, which might inhibit the growth and activity of the

methanogens (Boadi et al., 2004; Martin et al., 2010). This could explain the effect we observed for Roundup

and glyphosate IPA salt, due to the lowering of pH at 10 mg/ml. Nevertheless, when comparing these to

POEA, other factors might be involved, as pH increases in the presence of POEA. On the other hand,

production of acetate and butyrate leads to methane production, while propionate decreases it (Cunningham

and Klein, 2007). The decreased acetate to propionate ratio, we observed, could also have led to the

inhibition of the methane production. However, as archaea also possesses the shikimate pathway (Bult et al.,

45

1996; Daugherty et al., 2001; Graham et al., 2001), glyphosate can have a direct, inhibbiting effect on

methanogens.

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs

Objective 4

In addition to the direct effect of glyphosate on bacterial cultures, and the microflora, found in gut, we

wanted to investigate whether growth of a strain of Lactobacillus sobrius, in stomach content, was affected

by glyphosate IPA salt, when pH was held constant (pH=5). As this part of the experiment was conducted, as

a pilot study, no statistical tests were done.

In general, it seems like lactobacilli cell count were lowered at 10 mg/ml. Inhibition was due to the direct

effect of glyphosate IPA salt, and not pH, as it was constant, no matter concentration level of glyphosate IPA

salt. Changes in VFA’ concentrations were difficult to interpret, and no other clear effects, than an increase

in lactate – and succinate production over time, were evident. When L. sobrius were tested in lab media, the

MIC value for glyphosate acid were 0.23 mg/ml, considerably lower than the effect, observed here. The

differences between lab media and stomach content could, most likely, have had an influence on the effects

observed, as we do not know the exact composition of stomach content.

Comparisons of the three different experiments

Even though, especially glyphosate IPA salt, Roundup and POEA were able to inhibit growth of some bacterial

cultures, at considerably low concentrations, we were not able to demonstrate the same effects on gut

contents from pigs and cows, as well as on a single culture in stomach content. In cecum, colon and rumen

content, activity of microorganisms were measured through VFA production; the first effects of glyphosate

IPA salt and Roundup were observed at 1 mg/ml, and effects of POEA at 10 mg/ml. In addition, possible

effects of glyphosate IPA salt on L. sobrius, in stomach content, were observed at a considerably higher

concentration, than effects, observed in lab media. Some of the bacterial MIC - and MEC values found, would

be realistic to observe in vivo, based on the theoretical calculations made. However, none of the

concentrations, by which we observed an effect on gut content, can be found in vivo, at least not due to our

assumptions and calculations. This indicates that gut microbiota is complex, and that effects of treatments,

on individual bacteria in lab media, cannot be transferred to affecting bacteria in the same way, when present

in gut, as to a complex interaction pattern between the microorganisms (Madigan et al., 2009; McDonald et

al., 2011; Sjaastad et al., 2010). However, even though in vivo concentrations, would not affect activities of

microbiota, some changes in composition could be evident. Sequencing samples would have been helpful to

highlight, possible differences in composition of microbiota, but due to time limitations, activity was only

included in this experiment.

When tested on individual strains, bacteria were more or less equally sensitive towards glyphosate IPA salt,

Roundup and POEA. For glyphosate IPA salt and Roundup, these effects were also observable in gut contents,

even though concentrations, where effects were evident, increased considerable. However, when tested on

gut content, POEA was least toxic of all treatments. Compared to glyphosate, we do not know much about

the exact composition of POEA and therefore it is not clear, how it enters cells and exerts its mechanisms.

Other studies should be performed to investigate this further.

Even though pH decreased in both stomach, cecum, colon and rumen for glyphosate IPA salt and/or

Roundup, results for L. sobrius in stomach content, indicate that glyphosate IPA salt exert its mechanisms,

46

independent of pH, as it was held constant. However, as this experiment was only conducted, as a pilot study,

replicating the experiment could be advantageously.

Additional work, not included in the thesis

In total, I have run 73 plates and presented results from 44, in the thesis. The reason why I chose to exclude

the others is that they were used to investigate which glyphosate acid concentrations that were appropriate

to test, how the plates could be prepared as uniform as possible and which bacteria it was possible to grow

in the conditions, we were able to supply. As mentioned, some bacteria would not grow, even though they

were tested in different media.

To test the treatments on animal digesta, a pilot study was performed. This was both done to test how the

protocol worked, and at which concentrations, and in which compartments, it was relevant to further

investigate the effect of the treatments. To begin with, glyphosate acid was used instead of glyphosate IPA

salt, and this is the reason why there are missing values for glyphosate IPA salt in some of the datasets. In

total, five trials were performed with digesta from slaughtered pigs.

47

Conclusion Overall, we showed that glyphosate acid, glyphosate IPA salt, Roundup and POEA all had the ability to inhibit

bacterial growth, even though MIC values differed between treatments and bacteria (objective 1). In

addition, inhibiting effects of glyphosate acid, glyphosate IPA salt, Roundup and POEA differed between

gram-positive and gram-negative bacteria; gram-negative being more tolerant towards all treatments. We

cannot conclude that commensals and potential pathogens are affected differently, just due to this division,

but as most gram-negative bacteria are pathogens, this is a more reasonable explanation for observed

differences between commensals and potential pathogens. Instead of differentiating between commensals

and potential pathogens, differentiation between gram-positive and gram-negative would be a more correct

determination, according to our findings (objective 2).

Other than affecting growth of bacteria directly, glyphosate also has the potential to affect growth indirectly.

Some commensals are able to suppress growth of some potential pathogens, and if the commensals are

sensitive towards glyphosate, and the pathogens tolerant, addition of glyphosate has the potential to

eliminate the inhibiting effect, the commensal has towards the potential pathogen. This, in addition to the

direct effect of glyphosate, can create disturbances in the gut, possibly leading to health problems for the

animal.

Glyphosate IPA salt, Roundup and POEA, were able to change fermentation patterns in cecum, colon and

rumen, indicative of changes in microbial activity, even though effects failed to appear in stomach (objective

3). The earliest effects were evident at 1 mg/ml being considerably higher than the possible concentrations

we calculated theoretically, in the different compartments. Overall, addition of glyphosate IPA salt and

Roundup led to more changes, than POEA did.

Even though the results of methane production in rumen varied considerable, and no statistical analyses

were performed, no methane were produced at 10 mg/ml independent of the treatments. In addition, no

production was observed at 1 mg/ml for Roundup. The decreased acetate to propionate ratio, observed for

all treatments in rumen, could have led to the inhibition, but a decrease of pH might also play a role for

glyphosate IPA salt and Roundup.

The effect of glyphosate IPA salt on the growth of L. sobrius in stomach content, at pH 5, was investigated in

a pilot study. Due to time limitations, the experiment was only performed ones, and therefore, data were

not analyzed statistically. Overall, it seemed like, lactic acid cell count were lowered at 10 mg/ml, and no

other parameters were affected. However, the experiment have to be repeated, to detect eventual

differences (objective 4).

Overall, fermentation is the action of the entire microbial biomass and need to be considered like an overall

process, not focusing on individuals (Cunningham and Klein, 2007; McDonald et al., 2011). This means that

even though we found considerably effects of glyphosate IPA salt, Roundup and POEA, when treating

individual bacterial strains, effects on the overall microbiota, observed in the second part of the experiment,

is more important, as it better reflects how different treatments affect microbiota in vivo.

48

In conclusion, we have been able to accept, all the four of our working hypothesis. First of all, glyphosate

(Roundup) suppressed growth of gut bacteria, different gut bacteria had different susceptibilities towards

glyphosate (Roundup), glyphosate (Roundup) changed fermentation pattern in the gut of pigs and dairy cows

and the effect of glyphosate depended on the chemical formulation (acid, salt, mixture and surfactant) used.

49

Perspectives and future considerations Even though glyphosate does not have the ability to affect animals directly, it is now evident, through

different studies, that it has the potential to affect microbiota, and thereby the animals, they inhabits.

As our results were not conclusive, it would be interesting to further investigate, whether the effects of

glyphosate IPA salt and Roundup in gut contents, are attributed to the treatments directly, or indirectly, due

to a lowering of pH by the compounds. This can be done in bioreactors, by either keeping pH, or

concentrations, constants. In addition to VFA measurements, sequencing would help determine eventual

changes in microbial composition.

In this study, glyphosate etc. was added directly to gut contents. However, pigs and cows are not fed

glyphosate directly; plants, which have been sprayed with glyphosate, are fed to them. This could influence

the effects in vivo, and therefore, feeding trials should be performed, to give a better indication of how, and

if, the animals are affected. In addition, feeding trials has the ability to highlight long-term effect, as it is

possible that microorganisms can develop resistance towards glyphosate or that glyphosate, on the other

hand has an additive effect, increasing toxicity, the longer the period the animal are fed feed, containing

residues. Effects, of different residue levels, should also be determined. Other than measuring microbial

activity and composition, animal performance parameters are important to include. Through the effect on

microbiota, glyphosate can possibly effect livestock productions, which are the main interest for farmers.

50

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Appendix 1

Determination of viable counts by use of Drop Plate Procedure Add 250 µl sample to the first well in the first column (i), in a In 96-well microtiterplates

Add 225 µl medium in the wells, where dilutions are wanted

Transfer 25 µl sample from column i to 225 µl medium (0.1 M sodium phosphate buffer, pH 6.5) in

column (i+1)

Mixed sample and buffer 10 times

Repeat process until wanted dilutions are obtained

Plate 10 µl, from each of the dilutions of interest, for each sample, on MRS agar

Incubate MRS plates anaerobically overnight, at 37°C.

Count plates the next day

Calculate colony forming units (CFU)

Quantification of VFA concentrations Add 1 ml sample (1 ml standard mix and 1 ml blind (H2O) to SCFA tubes

Add 100 µl intern standard to each sample

Add 0,5 ml HCl to each sample

Add 2 ml ether to each sample

Mix samples for 30 sec on Vibrax

Centrifuge samples at 2000 g for 10 min (5°C)

Add 10 µl MTBSTFA to microvials

Add 50 µl of the supernatant (from the samples) to microvials

Close microvials

Mix microvials on vortex’er

Place microvials on heating block (80°C) for 20 min

Store microvials in room temperature for 48 hours

Run samples on the Gas Chromatograph (Thomas Rebsdorf)

Gas measurements Extract 1 ml gas from each sample of interest

Eject 0.5 ml of the gas into a Gas Chromatography

Analyze data using the appurtenant PEAK359 software.

Appendix 2

Influence of glyphosate acid, glyphosate IPA salt, Roundup and POEA on growth of bacterial

pure cultures

Preparation of MRS

Mix contents

Autoclave at 110°C for 20 min (sometimes 100° in 15 min, just to melt it, as it easily burns off)

Stir and cool media, while infusing CO2

Distribute media into VFA tubes while infusing CO2

Autoclave tubes at 121 °C for 15 min (118° for 15 min)

Preparation of BHI

Mix contents

Autoclave at 121°C for 15 min

Stir and cool media

If anaerobic, infuse CO2 while stirring

If microaerophilic, infuse a gas, with a low oxygen level

Distribute into VFA tubes

Autoclave tubes at 121 °C for 15 min

Preparation of RM02/Ruminococcus media

Kim (2012), 1Widdel et al. (1983), 2Tschech and Pfennig (1984)1

1Kim, C. C. (2012). "Identification of rumen methanogens, characterization of substrate requirements and measreument of hydrogen thresholds: a

thesis presented in partil fulfilment of the requirements for the degree of Master's in Microbiology." Massey University, New Zealand. Tschech, A., and Pfennig, N. (1984). Growth yield increase linked to caffeate reduction in Acetobacterium woodii. Archives of Microbiology 137, 163-

167. Widdel, F., Kohring, G. W., and Mayer, F. (1983). Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. Archives of

Microbiology 134, 286-294.

Broth media 52.4 g

Agar 0.5 g (0.05 % of 1 L)

Demineralized water 1 L

Broth media 37.1 g

Agar 0.5 g (0.05 % of 1 L)

Demineralized water 1 L

KH2PO4 1.4 g

(NH4)2SO4 0.6 g

KCl 1.5 g

Sodium acetate 1.6 g

Trace element solution (SL10)1 1 ml

Selenite/tungstate solution2 2 ml

Resazurin 5-7 drops

Distil led water 950 ml

Mix contents

Boil in the microwave - go on high for 4 minutes. Look for bubbles - once bubbles appear, keep

going for around a minute. Increase time if needed.

Place media on ice, while infusing CO2 until it reaches room temp or just a little warmer

After cooling, add 4 g NaHCO3 and 0.50 g L-cysteine·HCl·H2O

Adjust pH to 6.8 with 5.6 M NaOH

Disperse 7.6 ml media into each VFA tube while infusing CO2

Autoclave tubes at 121°C for 15 min

Store in the dark for 24 hours

Before use, add 0.4 ml CRF, yeast extract and vitamin mix, 0.4 ml glucose, cellobiose, xylose and

arabinose mix (40 mM each) and 0.2 ml reducing agent

Preparation of colon

Mix contents

Autoclave at 121°C for 10 min

Stir and infuse CO2

Add 0.200 ml Vitamin K1 and 1.0 g L-cysteinehydrochloride while stirring

Distribute media in VFA tubes while infusing CO2

Autoclave tubes at 121 °C for 15 min

Preparation of anaerobic basal broth

Mix contents

Autoclave at 121°C for 15 min

Stir and cool media while infusing CO2

Distribute media into VFA tubes under CO2 infusion

Autoclave tubes at 121 °C for 15 min

Solution A 167 ml

Solution B 167 ml

Colon-juice? 200 ml

Sugar solution 40 ml

Yeast extract (Merck 1.03753) 5.0 g

Trypticase peptone (Merck 1.07213) 5.0 g

NaHCO3 5.0 g

Resazurin 1 ml

Hemin 10 ml

Elga water Add up to 1 L

Broth media 35.4 g

Agar 0.5 g (0.05 % of 1 L)

Demineralized water 1 L

Preparation of microtiterplates

Bacteria cultures are inoculated to broth media and incubated overnight, at either 37°C or 41°C

Overnight culture is investigated in microscope, to check for purity

Overnight culture is diluted 100 times in broth media, if growth is found sufficient (density of approx.

109 cells per ml)

Overnight culture is diluted 10 times in broth media, if growth is less dense than approx. 109 cells per

ml

325 µl media + 1,4 µl POEA is added to each well; 1, 2 and 3, in row A, to make a replicate of three

Stock solution of glyphosate acid is made (10 mg/ml a.e.). 325 µl is added to each well; 4, 5 and 6 in

row A, to make a replicate of three

Stock solution of glyphosate IPA salt is made (10 mg/ml a.e.). 325 µl is added to each well; 7, 8 and 9

in row A, to make a replicate of three

Stock solution of Roundup is made (10 mg/ml a.e.). 325 µl is added to each well; 10,11 and 12 in row

A, to make a replicate of three

225 µl media is added to each well in row B-H

For each well in row A, solution is mixed 10 times with a pipette and 100 µl is transferred to the next

well (row B) and mixed 10 times. The procedure is repeated until row G, where the extra 100 µl is

thrown away. A 3.5 dilution is now made from each well to the next.

Row H corresponds to the control

In each well, 25µl bacterial dilution is added. Mix culture on vortex’er before use.

Bacteria and media is mixed, operating from high to low dilution

50 µl sterilized paraffin oil is added to each well

The microtiterplate is incubated in an ELISA spectrophotometer at 37°C for 18-24 h

Glyphosate acid: Add 80 mg glyphosate acid to 8 ml media to end with a final concentration of 10 mg/ml a.e.

Glyphosate IPA salt: Add 270 µl glyphosate IPA salt to 8 ml media to end with a final concentration of 10

mg/ml a.e.

Calculations:

Glyphosate IPA solutions: 40 wt % in H20, corresponds to 400 mg/ml.

0.74 ∗ IPA salt = glyphosate acid

glyphosate IPA salt = glyphosate acid

0.74=

10 mg/ml

0.74= 13,51 mg/ml

13.51 mg/ml

400 mg/ml= 0,0338 ml IPA pr ml media

0.0338 ml ∗ 8 ml = 0.270 ml = 270 µl

Roundup: Add 222 µl Roundup to 8 ml media to end with a final concentration of 10 mg/ml a.e.

Calculations:

Roundup contains 360 g/L glyphosate acid (a.e.):

10 mg/ml

360 mg/ml= 0.0278 ml Roundup pr ml media

0.0278 ml ∗ 8 ml = 0.222 ml = 222 µl

POEA: Add 1.4 µl POEA to 325 µl media to end with a final concentration of 4.17 mg/ml POEA.

Calculations:

We expect Roundup to contain 360 g/L glyphosate acid and 150 g/L POEA

150 mg/mL

360 mg/ml= 0.417

Based on the assumption that 1 mg POEA is the same as 1 ml, I have to add 1 µl POEA for every 250 µl.

Every time we have 10 mg/ml glyphosate acid, we have 4.17 mg/ml POEA.

Appendix 3

Influence of glyphosate IPA salt, Roundup and POEA on gut microbiota from pigs and cows

Preparation of 0.1 M sodium-phosphate buffer (pH 6.5)

Add 13,8 g NaH2PO4 * H2O to 1 L ELGA water

Stir the suspension on the magnetic stirrer, while measuring pH. Add 5.6 M NaOH to reach a final pH

of 6.5

Transfer 35 ml solution to 125 ml bottles and seal them with butyl rubber stoppers

Flush bottles with N2 by three successive cycles, where bottles are evacuated and refilled, using a

manifold, fitted to a vacuum pump and a gas tank.

Adjust pressure to atmospheric with a needle

Autoclave bottles at 121 °C for 15 min

Digesta samples from slaughter pigs and dairy cows:

Prepare glyphosate IPA salt, Roundup and POEA solutions according to Table 1. Mix on vortex’er

Pigs: Collect stomach, cecum and colon content at slaughter

Cows: Collect rumen content from fistulated dairy cows. Sieve content through a cheesecloth to

remove big particles

Place samples on ice, and take them to the laboratory immediately.

Add 10 ml content to each bottle, containing 35 ml 0.1 M sterile anaerobic sodium-phosphate

buffer (pH=6.5), with a cut-off syringe. A final suspension of 20 % wt/vol is reached.

Add 5 ml of glyphosate IPA salt/Roundup/POEA solution to each bottle, with a syringe, to reach

desired concentrations (Table 2, Table 3).

Flush bottles with CO2 by three successive cycles, where bottles are evacuated and refilled, using a

manifold, fitted to a vacuum pump and a gas tank.

Adjust pressure to atmospheric with a needle

Pigs: from each bottle, extract 1 ml for VFA measurements (store at -18°C until analysis), 1 ml for

DNA analysis (snap freeze in liquid nitrogen and store at -80°C), 1 ml for immediately pH

measurement and 0.5 ml for Drop Plating Procedure, at 0, 3/4 and 24 hours, respectively. Extraction

is done with a syringe to avoid contamination with air.

Cows: from each bottle, extract 1 ml for VFA measurements (store at -18°C until analysis), 1 ml for

DNA analysis (snap freeze in liquid nitrogen and store at -80°C) and 1 ml for immediately pH

measurement, at 0, 2, 4 and 24 hours, respectively. 1 ml gas samples, from each bottle are extracted

at 0, 2, 4, 6 and 24 hours, respectively. Extraction is done with a syringe to avoid contamination with

air.

POEA concentration

(mg/ml)Total volume (ml) Content (ml) 0.1 M sodium-phosphate buffer (ml) POEA (ml)

2.6 50 10 35 5 (26 mg/ml)

0.26 50 10 35 5 (2.6 mg/ml)

0.026 50 10 35 5 (0.26 mg/ml)

0.00026 50 10 35 5 (0.0026 mg/ml)

0 50 10 35 5 (0.00 mg/ml)

Concentration of glyphosate acid

equivalents (mg/ml)

Concentration of POEA

(mg/ml)

0.1 M sodium-phosphate

buffer (ml)

Glyphosate IPA salt 100 - 12 3945 µl IPA salt

10 - 10.8 1.2 ml of 100 mg/ml a.e.

1 - 10.8 1.2 ml of 10 mg/ml a.e.

0.1 - 10.8 1.2 ml of 1.0 mg/ml a.e.

0.01 - 10.8 1.2 ml of 0.1 mg/ml a.e.

Roundup 100 - 12 3333 µl Roundup

10 - 10.8 1.2 ml of 100 mg/ml a.e.

1 - 10.8 1.2 ml of 10 mg/ml a.e.

0.1 - 10.8 1.2 ml of 1.0 mg/ml a.e.

0.01 - 10.8 1.2 ml of 0.1 mg/ml a.e.

POEA - 26 12 51.4 µl POEA

- 2.6 10.8 1.2 ml of 26 mg/ml

- 0.26 10.8 1.2 ml of 2.6 mg/ml

- 0.026 10.8 1.2 ml of 0.26 mg/ml

- 0.0026 10.8 1.2 ml of 0.026 mg/ml

10 50 10 35 5 (100 mg/ml a.e.)

1 50 10 35 5 (10 mg/ml a.e.)

0.1 50 10 35 5 (1 mg/ml a.e.)

0.001 50 10 35 5 (0.01 mg/ml a.e.)

0 50 10 35 5 (0.00 mg/ml a.e.)

Concentration of glyphosate acid

equivalents (mg/ml)Total volume (ml) Content (ml) 0.1 M sodium-phosphate buffer (ml) Glyphosate IPA salt/Roundup (ml)

Table 1. Preparations of overall glyphosate IPA salt, Roundup and POEA solutions

Table 2. Overview of experimental setup for glyphosate IPA salt and Roundup

Table 3. Overview of experimental setup for POEA

Appendix 4

Influence of glyphosate IPA salt on growth of L. sobrius in stomach content from pigs

Preparation of anaerobic salt media

Mix contents

Autoclave at 121°C for 15 minutes.

Preparation of broth

Add yeast extract and peptone to 200 ml ELGA water

Add solution A, B, resazurin, hemin and tween 80 and the rest of the water

Autoclave at 110°C for 10 min.

Cool to room temperature while flushing with CO2.

Add 85 ml to 125 ml infusion serum bottles, while flushing with CO2

Flush sealed bottles with CO2

Autoclave the bottles at 121°C for 15 min.

The day of slaughter

Collect stomach content from pigs at slaughter

Pool stomach content

Store stomach content at -18°C until use

The day before start of experiment

Prepare an overnight culture of L. sobrius

Remove stomach content from freezer

On the day of the experiment

Add 5 ml R to the bottles with 85 ml broth

Solution A 167 ml

Solution B 167 ml

Hemin 5 ml

Elga water Add up to 1 L in total

Yeast extract 2.5 g

Peptone from casein (Merck 1.07213) 2.5 g

Solution A 167 ml

Solution B 167 ml

Resazurin 1 ml

Hemin 5 ml

Tween 80 1 ml

Elga water Add up to 1 L in total

Dilute 500 ml content in 500 ml sterile anaerobic salt media (1:1) and stomach the suspension. To

ease handling, each suspension is prepared in three successive steps, due to limitations of the

stomacher

Add 1000 ml of suspension to each bioreactor

Wait until the temperature has reached 37°C.

Add glyphosate IPA salt, to reach the desired concentrations (Tabel 4), in each bioreactor

Add 10 ml of L. sobrius overnight culture to each bioreactor

Suspension is stirred and kept under anoxic conditions by flushing with N2 gas in the headspace.

pH is maintained, using a pH-controller, regulated by 1M NaOH and 5M HCl.

Incubation temperature is kept at 37°C, by a circulating water bath.

Remove 12 ml samples from each bioreactors at 0, 2, 4 and 24 hours

Of the 12 ml, transfer 1 ml to SCFA tubes, for VFA determination and store at -18°C

Of the 12 ml, transfer 10 ml to sterile 125 ml serum bottles, with rubber stoppers, containing 85 ml

broth and 5 ml R. Pour each suspension from the bottles into a plastic bag and homogenize in a

stomacher for 2 minutes.

Perform the Drop Plate Procedure (Appendix 1)

Tabel 4. Preparation of glyphosate IPA salt concentrations

Bioreactor Glyphosate acid concentration (mg/ml) Glyphosate IPA salt (ml)

1 0 0

2 0.1 0.3375

3 1 3.375

4 10 33.75

Appendix 5

Statistical models

Required packages library(lme4)

library(lsmeans)

library(pbkrtest)

Example – Plates (B.adolescentis) adol=read.csv("C:/Users/Charlotte/Desktop/Speciale/Results/B. adolescentis .csv", header=T)

usedat1 <- reshape(adol,varying=names(adol)[3:ncol(adol)],

v.names = "resp",timevar="treat",

times=names(adol)[3:ncol(adol)],

direction = "long")

usedat1 <- usedat1[!is.na(usedat1$resp),]

adol.lmer <- lmer(resp ~ conc * treat + (1|plate),data=usedat1)

tmp <- lsmeans(adol.lmer,list(pairwise~conc:treat))

tmp[1]

Example - Cecum (representative for pigs)

pH ph = read.csv("C:/Users/Charlotte/Desktop/Speciale/Results/Statistics/Cecum/pH_all.csv",header=T)

ph$individ <- as.factor(ph$individ)

ph$timegrp <- as.factor(ph$time)

treatments <- unique(ph$treatment)

concentrations <- unique(ph$conc)

ph.lmer <- lmer(pH~1+treatment*conc+timegrp+(1|individ),data=ph)

ph.lsm <- lsmeans(ph.lmer,~conc|treatment)

ph.lsm

VFA all vfa = read.csv("C:/Users/Charlotte/Desktop/Speciale/Results/Statistics/Cecum/VFA_all.csv",header=T)

vfa$individ <- as.factor(vfa$individ)

vfa$timegrp <- as.factor(vfa$time)

treatments <- unique(vfa$treatment)

concentrations <- unique(vfa$conc)

all.lmer <- lmer(all~1+treatment*conc+timegrp+(1|individ),data=vfa)

all.lsm <- lsmeans(all.lmer,~conc|treatment)

all.lsm

Example - Rumen

pH ph = read.csv("C:/Users/Charlotte/Desktop/Speciale/Results/Rumen/pH_all.csv",header=T)

ph$day <- as.factor(ph$day)

ph$cow <- as.factor(ph$cow)

ph$timegrp <- as.factor(ph$time)

treatments <- unique(ph$treatment)

concentrations <- unique(ph$conc)

ph.lmer <- lmer(pH~1+treatment*conc+timegrp+(1|day)+(1|cow),data=ph)

ph.lsm <- lsmeans(ph.lmer,~conc|treatment)

ph.lsm

VFA vfa = read.csv("C:/Users/Charlotte/Desktop/Speciale/Results/Cow

trials/Statistics/Rumen/VFA_all.csv",header=T)

vfa$day <- as.factor(vfa$day)

vfa$cow <- as.factor(vfa$cow)

vfa$timegrp <- as.factor(vfa$time)

all.lmer <- lmer(all~1+treatment*conc+timegrp+(1|day)+(1|cow),data=vfa)

all.lsm <- lsmeans(all.lmer,~conc|treatment)

all.lsm

Appendix 6 Growth of commensal bacteria

Appendix 7 Growth of potential pathogenic bacteria

Appendix 8 Stomach

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Appendix 9 Cecum

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Appendix 10 Colon

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Appendix 11 Rumen

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Appendix 12 Bioreactors