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Glyphosate affects the spontaneous motoric activity of intestine at very low doses – In vitro study
Pesticide Biochemistry and Physiology 113 (2014) 25–30
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Glyphosate affects the spontaneous motoric activity of intestine at very low doses – In vitro study Magdalena Chłopecka, Marta Mendel ⇑, Natalia Dziekan, Wojciech Karlik Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Department of Preclinical Sciences, Division of Pharmacology and Toxicology, 8 Ciszewskiego St., 02-786 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 15 January 2014 Accepted 17 June 2014 Available online 24 June 2014 Keywords: Glyphosate Jejunum strips Motoric activity Biphasic reaction
a b s t r a c t Glyphosate is an active substance of the most popular herbicides worldwide. Its common use results from the belief that it affects exclusively plants. However, studies on glyphosate and its trade formulations reveal that it causes numerous morphological, physiological and biochemical disturbances in cells and organisms of animals, including mammals. Due to the fact that shortly after oral exposure glyphosate is detected in the highest amount in small intestine, the aim of this study was to evaluate the effect of this compound on the spontaneous motoric activity of intestine under in vitro conditions. The experiments were conducted on rat jejunum strips under isotonic conditions. The strips were incubated in buffered (pH 7.35) and non-buffered (pH 5.2) glyphosate solutions ranged from 0.003 to 1.7 g/L. The results indicate that glyphosate applied in buffered solution affects significantly the spontaneous motoric activity of rat isolated jejunum strips. The muscle response is biphasic (miorelaxation accompanied by contraction). The contraction is observed already at a dose of 0.003 g/L and the first significant biphasic reaction at a dose of 0.014 g/L. The incubation of jejunum strips with glyphosate in non-buffered solution (pH 5.2) results in a different reaction. The smooth muscle undergoes only persistent relaxation, which is stronger than the response to glyphosate solution in pH 7.35. Motility disturbances are also observed after glyphosate removal from the incubation solution. The gathered data suggests that glyphosate impairs gastrointestinal strips’ motility at concentration that are noticed in human exposed to non-toxic doses of glyphosate. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Glyphosate (N-phosphonomethyl-glycine) is an active substance in today’s most used herbicides for both professional and non-professional operators. The common usage of these products worldwide results from the belief that glyphosate-based pesticides are very safe as well for human as for animals. The main mechanism of phytotoxicity of glyphosate-containing herbicides includes the inhibition of aromatic amino acids metabolism what makes plant protein biosynthesis impossible. Glyphosate is an inhibitor of one of the fundamental enzymes involved in shikimate acid pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is present only in plants, fungi and bacteria [1]. The fact that glyphosate affects the metabolic process which is absent in animals became a reason for employing this active substance in the production of an effective and theoretically safe herbicide. The safety of glyphosate for animals was confirmed in numerous ⇑ Corresponding author. Fax: +48 225936065. E-mail address: [email protected] (M. Mendel). http://dx.doi.org/10.1016/j.pestbp.2014.06.005 0048-3575/Ó 2014 Elsevier Inc. All rights reserved.
studies, including acute toxicity experiments (in rats acute oral dose exceeds 5 g/kg b.w.) and long-term studies [1–3]. However, some recently published data indicates clearly that the belief in the safety of glyphosate and its trade formulations should be reconsidered and possibly verified. Numerous epidemiological data (referring to human and animals poisonings) points out the possibility of toxic, systemic effects of glyphosate products [2–6]. According to various experiments, glyphosate-based herbicides may interfere with different animal structures and biochemical pathways. For instance, glyphosate-containing products inhibit the activity of several enzymes: acetylcholinesterase in fish brain [7,8], muscle muscles [9] and in human erythrocytes [10]; butyrylcholinesterase, carboxylesterase and glutathione S-transferase in amphibian tadpoles tissues [11]; superoxide dismutase, glutathione peroxidase and glutathione S-transferase in fish hepatocytes [7]. Moreover, metabolism disturbances in hepatocytes [4,12] and enterocytes [4,13], as well as oxidative status imbalance leading to excessive membrane lipids’ peroxidation, cells and tissues damage are observed in animals exposed to glyphosate-based
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herbicides [12,14]. It was proved that glyphosate-containing products might be genotoxic in reptiles and mammals [15,16]. Studies on cells’ cultures and cell lines indicate the possibility of cancer development due to estrogenic receptors stimulation [17], and cell cycle regulation disturbances in sea urchin embryo model [18]. Toxic effects on embryo and placenta are demonstrated on human choriocarcinoma-derived placenta cell line/JEG3/ [19]. It is worth noting that the effects of glyphosate on non-target organisms were observed after application of glyphosate at large range of doses including concentrations of glyphosate used in the agriculture procedures [7–9,11,14,15,19–21], concentrations detected in blood samples collected from human showing no clinical signs of glyphosate toxicity [17,22–25], and concentrations at which only a limited risk for human health or environment was estimated [10,26]. The results of pharmacokinetic studies of Anadón et al. [27] and Brewster et al. [28] reveal that the bioavailability of glyphosate in rats amounts to about 23% and 36%, respectively. The majority of the administered dose is found in small intestine tissue but it is not associated with the intestinal contents and mucous layers. Small intestine is the tissue where the highest amount of glyphosate is detected (34% of the administered dose) and where glyphosate remains in the significant concentration for 7 days (above 1%) [28]. Taking into account the results of pharmacokinetic studies, the information that glyphosate exhibits different activities in wide range of doses and the fact that there is no available experimental data concerning the effect of glyphosate on the motoric activity of isolated gastrointestinal strips, the aim of this study was to verify the effect of glyphosate on the spontaneous motoric activity of rat intestine smooth muscle. 2. Materials and methods 2.1. Chemicals and media Acetylcholine chloride (ACh), isoproterenol hemisulfate (Isop), glyphosate (N-phosphonomethyl-glycine) (Sigma Chemicals Co, St. Louis, USA), CaCl2 (Merck, Darmstadt, Germany), NaH2PO4 (Fluka Chemie, AG, Buchs, Switzerland), NaCl, KCl, MgSO4, NaHCO3 and glucose (POCh, Gliwice, Poland) were used for preparing experiments. Modified Krebs–Henseleit solution (MK–HS) containing: NaCl (123.76 mM), KCl (5 mM), CaCl2 (2.5 mM), MgSO4 (1.156 mM), NaHCO3 (14.5 mM), KH2PO4 (2.75 mM) and glucose (12.5 mM) was used as incubation medium. The incubation medium maintained a pH value of 7.35 (7.30–7.40) throughout longterm experiments while heated up to 37 °C and bubbled with carbogen (95% O2 + 5% CO2). 2.2. Animals The experiments were carried out on intestinal strips isolated from male Wistar rats (weighting approx. 250 g). The use of animals, all procedures involving animals and their tissues were approved by the Local Ethics Committee (approval number 8/ 2011). After adaptation period (about 7 days), during which rats had free access to feed and water, the animals were euthanized in chambers filled with carbon dioxide (CO2) [29]. 2.3. Preparation of jejunum strips and registration of strips activity Immediately after rat’s euthanasia the abdominal cavity was opened and fragments of jejunum were incised. Intestine strips were placed in warm (37 °C) MK–HS and prepared as described somewhere else [30]. All jejunum stripes were incubated in
MK–HS in the chambers of Schuler Organ Bath set (Hugo Sachs Elektronic, Harvard Apparatus, USA). The experiments were carried out in isotonic conditions, under a load of 0.5 g. The registration of the data was performed through PowerLab (ADInstruments, Australia) and bridge amplifier (DBA, type 660, Hugo Sachs Elektronic, Harvard Apparatus, USA). Subsequently, the graphical records were analyzed by Chart v7.0 program and Excel (MS Office 2010). 2.4. Design of experiments Each experiment started with 60-min preincubation supplemented with 3 incubation medium exchanges, every 20 min. Subsequently, the strips were exposed to both reference substances, ACh and Isop, applied to the incubation medium in the reference concentrations of 1 lM and 0.1 lM, respectively [30]. The response of strips to the reference substances was registered and MK–HS was exchanged. Once the spontaneous motoric activity of jejunum strips stabilized after flushing with MK–HS, the incubation chambers were filled with glyphosate solution in MK–HS at concentrations amounted to 0.003, 0.014, 0.068, 0.34, and 1.7 g/L. As soon as glyphosate was dissolved, the pH of the MK–HS lowered, reaching the value of 5.2 at the concentration of 1.7 g/L. Thus, all glyphosate solutions were buffered with 0.1 M sodium hydroxide to obtain a pH value of 7.35. Additionally, non-buffered solutions of glyphosate and pure MK–HS of lowered pH value (5.2) were used in separate experiments. Irrespective of the treatment, jejunum strips were incubated in selected solutions for 20 min and then flushed with fresh MK–HS (pH 7.35). In case of experiments aimed at evaluating the effect of glyphosate on the jejunal motoric activity in pH 7.35, each solution containing tested substance at particular concentration was administered twice, in a non-cumulative manner (the strips were flushed with fresh MK–HS between individual treatments). At the end of each experiment, after stabilization of the spontaneous motoric activity, all preparations were re-exposed to ACh in the reference concentration. 2.5. Data analysis and statistics All data is expressed as the percent of the reaction caused by reference substances. The contraction and relaxation provoked by ACh and Isop, respectively, in the reference doses is defined as 100% (positive control). The negative control is expressed as contractile and relaxant response of jejunum strips to the filling of incubation chambers with pure MK–HS (pH 7.35). Results are expressed as mean values (±SD) from 6–7 separate experiments. The reaction of rat isolated jejunum strip to the tested compound is considered as significant, if its strength differs statistically from the force of the reaction to the negative control treatment. In the statistical analysis a one-way analysis of variance (ANOVA) with post hoc LSD Fisher test was used. Whenever it was reasonable t-Student test was employed. Values of p 6 0.05 are considered to be significant. Data was analyzed using Statistica 10 (Stat Soft., Inc.). 3. Results Irrespective of the employed concentration of the tested substance, the incubation of jejunum strips in buffered solutions containing glyphosate (pH 7.35) resulted always in significant disturbations of the spontaneous motoric activity. The observed reaction was transitory and usually biphasic; miorelaxation as well as contraction of smooth muscle were registered (Fig. 2A). It is noteworthy that statistically significant relaxant and contractile response occurred only after application of glyphosate solutions
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relaxation vs. Isop 0.1 uM (%)
120
*
100
*
80
B 140 contraction vs. ACh 1 uM (%)
*
A 140
60 40 20 0
120 100 80
*
60
*
40 20
*
*
*
0.068
0.34
1.7
0 0
0.003
0.014
0.068
0.34
1.7
glyphosate (g/L)
0
0.003
0.014
glyphosate (g/L)
Fig. 1. Effect of glyphosate solutions (pH 7.35) on the spontaneous motoric activity of isolated jejunum strips. Data is expressed as a mean of independent experiments (n = 6–7, ±SD). The strength of the reaction is expressed as a percentage of muscle strips response to isoproterenol (A) or acetylcholine (B) application in the reference concentration. The response of muscle strips to flushing with MK–HS without glyphosate was measured and expressed as control (without glyphosate).
A GLYPHOSATE 0.014 g/L, pH 7.35
F 2 mm
5 min
B MK-HS pH 5.2
GLYPHOSATE 1.7 g/L, pH 5.2
2 mm
5 min
Fig. 2. Representative sample recordings of isolated jejunum strip’s activity: (A) reaction to the application of glyphosate (0.014 g/L, pH 7.35); (B) reaction to the application of MK–HS pH 5.2 and non-buffered glyphosate solution (1.7 g/L, pH 5.2). MK–HS – modified Krebs–Henseleit solution F – flushing.
at doses which accounted to 0.014, 0.34 and 1.7 g/L (Fig. 1). In case of other concentrations of glyphosate, i.e., 0.068 and 0.003 g/L, a tendency of biphasic reaction was observed and only the force of contractile component of the response was significantly higher than the control treatment (Fig. 1). The observed changes of motoric activity, particularly the relaxant part, were not dose-dependent. The relaxation was significant and amounted to 60.6 ± 18.9%, 61.3 ± 28.0% and 100.2 ± 33.8% of the reaction induced by Isop after application of solutions containing 0.014, 0.34 and 1.7 g/L of glyphosate, respectively. In contrary, the strongest contraction was registered after application of the lowest dose of glyphosate, 0.003 g/L, and it accounted to approx. 30% of the reaction evoked by ACh (Fig. 1B). It is remarkable that the removal of glyphosate from the incubation medium (flushing with fresh, pure MK–HS) resulted always in a significant contraction of jejunum strips. This contractile response ranged from 18.9 ± 7% to 34.5 ± 14.3% of ACh-induced contraction, in case of removing
solutions containing glyphosate in the concentration of 0.068 and 0.014 g/L, respectively. The second (non-cumulative) application of glyphosate solution showed a tendency of enhancement of the contractile response and it was statistically significant in medium containing 0.014 g/L of glyphosate, and amounted to 51.8 ± 19.2% of the reaction induced by ACh (Fig. 3). The incubation of jejunum preparations in non-buffered glyphosate solution (1.7 g/L, pH 5.2) resulted in a very strong and permanent miorelaxation (Table 1, Fig. 2B). The incubation of intestine strips in pure MK–HS of lowered pH (pH 5.2) caused a relaxant response of a similar force to the one induced by non-buffered glyphosate solution, but the observed reaction was temporary, the strips recovered spontaneously during the incubation (about 20 min) and the basic motoric activity returned (Fig. 2B). The comparison of the force of miorelaxation response to glyphosate application in buffered and non-buffered solution containing the same
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140
140
120
120
contraction vs. ACh 1 uM (%)
relaxation vs. Isop 0.1 uM (%)
28
100 80 60 40 20 0
No 1
No 2 treatm ent
100 80 60 40 20 0
No 1
No 2 treatm ent
Fig. 3. Effect of glyphosate (0.014 g/L, pH 7.35), on the spontaneous motoric activity of isolated jejunum strips in repeated administration (non-cumulative manner). Data is expressed as a mean of independent experiments (n = 6–7, ±SD). The strength of the reaction is expressed as a percentage of muscle strips response to isoproterenol or acetylcholine application in the reference concentration ⁄p 6 0.05 vs. treatment 1.
Table 1 Effect of glyphosate (1.7 g/L in buffered and non-buffered solutions) and acidulated MK–HS (pH 5.2) on the spontaneous motoric activity of isolated jejunum strips. MK-HS pH 5.2
Relaxation vs. Isop 0.1 lM (%) Effect observed after 20 min incubation
256 ± 93.4 Transient
Solution of glyphosate 1.7 g/L Non-buffered (pH 5.2)
Buffered (pH 7.3)
261.8 ± 100.8 Constant
100.2 ± 33.7* Transient
Data is expressed as a mean of independent experiments (n = 6–7, ±SD). The strength of the reaction is expressed as a percentage of the muscle strips response to isoproterenol in the reference concentration p 6 0,05 vs. non-buffered glyphosate (pH 5.2).
dose of the active ingredient (1.7 g/L) revealed that the reaction strength was significantly higher in case of the latter treatment (Table 1).
4. Discussion Numerous biological effects observed in vitro refer to herbicides at concentrations that are used for agriculture procedures (and lower), concentrations measured in the environment and human organism. The results presented in this paper are in agreement with these observations. One should bear in mind that disturbances of the spontaneous motoric activity (smooth muscle contractions, Fig. 1B) were observed in this study when jejunum strips were incubated in the presence of glyphosate at concentration of 0.003 g/L. It means at a dose which is lower than concentrations measured in human without clinical signs of intoxication and approx. 650 times lower than the minimal concentration of glyphosate in field dilutions of pesticides used for agrochemical procedures. The first significant biphasic reaction (miorelaxation and contraction) was registered if intestine preparation were exposed to glyphosate at a dose of 0.014 g/L. It is remarkable that effective glyphosate concentrations presented herein are significantly lower than doses of this substance required to affect mammalian cells [10,19]. These discrepancies might suggest that tissue and organ models are usually more useful and reliable for estimation of toxic effects than cell models. The effective doses of glyphosate are similar to the concentrations of this substance found in different environmental elements on areas that undergo regular agrochemical procedures with glyphosate-based herbicides. The concentration of glyphosate may vary significantly dependent on the water bin character and ranges from 0.0005 to 1.7 mg/L and 0.0057 to
19 mg/L in the water and sediments, respectively [31,32]. The highest concentrations of glyphosate are characteristic for small water bins like ponds [21]. The most notable observation is the one that the results obtained herein refer to glyphosate concentrations measured in blood collected from people showing no clinical signs of glyphosate toxicity. The concentration of glyphosate detected in general population and in poisoned individuals with minor clinical outcomes amounts to approx. 0.074 and 40.8 mg/L, respectively [22,23]. Those ‘‘non-toxic’’ concentrations correspond to two lowest doses (0.003 and 0.014 g/L) which caused significant disturbances of intestine smooth muscle activity in vitro. It suggests that glyphosate may induce some subclinical motility alterations in human which are not recognized as consequences of the exposure to this herbicide. It is noteworthy that even slight motility alterations might cause serious health problems [33,34]. The risk of repetitive and chronic exposure to small quantities of glyphosate present in food and water due to the common use of glyphosate-containing products is very probable and significant. The hazard of health disturbances is even greater in case of farmers and professional pesticide operators and their families. Because of the high risk of multiple exposure to herbicides, it is worth to analyze the effect of glyphosate on jejunum smooth muscle after second (non-cumulative) application at the same dose. A double application of glyphosate in each experiment was conducted to verify the repeatability of the complex (biphasic) smooth muscle reaction. Unexpectedly, in case of some doses of glyphosate the second incubation of strips in the presence of the active substance differed from the first treatment (Fig. 3). The observed changes of smooth muscle response might suggest that multiple exposure at short time could cause progressive motility disorders. Although glyphosate probably does not accumulate in animal organisms
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[3,28], the repetitive exposure over several days might result in increased toxicity. This phenomenon is well recognized in other pesticides (e.g., 2,4 D, urea-derivatives) [35–37]. One should not forget that even slight recurrent disturbances of gastrointestinal motoric activity might affect absorption and secretion processes and the speed of gastrointestinal passage [33,34]. There is enormous amount of gastrointestinal disorders that occur without clear pathomorphological manifestations and which causes are not defined. It cannot be ruled out that simultaneous occurrence of several causes, including stress and exposure to substances in diet that affect smooth muscle functionality (e.g., nitrite), contributes to those disorders. The repetitive presence of glyphosate in low doses in intestine cells might play a crucial role in recurrent intestinal dysmotility. Besides, the interactions between glyphosate and other environmental contaminants or their metabolites as a result of concurrent exposure cannot be excluded. It was confirmed in in vivo studies on rats performed by Mariana et al. [38] who revealed that the toxic effect, involving oxidative stress mechanisms, was higher in case of the exposure to a mixture of agrochemicals (dimethoate, zineb and glyphosate) than after exposure to a single substance. The disturbances of intestine motoric activity that are not dosedependent and the biphasic character of strips’ response might suggest a complex mechanism of action of glyphosate on smooth muscle cells. The observed inconstancy of glyphosate effect is convergent with data gained in other experimental models. For instance, in human erythrocytes glyphosate at low concentration causes elevation of plasma membrane ATPase (PMCA) activity, whereas high doses of the same substance induces inhibition of enzyme activity [39]. Changes of PMCA activity in erythrocytes lead to calcium ions imbalance. This observation allows to hypothesize that a similar mechanism involving calcium ions movements comprises alterations of smooth muscle activity by glyphosate but this speculation requires further in-depth studies. Another aspect of glyphosate effect on smooth muscle, which might contribute to motility disturbances, is a very clear, but transient, smooth muscle contraction observed after the compound is removed from the incubation chambers (Fig. 2A). Possibly, due to elimination and diminishing concentration of glyphosate under in vitro conditions, some additional mechanism, which was previously inhibited by the presence of the compound in blood and tissues, became activated and resulted in a contraction of the gut smooth muscle. It is well known that the biological activity of glyphosate, is higher in lower pH. This may suggest that the character of reaction contributes to glyphosate’s mechanism of action. The results presented herein indicate that glyphosate significantly affects gastrointestinal smooth muscle (strong miorelaxation and significant contraction) even if it is used in buffered solutions which pH value amounts to 7.35. It allows to imply that after oral absorption, under well buffered in vivo conditions, glyphosate remains active. However, the obtained results clearly point out that the intensity of glyphosate activity is pH dependent and becomes stronger under acidic conditions. The incubation of jejunum strips in nonbuffered (acidic) glyphosate solution caused relaxant response that was more than twice greater than the response to the application of glyphosate at the same dose but used in buffered physiological pH (Table 1). Moreover, the reaction evoked by preparations’ incubation in acidic glyphosate solution consisted only of the persistent relaxant component whereas the response to buffered glyphosate solution as well as to acidified MK–HS (without the active substance) was always temporary and reversible by itself within 20 min of incubation. As mentioned before, the response to glyphosate in buffered solution evoked biphasic reaction, the relaxant component was accompanied by remarkable contraction. The increase of toxic effect of glyphosate in acidic conditions, pH 5.2
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(Table 1), can be explained by ionization state changes of the compound which binds an extra proton. It reduces the net charge of the molecule and, thus, glyphosate becomes more lipophilic [40,41]. It improves glyphosate’s penetration through membranes and the entry to cells and tissues. Inside the cells glyphosate converts probably into a bivalent ion due to the higher pH of cytoplasm. If the solution’s pH accounts to 7.3–7.4, physiological blood pH, the lipophilicity of glyphosate decreases and only a part of particles (the fraction of lower ion charge) penetrates into cells. Then those particles lose their charge due to lower pH (weak acid character of intracellular space) and molecules of lower net charge return into extracellular space. The proposed mechanism might explain the diminished and labile response of jejunum smooth muscle to glyphosate treatment when it is used in buffered solution. The acidification of glyphosate-based herbicides is a common procedure used to abolish glyphosate chelation with bivalent ions in aqueous solutions [42] in order to preserve their biological activity. Paradoxically, the mechanism involving bivalent ions chelation, including calcium ions, should be considered as one of the possible modes of action contributing to the observed disturbances of jejunum strips activity. In conclusion, glyphosate affects the spontaneous motoric activity of rat isolated jejunum strips, even if applied at very low concentrations. The effects are observed during the incubation of preparations in solutions of physiologic pH value and are biphasic (miorelaxant and contractile response). Upcoming studies should be focused on the estimation of the detailed mechanism of action of glyphosate on intestine smooth muscle and should definitely include manipulation of calcium ions concentration. The effect of commercial products containing glyphosate on gastrointestinal motoric activity should be evaluated. Acknowledgment The study was supported by a research Grant from the National Science Centre Poland No. UMO-2011/01/B/NZ7/01497. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pestbp.2014 06.005. References [1] M.G. Dill, R.D. Sammons, P.C.C. Feng, F. Kohn, K. Kretzmer, A. Mehrsheikh, M. Bleeke, J.L. Honedder, D. Farmer, D. Wright, E.A. Haupfear, Glyphosate: discovery, development, applications, and properties, in: V.K. Nandula (Ed.), Glyphosate Resistance in Crops and Leeds: History, Development, and Management, John Wiley and Sons Inc, Hoboken, 2010, pp. 1–33. [2] V. Burgat, G. Keck, P. Guerre, V. Bigorre, X. Pineau, Glyphosate toxicosis in domestic animals: a survey from the data of the Centre National d’Informations Toxicologiques Veterinaires (CNITV), Vet. Hum. Toxicol. 40 (1998) 363–367. [3] G.M. Williams, R. Kroes, I.C. Munro, Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans, Regul. Toxicol. Pharmacol. 31 (2000) 117–165, http://dx.doi.org/10.1006/ rtph.1999.1371. [4] S.M. Bradberry, A.T. Proudfoot, J.A. Vale, Glyphosate poisoning, Toxicol. Rev. 23 (2004) 159–167, http://dx.doi.org/10.2165/00139709-200423030-00003. [5] A.R. Talbot, M.H. Shiaw, J.S. Huang, S.F. Yang, T.S. Goo, S.H. Wasng, C.L. Chien, T.R. Sanford, Acute poisoning with a glyphosate-surfactant herbicide (Roundup): a review of 93 cases, Hum. Exp. Toxicol. 10 (1991) 1–8, http://dx.doi.org/ 10.1177/096032719101000101. [6] P. Bernya, F. Calonib, S. Croubelsc, M. Sachanad, V. Vandenbrouckec, F. Davanzo, R. Guitart, Animal poisoning in Europe. Part 2: Companion animals, Vet. J 183 (2010) 255–259, http://dx.doi.org/10.1016/j.tvjl.2009.03.034. [7] K.A. Modesto, C.B.R. Martinez, Effects of Roundup Transorb on fish: Hematology, antioxidant defenses and acetylcholinesterase activity, Chemosphere 81 (2010) 781–787, http://dx.doi.org/10.1016/ j.chemosphere.2010.07.005.
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Glyphosate affects the spontaneous motoric activity of intestine at very low doses – In vitro study Magdalena Chłopecka, Marta Mendel ⇑, Natalia Dziekan, Wojciech Karlik Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Department of Preclinical Sciences, Division of Pharmacology and Toxicology, 8 Ciszewskiego St., 02-786 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 15 January 2014 Accepted 17 June 2014 Available online 24 June 2014 Keywords: Glyphosate Jejunum strips Motoric activity Biphasic reaction
a b s t r a c t Glyphosate is an active substance of the most popular herbicides worldwide. Its common use results from the belief that it affects exclusively plants. However, studies on glyphosate and its trade formulations reveal that it causes numerous morphological, physiological and biochemical disturbances in cells and organisms of animals, including mammals. Due to the fact that shortly after oral exposure glyphosate is detected in the highest amount in small intestine, the aim of this study was to evaluate the effect of this compound on the spontaneous motoric activity of intestine under in vitro conditions. The experiments were conducted on rat jejunum strips under isotonic conditions. The strips were incubated in buffered (pH 7.35) and non-buffered (pH 5.2) glyphosate solutions ranged from 0.003 to 1.7 g/L. The results indicate that glyphosate applied in buffered solution affects significantly the spontaneous motoric activity of rat isolated jejunum strips. The muscle response is biphasic (miorelaxation accompanied by contraction). The contraction is observed already at a dose of 0.003 g/L and the first significant biphasic reaction at a dose of 0.014 g/L. The incubation of jejunum strips with glyphosate in non-buffered solution (pH 5.2) results in a different reaction. The smooth muscle undergoes only persistent relaxation, which is stronger than the response to glyphosate solution in pH 7.35. Motility disturbances are also observed after glyphosate removal from the incubation solution. The gathered data suggests that glyphosate impairs gastrointestinal strips’ motility at concentration that are noticed in human exposed to non-toxic doses of glyphosate. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Glyphosate (N-phosphonomethyl-glycine) is an active substance in today’s most used herbicides for both professional and non-professional operators. The common usage of these products worldwide results from the belief that glyphosate-based pesticides are very safe as well for human as for animals. The main mechanism of phytotoxicity of glyphosate-containing herbicides includes the inhibition of aromatic amino acids metabolism what makes plant protein biosynthesis impossible. Glyphosate is an inhibitor of one of the fundamental enzymes involved in shikimate acid pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is present only in plants, fungi and bacteria [1]. The fact that glyphosate affects the metabolic process which is absent in animals became a reason for employing this active substance in the production of an effective and theoretically safe herbicide. The safety of glyphosate for animals was confirmed in numerous ⇑ Corresponding author. Fax: +48 225936065. E-mail address: [email protected] (M. Mendel). http://dx.doi.org/10.1016/j.pestbp.2014.06.005 0048-3575/Ó 2014 Elsevier Inc. All rights reserved.
studies, including acute toxicity experiments (in rats acute oral dose exceeds 5 g/kg b.w.) and long-term studies [1–3]. However, some recently published data indicates clearly that the belief in the safety of glyphosate and its trade formulations should be reconsidered and possibly verified. Numerous epidemiological data (referring to human and animals poisonings) points out the possibility of toxic, systemic effects of glyphosate products [2–6]. According to various experiments, glyphosate-based herbicides may interfere with different animal structures and biochemical pathways. For instance, glyphosate-containing products inhibit the activity of several enzymes: acetylcholinesterase in fish brain [7,8], muscle muscles [9] and in human erythrocytes [10]; butyrylcholinesterase, carboxylesterase and glutathione S-transferase in amphibian tadpoles tissues [11]; superoxide dismutase, glutathione peroxidase and glutathione S-transferase in fish hepatocytes [7]. Moreover, metabolism disturbances in hepatocytes [4,12] and enterocytes [4,13], as well as oxidative status imbalance leading to excessive membrane lipids’ peroxidation, cells and tissues damage are observed in animals exposed to glyphosate-based
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herbicides [12,14]. It was proved that glyphosate-containing products might be genotoxic in reptiles and mammals [15,16]. Studies on cells’ cultures and cell lines indicate the possibility of cancer development due to estrogenic receptors stimulation [17], and cell cycle regulation disturbances in sea urchin embryo model [18]. Toxic effects on embryo and placenta are demonstrated on human choriocarcinoma-derived placenta cell line/JEG3/ [19]. It is worth noting that the effects of glyphosate on non-target organisms were observed after application of glyphosate at large range of doses including concentrations of glyphosate used in the agriculture procedures [7–9,11,14,15,19–21], concentrations detected in blood samples collected from human showing no clinical signs of glyphosate toxicity [17,22–25], and concentrations at which only a limited risk for human health or environment was estimated [10,26]. The results of pharmacokinetic studies of Anadón et al. [27] and Brewster et al. [28] reveal that the bioavailability of glyphosate in rats amounts to about 23% and 36%, respectively. The majority of the administered dose is found in small intestine tissue but it is not associated with the intestinal contents and mucous layers. Small intestine is the tissue where the highest amount of glyphosate is detected (34% of the administered dose) and where glyphosate remains in the significant concentration for 7 days (above 1%) [28]. Taking into account the results of pharmacokinetic studies, the information that glyphosate exhibits different activities in wide range of doses and the fact that there is no available experimental data concerning the effect of glyphosate on the motoric activity of isolated gastrointestinal strips, the aim of this study was to verify the effect of glyphosate on the spontaneous motoric activity of rat intestine smooth muscle. 2. Materials and methods 2.1. Chemicals and media Acetylcholine chloride (ACh), isoproterenol hemisulfate (Isop), glyphosate (N-phosphonomethyl-glycine) (Sigma Chemicals Co, St. Louis, USA), CaCl2 (Merck, Darmstadt, Germany), NaH2PO4 (Fluka Chemie, AG, Buchs, Switzerland), NaCl, KCl, MgSO4, NaHCO3 and glucose (POCh, Gliwice, Poland) were used for preparing experiments. Modified Krebs–Henseleit solution (MK–HS) containing: NaCl (123.76 mM), KCl (5 mM), CaCl2 (2.5 mM), MgSO4 (1.156 mM), NaHCO3 (14.5 mM), KH2PO4 (2.75 mM) and glucose (12.5 mM) was used as incubation medium. The incubation medium maintained a pH value of 7.35 (7.30–7.40) throughout longterm experiments while heated up to 37 °C and bubbled with carbogen (95% O2 + 5% CO2). 2.2. Animals The experiments were carried out on intestinal strips isolated from male Wistar rats (weighting approx. 250 g). The use of animals, all procedures involving animals and their tissues were approved by the Local Ethics Committee (approval number 8/ 2011). After adaptation period (about 7 days), during which rats had free access to feed and water, the animals were euthanized in chambers filled with carbon dioxide (CO2) [29]. 2.3. Preparation of jejunum strips and registration of strips activity Immediately after rat’s euthanasia the abdominal cavity was opened and fragments of jejunum were incised. Intestine strips were placed in warm (37 °C) MK–HS and prepared as described somewhere else [30]. All jejunum stripes were incubated in
MK–HS in the chambers of Schuler Organ Bath set (Hugo Sachs Elektronic, Harvard Apparatus, USA). The experiments were carried out in isotonic conditions, under a load of 0.5 g. The registration of the data was performed through PowerLab (ADInstruments, Australia) and bridge amplifier (DBA, type 660, Hugo Sachs Elektronic, Harvard Apparatus, USA). Subsequently, the graphical records were analyzed by Chart v7.0 program and Excel (MS Office 2010). 2.4. Design of experiments Each experiment started with 60-min preincubation supplemented with 3 incubation medium exchanges, every 20 min. Subsequently, the strips were exposed to both reference substances, ACh and Isop, applied to the incubation medium in the reference concentrations of 1 lM and 0.1 lM, respectively [30]. The response of strips to the reference substances was registered and MK–HS was exchanged. Once the spontaneous motoric activity of jejunum strips stabilized after flushing with MK–HS, the incubation chambers were filled with glyphosate solution in MK–HS at concentrations amounted to 0.003, 0.014, 0.068, 0.34, and 1.7 g/L. As soon as glyphosate was dissolved, the pH of the MK–HS lowered, reaching the value of 5.2 at the concentration of 1.7 g/L. Thus, all glyphosate solutions were buffered with 0.1 M sodium hydroxide to obtain a pH value of 7.35. Additionally, non-buffered solutions of glyphosate and pure MK–HS of lowered pH value (5.2) were used in separate experiments. Irrespective of the treatment, jejunum strips were incubated in selected solutions for 20 min and then flushed with fresh MK–HS (pH 7.35). In case of experiments aimed at evaluating the effect of glyphosate on the jejunal motoric activity in pH 7.35, each solution containing tested substance at particular concentration was administered twice, in a non-cumulative manner (the strips were flushed with fresh MK–HS between individual treatments). At the end of each experiment, after stabilization of the spontaneous motoric activity, all preparations were re-exposed to ACh in the reference concentration. 2.5. Data analysis and statistics All data is expressed as the percent of the reaction caused by reference substances. The contraction and relaxation provoked by ACh and Isop, respectively, in the reference doses is defined as 100% (positive control). The negative control is expressed as contractile and relaxant response of jejunum strips to the filling of incubation chambers with pure MK–HS (pH 7.35). Results are expressed as mean values (±SD) from 6–7 separate experiments. The reaction of rat isolated jejunum strip to the tested compound is considered as significant, if its strength differs statistically from the force of the reaction to the negative control treatment. In the statistical analysis a one-way analysis of variance (ANOVA) with post hoc LSD Fisher test was used. Whenever it was reasonable t-Student test was employed. Values of p 6 0.05 are considered to be significant. Data was analyzed using Statistica 10 (Stat Soft., Inc.). 3. Results Irrespective of the employed concentration of the tested substance, the incubation of jejunum strips in buffered solutions containing glyphosate (pH 7.35) resulted always in significant disturbations of the spontaneous motoric activity. The observed reaction was transitory and usually biphasic; miorelaxation as well as contraction of smooth muscle were registered (Fig. 2A). It is noteworthy that statistically significant relaxant and contractile response occurred only after application of glyphosate solutions
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relaxation vs. Isop 0.1 uM (%)
120
*
100
*
80
B 140 contraction vs. ACh 1 uM (%)
*
A 140
60 40 20 0
120 100 80
*
60
*
40 20
*
*
*
0.068
0.34
1.7
0 0
0.003
0.014
0.068
0.34
1.7
glyphosate (g/L)
0
0.003
0.014
glyphosate (g/L)
Fig. 1. Effect of glyphosate solutions (pH 7.35) on the spontaneous motoric activity of isolated jejunum strips. Data is expressed as a mean of independent experiments (n = 6–7, ±SD). The strength of the reaction is expressed as a percentage of muscle strips response to isoproterenol (A) or acetylcholine (B) application in the reference concentration. The response of muscle strips to flushing with MK–HS without glyphosate was measured and expressed as control (without glyphosate).
A GLYPHOSATE 0.014 g/L, pH 7.35
F 2 mm
5 min
B MK-HS pH 5.2
GLYPHOSATE 1.7 g/L, pH 5.2
2 mm
5 min
Fig. 2. Representative sample recordings of isolated jejunum strip’s activity: (A) reaction to the application of glyphosate (0.014 g/L, pH 7.35); (B) reaction to the application of MK–HS pH 5.2 and non-buffered glyphosate solution (1.7 g/L, pH 5.2). MK–HS – modified Krebs–Henseleit solution F – flushing.
at doses which accounted to 0.014, 0.34 and 1.7 g/L (Fig. 1). In case of other concentrations of glyphosate, i.e., 0.068 and 0.003 g/L, a tendency of biphasic reaction was observed and only the force of contractile component of the response was significantly higher than the control treatment (Fig. 1). The observed changes of motoric activity, particularly the relaxant part, were not dose-dependent. The relaxation was significant and amounted to 60.6 ± 18.9%, 61.3 ± 28.0% and 100.2 ± 33.8% of the reaction induced by Isop after application of solutions containing 0.014, 0.34 and 1.7 g/L of glyphosate, respectively. In contrary, the strongest contraction was registered after application of the lowest dose of glyphosate, 0.003 g/L, and it accounted to approx. 30% of the reaction evoked by ACh (Fig. 1B). It is remarkable that the removal of glyphosate from the incubation medium (flushing with fresh, pure MK–HS) resulted always in a significant contraction of jejunum strips. This contractile response ranged from 18.9 ± 7% to 34.5 ± 14.3% of ACh-induced contraction, in case of removing
solutions containing glyphosate in the concentration of 0.068 and 0.014 g/L, respectively. The second (non-cumulative) application of glyphosate solution showed a tendency of enhancement of the contractile response and it was statistically significant in medium containing 0.014 g/L of glyphosate, and amounted to 51.8 ± 19.2% of the reaction induced by ACh (Fig. 3). The incubation of jejunum preparations in non-buffered glyphosate solution (1.7 g/L, pH 5.2) resulted in a very strong and permanent miorelaxation (Table 1, Fig. 2B). The incubation of intestine strips in pure MK–HS of lowered pH (pH 5.2) caused a relaxant response of a similar force to the one induced by non-buffered glyphosate solution, but the observed reaction was temporary, the strips recovered spontaneously during the incubation (about 20 min) and the basic motoric activity returned (Fig. 2B). The comparison of the force of miorelaxation response to glyphosate application in buffered and non-buffered solution containing the same
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140
140
120
120
contraction vs. ACh 1 uM (%)
relaxation vs. Isop 0.1 uM (%)
28
100 80 60 40 20 0
No 1
No 2 treatm ent
100 80 60 40 20 0
No 1
No 2 treatm ent
Fig. 3. Effect of glyphosate (0.014 g/L, pH 7.35), on the spontaneous motoric activity of isolated jejunum strips in repeated administration (non-cumulative manner). Data is expressed as a mean of independent experiments (n = 6–7, ±SD). The strength of the reaction is expressed as a percentage of muscle strips response to isoproterenol or acetylcholine application in the reference concentration ⁄p 6 0.05 vs. treatment 1.
Table 1 Effect of glyphosate (1.7 g/L in buffered and non-buffered solutions) and acidulated MK–HS (pH 5.2) on the spontaneous motoric activity of isolated jejunum strips. MK-HS pH 5.2
Relaxation vs. Isop 0.1 lM (%) Effect observed after 20 min incubation
256 ± 93.4 Transient
Solution of glyphosate 1.7 g/L Non-buffered (pH 5.2)
Buffered (pH 7.3)
261.8 ± 100.8 Constant
100.2 ± 33.7* Transient
Data is expressed as a mean of independent experiments (n = 6–7, ±SD). The strength of the reaction is expressed as a percentage of the muscle strips response to isoproterenol in the reference concentration p 6 0,05 vs. non-buffered glyphosate (pH 5.2).
dose of the active ingredient (1.7 g/L) revealed that the reaction strength was significantly higher in case of the latter treatment (Table 1).
4. Discussion Numerous biological effects observed in vitro refer to herbicides at concentrations that are used for agriculture procedures (and lower), concentrations measured in the environment and human organism. The results presented in this paper are in agreement with these observations. One should bear in mind that disturbances of the spontaneous motoric activity (smooth muscle contractions, Fig. 1B) were observed in this study when jejunum strips were incubated in the presence of glyphosate at concentration of 0.003 g/L. It means at a dose which is lower than concentrations measured in human without clinical signs of intoxication and approx. 650 times lower than the minimal concentration of glyphosate in field dilutions of pesticides used for agrochemical procedures. The first significant biphasic reaction (miorelaxation and contraction) was registered if intestine preparation were exposed to glyphosate at a dose of 0.014 g/L. It is remarkable that effective glyphosate concentrations presented herein are significantly lower than doses of this substance required to affect mammalian cells [10,19]. These discrepancies might suggest that tissue and organ models are usually more useful and reliable for estimation of toxic effects than cell models. The effective doses of glyphosate are similar to the concentrations of this substance found in different environmental elements on areas that undergo regular agrochemical procedures with glyphosate-based herbicides. The concentration of glyphosate may vary significantly dependent on the water bin character and ranges from 0.0005 to 1.7 mg/L and 0.0057 to
19 mg/L in the water and sediments, respectively [31,32]. The highest concentrations of glyphosate are characteristic for small water bins like ponds [21]. The most notable observation is the one that the results obtained herein refer to glyphosate concentrations measured in blood collected from people showing no clinical signs of glyphosate toxicity. The concentration of glyphosate detected in general population and in poisoned individuals with minor clinical outcomes amounts to approx. 0.074 and 40.8 mg/L, respectively [22,23]. Those ‘‘non-toxic’’ concentrations correspond to two lowest doses (0.003 and 0.014 g/L) which caused significant disturbances of intestine smooth muscle activity in vitro. It suggests that glyphosate may induce some subclinical motility alterations in human which are not recognized as consequences of the exposure to this herbicide. It is noteworthy that even slight motility alterations might cause serious health problems [33,34]. The risk of repetitive and chronic exposure to small quantities of glyphosate present in food and water due to the common use of glyphosate-containing products is very probable and significant. The hazard of health disturbances is even greater in case of farmers and professional pesticide operators and their families. Because of the high risk of multiple exposure to herbicides, it is worth to analyze the effect of glyphosate on jejunum smooth muscle after second (non-cumulative) application at the same dose. A double application of glyphosate in each experiment was conducted to verify the repeatability of the complex (biphasic) smooth muscle reaction. Unexpectedly, in case of some doses of glyphosate the second incubation of strips in the presence of the active substance differed from the first treatment (Fig. 3). The observed changes of smooth muscle response might suggest that multiple exposure at short time could cause progressive motility disorders. Although glyphosate probably does not accumulate in animal organisms
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[3,28], the repetitive exposure over several days might result in increased toxicity. This phenomenon is well recognized in other pesticides (e.g., 2,4 D, urea-derivatives) [35–37]. One should not forget that even slight recurrent disturbances of gastrointestinal motoric activity might affect absorption and secretion processes and the speed of gastrointestinal passage [33,34]. There is enormous amount of gastrointestinal disorders that occur without clear pathomorphological manifestations and which causes are not defined. It cannot be ruled out that simultaneous occurrence of several causes, including stress and exposure to substances in diet that affect smooth muscle functionality (e.g., nitrite), contributes to those disorders. The repetitive presence of glyphosate in low doses in intestine cells might play a crucial role in recurrent intestinal dysmotility. Besides, the interactions between glyphosate and other environmental contaminants or their metabolites as a result of concurrent exposure cannot be excluded. It was confirmed in in vivo studies on rats performed by Mariana et al. [38] who revealed that the toxic effect, involving oxidative stress mechanisms, was higher in case of the exposure to a mixture of agrochemicals (dimethoate, zineb and glyphosate) than after exposure to a single substance. The disturbances of intestine motoric activity that are not dosedependent and the biphasic character of strips’ response might suggest a complex mechanism of action of glyphosate on smooth muscle cells. The observed inconstancy of glyphosate effect is convergent with data gained in other experimental models. For instance, in human erythrocytes glyphosate at low concentration causes elevation of plasma membrane ATPase (PMCA) activity, whereas high doses of the same substance induces inhibition of enzyme activity [39]. Changes of PMCA activity in erythrocytes lead to calcium ions imbalance. This observation allows to hypothesize that a similar mechanism involving calcium ions movements comprises alterations of smooth muscle activity by glyphosate but this speculation requires further in-depth studies. Another aspect of glyphosate effect on smooth muscle, which might contribute to motility disturbances, is a very clear, but transient, smooth muscle contraction observed after the compound is removed from the incubation chambers (Fig. 2A). Possibly, due to elimination and diminishing concentration of glyphosate under in vitro conditions, some additional mechanism, which was previously inhibited by the presence of the compound in blood and tissues, became activated and resulted in a contraction of the gut smooth muscle. It is well known that the biological activity of glyphosate, is higher in lower pH. This may suggest that the character of reaction contributes to glyphosate’s mechanism of action. The results presented herein indicate that glyphosate significantly affects gastrointestinal smooth muscle (strong miorelaxation and significant contraction) even if it is used in buffered solutions which pH value amounts to 7.35. It allows to imply that after oral absorption, under well buffered in vivo conditions, glyphosate remains active. However, the obtained results clearly point out that the intensity of glyphosate activity is pH dependent and becomes stronger under acidic conditions. The incubation of jejunum strips in nonbuffered (acidic) glyphosate solution caused relaxant response that was more than twice greater than the response to the application of glyphosate at the same dose but used in buffered physiological pH (Table 1). Moreover, the reaction evoked by preparations’ incubation in acidic glyphosate solution consisted only of the persistent relaxant component whereas the response to buffered glyphosate solution as well as to acidified MK–HS (without the active substance) was always temporary and reversible by itself within 20 min of incubation. As mentioned before, the response to glyphosate in buffered solution evoked biphasic reaction, the relaxant component was accompanied by remarkable contraction. The increase of toxic effect of glyphosate in acidic conditions, pH 5.2
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(Table 1), can be explained by ionization state changes of the compound which binds an extra proton. It reduces the net charge of the molecule and, thus, glyphosate becomes more lipophilic [40,41]. It improves glyphosate’s penetration through membranes and the entry to cells and tissues. Inside the cells glyphosate converts probably into a bivalent ion due to the higher pH of cytoplasm. If the solution’s pH accounts to 7.3–7.4, physiological blood pH, the lipophilicity of glyphosate decreases and only a part of particles (the fraction of lower ion charge) penetrates into cells. Then those particles lose their charge due to lower pH (weak acid character of intracellular space) and molecules of lower net charge return into extracellular space. The proposed mechanism might explain the diminished and labile response of jejunum smooth muscle to glyphosate treatment when it is used in buffered solution. The acidification of glyphosate-based herbicides is a common procedure used to abolish glyphosate chelation with bivalent ions in aqueous solutions [42] in order to preserve their biological activity. Paradoxically, the mechanism involving bivalent ions chelation, including calcium ions, should be considered as one of the possible modes of action contributing to the observed disturbances of jejunum strips activity. In conclusion, glyphosate affects the spontaneous motoric activity of rat isolated jejunum strips, even if applied at very low concentrations. The effects are observed during the incubation of preparations in solutions of physiologic pH value and are biphasic (miorelaxant and contractile response). Upcoming studies should be focused on the estimation of the detailed mechanism of action of glyphosate on intestine smooth muscle and should definitely include manipulation of calcium ions concentration. The effect of commercial products containing glyphosate on gastrointestinal motoric activity should be evaluated. Acknowledgment The study was supported by a research Grant from the National Science Centre Poland No. UMO-2011/01/B/NZ7/01497. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pestbp.2014 06.005. References [1] M.G. Dill, R.D. Sammons, P.C.C. Feng, F. Kohn, K. Kretzmer, A. Mehrsheikh, M. Bleeke, J.L. Honedder, D. Farmer, D. Wright, E.A. Haupfear, Glyphosate: discovery, development, applications, and properties, in: V.K. Nandula (Ed.), Glyphosate Resistance in Crops and Leeds: History, Development, and Management, John Wiley and Sons Inc, Hoboken, 2010, pp. 1–33. [2] V. Burgat, G. Keck, P. Guerre, V. Bigorre, X. Pineau, Glyphosate toxicosis in domestic animals: a survey from the data of the Centre National d’Informations Toxicologiques Veterinaires (CNITV), Vet. Hum. Toxicol. 40 (1998) 363–367. [3] G.M. Williams, R. Kroes, I.C. Munro, Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans, Regul. Toxicol. Pharmacol. 31 (2000) 117–165, http://dx.doi.org/10.1006/ rtph.1999.1371. [4] S.M. Bradberry, A.T. Proudfoot, J.A. Vale, Glyphosate poisoning, Toxicol. Rev. 23 (2004) 159–167, http://dx.doi.org/10.2165/00139709-200423030-00003. [5] A.R. Talbot, M.H. Shiaw, J.S. Huang, S.F. Yang, T.S. Goo, S.H. Wasng, C.L. Chien, T.R. Sanford, Acute poisoning with a glyphosate-surfactant herbicide (Roundup): a review of 93 cases, Hum. Exp. Toxicol. 10 (1991) 1–8, http://dx.doi.org/ 10.1177/096032719101000101. [6] P. Bernya, F. Calonib, S. Croubelsc, M. Sachanad, V. Vandenbrouckec, F. Davanzo, R. Guitart, Animal poisoning in Europe. Part 2: Companion animals, Vet. J 183 (2010) 255–259, http://dx.doi.org/10.1016/j.tvjl.2009.03.034. [7] K.A. Modesto, C.B.R. Martinez, Effects of Roundup Transorb on fish: Hematology, antioxidant defenses and acetylcholinesterase activity, Chemosphere 81 (2010) 781–787, http://dx.doi.org/10.1016/ j.chemosphere.2010.07.005.
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