Monitoring of glyphosate and AMPA in soil samples

Environ Monit Assess (2018) 190: 361 https://doi.org/10.1007/s10661-018-6728-x

Monitoring of glyphosate and AMPA in soil samples from two olive cultivation areas in Greece: aspects related to spray operators activities Evangelos Karanasios & Helen Karasali & Anna Marousopoulou & Antigoni Akrivou & Emilia Markellou Received: 23 February 2018 / Accepted: 11 May 2018 / Published online: 25 May 2018 # Springer International Publishing AG, part of Springer Nature 2018

Abstract The persistence of glyphosate and its primary metabolite AMPA (aminomethylphosphonic acid) was monitored in two areas in Southern Greece (Peza, Crete and Chora Trifilias, Peloponnese) with a known history of glyphosate use, and the levels of residues were linked to spray operators’ activities in the respective areas. A total of 170 samples were collected and analysed from both areas during a 3-year monitoring study. A new method (Impact Assessment Procedure - IAP) designed to assess potential impacts to the environment caused by growers’ activities, was utilised in the explanation of the results. The level of residues was compared to the predicted environmental concentrations in soil. The ratio of the measured concentrations to the predicted environmental concentrations (MCs/PECs) was > 1 in Chora the first 2 years of sampling and < 1 in the third year, whilst the MCs/PECs ratio was < 1 in Peza, throughout the whole monitoring period. The compliance to the instructions for best handling practices, which operators received during the monitoring period, was reflected in the amount of residues and the MCs/PECs ratio in the

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10661-018-6728-x) contains supplementary material, which is available to authorized users. E. Karanasios (*) : H. Karasali : A. Marousopoulou Department of Pesticides Control and Phytopharmacy, Benaki Phytopathological Institute, 8 St. Delta str, Kifissia, Greece e-mail: [email protected] A. Akrivou : E. Markellou Department of Phytopathology, Benaki Phytopathological Institute, 8 St. Delta str, Kifissia, Greece

second and especially the third sampling year. Differences in the level of residues between areas as well as sampling sites of the same area were identified. AMPA persisted longer than the parent compound glyphosate in both areas. Keywords Glyphosate . AMPA . Dissipation . Persistence . Soil . Training . Point-source pollution

Introduction The use of pesticides has contributed considerably to the total crop yield and the prevention of crop losses (Yadav et al. 2015) and allowed the major sources for human nutrition to double in the past 50 years (Oerke 2006). However, the heavy reliance of crop production on chemical solutions raised whistleblowing environmental concerns. Pesticides constitute a constant threat to both abiotic and biotic ecosystem components as they are directly released to the environment (Vega et al. 2005). The protection of aquatic and terrestrial ecosystems from contamination by pesticides is addressed within the European Union with the adoption of an extensive body of restrictive legislation, which includes the protection, management and the sustainable use of pesticides (Rathore and Nollet 2012; European Commission 2009; Kraemer et al. 2004). Pesticides applied to agricultural fields end up in soil and waterways via point and non-point (diffuse) entry routes. Diffuse sources include losses during the spray operation, whilst point sources are associated with

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improper disposal of pesticide wastes in restricted locations of the farm or farmyard usually in the pre- or postapplication period (filling, cleaning or emptying of spaying equipment, transport and storage of plant protection products) (Carter 2000; Cooper and Taylor 2008). The risk management from point source inputs is considered more feasible compared to diffuse sources (Bonicelli et al. 2008). Mitigation strategies include the implementation of best management practices, the integration of innovative techniques and upgraded infrastructure (Reichenberger et al. 2007; Cooper and Taylor 2008; Jaeken and Debaer 2005). Training and technical assistance in the form of stakeholder surveys, awareness raising programs and stewardship initiatives can play a decisive role (Roettele 2008; Jaeken and Debaer 2005). Previous initiatives aiming at the implementation of best management practices leaded to the restriction of total pesticide losses to the environment by 40–95% (Kreuger and Nilsson 2001; Reichenberger et al. 2007). Key elements of the training are the increased awareness on the potential hazards from misuse of pesticides, the dissemination and demonstration of best management practices and the certification of the competence of the operators (Carter 2000; Rose et al. 2000; Reichenberger et al. 2007; Balsari and Marucco 2007). Examples of successful training and stewardship programs are the EU-wide LIFE+ ENV Project ‘TOPPS’, the Dammbach Watershed Project in Germany and the EcoPest Project (LIFE+ ENV) in Greece. Glyphosate [N-(phosphonomethyl)glycine] is a nonselective, broad spectrum, systemic, post-emergence herbicide that is phloem mobile and is readily translocated throughout the plant (Spurrier 1973). It is used in farming, forestry, parks, public spaces and gardens over the last three decades and accounts for a significant share of the global herbicide market. The worldwide glyphosate use (including agricultural and non-agricultural use) rose more than 12-fold from about 67 million kg in 1995 to 826 million kg in 2014 (Benbrook 2016). Glyphosate has been extensively used over the past three decades in Greece for weed control in orchards, and it has been praised for its suitability in noor low-tillage systems, especially in areas vulnerable to soil erosion. However, its recurrent use increases the risk for downward movement and leaching and the off-target transfer of glyphosate residues from contaminated soil to nearby environmental compartments via run-off (Helander et al. 2012) and/or erosion-driven transport (Silva et al. 2018).

Environ Monit Assess (2018) 190: 361

Soil is the reservoir from which the initially released pesticide residues can be transferred to other environmental compartments (Goncalves and Alpendurada 2005). The behaviour of pesticides in soils is controlled by various dynamic processes, including sorption–desorption, volatilisation, chemical and biological degradation and uptake by plants (van der Werf 1996; AriasEstevez et al. 2008). Although the primary route of dissipation of glyphosate in soil is biodegradation via soil microorganisms under both aerobic and anaerobic conditions, other routes of decomposition (chemical, photolysis) are possible (Franz et al. 1997). It is normally utilised as a phosphorus, carbon or nitrogen source (Borggaard and Gimsing 2008), whilst the rate of dissipation is affected by temperature and soil moisture (Bento et al. 2016). Glyphosate is moderately persistent in soil. The dissipation half-life (DT50) in the field under a wide range of climatic conditions ranges from 1 to 130 days (European Commission 2002). The primary metabolite of glyphosate is aminomethylphosphonic acid (AMPA) which results from the cleavage of the C–N bond by the enzyme glyphosate oxidoreductase (Borggaard and Gimsing 2008). Another metabolic pathway includes the direct cleavage of the C–P bond of glyphosate and the formation of sarcosine as intermediate (Dick and Quinn 1995; Kishore and Jacob 1987; Liu et al. 1991). Degradation of AMPA is generally slower compared to the parent compound (DT50 76–240 days) which is partly attributed to the stronger sorption to soil (Grunewald et al. 2001). Glyphosate has a low potential to reach groundwater in most soil environments because of its strong adsorption to soil particles through the phosphonic acid moiety (Morillo et al. 2000). Organic matter and clay minerals like amorphous Fe- and Aloxides are possible sites for sorption (Borggaard and Gimsing 2008; Roy et al. 1989; Piccolo et al. 1994). In addition to specific surface area and mineral group, glyphosate sorption also depends on pH (Borggaard and Gimsing 2008). Despite its relevant immobility to soil, monitoring data obtained from 13 countries across Europe indicated that glyphosate and AMPA can be occasionally detected in groundwater (1.3 and 1.7% of total samples, respectively) with AMPA being detected usually at higher concentrations and in a larger proportion of samples (Horth and Blackmore 2009). Despite the extensive use of glyphosate, information on the occurrence and levels of glyphosate and AMPA in soil is infrequent and out of date, especially in the EU (Silva et al. 2018). The extensive and continual use of


glyphosate products explains the frequency of occurrence of glyphosate residues in soil environments (Pyne 2015; Battaglin et al. 2014; Aparicio et al. 2013; Silva et al. 2018) and the transient accumulation of AMPA in glyphosate-treated soils (Torstensson 1985). Trace levels of glyphosate and AMPA in the soil may be carried over from year to year after repeated use of glyphosate (Scribner et al. 2007) and can be traced even years after the last spraying under unfavourable conditions for dissipation (Helander et al. 2012; Torstensson et al. 1989; Laitinen et al. 2009). The objective of this investigation was to monitor any temporal or spatial patterns of glyphosate and AMPA occurrence in soil in two target olivecultivation areas in Southern Greece, with a known history of use of glyphosate, and to assess whether changes in operators’ activities after training on the rational use of herbicides could be linked to changes in glyphosate and AMPA levels in soil.

Materials and methods Field sites Sampling was carried out between 2012 and 2014 in two typical olive-growing areas of Southern Greece (Peza, Crete and Chora Trifilias, Peloponnese) (Fig. 1).

Chora

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The first year of the monitoring program, sampling was carried out at each site in order to quantify the background pollution levels. A total of 51 sites were selected in Peza, 16 of which did not receive any glyphosate during the 3-year sampling period. Further, soil from 27 sites from conventional farms (6 of which were not treated with glyphosate) and 13 sites from organic farms in Chora Trifilias were collected and analysed. The selection of the study sites was based on the following criteria: (i) the spatial distribution within the studied areas and the landscape variability, (ii) the soil texture and properties and (iii) the farming practices/production schemes. Soil types varied between target areas and within sampling sites of the same area (Table 1). The physiochemical characteristics of soils are presented in Table 2. Soil samples Samples for residual analysis were taken from the 0- to 30-cm topsoil layer using a soil sampler. At least four soil sub-samples were collected per plot and pooled to obtain a representative sample for each site. Each soil sample consisted of 1 kg stored in labelled clean plastic bags and sent for analysis to the Laboratory of Chemical Control of Pesticides of Benaki Phytopathological Institute in portable containments under low temperature conditions and constant darkness. For practical reasons,

Peza

Fig. 1 Map of sampling locations. Sites where glyphosate was applied are identified by blue pins. Sites without glyphosate application identified by red pins. Sites belonging to organic farms or farms in transition to organic farming are identified by yellow pins

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Table 1 Characterisation of soil in sampling sites in Chora and Peza

combinations and/or sequential applications of herbicides were required to provide effective weed control.

Number of sites at each soil type Choraa Clay Clay-loam

2

23

11

16

Loam

7

6

Sandy-loam

5

2

Sandy-clay

0

3

Sandy-clay-loam

9

1

Silty-clay-loam

5

0

a

Analytical standards

Peza

Not determined for two soils

sampling was carried out at variable dates after application of glyphosate (Table 3). Glyphosate applications Glyphosate was applied once after the onset of rainfalls in both regions between mid-February and early May, after weeds had emerged or were actively growing at the time of spraying, except for three soil sampling sites in Chora where a complementary application of glyphosate, at a much lower dose, was carried out in the middle of summer (Table 3). Application rate of glyphosate varied between and within areas due to differences in the target weeds and local practicalities. Weed management differed among olive groves and depended on weed species present, parcel’s soil type, application of irrigation and various other factors related to the farming system applied. At least two weed surveys per year (late winter and end of spring) were conducted by agronomists and included identification of the weed species and determination of weed density. These surveys were the basis for weed management advices provided by agronomists to operators related to herbicide choice and practices on rational handling, spraying and herbicides remnants management. In few olive groves,

High purity analytical standards of glyphosate (98%) and aminomethyl phosphonic acid (AMPA) (99.8%) were purchased from ChemService (USA). Analytical standards of glyphosate-FMOC (97%) and aminomethyl phosphonic acid-FMOC (97.5%) were obtained from Dr. Ehrenstorfer. Individual stock solutions of glyphosate and AMPA were prepared by gravimetric weighing of high purity standards at concentrations of approximately 1000 mg L−1 in water (HPLC grade). Working solutions of individual compounds, their mixtures and spiked samples were prepared at different concentration levels, by appropriate dilutions of the stock solutions in water. Glyphosate and AMPA mixture working solutions were used for the estimation of recovery. Individual stock solutions of glyphosate-FMOC and AMPA-FMOC were prepared by gravimetric weighing of the high purity analytical standards at concentrations 492.76 and 970 μg mL−1 respectively, in an appropriate mixture of water:methanol (75:25) (HPLC grade). Working solutions of their mixtures were prepared in methanol at the concentrations of 0.01, 0.05, 0.1, 0.5 and 1 μg mL−1 and were used for establishing the linearity of the chromatographic system. All the standard and working solutions were stored in amber nonsilanized glasses at 0–1 °C in dark. Before each use, the standard solutions were equilibrated at room temperature and weighed to check for evaporation losses (Kmellar et al. 2011). Solvents and reagents Analytical reagent-grade sodium tetraborate decahydrate of 100% purity and 9-fluorenylmethylchloroformate (FMOC-Cl) of 98% purity were obtained form LACH-

Table 2 Physicochemical characteristics of soils No of samples

Organic carbon

Clay

CEC

pH

P (mg kg−1)

N (mg kg−1)

Peza

51

1.57 (0.6–3.02)

37.1 (16.8–61.6)

20.8 (6.8–41.8)

7.6 (7.2–7.9)

11.64 (0.94–62.6)

16.37 (6.6–77)

Chora

39a

1.42 (0.7–2.7)

28.9 (10.4–42.4)

17.3 (7.9–23.9)

7.2 (4.7–7.8)

20.62 (1.10–162.9)

14.68 (4.2–66.7)

CEC cation exchange capacity, P P-Olsen a

Data for two sites are not available


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Table 3 Application of glyphosate in the two target areas Date of application

2012

2013

2014

Application rate (g glyphosate ha−1)

Interval between application and sampling (days)

2012

2012

2013

2014

2013

2014

Peza

5 Mar–22 Apr

10 Feb–25 Apr

15 Mar–5 Apr

973–4186

900–4003

1362–4153

13–23

47–134

15–21

Chora

27 Mar–30 Apra

28 Feb–26 Apr

5 Mar–5 May

216–3750

83.9–3428

360–3333

44–107

58–276

33–137

a

Complementary applications in three sites during July–August at rates between 100 and 240 g a.i./ha

NER (Czech Republic) and ACROS ORGANICS respectively. Reagent grade hydrochloric acid and potassium hydroxide (KOH) was purchased from Panreac Quimica S.A. (Spain), and ammonium acetate from (NH4Ac) of 98% purity was obtained from Merck (Germany). Hydrochloric acid 11.65N, LC-MS grade water and acetonitrile and HPLC water used in this study were supplied by Fisher Scientific (UK). Solution of 5% borate buffer at approximately pH 9 in water of HPLC grade and solution containing 12,000 mg L −1 of FMOC-Cl in acetonitrile were used for the derivatization step of the samples. Argon (Ar), used as collisioninduced gas (CID gas) in the triple quadrupole, was obtained from Air Liquid (Greece). Sample preparation and extraction method Sample preparation was based on the method proposed by Ibanez et al. (2005) with minor modifications as described below. Soil samples were air dried at room temperature in the dark, sieved through 2-mm sieve and frozen at − 40 °C till extraction. Soil samples were allowed to reach ambient temperature and after thorough mixing of the sample, a subsample of 5 g (± 0.1) was transferred to a centrifuge tube (50 mL) with 10 mL of 0.6 M KOH, shaken mechanically in a horizontal shaker for 30 min and then centrifuged at 3000 rpm for 30 min. The alkaline supernatant was separated and neutralised by adding drops of 6N and 0.6N HCL until approximately pH 7.0. After that, the neutralised supernatant was tenfold diluted with water of HPLC grade. The next step concerns the derivatisation step in which 2 mL of the tenfold diluted supernatant was pipetted into a glass tube together with 120 μL HPLC water, 120 μL of borate buffer (pH 9) and 120 μL of FMOC–Cl reagent (12,000 mg L−1). The tube was swirled and left overnight at room temperature, and then the samples were acidified with hydrochloric acid until pH 1.5, filtered through 0.45 μm syringe filter and injected

directly to LC-ESI-MS/MS system. It should be mentioned that the tenfold dilution of soil samples with water was assayed as a simple and fast way to minimise matrix interferences (Ibanez et al. 2005). Instrumental The high-performance liquid chromatograph used for the separation glyphosate and AMPA was a Varian (USA) system (working pressure maximum 400 bar), composed of two Prostar pumps (VARIAN, Prostar 210), a vacuum degasser (Metachem Technologies Inc), an autosampler (Varian, Prostar 420) with a 10-μL sample loop and a column oven (Varian, Prostar 510). The analytical column employed was a reversedphase C18 of 50 mm × 2 mm × 5 μm particle size (Agilent Zorbax Eclipse Plus). The mobile phases, A and B, consisted of water 5 mM acetic acid/ammonium acetate adjusted at pH 4.6 and acetonitrile at a ratio 10:90 respectively. The flow rate was set at 0.2 mL min−1 and the column gradient program consisted of 90 vol.% of A and 10 vol.% of B where it remained for 5.06 min. Next, at 5.1 min, it was reversed to 10 vol.% of A and 90 vol.% of B where it remained for 10 min. At 10.01 min, the gradient was returned to the initial conditions (90 vol.% A) where it maintained up to the end of the analysis at 20 min. After the 20 min run time, the column was re-equilibrated for 10 min at the initial mobile phase composition. The column temperature was maintained at 30 °C during all runs and the injection volume was 5 μL. In order to avoid carry over, the autosampler was purged with a mixture methanol/ water (50:50 v/v) before sample injection. The triple quadrupole system used was a Varian 1200 L (VARIAN, USA) Quadrupole MS–MS spectrometer fitted with an electrospray ionisation (ESI) interface. The ESI–MS interface was operated in the positive ion detection mode. The ESI source conditions were capillary voltage, 5000 V in positive-ion (PI)

361 Page 6 of 15


mode; drying gas temperature, 300 °C; nebuliser gas pressure, 45 psi (both nebuliser and drying gas were high purity nitrogen, produced by a high purity generator) and electron multiplier voltage, 1600 V. MS/MS experiments were carried out with Argon (purity 99.9%) at pressure of approximately 1.5 mTorr in the collision cell. Cone voltage and collision energy values optimised for each of the two compounds selected, were used. For selected ion monitoring (SIM) experiments, both Q1 and Q3 were set at fixed m/z values. For each analyte, the most abundant and characteristic fragment ion was chosen for quantization and two fragment ions selected for confirmation (Table 4). Dwell times of 0.1 ms were set. For instrument control, data acquisition and processing, the Varian MS Workstation software version 6.8 was used. The selected ion monitoring (SIM) mode was applied, and the selected characteristic ions are presented at Table 4. The transition of the most abundant product ion was used for quantitation and the second one in abundance for identification. The first step involved selection of the precursor ion for each compound. Although glyphosate and AMPA have been traditionally recorded at negative ion mode, Ibanez et al. (2005) found that positive ion mode is much more sensitive. For that reason, the method proposed in that work has been followed. For both compounds glyphosate and AMPA and in the positive-ion electrospray full scan spectrum, the protonated derivatized molecule [M + H] + was recorded at m/z 392 and 334, respectively. In the case of glyphosate, the MS/MS spectra showed two abundant fragments at m/z 214 and 88, whereas in the case of AMPA the respective abundant fragments were at m/z 112 and 179.

Table 4 Mass spectrometry parameters for glyphosate and AMPA Mass spectrometry and chromatography parameters

GlyphosateFMOC

AMPA-FMOC

Quantification transition (m/z) Capillary voltage (V)

392 ➔ 88.1

334 ➔ 179.1

50

60

Collision energy (eV)

20

15

Qualifier transition (m/z)

392 ➔ 214.1

334 ➔ 112.1

Capillary voltage (V)

50

60

Collision energy 2 (eV)

10

10

Rt (min)

7.8

8.2

Validation study The method has been fully validated following the European Union SANCO guidelines (SANCO 2010). The precision (repeatability, in terms of %RSD) and the accuracy (percentage recoveries) of the method were estimated by recovery experiments in soil which was free of glyphosate and AMPA at three fortification levels. Linearity Linearity for glyphosate and AMPA was evaluated using calibration curves at five concentration levels covering concentrations at three orders of magnitude: 0.01– 1 μg g−1, based on the linear regression and squares correlation coefficients, R2. Regression analysis exhibited an excellent relationship, as correlation coefficients (R2) were 0.9987 for AMPA and 0.9978 for glyphosate. Precision The repeatability of the method was determined at the concentration level of 0.05 μg g−1 dry weight, by the analysis of five spiked matrix extracts (n = 5). The calculated RSDs ranged between 5 and 15%. Inter-day RSDs were calculated for 5 days and varied between 7 and 19%. According to BGuidance document on pesticide residue analytical methods^, these results were considered to be acceptable and demonstrated a satisfactory repeatability of the method and therefore its effectiveness for quantitative purposes (SANCO 2010). The accuracy of the method was verified by measuring from spiked blank samples at three concentration levels, i.e. at 0.01, 0.05 and 0.5 μg g−1 dry weight. All experiments were performed five times, and the relative standard deviation (RSD%) was calculated, and the values obtained were used for the estimation of the precision of the extraction method. Recovery and limit of quantitation The accuracy of the method was verified by measuring recoveries form spiked blank samples at three concentrations levels, i.e. at 0.01, 0.05 and 0.5 μg g−1 dry weight. All experiments were performed five times, and the relative standard deviation (RSD) was calculated. Recovery ranged between 89.6 and 118.8% for glyphosate and between 67.9 and 94.6% for AMPA


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whereas the RSD was 15.35% for glyphosate and 11.9% for AMPA in all cases. The validated LOQs were defined as the lowest validated spike level (expressed in μg g−1 dry weight) for which a recovery in the 70–120% range could be obtained, with a corresponding RSD ≤ 20%, according to the EU SANCO document on validation and QC procedures (SANCO 2010). Based on the EU SANCO, the validated LOQs were defined as the lowest calibrated spiked level and were 0.01 μg g−1 soil dry weight for both compounds. Recoveries for the studied compounds were in the range 75.62–113.65%, thus, the concentration of pesticides in soil samples was not corrected for recovery (Bozlaker et al. 2009). A soil sample free from glyphosate and AMPA residues was used for recovery experiments. The specific sample was previously analysed to ensure that it did not contain the studied compounds and was used as blank soil sample. This blank soil sample used for the estimation of recovery was treated as follows: 10 g of the sample (blank soil sample) was placed in a centrifuge tube (50 mL) along with 1 mL of the standard mixture of the desired pesticide concentration in water. It was homogenised by mechanical shaking for 60 min for better analyte distribution, and the bulk of the solvent was left to evaporate at ambient temperature and controlled by weight. This is a procedure able to mimic weathered residues (Diez et al. 2008). Then, spiked samples were extracted in the same way as described in the sample preparation and extraction method. Predicted environmental concentration The concentration of glyphosate and AMPA in soil was estimated with the soil persistence model of the Soil Modelling Work group of FOCUS (FOCUS 1997): PECs ðt 0 Þ ¼

A ð1− f int Þ 100 depth bd

PECs ðafter n applicationsÞ ¼ PECs ðt 0 Þ k¼

ln2 DT50

ð1Þ 1−e−nki ð2Þ 1−e−ki ð3Þ

where A is the application dose (g ha−1); fint is the fraction intercepted by crop canopy; depth is the mixing depth (cm) and bd is the dry soil bunk density (g cm3); k is the dissipation rate constant and DT50 the time for

disappearance of half the chemical. The following assumptions were made: the fint was set to 0, the mixing depth to 15 cm, the DT50 of glyphosate and AMPA were 8.2 και 137.2 days, respectively (geomean of available EU data); and the formation factor of AMPA was set to 27.5% (European Commission 2002). Scoring of environmental impact-IAP method The results of the IAP (Impact Assessment Procedure) method (under publication), which was implemented in the two target areas (Chora and Peza) in the context of the LIFE09 ENV/GR/000302 SAGE10 project, were used to explain the results from the soil monitoring studies. According to the IAP concept, each impact is expressed as a combination of three elements (called in IAP Triplet): Aspect (growers’ activities)-Impact-Compartment (soil, water, humans, biodiversity). Several parameters were utilised for the assessment of the environmental impacts in the two target areas. Parameters, which can be related to the farmers’ practices and choices or to the resilience of the environment to contamination, were recorded and weighted. Each of the 200 olive-groves which were randomly selected in each area received a score, was based on data collected by agronomists. Data for the value or class of parameters were collected annually for three consecutive years (starting 1 year prior to the initiation of the monitoring). The score of each triplet was normalised to a 0–1 scale, where 0 represents the absence of expected impact and 1 the possibility of significant impact. The four triplets which are related to point source pollution with pesticides are the handling of wastewater loads from pesticide use (emptying, filling and cleaning of equipment), the management of empty containers, the transport and the storage of agrochemicals. In each of these triplets, the impact was pollution and the compartment was the abiotic environment. Groves under organic farming system or groves where chemicals were not used for weed control were excluded.

Results and discussion Monitoring of glyphosate and AMPA residues in conventional olive farms of Chora and Peza with long history of glyphosate use The analysis of the soil residues was restricted to glyphosate which was extensively used in both studied areas.

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Its major metabolite AMPA was also determined in all analysed soil samples. The analyses results for glyphosate and AMPA in soil samples during the three sampling years (2012–2014) are given in Tables 5 and 6. For practical reasons (workload, distance between parcels, number of sampling sites etc), sampling was conducted at various intervals after glyphosate application as presented in Table 3, thus, the side-by-side comparison of the residue levels between years and sampling sites is not possible. In order to compare the level of glyphosate and AMPA residues in different sites, the measured concentration in soil (MCs) was associated with the estimated PECs which corresponds to the time of sampling, using the initially applied dose and the theoretical dissipation rate constants for glyphosate and AMPA (MCs/PECs ratio; Fig. 2). Glyphosate and AMPA concentrations from soil samples collected in Peza ranged from < LOQ to 240 μg kg−1 and from < LOQ to 100 μg kg−1, respectively. Glyphosate residues exceeding the LOQ were determined in 6 out of 35 glyphosate-treated sites. Concentrations of glyphosate and AMPA were generally far lower than the theoretically estimated levels (PECs) except for three sampling occasions (Table 5), which suggest that glyphosate was rapidly degraded in this area. The MCs/PECs ratio was 0.35 in the first sampling event and reduced further at subsequent samplings (Fig. 2). The decrease of the MCs/PECs ratio can be linked to the reduction of glyphosate losses from improper pre- and postapplication handling as suggested by the improvement of the triplet score through the application of IAP Method. The targeted training that operators received led to the lowering of the mean score for two of the four examined aspects (remnant handling and transport) (Table 7). The change in score was significant in groves which received the highest score in the baseline year (score class 0.3–0.4). The reduction of transport distance, the selection of zero-slope spots for handling and disposal of spay leftovers and the frequency of use of spraying equipment are the associated parameters which were refined in the 2012–2013 period. The adoption of environmentally sound practices was mirrored in the slight improvement of specific indicators: the proportion of operators which accurately performed the triple rinsing of empty containers (increase from 55% in the baseline year to 63% in 2013; data not shown) and the proportion of spraying equipment without visible leakages (increased by 9.5% in the same period). It is noticeable that glyphosate remained one


of the prevalent weed control practices in the area as the total glyphosate load was reduced by only 9.9% between 2011 and 2013 in the area. Based on the results of the first sampling year in Peza, an estimation of the rate of degradation of glyphosate was done, assuming that residue decline follows simple first-order kinetics. Residues of glyphosate reached the limit of quantification (LOQ) within 2– 3 weeks after application in 15 out of 18 samples in 2012. In the three remaining sites from the 2012 sampling, some glyphosate residues were traced (31– 240 μg kg−1). If first order degradation is assumed, the estimated half-lives for AMPA in these three soils could be approximated to range from 3.6 to 5.7 days. Thus, DT50 can be anticipated to be close to the lowest recorded values for this active substance (European Commission 2002). The absence of substantial residual amounts of glyphosate and AMPA indicates that builtup of residues after repeated use of glyphosate products is not expected in this area. The concentration of glyphosate and AMPA in soil received from Chora ranged from < LOQ to 350 μg kg−1 and < LOQ to 650 μg kg−1, respectively. The variation in the level of residues between sites may be explained by differences in the application rates, the frequency of application events and the interval between last application and sampling. The analysed concentrations of glyphosate and AMPA in Chora exceeded the theoretically estimated values in a number of sites, especially at the first sampling year. The maximum measured concentration of AMPA is soil (650 μg kg−1) is, however, lower that the theoretical worst-case plateau concentration of AMPA in permanent crops (4140 μg kg−1) after 10 years of continuous glyphosate applications (EFSA 2015). The proportion of sampling sites with residues exceeding the theoretically estimated values was reduced from 88% in 2012 to 36% in 2013 and 21% in 2014. To be noted that residual AMPA residues from applications before 2012 were not included (the baseline concentration of AMPA was not set). Various sources of contamination linked to the handling of pesticide equipment and the management of the application leftovers are possible to have contributed to the exceeding of the theoretical values. Point source contamination of soil as a result of improper handling of pesticides e.g. losses during filling, emptying and cleaning of equipment in the edge-of-field area were seen elsewhere (Müller et al. 2002). To be noted, that 43% of the glyphosate-treated sites in Chora have a slope of > 2% which was


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Table 5 Measured concentrations (MCs; μg kg−1) and predicted environmental concentration (PECs; μg kg−1) of glyphosate and AMPA in treated sites in Peza Site code

2012

2013

Glyphosate

AMPA

PECs

MCs

PECs

CFP1

137

< LOQ

CFP2

555

< LOQ

2014

Glyphosate

AMPA

Glyphosate

AMPA

MCs

PECs

MCs

PECs

MCs

PECs

MCs

PECs

MCs

79

32

0

< LOQ

82

20

ndd

ndd

ndd

ndd

297

71

3

< LOQ

172

< LOQ

ndd

ndd

ndd

ndd

< LOQ

d

d

d

CFP3

149

< LOQ

127

13

3

< LOQ

CFP4

213

< LOQ

123

80

1

19

138

CFP5

291

< LOQ

168

26

1

< LOQ

128

CFP6

351

< LOQ

203

24

1

CFP7

257

< LOQ

161

< LOQ

0 a

147

nd

nd

nd

ndd

26

289

< LOQ

268

47

17

ndd

ndd

ndd

ndd

d

d

d

< LOQ

137

< LOQ

nd

nd

nd

ndd

< LOQ

210

< LOQ

ndd

ndd

ndd

ndd

d

d

d

CFP8

678

< LOQ

336

30

nd

12

nd

10

nd

nd

nd

ndd

CFP9

399

140

249

27

1

23

156

< LOQ

271

< LOQ

231

96

CFP10

386

31

415

65

1

14

167

14

ndd

ndd

ndd

ndd

a

d

d

d

a

a

CFP11

361

< LOQ

285

12

nd

14

nd

26

nd

nd

nd

ndd

CFP12

441

< LOQ

240

70

60

< LOQ

306

< LOQ

ndd

ndd

ndd

ndd

d

d

d

CFP13

462

< LOQ

289

32

1

< LOQ

194

22

nd

nd

nd

ndd

CFP14

379

240

174

21

1

< LOQ

214

15

311

< LOQ

227

< LOQ

CFP15

114

< LOQ

66

34

0

< LOQ

81

< LOQ

348

< LOQ

340

< LOQ

CFP16

629

< LOQ

312

62

0

< LOQ

169

27

ndd

ndd

ndd

ndd

CFP17

629

< LOQ

312

22

ndd

ndd

ndd

ndd

ndd

ndd

ndd

ndd

CFP18

b

< LOQ

b

d

d

d

nd

d

d

nd

d

< LOQ d

2 c

< LOQ

198

< LOQ

nd

nd

nd

ndd

c

CFP19

nd

nd

nd

nd

nd

< LOQ

nd

< LOQ

263

< LOQ

234

100

CFP20

ndd

ndd

ndd

ndd

9

< LOQ

62

< LOQ

ndd

ndd

ndd

ndd

CFP21

d

nd

d

nd

d

nd

d

nd

0

< LOQ

186

< LOQ

d

nd

d

nd

d

nd

ndd

CFP22

ndd

ndd

ndd

ndd

3

< LOQ

119

< LOQ

ndd

ndd

ndd

ndd

CFP23

d

d

d

d

< LOQ

d

d

d

ndd

d

ndd

d

ndd

d

CFP24 CFP25

nd

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

< LOQ

1

< LOQ

0

< LOQ

108 112 169

< LOQ < LOQ

nd

d

nd

d

nd

d

nd

d

nd

nd nd

nd

0

< LOQ

169

< LOQ

nd

nd

nd

ndd

CFP27

ndd

ndd

ndd

ndd

1

< LOQ

162

15

ndd

ndd

ndd

ndd

CFP28

d

d

d

d

18

d

d

d

nd

d

nd

d

9

< LOQ d

d

59 d

d

nd

nd

d

d

nd

nd nd

d

1

nd

d

d

nd

CFP26

nd

d

nd

nd

nd

nd

ndd

d

CFP29

nd

nd

nd

nd

nd

nd

nd

nd

156

< LOQ

143

< LOQ

CFP30

ndd

ndd

ndd

ndd

ndd

ndd

ndd

ndd

160

< LOQ

147

< LOQ

CFP31

ndd

ndd

ndd

ndd

ndd

ndd

ndd

ndd

178

< LOQ

164

60

CFP32

ndd

ndd

ndd

ndd

ndd

ndd

ndd

ndd

121

< LOQ

112

10

CFP33

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

d

nd

172

< LOQ

158

< LOQ

CFP34

ndd

ndd

ndd

ndd

ndd

ndd

ndd

ndd

143

< LOQ

132

< LOQ

CFP35

ndd

ndd

ndd

ndd

ndd

ndd

ndd

ndd

223

< LOQ

211

< LOQ

nd not determined a

Application in 2013 is conducted but no information on the application rate or date is available

b

Applications with glyphosate was not carried out in this year

c

No information on sampling date is available

d

No sampling was carried out

361 Page 10 of 15


Table 6 Measured concentrations (MCs; μg kg−1) and predicted environmental concentration (PECs; μg kg−1) of glyphosate and AMPA in treated sites in Chora Trifilias Site code

2012

2013

2014

Glyphosate

AMPA

Glyphosate

AMPA

PECs

MCs

PECs

MCs

PECs

MCs

PECs

CFC1

41

190

CFC2

39

150

72

210

0

< LOQ

70

180

0

< LOQ

CFC3

0

< LOQ

74

< LOQ

0

< LOQ

50

< LOQ

1

< LOQ

102

29

CFC4

nda

< LOQ

nda

< LOQ

ndb

ndb

ndb

ndb

0

< LOQ

67

100

CFC5

0

< LOQ

176

250

0

< LOQ

62

10

0

< LOQ

44

150

CFC6

0

230

176

260

0

< LOQ

62

19

0

< LOQ

35

34

CFC7

0

350

177

650

0

< LOQ

63

14

0

< LOQ

35

16

CFC8

0

< LOQ

6

260

0

< LOQ

27

26

35

< LOQ

152

10

CFC9

0

< LOQ

35

100

0

< LOQ

58

26

0

< LOQ

109

< LOQ

CFC10

0

< LOQ

32

330

0

< LOQ

58

170

0

< LOQ

107

< LOQ

CFC11

0

< LOQ

87

310

ndb

ndb

ndb

ndb

42

< LOQ

118

< LOQ

CFC12

ndb

ndb

ndb

ndb

ndb

< LOQ

11

< LOQ

0

< LOQ

102

< LOQ

CFC13

0

< LOQ

93

180

0

70

18

30

0

< LOQ

162

< LOQ

CFC14

10

140

52

310

ndb

< LOQ

25

50

0

< LOQ

76

47

CFC15

0

ndb

11

ndb

ndb

ndb

ndb

ndb

0

< LOQ

36

< LOQ

CFC16

0

< LOQ

53

< LOQ

ndb

ndb

ndb

ndb

0

< LOQ

60

34

CFC17

ndb

ndb

ndb

ndb

0

< LOQ

26

70

ndc

< LOQ

ndc

< LOQ

b

b

b

Glyphosate

AMPA

MCs

PECs

MCs

PECs

MCs

16

140

22

< LOQ

227

< LOQ

40

< LOQ

1

< LOQ

81

80

b

CFC18

39

< LOQ

54

320

nd

nd

nd

nd

0

< LOQ

22

80

CFC19

28

< LOQ

40

140

ndb

ndb

ndb

ndb

0

< LOQ

16

140

CFC20

0

< LOQ

19

20

0

< LOQ

3

< LOQ

ndc

< LOQ

ndc

20

CFC21

ndb

ndb

ndb

ndb

ndb

ndb

ndb

ndb

0

< LOQ

35

18

nd not determined a

Application with glyphosate was not carried out in this year

b

No sampling was carried out

c

No information on glyphosate application is available

previously reported to increase the risk for soil contamination after improper pesticide use at nearby areas (Dabrowski et al. 2002). The results of a survey in Chora showed a perceivable improvement in mean score of triplets and more specifically for aspects linked to point source contamination of soil with pesticides in 2012 and especially 2013 compared to 2011 (Table 7). The adoption of environmentally friendlier aptitude at the second and especially the third year of monitoring is mirrored in the steep decrease of MCs/PECs values in Chora between 2013 and 2014 (Fig. 2). The mean MCs/PECs ratio was 6.95 in the first sampling year and reduced to < 1 in the third year. The aspects which were improved

are the handling of remnants from application, the safe transport of pesticide loads and the management of obsolete containers. On the contrary, the safety of storage practices was not practically improved in the monitoring period. The improvement of the environment impact score between 2011 and 2013 is directly linked to the training which operators received by experts during the same period in the context of the program SAGE10. Further scrutiny of the parameters linked to the triplet score revealed that the most significant contributing factors to the year-by-year decrease of the score in the area are the quantity of pesticides in transport and the transport distance, the lowering of the distance between handling areas and surface water


Page 11 of 15 361

10.00

Peza

1.00 MCs/PECs

Chora

0.10 2012 2013 2014

0.01 Fig. 2 The measure concentration (MCs) to predicted environmental concentration (PEC) ratio of AMPA residues in 2012–2014 in 11 sites from Chora and four sites in Peza (only sites for which data on all 3 years are presented)

bodies and the improvement in the frequency of the visual examination and calibration of spraying equipment before use. Further, in-site inspections and interviews revealed a shift to environmentally friendlier practices. The number of operators which are considered to accurately perform the triple-rinsing increased from 57 to 64%, and the proportion of spraying equipment without visible leakages increased from 60 to 64% in the same period (data not presented). Other contributing factor is the reduction of total glyphosate load in the catchment between 2011 and 2013. The total amount of glyphosate was reduced by 61.2%, due to the gradual shifting to other chemical solutions (oxyfluorfen, glufosinate-ammonium) as part of the Conyza spp. resistance management (data under publication). The mean level of AMPA residues in Chora for all sampling years was higher compared to Peza despite the fact that the mean application dose was higher in Peza, and the interval between application and sampling was narrower (Table 3). Differences were more striking in the first year of application. Variances in residue levels may reflect differences in pesticide residue management or the dissipation potential of soils. Further, differences in the application technique may have influenced the residual amount of glyphosate in soil. In a significant proportion of olive groves in the Peza Region (46.9– 63.5%, depending on the year), glyphosate is carried out

as spot application, whilst most spray operations in Chora are usually performed by broadcast spraying (73.0–99.1% of groves in the 2001–2003 period). Further, the two regions belong to different climatic zones: Chora has a subhumid climate whilst Peza belongs to the semi-arid zone, which may affect the dissipation potential. Compared to Chora, a more favourable environmental profile was observed in Peza as regards the handling of pesticide leftovers and the management of empty containers. On the contrary, a lower mean score was recorded for Chora as regards storage, irrespectively of the year and transport in 2012–2013. However, it should be noted that the initial mean score was generally low in both areas as only a few triplets received a score of higher than 0.3. Only slight differences in the physicochemical characteristics of soils in the two sites were seen, except for Olsen-P content. The presence of phosphate in soil has been reported to compete with glyphosate and AMPA for sorption sites and thus can affect the bioavailability of the both substances (Simonsen et al. 2008) as well as stimulate the glyphosate degradation (Torstensson 1985). However, due to the lack of relevant data, it is not possible to correlate the higher levels of P in the Chora region with the presence of naturally occurred phosphates and/or phosphate fertilisation. Despite the significant number of samples taken for analysis,

361 Page 12 of 15


Table 7 Percentage of parcels in Chora and Peza in various score classes for each of the three triplets which are associated with the pesticide handling and the risk for contamination of the environment via point sources in the years 2011 to 2013 Triplet

Score class

Percentage of sites Chora 2011

Pesticide wastewater handling

Pesticide transport

Storage

Management of obsolete containers

0–0.1

Peza 2012

2013

2011

2012

2013

8.4

7.9

6.3

0

6.6

5.5

0.1–0.2

33.6

47.4

59.4

80.0

71.9

72.4

0.2–0.3

53.8

41.2

32.0

16.4

18.2

21.3

0.3–0.4

4.2

3.5

2.3

3.6

3.3

0.8

0.4–0.5

0

0

0

0

0

0

> 0.5

0

0

0

0

0

0

0–0.1

13.4

23.7

38.3

25.5

7.4

5.5

0.1–0.2

58.0

46.5

48.4

52.7

70.3

76.4

0.2–0.3

28.6

29.8

13.3

13.6

16.5

18.1

0.3–0.4

0

0

0

7.3

5.8

0

> 0.5

0

0

0

0.9

0

0

0–0.1

77.0

76.5

80.5

2.0

2.0

2.0

0.1–0.2

23.0

23.5

19.5

54.5

54.5

54.0

0.2–0.3

0

0

0

1.0

1.0

1.0

0.3–0.4

0

0

0

0.5

0.5

1.0

0.4–0.5

0

0

0

14.5

14.5

14.5

> 0.5

0

0

0

27.5

27.5

27.5

0–0.1

40.3

50.9

57.0

79.1

79.3

78.0

0.1–0.2

51.3

44.7

39.1

20.9

20.6

22.1

0.2–0.3

8.4

4.4

3.9

0

0

0

0.3–0.4

0

0

0

0

0

0

0.4–0.5

0

0

0

0

0

0

> 0.5

0

0

0

0

0

0

correlation analysis performed did not reveal association between detected residue levels and pH, soil type or any of the physicochemical soil properties probably due to the fact that the interval between application and sampling differed significantly between sites. Monitoring of glyphosate and AMPA in organic farms in Chora and conventional farms in Chora and Peza where glyphosate is not used A number of soil samples were collected from certified organic farms of Chora (OF1-OF13) and conventionally cultivated groves in both target areas, where glyphosate is not used for weed control (Supplementary Tables 1– 3). The analysis of soil samples aimed at the examination of possible occurrence of glyphosate residues

transferred from bordering sites, where glyphosate is used for weed control, or from other unpredictable routes of entry. Except for one site in which glyphosate was traced at levels of 27 μg kg−1 in 2013, no glyphosate was detected at any sampling event in the OF sites. The metabolite AMPA was detected in five sites, in at least one sampling event, and at concentrations ranging from 13 to 440 μg kg−1 (Fig. 3). It is possible that glyphosate and AMPA residues were derived from neighbouring sites via drift and run-off. Glyphosate and AMPA have been previously found in soil environments in which glyphosate had never been used as a result of surface run-off from zones where it was initially applied (Aparicio et al. 2013). However, this route of entry cannot explain the elevated concentrations of AMPA in OF5 and OF7 sites. Further scrutiny revealed


Page 13 of 15 361

Fig. 3 Glyphosate and AMPA residues in 2012–2014 in nine sites from organic farms in Chora (only samples with data on more than 1 year are presented)

that the two sites were used as spots for washing of application equipment after use in nearby fields in 2012. The high AMPA levels can thus be considered as a result of point source pollution. The improper disposal of spraying remnants was not repeated at subsequent years. The quantified levels of AMPA in these two sites significantly decreased in 2013, resulting in 93–100% dissipation of the initial amount within 1 year. Except for one site in which AMPA amounted to 25 μg kg−1, no glyphosate or AMPA residues was traced in the 16 sites in Peza in which no chemical weed control was carried out the year of sampling (Supplementary Table 1). Furthermore, AMPA residues ranging from 16 to 21 μg kg−1 were quantified in the six sites in Chora where glyphosate was not used for weed control (Supplementary Table 2). Comparison of residue levels with results from other monitoring studies The frequency of quantification and the concentration levels of glyphosate and AMPA were compared to results from other studies conducted in the EU (Silva et al. 2018; Todorovic et al. 2013) and elsewhere (Battaglin et al. 2014; Primost et al. 2017; Aparicio et al. 2013; Lupi et al. 2015). In a recent EU-wide survey, the highest frequency of glyphosate and AMPA detection in soil was found in sites with permanent crops, including olive trees (Silva et al. 2018). In our study, the predominant form of soil residues was AMPA (the metabolite was present in 61%, whilst glyphosate was detected in 14% of the glyphosate-treated sites). These findings are consistent with the those of Silva et al.

(2018), where AMPA and glyphosate were present in 45 and 21% of the total samples, respectively. Although, direct comparison between values reported in different studies entails a certain level of uncertainty associated with the differences on application (e.g. dose and timing) and sampling (e.g. soil sampling depth) procedures in the respective studies, it may provide a general indication of the level of AMPA and glyphosate contamination in different regions. The maximum concentration of contaminants in the monitoring sites of our study was 1000 μg kg−1 (summed glyphosate and AMPA residues). This value is comparable to the maximum values of 2000 and > 500 μg kg−1 which were reported in other EU studies (Silva et al. 2018; Todorovic et al. 2013). Even higher values of the two contaminants were reported in other regions. Specifically, a mean of 2299 and 4200 μg kg−1 for glyphosate and AMPA, respectively, were found in Mesopotamic Pampas soils and a maximum summed concentration of 1800 μg kg−1 in agricultural basins in Argentina (Primost et al. 2017; Aparicio et al. 2013).

Conclusions Glyphosate and the primary metabolite AMPA were present at maximum concentrations of 350 and 650 μg kg−1, respectively, in soil sampled from olive groves in two monitoring areas in Greece. The residual amount of both contaminants differed between areas. Reduction of pesticide losses in the environment, which was one of the objectives of the SAGE10 project, was achieved by a combination of reduced glyphosate loads

361 Page 14 of 15

(especially in Chora, Trifilias, Peloponnese) and decreased glyphosate point source entries. The steep reduction of MCs/PECs values at the second and third year of monitoring was mirrored in the IAP Method triplet score, where aspects related to point source contamination were decreased, which in turn can be considered as a result of the targeted training of operators. Acknowledgements This work was carried out in the frame of the co-funded project by the European Commission and BPI, (LIFE09 ENV/GR/000302 SAGE 10) BEstablishment of Impact Assessment Procedure as the tool for the sustainability of agro-ecosystem: the case of Mediterranean olives^ (http://www.sage10.gr/index.php/en/). Data summarised in Tables 1 and 2 were kindly provided by the Land Reclamation Institute (LRI), Thessaloniki, Greece.

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